Reliable N-Glycan Analysis-Removal of Frequently Occurring Oligosaccharide Impurities by Enzymatic Degradation.

Glycosylation, especially N-glycosylation, is one of the most common protein modifications, with immense importance at the molecular, cellular, and organismal level. Thus, accurate and reliable N-glycan analysis is essential in many areas of pharmaceutical and food industry, medicine, and science. However, due to the complexity of the cellular glycosylation process, in-depth glycoanalysis is still a highly challenging endeavor. Contamination of samples with oligosaccharide impurities (OSIs), typically linear glucose homo-oligomers, can cause further complications. Due to their physicochemical similarity to N-glycans, OSIs produce potentially overlapping signals, which can remain unnoticed. If recognized, suspected OSI signals are usually excluded in data evaluation. However, in both cases, interpretation of results can be impaired. Alternatively, sample preparation can be repeated to include an OSI removal step from samples. However, this significantly increases sample amount, time, and effort necessary. To overcome these issues, we investigated the option to enzymatically degrade and thereby remove interfering OSIs as a final sample preparation step. Therefore, we screened ten commercially available enzymes concerning their potential to efficiently degrade maltodextrins and dextrans as most frequently found OSIs. Of these enzymes, only dextranase from Chaetomium erraticum and glucoamylase P from Hormoconis resinae enabled a degradation of OSIs within only 30 min that is free of side reactions with N-glycans. Finally, we applied the straightforward enzymatic degradation of OSIs to N-glycan samples derived from different standard glycoproteins and various stem cell lysates.

Glycosyltransferase POMGNT1 deficiency strengthens N-cadherin-mediated cell-cell adhesion.

Defects in protein O-mannosylation lead to severe congenital muscular dystrophies collectively known as α-dystroglycanopathy. A hallmark of these diseases is the loss of the O-mannose-bound matriglycan on α-dystroglycan, which reduces cell adhesion to the extracellular matrix. Mutations in protein O-mannose ß1,2-N-acetylglucosaminyltransferase 1 (POMGNT1), which is crucial for the elongation of O-mannosyl glycans, have mainly been associated with muscle-eye-brain (MEB) disease. In addition to defects in cell-extracellular matrix adhesion, aberrant cell-cell adhesion has occasionally been observed in response to defects in POMGNT1. However, specific molecular consequences of POMGNT1 deficiency on cell-cell adhesion are largely unknown. We used POMGNT1 knockout HEK293T cells and fibroblasts from an MEB patient to gain deeper insight into the molecular changes in POMGNT1 deficiency. Biochemical and molecular biological techniques combined with proteomics, glycoproteomics, and glycomics revealed that a lack of POMGNT1 activity strengthens cell-cell adhesion. We demonstrate that the altered intrinsic adhesion properties are due to an increased abundance of N-cadherin (N-Cdh). In addition, site-specific changes in the N-glycan structures in the extracellular domain of N-Cdh were detected, which positively impact on homotypic interactions. Moreover, in POMGNT1-deficient cells, ERK1/2 and p38 signaling pathways are activated and transcriptional changes that are comparable with the epithelial-mesenchymal transition (EMT) are triggered, defining a possible molecular mechanism underlying the observed phenotype. Our study indicates that changes in cadherin-mediated cell-cell adhesion and other EMT-related processes may contribute to the complex clinical symptoms of MEB or α-dystroglycanopathy in general and suggests that the impact of changes in O-mannosylation on N-glycosylation has been underestimated.

Serum N-glycomics of a novel CDG-IIb patient reveals aberrant IgG glycosylation.

