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Background In plants, a large diversity of polysaccharides comprise the cell wall. Each major type of plant cell wall polysaccharide, including cellulose, hemicellulose, and pectin, has distinct structures and functions that contribute to wall mechanics and influence plant morphogenesis. In recent years, pectin valorization has attracted much attention due to its expanding roles in biomass deconstruction, food and material science, and environmental remediation. However, pectin utilization has been limited by our incomplete knowledge of its structure. Herein, we present a workflow of principles relevant for the characterization of polysaccharide primary structure using nature’s most complex polysaccharide, rhamnogalacturonan-II (RG-II), as a model. Results We outline how to isolate RG-II from celery and duckweed cell walls and from red wine using chemical or enzymatic treatments coupled with size-exclusion chromatography. From there, we applied mass spectrometry (MS)-based techniques to determine the glycosyl residue and linkage compositions of the intact RG-II and derived oligosaccharides including special considerations for labile monosaccharides. In doing so, we demonstrated that in the duckweed Wolffiella repanda the arabinopyranosyl (Arap) residue of side chain B is substituted at O-2 with rhamnose. We used electrospray-MS techniques to identify non-glycosyl modifications including methyl-ethers, methyl-esters, and acetyl-esters on RG-II-derived oligosaccharides. We then showed the utility of proton nuclear magnetic resonance spectroscopy (1H-NMR) to investigate the structure of intact RG-II and to complement the RG-II dimerization studies performed using size-exclusion chromatography. Conclusions The complexity of pectic polysaccharide structures has hampered efforts aimed at their valorization. In this work, we used RG-II as a model to demonstrate the steps necessary to isolate and characterize polysaccharides using chromatographic, MS, and NMR techniques. The principles can be applied to the characterization of other saccharide structures and will help inform researchers on how saccharide structure relates to functional properties in the future.

Xylans are a major component of plant cell walls. O-Acetyl moieties are the dominant backbone substituents of glucuronoxylan in dicots and play a major role in the polymer-polymer interactions that are crucial for wall architecture and normal plant development. Here, we describe the biochemical, structural, and mechanistic characterization of Arabidopsis (Arabidopsis thaliana) xylan O-acetyltransferase 1 (XOAT1), a member of the plant-specific Trichome Birefringence Like (TBL) family. Detailed characterization of XOAT1-catalyzed reactions by real-time NMR confirms that it exclusively catalyzes the 2-O-acetylation of xylan, followed by nonenzymatic acetyl migration to the O-3 position, resulting in products that are monoacetylated at both O-2 and O-3 positions. In addition, we report the crystal structure of the catalytic domain of XOAT1, which adopts a unique conformation that bears some similarities to the α/β/α topology of members of the GDSL-like lipase/acylhydrolase family. Finally, we use a combination of biochemical analyses, mutagenesis, and molecular simulations to show that XOAT1 catalyzes xylan acetylation through formation of an acyl-enzyme intermediate, Ac–Ser-216, by a double displacement bi-bi mechanism involving a Ser-His-Asp catalytic triad and unconventionally uses an Arg residue in the formation of an oxyanion hole.

Human breast milk is an incredibly rich and complex biofluid composed of proteins, lipids and complex carbohydrates, including a diverse repertoire of free human milk oligosaccharides (HMOs). Strikingly, HMOs are not digested by the infant but function as prebiotics for bacterial strains associated with numerous benefits. Considering the broad variety of beneficial effects of HMOs, and the vast number of factors that affect breast milk composition, the analysis of HMO diversity and complexity is of utmost relevance. Using human milk samples from a cohort of Bangladeshi mothers participating in a study on malnutrition and stunting in children, we have characterized breast milk oligosaccharide composition by means of permethylation followed by liquid chromatography coupled with high-resolution tandem mass spectrometry (LC-MS/MS) analysis. This approach identified over 100 different glycoforms and showed a wide diversity of milk composition, with a predominance of fucosylated and sialylated HMOs over nonmodified HMOs. We observed that these samples contain on average 80 HMOs, with the highest permethylated masses detected being >5000 mass units. Here we report an easily implemented method developed for the separation, characterization and relative quantitation of large arrays of HMOs, including higher molecular weight sialylated HMOs. Our ultimate goal is to create a simple, high-throughput method, which can be used for full characterization of sialylated and/or fucosylated HMOs. These results demonstrate how current analytical techniques can be applied to characterize human milk composition, providing new tools to help the scientific community shed new light on the impact of HMOs during infant development.

