A novel PDPN antagonist peptide CY12-RP2 inhibits melanoma growth via Wnt/ß-catenin and modulates the immune cells.

BACKGROUND: Podoplanin (PDPN) is a highly conserved, mucin-type protein specific to the lymphatic system. Overexpression of PDPN is associated with the progression of various solid tumors, and plays an important roles in the tumor microenvironment by regulating the immune system. However, the role of PDPN-mediated signal activation in the progression of melanoma is still unknown. METHODS: PDPN expression was first analyzed in 112 human melanoma tissue microarrays and melanoma cell lines. Functional experiments including proliferation, clone formation, migration, and metastasis were utilized to identify the suppressive effects of PDPN. The Ph.D.TM-12 Phage Display Peptide Library was used to obtain a PDPN antagonist peptide, named CY12-RP2. The immunofluorescence, SPR assay, and flow cytometry were used to identify the binding specificity of CY12-RP2 with PDPN in melanoma cells. Functional and mechanistic assays in vivo and in vitro were performed for discriminating the antitumor and immune activation effects of CY12-RP2. RESULTS: PDPN was overexpressed in melanoma tissue and cells, and inhibited melanoma cells proliferation, migration, and metastasis by blocking the EMT and Wnt/ß-catenin pathway. PDPN antagonistic peptide, CY12-RP2, could specifically bind with PDPN, suppressing melanoma various functions inducing apoptosis in both melanoma cells and 3D spheroids. CY12-RP2 also enhanced the anti-tumor capacity of PBMC, and inhibited melanoma cells growth both in xenografts and allogeneic mice model. Moreover, CY12-RP2 could inhibit melanoma lung metastasis, and abrogated the immunosuppressive effects of PDPN by increasing the proportion of CD3 + CD4 + T cells, CD3 + CD8 + T cells, CD49b + Granzyme B + NK cells, and CD11b + CD86 + M1-like macrophages and the levels of IL-1ß, TNF-α, and IFN-γ. CONCLUSIONS: This study has demonstrated the important role of PDPN in the progression of melanoma and formation of immunosuppressive environment, and provided a potential approach of treating melanoma using the novel CY12-RP2 peptide. In melanoma, PDPN is overexpressed in the cancer cells, and promotes melanoma cells growth and metastasis through activating the Wnt/ß-catenin pathway. Treatment with the PDPN antagonistic peptide CY12-RP2 could not only inhibit the melanoma growth and metastasis both in vitro and in vivo through Wnt/ß-catenin pathway blockade, but also abrogate the immunosuppressive effects of PDPN through modulating immune cells.

Effects of high manganese-cultivated seedlings on cadmium uptake by various rice (Oryza sativa L.) genotypes.

Cadmium (Cd) contamination in paddy soil threatens rice growth and food safety, enriching manganese (Mn) in rice seedlings is expected to reduce Cd uptake by rice. The effects of 250 µM Mn-treated seedlings on reducing Cd uptake of four rice genotypes (WYJ21, ZJY1578, HHZ, and HLYSM) planted in 0.61 mg kg-1 Cd-contaminated soil, were studied through the hydroponic and pot experiments. The results showed that the ZJY1578 seedling had the highest Mn level (459 µg plant-1), followed by WYJ21 (309 µg plant-1), and less Mn accumulated in the other genotypes. The relative expression of OsNramp5 (natural resistance-associated macrophage protein) was reduced by 42.7 % in ZJY1578 but increased by 23.3 % in HLYSM. The expressions of OsIRT1 (iron-regulated transporter-like protein) were reduced by 24.0-56.0 % in the four genotypes, with the highest reduction in ZJY1578. Consequently, a greater reduction of Cd occurred in ZJY1578 than that in the other genotypes, i.e., the root and shoot Cd at the tillering were reduced by 27.8 % and 48.5 %, respectively. At the mature stage, total Cd amount and distribution in the shoot and brown rice were also greatly reduced in ZJY1578, but the inhibitory effects were weakened compared to the tillering stage. This study found various responses of Cd uptake and transporters to Mn-treated seedlings among rice genotypes, thus resulting in various Cd reductions. In the future, the microscopic transport processes of Cd within rice should be explored to deeply explain the genotypic variation.

