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T cells exhibit heterogeneous functional states, which correlate with responsiveness to immune checkpoint blockade and prognosis of tumor patients. However, the molecular regulatory mechanisms underlying the dynamic process of T cell state transition remain largely unknown. Based on single-cell transcriptome data of T cells in non-small cell lung cancer, we combined cell states and pseudo-times to propose a pipeline to construct dynamic regulatory networks for dissecting the process of T cell dysfunction. Candidate regulators at different stages were revealed in the process of tumor-infiltrating T cell dysfunction. Through comparing dynamic networks across the T cell state transition, we revealed frequent regulatory interaction rewiring and further refined critical regulators mediating each state transition. Several known regulators were identified, including TCF7, EOMES, ID2, and TOX. Notably, one of the critical regulators, TSC22D3, was frequently identified in the state transitions from the intermediate state to the pre-dysfunction and dysfunction state, exerting diverse roles in each state transition by regulatory interaction rewiring. Moreover, higher expression of TSC22D3 was associated with the clinical outcome of tumor patients. Our study embedded transcription factors (TFs) within the temporal dynamic networks, providing a comprehensive view of dynamic regulatory mechanisms controlling the process of T cell state transition., Graphical abstract, Antigen recognition in the tumor microenvironment drives T cell dysfunction, which is governed by a complex regulatory network. We constructed dynamic regulatory networks and identified critical regulators during state transition processes along T cell dysfunction. TSC22D3 was frequently identified in different state transition stages, and it exhibited prognostic significance for NSCLC patients.

The presence of sulfate groups in agar compromises the agar quality by affecting the crosslinking during gelling process. Some arylsulfatases can catalyze the hydrolysis of sulfate bonds in agar to improve the agar quality. Immobilized arylsulfatases prove beneficial advantages for their industrial applications. Here, a previously characterized mutant arylsulfatase K253H/H260L was immobilized on the synthesized magnetic Fe3O4 nanoparticles after functionalization by tannic acid (MNPs@TA). The surface properties and molecular structures of the immobilized arylsulfatase (MNPs@TA@ARS) were examined by scanning electron microscopy and Fourier transform infrared spectroscopy. Enzymatic characterization showed that MNPs@TA@ARS exhibited shifted optimal temperature and pH with deviated apparent Km and Vmax compared to its free counterpart. The immobilized arylsulfatase demonstrated improved thermal and pH stability and enhanced storage stability with modest reusability. In addition, MNPs@TA@ARS displayed enhanced tolerance to various inhibitors and detergents. The utilization of the immobilized arylsulfatase for agar desulfation brought the treated agar with improved quality.

Accurately classifying patients with non‐small cell lung cancer (NSCLC) from the perspective of tumor evolution has not been systematically studied to date. Here, we reconstructed phylogenetic relationships of somatic mutations in 100 early NSCLC patients (327 lesions) through reanalyzing the TRACERx data. Based on the genomic evolutionary patterns presented on the phylogenetic trees, we grouped NSCLC patients into three evolutionary subtypes. The phylogenetic trees among three subtypes exhibited distinct branching structures, with one subtype representing branched evolution and another reflecting the early accumulation of genomic variation. However, in the evolutionary pattern of the third subtype, some mutations experienced selective sweeps and were gradually replaced by multiple newly formed subclonal populations. The subtype patients with poor prognosis had higher intra‐tumor heterogeneity and subclonal diversity. We combined genomic heterogeneity with clinical phenotypes analysis and found that subclonal expansion results in the progression and deterioration of the tumor. The molecular mechanisms of subtype‐specific Early Driver Feature (EDF) genes differed across the evolutionary subtypes, reflecting the characteristics of the subtype itself. In summary, our study provided new insights on the stratification of NSCLC patients based on genomic evolution that can be valuable for us to understand the development of pulmonary tumor profoundly., The NSCLC patients were grouped into three subtypes based on the genomic evolutionary patterns. The phylogenetic trees among subtypes exhibited distinct branching structures. The molecular mechanisms of subtype‐specific EDF genes differed across the evolutionary subtypes.

α-L-Rhamnosidase has attracted lots of attention due to its industrial potential applications. The applicability of α-L-rhamnosidase, however, was limited by their low ligand affinity on the industrial scale. In order to improve the affinity of α-L-rhamnosidase for industrial use, we investigated the variation of its affinity by amino acid replacement. Particularly, the enzyme affinity of a α-L-rhamnosidase from Aspergillus niger JMU-TS528 (rRha1) was measured with the semi-conservative amino acid (homology between 30% -80%) replaced. As a result, the enzyme affinity of the two mutants, R404S and N578D, were increased by 1.45-fold and 2.3-fold, respectively, showing that these two mutants could be the promising candidates for industrial use. To test if these mutations bring negative effect on the enzyme properties, we also determined the other enzymatic properties of these mutants and showed no negative effect. To understand the improvement of enzyme affinity, the conformational flexibility of (α/α)6-barrel catalytic domain were examined by molecular dynamics (MD) simulation, and demonstrated that the conformations of these mutants are more flexible, which could influence the affinity of substrates to the enzyme and hence the enzyme activity. This work not only enhanced the enzyme affinity of a α-L-rhamnosidase, making rRha1 a promising candidate for industrial processes, but also provided an effective technical strategy for improving affinity of other enzymes.

The infiltration of tumor-reactive T cells in the tumor site is associated with better survival and immunotherapy response. However, tumor-reactive T cells were often represented by the infiltration of total CD8+ T cells, which was confounded by the presence of bystander T cells. To identify tumor-reactive T cells at the cancer lesion, we performed integration analyses of three scRNA-seq data sets of T cells in melanoma. Extensive heterogeneous functional states of T cells were revealed in the tumor microenvironment. Among these states, we identified a subset of tumor-reactive T cells which specifically expressed tumor-reactive markers and T cell activation signature, and were strongly enriched for larger T cell receptor (TCR) clones. We further identified and validated a tumor-reactive T cell signature (TRS) to evaluate the tumor reactivity of T cells in tumor patients. Patients with high TRS scores have strong immune activity and high mutation burden in the TCGA-SKCM cohort. We also demonstrated a significant association of the TRS with the clinical outcomes of melanoma patients, with higher TRS scores representing better survival, which was validated in four external independent cohorts. Furthermore, the TRS scores exhibited greater performance on prognosis prediction than infiltration levels of CD8+ T cells and previously published prognosis-related signatures. Finally, we observed the capability of TRS to predict immunotherapy response in melanoma. Together, based on integrated analysis of single-cell RNA-sequencing, we developed and validated a tumor-reactive-related signature that demonstrated significant association with clinical outcomes and response to immunotherapy.