Rare genetic mutations of the mannosyl-oligosaccharide glucosidase (MOGS) gene affecting the function of the mannosyl-oligosaccharide glucosidase (glucosidase I) are the cause of the congenital disorder of glycosylation IIb (CDG-IIb). Glucosidase I specifically removes the distal α1,2-linked glucose from the protein bound precursor N-glycan Glc3Man9GlcNAc2, which is the initial step of N-glycan maturation. Here, we comparatively analyzed N-glycosylation of the whole serum proteome, serum-derived immunoglobulin G (IgG), transferrin (TF), and α-1-antitrypsin (AAT) of a female patient who is compound heterozygous for 2 novel missense mutations in the MOGS gene, her heterozygous parents, and a sibling with wildtype genotype by multiplexed capillary gel electrophoresis coupled to laser induced fluorescence detection (xCGE-LIF) at unprecedented depth. Thereby, we detected the CDG-IIb-characteristic non-de-glucosylated N-glycans Glc3Man7-9GlcNAc2 as well as the free tetrasaccharide Glc3-Man in whole serum of the patient but not in the other family members. The N-glycan analysis of the serum proteome further revealed that relative intensities of IgG-specific complex type di-antennary N-glycans with core-fucosylation were considerably reduced in the patient's serum whereas TF- and AAT-characteristic sialylated di- and tri-antennary N-glycans were increased. This finding reflected the hypogammaglobulinemia diagnosed in the patient. We further detected aberrant oligo-mannose (Glc3Man7GlcNAc2) and hybrid type N-glycans on patient-derived IgGs and we attributed this defective glycosylation to be the reason for an increased IgG clearance. This mechanism can explain the hypogammaglobulinemia that is associated with CDG-IIb.

NIST Interlaboratory Study on Glycosylation Analysis of Monoclonal Antibodies: Comparison of Results from Diverse Analytical Methods.

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.

Exploring the Chemical Space of Protein Glycosylation in Noncovalent Protein Complexes: An Expedition along Different Structural Levels of Human Chorionic Gonadotropin by Employing Mass Spectrometry.

Modern analytical approaches employing high-resolution mass spectrometry (MS) facilitate the generation of a vast amount of structural data of highly complex glycoproteins. Nevertheless, systematic interpretation of this data at different structural levels remains an analytical challenge. The glycoprotein utilized as a model system in this study, human chorionic gonadotropin (hCG), exists as a heterodimer composed of two heavily glycosylated subunits. In order to unravel the multitude of glycoforms of recombinant hCG (drug product Ovitrelle), we combine established techniques, such as released glycan and glycopeptide analysis, with novel approaches employing high-performance liquid chromatography-mass spectrometry (HPLC-MS) to characterize protein subunits and native MS to analyze the noncovalent hCG complex. Starting from the deconvoluted mass spectrum of dimeric hCG comprising about 50 signals, it was possible to explore the chemical space of hCG glycoforms and elucidate the complexity that hides behind just 50 signals. Systematic, stepwise integration of data obtained at the levels of released glycans, glycopeptides, and subunits using a computational annotation tool allowed us to reveal 1031 underlying glycoforms. Additionally, critical quality attributes such as sialylation and core fucosylation were compared for two batches of Ovitrelle to assess the potential product variability.

N-glycosylation analysis of mouse immunoglobulin G isolated from dried blood spots.

The association of immunoglobulin G (IgG) glycosylation changes with various human diseases and physiological conditions is well established. Since the mechanistical explanation of the regulation of IgG glycosylation and its functional role in these various states is still missing, the eyes of the biomedical community are now turned towards animal models, which enable intervention studies necessary for conclusions on causality. Mice are recognized and used as a good experimental model for human IgG glycosylation. However, smaller blood volumes, low IgG concentrations at young ages (which are most often used in mice experiments) and multiple sampling protocols during the course of longitudinal studies would profit from a robust workflow for mouse IgG glycome analysis from minute amounts of starting material, collected through a simple sampling procedure. For this purpose, we have developed a protocol for analysis of total N-glycans of IgG isolated from mouse dried blood spots (DBS), which we report here. We show that mouse DBS are a good source of material for IgG N-glycan analysis by multiplexed capillary gel electrophoresis with laser-induced fluorescence (xCGE-LIF).

Combining functional metagenomics and glycoanalytics to identify enzymes that facilitate structural characterization of sulfated N-glycans.

BACKGROUND: Sulfate modification of N-glycans is important for several biological functions such as clearance of pituitary hormones or immunoregulation. Yet, the prevalence of this N-glycan modification and its functions remain largely unexplored. Characterization of N-glycans bearing sulfate modifications is hampered in part by a lack of enzymes that enable site-specific detection of N-glycan sulfation. In this study, we used functional metagenomic screening to identify enzymes that act upon sulfated N-acetylglucosamine (GlcNAc). Using multiplexed capillary gel electrophoresis with laser-induced fluorescence detection (xCGE-LIF) -based glycoanalysis we proved their ability to act upon GlcNAc-6-SO4 on N-glycans. RESULTS: Our screen identified a sugar-specific sulfatase that specifically removes sulfate from GlcNAc-6-SO4 when it is in a terminal position on an N-glycan. Additionally, in the absence of calcium, this sulfatase binds to the sulfated glycan but does not remove the sulfate group, suggesting it could be used for selective isolation of sulfated N-glycans. Further, we describe isolation of a sulfate-dependent hexosaminidase that removes intact GlcNAc-6-SO4 (but not asulfated GlcNAc) from a terminal position on N-glycans. Finally, the use of these enzymes to detect the presence of sulfated N-glycans by xCGE-LIF is demonstrated. CONCLUSION: The present study demonstrates the feasibility of using functional metagenomic screening combined with glycoanalytics to discover enzymes that act upon chemical modifications of glycans. The discovered enzymes represent new specificities that can help resolve the presence of GlcNAc-6-SO4 in N-glycan structural analyses.