Natural immunomodulating substances used for alleviating food allergy.

Food allergy is a serious health problem affecting more than 10% of the human population worldwide. Medical treatments for food allergy remain limited because immune therapy is risky and costly, and anti-allergic drugs have many harmful side effects and can cause drug dependence. In this paper, we review natural bioactive substances capable of alleviating food allergy. The sources of the anti-allergic substances reviewed include plants, animals, and microbes, and the types of substances include polysaccharides, oligosaccharides, polyphenols, phycocyanin, polyunsaturated fatty acids, flavonoids, terpenoids, quinones, alkaloids, phenylpropanoids, and probiotics. We describe five mechanisms involved in anti-allergic activities, including binding with epitopes located in allergens, affecting the gut microbiota, influencing intestinal epithelial cells, altering antigen presentation and T cell differentiation, and inhibiting the degranulation of effector cells. In the discussion, we present the limitations of existing researches as well as promising advances in the development of anti-allergic foods and/or immunomodulating food ingredients that can effectively prevent or alleviate food allergy. This review provides a reference for further research on anti-allergic materials and their hyposensitizing mechanisms.

N-glycosylation of PD-1 promotes binding of camrelizumab.

PD-1 is a highly glycosylated inhibitory receptor expressed mainly on T cells. Targeting of PD-1 with monoclonal antibodies (MAbs) to block the interaction with its ligand PD-L1 has been successful for the treatment of multiple tumors. However, polymorphisms at N-glycosylation sites of PD-1 exist in the human population that might affect antibody binding, and dysregulated glycosylation has been observed in the tumor microenvironment. Here, we demonstrate varied N-glycan composition in PD-1, and show that the binding affinity of camrelizumab, a recently approved PD-1-specific MAb, to non-glycosylated PD-1 proteins from E. coli is substantially decreased compared with glycosylated PD-1. The structure of the camrelizumab/PD-1 complex reveals that camrelizumab mainly utilizes its heavy chain to bind to PD-1, while the light chain sterically inhibits the binding of PD-L1 to PD-1. Glycosylation of asparagine 58 (N58) promotes the interaction with camrelizumab, while the efficiency of camrelizumab to inhibit the binding of PD-L1 is substantially reduced for glycosylation-deficient PD-1. These results increase our understanding of how glycosylation affects the activity of PD-1-specific MAbs during immune checkpoint therapy.

Anti-fibrotic effect of melittin on TRIM47 expression in human embryonic lung fibroblast through regulating TRIM47 pathway.

AIMS: To investigate the effect and underlying mechanism of melittin and tripartite motif (TRIM) family in human embryonic lung fibroblast (HELF). MATERIALS AND METHODS: Lentiviral RNA interference vector and lentiviral overexpression vector were constructed and packaged by transfecting 293T cells; the proliferation of HELF was examined using Cell Counting Kit 8; Western blot and qRT-PCR were performed to examine protein and mRNA expression; the interaction with protein phosphatase magnesium-dependent 1A (PPM1A) was examined by Co-immunoprecipitation. KEY FINDINGS: Compared with the control group, the mRNA expression of the TRIM6, TRIM8 and TRIM47 in the IPF group significantly increased. Melittin inhibited the mRNA expression and protein expression levels of TRIM47, the HELF proliferation, the hydroxyproline levels, and the phosphorylation of Smad2/3; the interference of TRIM47 inhibited the protein expression of Vimentin, α-SMA, CTGF, the phosphorylation of Smad2/3 and the synthesis of hydroxyproline; TRIM47 overexpression elevated the phosphorylation of Smad2/3, induced ubiquitination of PPM1A and decreased the expression level of PPM1A, while TRIM47 RNA interference reversed this result. SIGNIFICANCE: Melittin has anti-fibrotic effect in HELF by directly reducing the phosphorylation of Smad2/3 or indirectly reducing the phosphorylation of Smad2/3 by decreasing the expression levels of TRIM47 whose overexpression induces ubiquitination of PPM1A.