In this study, a sulfated polysaccharide (BFP) was isolated from the edible red alga Bangia fusco-purpurea. Gel-filtration and thin layer chromatographically analyses suggested that BFP was a homogenous polysaccharide. The chemical structural analysis revealed that BFP mainly consisted of galactose together with a small amount of uronic acid, mannose, and glucose. Its molecular mass was estimated to be 133.18 kDa by high-performance liquid chromatography (HPLC) analysis. BFP inhibited α-amylase and α-glucosidase in a concentration-dependent manner. The IC50 values of BFP against α-amylase and α-glucosidase were estimated to be 1.26 ± 0.11 mg/mL and 1.34 ± 0.07 mg/mL, respectively. Kinetic analyses suggested that BFP showed competitive and non-competitive inhibition against α-amylase and α-glucosidase, respectively. Circular dichroism spectral and fluorescence spectral analyses suggested that BFP affects the conformational structures of these enzymes, which may lead to the inhibition of the enzymatic activities. Abbreviations: Ara: D-arabinose; AnGal: anhydro-L-galactose residues; CD spectroscopy: Circular Dichroism spectroscopy; DNS: dinitrosalicylic acid; FT-IR: fourier transform infrared spectra; Fuc: L-fucose; Gal: D-galactose; Glc: D-glucose; GlcA: D-Glucuronic acid; HPLC: high performance liquid chromatography; Man: D-mannose; pNPG: p-nitrophenyl-α-D-glucoside; TFA: trifluoroacetic acid; TLC: thin-layer chromatography; PMP: 1-phenyl-3-methyl-5-pyrazolone; Xyl: D-xylose

A new alginate lyase gene of algl17 was cloned from an alginate-degrading marine bacterium Microbulbifer sp. ALW1. The gene contained 2220 bp and encoded a 739-amino acid protein classified into the PL-17 family. The recombinant alginate lyase AlgL17 was overexpressed and purified from Escherichia coli BL21 (DE3) with a molecular mass of 84.9 kDa. This enzyme showed activities towards sodium alginate, polyM and alginate oligosaccharide, but very low activity towards polyG. These results indicate that AlgL17 is a polyM-specific oligoalginate lyase. When sodium alginate was used as a substrate, the optimum reaction temperature and pH for the enzyme were 35 °C and pH 8.0, respectively. Recombinant AlgL17 was stable at 25 °C, but not stable at 30 °C and 35 °C. It showed good stability over a pH range of 5.0–8.0. The enzyme activity was increased to 1.7 times by adding NaCl to a final concentration of 0.7 M. The ability of the recombinant AlgL17 producing 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH) from sodium alginate and polyM block indicates that AlgL17 is an exo-type alginate lyase.

Isoquercitrin is a flavonoid with important applications in the pharmaceutical and nutraceutical industries. However, a low yield and high production cost hinders the industrial preparation...

α-L-Rhamnosidase is a glycoside hydrolase capable of removing naringin from citrus juice. However, α-L-rhamnosidases always have broad substrate spectra, causing negative effects on citrus juice. In this study, a α-L-rhamnosidase-expressing fungal strain, JMU-TS529, was identified, and its α-L-rhamnosidase was characterized. As a result, JMU-TS529 was identified as Aspergillus tubingensis via morphological and molecular characteristics. The predicted protein sequence shared an amino acid identity of less than 30% with previously characterized α-L-rhamnosidases. The optimal pH and temperature were 4.0 and 50-60 °C, respectively. Most importantly, the α-L-rhamnosidase showed a strong ability to hydrolyze naringin but scarcely acted on other substrates. Furthermore, the enzyme could efficiently remove naringin from pomelo juice without changing its attractive aroma. These results indicate that the present enzyme represents a new clade of Aspergillus α-L-rhamnosidase that is desirable for debittering citrus juice, providing a better alternative for improving the quality of citrus juice.

β-Agarase was immobilized by using tannic acid modified-Fe3O4 magnetic nanoparticles (TA-MNPs) as a support material. The MNPs were synthesized by improved chemical coprecipitation method and modified with TA for agarase immobilization. TA-MNPs and immobilized β-agarase were characterized by transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), and thermal gravimetric analysis (TGA), all of which indicated the successful surface modification of MNPs with TA and the immobilization of β-agarase. The optimal immobilization conditions for 25 mg TA-MNPs included 100 r/min oscillation speed, immobilization time of 2 h, immobilization temperature of 15 °C, and initial β-agarase concentration of 3 mL (480 U). Immobilized β-agarase showed better pH and thermal stability and excellent reusability than the free enzyme. Results revealed the promising application of β-agarase-TA-MNPs for the preparation of neoagaro-oligosaccharides with different average polymerization degrees and varying activities in the antioxidant.

The bacterium Microbulbifer sp. ALW1 was previously characterized with the capability to break down the cell wall of brown algae into fine pieces. The biological functions of strain ALW1 were yet to be elucidated. In this study, a gene, namely MaCel5A, was isolated from the ALW1 strain genome, encoding an endo-β-1,4-glucanase. MaCel5A was phylogenetically categorized under the glycoside hydrolase family GH5, with the highest identity to a putative cellulase of Microbulbifer thermotolerans. The recombinant MaCel5A protein purified from heterologous expression in E. coli exhibited maximum activity at 50 °C and pH 6.0, respectively, and functioned selectively toward carboxymethyl cellulose and barley β-glucan. Recombinant MaCel5A demonstrated considerable tolerance to the exposure to high temperature up to 80 °C for 30 min retaining 49% residual activity. In addition, MaCel5A showed moderate stability against pH 5.0–11.0 and strong stability in the presence of nonionic surfactant. MaCel5A exhibited strong halo-stability and halotolerance. The activity of the enzyme increased about tenfold at 0.5 M NaCl, and about fivefold even at 4.0 M NaCl compared to the enzyme activity without the addition of salt. The two conserved glutamic acid residues in MaCel5A featured the typical catalytic acid/base and nucleophile machinery of glycoside hydrolases. These characteristics highlight the industrial application potential of MaCel5A.

The present study aims to evaluate the deodorization of Bangia fusco-purpurea using microorganism fermentation with commercial starter cultures of Saccharomyces cerevisiae, Acetobacter pasteurianus, and Lactobacillus plantarum. The results showed the fermentation with S. cerevisiae, A. pasteurianus, and L. plantarum resulted in significantly decreases (p < .05) of the fishy malodor in B. fusco-purpurea. Among the three strains, S. cerevisiae was the best for reducing the fishy malodor. The optimal inoculum size and fermentation time were 0.2% and 4 hr, respectively. After the fermentation with the S. cerevisiae, the content of 1-octen-3-ol, (E)-2-octen-1-ol, hexanal, non-(2E)-enal, (E,E)-2,4-decadienal, 3,5-octadien-2-one, and 2-pentyl-furan were hard to be detected in the seaweed, whereas increases were observed in the concentrations of 2-butyl-1-octanol, cedrol, diisobutyl phthalate, and 2,4-di-t-butylphenol. The odor active value analysis indicated the removal of fishy odor was related to the reduction, dehydrogenation, and deformylating oxygenation of hexanal, nonanal, non-(2E)-enal, and (E,E)-2,4-decadienal and esterification of 1-octen-3-ol and (E)-2-octen-1-ol. Our findings provide a technical and scientific basis for the removal of fishy odor from B. fusco-purpurea. PRACTICAL APPLICATIONS: Bangia fusco-purpurea is a seaweed that can reduce the risks of cardiovascular and chronic metabolic diseases in human body. However, the seaweed has a strong fishy malodor, which largely declines consumer's acceptance. In this study, the commercial starters of Saccharomyces cerevisiae, Acetobacter pasteurianus, and Lactobacillus plantarum were shown to reduce the fishy malodor in B. fusco-purpurea via fermentation. After the fermentation with the microorganisms especially with the S. cerevisiae, the fishy malodor significantly reduced, and the overall aroma acceptance of B. fusco-purpurea products greatly improved. Therefore, this study provides a technical basis for the removal of fishy odor from B. fusco-purpurea and processing value-added products from it and facilitating its health benefits for human.