High-throughput Serum N-Glycomics: Method Comparison and Application to Study Rheumatoid Arthritis and Pregnancy-associated Changes.

N-Glycosylation is a fundamentally important protein modification with a major impact on glycoprotein characteristics such as serum half-life and receptor interaction. More than half of the proteins in human serum are glycosylated, and the relative abundances of protein glycoforms often reflect alterations in health and disease. Several analytical methods are currently capable of analyzing the total serum N-glycosylation in a high-throughput manner.Here we evaluate and compare the performance of three high-throughput released N-glycome analysis methods. Included were hydrophilic-interaction ultra-high-performance liquid chromatography with fluorescence detection (HILIC-UHPLC-FLD) with 2-aminobenzamide labeling of the glycans, multiplexed capillary gel electrophoresis with laser-induced fluorescence detection (xCGE-LIF) with 8-aminopyrene-1,3,6-trisulfonic acid labeling, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) with linkage-specific sialic acid esterification. All methods assessed the same panel of serum samples, which were obtained at multiple time points during the pregnancies and postpartum periods of healthy women and patients with rheumatoid arthritis (RA). We compared the analytical methods on their technical performance as well as on their ability to describe serum protein N-glycosylation changes throughout pregnancy, with RA, and with RA disease activity.Overall, the methods proved to be similar in their detection and relative quantification of serum protein N-glycosylation. However, the non-MS methods showed superior repeatability over MALDI-TOF-MS and allowed the best structural separation of low-complexity N-glycans. MALDI-TOF-MS achieved the highest throughput and provided compositional information on higher-complexity N-glycans. Consequentially, MALDI-TOF-MS could establish the linkage-specific sialylation differences within pregnancy and RA, whereas HILIC-UHPLC-FLD and xCGE-LIF demonstrated differences in α1,3- and α1,6-branch galactosylation. While the combination of methods proved to be the most beneficial for the analysis of total serum protein N-glycosylation, informed method choices can be made for the glycosylation analysis of single proteins or samples of varying complexity.

The transverse coloplasty pouch is technically easy and safe and improves functional outcomes after low rectal cancer resection-a single center experience with 397 patients.

BACKGROUND: Following resection for low rectal cancer, numerous patients suffer from frequent bowel movements, fecal urgency, and incontinence. Although there is good evidence that colonic J-pouch reconstruction, side-to-end anastomosis, or a transverse coloplasty pouch (TCP) improves functional outcome, many surgeons still prefer straight coloanal anastomosis because it is technically easier and lacks the risk of pouch-associated complications. The present single-center study aimed to evaluate the practicability of TCPs in routine clinical practice as well as pouch-related complications. METHOD: All consecutive patients who underwent low anterior rectal resection with restoration of bowel continuity for cancer during the period September 2008 to June 2018 were included. A TCP in combination with a diverting ileostomy was defined as the hospital standard. The feasibility and safety of TCPs were assessed in a retrospective single-center study. RESULTS: A total of 397 patients were included in the study. A total of 328/397 patients underwent TCP construction (82.6%). Two pouch-related surgical complications occurred (0.6%); one case of pouch-related stenosis and one case of sutural insufficiency. Overall, leakage of the coloanal anastomosis was reported in 14.1% of patients with a TCP and in 18.8% of patients without a pouch (p=0.252). Diverting ileostomy was applied in 378/397 patients (95.2%). The 30-day mortality was 0.25%. CONCLUSION: The present study is by far the largest single-center experience with TCP construction for low rectal cancer resection. The study shows that a TCP is technically applicable in the vast majority of cases (82.6%). Pouch-associated surgical complications are sporadic events. In our opinion, the TCP can be considered an alternative to J-pouch construction after low anterior rectal resection.

