Separation, purification and structural characterization of marine oligosaccharides: A comprehensive and systematic review of chromatographic methods.

Marine oligosaccharides have now been applied in a wide range of industry due to various kinds of physiological activities. However, the oligosaccharides with different polymeric degrees (Dps) differed in physiological activities and applicable fields. So it is promising and essential to separate, purify and structurally characterize these oligosaccharides for understanding their structure-function relationship. This review will summarize the lasted developments in the separation, purification and structural characterization of marine oligosaccharides, including the alginate oligosaccharides, carrageenan oligosaccharides, agar oligosaccharides, chitin oligosaccharides and chitosan oligosaccharides, emphasizing the successful examples of methods for separation and purification. Furthermore, an outlook for preparation of functional oligosaccharides in food biotechnology and agriculture fields is also included. This comprehensive review could definitely promote the utilization of marine functional polysaccharides for food and agriculture.

Ulvan and Ulva oligosaccharides: a systematic review of structure, preparation, biological activities and applications.

Ulva is one of the main green algae causing green tide disasters. Ulvan is the primarily component polysaccharide of the cell wall of Ulva and its complex structure and monosaccharide composition resulted in various biological activities. However, the high-value and effective utilization of extracted ulvan have been obstructed by limitations ranging from large molecular weight and low solubility to poor bioavailability. Ulva oligosaccharide obtained by degrading ulvan can not only ideally retain the various biological activities of ulvan very well but also effectively solve the problems of low solubility and poor bioavailability. The preparation and biological activity studies of ulvan and Ulva oligosaccharides have become a hot spot in the field of marine biological resources development research. At present, the comprehensive reviews of ulvan and Ulva oligosaccharides are still scarce. What are overviewed in this paper are the chemical composition, structure, extraction, and purification of ulvan and Ulva oligosaccharides, where research progress on the biological activities of ulvan and Ulva oligosaccharides is summarized and prospected. A theoretical and practical basis has been provided for further research on ulvan and Ulva oligosaccharides, as well as the high-value development and effective utilization of marine algae resources.

Ulva (Enteromorpha) Polysaccharides and Oligosaccharides: A Potential Functional Food Source from Green-Tide-Forming Macroalgae.

The high-valued utilization of Ulva (previously known as Enteromorpha) bioresources has drawn increasing attention due to the periodic blooms of world-wide green tide. The polysaccharide is the main functional component of Ulva and exhibits various physiological activities. The Ulva oligosaccharide as the degradation product of polysaccharide not only possesses some obvious activities, but also possesses excellent solubility and bioavailability. Both Ulva polysaccharides and oligosaccharides hold promising potential in the food industry as new functional foods or food additives. Studies on Ulva polysaccharides and oligosaccharides are increasing and have been the focus of the marine bioresources field. However, the comprehensive review of this topic is still rare and do not cover the recent advances of the structure, isolation, preparation, activity and applications of Ulva polysaccharides and oligosaccharides. This review systematically summarizes and discusses the recent advances of chemical composition, extraction, purification, structure, and activity of Ulva polysaccharides as well as oligosaccharides. In addition, the potential applications as new functional food and food additives have also been considered, and these will definitely expand the applications of Ulva oligosaccharides in the food and medical fields.

Evolving strategies for marine enzyme engineering: recent advances on the molecular modification of alginate lyase.

Alginate, an acidic polysaccharide, is formed by ß-d-mannuronate (M) and α-l-guluronate (G). As a type of polysaccharide lyase, alginate lyase can efficiently degrade alginate into alginate oligosaccharides, having potential applications in the food, medicine, and agriculture fields. However, the application of alginate lyase has been limited due to its low catalytic efficiency and poor temperature stability. In recent years, various structural features of alginate lyase have been determined, resulting in modification strategies that can increase the applicability of alginate lyase, making it important to summarize and discuss the current evidence. In this review, we summarized the structural features and catalytic mechanisms of alginate lyase. Molecular modification strategies, such as rational design, directed evolution, conserved domain recombination, and non-catalytic domain truncation, are also described in detail. Lastly, the application of alginate lyase is discussed. This comprehensive summary can inform future applications of alginate lyases.

A Multicenter Study of Viral Aetiology of Community-Acquired Pneumonia in Hospitalized Children in Chinese Mainland.