Quantitative Localized Analysis Reveals Distinct Exosomal Protein-Specific Glycosignatures: Implications in Cancer Cell Subtyping, Exosome Biogenesis, and Function.

Protein-specific glycoform analysis is essential for the thorough understanding of cellular chemistry and signaling but presents a significant assay challenge for small-sized, free-floating exosomes, ubiquitous regulators of cellular physiological functions and mediators of intercellular communication. We report herein a quantitative localized analysis (QLA) method for the first-time achievement of a protein-specific glycosignature assay on these important extracellular vesicles. The integration of localized chemical remodeling and quantitative electrochemistry allows the proof-of-concept QLA examination of exosomal mucin 1 (MUC1)-specific terminal galactose/N-acetylgalactosamine (Gal/GalNAc). In combination with sialic acid (Sia) cleavage manipulation for the exposure of originally capped Gal/GalNAc, QLA has revealed distinct MUC1-specific sialylation capping ratios for MCF-7 and MDA-MB-231 exosomes, as well as when compared to parent cells. These findings suggest a useful noninvasive indicator for subtyping cancer cells and exosome secretion as a likely venue for the preservation of cellular compositional and functional integrity. The QLA method also permits dynamic monitoring of changes in the exosomal MUC1-specific sialylation capping ratio, enabling the distinction of biogenesis pathways of exosomes.

Arabinogalactan derived from Lycium barbarum fruit inhibits cancer cell growth via cell cycle arrest and apoptosis.

Previous studies have shown that crude polysaccharides from the Lycium barbarum fruit could inhibit cancer cell growth, but the major effective constituents are yet to be identified. In this study, we compared the effects of L. barbarum fruit polysaccharide fractions on the growth of hepatoma cells (SMMC-7721 and HepG2), cervical cancer cells (HeLa), gastric carcinoma cells (SGC-7901), and human breast cancer cells (MCF-7). LBGP-I-3 showed stronger inhibitory effects on MCF-7 cells (cell viability of 48.96%) than SMMC-7721 (cell viability of 78.91%) and HeLa cells (cell viability of 55.94%), and had no effect on HepG2 and SGC-7901 cells. In addition, LBGP-I-3 had no inhibitory effect on normal liver cells (L02, cell viability of 115.58%). Investigation of the underlying mechanism suggested that LBGP-I-3 inhibited the growth of cancer cells by cell cycle arrest and apoptosis. LBGP-I-3 arrested the cell cycle at the G0/G1 phase, altered mitochondrial function, activated oxidative stress, and regulated the MAPK signaling pathway to induce apoptosis. Thus, LBGP-I-3 may be a potential functional food ingredient for the prevention of cancer without toxicity to normal cells in vitro. These results could help further elucidate the structure-activity relationship of L. barbarum fruit polysaccharides and functional food development.

Preparation of chondroitin sulfates with different molecular weights from bovine nasal cartilage and their antioxidant activities.

Biological functions of chondroitin sulfate, including anti-oxidation and anti-inflammation, are associated with its molecular weight. This study aimed to evaluate the correlation between antioxidant activity and molecular weights of chondroitin sulfate derived from bovine nasal cartilage (BCS). BCS extracted by compound enzymatic method was further purified via DEAE-cellulose column separation to obtain BCS-II (129.4 kDa), which was further degraded by H2O2-Vc to obtain four subfractions: BCS-II-1 (92.7 kDa), BCS-II-2 (54.1 kDa), BCS-II-3 (26.3 kDa), and BCS-II-4 (19.7 kDa). Changes in the physicochemical properties of BCS-II before and after degradation were compared via FT-IR, NMR and monosaccharide composition analysis. Finally, antioxidant activities of BCS-II and its subfractions BCS-II-1-4 were compared. Our results showed that the H2O2-Vc system did not disrupt the primary functional group of BCS-II, with no significant change in sulfate content between BCS-II and its degraded fractions; however, uronic acid levels increased in degraded fractions when compared with BCS-II. In vitro, BCS-II-4 displayed the lowest molecular weight and had the strongest antioxidant activity. Therefore, the antioxidant activity of chondroitin sulfate in vitro is robustly associated with its molecular weight, and low-molecular-weight chondroitin sulfate can be used as an antioxidant in the food and pharmaceutical industries and other sectors.