Glucose-tolerant and/or glucose-stimulated β-glucosidase is of great interest for its industrial utilization in enzymatic digestion of lignocellulosic biomass for biofuel production. In this study, a new gene of β-glucosidase MaGlu1A was cloned from an alginate-degrading marine bacterium Microbulbifer sp. ALW1. The gene of MaGlu1A encoded a 472-amino acid protein classified into the glycosyl hydrolase family 1 (GH1). The recombinant β-glucosidase was overexpressed and purified from Escherichia coli with a molecular mass of 65.0 kDa. Structure analysis illustrated the catalytic acid/base residue Glu186 and nucleophilic residue Glu370 in the enzyme. MaGlu1A displayed optimal activity at 40 °C and pH 4.5, respectively. It had substrate preference to the aryl-β-glycosidic bonds with glucose, fucose, and galactose moieties, in addition to cellobiose. MaGlu1A demonstrated strong stimulation to the supplemental glucose. Site-directed mutagenesis suggested an essential role of Asn242 in glucose stimulation. The enzymatic characterization of MaGlu1A provides general information about its catalytic properties facilitating its practical applications.

Background: Somatic mutations accumulate over time in cancer cells as a consequence of mutational processes. However, the role of mutational processes in carcinogenesis remains poorly understood. Methods: We infer the causal relationship between mutational processes and somatic mutations in 5,828 samples spanning 34 cancer subtypes based on the patterns of the mutational signatures and their contributions. A ranked hypergeometric test is used to identify pathways affected by a mutational process. The clinical actionability of APOBEC mutagenesis is evaluated in bladder cancer patients who accepted platinum-based therapy. Finding: Most mutational processes cause abundant recurrent mutations in cancer genes, while exceptionally ultraviolet exposure and altered activity of the error-prone polymerase bring a large number of recurrent non-driver mutations. Furthermore, some mutations are specifically induced by a certain mutational process, such as IDH1 p.R132H which is mainly caused by spontaneous deamination of 5-methylcytosine. At the pathway level, clock-like mutational processes extensively trigger mutations to dysregulate cancer signal transduction pathways. In addition, APOBEC mutational process destroys DNA double strand break repair pathway, and bladder cancer patients with high APOBEC activity, though with homologous recombination proficient, show a significantly longer overall survival with platinum regimens. Interpretation: These findings help to understand how mutational processes act on the genome to promote carcinogenesis, and further, presents novel insights for cancer prevention and treatment, as our results showing, APOBEC mutagenesis and HRD synergistically contributed to the clinical benefits of platinum-based treatment. Funding Information: National Key RD the National Natural Science Foundation of China (61873075, 32070673 and 31871336); the Heilongjiang Provincial Natural Science Foundation (YQ2019C012); the Heilongjiang Postdoctoral Foundation (LBH-Q18099); Heilongjiang Touyan Innovation Team Program and Foundation of Harbin Medical University (QMPT-1905). Declaration of Interests: The authors declare no competing financial interests.

10 pages, 5 figures, 1 table, Neon flying squid (OB) and jumbo squid (DG) mantles were evaluated to reveal the similarities and differences in their physicochemical features and protein abundances. Microstructural results indicated that the OB mantle exhibited numerous myofibril fragments and disordered microstructures after frozen storage compared with DG tissues. Chemical analysis suggested that freezing resulted in a rapid decrease in myofibrillar protein (MP) content, Ca2+-ATPase activity, and total sulfhydryl content, and promoted the increase in carbonyl content of MPs in both OB and DG. While, DG presented better MP stability than OB muscle after 120 days of frozen storage. Label-free proteomic analysis detected 24 down- and 33 up-regulated differentially abundant proteins (DAPs) in OB and DG mantles. Identified DAPs including isocitrate dehydrogenase and malic enzyme initiated a rapid decrease in the MP properties in OB samples. Moreover, DAPs were related to cytoskeleton function, including paramyosin, tropomyosin, and troponin C, which improved the stability of DG in response to freezing-induced changes, This study was funded by the National Natural Science Foundation of China (No. 32072146), the Zhejiang Natural Science Foundation of China (No. LR21C200001), and the Zhejiang Leading Training Program of China (2020R52027)

In this study, we found that a sulfated polysaccharide isolated from the brown alga Ascophyllum nodosum, ascophyllan, showed suppressive effects on stimulated RAW264.7 cells. Ascophyllan significantly inhibited expression of inducible nitric oxide synthase mRNA and excessive production of nitric oxide (NO) in lipopolysaccharide (LPS)-stimulated RAW264.7 cells in a dose-dependent manner without affecting the viability of RAW264.7 cells. Ascophyllan also reduced the elevated level of intracellular reactive oxygen species (ROS) in LPS-stimulated RAW264.7 cells. Furthermore, preincubation with ascophyllan resulted in concentration-dependent decrease in ROS production in phorbol 12-myristate-13-acetate-stimulated RAW264.7 cells. Our results suggest that ascophyllan can exhibit anti-inflammatory effects on stimulated macrophages mainly through the attenuation of NO and ROS productions.

In this study, the polysaccharide ascophyllan, isolated from Ascophyllum nodosum, was degraded using alginate lyase (EC 4.2.2.3) to prepare low-molecular-weight fragments. LMWAs-L, an extremely low-molecular-weight saccharide (6.71 kDa) with low fucose- and sulphate-contents, was purified by gel filtration chromatography. LMWAs-L exhibited significant macrophage-stimulating activities, similar to ascophyllan, through intracellular signalling pathways. Methylation and GC/MS analyses indicated that the main glycosidic linkages of LMWAs-L were 1,2-linked-GlcpA, followed by T-linked-Glcp, 1,3,6-linked-Galp, 1,3-linked-Fucp, 1,4-linked-Xylp, and 1,4-linked-ManpA at a molar ratio of 3.37: 1.13: 1.11: 1.00: 0.96: 0.61. The proposal repeating structure of LMWAs-L was shown to be →2)-α-D-GlcpA-(1 → 2)-α-D-GlcpA-(1 → 6)-α-D-Galp-(1 → 2)-α-D-GlcpA-(1→ as main chain, branched by T-α-D-Glcp-(1 → 4)-β-D-Xylp-(1 → 3)-α-L-Fucp4S-(1→ at the O-3 of 3,6)-α-D-Galp-(1→ residues. 4-deoxy-L-erythro-hex-4-enuronosyluronate-4-β-D-ManpA-(1→ and →4)-β-D-ManpAred residues were attached to the ends of main chain as non-reducing end residue and reducing end residue, respectively. Our results suggest that the fragment LMWAs-L contain an essential structural element of ascophyllan that is responsible for the macrophage-stimulating activities.