A broad-based interlaboratory study of glycosylation profiles of a reference and modified IgG antibody involving 103 reports from 76 laboratories., Graphical Abstract Highlights A broad-based interlaboratory study of the glycosylation of a reference antibody: NISTmAb. 103 reports were received from 76 diverse laboratories worldwide. Analysis involved two samples, the NISTmAb and an enzymatically modified sample, enabling within-lab separation of random and systematic errors using the “Youden two-sample” method. Consensus values were derived and similar performance across all experimental methods was noted., Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide community-derived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods.

The proteoglycan Syndecan-2 (Sdc2) has been implicated in regulation of cytoskeleton organization, integrin signaling and developmental angiogenesis in zebrafish. Here we report that mice with global and inducible endothelial-specific deletion of Sdc2 display marked angiogenic and arteriogenic defects and impaired VEGFA165 signaling. No such abnormalities are observed in mice with deletion of the closely related Syndecan-4 (Sdc4) gene. These differences are due to a significantly higher 6-O sulfation level in Sdc2 versus Sdc4 heparan sulfate (HS) chains, leading to an increase in VEGFA165 binding sites and formation of a ternary Sdc2-VEGFA165-VEGFR2 complex which enhances VEGFR2 activation. The increased Sdc2 HS chains 6-O sulfation is driven by a specific N-terminal domain sequence; the insertion of this sequence in Sdc4 N-terminal domain increases 6-O sulfation of its HS chains and promotes Sdc2-VEGFA165-VEGFR2 complex formation. This demonstrates the existence of core protein-determined HS sulfation patterns that regulate specific biological activities., Proteoglycans are glycosylated proteins that play a number of structural and signalling functions. Here, Corti, Wang et al. show that the N-terminal sequence of proteoglycan Syndecan-2 selectively increases 6-O sulfation of its heparan sulfate chains, and that this promotes formation of a ternary Sdc2/VEGFA/VEGFR2 complex leading to increased angiogenesis.

Offspring of obese mothers suffer higher risks of type 2 diabetes due to increased adiposity and decreased β cell function. To date, the sex-differences in offspring islet insulin secretion during early life has not been evaluated extensively, particularly prior to weaning at postnatal day 21 (P21). To determine the role of maternal obesity on offspring islet insulin secretion, C57BL/6J female dams were fed chow or western diet from 4 weeks prior to mating to induce maternal obesity. First, offspring of chow-fed and obese dams were evaluated on postnatal day 21 (P21) prior to weaning for body composition, glucose and insulin tolerance, and islet phasic insulin-secretion. Compared to same-sex controls, both male and female P21 offspring born to obese dams (MatOb) had higher body adiposity and exhibited sex-specific differences in glucose tolerance and insulin secretion. The male MatOb offspring developed the highest extent of glucose intolerance and lowest glucose-induced insulin secretion. In contrast, P21 female offspring of obese dams had unimpaired insulin secretion. Using SAX-HPLC, we found that male MatOb had a decrease in pancreatic heparan sulfate glycosaminoglycan, which is a macromolecule critical for islet health. Notably, 8-weeks-old offspring of obese dams continued to exhibit a similar pattern of sex-differences in glucose intolerance and decreased islet insulin secretion. Overall, our study suggests that maternal obesity induces sex-specific changes to pancreatic HSG in offspring and a lasting effect on offspring insulin secretion, leading to the sex-differences in glucose intolerance.