High-sensitivity quantification of glycosphingolipid glycans by ESI-MS utilizing ozonolysis-based release and isotopic Girard's reagent labeling.

Quantitative analysis of glycosphingolipids (GSLs) has been hindered by the lack of chromogenic groups for spectral detection or active functional groups for specific derivatization. In this study, a highly sensitive method based on ozonolysis-induced release and isotopic Girard's reagent P labeling of GSL glycans coupled with mass spectrometric detection for the quantification of animal tissue GSLs is developed. First, different approaches for the release of glycans from GSLs were compared with each other and the ozonolysis-based method was found to have the highest glycan yield under relative mild reaction conditions. Then a relative quantification method of ozonolysis-released GSL glycans based on stable isotope labeling using nondeuterated (d0-) and 2,3,4,5,6-pentadeuterated (d5-) Girard's reagent P (GP) was established, and its good linearity, accuracy and reproducibility were statistically verified. Finally, the new method was successfully applied to revealing the difference between porcine brain and liver as well as between the brain of normal and aging rats in GSL glycome by online hydrophilic interaction liquid chromatography coupling with ultraviolet detection and tandem mass spectrometry (HILIC-UV-MS/MS). This novel method is versatile and sensitive, enabling accurate quantitative analysis of tissue GSLs and showing great significance for glycomic studies.

Glycoqueuing: Isomer-Specific Quantification for Sialylation-Focused Glycomics.

Changes of α-2,3-/α-2,6-linked sialic acids (SAs) in sialylglycans have been found to be closely related with some diseases. However, accurate quantification of sialylglycans at the isomeric level remains challenging due to their instability, structural complexity, and low mass spectrometry (MS) detection sensitivity. Herein, we propose an analytical strategy named "glycoqueuing", which allows sequential chromatographic elution and high-sensitivity MS quantification of various sialylglycan isomers based on isotopic labeling followed by analysis via online reversed-phase high performance liquid chromatography coupling with MS (RP-HPLC-MS). The new method was validated by detailed structural identification and quantification of fetal bovine serum (FBS) N-linked sialylglycan isomers, during which many branching isomers were successfully differentiated, and 28 sialylglycan compositions with Neu5Gc residues were analyzed. The method was successfully applied to isomer-specific, quantitative comparison of sialylated N-glycans between bovine and rabbit immunoglobulin G (IgG) and the search for serum sialylated N-glycan biomarker candidates of hepatocellular carcinoma, during which a 55% increase of α-2,6-sialylated fucosylated N-glycans was revealed, demonstrating the great applicability and potential clinical usage of the method.

Preparation, structural characterization and bioactivity of 4-O-Methylglucuronoxylan from Artemisia sphaerocephala Krasch.

We obtained four soluble acid xylan fractions AGP-III-A, AGP-III-B, AGP-III-C and AGP-III-D from the insoluble Artemisia sphaerocephala Krasch gum (ASKG) polysaccharide by weak alkali treatment combined with H2O2-Vc oxidative degradation. Activity studies showed that the degradation components could reduce the cell viability of several cancer cell lines in a concentration-dependent manner, especially 4-O-Methylglucuronoxylan AGP-III-C with specific molecular weight and branching degree significantly reduced cancer cells viability and induced HepG2 apoptosis, also caused mitochondrial membrane dysfunction upregulated ROS levels, and induced G0/G1 arrest in HepG2 cells by cell cycle assay. Further, AGP-III-C mediates apoptosis in HepG2 cells by upregulating MAPK phosphorylation. The structure of AGP-III-C was characterized by uronic acid reduction, permethylation with GC-MS, and 2D-NMR analysis. The structure of AGP-III-C had a linear (1→4)-linked ß-Xylf residue backbone with one branched 4-O-Me-α-GlcAp attached to the main chain by a (1→2)-glycosidic bond at every two ß-(1→4)-Xylf units.

Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Analysis of Human Milk Neutral and Sialylated Free Oligosaccharides Using Girard's Reagent P On-Target Derivatization.

