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Antioxidant biomaterials have attracted much attention in various biomedical fields because of their effective inhibition and elimination of reactive oxygen species (ROS) in pathological tissues. However, the difficulty in ensuring biocompatibility, biodegradability and bioavailability of antioxidant materials has limited their further development. Novel bioavailable antioxidant materials that are derived from natural resources are urgently needed. Here, an integrated multi-omics method was applied to fabricate antioxidant biomaterials. A key cysteine-rich thrombospondin-1 type I repeat-like (TSRL) protein was efficiently discovered from among 1262 adhesive components and then used to create a recombinant protein with a yield of 500 mg L−1. The biocompatible TSRL protein was able to self-assemble into either a water-resistant coating through Ca2+-mediated coordination or redox-responsive hydrogels with tunable physical properties. The TSRL-based hydrogels showed stronger 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging rates than glutathione (GSH) and ascorbic acid (Aa) and protected cells against external oxidative stress significantly more effectively. When topically applied to mice skin, TSRL alleviated epidermal hyperplasia and suppressed the degradation of collagen and elastic fibers caused by ultraviolet radiation B (UVB) irradiation, confirming that it enhanced antioxidant activity in vivo. This is the first study to successfully characterize natural antioxidant biomaterials created from marine invertebrate adhesives, and the findings indicate the excellent prospects of these biomaterials for great applications in tissue regeneration and cosmeceuticals., Graphical abstract Image 1, Highlights • A cysteine-rich TSRL protein was efficiently discovered from 1262 adhesive components. • The TSRL-based hydrogels exhibited significantly better protection for cells against external oxidative stress. • It is the first time to identify and characterize natural antioxidant biomaterials from marine invertebrate adhesives.

The extensive use of proteases in many areas, especially in the textile, food, and detergent industries, has led increases in the discovery and characterization of novel proteolytic enzymes. Microorganism from the Antarctic may contain novel proteases because of the extreme environment; however, to our knowledge, few relevant studies have been reported to date. Here, an Antarctic soil metagenomic fosmid library was successfully constructed, and a 36 kb positive fosmid clone was screened using lactose-free skim milk agar. Sequence alignment and phylogenetic analysis indicated that the positive fosmid clone encoded a new protein belonging to the metalloprotease subfamily M43B (36.6 % identity), which was named as Pr18H-2. Enzymatic analysis revealed that Pr18H-2 possess several features: (1). it demonstrated high thermal stability with the optimal temperature was 60℃; (2). it was active in the broad pH range of 5.0–10.0, with the optimal pH of 8.0 which allowed it to be an alkaline protease; (3). it was able to degrade a wide range of protein substrates and exhibit the substrate preferences for small substrates, such as casein hydrolysate and fibrin; (4). it showed that the enzymatic activity was increased by approximately four times in the presence of 5 mM Co2+.

Recently, we reported alginate lyase AlyF that predominantly produced trisaccharides (the trisaccharide content is 87.0%), and the determination of its substrate-binding mode facilitated its protein engineering for new product distribution. To clarify the relationship between the substrate-binding pocket and end-product distribution, the open binding pocket change was initially designed. The resulting F128T_W172R mutant of AlyF exhibited different intermediate-product distributions but still similar end-product distributions. However, these observations suggested that cleavage pattern changes for intermediate products might contribute to an altered end-product distribution. Structural analysis indicated that the sugar-binding affinity at subsite -2 should be redesigned to achieve this goal. Thus, residue Arg266, which is involved in sugar binding at subsite -2, was selected for site-saturation mutagenesis in the F128T_W172R mutant. The dominant end products of the F128T_W172R_R226H mutant were altered to disaccharides and trisaccharides (the disaccharide content increased to 40.5%).

Panzerina lanata is a Chinese medicine with the bioactivity of detumescence and detoxification. In this study, novel qualitative and quantitative methods were established by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry and ultra-high-performance liquid chromatography-triple quadrupole linear ion trap mass spectrometry, respectively. As a result, 20 compounds were identified or tentatively characterized including flavonoids, organic acids, alkaloids, and lignans, five of which were identified for the first time based on the reference standards. The quantitative approach exhibited good linearity (R2 > 0.995), precision (RSDs

To improve the weak strength of biodegradable poly(propylidene carbonate), blending with the both biodegradable polylactide is very effective if they can form good interfaces. Nanoparticles can impart their superior properties on the matrix as well as enhance the interface of the blends. However, anchoring nanoparticles at the interface and avoidance of nanoparticles transferring into the continuous phase or dispersed phase during varied processing are still a challenge. Poly(D-lactide) (PDLA) was grafted onto GO and added into poly(propylidene carbonate)/polylactide (70/30) blend to solve this problem. By investigating the structures of solution cast film samples and melt processing samples, it was demonstrated that GO was anchored at the interface of the blends, which decreased the sizes of dispersed phase. Atomic force microscope investigations found that the interfacial width increased with the increase in PDLA-g-GO contents. Consequently, the mechanical properties were significantly improved. In particular, the tensile strength and Young’s modulus of the solution cast film samples with loading 10 wt% PDLA-g-GO increased by 310% and 226%, respectively. According to the shifting glass transition temperature (Tg) of PPC and PLLA phase, it was deduced that the rigid interfacial phase of PDLA-g-GO linked both PPC and PLLA and played compatibilizer. Therefore, it is expected to deepen the understanding of the compatibilization mechanism of nanoparticles and promote the usages of biodegradable materials.