This study aimed to improve the thermostability of arylsulfatase from Pseudoalteromonas carrageenovora. A total of 10 single-site mutants were chosen using the PoPMuSiC program, and two mutants of K253N and P314T showed enhanced thermal stability. By saturation mutagenesis and thermostability analysis, K253H and P314T were the best mutants at the two sites. Combinational mutations of K253H, P314T and H260L were subsequently introduced, and the best mutant of K253H/H260L was selected. Thermal inactivation analysis showed the half-life (t1/2) value at 55°C for K253H/H260L was 7.7-fold that of the wild-type enzyme (WT), meanwhile this mutant maintained the specific enzyme activity. Structure modeling demonstrated that the additional hydrogen bonds, optimization of surface charge-charge interactions, and increasing of hydrophobic interaction could account for the improved thermostability imparted by K253H/H260L.

Alginate is a naturally acidic polysaccharide consisting alternately of β-d-mannuronic acid and α-l-guluronic acid with 1, 4-glycosidic linkages and is derived from brown seaweeds. Herein, the effect of alginate on the promotion of macrophage phagocytosis and the corresponding molecular mechanisms were investigated in murine RAW264.7 cells. Alginate could enhance the intracellular phagocytosis of gold nanoparticles (AuNPs), fluorescent microspheres and immunoglobulin G (IgG)-opsonized Staphylococcus aureus (S. aureus). Moreover, alginate increased Toll-like receptor 4 (TLR4) expression and activated the Akt/nuclear factor-κB (NF-κB) and p38 mitogen-activated protein kinase (MAPK) signalling pathways. Alginate-promoted phagocytosis was suppressed by the addition of inhibitors of TLR4, NF-κB and p38 MAPK and by TLR4 gene knockdown, indicating the involvement of these key components. This work is the first to propose that alginate promotes phagocytosis via upregulating TLR4 expression and stimulating the Akt/NF-κB and p38 MAPK signalling pathways, which may contribute to the capacity of alginate to activate macrophages.

A library of Pseudoalteromonas carrageenovora arylsulfatase mutants was constructed by introducing random mutagenesis using error-prone PCR. After screening, one mutant strain was obtained whose arylsulfatase had improved thermal stability. Protein sequence analysis revealed one amino acid substitution of H260L. The mutant arylsulfatase (named H260L) retained higher residual activity than wild-type enzyme (named WT) after incubation at 45, 50, 55 and 60°C for 60min. Thermal inactivation analysis showed that the half-life (t1/2) value at 55°C for H260L was 40.6min, while that of WT was 9.1min. When p-nitrophenyl sulfate was used as a substrate, the optimal reaction temperature and pH for the mutant enzyme were 55°C and pH 8.0, respectively. H260L was stable over the pH range of 6.0-9.0. Inhibition assay with EDTA indicated that metal ions play an important role during the catalytic process of the mutant enzyme. The desulfation ratio against agar of Gracilaria lemaneiformis was 82%.

In this study, a low-molecular-weight saccharide fragment (LMWAs-L) was prepared from alginate lyase (EC 4.2.2.3) hydrolyzed ascophyllan by ultra-filtration separation method. LMWAs-L was a homogeneous saccharide fraction with an average molecular weight of 6.96 kDa. Enzymolysis process optimization experiments revealed that the optimum process parameters for preparing LMWAs-L were the enzyme concentration 0.02 U/mL, initial pH 6.8, and enzymolysis temperature 43 °C. After optimization, the yield of LMWAs-L was increased to 9.74% higher than that without optimization. Interestingly, LMWAs-L exhibited stronger enhancing activities on the proliferation and migration of human skin fibroblasts cells in vitro and better antibacterial activities as compared to native ascophyllan at the same mass concentration. Our study establishes a simple way to prepare low-molecular-weight saccharide with beneficial bioactivities from ascophyllan efficiently. This is the first report to reveal that ascophyllan and its low-molecular-weight saccharide have the potentials to be developed as natural biological dressing and antibacterial agents.

The unpleasant stale note is a negative factor hindering the consumption of instant ripened Pu-erh tea products. This study focused on investigating volatile chemicals in instant ripened Pu-erh tea that could mask the stale note via sensory evaluation, gas chromatography-mass spectrometry (GC-MS), and gas chromatography-olfactometry (GC-O) analyses. GC-MS and GC-O analyses showed that linalool, linalool oxides, trans-&beta, ionone, benzeneacetaldehyde, and methoxybenzenes were the major aroma contributors to the simultaneous distillation and extraction (SDE) extract of instant ripened Pu-erh tea. Sensory evaluation showed that the SDE extract had a strong stale note, which was due to methoxybenzenes. By investigating suppressive interaction among flavour components, the stale note from methoxybenzenes was shown to have reciprocal masking interactions with sweet, floral, and green notes. Moreover, the validation experiment showed that the addition of 40 &mu, g/mL of trans-&beta, ionone in the instant ripened Pu-erh tea completely masked the stale note and improved the overall aromatic acceptance. These results elucidate the volatile chemicals that could mask the stale note of instant ripened Pu-erh tea products, which might help to develop high quality products made from instant ripened Pu-erh tea.

Agricultural or food processing wastes cause serious environmental burden and economic losses. Solid-state fermentation using these wastes is an attractive option to valorize these wastes. However, conventional autoclaving of substrate may degrade nutrients and generate toxins. Unsterilization of the substrate will cause undesired microbial contamination. Therefore, we compared irradiation with autoclaving to treat citrus wastes as substrate for solid-state fermentation by Aspergillus aculeatus. By comparing microbial growth, enzymes tested and medium consumption, irradiated substrate had higher biomass and extracellular protein, more sugar consumption and higher enzyme production than those with autoclaved substrate. Irradiation prevented the generation of cell-inhibiting components such as 5-hydroxymethylfurfural (5-HMF) whereas preserved the flavonoids well that are often enzyme inducers. These findings suggest that irradiation of agricultural and food processing wastes as substrate has advantages over autoclaving for solid-state fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: This study proposes irradiation as an alternative to sterilize agricultural residues rich in nutrients and thermosensitive compounds, such as citrus wastes for fungal solid-state fermentation and production of enzymes.

Bee pollen is an important bee-product that contains larger amount of bioactive compounds. In this paper, a tyrosinase inhibitor was separated from camellia pollen by the aid of high-speed counter-current chromatography (HSCCC), macroporous adsorptive resin chromatography and solvent fraction. The tyrosinase inhibitor was identified to be kaempferol by absorption spectrum, nuclear magnetic resonance and mass spectrum. The purity and recovery were 98.47% and 41.94%, respectively. The kaempferol was detected to have the IC50 of 0.05 mg/mL and mainly exists in glycoside forms. It had a concentration of 1.03 mg/g in the acidic hydrolysis of the ethanol extract (HEE), and contributed 32.41% to the total tyrosinase inhibition activity of HEE. This study demonstrates an effective procedure to purify bioactive compounds from bee pollen, and reveals kaempferol as a major tyrosinase inhibitor of camellia pollen, providing interesting information for in-depth study of bioactive compounds of pollen.