The subcommissural organ and the Reissner fiber: old friends revisited.

The subcommissural organ (SCO) is an ancient and conserved brain gland secreting into cerebrospinal fluid (CSF) glycoproteins that form the Reissner fiber (RF). The present investigation was designed to further investigate the dynamic of the biosynthetic process of RF glycoproteins prior and after their release into the CSF, to identify the RF proteome and N-glycome and to clarify the mechanism of assembly of RF glycoproteins. Various methodological approaches were used: biosynthetic labelling injecting 35S-cysteine and 3H-galactose into the CSF, injection of antibodies against galectin-1 into the cerebrospinal fluid, light and electron microscopical methods; isolated bovine RF was used for proteome analyses by mass spectrometry and glycome analysis by xCGE-LIF. The biosynthetic labelling study further supported that a small pool of SCO-spondin molecules rapidly enter the secretory pathways after its synthesis, while most of the SCO-spondin molecules are stored in the rough endoplasmic reticulum for hours or days before entering the secretory pathway and being released to assemble into RF. The proteomic analysis of RF revealed clusterin and galectin-1 as partners of SCO-spondin; the in vivo use of anti-galectin-1 showed that this lectin is essential for the assembly of RF. Galectin-1 is not secreted by the SCO but evidence was obtained that it would be secreted by multiciliated ependymal cells lying close to the SCO. Further, a surprising variety and complexity of glycan structures were identified in the RF N-glycome that further expands the potential functions of RF to a level not previously envisaged. A model of the macromolecular organization of Reissner fiber is proposed.

Continuous low flow ascites drainage through the urinary bladder via the Alfapump system in palliative patients with malignant ascites.

BACKGROUND: Malignant Ascites (MA) is a therapeutic dilemma significantly impairing patients' quality of life (QoL). The Sequana Medical alfapump System (AP), a subcutaneous, externally rechargeable, implantable device, continually draining ascites via the urinary bladder, has been well established in liver cirrhosis, but not yet in MA. The AP-system was evaluated in cancer patients in reducing the need for large volume paracentesis (LVP). METHODS: A retrospective multicentre evaluation of all eligible patients who received an AP for MA-palliation was performed. AP was evaluated for its ability to reduce LVP and cross-correlated with adverse events (AE), survival and retrospective physician-reported QoL. RESULTS: Seventeen patients with median age of 63 years (range: 18-81), 70.6% female, across 7 primary tumour types were analysed. Median duration of AP-implantation was 60 min (range: 30-270) and median post-implantation hospital stay: 4 days (range: 2-24). Twelve protocol-defined AE occurred in 5 patients (29.4%): 4 kidney failures, 4 pump/catheter-related blockages, 3 infections/peritonitis and 1 wound dehiscence. Median ascitic volume (AV) pumped daily was 303.6 ml/day (range:5.6-989.3) and median total AV drained was 28 L (range: 1-638.6). Median patient post-AP-survival was 111 days (range:10-715) and median pump survival was 89 days (range: 0-715). Median number of paracenteses was 4 (range: 1-15) per patient pre-implant versus 1 (range: 0-1) post-implant (p = 0.005). 71% of patients were reported to have an improvement of at least one physician reported QoL-parameters. CONCLUSIONS: AP appears to be effective in palliating patients with MA by an acceptable morbidity profile. Its broader implementation in oncology services should be further explored. TRIAL REGISTRATION: NCT03200106; June 27, 2017.

The Fine Art of Destruction: A Guide to In-Depth Glycoproteomic Analyses-Exploiting the Diagnostic Potential of Fragment Ions.

The unambiguous mass spectrometric identification and characterization of glycopeptides is crucial to elucidate the micro- and macroheterogeneity of glycoproteins. Here, combining lower and stepped collisional energy fragmentation for the in-depth and site-specific analysis of N- and O-glycopeptides is proposed. Using a set of four representative and biopharmaceutically relevant glycoproteins (IgG, fibrinogen, lactotransferrin, and ribonuclease B), the benefits and limitations of the developed workflow are highlighted and a state-of-the-art blueprint for conducting high-quality in-depth N- and O-glycoproteomic analyses is provided. Further, a modified and improved version of cotton hydrophilic interaction liquid chromatography-based solid phase extraction for glycopeptide enrichment is described. For the unambiguous identification of N-glycopeptides, the use of a conserved yet, rarely employed-fragmentation signature [Mpeptide +H+0,2 X GlcNAc]+ is proposed. It is shown for the first time that this fragmentation signature can consistently be found across all N-glycopeptides, but not on O-glycopeptides. Moreover, the use of the relative abundance of oxonium ions to retrieve glycan structure information, for example, differentiation of hybrid- and high-mannose-type N-glycans or differentiation between antenna GlcNAc and bisecting GlcNAc, is systematically and comprehensively evaluated. The findings may increase confidence and comprehensiveness in manual and software-assisted glycoproteomics.