The poor thermal stability of cellulose nano-crystals (CNCs) and dispersion in polymer matrices are significant obstacles limiting their applications in modification of polymers, especially during melt processing. The self-assembly nucleation agent, decamethylene dicarboxylic dibenzoyl hydrazide (TMC300), was used to modify CNCs via physical adsorption methods. Because TMC300 adsorbed on the surface of CNCs shields the sulfonate on CNCs, the thermal stability of CNCs was significantly improved. Meanwhile, modified CNCs formed different self-assembly morphologies in confined and unconfined spaces, which influenced the crystallization behaviors and microstructures of poly(L-lactide) (PLLA). It improved the crystallization rate of PLLA, which increased the crystallinities and heat resistances of PLLA/CNC composites. More importantly, the self-assembly nucleation agent induced the shish-kebab structures of PLLA, which enhanced the interfacial structure as mechanically interfacial lock and simultaneously improved tensile strength and toughness. This physical and economical modification method of CNCs is anticipated to take full advantage of CNCs to modify PLLA or other semi-crystalline polymers via melt processing.

Cardiovascular diseases (CVDs) are the leading causes of premature death and disability in people around the world. Therefore, the prevention and treatment of CVDs has become an important subject. In this study, we verified the thrombolytic activities of a nattokinase‐like protease named NK‐01 in vivo. Label‐free liquid chromatography–tandem mass spectrometry (LC‐MS/MS) technique was used in our study. NK‐01 could inhibit the activity of coagulation factors though the up‐regulation of proteinase C inhibitors and protein S. NK‐01 also could inhibit the angiotensinogen conversion to AngII and promote the degradation of kininogen to reduce the blood pressure. In addition, NK‐01 could increase the content of paraoxonase 1, which could prevent atherosclerosis. In our study, we found that NK‐01 cloud effect some key proteins which participant in CVDs associated metabolic processes such as coagulation system, blood pressure, and atherosclerosis. Taken together, the underlying molecular mechanisms for the biological beneficial of NK‐01 were investigated. Our proteomic study will provide further theoretical basis for application of NK in prevention or adjuvant treatment in biomedicine areas.

Alginate lyases have a wide range of industrial applications, such as oligosaccharide preparation, medical treatment, and bioconversion. Therefore, the discovery and characterization of novel alginate lyases are extremely important. PL-6 alginate lyases are classified into two groups: those with a single domain or two domains. However, only one structure of a two-domain alginate lyase has been determined to date. In this study, we characterized a novel single-domain PL-6 alginate lyase (named AlyF). According to the biochemical analysis, AlyF possesses unique features compared with other PL-6 enzymes, including (1) a Ca2+-independent catalytic mechanism and (2) a PolyG-specific cleavage specificity that predominantly produces trisaccharides. The structures of AlyF and its complexes described here reveal the structural basis for these unique features and substrate binding mechanisms, which were further confirmed using mutagenesis. More importantly, we determined the possible subsites specifying the predominantly trisaccharide products of AlyF, which may facilitate the rational design of AlyF for potential applications in preparing a single alginate oligomer.

As the third-generation nucleic acid amplification technology, digital polymerase chain reaction (PCR) has been widely adopted in the analysis of nucleic acids. However, further application of this powerful technology is hindered due to the limitation of surfactants. Here, for the first time, we propose the use of colloidosomes self-assembled from fluorinated silica nanoparticle for digital PCR to address this limitation. A one-step fluorinated silica nanoparticle synthesis method is proposed, which is much more convenient and reproducible compared with the synthesis of conventional fluorine-based surfactants. Fluorinated silica nanoparticles facilitate the formation of colloidosomes with excellent stability capable of enduring the rapid temperature changes associated with the PCR and avoiding material exchange (cross-talk) between droplets for high-fidelity analysis. The colloidosome digital PCR method was developed using these colloidosomes as highly parallel reactors for single-copy nucleic acid amplification and rare mutant detection. The method is robust and accurate, and it offers possibilities for a great variety of applications, such as gene expression studies, single cell analysis, and circulating tumor DNA detection.

Chitinase, as one of the most important extracellular enzymes in the marine environment, has great ecological and applied values. In this study, two chitinases (Chi1557 and Chi4668) with 97.33% amino acid sequences identity were individually found in Vibrio rotiferianus and Vibrio harveyi. They both were encoding by 561 amino acids, but differed in 15 amino acids and showed different enzymatic properties. The optimal temperature and pH ranges were 45&ndash, 50 &deg, C and pH 5.0&ndash, 7.0 for Chi1557, while ~50 &deg, C and pH 3.0&ndash, 6.0 for Chi4668. K+, Mg2+, and EDTA increased the enzymatic activity of Chi4668 significantly, yet these factors were inhibitory to Chi1557. Moreover, Chi1557 degraded colloidal chitin to produce (GlcNAc)2 and minor GlcNAc, whereas Chi4668 produce (GlcNAc)2 with minor (GlcNAc)3 and (GlcNAc)4. The Kcat/Km of Chi4668 was ~4.7 times higher than that of Chi1557, indicating that Chi4668 had stronger catalytic activity than Chi1557. Furthermore, site-directed mutagenesis was performed on Chi1557 focusing on seven conserved amino acid residues of family GH18 chitinases. Chi1557 was almost completely inactive after Glu154, Gln219, Tyr221, or Trp312 was individually mutated, retained ~50% activity after Tyr37 was mutated, and increased two times activity after Asp152 was mutated, indicating that these six amino acids were key sites for Chi1557.