Community-acquired pneumonia (CAP) is one of the leading causes of morbidity and mortality in children worldwide. In this study, we aimed to describe the aetiology of viral infection of pediatric CAP in Chinese mainland. During November 2014 to June 2016, the prospective study was conducted in 13 hospitals. The hospitalized children under 18 years old who met the criteria for CAP were enrolled. The throat swabs or nasopharyngeal aspirates (NPAs) were collected which were then screened 18 respiratory viruses using multiplex PCR assay. Viral pathogens were present in 56.6% (1539/2721) of the enrolled cases, with the detection rate of single virus in 39.8% of the cases and multiple viruses in 16.8% of the cases. The most frequently detected virus was respiratory syncytial virus (RSV) (15.2%, 414/2721). The highest detection rate of virus was in < 6-month-age group (70.7%, 292/413). RSV, human metapneumovirus (HMPV), human parainfluenza viruses (HPIVs) and influenza B virus (Flu B) showed the similar prevalence patterns both in north and south China, but HPIVs, Flu A, human bocavirus (HBoV), human adenovirus (HAdV) and human coronaviruses (HCoVs) showed the distinct circulating patterns in north and south China. Human enterovirus/human rhinovirus (HEV/HRV) (27.6%, 27/98), HBoV (18.4%, 18/98), RSV (16.3%, 16/98) and HMPV (14.3%, 14/98) were the most commonly detected viruses in severe pneumonia cases with single virus infection. In conclusion, viral pathogens are frequently detected in pediatric CAP cases and may therefore play a vital role in the aetiology of CAP. RSV was the most important virus in hospitalized children with CAP in Chinese mainland.

Rapid Naked-Eye Tracking of On-Cell Phenotype Based on Dual-Aptamer-Weaved Cascade Assembly of Nanostructures.

Phenotypic plasticity is an emerging paradigm for providing biological and clinical insights into cancer initiation, progression, and resistance to therapy. However, it is a great challenge to track phenotypic information on live cells with high levels of sensitivity, specificity, and simplicity, when a specific cancer-cell subset is being targeted. In this work, we have successfully achieved cascade assembly of nanoparticles on the surface of specific cancer cells by designing a dual-aptamer-weaved molecular AND logic system. Taking advantage of spatial addressability, precise controllability, and targeting recognition of the nanostructure assemblies, we can precisely label the target-cell subset in a large population of similar cells and rapidly obtain phenotypic information in response to the surface changes of captured cancer cells. Without sophisticated instruments, we can know the phenotypic information on HepG2 cells in whole blood with a high level of sensitivity and rapid naked-eye tracking of on-cell phenotype changes of HepG2 cells undergoing epithelial-mesenchymal transition.

Molecular Characteristics of Human Adenovirus Type 3 Circulating in Parts of China During 2014-2018.

Human adenoviruses (HAdVs) are important pathogens causing respiratory infections; 3.5-11% of childhood community-acquired pneumonia is associated with HAdV infection. Human adenovirus type 3 (HAdV-3), leading to severe morbidity and mortality, is one of the most prevalent genotype among adenoviruses responsible for acute respiratory infections (ARIs) in children in China. To identify the genetic variation of HAdV-3 in children with ARIs in China, a molecular epidemiological study was conducted. A total of 54 HAdV-3 isolated strains were obtained from children with ARIs in Beijing, Wenzhou, Shanghai, Shijiazhuang, Hangzhou, Guangzhou, and Changchun from 2014 to 2018. Thirty-two strains of which were selected for whole-genome sequencing, while the hexon, penton base, and fiber genes were sequenced for remaining strains. Bioinformatics analysis was performed on the obtained sequences. The phylogenetic analyses based on whole-genome sequences, major capsid protein genes (hexon, penton base, and fiber), and early genes (E1, E2, E3, and E4) showed that the HAdV-3 strains obtained in this study always clustered together with the reference strains from Chinese mainland, while the HAdV-3 prototype strain formed a cluster independently. Compared with the prototype strain, all strains possessed nine amino acid (AA) substitutions at neutralization antigenic epitopes of hexon. The homology models of the hexon protein of the HAdV-3 prototype and strain BJ20160214 showed that there was no evident structural change at the AA mutation sites. Two AA substitutions were found at the Arg-Gly-Asp (RGD) loop and hypervariable region 1 (HVR1) region of the penton base. A distinct AA insertion (20P) in the highly conserved PPPSY motif of the penton base that had never been reported before was observed. Recombination analysis indicated that partial regions of protein IIIa precursor, penton base, and protein VII precursor genes among all HAdV-3 strains in this study were from HAdV-7. This study showed that the genomes of the HAdV-3 strains in China were highly homologous. Some AA mutations were found at antigenic sites; however, the significance needs further study. Our data demonstrated the molecular characteristics of HAdV-3 circulating in China and was highly beneficial for further epidemiological exploration and the development of vaccines and drugs against HAdV-3.