Engineered glycomaterial implants orchestrate large-scale repair after severe traumatic brain injury., Severe traumatic brain injury (sTBI) survivors experience permanent functional disabilities due to significant volume loss and the brain’s poor capacity to regenerate. Chondroitin sulfate glycosaminoglycans (CS-GAGs) are key regulators of growth factor signaling and neural stem cell homeostasis in the brain. However, the efficacy of engineered CS (eCS) matrices in mediating structural and functional recovery chronically after sTBI has not been investigated. We report that neurotrophic factor functionalized acellular eCS matrices implanted into the rat M1 region acutely after sTBI significantly enhanced cellular repair and gross motor function recovery when compared to controls 20 weeks after sTBI. Animals subjected to M2 region injuries followed by eCS matrix implantations demonstrated the significant recovery of “reach-to-grasp” function. This was attributed to enhanced volumetric vascularization, activity-regulated cytoskeleton (Arc) protein expression, and perilesional sensorimotor connectivity. These findings indicate that eCS matrices implanted acutely after sTBI can support complex cellular, vascular, and neuronal circuit repair chronically after sTBI.

Emergence of COVID-19 pandemic caused by SARS-CoV-2 demanded development of new therapeutic strategies and thus the understanding the mode of viral attachment, entry and replication has become key aspect for such interventions. The coronavirus surface features a trimeric spike (S) protein that is essential in viral attachment, entry and membrane fusion. The S protein of SARS-CoV-2 binds to the human angiotensin converting enzyme 2 (hACE2) for the entry and the serine protease TMPRSS2 for S protein priming. The heavily glycosylated S protein is comprised of two protein subunits (S1 and S2), and the receptor binding domain within S1 subunit binds with to the hACE2 receptor. Even though hACE2 has been known for two decades and has been recognized as the entry point of several human coronaviruses, no comprehensive glycosylation characterization of hACE2 has been reported. Herein, we describe the quantitative glycosylation mapping on hACE2 expressed on human cells by both glycoproteomics and glycomics. We observed heavy glycan occupancy at all the seven possible N-glycosylation sites and surprisingly, observed three novel O-glycosylation sites. In order to deduce the key full detail of structure of the glycan epitopes on hACE2 involved with viral binding, we have characterized the terminal sialic acid linkages, presence of bisecting GlcNAc and also the pattern of N-glycan fucosylation. We have conducted extensive manual interpretation of each glycopeptide and glycan spectra in addition to the use of bioinformatics tools to validate the hACE2 glycosylation. Elucidation of the site-specific glycosylation and its terminal orientations on the hACE2 receptor can aid in understanding the intriguing virus-receptor interactions and help in the development of novel therapeutics to circumvent the viral entry.

Acetylation of biomolecules is gaining increased attention due to both the abundance and importance of this modification across all kingdoms of life. Xylans are a major component of plant cell walls and are the third most abundant biopolymer in Nature. O-Acetyl moieties are the dominant backbone substituents of glucuronoxylan in dicots and play a major role in the polymer-polymer interactions that are crucial for proper wall architecture and normal plant development. Here, we describe the biochemical, structural, and mechanistic characterization of Arabidopsis thaliana xylan O-acetyltransferase 1 (AtXOAT1), a member of the plant-specific Trichome Birefrigence Like (TBL) family that catalyzes the 2-O-acetylation of xylan. A multipronged approach involving X-ray crystallography, biochemical analyses, mutagenesis, and molecular simulations show that XOAT1 catalyzes xylan acetylation through formation of an acyl-enzyme intermediate by a double displacement bi-bi mechanism involving a Ser-His-Asp catalytic triad and unconventionally employs an arginine residue in formation of an oxyanion hole.