Glycomic Characterization of Induced Pluripotent Stem Cells Derived from a Patient Suffering from Phosphomannomutase 2 Congenital Disorder of Glycosylation (PMM2-CDG).

PMM2-CDG, formerly known as congenital disorder of glycosylation-Ia (CDG-Ia), is caused by mutations in the gene encoding phosphomannomutase 2 (PMM2). This disease is the most frequent form of inherited CDG-diseases affecting protein N-glycosylation in human. PMM2-CDG is a multisystemic disease with severe psychomotor and mental retardation. In order to study the pathophysiology of PMM2-CDG in a human cell culture model, we generated induced pluripotent stem cells (iPSCs) from fibroblasts of a PMM2-CDG-patient (PMM2-iPSCs). Expression of pluripotency factors andin vitrodifferentiation into cell types of the three germ layers was unaffected in the analyzed clone PMM2-iPSC-C3 compared with nondiseased human pluripotent stem cells (hPSCs), revealing no broader influence of the PMM2 mutation on pluripotency in cell culture. Analysis of gene expression by deep-sequencing did not show obvious differences in the transcriptome between PMM2-iPSC-C3 and nondiseased hPSCs. By multiplexed capillary gel electrophoresis coupled to laser induced fluorescence detection (xCGE-LIF) we could show that PMM2-iPSC-C3 exhibit the common hPSC N-glycosylation pattern with high-mannose-type N-glycans as the predominant species. However, phosphomannomutase activity of PMM2-iPSC-C3 was 27% compared with control hPSCs and lectin staining revealed an overall reduced protein glycosylation. In addition, quantitative assessment of N-glycosylation by xCGE-LIF showed an up to 40% reduction of high-mannose-type N-glycans in PMM2-iPSC-C3, which was in concordance to the observed reduction of the Glc3Man9GlcNAc2 lipid-linked oligosaccharide compared with control hPSCs. Thus we could model the PMM2-CDG disease phenotype of hypoglycosylation with patient derived iPSCsin vitro Knock-down ofPMM2by shRNA in PMM2-iPSC-C3 led to a residual activity of 5% and to a further reduction of the level of N-glycosylation. Taken together we have developed human stem cell-based cell culture models with stepwise reduced levels of N-glycosylation now enabling to study the role of N-glycosylation during early human development.

Pylorus drainage procedures in thoracoabdominal esophagectomy - a single-center experience and review of the literature.

BACKGROUND: Pylorotomy and pyloroplasty in thoracoabdominal esophagectomy are routinely performed in many high-volume centers to prevent delayed gastric emptying (DGE) due to truncal vagotomy. Currently, controversy remains regarding the need for these practices. The present study aimed to determine the value and role of pyloric drainage procedures in esophagectomy with gastric replacement. METHODS: A retrospective review of prospectively collected data was performed for all consecutive patients who underwent thoracoabdominal resection of the esophagus between January 2009 and December 2016 at the Katharinenhospital in Stuttgart, Germany. Clinicopathologic features and surgical outcomes were evaluated with a focus on postoperative nutrition and gastric emptying. RESULTS: The study group included 170 patients who underwent thoracoabdominal esophageal resection with a gastric conduit using the Ivor Lewis approach. The median age of the patients was 64 years. Most patients were male (81%), and most suffered from adenocarcinoma of the esophagus (75%). The median hospital stay was 20 days, and the 30-day hospital death rate was 2.9%. According to the department standard, pylorotomy, pyloroplasty, or other pyloric drainage procedures were not performed in any of the patients. Overall, 28/170 patients showed clinical signs of DGE (16.5%). CONCLUSIONS: In the literature, the rate of DGE after thoracoabdominal esophagectomy is reported to be approximately 15%, even with the use of pyloric drainage procedures. This rate is comparable to that reported in the present series in which no pyloric drainage procedures were performed. Therefore, we believe that pyloric drainage procedures may be unwarranted in thoracoabdominal esophagectomy. However, future randomized trials are needed to ultimately confirm this supposition.