An extracellular agarase from marine bacterium Stenotrophomonas sp. NTa was purified to homogeneity. By size exclusion chromatography and SDS-PAGE analysis, the enzyme was determined to be a homodimer with monomeric molecular mass of 89.0 kDa. The optimal temperature and pH of strain NTa agarase were 40 °C and 10.0, respectively. It exhibited striking stability across a wide pH range of 5.0-11.0. Agarase from Stenotrophomonas sp. NTa had a relatively good resistance against the detected inhibitors, detergents and urea denaturant. The Km and Vmax for agar were 11.3mg/ml and 25.4 U/mg, respectively. Thin layer chromatography analysis, mass spectrometry, and enzyme assay using p-nitrophenyl-α/β-D-galactopyranoside revealed that strain NTa agarase was a β-agarase that degraded agarose into neoagarobiose, neoagarotetraose and neoagarohexaose as the predominant products, as well as a small amount of 3,6-anhydro-α-L-galactose. This is the first to present evidence of agarolytic activity in strain from genus Stenotrophomonas.

Enzymatic desulfation using arylsulfatase provides an attractive approach to improve agar quality. We have previously characterized a functional arylsulfatase from Pseudoalteromonas carrageenovora. To further improve its enzymatic performance, we isolated a mutant arylsulfatase of K253Q with improved enzyme activity from a random mutant library. Compared to wild-type arylsulfatase (WT), K253Q showed 33% increase in enzyme activity, with optimal temperature and pH of 55 °C and 8.0, respectively. K253Q demonstrated better substrate binding ability with lower Km value. Structure analysis indicated that a combination of the additional hydrogen bond and the enhanced substrate binding affinity could account for the improved enzyme activity of K253Q. K253Q exhibited about 54% sulfate removal against agar, resulting in additional 8% increase in 3,6-AG content and 20% increase in gel strength compared to WT. Scanning electron microscopy showed that K253Q treatment led to a stronger crosslinking structure of agar.

The anti-inflammatory properties of porphyrans (D1-D4) obtained from four discolored nori (Pyropia yezoensis) with different growth backgrounds were studied to examine possible variations in their bioactivities. Elution profiles of the porphyrans on Sepharose 4B indicated that D2-porphyran had relatively lower-molecular-size porphyrans than the other porphyrans. Inhibitory activities of the four porphyrans against nitric oxide (NO) and tumor necrosis factor-α (TNF-α) secretion by lipopolysaccharide (LPS)-stimulated RAW264.7 cells were different, whereas no significant differences were observed in the sulfate and anhydrogalactose levels. D2-porphyran showed the highest inhibitory activity against NO and TNF-α secretion by LPS-stimulated RAW264.7 cells, whereas D3- and D4-porphyrans had almost no activity. All porphyrans were efficiently degraded by free radical generated with ascorbate and hydrogen peroxide. The free-radical degradation resulted in a significant increase in the inhibitory activities of the four porphyrans against NO and TNF-α secretion, with varying rates depending on the porphyrans. The ability of D2-porphyran to suppress the receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclastogenesis in RAW264.7 cells was also significantly enhanced after degradation. Our results suggest that molecular size is an important factor affecting the anti-inflammatory activity of porphyrans, and radical degradation might be a promising procedure to obtain active low-molecular-size porphyrans.

Objective: To investigate effects of oxygen and heating on aromas and volatile components of pummelo essential oil (PEO). Method: The PEOs were analyzed by sensory evaluation, gas chromatography coupled with mass spectrometry (GC-MS), GC-olfactometry (GC-O) and sensory reconstruction analysis. Results: The PEO co-treated by oxygen and heating exhibited significant differences in both aromas and volatile compositions from those of the fresh, the nitrogen-protected, the heated and the oxygen-exposed PEOs. The strong sweet and floral notes of the oxygen and heating co-treated PEO were attributed to high concentration of limonene oxides; the strong minty note was resulted from the increase of L-carvone. Conclusion: The results indicate that limonene oxides and L-carvone could significantly change the aroma of PEO co-treatment by oxygen and heating, providing valuable information for the production and storage of aromatic products of PEO.

We previously found that ascophyllan, a sulfated polysaccharide isolated from brown seaweed Ascophyllum nodosum, exhibited antitumor activity in sarcoma-180 tumor-bearing mice. In this study, we found that ascophyllan inhibited the migration and adhesion of B16 melanoma cells by reducing the expression of N-cadherin and enhancing the expression of E-cadherin in a concentration-dependent manner. Transwell invasion assay revealed that ascophyllan suppressed the invasion ability of B16 cells. It also inhibited the expression of matrix metalloprotease-9 (MMP-9) mRNA and the secretion of MMP-9 protein in B16 cells, a process that may involve the extracellular signal-regulated kinase (ERK) signaling pathway. Furthermore, ascophyllan administered intraperitoneally at 25 mg/kg showed anti-metastatic activity in a mouse model of metastasis induced by intravenous injection of B16 cells, and the number of lung surface metastatic nodules in ascophyllan-treated mice was significantly reduced compared to that in the untreated control mice. Since splenic natural killer cell activity enhanced in the mice injected with ascophyllan intraperitoneally, we suggest that ascophyllan may exhibit in vivo anti-metastatic activity on B16 melanoma cells through activation of the host immune system in addition to a direct action on cancer cells.

A tannase that was prepared from Aspergillus niger by a solid-state fermentation on tea byproducts was investigated to transform the catechins in tea infusion. The tannase could effectively hydrolyze epigallocatechin gallate (EGCG), gallocatechin gallate (GCG) and epicatechin gallate (ECG) of tea infusion, which showed pH values ranged from 4.8 to 5.3 during the tannase treatment. The enzyme was active and stable within pH 3.0–7.0 and 4–60 °C. The enzyme had Kms of 14.89 mmol/L, 16.13 mmol/L and 3.57 mmol/L for EGCG, GCG and ECG, respectively in pure solutions. However, the hydrolysis of individual EGCG, GCG and ECG in the tea infusion showed different kinetic parameters from those in pure solutions. The transformation of EGCG was the dominant reaction in tea infusion. After the tannase treatment, tea infusion was detected to have significant increases in the clarity and activities for scavenging 2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonate) (ABTS) radical, 2, 2-diphenyl-1-picryl-hydrazyl (DPPH) radical and hydroxyl radical. The results indicated the tannase was effective to transform the catechins, which could be possibly used for commercial extraction of catechins. In addition, the results suggested it is more desirable to follow the dynamic model to control the tannase treatment of tea infusion for a better processing.

To improve the thermostability of α-L-rhamnosidase (r-Rha1), an enzyme previously identified from Aspergillus niger JMU-TS528, multiple arginine (Arg) residues were introduced into the r-Rha1 sequence to replace several lysine (Lys) residues that located on the surface of the folded r-Rha1. Hinted by in silico analysis, five surface Lys residues (K134, K228, K406, K440, K573) were targeted to produce a list of 5 single-residue mutants and 4 multiple-residue mutants using site-directed mutagenesis. Among these mutants, a double Lys to Arg mutant, i.e. K406R/K573R, showed the best thermostability improvement. The half-life of this mutant's enzyme activity increased 3 h at 60 °C, 23 min at 65 °C, and 3.5 min at 70 °C, when compared with the wild type. The simulated protein structure based interaction analysis and molecular dynamics calculation indicate that the thermostability improvement of the mutant K406R-K573R was possibly due to the extra hydrogen bonds, the additional cation-π interactions, and the relatively compact conformation. With the enhanced thermostability, the α-L-rhamnosidase mutant, K406R-K573R, has potentially broadened the r-Rha1 applications in food processing industry.

