In-depth investigation of altered glycosylation in human haptoglobin associated cancer by mass spectrometry.

Serum haptoglobin (Hp), a highly sialylated biomolecule with four N-glycosylation sites, is a positive acute-phase response glycoprotein that acts as an immunomodulator. Hp has gained considerable attention due to its potential as a signature molecule that exhibits aberrant glycosylation in inflammatory disorders and malignancies. Its glycosylation can be analyzed qualitatively and quantitatively by various methods using mass spectrometry. In this review, we have provided a brief overview of Hp structure and biological function and described mass spectrometry-based techniques for analyzing glycosylation ranging from macroheterogeneity to microheterogeneity of Hp in diseases and cancer. The sugars on haptoglobin can be a sweet bridge to link the potential of cancer-specific biomarkers to clinically relevant applications.

Targeted polyelectrolyte complex micelles treat vascular complications in vivo.

Vascular disease is a leading cause of morbidity and mortality in the United States and globally. Pathological vascular remodeling, such as atherosclerosis and stenosis, largely develop at arterial sites of curvature, branching, and bifurcation, where disturbed blood flow activates vascular endothelium. Current pharmacological treatments of vascular complications principally target systemic risk factors. Improvements are needed. We previously devised a targeted polyelectrolyte complex micelle to deliver therapeutic nucleotides to inflamed endothelium in vitro by displaying the peptide VHPKQHR targeting vascular cell adhesion molecule 1 (VCAM-1) on the periphery of the micelle. This paper explores whether this targeted nanomedicine strategy effectively treats vascular complications in vivo. Disturbed flow-induced microRNA-92a (miR-92a) has been linked to endothelial dysfunction. We have engineered a transgenic line (miR-92aEC-TG /Apoe-/- ) establishing that selective miR-92a overexpression in adult vascular endothelium causally promotes atherosclerosis in Apoe-/- mice. We tested the therapeutic effectiveness of the VCAM-1-targeting polyelectrolyte complex micelles to deliver miR-92a inhibitors and treat pathological vascular remodeling in vivo. VCAM-1-targeting micelles preferentially delivered miRNA inhibitors to inflamed endothelial cells in vitro and in vivo. The therapeutic effectiveness of anti-miR-92a therapy in treating atherosclerosis and stenosis in Apoe-/- mice is markedly enhanced by the VCAM-1-targeting polyelectrolyte complex micelles. These results demonstrate a proof of concept to devise polyelectrolyte complex micelle-based targeted nanomedicine approaches treating vascular complications in vivo.

Pathological features in 'humanized' neonatal pig.

Cytidine monophosphate-Nacetylneuraminic acid (Neu5Ac) hydroxylase (CMAH) and glycoprotein, alpha1, 3-galactosyltransferase (GGTA1) double knockout (DKO) pig models were produced to reduce immune reaction for xenotransplantation. However, the role of Neu5Gc and α-Gal in pigs has not been fully elucidated and it is necessary to consider the after-effect of inactivation of GGTA1 and CMAH in pigs. Hematological profiles of DKO pigs were analyzed through complete blood count (CBC). Histology of liver and spleen of DKO were investigated, and lectin blotting and mass spectrometry (MS) were performed to explore glycosylation changes in red blood cell (RBC) membranes of DKO pigs. DKO pigs showed common clinical signs such as weakness (100%), dyspnea (90%) and constipation (65%). DKO pigs revealed a significant decrease in RBC, hemoglobin (HGB) and hematocrit (HGB), and an increase in white blood cell (WBC), lymphocyte (LYM), monocyte (MON), and erythrocyte mean corpuscular volume (MCV). DKO piglets showed swollen liver and spleen, and exhibited raised deposition of hemosiderin and severe bleeding. Lectin assay and MS proved variations in glycosylation on RBC membranes. GGTA1/CMAH DKO pigs developed pathological features which are similar to anemic symptoms, and the variations in glycosylation on RBC membranes of DKO pigs may be attributed to the pathologies observed.

In-depth characterization of non-human sialic acid (Neu5Gc) in human serum using label-free ZIC-HILIC/MRM-MS.