Cerebral cavernous malformations (CCM) are dysplasias that primarily occur in the neurovasculature, and are associated with mutations in three genes: KRIT1, CCM2, and PDCD10, the protein products of which are KRIT1 (Krev/Rap1 Interaction Trapped 1; CCM1, cerebral cavernous malformations 1), CCM2 (cerebral cavernous malformations 2; OSM, osmosensing scaffold for MEKK3), and CCM3 (cerebral cavernous malformations 3; PDCD10, programmed cell death 10). Until recently, these proteins were relatively understudied at the molecular level, and only three folded domains were documented. These were a band 4.1, ezrin, radixin, moesin (FERM), and an ankyrin repeat domain (ARD) in KRIT1, and a phosphotyrosine-binding (PTB) domain in CCM2. Over the past 10 years, a crystallographic approach has been used to discover a series of previously unidentified domains within the CCM proteins. These include a non-functional Nudix (or pseudonudix) domain in KRIT1, a harmonin homology domain (HHD) in CCM2, and dimerization and focal adhesion targeting (FAT)-homology domains within CCM3. Many of the roles of these domains have been revealed by structure-guided studies that show the CCM proteins can directly interact with one another to form a signaling scaffold, and that the "CCM complex" functions in signal transduction by interacting with other binding partners, including ICAP1, RAP1, and MEKK3. In this chapter, we describe the crystallization of CCM protein domains alone, and with their interaction partners.

Charge recombination loss caused by defects and interfaces in the inverted perovskite solar cells (PSCs) significantly impact cell performance and stability. Here, a kind of quaternary ammonium salt of tetra-n-octadecyl ammonium bromide (TODB) with an symmetrical structure is introduced into inverted PSCs to suppress the charge recombination loss. We have comprehensively studied the impact of TODB on the physical properties of the perovskite films and corresponding the performance of the devices. TODB post-treatment results in defect passivation, the built-in electric field enhancement as well as hydrophobicity improvement in the inverted PSCs, which correspondingly enhance device performance and stability. The highest power conversion efficiency (PCE) reaches 20.36 %, which is one of the highest PCE of the inverted PSCs based on methylammonium lead iodide (MAPbI3)/phenyl-C61-butyric acid methyl ester (PCBM) planar heterojunction. The device with TODB modification can maintain 80 % of its initial PCE value in the air for 384 h, while the control device only maintains about 52 % of its initial PCE under the same exposure conditions. This work presents an efficient strategy to improve the performance and stability of the PSCs by passivating the trap states at the interfaces and increasing the hydrophilicity of the perovskite films.

Noncatalytic carbohydrate binding modules (CBMs) have been demonstrated to play various roles with cognate catalytic domains. However, for polysaccharide lyases (PLs), the roles of CBMs remain mostly unknown. AlyB is a multidomain alginate lyase that contains CBM32 and a PL7 catalytic domain. The AlyB structure determined herein reveals a noncanonical alpha helix linker between CBM32 and the catalytic domain. More interestingly, CBM32 and the linker does not significantly enhance the catalytic activity but rather specifies that trisaccharides are predominant in the degradation products. Detailed mutagenesis, biochemical and cocrystallization analyses show "weak but important" CBM32 interactions with alginate oligosaccharides. In combination with molecular modeling, we propose that the CBM32 domain serves as a "pivot point" during the trisaccharide release process. Collectively, this work demonstrates a novel role of CBMs in the activity of the appended PL domain and provides a new avenue for the well-defined generation of alginate oligosaccharides by taking advantage of associated CBMs.

Floridean starch and floridoside are the main storage carbohydrates of red algae. However, their complete metabolic pathways and the origin, function, and regulatory mechanism of their pathway genes have not been fully elucidated. In this study, we identified their metabolic pathway genes and analyzed the changes in related gene expression and metabolite content in Neoporphyra haitanensis under continuous dark conditions. Our results showed that genes from different sources, including eukaryotic hosts, cyanobacteria, and bacteria, were combined to construct floridean starch and floridoside metabolic pathways in N. haitanensis. Moreover, compared with those in the control, under continuous dark conditions, floridean starch biosynthesis genes and some degradation genes were significantly upregulated with no significant change in floridean starch content, whereas floridoside degradation genes were significantly upregulated with a significant decrease in floridoside content. This implies that floridean starch content is maintained but floridoside is consumed in N. haitanensis under dark conditions. This study elucidates the “floridean starch–floridoside” metabolic network and its gene origins in N. haitanensis for the first time.

This paper describes a centrifugal micropipette-tip method for ELISA sample processing combined with a pressure meter for portable quantitative detection of acute myocardial infarction (AMI) biomarker myoglobin (Myo). The method enables sensitive and reliable quantification of Myo in 35 minutes. With the advantages of simplicity, speed, user friendliness, low cost, and small sample consumption, centrifugal micropipette-tip ELISA shows great potential for quantitative POC diagnostics for AMI.

