Structural and functional characterization of the novel endo-α(1,4)-fucoidanase Mef1 from the marine bacterium Muricauda eckloniae.

Fucoidanases (EC 3.2.1.-) catalyze the hydrolysis of glycosidic bonds between fucose residues in fucoidans. Fucoidans are a compositionally and structurally diverse class of fucose-containing sulfated polysaccharides that are primarily found in brown seaweeds. Here, the structural characterization of a novel endo-α(1,4)-fucoidanase, Mef1, from the marine bacterium Muricauda eckloniae is presented, showing sequence similarity to members of glycoside hydrolase family 107. Using carbohydrate polyacrylamide gel electrophoresis and nuclear magnetic resonance analyses, it is shown that the fucoidanase Mef1 catalyzes the cleavage of α(1,4)-linkages between fucose residues sulfated on C2 in the structure [-3)-α-L-Fucp2S-(1,4)-α-L-Fucp2S-(1-]n in fucoidan from Fucus evanescens. Kinetic analysis of Mef1 activity by Fourier transform infrared spectroscopy revealed that the specific Mef1 fucoidanase activity (Uf) on F. evanescens fucoidan was 0.1 × 10-3 Uf µM-1. By crystal structure determination of Mef1 at 1.8 Šresolution, a single-domain organization comprising a (ß/α)8-barrel domain was determined. The active site was in an extended, positively charged groove that is likely to be designed to accommodate the binding of the negatively charged, sulfated fucoidan substrate. The active site of Mef1 comprises the amino acids His270 and Asp187, providing acid/base and nucleophile groups, respectively, for the hydrolysis of glycosidic bonds in the fucoidan backbone. Electron densities were identified for two possible Ca2+ ions in the enzyme, one of which is partially exposed to the active-site groove, while the other is very tightly coordinated. A water wire was discovered leading from the exterior of the Mef1 enzyme into the active site, passing the tightly coordinated Ca2+ site.

Pectins from the sea grass Enhalus acoroides (L.f.) Royle: Structure, biological activity and ability to form nanoparticles.

Two pectins from the seagrass Enhalus acoroides (L.f.) Royle were isolated for the first time. Their structures and biological activities were investigated. NMR spectroscopy showed one of them to consist exclusively from the repeating →4-α-d-GalpUA→ residue (Ea1), while the other had a much more complex structure that also included 1→3-linked α-d-GalpUA residues, 1→4-linked ß-apiose residues and small amounts of galactose and rhamnose (Ea2). The pectin Ea1 showed noticeable dose-dependent immunostimulatory activity, the Ea2 fraction was less effective. Both pectins were used to create pectin-chitosan nanoparticles for the first time, and the influence of pectin/chitosan mass ratio on their size and zeta potential was investigated. Ea1 particles were slightly smaller than Ea2 particles (77 ± 16 nm vs 101 ± 12 nm) and less negatively charged (-23 mV vs -39 mV). Assessment of their thermodynamic parameters showed that only the second pectin could form nanoparticles at room temperature.

Structure and chemopreventive activity of fucoidans from the brown alga Alaria angusta.

Six fucoidan fractions were isolated from the brown alga Alaria angusta. Structures of enzymatic hydrolysis products of the fraction 1AaF2 (Fuc:Gal ~ 1:1; 33 % of sulfates) by fucanase from Wenyingzhuangia fucanilytica were studied by chemical and instrumental (NMR spectroscopy and mass-spectrometry) methods. It was shown that 1AaF2 consisted of two structurally different fucoidans: a sulfated 1,3;1,4-α-L-fucan and an enzyme-resistant sulfated and acetylated complex fucogalactan (Fuc:Gal ~ 1:2; 19 % of sulfates) 1AaF2_HMP containing extended 1,3-linked fucose and 1,3/1,4-linked galactose fragments (up to 5 residues). The fractions 1AaF2 and 1AaF2_HMP were a non-cytotoxic, possessed dose-dependent chemopreventive effect on EGF-induced neoplastic cell transformation of mouse normal epidermal JB6 Cl41 cells and inhibited the colony formation of human melanoma SK-MEL-28 cells.

Fucose-Rich Sulfated Polysaccharides from Two Vietnamese Sea Cucumbers Bohadschia argus and Holothuria (Theelothuria) spinifera: Structures and Anticoagulant Activity.