Polyvalent Biotinylated Aptamer Scaffold for Rapid and Sensitive Detection of Tau Proteins.

Biomimetic construction of artificial scaffolds has attracted increasing attention. However, the construction methods usually require redundant materials and procedures, which is inconvenient for further application. Herein, inspired by the polyvalent multifunctional structure in nature, we have designed a polyvalent biotinylated aptamer scaffold (PBAS) which can conduct analytical performance with high sensitivity and simplified procedures. To construct a PBAS, the aptamers are designed to hybridize with prepared linker probes to form polyvalent biotinylated scaffolds, which contain both multiple aptamers and signal labels. Therefore, multifunctional scaffolds can be constructed with high recognition and capture efficiency as well as significant signal amplification. Furthermore, the scaffold can be used for the assay of some disease marker proteins. By taking tau proteins as an example, the proposed aptasensor can exhibit excellent performance with a low detection limit of 153 pg mL-1 and a short assay time of 50 min, which is much better than most of the previous methods. By assays of tau proteins in both serum and artificial cerebro spinal fluid, the PBAS-based aptasensor can work well. Therefore, the scaffold may be expected to be a powerful analytical tool which may have wide applications in the detection of a variety of analytes.

Recent advances in optical aptasensor technology for amplification strategies in cancer diagnostics.

Aptamers are chemically synthetic single-stranded DNA or RNA molecules selected by molecular evolution. They have been widely used as attractive tools in biosensing and bioimaging because they can bind to a large variety of targets with high sensitivity and high affinity and specificity. As recognition elements, aptamers contribute in particular to cancer diagnostics by recognizing different cancer biomarkers, while they can also facilitate ultrasensitive detection by further employing signal amplification elements. Optical techniques have been widely used for direct and real-time monitoring of cancer-related biomolecules and bioprocesses due to the high sensitivity, quick response, and simple operation, which has greatly benefited cancer diagnostics. In this review, we highlight recent advances in optical platform-based sensing strategies for cancer diagnostics aided by aptamers. Limitations and current challenges are also discussed.

Dual-Responsive DNA Nanodevice for the Available Imaging of an Apoptotic Signaling Pathway in Situ.

Fluorescence imaging tools enable the in situ visualization of those molecules involved in various cell signaling pathways, but directly describing these pathways instead of the separate mediators in situ is much more meaningful and still full of challenges. In this work, a dual-responsive DNA nanodevice that allows the available imaging of an apoptotic signaling pathway in living cells has been developed. The nanodevice is constructed through assembling an elaborately designed Y-shaped DNA (Y-DNA) layer on gold nanoparticles (AuNPs). Only if an apoptotic signaling pathway involving the manganese superoxide dismutase (MnSOD) mRNA and downstream cytochrome c (Cyt c) is presented to serve as the input, the nanodevice can perform an "AND" logic gate operation, disassembling the Y-DNA from the AuNP surface and thereafter outputting a fluorescence signal. In comparison with the fluorescence imaging methods that target individual specific molecules, our strategy allows direct profiling of a specific signaling pathway by connected characterization of two correlative targets. The programmable feature of this strategy also shows the potential for the profiling of other signaling pathways. The concept of studying the line connecting two points will contribute to the systematic interrogation on the signaling networks in situ as the networks are composed of lines instead of individual points.

Preparation of trisaccharides from alginate by a novel alginate lyase Alg7A from marine bacterium Vibrio sp. W13.

Enzymatic digestion of sodium alginate to produce specific oligosaccharides has attracted great attention. However, commercial enzymes that efficiently produce specific oligosaccharides are still unavailable. In the present study, a novel gene encoding an alginate lyase (designated alg7A) was cloned from the marine bacterium Vibrio sp. W13 and expressed in E. coli. The recombinant Alg7A shows high activities toward alginate, poly-α-l-guluronate (polyG), poly-ß-d-mannuronate (polyM) and polyMG, and more preferred to polyMG. Moreover, the enzyme contains a highly conserved domain of the Polysaccharide Lyase (PL) 7 family (R*E*R, Q*H and Y*KAG*Y*Q), which indicates that it belongs to PL7. Furthermore, the thin layer chromatography and ESI-MS analysis showed that Alg7A mainly releases trisaccharides from alginate. These results demonstrated that Alg7A has a great potential to be used to produce oligosaccharides from alginate.

Bridging exosome and liposome through zirconium-phosphate coordination chemistry: a new method for exosome detection.

We have proposed a new exosomes-zirconium-liposomes sandwich structure to detect exosomes by using zirconium-phosphate coordination chemistry. The combined use of the intrinsic property of phosphate in both exosomes and liposomes as well as zirconium ions can endow the method lower cost, no modified label, simplicity and high efficiency.