Protein phosphorylation is a widespread modification that and plays a significant role in marine bioadhesion. The phosphorylated proteins of the barnacle Amphibalanus amphitrite can form strong ionic bonds with mineral surfaces to adapt to marine environments. The adhesion protein PC-3 in the sandcastle worm Phragmatopoma californica contains multipleserine phosphorylations. Interactions between these phosphate groups and the Mg/Ca2+ ions are less soluble at seawater pH, making the cement less fluid and more gel-like. The scallop byssus is characterized by strong wet adhesion performance and substantial byssus secretions. Thus, the excellent underwater adhesion properties of the byssus make it an ideal candidate for studies related to the development of new and versatile composite materials. However, phosphoproteins have not been identified or studied in the scallop Chlamys farreri. Phosphorylated proteins in the C. farreri byssus protein were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and further confirmed by phosphorylation staining and in-gel digestion coupled with mass spectrometric analysis (GeLC-MS/MS). Finally, sequence analyses and potential functional analyses were performed for these newly identified proteins. We have identified previously unreported phosphorylation sites within the C. farreri byssus protein. The results show phosphorylation modifications in all parts of the byssus structure and four foot-specific phosphorylated proteins were verified by two types of mass spectrometry and staining. The annotation of biological functions, based on sequence alignments shows that the protein 40,215.25 is homologous with TIMP-2. Similar to the previously identified TIMP-2-like protein Sbp8-1 in the scallop byssus, it contains an abundance of phosphorylated Cys, which may promote protein polymerization. We speculate that protein 40,215.25 may play an important role in cross-linking and adhesion of the scallop byssus. The phosphorylated protein we have identified in the C. farreri byssus may be related to the formation of protein cross-linkings and adhesion of the scallop foot. Our study lays the groundwork for a better understanding of the adhesion mechanism of the scallop byssus.

Due to the wide applications of chitooligosaccharides and its monomer in various fields, the approaches to produce the chitooligosaccharides and its monomer have attracted much attention. The chemical-based approach generated a series of environmental pollution problems. In contrast, the enzymatic-based approach is able to make up this deficiency. However, the cost of the enzymes is still relative high, and the lack of fundamental understanding of the enzyme catalytic mechanisms hampers the preparation of chitooligosaccharides and its monomer by enzymatic-based approach. This review not only covers the typical chemical-based approach, also pays more attention to the description of the enzymatic-based approach used for chitooligosaccharides and its monomer generation. Both the nonspecific enzymes and specific enzymes are able to be utilized. Recently, more and more investigations are carried out to reveal the underlying catalytic mechanisms for the chitosanase, especially for the chitosanase GH46. These findings significantly facilitate the applications of chitosanase and protein-engineering to improve the chitosanase degradation efficiency. In addition, the metabolic engineering of model microorganism for microbial production of GlcNAc/GlcN is summarized. This review provides the viewpoints for further bio-manufacturing to achieve the industrial production of chitooligosaccharides and its monomer.

Inverted perovskite solar cells (PSCs) have significant potential in large-scale production due to their low cost and low-temperature process-ability. Nevertheless, the efficiency of the inverted PSCs suffers from non-ideal light-harvesting, surface recombination, and charge extraction loss. Here, tetraoctylammonium bromide (TOAB) is introduced by a post-treatment strategy to address these challenges. The film properties and the corresponding device performance are systemically studied. The results show that TOAB molecules distribute throughout the perovskite films and thus effectively passivate the trap states at grain boundaries (GBs) and surface. The built-in electrical field (Vbi) of the devices and hydrophobic properties of the perovskite films are enhanced by TOAB molecular arrangement at the perovskite surface. Moreover, the light-harvesting efficiency is also improved via the redistribution of the light field in the PSCs. Benefiting from trap sates passivation, enhanced Vbi and light-harvesting, the champion power conversion efficiency (PCE) of 21.47% is achieved, which is among the reported high efficiencies for inverted PSCs. In addition, device stability has been greatly improved. The PCE of PSCs treated by TOAB can retain 81% of its initial value after 264 h in the air and can retain 94% of its initial value after 71 days in N2. This work represents a novel passivation strategy by post-treatment method without disturbing the perovskite film growth to understand the sole defect effects on the device performance of PSCs.

Endo-1,3-&beta, glucanases derived from marine mollusks have attracted much attention in recent years because of their unique transglycosylation activity. In this study, a novel endo-1,3-&beta, glucanase from the scallop Chlamys farreri, named Lcf, was biochemically characterized. Unlike in earlier studies on marine mollusk endo-1,3-&beta, glucanases, Lcf was expressed in vitro first. Enzymatic analysis demonstrated that Lcf preferred to hydrolyze laminarihexaose than to hydrolyze laminarin. Furthermore, Lcf was capable of catalyzing transglycosylation reactions with different kinds of glycosyl acceptors. More interestingly, the transglycosylation specificity of Lcf was different from that of other marine mollusk endo-1,3-&beta, glucanases, although they share a high sequence identity. This study enhanced our understanding of the diverse enzymatic specificities of marine mollusk endo-1,3-&beta, glucanases, which facilitated development of a unique endo-1,3-&beta, glucanase tool in the synthesis of novel glycosides.