Sialic acid Neu5Gc, a non-human glycan, is recognized as a new harmful substance that can cause vascular disease and cancer. Humans are unable to synthesize Neu5Gc due to a genetic defect that converts Neu5Ac to Neu5Gc, but Neu5Gc is often observed in human biological samples. Therefore, the demand for accurately measuring the amount of Neu5Gc present in human blood or tissues is rapidly increasing, but there is still no method to reliably quantify trace amounts of a non-human sugar. In particular, selective isolation and detection of Neu5Gc from human serum is analytically challenging due to the presence of excess sialic acid Neu5Ac, which has physicochemical properties very similar to Neu5Gc. Herein, we developed the label-free approach based on ZIC-HILIC/MRM-MS that can enrich sialic acids released from human serum and simultaneously monitor Neu5Ac and Neu5Gc. The combination of complete separation of Neu5Gc from abundant Neu5Ac by hydrophilic and electrostatic interactions with selective monitoring of structure-specific cross-ring cleavage ions generated by negative CID-MS/MS was remarkably effective for quantification of Neu5Ac and Neu5Gc at the femtomole level. Indeed, we were able to successfully determine the absolute quantitation of Neu5Gc from 30 healthy donors in the range of 3.336 ± 1.252 pg/µL (mean ± SD), 10,000 times lower than Neu5Ac. In particular, analysis of sialic acids in protein-free serum revealed that both Neu5Ac and Neu5G are mostly bound to proteins and/or lipids, but not in free form. In addition, the correlation between expression level of Neu5Gc and biological factors such as BMI, age, and sex was investigated. This method can be widely used in studies requiring sialic acid-related measurements such as disease diagnosis or prediction of immunogenicity in biopharmaceuticals as it is both fast and highly sensitive.

LDL induces cholesterol loading and inhibits endothelial proliferation and angiogenesis in Matrigels: correlation with impaired angiogenesis during wound healing.

Hypercholesterolemia is a major risk factor for adverse cardiovascular outcomes, but its effect on angiogenesis and wound healing is not well understood. In this study, using a combination of mass spectrometry and laurdan two-photon imaging, we show that elevated levels of low-density lipoprotein (LDL), like those seen in hypercholesterolemic patients, lead to an increase in both free cholesterol and cholesterol esters, as well as increase in lipid order of endothelial cell membranes. Notably, these effects are distinct and opposite to the lack of cholesterol loading and the disruption of lipid order observed in our earlier studies in response to oxidized LDL (oxLDL). The same pathological level of LDL leads to a significant inhibition of endothelial proliferation and cell cycle arrest in G2/M phase, whereas oxLDL enhances endothelial proliferation in S phase of the cycle. LDL but not oxLDL suppresses the expression of vascular endothelial growth factor receptor-2 while enhancing the expression of vascular endothelial growth factor (VEGF). Furthermore, we show that aged (8-10 mo) hypercholesterolemic apolipoprotein E-deficient (ApoE-/-) mice display delayed wound closure compared with age-matched C57/BL6 wild-type controls following a skin punch biopsy. The delay in wound healing is associated with a decreased expression of cluster of differentiation 31 platelet endothelial cell adhesion molecule endothelial marker and decreased angiogenesis within the wound bed. Furthermore, decreased endothelial responsiveness to the growth factors VEGF and basic fibroblast growth factor is observed in ApoE-/- mice in Matrigel plugs and in Matrigels with high levels of LDL in wild-type mice. We propose that plasma hypercholesterolemia is antiangiogenic due to elevated levels of LDL.

Glycosylation of serum haptoglobin as a marker of gastric cancer: an overview for clinicians.

Introduction: Gastric cancer (GC) is one of the leading causes of cancer-related death worldwide because of difficulties in early diagnosis. Aberrant glycosylation in serum proteins has been associated with many human diseases. Serum haptoglobin, a highly sialylated glycoprotein with four N-glycosylation sites, has gained considerable attention due to its potential as a signature molecule to display aberrant glycosylation in inflammatory disorders and various types of cancer. In particular, the relevance of haptoglobin glycosylation in GC has been investigated in a multifaceted way.Areas covered: The screening of haptoglobin glycosylation could offer an alternative approach toward GC diagnosis and detection. In this report, various assay platforms such as glycan profiling, site-specific glycopeptide profiling, and intact protein profiling are introduced for the detection of abnormal glycosylation of serum haptoglobin.Expert opinion: Although aberrant glycosylation of serum haptoglobin is associated with gastric cancer patients and might be a promising marker of GC screening, the development of a diagnosis platform to increase specificity and sensitivity for clinical use is still an analytical challenge. However, the continuous advancement of analytical technologies and methods will spur the paradigm shift from traditional serum markers, enabling the effective mining of human glycoproteome for GC diagnostic markers.