Polysaccharides prepared from marine algae sometimes contain contaminants such as polyphenols and endotoxins that may mislead their bona fide biological activities. In this study, we examined bioactive contaminants in commercially available fucoindan from Fucus vesiculosus, along with ascophyllan and fucoidan from Ascophyllum nodosum. F. vesiculosus fucoidan inhibited the growth of Vibrio alginolyticus in a concentration-dependent manner (0-1,000 μg mL?1). However, the antibacterial activity of the fucoidan significantly reduced after methanol-extraction, and the methanol-extract showed a potent antibacterial activity. The extract also showed cytotoxicity to RAW264.7 and U937 cells, and induced apoptotic nuclear morphological changes in U937 cells. These results suggest that the antibacterial activity of the fucoidan is partly due to the methanol-extractable contaminants that can also contribute to the cytotoxicity on RAW264.7 and U937 cells. On the other hand, the activities to induce secretion of nitric oxide and tumor necrosis factor-α from RAW264.7 cells were observed in the fucoidan even after methanol extraction, and the extract had no such activities. Our observations suggest that commercially available fucoidan should be purified prior to biochemical use., ALGAE, 29(3), pp.237-247; 2014

Marine-derived sulfated polysaccharides have been shown to possess certain anti-virus, anti-tumor, anti-inflammatory and anti-coagulant activities. However, the in vivo immunomodulatory effects of marine-derived pure compounds have been less well characterized. In this study, we investigated the effect of ascophyllan, a sulfated polysaccharide purified from Ascophyllum nodosum, on the maturation of mouse dendritic cells (DCs) in vitro and in vivo. Ascophyllan induced up-regulation of co-stimulatory molecules and production of pro-inflammatory cytokines in bone marrow-derived DCs (BMDCs). Moreover, in vivo administration of ascophyllan promotes up-regulation of CD40, CD80, CD86, MHC class I and MHC class II and production of IL-6, IL-12 and TNF-α in spleen cDCs. Interestingly, ascophyllan induced a higher degree of co-stimulatory molecule up-regulation and pro-inflammatory cytokine production than fucoidan, a marine-derived polysaccharide with well-defined effect for promoting DC maturation. Ascophyllan also promoted the generation of IFN-γ-producing Th1 and Tc1 cells in the presence of DCs in an IL-12-dependent manner. Finally, myeloid differentiation primary response 88 (MyD88) signaling pathway was essential for DC maturation induced by ascophyllan. Taken together, these results demonstrate that ascophyllan induces DC maturation, and consequently enhances Th1 and Tc1 responses in vivo. This knowledge could facilitate the development of novel therapeutic strategies to combat infectious diseases and cancer., Marine Drugs, 12(7), pp.4148-4164; 2014

This study evaluated the immunomodulatory activities, including regulation of nitric oxide (NO), reactive oxygen species (ROS), and tumor necrosis factor (TNF)-α production in RAW264.7 murine macrophages, of alginate oligosaccharides (AOS) and investigated their structure-activity relationships. Our results revealed that unsaturated guluronate oligosaccharide prepared by enzymatic degradation (GOS-ED) induced NO production and inducible nitric oxide synthase (iNOS) expression, dose and time dependently, and stimulated ROS and TNF-α production; however, other AOS prepared by different ways or polymers showed very low and even no such effects. Moreover, GOS-ED induced macrophage activation to release the above-mentioned mediators partly involved in nuclear factor (NF)-κB and mitogen-activated protein (MAP) kinase signaling pathways. We also show that the structural characteristics of AOS, especially the unsaturated terminal structure, molecular size, and M/G ratio, play important roles in determining the macrophage-activating effects. GOS-ED could be applicable for agriculture, drug, and food industry as a potent immune-modulatory agent.

Alginate from marine seaweeds is receiving continuous attention owing to its wide physiological activities. Herein, we sought to elucidate possible effects of alginate-derived polyguluronate (PG) and unsaturated guluronate oligosaccharide (GOS) on antibacterial activities of macrophages. Our results showed that, in contrast to PG, GOS markedly increased the phagocytosis of IgG-opsonized Escherichia coli and Staphylococcus aureus and further inhibited the survival of intracellular bacteria in macrophages. In line with this, GOS treatment resulted in the enhanced expression of Fcγ receptors on macrophages. In addition, GOS activated NF-κB pathway, induced TNF-α secretion, and elevated the expression of inducible nitric oxide synthase and the production of nitric oxide. Meanwhile, GOS stimulated the production of reactive oxygen species in macrophages. Moreover, guluronate trimer to hexamer (G3-G6) in GOS exhibited significant activity that increased the bacterial phagocytosis of macrophages, with the pentamer (G5), displaying the highest activity. Finally, our in vivo results further confirmed that GOS but not PG significantly improved bacterial clearance in murine acute peritonitis. In conclusion, GOS enhances antibacterial activities of macrophages via modulating signaling pathways related to innate immunity, suggesting that GOS might be a promising therapeutic candidate to improve the host defense against bacterial infection.

To investigate the role of sulfate groups on the macrophage-stimulating activities of ascophyllan, we prepared desulfated ascophyllan, and its effects on RAW264.7 cells were compared with native ascophyllan. The chemical structural analysis revealed that nearly 21% of sulfate groups of ascophyllan were removed by desulfation reaction, while no significant changes in the molecular mass and monosaccharide composition occurred after desulfation. NO- and cytokine- (TNF-α and G-CSF) inducing activities of the desulfated ascophyllan on RAW264.7 cells were significantly decreased as compared to native ascophyllan. Furthermore, the activity of desulfated ascophyllan to induce reactive oxygen species (ROS) generation from RAW264.7 cells decreased to almost negligible level. Our results suggest that the level of sulfate groups of ascophyllan is an important structural element responsible for the macrophage-stimulating activities. Probably, even the limited removal of sulfate residues sensitive to desulfation reaction may result in significant decrease in the bioactivities of ascophyllan.

Antioxidant activities of sulfated polysaccharide ascophyllan from Ascophyllum nodosum was investigated in vitro by various assays, and compared with those of fucoidan. A chemiluminescence (CL) analysis using a luminol analog, L-012, showed that ascophyllan scavenges superoxide, and the activity is greater than fucoidan. However, in the presence of 10μg/ml of ascophyllan or 10μg/ml and 100μg/ml of fucoidan, slightly enhanced CL-responses were observed. Since EDTA-treatment resulted in disappearance of the enhancement effects, it was suggested that metal ions especially iron ions in the polysaccharides might be involved in this phenomenon. In fact, metal element analysis revealed that ascophyllan and fucoidan inherently contain iron and other metal elements. EDTA-treatment resulted in significant increase in Fe2+-chelating activities of these polysaccharides. In an electron spin resonance (ESR)-spin trapping analysis in which direct UV-radiation to hydrogen peroxide was used as a source of hydroxyl radical, ascophyllan and fucoidan showed potent hydroxyl radical scavenging activity with similar extent. Reducing power of ascophyllan was stronger than that of fucoidan. Our results indicate that ascophyllan can exhibit direct and potent antioxidant activity., International Journal of Biological Macromolecules, 59, pp.305-312; 2013

A time-course analysis of reactive oxygen species (ROS) generation in fertilized eggs of the devil stinger (Inimicus japonicus) from 0 h post-fertilization (hpf) to the early larval stage indicated that the ROS level was highest in the 22 hpf embryo, and declined thereafter. Phorbol myristate acetate (PMA) had no effect on ROS generation by the 22 hpf embryo, whereas PMA significantly increased larval ROS generation, suggesting that the ROS generation mechanisms of the 22 hpf embryo and larva are different at least in terms of PMA-responsiveness. Our results suggest the presence of a specific ROS generation system in devil stinger embryo which can be transitionally activated during embryogenesis.