Inspired by the adhesion behavior of scallops, we grafted dopamine hydrochloride onto hyaluronic acid to prepare hydrogels with horseradish peroxidase and H2O2 as triggers. We tested the performance of the hydrogels as tissue adhesives, the results showed that with increasing concentration of horseradish peroxidase and H2O2, the gelation time of these crosslinked hydrogels decreased to about 5 s; at the same time, mechanical strength and wet adhesion strength increased. Because of the outstanding mechanical properties and wet adhesion properties, and biocompatibility on a cellular level, we further investigated the hydrogels for hemostatic testing and skin healing tests. We observed a shortened hemostasis time and significant hemostatic effect in the enzymatically active hyaluronic acid hydrogels relative to the control in rat liver defect and rat artery defect models. In a rat skin injury model, we also observed a promoting effect on skin wound repair by collagen metabolism and the formation of granulation tissue. Therefore, the hydrogel inspired by the adhesion behavior of scallops has the potential to replace sutures in wound healing.

Hydroxypropyl chitosan (HP-chitosan) has been shown to have promising applications in a wide range of areas due to its biocompatibility, biodegradability and various biological activities, especially in the biomedical and pharmaceutical fields. However, it is not yet known about its pharmacokinetics and biodegradation performance, which are crucial for its clinical applications. In order to lay a foundation for its further applications and exploitations, here we carried out fluorescence intensity and GPC analyses to determine the pharmacokinetics mode of fluorescein isothiocyanate-labeled HP-chitosan (FITC-HP-chitosan) and its biodegradability. The results showed that after intraperitoneal administration at a dose of 10 mg per rat, FITC-HP-chitosan could be absorbed rapidly and distributed to liver, kidney and spleen through blood. It was indicated that FITC-HP-chitosan could be utilized effectively, and 88.47% of the FITC-HP-chitosan could be excreted by urine within 11 days with a molecular weight less than 10 kDa. Moreover, our data indicated that there was an obvious degradation process occurred in liver (< 10 kDa at 24 h). In summary, HP-chitosan has excellent bioavailability and biodegradability, suggesting the potential applications of hydroxypropyl-modified chitosan as materials in drug delivery, tissue engineering and biomedical area.

Diacetyl chitin (DAC) is an acidylated chitin obtained using acetic anhydride mixed perchloric acid system. By wet spinning and weaving technique, DAC has been successfully developed into a novel absorbable surgical suture. Thanks to the unique properties of chitins, the potential application of this novel monocomponent multifilament DAC suture may break the monopoly of synthetic polymer sutures in wound closure area. In this study, DAC was synthesized and characterized by multiple approaches including elemental analysis, Fourier transform infrared spectrometry (FTIR), and X-ray diffraction (XRD). In addition, we performed the feasibility assessment of DAC suture (USP 2-0) as absorbable suture for wound healing. Several lines of evidences suggested that DAC suture had comparable mechanical properties as synthetic polymer sutures. Moreover, DAC suture retained approximately 63% of the original strength at 14 days and completely absorbed in 42 days with no remarkable tissue reaction in vivo. Most important of all, DAC suture significantly promoted skin regeneration with faster tissue reconstruction and higher wound breaking strength on a linear incisional wound model. All these results demonstrated the potential use of DAC suture in short- or middle-term wound healing, such as epithelial and connective tissue. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2015.

Chitosanase is able to specifically cleave β-1,4-glycosidic bond linkages in chitosan to produce a chito-oligomer product, which has found a variety of applications in many areas, including functional food and cancer therapy. Although several structures for chitosanase have been determined, the substrate-binding mechanism for this enzyme has not been fully elucidated because of the lack of a high-resolution structure of the chitosanase–substrate complex. In the present study we show the crystal structure of a novel chitosanase OU01 from Microbacterium sp. in complex with its substrate hexa-glucosamine (GlcN)6, which belongs to the GH46 (glycoside hydrolyase 46) family in the Carbohydrate Active Enzymes database (http://www.cazy.org/). This structure allows precise determination of the substrate-binding mechanism for the first time. The chitosanase–(GlcN)6 complex structure demonstrates that, from the −2 to +1 position of the (GlcN)6 substrate, the pyranose rings form extensive interactions with the chitosanase-binding cleft. Several residues (Ser27, Tyr37, Arg45, Thr58, Asp60, His203 and Asp235) in the binding cleft are found to form important interactions required to bind the substrate. Site-directed mutagenesis of these residues showed that mutations of Y37F and H203A abolish catalytic activity. In contrast, the mutations T58A and D235A only lead to a moderate loss of catalytic activity, whereas the S27A mutation retains ~80% of the enzymatic activity. In combination with previous mutagenesis studies, these results suggest that the −2, −1 and +1 subsites play a dominant role in substrate binding and catalysis. DSF (differential scanning fluorimetry) assays confirmed that these mutations had no significant effect on protein stability. Taken together, we present the first mechanistic interpretation for the substrate (GlcN)6 binding to chitosanase, which is critical for the design of novel chitosanase used for biomass conversion.

Marine polysaccharides are used in a variety of applications, and the enzymes that degrade these polysaccharides are of increasing interest. The main food source of herbivorous marine mollusks is seaweed, and several polysaccharide-degrading enzymes have been extracted from mollusk digestive glands (hepatopancreases). Here, we used a comprehensive proteomic approach to examine the hepatopancreatic proteins of the Zhikong scallop (Chlamys farreri). We identified 435 proteins, the majority of which were lysosomal enzymes and carbohydrate and protein metabolism enzymes. However, several new enzymes related to polysaccharide metabolism were also identified. Phylogenetic and structural analyses of these enzymes suggest that these polysaccharide-degrading enzymes may have a variety of potential substrate specificities. Taken together, our study characterizes several novel polysaccharide-degrading enzymes in the scallop hepatopancreas and provides an enhanced view of these enzymes and a greater understanding of marine polysaccharide digestion.