Anti-inflammatory effect of bee venom in phthalic anhydride-induced atopic dermatitis animal model.

Globally, many people have been affected with atopic dermatitis (AD), a chronic inflammatory skin disease. AD is associated with multiple factors such as genetic, inflammatory, and immune factors. Bee venom (BV) is now widely used for the treatment of several inflammatory diseases. However, its effect on 5% phthalic anhydride (PA)-induced AD has not been reported yet. We investigated the anti-inflammatory and anti-AD effects of BV in a PA-induced animal model of AD. Balb/c mice were treated with topical application of 5% PA to the dorsal skin and ears for induction of AD. After 24 h, BV was applied on the back and ear skin of the mice three times a week for 4 weeks. BV treatment significantly reduced the PA-induced AD clinical score, back and ear epidermal thickness, as well as IgE level and infiltration of immune cells in the skin tissues compared to those of control mice. The levels of inflammatory cytokines in the serum were significantly decreased in BV-treated group compared to PA-treated group. In addition, BV inhibited the expression of iNOS and COX-2 as well as the activation of mitogen-activated protein kinase (MAPK) and NF-Ò¡B induced by PA in the skin tissues. We also found that BV abrogated the lipopolysaccharide or TNF-α/IFN-γ-induced NO production, expression of iNOS and COX-2, as well as MAPK and NF-Ò¡B signaling pathway in RAW 264.7 and HaCaT cells. These results suggest that BV may be a potential therapeutic macromolecule for the treatment of AD.

Comprehensive Characterization of Biotherapeutics by Selective Capturing of Highly Acidic Glycans Using Stepwise PGC-SPE and LC/MS/MS.

The glycosylation of biologics is an important factor in pharmacological functions such as efficacy, safety, and biological activity and is easily affected by subtle changes in the cellular environment. Hence, comprehensive and in-depth glycomic characterization of biological products should be performed to ensure product quality and process consistency prior to regulatory approval, but it is still highly challenging due to glycan microheterogeneity produced by enzymatic machinery. In this study, we have developed a systematic methodology for the separation and characterization of various glycans of biotherapeutics using the combination of solid-phase extraction (SPE) and high resolution LC/MS. Neutral and multiple-acidic glycans were selectively fractionated by SPE with a porous graphitized carbon (PGC) cartridge according to their molecule size and polarity (acidity, p Ka). Subsequent LC-MS and -MS/MS analyses enabled us to obtain glycan compositions, structures, and quantitative information. Indeed, we have successfully performed glycomic characterization of agalsidase-beta, a representative therapeutic enzyme containing both phosphorylated and sialylated glycans. In addition, a comparative analysis of functional glycans released from different batches of enzymes was performed to verify our method. These results suggest that stepwise PGC-SPE and LC/MS/MS pairwise assays can be used as an efficient tool to detect glycosylation changes of therapeutic glycoproteins including abundant acidic species in biologics or biosimilar development.

Designation of fingerprint glycopeptides for targeted glycoproteomic analysis of serum haptoglobin: insights into gastric cancer biomarker discovery.

Gastric cancer (GC) is one of the leading causes of cancer-related death worldwide, largely because of difficulties in early diagnosis. Despite accumulating evidence indicating that aberrant glycosylation is associated with GC, site-specific localization of the glycosylation to increase specificity and sensitivity for clinical use is still an analytical challenge. Here, we created an analytical platform with a targeted glycoproteomic approach for GC biomarker discovery. Unlike the conventional glycomic approach with untargeted mass spectrometric profiling of released glycan, our platform is characterized by three key features: it is a target-protein-specific, glycosylation-site-specific, and structure-specific platform with a one-shot enzyme reaction. Serum haptoglobin enriched by immunoaffinity chromatography was subjected to multispecific proteolysis to generate site-specific glycopeptides and to investigate the macroheterogeneity and microheterogeneity. Glycopeptides were identified and quantified by nano liquid chromatography-mass spectrometry and nano liquid chromatography-tandem mass spectrometry. Ninety-six glycopeptides, each corresponding to a unique glycan/glycosite pairing, were tracked across all cancer and control samples. Differences in abundance between the two groups were marked by particularly high magnitudes. Three glycopeptides exhibited exceptionally high control-to-cancer fold changes along with receiver operating characteristic curve areas of 1.0, indicating perfect discrimination between the two groups. From the results taken together, our platform, which provides biological information as well as high sensitivity and reproducibility, may be useful for GC biomarker discovery. Graphical abstract ᅟ.