The functional role of human milk oligosaccharides (HMOs) is closely associated with their type, composition, and structure. However, a detailed analysis of HMOs is difficult because neutral oligosaccharides (NHMOs) are mixed with sialylated oligosaccharides (SHMOs) in milk. Here, NHMOs were separated from SHMOs by DEAE-52 anion chromatography, and lactose was removed by graphite carbon solid-phase extraction. Lactose-free NHMOs were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) based on Girard's reagent P on-target derivatization (GPOD), and SHMOs were analyzed by MALDI-TOF-MS following selective sialic acid derivatization and GPOD. Sixty-four oligosaccharides were detected: 36 NHMOs, of which 28 were fucosylated, and 28 SHMOs, of which 8 with α-2,3-linked monosialic acid, 2 with α-2,3-linked disialic acid, 10 with α-2,6-linked monosialic acid, 2 with α-2,6-linked disialic acid, and 5 with both α-2,3- and α-2,6-linked disialic acid. These findings provide the groundwork for further characterization of the structure and activity of HMOs.

Physicochemical properties, antioxidant activity and immunological effects in vitro of polysaccharides from Schisandra sphenanthera and Schisandra chinensis.

Schisandra sphenanthera and Schisandra chinensis are widely consumed either as food or for medicinal purposes. Nevertheless, no detailed comparative assessments of their physicochemical properties and biological activity have been reported. In this paper, using hot-water extraction, alcohol precipitation, and deproteinization, we obtained polysaccharidic extracts from Schisandra sphenanthera and Schisandra chinensis (denoted as SSP and SCP, respectively) and investigated their antioxidant and immunological activities. The extracts were different from each other with regard to sugar, protein, and uronic acid contents. Both extracts were mainly composed of arabinose, glucose, and galactose, but their contents varied greatly; SSP had more galacturonic acid. Compared with SCP, SSP had stronger free radical scavenging ability, protective effects on biomolecules, cellular antioxidant activity, owing to its higher protein (35.35 ±â€¯1.73%) and uronic acid (12.81 ±â€¯1.15%) contents. With respect to cell viability, neutral red phagocytosis, NO production, and acid phosphatase activity, SCP had stronger effects than SSP; this was largely due to its high levels of mannose, galactose, arabinose, and glucose. These results provide evidence to support the use Schisandra-derived polysaccharides for several purposes, including clinical, agricultural, and industrial applications.

Sensitive and robust MALDI-TOF-MS glycomics analysis enabled by Girard's reagent T on-target derivatization (GTOD) of reducing glycans.

Sensitive glycomics analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is of great importance but significantly hampered by their low ionization efficiency and labile sialic acid moieties. Chemical derivatization offers a viable way to improve both the ionization efficiency and analytical sensitivity of the glycans in MS analysis by altering their hydrophobicity or charge property. Here we employed Girard's reagent T (GT) for on-target derivatization (GTOD) of reducing glycan under mild acid condition to form stable hydrazones at room temperature, allowing rapid and sensitive identification of neutral and sialylated glycans in positive-ion mode as only permanently positive charged molecular ions without multiple ion adducts by MALDI-TOF-MS. The MS signal intensities of lactose, sialylated N-glycans derived from bovine fetuin and neutral N-glycans derived from RNaseB and ovalbumin were boosted by 7.44, 9.13, 12.96 and 13.47 folds on average (n = 3), respectively. More importantly, after GTOD strategy, unwanted desialylation of sialylated glycans during MS was suppressed. The detection limit of the assay is desirable since the nanogram of N-glycans derived from 0.16 µg ovalbumin could be detected. The assay demonstrated good stability (RSD≤2.95%, within 10 days), reliable reproducibility (RSD = 2.96%, n = 7) and a desirable linear dynamic range from 78 nmol/mL to 10 µmol/mL. The strategy has been successfully applied to MS analysis of reducing glycans from human milks, neutral and sialylated O-, N-glycans from glycoproteins, and reducing glycans derived from glycosphingolipids, presenting neater [M]+ signals which allow detection of more low-abundance glycans and assignation of Neu5Ac vs. Neu5Gc or fucose vs. hexose in glycans due to the absence of the ambiguous interpretation from multiple peaks (ion adducts [M+Na]+ and [M+K]+). Moreover, the GTOD assay prevents desialylation during MALDI-TOF-MS profiling and enables distinct linkage-specific characterization of terminal sialic acids of N-glycans derived from human serum protein when combines with an esterification.