Crystallization of contaminating proteins is a frequently encountered problem for macromolecular crystallographers. In this study, an attempt was made to obtain a binary cocrystal structure of the SH3 domain of cortactin and a 17-residue peptide from the Arg nonreceptor tyrosine kinase encompassing a PxxPxxPxxP (PxxP1) motif. However, cocrystals could only be obtained in the presence of trace amounts of a contaminating protein. A structure solution obtained by molecular replacement followed byARP/wARPautomatic model building allowed a `sequence-by-crystallography' approach to discover that the contaminating protein was lysozyme. This 1.65 Å resolution crystal structure determination of a 1:1:1 heterotrimeric complex of Arg, cortactin and lysozyme thus provides an unusual `caveat emptor' warning of the dangers that underpurified proteins harbor for macromolecular crystallographers.

Metazoan spliced leader (SL) trans-splicing generates mRNAs with an m(2,2,7)G-cap and a common downstream SL RNA sequence. The mechanism for eIF4E binding an m²²⁷G-cap is unknown. Here, we describe the first structure of an eIF4E with an m(2,2,7)G-cap and compare it to the cognate m⁷G-eIF4E complex. These structures and Nuclear Magnetic Resonance (NMR) data indicate that the nematode Ascaris suum eIF4E binds the two different caps in a similar manner except for the loss of a single hydrogen bond on binding the m(2,2,7)G-cap. Nematode and mammalian eIF4E both have a low affinity for m(2,2,7)G-cap compared with the m⁷G-cap. Nematode eIF4E binding to the m⁷G-cap, m(2,2,7)G-cap and the m(2,2,7)G-SL 22-nt RNA leads to distinct eIF4E conformational changes. Additional interactions occur between Ascaris eIF4E and the SL on binding the m(2,2,7)G-SL. We propose interactions between Ascaris eIF4E and the SL impact eIF4G and contribute to translation initiation, whereas these interactions do not occur when only the m(2,2,7)G-cap is present. These data have implications for the contribution of 5'-UTRs in mRNA translation and the function of different eIF4E isoforms.

Alginate lyases are enzymes that degrade alginate into oligosaccharides which possess a variety of biological activities. Discovering and characterizing novel alginate lyases has great significance for industrial and medical applications. In this study, we reported a novel alginate lyase, AlyA-OU02, derived from the marine Vibrio splendidus OU02. The BLASTP searches showed that AlyA-OU02 belonged to polysaccharide lyase family 7 (PL7) and contained two consecutive PL7 domains, which was rare among the alginate lyases in PL7 family. Both the two domains, AlyAa and AlyAb, had lyase activities, while AlyAa exhibited polyM preference, and AlyAb was polyG-preferred. In addition, the enzyme activity of AlyAa was much higher than AlyAb at 25 &deg, C. The full-length enzyme of AlyA-OU02 showed polyM preference, which was the same as AlyAa. AlyAa degraded alginate into di-, tri-, and tetra-alginate oligosaccharides, while AlyAb degraded alginate into tri-, tetra-, and penta-alginate oligosaccharides. The degraded products of AlyA-OU02 were similar to AlyAa. Our work provided a potential candidate in the application of alginate oligosaccharide production and the characterization of the two domains might provide insights into the use of alginate of this organism.

beta-Hydroxyacyl-acyl carrier protein dehydratase (FabZ) is an important enzyme for the elongation cycles of both saturated and unsaturated fatty acids biosyntheses in the type II fatty acid biosynthesis system (FAS II) pathway. FabZ has been an essential target for the discovery of compounds effective against pathogenic microbes. In this work, to characterize the catalytic and inhibitory mechanisms of FabZ, the crystal structures of the FabZ of Helicobacter pylori (HpFabZ) and its complexes with two newly discovered inhibitors have been solved. Different from the structures of other bacterial FabZs, HpFabZ contains an extra short two-turn alpha-helix (alpha4) between alpha3 and beta3, which plays an important role in shaping the substrate-binding tunnel. Residue Tyr-100 at the entrance of the tunnel adopts either an open or closed conformation in the crystal structure. The crystal structural characterization, the binding affinity determination, and the enzymatic activity assay of the HpFabZ mutant (Y100A) confirm the importance of Tyr-100 in catalytic activity and substrate binding. Residue Phe-83 at the exit tunnel was also refined in two alternative conformations, leading the tunnel to form an L-shape and U-shape. All these data thus contributed much to understanding the catalytic mechanism of HpFabZ. In addition, the co-crystal structures of HpFabZ with its inhibitors have suggested that the enzymatic activity of HpFabZ could be inhibited either by occupying the entrance of the tunnel or plugging the tunnel to prevent the substrate from accessing the active site. Our study has provided some insights into the catalytic and inhibitory mechanisms of FabZ, thus facilitating antibacterial agent development.