Ticks are important ectoparasites and vectors of numerous human and animal pathogens. Ticks secrete saliva that contains various bioactive materials to evade the host defense system, and often facilitates the pathogen transmission. In addition, the Lone star tick saliva is thought to be the sensitizer in red meat allergy that is characterized by an allergic reaction to glycan moieties carrying terminal galactose-alpha-1,3-galactose (aGal). To assess N-glycome of Amblyomma americanum, we examined the N-glycan structures in male and female salivary glands at three different feeding stages and in carcasses of partially fed lone star ticks. We also surveyed the genes involved in the N-glycosylation in the tick species. The aGal epitopes and cross-reactive carbohydrate determinants (CCD) increases over time after the onset of blood feeding in both male and female A. americanum. These CCDs include xylosylation of the core mannose, 1,3-mono and 1,3- and 1,6-difucosylations of the basal GlcNac and mono- or diantennary aGal. Combinations of both xylosylation and aGal and fucosylation and aGal were also found on the N-glycan structures. While the enzymes required for the early steps of the N-glycosylation pathway are quite conserved, the enzymes involved in the later stages of N-glycan maturation in the Golgi apparatus are highly diverged from those of insects. Most of all, we propose that the aGal serves as a molecular mimicry of bioactive proteins during tick feedings on mammalian hosts, while it contributes as a sensitizer of allergy in atypical host human.

The original version of this Article contained errors in Figures 1, 3 and 4. In panels b and d of Figure 1, the labels ‘Sdc4-/-’ were inadvertently replaced by ‘Sdc4+/+‘. In panels c and d of Figure 3, the labels ‘Sdc4-/-’ were replaced by ‘Sdc2-/-’. In panel f of Figure 3, the labels ‘FGF2’ were replaced by ‘VEGFA165’. In panel e of Figure 6, a ‘Sdc2-/-‘ label was inadvertently included. This has now been corrected in the PDF and HTML versions of the Article.

Contains fulltext : 200468.pdf (Publisher’s version ) (Closed access) Heparan sulfate (HS) is a complex linear polysaccharide that modulates a wide range of biological functions. Elucidating the structure-function relationship of HS has been challenging. Here we report the generation of an HS-mutant mouse lung endothelial cell library by systematic deletion of HS genes expressed in the cell. We used this library to (1) determine that the strictly defined fine structure of HS, not its overall degree of sulfation, is more important for FGF2-FGFR1 signaling; (2) define the epitope features of commonly used anti-HS phage display antibodies; and (3) delineate the fine inter-regulation networks by which HS genes modify HS and chain length in mammalian cells at a cell-type-specific level. Our mutant-cell library will allow robust and systematic interrogation of the roles and related structures of HS in a cellular context.

Early damage to transplanted organs initiates excess inflammation that can cause ongoing injury, a leading cause for late graft loss. The endothelial glycocalyx modulates immune reactions and chemokine-mediated haptotaxis, potentially driving graft loss. In prior work, conditional deficiency of the glycocalyx-modifying enzyme N-deacetylase-N-sulfotransferase-1 (Ndst1f/f TekCre+) reduced aortic allograft inflammation. Here we investigated modification of heparan sulfate (HS) and chemokine interactions in whole-organ renal allografts. Conditional donor allograft Ndst1 deficiency (Ndst1−/−; C57Bl/6 background) was compared to systemic treatment with M-T7, a broad-spectrum chemokine-glycosaminoglycan (GAG) inhibitor. Early rejection was significantly reduced in Ndst1−/− kidneys engrafted into wildtype BALB/c mice (Ndst1+/+) and comparable to M-T7 treatment in C57Bl/6 allografts (P Ndst1−/− allografts, while M-T7 point mutants with modified GAG-chemokine binding displayed a range of anti-rejection activity. CD3+ T cells (P Ndst1 deficiency exhibit significantly reduced acute rejection, comparable to systemic chemokine-GAG inhibition. Modified disaccharides in engrafted organs correlate with reduced rejection. Altered disaccharides in engrafted organs provide markers for rejection with potential to guide new therapeutic approaches in allograft rejection.