Experimental Lung Injury Reduces Krüppel-like Factor 2 to Increase Endothelial Permeability via Regulation of RAPGEF3-Rac1 Signaling.

RATIONALE: Acute respiratory distress syndrome (ARDS) is caused by widespread endothelial barrier disruption and uncontrolled cytokine storm. Genome-wide association studies (GWAS) have linked multiple genes to ARDS. Although mechanosensitive transcription factor Krüppel-like factor 2 (KLF2) is a major regulator of endothelial function, its role in regulating pulmonary vascular integrity in lung injury and ARDS-associated GWAS genes remains poorly understood. OBJECTIVES: To examine KLF2 expression in multiple animal models of acute lung injury and further elucidate the KLF2-mediated pathways involved in endothelial barrier disruption and cytokine storm in experimental lung injury. METHODS: Animal and in vitro models of acute lung injury were used to characterize KLF2 expression and its downstream effects responding to influenza A virus (A/WSN/33 [H1N1]), tumor necrosis factor-α, LPS, mechanical stretch/ventilation, or microvascular flow. KLF2 manipulation, permeability measurements, small GTPase activity, luciferase assays, chromatin immunoprecipitation assays, and network analyses were used to determine the mechanistic roles of KLF2 in regulating endothelial monolayer integrity, ARDS-associated GWAS genes, and lung pathophysiology. MEASUREMENTS AND MAIN RESULTS: KLF2 is significantly reduced in several animal models of acute lung injury. Microvascular endothelial KLF2 is significantly induced by capillary flow but reduced by pathologic cyclic stretch and inflammatory stimuli. KLF2 is a novel activator of small GTPase Ras-related C3 botulinum toxin substrate 1 by transcriptionally controlling Rap guanine nucleotide exchange factor 3/exchange factor directly activated by cyclic adenosine monophosphate, which maintains vascular integrity. KLF2 regulates multiple ARDS GWAS genes related to cytokine storm, oxidation, and coagulation in lung microvascular endothelium. KLF2 overexpression ameliorates LPS-induced lung injury in mice. CONCLUSIONS: Disruption of endothelial KLF2 results in dysregulation of lung microvascular homeostasis and contributes to lung pathology in ARDS.

Color Doppler Sonography Accompanied by Dynamic Scanning for the Diagnosis of Ankle and Foot Fractures.

Sonography is conventionally used to diagnose fractures by identifying cortical discontinuity of the bone. In this study, fracture sonography in addition to color Doppler and dynamic scanning was performed in settings with limited or no access to radiography. We describe 5 cases of ankle and foot fractures with the use of sonography to identify changes in the fractured site. The width of the fracture space increased on dynamic scanning, and the Doppler signals were generated inside the fracture space on dynamic scanning. In conclusion, color Doppler sonography accompanied by dynamic scanning is a useful adjunctive diagnostic tool in addition to previously described sonographic fracture findings.

oxLDL induces endothelial cell proliferation via Rho/ROCK/Akt/p27kip1 signaling: opposite effects of oxLDL and cholesterol loading.

Oxidized modifications of LDL (oxLDL) play a key role in the development of endothelial dysfunction and atherosclerosis. However, the underlying mechanisms of oxLDL-mediated cellular behavior are not completely understood. Here, we compared the effects of two major types of oxLDL, copper-oxidized LDL (Cu2+-oxLDL) and lipoxygenase-oxidized LDL (LPO-oxLDL), on proliferation of human aortic endothelial cells (HAECs). Cu2+-oxLDL enhanced HAECs' proliferation in a dose- and degree of oxidation-dependent manner. Similarly, LPO-oxLDL also enhanced HAEC proliferation. Mechanistically, both Cu2+-oxLDL and LPO-oxLDL enhance HAEC proliferation via activation of Rho, Akt phosphorylation, and a decrease in the expression of cyclin-dependent kinase inhibitor 1B (p27kip1). Both Cu2+-oxLDL or LPO-oxLDL significantly increased Akt phosphorylation, whereas an Akt inhibitor, MK2206, blocked oxLDL-induced increase in HAEC proliferation. Blocking Rho with C3 or its downstream target ROCK with Y27632 significantly inhibited oxLDL-induced Akt phosphorylation and proliferation mediated by both Cu2+- and LPO-oxLDL. Activation of RhoA was blocked by Rho-GDI-1, which also abrogated oxLDL-induced Akt phosphorylation and HAEC proliferation. In contrast, blocking Rac1 in these cells had no effect on oxLDL-induced Akt phosphorylation or cell proliferation. Moreover, oxLDL-induced Rho/Akt signaling downregulated cell cycle inhibitor p27kip1 Preloading these cells with cholesterol, however, prevented oxLDL-induced Akt phosphorylation and HAEC proliferation. These findings provide a new understanding of the effects of oxLDL on endothelial proliferation, which is essential for developing new treatments against neovascularization and progression of atherosclerosis.