Background A detailed knowledge about the degradation mechanism of chitosanase hydrolysis is critical for the design of novel enzymes to produce well-defined chito-oligosaccharide products. Methods Through the combination of structural and biochemical analysis, we present new findings that provide novel insights into the degradation mechanism of chitosanase OU01. Results We have determined the crystal structure of Asp 43 /Ala mutant of OU01, and have trapped the hydrolyzed product of the reaction. This structure reveals the role of the general acid (Glu 25 ) in catalysis. Two structural features about the mechanisms of the non-processive chitosanases are described for the first time. 1). Structural comparison reveals that the enzyme goes through an open–closed–open conformational transition upon substrate binding and product release; 2). polar residues constitute the substrate binding cleft. Additional site important for polymeric substrate recognition is identified and a three-step polymeric substrate recognition mechanism is proposed. Conclusions Detailed substrate recognition mechanism is described for non-processive chitosanase for the first time. General significance These findings provide new structural insights into the understanding of overall hydrolysis mechanism for non-processive chitosanase, and also will facilitate the design of new enzymes used for industrial purpose.

A mitochondria-targetable fluorescence probe, methyl(4-hydroxyphenyl)amino-substituted pyronin (1), was exploited, which could highly selectively sense peroxynitrite (ONOO−) within seconds.

Helicobacter pylori is a gram-negative pathogenic bacterium that causes peptic ulcer disease and gastric cancer, and studies of the related potent enzymes associated with this bacterium are urgent for the discovery of novel drug targets. In bacteria, β-hydroxyacyl-acyl carrier protein (ACP) dehydratase (FabZ) is a potent enzyme in fatty acid biosynthesis and catalyzes the dehydration of β-hydroxyacyl-ACP to trans-2-acyl-ACP. In this study, the cloning and enzymatic characterization of FabZ from H. pylori strain SS1 (HpFabZ) were reported, and the gene sequence of HpfabZ was deposited in the GenBank database (Accession No. AY725427 ). Enzyme dynamic analysis showed that HpFabZ had a Km of 82.6 ± 4.3 μM toward its substrate analog crotonoyl-CoA. Dynamic light scattering and native-PAGE investigations suggested that HpFabZ exists as hexamer in native state. Enzymatic characterization and thermal-induced unfolding analysis based on circular dichroism spectral measurements indicated that HpFabZ is very stable against high temperature (90 °C). Such a high stability of HpFabZ was well elucidated by the strong H-bonds and hydrophobic interactions among the HpFabZ hexamer as investigated in the modeled HpFabZ hexamer structure. Our current study is hoped to provide useful information in better understanding the FabZ of H. pylori strain and further supply possible hints in the discovery of anti-bacterial compounds using HpFabZ as target.

The in vivo and in vitro immunostimulating properties of chitosan oligosaccharide (COS) prepared by enzymatic hydrolysis of chitosan and the mechanisms mediating the effects were investigated. Our data showed that the highly active chitosanase isolated could hydrolyze chitosan to the polymerization degree of 3-8. The resulting COS was an efficient immunostimulator. COS markedly enhanced the proliferation and neutral red phagocytosis by RAW 264.7 macrophages. The production of nitric oxide (NO) and tumor necrosis factor alpha (TNF-α) by macrophages was significantly increased after incubation with COS. Oral administration of COS in mice could increase spleen index and serum immunoglobin G (IgG) contents. COS was labeled with FITC to study the pinocytosis by macrophages. Results showed that FITC-COS was phagocyted by macrophages and anti-murine TLR4 antibody completely blocked FITC-COS pinocytosis. RT-PCR indicated that COS treatment of macrophages significantly increased TLR4 and inducible nitric oxide synthase (iNOS) mRNA levels. When cells were pretreated with anti-murine TLR4 antibody, the effect of COS on TLR4 and iNOS mRNA induction was decreased. COS-induced NO secretion by macrophages was also markedly decreased by anti-murine TLR4 antibody pretreatment. In conclusion, the present study revealed that COS possesses potent immune-stimulating properties by activating TLR4 on macrophages.

Integrin cytoplasmic domain-associated protein-1 (ICAP1) is a suppressor of integrin activation and directly binds to the cytoplasmic tail of β1 integrins; its binding suppresses integrin activation by competition with talin. Krev/Rap1 interaction trapped-1 (KRIT1) releases ICAP1 suppression of integrin activation by sequestering ICAP1 away from integrin cytoplasmic tails. Here, the cocrystal structure of the PTB domain of ICAP1 in complex with a 29-­amino-acid fragment (residues 170–198) of KRIT1 is presented to 1.7 A resolution [the resolution at which 〈I/σ(I)〉 = 2.9 was 1.83 A]. In previous studies, the structure of ICAP1 with integrin β1 was determined to 3.0 A resolution and that of ICAP1 with the N-terminal portion of KRIT1 (residues 1–­198) was determined to 2.54 A resolution; therefore, this study provides the highest resolution structure yet of ICAP1 and allows further detailed analysis of the interaction of ICAP1 with its minimal binding region in KRIT1.

Integration of the retroviral genome into host DNA is a critical step in the life cycle of a retrovirus. Although assays for in vitro integration have been developed, the actual DNA sequences targeted by murine leukemia retrovirus (MLV) during in vitro reproduction are unknown. While previous studies used artificial target sequences, we developed an assay using target DNA sequences from common MLV integration sites in Stat5a and c-myc in the genome of murine lymphomas and successfully integrated MLV into the target DNA in vitro. We calculated the free energy change during folding of the target sequence DNA and found a close correlation between the calculated free energy change and the number of integrations. Indeed, the integrations closely correlated with fluctuation of the structure of the target DNA segment. These data suggest that the fluctuation may generate a DNA structure favorable for in vitro integration into the target DNA. The approach described here can provide data on the biochemical properties of the integration reaction to which the target DNA structure may contribute.