Fucosylated chondroitin sulfates (FCSs) FCS-BA and FCS-HS, as well as fucan sulfates (FSs) FS-BA-AT and FS-HS-AT were isolated from the sea cucumbers Bohadschia argus and Holothuria (Theelothuria) spinifera, respectively. Purification of the polysaccharides was carried out by anion-exchange chromatography on DEAE-Sephacel column. Structural characterization of polysaccharides was performed in terms of monosaccharide and sulfate content, as well as using a series of non-destructive NMR spectroscopic methods. Both FCSs were shown to contain a chondroitin core [→3)-ß-d-GalNAc-(1→4)-ß-d-GlcA-(1→]n bearing sulfated fucosyl branches at O-3 of every GlcA residue in the chain. These fucosyl residues were different in pattern of sulfation: FCS-BA contained Fuc2S4S, Fuc3S4S and Fuc4S at a ratio of 1:8:2, while FCS-HS contained these residues at a ratio of 2:2:1. Polysaccharides differed also in content of GalNAc4S6S and GalNAc4S units, the ratios being 14:1 for FCS-BA and 4:1 for FCS-HS. Both FCSs demonstrated significant anticoagulant activity in clotting time assay and potentiated inhibition of thrombin, but not of factor Xa. FS-BA-AT was shown to be a regular linear polymer of 4-linked α-L-fucopyranose 3-sulfate, the structure being confirmed by NMR spectra of desulfated polysaccharide. In spite of considerable sulfate content, FS-BA-AT was practically devoid of anticoagulant activity. FS-HS-AT cannot be purified completely from contamination of some FCS. Its structure was tentatively represented as a mixture of chains identical with FS-BA-AT and other chains built up of randomly sulfated alternating 4- and 3-linked α-L-fucopyranose residues.

Veno-Venous Extracorporeal Rewarming Using Dual-Lumen Cannula in Accidental Hypothermia.

Accidental hypothermia with a core temperature below 28°C is associated with an increased risk of hemodynamic instability. It is difficult to predict which patients will survive with a favorable neurologic outcome; therefore, decision-making regarding extracorporeal support is not straightforward. We report a case of rewarming using veno-venous dual-lumen cannula as an alternative to veno-arterial support with full recovery and normal neurologic examination. In centers where extracorporeal membrane oxygenation is available, rewarming using veno-venous dual-lumen extracorporeal support may be a useful strategy to mitigate the risks associated with veno-arterial extracorporeal support.

The structure of fucoidan from Sargassum oligocystum and radiosensitizing activity of galactofucans from some algae of genus Sargassum.

The aim of this study was to establish the fine structure of fucoidan from Sargassum oligocystum and to study the radiosensitizing effect of fucoidans from three algae of genus Sargassum (S. oligocystum, S. duplicatum, and S. feldmannii) with different structures. The fucoidan SoF2 from S. oligocystum was sulfated (32%) galactofucan (Fuc:Gal = 2:1), with a Mw of 183 kDa (Mw/Mn = 2.0). Its supposed structure was found to be predominantly 1,3-linked fucose as the main chain, with branching points at C2 and C4. The branches could be single galactose and/or fucose short chains with terminal galactose residues. Sulfate groups were found at positions C3, C2, and/or C4 of fucose residues and at C2 and/or C4 of galactose residues. The radiosensitizing effect of galactofucans from S. oligocystum, S. duplicatum, and S. feldmannii against human melanoma SK-MEL-28, colon HT-29, and breast MDA-MB-231 cancer cells was investigated. The influence of all investigated polysaccharides treatments with/without X-ray radiation on colony formation of human melanoma cells SK-MEL-28 was weak. Fucoidan from S. feldmannii has been shown to be the most promising radiosensitizing compound against human colon HT-29 and breast MDA-MB-231 cancer cells.

Features and Advantages of Supercritical CO2 Extraction of Sea Cucumber Cucumaria frondosa japonica Semper, 1868.

Extraction process of Cucumaria frondosa japonica Semper, 1868, which are subspecies of Cucumaria frondosa (Gunnerus, 1767), were studied. It was shown that supercritical carbon dioxide extraction of holothuria was more effective than conventional solvent extraction. Step-by-step extraction with carbon dioxide followed by supercritical extraction with the addition of a co-solvent of ethanol can almost double the yields of extracts of triterpene glycosides, styrenes and carotenoids. Moreover, the fraction of triterpene glycosides practically does not contain colored impurities, in contrast to traditional ethanol extraction. The obtained extracts by HPLC in combination with tandem mass spectrometry (HPLC-MS/MS) identified 15 triterpene glycosides, 18 styrene compounds and 14 carotenoids. Supercritical extraction made it possible to obtain extracts with yields superior to conventional hexane and alcohol extracts. Moreover, such an approach with the use of supercritical fluid extraction (SFE) and subsequent profiling of metabolites can help with the study of holothuria species that are not as well studied.