N-Glycans of human proteins possess both α2,6- and α2,3-linked terminal sialic acid (SA). Recombinant glycoproteins produced in Chinese hamster overy (CHO) only have α2,3-linkage due to the absence of α2,6-sialyltransferase (St6gal1) expression. The Chinese hamster ST6GAL1 was successfully overexpressed using a plasmid expression vector in three recombinant immunoglobulin G (IgG)-producing CHO cell lines. The stably transfected cell lines were enriched for ST6GAL1 overexpression using FITC-Sambucus nigra (SNA) lectin that preferentially binds α2,6-linked SA. The presence of α2,6-linked SA was confirmed using a novel LTQ Linear Ion Trap Mass Spectrometry (LTQ MS) method including MSn fragmentation in the enriched ST6GAL1 Clone 27. Furthermore, the total SA (mol/mol) in IgG produced by the enriched ST6GAL1 Clone 27 increased by 2-fold compared to the control. For host cell engineering, the CHOZN(®) GS host cell line was transfected and enriched for ST6GAL1 overexpression. Single-cell clones were derived from the enriched population and selected based on FITC-SNA staining and St6gal1 expression. Two clones ("ST6GAL1 OE Clone 31 and 32") were confirmed for the presence of α2,6-linked SA in total host cell protein extracts. ST6GAL1 OE Clone 32 was subsequently used to express SAFC human IgG1. The recombinant IgG expressed in this host cell line was confirmed to have α2,6-linked SA and increased total SA content. In conclusion, overexpression of St6gal1 is sufficient to produce recombinant proteins with increased sialylation and more human-like glycoprofiles without combinatorial engineering of other sialylation pathway genes. This work represents our ongoing effort of glycoengineering in CHO host cell lines for the development of "bio-better" protein therapeutics and cell culture vaccine production.

Synapses are fundamental units of communication in the brain. The prototypical synapse-organizing complex neurexin-neuroligin mediates synapse development and function and is central to a shared genetic risk pathway in autism and schizophrenia. Neurexin’s role in synapse development is thought to be mediated purely by its protein domains, but we reveal a requirement for a rare glycan modification. Mice lacking heparan sulfate (HS) on neurexin-1 show reduced survival, as well as structural and functional deficits at central synapses. HS directly binds postsynaptic partners neuroligins and LRRTMs, revealing a dual binding mode involving intrinsic glycan and protein domains for canonical synapse-organizing complexes. Neurexin HS chains also bind novel ligands, potentially expanding the neurexin interactome to hundreds of HS-binding proteins. Because HS structure is heterogeneous, our findings indicate an additional dimension to neurexin diversity, provide a molecular basis for fine-tuning synaptic function, and open therapeutic directions targeting glycan-binding motifs critical for brain development.

The binding affinity of 17β-estradiol with an immobilized DNA aptamer was measured using capillary electrophoresis. Estradiol captured by the immobilized DNA was injected into the separation capillary using pH-mediated sample stacking. Stacked 17β-estradiol was then separated using micellar electrokinetic capillary chromatography and detected with UV-visible absorbance. Standard addition was used to quantify the concentration of estradiol bound to the aptamer. Following incubation with immobilized DNA, analysis of free and bound estradiol yielded a dissociation constant of 70 ± 10 μM. The method was also used to screen binding affinity of the aptamer for estrone and testosterone. This study demonstrates the effectiveness of capillary electrophoresis to assess the binding affinity of DNA aptamers.