Towards personalized diagnostics via longitudinal study of the human plasma N-glycome.

Facilitated by substantial advances in analytical methods, plasma N-glycans have emerged as potential candidates for biomarkers. In the recent years, several investigations could link aberrant plasma N-glycosylation to numerous diseases. However, due to often limited specificity and sensitivity, only a very limited number of glycan biomarkers were approved by the authorities up to now. The inter-individual heterogeneity of the plasma N-glycomes might mask disease related changes in conventional large cross-sectional cohort studies, with a one-time sampling approach. But, a possible benefit of longitudinal sampling in biomarker discovery could be, that already small changes during disease progression are revealed, by monitoring the plasma N-glycome of individuals over time. To evaluate this, we collected blood plasma samples of five healthy donors over a time period of up to six years (min. 1.5 years). The plasma N-glycome was analyzed by xCGE-LIF, to investigate the intra-individual N-glycome variability over time. It is shown, that the plasma N-glycome of an individual is remarkably stable over a period of several years, and that observed small longitudinal changes are independent from seasons, but significantly correlated with lifestyle and environmental factors. Thus, the potential of future longitudinal biomarker discovery studies could be demonstrated, which is a further step towards personalized diagnostics. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.

Quantitative Assessment of Sialo-Glycoproteins and N-Glycans during Cardiomyogenic Differentiation of Human Induced Pluripotent Stem Cells.

Human induced pluripotent stem-cell-derived cardiomyocytes (hiPSC CMs) may be used in regenerative medicine for individualized tissue transplants in the future. For application in patients, the generated CMs have to be highly pure and well characterized. In order to overcome the prevalent scarcity of CM-specific markers, we quantitatively assessed cell-surface-exposed sialo-glycoproteins and N-glycans of hiPSCs, CM progenitors, and CMs. Applying a combination of metabolic labeling and specific sialo-glycoprotein capture, we could highly enrich and quantify membrane proteins during cardiomyogenic differentiation. Among them we identified a number of novel, putative biomarkers for hiPSC CMs. Analysis of the N-glycome by capillary gel electrophoresis revealed three novel structures comprising ß1,3-linked galactose, α2,6-linked sialic acid and complex fucosylation; these were highly specific for hiPSCs. Bisecting GlcNAc structures strongly increased during differentiation, and we propose that they are characteristic of early, immature CMs.

Comparative performance of four methods for high-throughput glycosylation analysis of immunoglobulin G in genetic and epidemiological research.

The biological and clinical relevance of glycosylation is becoming increasingly recognized, leading to a growing interest in large-scale clinical and population-based studies. In the past few years, several methods for high-throughput analysis of glycans have been developed, but thorough validation and standardization of these methods is required before significant resources are invested in large-scale studies. In this study, we compared liquid chromatography, capillary gel electrophoresis, and two MS methods for quantitative profiling of N-glycosylation of IgG in the same data set of 1201 individuals. To evaluate the accuracy of the four methods we then performed analysis of association with genetic polymorphisms and age. Chromatographic methods with either fluorescent or MS-detection yielded slightly stronger associations than MS-only and multiplexed capillary gel electrophoresis, but at the expense of lower levels of throughput. Advantages and disadvantages of each method were identified, which should inform the selection of the most appropriate method in future studies.

Effectiveness of triclosan-coated PDS Plus versus uncoated PDS II sutures for prevention of surgical site infection after abdominal wall closure: the randomised controlled PROUD trial.