Elucidation of degrading pattern and substrate recognition of a novel bifunctional alginate lyase from Flammeovirga sp. NJ-04 and its use for preparation alginate oligosaccharides.

BACKGROUND: The alginate oligosaccharides have been widely used in agriculture, medicine, and food industries due to their versatile physiological functions such as antioxidant, anticoagulant, and antineoplastic activities. The bifunctional alginate lyases can degrade the alginate polysaccharide more efficiently into alginate oligosaccharides. Therefore, it is crucial to discover new bifunctional alginate lyase for alginate oligosaccharide production. RESULTS: Herein, a novel bifunctional alginate lyase FsAlgB was cloned and identified from deep-sea bacterium Flammeovirga sp. NJ-04, which exhibited broad substrate specificity and the highest activity (1760.8 U/mg) at pH 8.0 and 40 °C. Furthermore, the K m values of FsAlgB towards polyG (0.69 mM) and polyMG (0.92 mM) were lower than that towards sodium alginate (1.28 mM) and polyM (2.06 mM). Recombinant FsAlgB was further characterized as an endolytic alginate lyase, and it can recognize the tetrasaccharide as the minimal substrate and cleave the glycosidic bonds between the subsites of - 3 and + 1. CONCLUSION: This study provided extended insights into the substrate recognition and degrading pattern of alginate lyases with broad substrate specificity.

Modularization of three-dimensional gold nanoparticles/ferrocene/liposome cluster for electrochemical biosensor.

For electrochemical biosensors, just like a computer, the modularization and coordinated operation of different components will facilitate the development of versatile biosensors and effectively reduce costs. However, the efficient synergy between different modules is always difficult. It would be a beneficial way to construct the multi-functional module. In this work, a three-dimensional gold nanoparticles/ferrocene/liposome cluster (GFLC) is fabricated and explored as a building block for the fabrication of an electrochemical biosensor, in which gold nanoparticles, ferrocene and liposome cluster work as a signal amplification component, a signal output component and a molecular recognition component, respectively. With the synergy of multi-functions, GFLC has been successfully applied for electrochemical analysis of lipopolysaccharide (LPS) in food samples. LPS can be linearly assayed in the range from 2 × 10-9 µg/mL to 8 µg/mL with a detection limit of 0.51 × 10-10 µg/mL. In view of the favorable modularization effect, GFLC shows a great potential in the development of electrochemical biosensor with considerable versatility and cost-efficiency.

Cucurbit[8]uril-assisted peptide assembly for feasible electrochemical assay of histone acetyltransferase activity.

Accumulating findings demonstrate the importance of histone acetyltransferases (HATs) in regulating the acetylation of histones and reveal that their aberrant catalytic activities are involved in the occurrence and progress of numerous diseases. Herein, a feasible electrochemical method is proposed to assay the activity of HAT. The critical elements of the assay method are the hindrance of HAT-catalyzed acetylation against carboxypeptidase Y-catalyzed digestion and cucurbit[8]uril-assisted peptide assembly, which may recruit peptide-templated silver nanoparticles onto the electrode surface, producing significant electrochemical signals. Taking p300 as a model HAT, the assay method is validated to exhibit desirable selectivity, reproducibility, and usability in inhibitor analysis, and allow absolute activity determination in a linear range from 0.1 to 50 nM with a detection limit of 0.055 nM, which is lower than those of previous reports. Therefore, this work may provide an effective tool for HAT activity assay, which will be of great potential in HAT-related fundamental research, disease diagnosis, and drug development in the future. Graphical abstract ᅟ.

Biochemical Characterization and Degradation Pattern of a Novel Endo-Type Bifunctional Alginate Lyase AlyA from Marine Bacterium Isoptericola halotolerans.

Alginate lyases are important tools to prepare oligosaccharides with various physiological activities by degrading alginate. Particularly, the bifunctional alginate lyase can efficiently hydrolyze the polysaccharide into oligosaccharides. Herein, we cloned and identified a novel bifunctional alginate lyase, AlyA, with a high activity and broad substrate specificity from bacterium Isoptericola halotolerans NJ-05 for oligosaccharides preparation. For further applications in industry, the enzyme has been characterized and its action mode has been also elucidated. It exhibited the highest activity (7984.82 U/mg) at pH 7.5 and 55 °C. Additionally, it possessed a broad substrate specificity, showing high activities towards not only polyM (polyß-d-mannuronate) (7658.63 U/mg), but also polyG (poly α-l-guluronate) (8643.29 U/mg). Furthermore, the Km value of AlyA towards polyG (3.2 mM) was lower than that towards sodium alginate (5.6 mM) and polyM (6.7 mM). TLC (Thin Layer Chromatography) and ESI-MS (Electrospray Ionization Mass Spectrometry) were used to study the action mode of the enzyme, showing that it can hydrolyze the substrates in an endolytic manner to release a series of oligosaccharides such as disaccharide, trisaccharide, and tetrasaccharide. This study provided extended insights into the substrate recognition and degrading pattern of the alginate lyases, with a broad substrate specificity.