CE separations of glycans taken from the cancer drug, Trastuzumab (Herceptin(®)), were accomplished using phospholipid additives. Glycans were labeled with 1-aminopyrene-3,6,8-trisulfonic acid and were separated with efficiencies as high as 510000 theoretical plates in a 60.2 cm 25 μm id fused-silica capillary. The thermally tunable phospholipid was loaded into the capillary when it possessed a viscosity similar to that of water. The temperature was increased, and the separations were performed when the material exhibited higher viscosity. Enzymes were integrated into the separation with the phospholipid additive. Neuraminidase, β1-4 galactosidase, and β-N-acetylglucosaminidase were injected into the capillary without covalent modification and used for enzyme hydrolysis. Exoglycosidase enzymes cleaved the terminal glycan residues. The glycan sequence could be verified based on enzyme specificity. Neuraminidase was used to determine total glycan content of the low-abundance glycans containing sialic acid. β1-4 Galactosidase and β-N-acetylglucosaminidase were used sequentially in-capillary, to determine the structure of the high-abundance glycans.

Phospholipids are used as an additive in capillary electrophoresis to enhance the separation of glycans derived from alpha1-acid glycoprotein, fetuin, and ribonuclease B. The properties of phospholipid preparations are dependent upon composition, hydration, and temperature. Separation performance is evaluated as a function of these variables. A preparation of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC), with [DMPC]/[DHPC] = 2.5, in 10% lipid/aqueous buffer at 25 degrees C provides the best separation efficiency at an electric field strength of 400 V/cm. Resolution is enhanced with the additive. Concanavalin A, a lectin selective for high mannose and mannose branching glycans, and alpha1-2,3 mannosidase, an enzyme that cleaves 1-2 and 1-3 mannopyranosyl residues, are incorporated in the separation to provide additional selectivity and to expand the application of phospholipid additives for glycan separation.

The applicability of dual injection CE for affinity selection of biopolymers that contain multiple binding sites is demonstrated. The efficient analysis of biomolecules such as carbohydrates and proteins, as well as pharmaceuticals by CE requires the reduction or elimination of nonspecific interactions with the capillary surface. Phospholipids are integral components of cell membranes and aqueous phospholipid liquid crystals adopt a bilayer structure on fused-silica. This phospholipid surface does not interact significantly with the following biomolecules: serum albumin, the 96-110 heparin binding domain of amyloid precursor protein (APP), polydisperse glycosaminoglycans, and variable chain-length oligosaccharides. Pharmaceuticals including five anionic nonsteroidal anti-inflammatory drugs, three cationic analgesics, and two cationic beta-blockers, also show minimal interaction with the surface. In addition, the use of a phospholipid coating suppresses EOF, which enables reversed-polarity separations, dual opposite injection CE, affinity screening via CE by dual opposite injection, and serial target-ligand injections.

Asialo-erythropoietin, a desialylated form of human erythropoietin (EPO) lacking hematopoietic activity, is receiving increased attention because of its broader protective effects in preclinical models of tissue injury. However, attempts to translate its protective effects into clinical practice is hampered by unavailability of suitable expression system and its costly and limit production from expensive mammalian cell-made EPO (rhuEPOM) by enzymatic desialylation. In the current study, we took advantage of a plant-based expression system lacking sialylating capacity but possessing an ability to synthesize complex N-glycans to produce cytoprotective recombinant human asialo-rhuEPO. Transgenic tobacco plants expressing asialo-rhuEPO were generated by stably co-expressing human EPO and β1,4-galactosyltransferase (GalT) genes under the control of double CaMV 35S and glyceraldehyde-3-phosphate gene (GapC) promoters, respectively. Plant-produced asialo-rhuEPO (asialo-rhuEPOP) was purified by immunoaffinity chromatography. Detailed N-glycan analysis using NSI-FTMS and MS/MS revealed that asialo-rhuEPOP bears paucimannosidic, high mannose-type and complex N-glycans. In vitro cytoprotection assays showed that the asialo-rhuEPOP (20 U/ml) provides 2-fold better cytoprotection (44%) to neuronal-like mouse neuroblastoma cells from staurosporine-induced cell death than rhuEPOM (21%). The cytoprotective effect of the asialo-rhuEPOP was found to be mediated by receptor-initiated phosphorylation of Janus kinase 2 (JAK2) and suppression of caspase 3 activation. Altogether, these findings demonstrate that plants are a suitable host for producing cytoprotective rhuEPO derivative. In addition, the general advantages of plant-based expression system can be exploited to address the cost and scalability issues related to its production. Published version