Oxidized LDL signals through Rho-GTPase to induce endothelial cell stiffening and promote capillary formation.

Endothelial biomechanics is emerging as a key factor in endothelial function. Here, we address the mechanisms of endothelial stiffening induced by oxidized LDL (oxLDL) and investigate the role of oxLDL in lumen formation. We show that oxLDL-induced endothelial stiffening is mediated by CD36-dependent activation of RhoA and its downstream target, Rho kinase (ROCK), via inhibition of myosin light-chain phosphatase (MLCP) and myosin light-chain (MLC)2 phosphorylation. The LC-MS/MS analysis identifies 7-ketocholesterol (7KC) as the major oxysterol in oxLDL. Similarly to oxLDL, 7KC induces RhoA activation, MLCP inhibition, and MLC2 phosphorylation resulting in endothelial stiffening. OxLDL also facilitates formation of endothelial branching networks in 3D collagen gels in vitro and induces increased formation of functional blood vessels in a Matrigel plug assay in vivo. Both effects are RhoA and ROCK dependent. An increase in lumen formation was also observed in response to pre-exposing the cells to 7KC, an oxysterol that induces endothelial stiffening, but not to 5α,6α epoxide that does not affect endothelial stiffness. Importantly, loading cells with cholesterol prevented oxLDL-induced RhoA activation and the downstream signaling cascade, and reversed oxLDL-induced lumen formation. In summary, we show that oxLDL-induced endothelial stiffening is mediated by the CD36/RhoA/ROCK/MLCP/MLC2 pathway and is associated with increased endothelial angiogenic activity.

Assessing the Risk of Normal Weight Obesity in Korean Women across Generations: A Study on Body Composition and Physical Fitness.

Normal weight obesity (NWO) refers to a condition in which the body mass index falls within the normal range, but the percent of body fat is excessive. Although there are reports of a high prevalence of cardiovascular and metabolic diseases in NWO, analyses regarding physical fitness have been lacking. Therefore, the purpose of this study was to analyze the age-related prevalence of NWO and to examine physical fitness across generations. Our study utilized a dataset comprising 119,835 participants for analysis. The prevalence of NWO across ages was examined using cross-tabulation analysis. For body composition and physical fitness, medians and group differences were assessed by generation through Kruskal-Wallis and Bonferroni post hoc tests. Additionally, univariate logistic regression was adopted to analyze the odds ratio. The prevalence of NWO in Korean women was 18.3%. The fat-free mass of the NWO group was consistently lower than that of both the group with normal body mass indexes (Normal) and obese body mass indexes (Obesity) across all generations. Additionally, the waist circumference and blood pressure were greater in the now group than in the Normal group. When considering maximal strength, muscle endurance, power, balance, and coordination, the NWO group exhibited lower levels compared to the Normal group. The NWO group showed lower muscle mass than both the Normal and Obesity groups, resulting in significantly reduced physical fitness compared to that of the Normal group, similar to the Obesity group. This condition may increase not only the risk of posing a potentially more serious health concern than obesity but also the risk of falls in elderly people. Therefore, based on this study, it is crucial to not only define obesity using BMI criteria but also to diagnose NWO. Public health policies and preventive measures must be implemented accordingly.

Glyco-analytical multispecific proteolysis (Glyco-AMP): a simple method for detailed and quantitative Glycoproteomic characterization.