Cloning and characterization of a new pH-stable alginate lyase with high salt tolerance from marine Vibrio sp. NJ-04.

Marine polysaccharide-degrading enzymes play an important role in marine algae degradation and carbon cycling, especially the alginate lyases. Although many alginate lyases have been characterized, the enzymes with industrial potential are still rather rare. A gene, encoding a new alginate lyase AlgNJ04, has been cloned from the marine bacterium Vibrio sp. NJ04. The recombinant alginate lyase was characterized followed by purification on Ni-NTA Sepharose. It exhibited an optimum activity (2416 U/mg) at pH 7.0 and 40 °C. Notably, the AlgNJ04 retained more than 80% of its maximum activity at a broad pH range of pH 4.0 and 10.0, which exhibited excellent pH stability. Additionally, it possessed broader substrate specificity, showing activities towards both poly ß-D-mannuronate (polyM) and poly α-L-guluronate (polyG). Furthermore, the Km values of AlgNJ04 towards sodium alginate (0.49 mM) and polyG (0.24 mM) were lower than that towards polyM (0.86 mM). Notably, the activity of AlgNJ-04 could be activated by NaCl with certain concentrations, which was partly caused by the removal of bound water from sodium alginate molecules or by the effects of charges in forming the alginate-enzyme complex. The ESI-MS analysis suggested that it mainly released oligosaccharides with DP of 2-5 as end products in an endolytic manner. Therefore, it may be a potent tool to produce alginate oligosaccharides with lower DPs.

Characterization of a new endo-type alginate lyase from Vibrio sp. NJU-03.

A gene, encoding a new alginate lyase AlgNJU-03, was cloned from marine bacteria Vibrio sp. NJU-03. The recombinant alginate lyase was characterized followed by being purified by NTA-Ni Sepharose. It exhibited the highest activity (6468.99U/mg) at pH 7.0 and 30°C. Interestingly, AlgNJU-03 possessed broader substrate specificity and high activity toward both polyM (poly ß-d-mannuronate) and polyG (poly α-l-guluronate), indicating that it is a bifunctional alginate lyase. Furthermore, Km of AlgNJU-03toward polyG (4.00mM) is lower than those toward alginate (8.50mM) and polyM (10.94mM), demonstrating that the enzyme has a higher affinity to polyG. Meanwhile, the catalytic efficiency (Kcat/Km) toward polyG (11.47s-1/mM) is much higher than those toward sodium alginate (3.60s-1/mM) and polyM (0.50s-1/mM). ESI-MS analysis suggested that AlgNJU-03 mainly released disaccharides, trisaccharides and tetrasaccharides from the three kinds of substrates in an endolytic manner. Therefore, it may be a potential tool to produce alginate oligosaccharides with low DP.

Cloning and biochemical characterization of a novel κ-carrageenase from newly isolated marine bacterium Pedobacter hainanensis NJ-02.

Enzymatic preparation of carrageenan oligosaccharides has drawn increasing attention due to its advantages of mild reaction conditions and excellent product-specificity. A novel gene (CgkA) encoding a new κ-carrageenase was cloned, heterogeneously expressed and characterized from a newly isolated marine bacterium Pedobacter hainanensis NJ-02. It consisted of 1539bp and encoded 512 amino acid residues with a molecular weight of 57.12kDa. Multiple alignment analysis indicated that CgkA belongs to glycoside hydrolase (GH) family 16 and was most homologous to κ-carrageenase of Zobellia sp. M-2 with identity of 50%. The recombinant enzyme showed maximal activity of 3659.72U/mg at 40°C and pH 7.0. Additionally, it could retain more than 80% of its maximal activity after being incubated at pH of 5.0-9.0 below 40°C.K+ and Na+ with a wide range of concentration can activate the enzyme, while other divalent ions such as Cu2+, Zn2+ showed inhibitory effect on the enzyme. The ESI-MS analysis of hydrolysates indicated that the enzyme can endolytically depolymerize the carrageenan into tetrasaccharides and hexasaccharides. The results suggest that it is an endo-type carrageenase and could be a valuable enzyme tool to produce carrageenan oligosaccharides with higher Dps.