Comprehensive quali-quantitative profiling of neutral and sialylated O-glycome by mass spectrometry based on oligosaccharide metabolic engineering and isotopic labeling.

Mass spectrometry (MS) analysis combined with stable isotopic labeling is of great importance for quantitatively profiling abnormal sialylated O-glycans associated with disease development, but technically hindered by the poor releasing efficiency of O-glycans from glycoprotein or the labile nature of sialic acid residues at glycans. Herein, we developed an isotopic precursor based metabolic amplification and labeling (IPMAL) technique for relative quantitative profiling of the repertoire O-glycans between normal and tumor cells by ESI-MS. Two groups of cells were incubated with peracetylated benzyl-α-N-acetylgalactosamine (Ac3GalNAc-α-Bnd0) or a heavy labeled peracetylated benzyl-α-N-acetylgalactosamine (Ac3GalNAc-α-Bnd5) precursor respectively to amplify the repertoire of O-glycans as Bnd0/d5-O-glycans which could achieve the quantitative O-glycome analysis by ESI-MS after derivatization. The established method demonstrates desirable feasibility, accuracy (relative error (RE) ≤ 4.20%), reproducibility (coefficient of variation (CV) ≤ 7.61%, n = 3) and good quantitation linearity (R 2 > 0.99, n = 3) for five Bn-O-glycans with 2 orders of magnitude. Finally, the method has been successfully applied to quantitative analysis of the repertoire O-glycome changes between normal human liver cell line L02 and human hepatoma cell line SMMC-7721. Moreover, the α-2,3/2,6 sialic acid isomers of Bn-O-glycans from these two cells have been further quantitatively distinguished when involved a sialic acid specific derivatization procedure.

FGF2 and FGFR2 in patients with idiopathic pulmonary fibrosis and lung cancer.

The aim of this study was to investigate the expression of FGF2 and FGFR2 in patients with idiopathic pulmonary fibrosis (IPF) and lung cancer (LC) as well as their clinical significance. Reverse transcription-quantitative PCR (RT-qPCR) and western blotting were used to detect FGF2 and FGFR2 expression in LC and adjacent normal tissues of LC patients and lavage fluid of idiopathic pulmonary fibers patients and normal controls (confirmed by bronchoalveolar lavage examination). The expression levels of FGF2 mRNA and protein in the non-small cell LC tissues were significantly higher than those in the adjacent normal tissues (P<0.001). The expression level of FGF2 protein in lavage fluid of patients with IPF was higher than that of the control group (P<0.001). The expression level of FGFR2 mRNA in the non-small cell LC tissues was significantly higher than that in the adjacent normal tissues (P<0.001). The expression level of FGFR2 protein in the non-small cell LC tissues was higher than that in the adjacent normal lung tissues (P<0.001). The expression levels of FGF2 mRNA and FGFR2 mRNA in cancer tissues were not significantly correlated with age, sex and history of smoking (P>0.05), but were significantly correlated with lymph node metastasis, tumor differentiation and TNM staging. FGF2 and FGFR2 proteins were highly expressed in cancer tissues of LC patients and lavage fluid of patients with IPF. The expression of FGF2 mRNA and FGFR2 mRNA was correlated with lymph node metastasis and TNM stage. The high expression levels of FGF2 mRNA and FGFR2 mRNA were associated with tumor metastasis and poor prognosis of LC patients.

Purification and activity characterization of polysaccharides in the medicinal lichen Umbilicaria tornata from Taibai Mountain, China.