The binding affinity of 17β-estradiol with an immobilized DNA aptamer was measured using capillary electrophoresis. Estradiol captured by the immobilized DNA was injected into the separation capillary using pH-mediated sample stacking. Stacked 17β-estradiol was then separated using micellar electrokinetic capillary chromatography and detected with UV-visible absorbance. Standard addition was used to quantify the concentration of estradiol bound to the aptamer. Following incubation with immobilized DNA, analysis of free and bound estradiol yielded a dissociation constant of 70 ± 10 μM. The method was also used to screen binding affinity of the aptamer for estrone and testosterone. This study demonstrates the effectiveness of capillary electrophoresis to assess the binding affinity of DNA aptamers.

A capillary separation method that incorporates pH-mediated stacking is employed for the simultaneous determination of circulating steroid hormones in plasma from Perca flavescens (yellow perch) collected from natural aquatic environments. The method can be applied to separate eight steroid standards: progesterone, 17alpha,20beta-dihydroxypregn-4-en-3-one, 17alpha-hydroxyprogesterone, testosterone, estrone, 11-ketotestosterone, ethynyl estradiol, and 17beta-estradiol. Based on screening of plasma, the performance of the analytical method was determined for 17alpha,20beta-dihydroxypregn-4-en-3-one, testosterone, 11-ketotestosterone, and 17beta-estradiol. The within-day reproducibility in migration time for these four steroids in aqueous samples was < or =2%. Steroid quantification was accomplished using a calibration curve obtained with external standards. Plasma samples from fish collected from the Choptank and Severn Rivers, Maryland, USA, stored for up to one year were extracted with ethyl acetate and then further processed with anion exchange and hydrophobic solid phase extraction cartridges. The recovery of testosterone and 17beta-estradiol from yellow perch plasma was 84 and 85%, respectively. Endogenous levels of testosterone ranged from 0.9 to 44 ng/ml, and when detected 17alpha,20beta-dihydroxypregn-4-en-3-one ranged from 5 to 34 ng/ml. The reported values for testosterone correlated well with the immunoassay technique. Endogenous concentrations of 17beta-estradiol were < or =1.7 ng/ml. 11-Ketotestosterone was not quantified because of a suspected interferant. Higher levels of 17alpha,20beta-dihydroxypregn-4-en-3-one were found in male and female fish in which 17beta-estradiol was not detected. Monitoring multiple steroids can provide insight into hormonal fluctuations in fish.

For the rapid production of influenza vaccine antigens in unlimited quantities, a transition from conventional egg-based production to cell-based and recombinant systems is required. The need for higher-yield, lower-cost, and faster production processes is critical to provide adequate supplies of influenza vaccine to counter global pandemic threats. In this study, recombinant hemagglutinin proteins of influenza virus were expressed in the microalga Schizochytrium sp., an established, fermentable organism grown in large scale for the manufacture of polyunsaturated fatty acids for animal and human health applications. Schizochytrium was capable of exporting the full-length membrane-bound proteins in a secreted form suitable for vaccine formulation. One recombinant hemagglutinin (rHA) protein derived from A/Puerto Rico/8/34 (H1N1) influenza virus was evaluated as a vaccine in a murine challenge model. Protective immunity from lethal challenge with homologous virus was elicited by a single dose of 1.7, 5 or 15 µg rHA with or without adjuvant at survival rates between 80-100%. Full protection (100%) was established at all dose levels with or without adjuvant when mice were given a second vaccination. These data demonstrate the potential of Schizochytrium sp. as a platform for the production of recombinant antigens useful for vaccination against influenza.