Despite recent advances, site-specific profiling of protein glycosylation remains a significant analytical challenge for conventional proteomic methodology. To alleviate the issue, we propose glyco-analytical multispecific proteolysis (Glyco-AMP) as a strategy for glycoproteomic characterization. Glyco-AMP consists of rapid, in-solution digestion of an analyte glycoprotein (or glycoprotein mixture) by a multispecific protease (or protease cocktail). Resulting glycopeptides are chromatographically separated by isomer-specific porous graphitized carbon nano-LC, quantified by high-resolution MS, and structurally elucidated by MS/MS. To demonstrate the consistency and customizability of Glyco-AMP methodology, the glyco-analytical performances of multispecific proteases subtilisin, pronase, and proteinase K were characterized in terms of quantitative accuracy, sensitivity, and digestion kinetics. Glyco-AMP was shown be effective on glycoprotein mixtures as well as glycoproteins with multiple glycosylation sites, providing detailed, quantitative, site- and structure-specific information about protein glycosylation.

Vimentin phosphorylation by Cdc2 in Schwann cell controls axon growth via ß1-integrin activation.

Although preconditioning injury on the peripheral nerve induces axonal regenerative capacity in neurons, it is not known whether similar lesion effects occur in glial cells. Here we demonstrate that Schwann cells are activated by peripheral nerve preinjury and primed to mediate axon regeneration. Cdc2, which was induced from Schwann cells after sciatic nerve injury, phosphorylated vimentin almost exclusively in the distal nerve area. Phospho-vimentin-positive Schwann cells showed increased migration activity and were in close contact with process outgrowth of co-cultured neurons. Vimentin phosphorylation by Cdc2 was involved in ß1-integrin activation leading to FAK phoshorylation and associated with Erk1/2 activation in Schwann cells. Neurite outgrowth of dorsal root ganglion neurons was increased by co-culture with activated Schwann cells, in which phospho-vimentin signaling was transmitted into ß1-integrin activation. Then neurite outgrowth was suppressed by genetic depletion of phospho-vimentin and ß1 integrin as well as inhibition of vimentin phosphorylation by Cdc2 inhibitor purvalanol A. The sciatic nerve graft harboring activated Schwann cells into the spinal cord induced Schwann cell migration beyond the graft-host barrier and facilitated regeneration of spinal axons, which was inhibited by purvalanol A pretreatment of the graft. This is the first report to our knowledge demonstrating that activation of phospho-vimentin linked to ß1-integrin pathway may mediate transcellular signaling to promote axon growth.

Equivalence assessment of biotherapeutics with N- and O-glycosylation sites by sequential intact glycoform mass spectrometry (IGMS).

Glycosylation is a crucial attribute for biotherapeutics with significant impacts on quality, stability, safety, immunogenicity, pharmacokinetics, and efficacy. Therefore, to ensure consistent glycosylation, a systematic review of biotherapeutics is absolutely required including the variable glycan structure (micro-heterogeneity) and different occupancy at individual site (macro-heterogeneity) from drug design to upstream and downstream bioprocesses. Various methods have been used for glyco-characterization of biotherapeutics at the glycan, glycopeptide, and intact protein levels. In particular, intact protein analysis is considered a facile and rapid glycoform monitoring approach used throughout the product development lifecycle to determine suitable glycosylation lead candidates and reproducible product quality. However, intact glycoform characterization of diverse and complex biotherapeutics with multiple N- and O-glycosylation sites can be very challenging. To address this, a robust analytical platform that enables rapid and accurate characterization of a biotherapeutics with highly complex multiple glycosylation using two-step intact glycoform mass spectrometry has been developed. We used darbepoetin alfa, a second-generation EPO bearing multiple N- and O-glycosylation sites, as a model biotherapeutics to obtain integrated information on glycan heterogeneity and site occupancy through step-by-step MS of intact protein and enzyme-treated protein. In addition, we performed a comparative assessment of the heterogeneity from different products, confirming that our new method can efficiently evaluate glycosylation equivalence. This new strategy provides rapid and accurate information on the degree of glycosylation of a therapeutic glycoprotein with multiple glycosylation, which can be used to assess glycosylation similarity between batches and between biosimilar and reference during development and production.

An enhanced LC-MRM-MS platform for sensitive and simultaneous quantification of cyclic imines in shellfish.