Water-soluble polysaccharides from Umbilicaria tornata (UTP) were purified and preliminarily characterized. The antioxidant and antitumor activities of crude UTP and two purified fractions (UTP-1 and UTP-2) were evaluated using in vitro experiments. The results showed that the molecular weights of UTP-1 and UTP-2 were 84.86 and 28.66 kDa, respectively. Both UTP-1 and UTP-2 were composed of glucose and xylose, with their molar ratios being 1.3:0.9 and 0.9:4.6, respectively. In addition, crude UTP, UTP-1 and UTP-2 showed dose-dependent DPPH and hydroxyl radical scavenging and reducing activities. However, crude UTP exhibited stronger antioxidant activity than UTP-1 and UTP-2, particularly in terms of DPPH radicals. Crude UTP and the two purified fractions inhibited the growth of HeLa, HepG2, A375, MCF-7, SGC7901 and Caco2 cancer cells in vitro. Compared with UTP-1 and UTP-2, crude UTP presented significantly higher antitumor activity in vitro against HeLa and HepG2 cells (p < 0.05). These findings provide a scientific basis for the deeper exploration and resource development of U. tornata.

Purification, structural features and immunostimulatory activity of novel polysaccharides from Caulerpa lentillifera.

In this study, four purified fractions (CLGP1, CLGP2, CLGP3 and CLGP4) were prepared from green seaweed Caulerpa lentillifera. They were identified to be a novel kind of xylogalactomanans, differed in molecular weight, monosaccharide composition, and the content of uronic acids and sulfate groups, leading to various ζ-potential, ultrastructure and immunostimulatory activity. Especially, CLGP4 was quite different from the others, as it was found to be a homogeneous heteropolysaccharide composed of Xyl, Man and Gal in a percentage ratio of 1.00:2.15:2.40 with 3877.8kDa. Moreover, CLGP4 contained minor uronic acids (2.37%±0.94%) and the highest sulfate content (21.26%±1.22%). These differences in structural features had an effect on the ζ-potential and ultrastructure of CLGP4, showing rod-, rubble- and ellipsoid-shaped particles with largest negatively charge. In vitro immunostimulatory activity evaluation revealed that all the four fractions significantly stimulated macrophages, but CLGP4 showed more potent immunostimulatory activity due to its stronger function on promoting proliferation of macrophages, enhancing phagocytosis, NO production and acid phosphatase activity in macrophages. Therefore, CLGP4 could be explored as a natural immunomodulator. These results would help a fully exploition of Caulerpa lentillifera polysaccharides recognized as health-improving ingredients in functional foods.

An unexpected N-terminal loop in PD-1 dominates binding by nivolumab.

Cancer immunotherapy by targeting of immune checkpoint molecules has been a research 'hot-spot' in recent years. Nivolumab, a human monoclonal antibody targeting PD-1, has been widely used clinically since 2014. However, the binding mechanism of nivolumab to PD-1 has not yet been shown, despite a recent report describing the complex structure of pembrolizumab/PD-1. It has previously been speculated that PD-1 glycosylation is involved in nivolumab recognition. Here we report the complex structure of nivolumab with PD-1 and evaluate the effects of PD-1 N-glycosylation on the interactions with nivolumab. Structural and functional analyses unexpectedly reveal an N-terminal loop outside the IgV domain of PD-1. This loop is not involved in recognition of PD-L1 but dominates binding to nivolumab, whereas N-glycosylation is not involved in binding at all. Nivolumab binds to a completely different area than pembrolizumab. These results provide the basis for the design of future inhibitory molecules targeting PD-1.

Structural characterization and antioxidant activities of κ-carrageenan oligosaccharides degraded by different methods.

In the present study, four kinds of κ-carrageenan oligosaccharides were obtained by the degradation of parent κ-carrageenan using free radical depolymerization, mild acid hydrolysis, κ-carrageenase digestion and partial reductive hydrolysis, respectively. Their structure types were accurately and comparatively elucidated by ESI-MS and CID MS/MS. The antioxidant activities of different degraded products were investigated by four different antioxidant assays, including superoxide radical scavenging activity, hydroxyl radical scavenging activity, reducing power and DPPH radical scavenging activity. The methods of depolymerization had an influence on the antioxidant activities of κ-carrageenan oligosaccharides obtained from κ-carrageenan. These results indicated that the antioxidant activities of κ-carrageenan oligosaccharides could be related to the degree of polymerization, the content of reducing sugar and sulfate groups, and the structure of reducing terminus.