Comparison of structure and in vitro anticancer activity of native and modified fucoidans from Sargassum feldmannii and S. duplicatum.

Fucoidans are valuable biologically active polysaccharides of brown algae. The aim of this study was to investigate the structure of fucoidan from Sargassum feldmannii and the anticancer effects of native and modified polysaccharides from S. feldmannii and S. duplicatum. The structure of sulfated (25.3%) galactofucan SfF2 (Fuc/Gal = 72/28 mol%) from S. feldmannii was investigated by NMR spectroscopy of desulfated derivative and mass spectrometry of fucoidan fragments labelled with 18O. SfF2 was shown to contain the main chain from 1,3-linked α-l-fucopyranose and ß-d-galactopyranose residues with fucose branches at C4 and C6 of galactose residues and C2 of fucose residues. The following fragments were also identified in SfF2: Fuc-(1,4)-Fuc, Gal-(1,3)-Gal, and Gal-(1,4)-Gal. The sulfate groups occupied positions C2, C3, and C4 of fucose residues and C2, C3, C4, and C6 of galactose residues. The galactofucans from S. feldmannii, S. duplicatum, and their derivatives exhibited no cytotoxicity in vitro. The native and deacetylated fucoidans (200 µg/mL) inhibited colony formation of human colon cancer cells (DLD-1, HT-29, and HCT-116). Both desulfated fucoidans possessed weak anticancer activity.

Interpreting Neurologic Outcomes in a Changing Trial Design Landscape: An Analysis of HeartWare Left Ventricular Assist Device Using a Hybrid Intention to Treat Population.

Randomized controlled trials can provide optimal clinical evidence to assess the benefits of new devices, and it is these data that often shape device usage in real-world practice. However, individual clinical trial results sometimes appear discordant for the same device, and alternative devices are sometimes not employed in similar patient populations. To make sound evidence-based decisions, clinicians routinely rely on cross-trial comparisons from different trials of similar but not identical patient populations to assess competing technology when head-to-head randomized comparisons are unavailable.

A novel sulfated fucan from Vietnamese sea cucumber Stichopus variegatus: Isolation, structure and anticancer activity in vitro.

In the present study, three sulfated polysaccharides, two fractions of fucosylated chondroitin sulfates, and one sulfated fucan were isolated from the body wall of the Vietnamese sea cucumber Stichopus variegatus. The structure of the sulfated fucan fraction SvF3 from S. variegatus was investigated for the first time. According to NMR spectroscopy data, the sulfated fucan SvF3 contained 1,2- and 1,3-linked α-l-fucopyranose residues. Sulfate groups were found at the 2 and/or 4 positions. The structural analysis of fucoidan was assisted by tandem mass spectrometry; the recently-developed technique of autohydrolysis in heavy­oxygen water for the obtaining of selectively labeled fucoidan fragments was applied. The labeling (+2 Da mass shift at the reducing end) allowed us to assign MS/MS data unambiguously, and thus to confirm the NMR data and revealed minor sulfation at position 3. It was shown that the sulfated fucan SvF3 was not cytotoxic to human breast cancer T-47D and MDA-MB-231 cell lines, and it inhibited colony formation of those cells in vitro. SvF3 also possessed slight activity against migration of MDA-MB-231 cells in vitro.

Structural characteristics and anticancer activity in vitro of fucoidan from brown alga Padina boryana.

The sulfated and acetylated fucoidan fraction, containing fucose, galactose, mannose, glucose and uronic acid residues, was isolated from the brown alga Padina boryana. The structure of galactofucan part was studied after different modifications by NMR spectroscopy and mass spectrometry. It was shown that galactofucan contained the main chain of alternating 1,4-linked α-l-fucopyranose and 1,3-linked ß-d-Galactopyranose. Single fucose residues were found as branches at C4 of galactose residues. Also, fucoidan contained 1,3- or 1,4-linked Fuc-Fuc and Gal-Gal fragments. The sulfate groups occupied positions C2, C3 and C4 of both fucose and galactose residues, which was shown by tandem mass spectrometry of fragments, labeled with heavy-oxygen. The anticancer effect of native and modified fucoidan fractions was studied in vitro on the colorectal carcinoma cells DLD-1 and HCT-116. All fucoidans had no cytotoxicity under 400 µg/mL and inhibited colony formation of cancer cells at concentration of 200 µg/mL.