Polybrominated diphenyl ethers (PBDEs) were measured in atmosphere and soil samples taken in winter and summer at a PBDE production area of Laizhou Bay in China. The concentrations of σ11PBDE were 0.017–1.17 ng/m3 in gaseous phase, 0.5–161.1 ng/m3 in particulate phase, and 73–2629 ng/g dry weight in soil samples. The PBDE congener pattern in the gaseous phase differed from that in the particulate phase, and the PBDE congener pattern in the particulate phase was similar with that in soil. This demonstrated that there was little difference with atmospheric particle-soil transfer efficiency among PBDE congeners. In addition, there were seasonal variations in percentages on particle for lower brominated congeners. The BDE-28 was mostly in the gaseous phase in summer (88.3%), whereas the average proportion of BDE-28 in gaseous phase in winter was 38.9%. Higher brominated congeners (i.e., BDE-206, BDE-207, BDE-208, and BDE-209) were bound to the atmospheric particulate phase, and their potentials for long-range migration were mainly affected by the environmental behavior of atmospheric particles. Results indicated that PBDE congeners in summer were closer to gas-particle partition equilibrium than in winter. Temperature should be considered the main factor causing nonequilibrium in winter.

Acyl carrier protein (ACP) is an essential component in the type II fatty acid biosynthesis (FAS II) process and is responsible for the acyl group transfer within a series of related enzymes. In this work, the ACP from Helicobacter pylori strain SS1 was cloned and the gene sequence of Hpacp was deposited in the GenBank database (Accession No.: AY904356 ). Two forms of Hp ACP (apo, holo) were successfully purified and characterized. The thermal stability of these two forms was quantitatively investigated by CD spectral analyses. The results revealed that the holo- Hp ACP was more stable than apo- Hp ACP according to the transition midpoint temperature( T m ). Moreover, the interaction of Hp ACP with the related enzyme (β-hydroxyacyl-ACP dehydratase, Hp FabZ) was determined by GST-pull down assay and surface plasmon resonance (SPR) technique in vitro , the results showed that Hp ACP displays a strong binding affinity to Hp FabZ ( K D = 1.2 × 10 −8 M). This current work is hoped to supply useful information for better understanding the ACP features of Helicobacter pylori SS1 strain.

Type II fatty acid synthesis (FAS II) is an essential process for bacteria survival, and malonyl-CoA:acyl carrier protein transacylase (MCAT) is a key enzyme in FAS II pathway, which is responsible for transferring the malonyl group from malonyl-CoA to the holo-ACP by forming malonyl-ACP. In this work, we described the cloning, characterization and enzymatic inhibition of a new MCAT from Helicobacter pylori strain SS1 (HpMCAT), and the gene sequence of HpfabD was deposited in the GenBank database (Accession No. AY738332 ). Enzymatic characterization of HpMCAT showed that the K(m) value for malonyl-CoA was 21.01+/-2.3 microM, and the thermal- and guanidinium hydrochloride-induced unfolding processes for HpMCAT were quantitatively investigated by circular dichroism spectral analyses. Moreover, a natural product, corytuberine, was discovered to demonstrate inhibitory activity against HpMCAT with IC(50) value at 33.1+/-3.29 microM. Further enzymatic assay results indicated that corytuberine inhibits HpMCAT in an uncompetitive manner. To our knowledge, this is the firstly reported MCAT inhibitor to date. This current work is hoped to supply useful information for better understanding the MCAT features of H. pylori strain, and corytuberine might be used as a potential lead compound in the discovery of the antibacterial agents using HpMCAT as target.

Microcapsules made of polyelectrolyte multilayers exhibit no or low toxicity, appropriate mechanical stability, variable controllable degradation and can incorporate remote release mechanisms triggered by various stimuli, making them well suited for targeted drug delivery to live cells. This study investigates interactions between microcapsules made of synthetic (i.e. polystyrenesulfonate sodium salt/polyallylamine hydrochloride) or natural (i.e. dextran sulfate/poly- l -arginine) polyelectrolyte and human umbilical vein endothelial cells with particular focus on the effect of the glycocalyx layer on the intake of microcapsules by endothelial cells. Neuraminidase cleaves N -acetyl neuraminic acid residues of glycoproteins and targets the sialic acid component of the glycocalyx on the cell membrane. Three-dimensional confocal images reveal that microcapsules, functionalized with neuraminidase, can be internalized by endothelial cells. Capsules without neuraminidase are blocked by the glycocalyx layer. Uptake of the microcapsules is most significant in the first 2 h. Following their internalization by endothelial cells, biodegradable DS/PArg capsules rupture by day 5; however, there is no obvious change in the shape and integrity of PSS/PAH capsules within the period of observation. Results from the study support our hypothesis that the glycocalyx functions as an endothelial barrier to cross-membrane movement of microcapsules. Neuraminidase-loaded microcapsules can enter endothelial cells by localized cleavage of glycocalyx components with minimum disruption of the glycocalyx layer and therefore have high potential to act as drug delivery vehicles to reach tissues beyond the endothelial barrier of blood vessels.