Ascophyllan isolated from the brown alga Ascophyllum nodosum is a fucose-containing sulfated polysaccharide, which has similar but distinct characteristic monosaccharide composition and entire chemical structure to fucoidan. In this study, we examined the effects of ascophyllan, fucoidan isolated from A. nodosum (A-fucoidan), and fucoidan from Sigma (S-fucoidan) as a representative fucoidan derived from other source (Fucus vesiculosus) on mouse macrophage cell line RAW264.7 cells. No significant cytotoxic effects of ascophyllan and A-fucoidan on RAW264.7 cells were observed up to 1000μg/ml, while S-fucoidan showed cytotoxic effect in a concentration-dependent manner. Ascophyllan induced extremely higher level of nitric oxide (NO) production from RAW264.7 cells than those induced by fucoidans over the concentration range tested (0-200μg/ml). Reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis revealed that expression level of inducible NO synthase (iNOS) in ascophyllan-treated RAW264.7 cells was much higher than the levels detected in the cells treated with fucoidans. Furthermore, the activities of ascophyllan to induce the secretion of tumor necrosis factor-α (TNF-α) and granulocyte colony-stimulating factor (G-CSF) from RAW264.7 cells were also greater than those induced by fucoidans especially at lower concentration range (3.1-50μg/ml). The activities of ascophyllan to induce NO and cytokine production in mouse peritoneal macrophages were also stronger than those of fucoidans. Electrophoretic mobility shift assay (EMSA) using infrared dye labeled nuclear factor-kappa B (NF-κB) and AP-1 consensus sequences suggested that ascophyllan can strongly activate these transcription factors. Marked increase in the nuclear translocation of p65, and the phosphorylation and degradation of IκB-α were also observed in ascophyllan-treated RAW264.7 cells. Analysis using mitogen-activated protein (MAP) kinase inhibitors and western blot analysis suggested that c-Jun N-terminal kinase (JNK) and p38 MAP kinase are mainly involved in ascophyllan-induced NO production., Nitric Oxide, 25(4), pp.407-415; 2011

The effects of fucose-containing sulfated polysaccharides, ascophyllan and fucoidan, isolated from the brown alga Ascophyllum nodosum, on the growth of various cell lines (MDCK, Vero, PtK(1), CHO, HeLa, and XC) were investigated. In a colony formation assay, ascophyllan and fucoidan showed potent cytotoxic effects on Vero and XC cells, while other cell lines were relatively resistant to these polysaccharides. Almost no significant effects of these polysaccharides were observed in the cell lines tested using the Alamar blue cytotoxicity assay over 48 h with varying initial cell densities (2500-20,000 cells/well) in growth medium. Interestingly, a significant growth promoting effect of ascophyllan on MDCK cells was observed, whereas treatment with fucoidan showed growth suppressive effects on this cell line under the same experimental conditions. These results suggest that ascophyllan is distinguishable from fucoidan in terms of their bioactivities. This is the first report of the growth promoting effects of a sulfated fucan on a mammalian cell line under normal growth conditions., Journal of bioscience and bioengineering, 110(1), pp.113-117; 2010

We examined the growth and interactions between the bloom-forming flagellates Prorocentrum minimum and Heterosigma akashiwo using bi-algal culture experiments. When both species were inoculated at high cell densities, growth of H. akashiwo was inhibited by P. minimum. In other combinations of inoculation densities, the species first reaching the stationary phase substantially suppressed maximum cell densities of the other species, but the growth inhibition effect of P. minimum was stronger than that of H. akashiwo. We used a mathematical model to simulate growth and interactions of P. minimum and H. akashiwo in bi-algal cultures. The model indicated that P. minimum always out-competed H. akashiwo over time. Additional experiments showed that crude extracts from P. minimum and H. akashiwo cultures did not affect the growth of either species, but both strongly inhibited the growth of the bloom-forming diatom Skeletonema costatum. Further experiments showed that it was unlikely that reactive oxygen species produced by H. akashiwo were responsible for the inhibition of P. minimum growth., Hydrobiologia, 641(1), pp.33-44; 2010

A putative cDNA of α-l-rhamnosidase was PCR-cloned from Aspergillus niger JMU-TS528 and further extracellular over-expressed in Pichia pastoris GS115. The activity of the recombinant α-l-rhamnosidase r-Rha1 was 711.9U/mL, eightfold higher than the native α-l-rhamnosidase from A. niger JMU-TS528. r-Rha1 is a N-glycosylated protein of 90kDa and possesses broad substrate specificities by hydrolyzing α-1,2, α-1,3 α-1,4, and α-1,6 linkages to β-d-glucosides. This is the first report presenting that α-l-rhamnosidase showed activity on four kinds of glucosidic linkages. Compared with other previously characterized α-l-rhamnosidases, r-Rha1 showed a good thermostability and wide range of pH-stability with the optimum pH of 5.0 and temperature of 60°C. r-Rha1 activity was not greatly affected by representative metal ions and other detected effectors and showed excellent tolerance abilities against glucose and ethanol. These beneficial characteristics of r-Rha1 suggest that r-Rha1 should be considered a potential new biocatalyst for food and drug industrial applications.

Sensory evaluation, GC–MS and omission test were used to characterize aromas of noninstant Oolong tea (NIOT), original instant Oolong tea (OIOT) and its counterparts treated by two enzyme extracts from Aspergillus niger. The results showed that OIOT had stronger sweet, fruity, and roasted notes, but weaker grass and floral notes than NIOT. After treated by the crude enzymes extracted from the fermented broth from tea waste (TW) medium, the treated-OIOT had an improved aroma similar to that of NIOT, due to the increases of 2-pentyl-furan and 3-cis-hexen-1-ol. In comparison, after the treatment of crude enzymes from wheat bran and orange peel (WBOP) medium, the treated-OIOT showed significantly aroma difference from NIOT and OIOT, which was attributed to the increases of 3-cis-hexen-1-ol, γ-terpinene, and β-trans-farnesene. These results revealed that the original aroma of OIOT could be effectively modified and improved by the enzyme extracts, particularly by that from the TW medium. Practical applications Tea is one of the most popular beverages all over the world. Instant tea is an important tea product that is processed from teas by extraction, concentration and drying. Instant tea is more convenient than teas for industrially producing tea foods such as tea beverage, tea break, and tea ice cream. However, the aroma profile of instant tea is always inferior to those of the original unprocessed noninstant teas, because the characteristic aroma of the original unprocessed tea could undergo significant changes during the preparation of instant tea. The present manuscript shows two enzyme extracts from Aspergillus niger could improve the aroma of instant Oolong tea, providing interesting information to promote the aroma of instant tea via enzymatic treatments, which may help to develop effective approaches to enhancing the quality of instant teas.