Polysaccharides from brown algae Sargassum duplicatum: the structure and anticancer activity in vitro.

The laminaran SdL and fucoidan SdF were isolated from brown algae Sargassum duplicatum. SdL was 1,3;1,6-ß-d-glucan (1,3:1,6=6:1) with a main chain, represented by 1,3-linked glucose residues, due to NMR spectroscopy data. Single glucose residues could form branches at C6. Unusual structure of fucoidan SdF was studied by chemical and enzymatic methods, NMR spectroscopy of desulfated and deacetylated polysaccharide and mass spectrometry of fucoidan fragments labeled with 18O. Fucoidan was sulfated (31.7%) and acetylated galactofucan (Fuc:Gal∼1:1) with a main chain of 1,4-linked alternating α-l-fucose and ß-d-galactose residues. Side chains were represented by extensive (DP≥5) 1,3-linked 2,4-disulfated α-l-fucose residues with branching points at C2. Fucose residues in the main chain were sulfated at C2 and less at C3, while galactose residues were sulfated at C2, C3, and less at C4, C6. The fucoidan SdF was effective against colony formation of colon cancer cells in vitro.

Perioperative management of patients with left ventricular assist devices undergoing noncardiac procedures: a survey of current practices.

OBJECTIVES: To describe perioperative management of patients with left ventricular assist devices (LVAD) in noncardiac procedures. DESIGN: Survey of (1) respondent demographic characteristics, (2) anesthetic practices for LVAD patients having endoscopies, and (3) low-risk surgeries requiring general anesthesia. SETTING: Internet-based. PARTICIPANTS: Society of Cardiovascular Anesthesiologists membership. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Inpatient endoscopic procedures were done mainly in the endoscopy suite (71.7%) by a solo practitioner or 1:1 staffing ratio 59% of the time. LVAD-specific support personnel were present in more than 80% of all procedures. Both endoscopy and surgical patients used post-anesthesia recovery units and intensive care units for recovery; however, compared with endoscopy patients, surgical patients recovered in the ICU more frequently (45.5% v 29.1%, p<0.001). In addition, 18% of endoscopy patients recovered on site. Regarding patient monitoring, more than 90% of responders used electrocardiogram, pulse oximetry, end-tidal CO2, and blood pressure monitors on LVAD patients. Responders reported using arterial catheters to monitor blood pressure in 49% of endoscopy cases and 71% of surgical patients. The reported use of invasive monitors by individual clinicians was related inversely to institutional LVAD volume (p = 0.04 and p = 0.01 in endoscopy and surgical procedures, respectively). CONCLUSIONS: This survey found heterogeneity in hospital resource utilization for noncardiac LVAD procedures. There was a decrease in the use of invasive monitors with increased institutional LVAD volume in both endoscopy and surgical procedures.

Structural characteristics and anticancer activity of fucoidan from the brown alga Sargassum mcclurei.

Three different fucoidan fractions were isolated and purified from the brown alga, Sargassum mcclurei. The SmF1 and SmF2 fucoidans are sulfated heteropolysaccharides that contain fucose, galactose, mannose, xylose and glucose. The SmF3 fucoidan is highly sulfated (35%) galactofucan, and the main chain of the polysaccharide contains a →3)-α-L-Fucp(2,4SO3⁻)-(1→3)-α-L-Fucp(2,4SO3⁻)-(1→ motif with 1,4-linked 3-sulfated α-L-Fucp inserts and 6-linked galactose on reducing end. Possible branching points include the 1,2,6- or 1,3,6-linked galactose and/or 1,3,4-linked fucose residues that could be glycosylated with terminal ß-D-Galp residues or chains of alternating sulfated 1,3-linked α-L-Fucp and 1,4-linked ß-D-Galp residues, which have been identified in galactofucans for the first time. Both α-L-Fucp and ß-D-Galp residues are sulfated at C-2 and/or C-4 (and some C-6 of ß-D-Galp) and potentially the C-3 of terminal ß-D-Galp, 1,4-linked ß-D-Galp and 1,4-linked α-L-Fucp residues. All fucoidans fractions were less cytotoxic and displayed colony formation inhibition in colon cancer DLD-1 cells. Therefore, these fucoidan fractions are potential antitumor agents.