BACKGROUND: Postoperative surgical site infections are one of the most frequent complications after open abdominal surgery, and triclosan-coated sutures were developed to reduce their occurrence. The aim of the PROUD trial was to obtain reliable data for the effectiveness of triclosan-coated PDS Plus sutures for abdominal wall closure, compared with non-coated PDS II sutures, in the prevention of surgical site infections. METHODS: This multicentre, randomised controlled group-sequential superiority trial was done in 24 German hospitals. Adult patients (aged ≥18 years) who underwent elective midline abdominal laparotomy for any reason were eligible for inclusion. Exclusion criteria were impaired mental state, language problems, and participation in another intervention trial that interfered with the intervention or outcome of this trial. A central web-based randomisation tool was used to randomly assign eligible participants by permuted block randomisation with a 1:1 allocation ratio and block size 4 before mass closure to either triclosan-coated sutures (PDS Plus) or uncoated sutures (PDS II) for abdominal fascia closure. The primary endpoint was the occurrence of superficial or deep surgical site infection according to the Centers for Disease Control and Prevention criteria within 30 days after the operation. Patients, surgeons, and the outcome assessors were masked to group assignment. Interim and final analyses were by modified intention to treat. This trial is registered with the German Clinical Trials Register, number DRKS00000390. FINDINGS: Between April 7, 2010, and Oct 19, 2012, 1224 patients were randomly assigned to intervention groups (607 to PDS Plus, and 617 to PDS II), of whom 1185 (587 PDS Plus and 598 PDS II) were analysed by intention to treat. The study groups were well balanced in terms of patient and procedure characteristics. The occurrence of surgical site infections did not differ between the PDS Plus group (87 [14·8%] of 587) and the PDS II group (96 [16·1%] of 598; OR 0·91, 95% CI 0·66-1·25; p=0·64). Serious adverse events also did not differ between the groups-146 of 583 (25·0%) patients treated with PDS Plus had at least one serious adverse event, compared with 138 of 602 (22·9%) patients treated with PDS II; p=0·39). INTERPRETATION: Triclosan-coated PDS Plus did not reduce the occurrence of surgical site infection after elective midline laparotomy. Innovative, multifactorial strategies need to be developed and assessed in future trials to reduce surgical site infections. FUNDING: Johnson & Johnson Medical Limited.

Development of a high-throughput glycoanalysis method for the characterization of oligosaccharides in human milk utilizing multiplexed capillary gel electrophoresis with laser-induced fluorescence detection.

During the last decade, enormous progress regarding knowledge about composition and properties of human milk (HM) has been made. Besides nutrition, the three macro-nutrients: proteins, lipids, and carbohydrates combine a large variety of properties and functions. Especially, complex oligosaccharides emerge as important dietary factors during early life with multiple functions. The characterization of these HM oligosaccharides (HMOS) within the total carbohydrate fraction is prerequisite to understand the relationship between milk composition and biological effects. Therefore, extended studies of large donor cohorts and thus, new high-throughput glycoanalytical methods are needed. The developed method comprises sample preparation, as well as analysis of HMOS by multiplexed CGE with LIF detection (xCGE-LIF). Via a respective database the generated "fingerprints" (normalized electropherograms) could be used for structural elucidation of HMOS. The method was tested on HM samples from five different donors, partly sampled as a series of lactation time points. HMOS could be easily identified and quantified. Consequently, secretor and Lewis status of the donors could be determined, milk typing could be performed and quantitative changes could be monitored along lactation time course. The developed xCGE-LIF based "real" high-throughput HMOS analysis method enables qualitative and quantitative high-performance profiling of the total carbohydrate fraction composition of large sets of samples.

Red-emitting rhodamines with hydroxylated, sulfonated, and phosphorylated dye residues and their use in fluorescence nanoscopy.

Fluorescent dyes emitting red light are frequently used in conventional and super-resolution microscopy of biological samples, although the variety of the useful dyes is limited. We describe the synthesis of rhodamine-based fluorescent dyes with absorption and emission maxima in the range of 621-637 and 644-660 nm, respectively and demonstrate their high performance in confocal and stimulated emission depletion (STED) microscopy. New dyes were prepared by means of reliable chemical transformations applied to a rhodamine scaffold with three variable positions. They feature polarity, water solubility, variable net charges, improved stabilities of N-hydroxysuccinimidyl (NHS) esters, as well as large fluorescence quantum yields in dye solutions and antibody conjugates. The photophysical and imaging properties of dyes containing three different polar groups, namely primary phosphate, sulfonic acid (in two different positions), and hydroxyl were compared. A dye with two primary phosphate groups was explored as a valuable alternative to dyes with "classical" sulfonic acid groups. Due to the increased net charge of the phosphorylated dye (q=-4 at pH 8), it demonstrated a far better electrophoretic mobility compared with analogues with two sulfonic acid groups (q=-2). As an example, one fluorescent dye was designed to be especially convenient for practical use. It is characterized by sufficiently high chemical stability of the NHS ester, its simple isolation, handling, and solubility in aqueous buffers, as well as in organic solvents. All these features, accompanied by a zero net charge in conjugates, were accomplished by the introduction of hydrophilic groups of two types: two hydroxyl groups and one sulfonic acid residue.