Alginate has notably diverse pharmacological activities. The present study investigated the anti-inflammatory activity of the guluronate oligosaccharides prepared by oxidative degradation (GOS-OD) from alginate. GOS-OD significantly attenuated the production of nitric oxide (NO), prostaglandin E2 (PGE2), and reactive oxygen species (ROS), the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2, and the secretion of pro-inflammatory cytokines in lipopolysaccharide (LPS)-activated murine macrophage RAW 264.7 cells. Moreover, GOS-OD potently decreased the binding of LPS to the cell surface and LPS-induced Toll-like receptor 4 (TLR4) and cluster of differentiation (CD) 14 expression. Additionally, GOS-OD could remarkably inhibit the LPS-induced activation of nuclear factor (NF)-κB and mitogen-activated protein (MAP) kinase pathways in RAW 264.7 cells. These results indicate that GOS-OD may reduce the LPS-stimulated inflammatory responses through blocking the activation of NF-κB and MAP kinases, suggesting that GOS-OD may be considered as a potential nutraceutical for inflammation.

Splenic natural killer (NK) cell activity against YAC-1 cells increased in mice intraperitoneally injected with ascophyllan. Ascophyllan enhanced the cytotoxicity of RAW264.7 cells toward YAC-1 cells in a concentration-dependent manner. The cytotoxicity of ascophyllan-stimulated RAW264.7 cells as to YAC-1 cells was suppressed with N(G)-nitro-L-arginine methyl ester hydrochloride (L-NAME), an inhibitor of nitric oxide (NO) synthase, suggesting the involvement of NO in the cytotoxicity of ascophyllan-stimulated RAW264.7 cells.

In this study, we investigated the bioactivity of ascophyllan in terms of reactive oxygen species (ROS) generation. In RAW264.7 cells, we found that ascophyllan induced ROS generation in a concentration-dependent manner, but with bell-shaped profile. Immunoblot analysis demonstrated that ascophyllan promoted the translocation of cytosolic subunits (p67phox and p47phox) of NADPH oxidase to the plasma membrane. Among mitogen activated protein (MAP) kinase inhibitors tested, JNK inhibitor showed the most potent inhibitory effect on ascophyllan-induced ROS production. Consistently, significant level of phosphorylated JNK MAP kinase was detected in ascophyllan-treated RAW264.7 cells. Our findings suggest for the first time that ascophyllan can stimulate macrophages to produce ROS through the activations of NADPH oxidase and JNK MAP kinase., International Journal of Biological Macromolecules, 52, pp.164-169; 2013

Heterocapsa circularisquama showed much higher toxic effects on short-necked clams than Chattonella marina. Clams exposed to H. circularisquama exhibited morphological changes concomitant with an accumulation of mucus-like substances in the gills, a profound reduction in filtration activity, and lysosomal destabilization in hemocytes. Chattonella marina was less effective than H. circularisquama, and Heterocapsa triquetra was almost harmless in all these criteria. These results suggest that H. circularisquama exerted its lethal effect on short-necked clams through gill tissue damage and subsequent induction of physiological stress.

Alginate is a natural acidic linear polysaccharide that is produced by brown seaweeds. It is currently used in a broad range of commercial enterprises, such as the food and medical products industries. Recent evidence has demonstrated that alginate oligosaccharides may function as growth promoting agents for certain plant cells, including those of some green algae. Chlamydomonas reinhardtii is a green alga that is used as a model organism in fundamental molecular biology studies; it is also a producer of biohydrogen. In the present study, we examined effects of two types of alginate oligosaccharide mixtures (AOMs), which were prepared by either enzymatic degradation (ED) or acid hydrolysis (AH), on the growth of C. reinhardtii. Growth was significantly promoted by AOM (ED) in a concentration-dependent manner. The maximum effect was observed on day 4 of treatment. The fatty acid composition of C. reinhardtii was also influenced by AOM (ED); the levels of C16:0, C18:2 cis and C18:3 n-3 increased in treated cells. AOM (AH) and the other saccharides that we tested did not affect the growth of C. reinhardtii. The effects that we identified could promote efficient biomass production by reducing culture times and by changing cellular fatty acid levels., Journal of Bioscience and Bioengineering, 113(1), pp.112-116; 2012

The effects of cytotoxic lectins, modeccin and phytohemagglutinin (PHA) on mouse macrophage cell line RAW264.7 was studied by detecting the induction of inflammatory mediators. Results showed that modeccin induced the release of tumor necrosis factor-α (TNF-α) from RAW264.7 cells with a bell-shape concentration-dependent profile. PHA that showed no significant cytotoxicity on RAW264.7 cells up to 100,000 ng/ml induced much higher level of TNF-α than modeccin. PHA simultaneously induced the secretion of granulocyte colony stimulation factor (G-CSF) from RAW264.7 cells with even much higher level than that of TNF-α, whereas modeccin did not. Furthermore, PHA induced the secretion of nitric oxide (NO) in RAW264.7 cells, while no significant level of NO was detected in the modeccin-treated cells. NH₄Cl (a lysomotoropic agent) and cycloheximide (a ribosome inhibitor) strongly inhibited modeccin-induced TNF-α secretion, but no significant inhibitory effects of these reagents on the PHA-induced TNF-α secretion were observed. Contrary to modeccin-induced TNF-α secretion, even slightly increased TNF-α secretion was observed in PHA-treated cells in the presence of 10 mM NH₄Cl. In addition, the inhibition profiles of modeccin-induced TNF-α secretion by various kinase inhibitors were different from those of PHA. These results suggested that the action mode of modeccin to stimulate RAW264.7 cells leading to the secretion of inflammatory molecules, including TNF-α, is distinct from that of PHA. On the other hand, significantly increased translocation of activator protein-1 (AP-1), a crucial transcription factor involved in expression of inflammatory molecules, into nucleus was observed in RAW264.7 cells treated with PHA and modeccin., Acta biochimica et biophysica Sinica, 43(1), pp.52-60; 2011

Genetic polymorphisms of methylenetetrahydrofolate reductase (MTHFR) have been suggested as being associated with cerebral palsy (CP) but the evidence is uncertain. The purpose of this study was to investigate whether MTHFR gene polymorphisms contribute to the development of CP in Chinese infants. For this study, 169 health controls and 159 infants with CP including 43 cases also suffering from mental retardation (MR) were recruited. Genomic DNA was prepared from venous blood and all five single nucleotide polymorphisms in MTHFR (rs4846049, rs1476413, rs1801131, rs1801133 and rs9651118) were genotyped using TaqMan technology. There were no significant differences in allele or genotype frequencies between the CP patients and controls at any of the five genetic polymorphisms. Subgroup analysis found statistically significant difference in allele and genotype frequencies between cases with both CP and MR (CP + MR) compared with both CP-only cases and controls at rs4846049, rs1476413 and rs1801131. The frequencies of the T alleles of rs4846049, rs1476413 and the G allele of rs1801131 were greater in the CP + MR patients than in the CP-only patients and controls. This study provides the first evidence pointing to a MTHFR gene polymorphism as a potential risk factor for CP combined with MR.