Cyclic imines (CIs) produced by microalgae species and accumulating in the food chain of marine organisms are novel biotoxins that do not belong to the classical group of marine biotoxins. In the past, CIs were found only in limited areas, but in recent years, rapid changes in marine ecosystems have led to widespread CIs, increasing exposure to toxic risks. Monitoring of CIs is therefore required, but still analytically challenging due to the presence of high levels of analogues and interference from other lipophilic substances. Herein, we developed the LC/MRM-MS-based quantitative platform that can selectively enrich for marine-derived CIs and monitor seven CIs simultaneously: pinnatoxin (PnTX E, PnTX F, PnTX G), gymnodimine (GYM A), and spirolide (13-desMe SPX C, 13,19-didesMe SPX C, 20-Me SPX G). In particular, the combination of chromatographic separation by the hydrophobic nature of intrinsic residues of CIs with monitoring of CI structure-specific product ions generated by CID-MS/MS significantly improves the selectivity and sensitivity for quantitative analysis. Indeed, three CIs corresponding to PnTX G, GYM A, and 13-desMe SPX C could be successfully determined at the level of part-per-trillion (ppt) in three species of shellfish collected around the Korean Peninsula. Our analysis revealed that the expression of CIs in the Korean Peninsula was more influenced by the season rather than the species. This analytical platform with high sensitivity can be applied not only to marine biology but also to various other fields requiring CI analysis. Key Contribution: A highly sensitive analytical method for the simultaneous quantitation of cyclic imines based on LC/MRM-MS has been developed.

The role of oxysterols in control of endothelial stiffness.

Endothelial dysfunction is a key step in atherosclerosis development. Our recent studies suggested that oxLDL-induced increase in endothelial stiffness plays a major role in dyslipidemia-induced endothelial dysfunction. In this study, we identify oxysterols, as the major component of oxLDL, responsible for the increase in endothelial stiffness. Using Atomic Force Microscopy to measure endothelial elastic modulus, we show that endothelial stiffness increases with progressive oxidation of LDL and that the two lipid fractions that contribute to endothelial stiffening are oxysterols and oxidized phosphatidylcholines, with oxysterols having the dominant effect. Furthermore, endothelial elastic modulus increases as a linear function of oxysterol content of oxLDL. Specific oxysterols, however, have differential effects on endothelial stiffness with 7-ketocholesterol and 7α-hydroxycholesterol, the two major oxysterols in oxLDL, having the strongest effects. 27-hydroxycholesterol, found in atherosclerotic lesions, also induces endothelial stiffening. For all oxysterols, endothelial stiffening is reversible by enriching the cells with cholesterol. oxLDL-induced stiffening is accompanied by incorporation of oxysterols into endothelial cells. We find significant accumulation of three oxysterols, 7α-hydroxycholesterol, 7ß-hydroxycholesterol, and 7-ketocholesterol, in mouse aortas of dyslipidemic ApoE⁻/⁻ mice at the early stage of atherosclerosis. Remarkably, these are the same oxysterols we have identified to induce endothelial stiffening.

Prospective comparative study of endoscopic papillary large balloon dilation and endoscopic sphincterotomy for removal of large bile duct stones in patients above 45 years of age.

OBJECTIVE: Although endoscopic papillary large balloon dilation (EPLBD) with limited endoscopic sphincterotomy (ES) showed excellent outcomes for treatment of large bile duct stones, hemorrhage and recurrence of stones are problematic complications. Recent studies suggest that EPLBD alone is safe and effective for removal of large bile duct stones. This study aimed to determine the therapeutic outcomes and safety of EPLBD, compared with ES, for removal of large bile duct stones. MATERIAL AND METHODS: Eighty-three patients above 45 years of age with bile duct stones >1 cm in diameter were randomized to EPLBD and ES groups for removal of common bile duct stones from September 2010 to August 2011. Prophylactic gabexate mesilate was given to all patients. RESULTS: Baseline characteristics were not significantly different, except diabetes and gallbladder stones between the EPLBD group (n = 40) and ES group (n = 43). The overall complete stone removal rate in each group was 97.5% (39/40) and 95.3% (41/43), respectively (p = 0.600). Requirement of mechanical lithotripsy was not significantly different between the EPLBD and ES group (10% vs. 21%, p = 0.171). Complete ductal clearance in one session was achieved in 82.4% and 81.4% of cases in each group, respectively (p = 0.577). There were no differences in complication rates between the EPLBD and ES group; pancreatitis, 5.0% vs. 7.0%; hemorrhage, 10.0% vs. 16.3%; acute cholangitis, 5.0% vs. 2.3%, and perforation, 2.5% vs. 0%. CONCLUSIONS: The therapeutic outcomes and complications of EPLBD for removal of large bile duct stones are comparable to those of ES.