Hemostatic balance between pro- and anticoagulant is maintained during glucocorticoid treatment.

BACKGROUND: Glucocorticoids are associated with an increased risk of venous thrombosis. Glucocorticoid treatment increases coagulation factor and anticoagulant levels; however, its effect on hemostatic function remains unclear. This study aimed to investigate the changes in comprehensive coagulation profiles after glucocorticoid treatment in noninflammatory diseases to elucidate the direct contribution of glucocorticoids to hemostatic function. PROCEDURE: Patients diagnosed with primary immune thrombocytopenia requiring glucocorticoid treatment were prospectively enrolled in this study. Changes in coagulation factors and anticoagulants during glucocorticoid treatment and changes in thrombin generation potential were determined in the absence and presence of soluble thrombomodulin (sTM). RESULTS: Seven treatment cases (four for steroid pulse therapy and three for oral glucocorticoid therapy) in six patients with immune thrombocytopenia were examined. After glucocorticoid treatment, activated partial thromboplastin time significantly shortened, and activities of factor VIII, IX, XI, and XII significantly increased, except for von Willebrand factor antigen. Moreover, antithrombin and protein C (PC) activities significantly increased after glucocorticoid treatment. Two major parameters of thrombin generation potential, endogenous thrombin potential (ETP) and peak thrombin (Peak), significantly increased in the absence of sTM after glucocorticoid treatment. However, no significant increases in either parameter were observed in the presence of sTM. ETP-TM and Peak-TM ratios, which represent resistance to the anticoagulant effect of the PC pathway, significantly decreased after glucocorticoid treatment, suggesting that anticoagulant function via the PC pathway is elevated after glucocorticoid treatment. CONCLUSIONS: As glucocorticoids increase intrinsic coagulation factor and anticoagulant levels, hemostatic balance between pro- and anticoagulant functions is maintained.

Right ventricular systolic function as a predictor of appropriate ICD therapy.

BACKGROUND: Recent studies have shown that right ventricular dysfunction is associated with a significantly increased risk of sudden cardiac death. The purpose of this study was to evaluate the association of the right ventricular fractional area change (RVFAC) and appropriate implantable cardioverter-defibrillator (ICD) therapy to determine the cutoff value of the RVFAC. METHODS: Consecutive patients who underwent initial ICD implantations except those with hypertrophic cardiomyopathy, Brugada syndrome, and long QT syndrome were retrospectively enrolled. The primary endpoint was defined as any appropriate ICD therapy. The right ventricular dimensions and function on transthoracic echocardiography were measured for analysis. RESULTS: In total, 172 patients (60.3 ± 13.6 years, 131 males) were enrolled. Ninety patients received an ICD as a secondary prophylaxis. The mean LV ejection fraction and RVFAC were 38.3 ± 14.3% and 35.8 ± 8.8%, respectively. Regarding appropriate ICD therapy events, the best cutoff value of the RVFAC was 34.8%, while 74 patients had an RVFAC < 34.8%. Regarding the primary endpoint, the hazard ratio of a low RVFAC was 2.73 (95% CI 1.46-5.12, P < 0.01). In the multivariate analysis, a low RVFAC was an independent predictor of appropriate ICD therapy (HR: 3.40, 95% CI 1.74-6.64, P < 0.01). The secondary prophylactic cohort with a low RVFAC had the highest incidence of appropriate ICD therapy. Among the patients with RV dysfunction, the RVFAC normalized in 39% of patients during follow-up. This recovered RVFAC group had a significantly lower incidence of appropriate ICD therapy than the unrecovered RVFAC group (P = 0.043). CONCLUSION: A low RVFAC might be associated with increased appropriate ICD therapy.

Effectiveness of non-pharmacological interventions to prevent anemia in pregnant women: a quantitative systematic review protocol.

OBJECTIVE: This review aims to identify the effectiveness of non-pharmacological interventions in preventing iron deficiency anemia in pregnant women with a normal course of pregnancy. INTRODUCTION: The global prevalence of anemia among pregnant women is 36.5%, posing risks to women and fetuses. This underscores the need for effective prevention; however, the effectiveness of non-pharmacological approaches in preventing pregnancy anemia remains unclear. INCLUSION CRITERIA: This review will encompass experimental and quasi-experimental studies on the following approaches to prevent anemia during pregnancy: recommendations for dietary supplements, oral iron supplements (over the counter), provision of supplements to promote iron absorption, participation in anemia prevention education, and provision of information. There will be no restrictions on the duration or frequency of intervention, and longitudinal intervention studies will be included. In studies with a control group, the comparator may be usual care or pharmacological interventions; in studies without, it may involve no intervention, temporal comparisons, or baseline periods without non-pharmacological interventions. Evaluation of hemoglobin, hematocrit, and ferritin will be included as primary outcomes. Low birth weight, preterm birth, amount of blood loss at delivery, small for gestational age, and Apgar scores will be included as secondary outcomes. METHODS: A search will be conducted of MEDLINE (Ovid), Embase, CINAHL (EBSCOhost), Scopus, Australian New Zealand Clinical Trials Registry, Cochrane Central Register of Controlled Trials, and ICHUSHI-Web. Researchers will screen studies, extract data, assess the quality of studies, and analyze the data in accordance with the JBI guidance for systematic reviews of effectiveness. GRADE will be used to assess the certainty of the findings. REVIEW REGISTRATION NUMBER: PROSPERO CRD42022344155.

Endoscopic management of patients with high-risk colorectal colitis-associated neoplasia: a Delphi study.

BACKGROUND AND AIMS: Current guidelines recommend endoscopic resection of visible and endoscopically resectable colorectal colitis-associated neoplasia (CAN) in patients with inflammatory bowel disease (IBD). However, patients with high-risk CAN (HR-CAN) are often not amenable to conventional resection techniques, and a consensus approach for the endoscopic management of these lesions is presently lacking. This Delphi study aims to reach consensus among experts on the endoscopic management of these lesions. METHODS: A 3-round modified Delphi process was conducted to reach consensus among worldwide IBD and/or endoscopy experts (n = 18) from 3 continents. Consensus was considered if ≥75% agreed or disagreed. Quality of evidence was assessed by the criteria of the Cochrane Collaboration group. RESULTS: Consensus was reached on all statements (n = 14). Experts agreed on a definition for CAN and HR-CAN. Consensus was reached on the examination of the colon with enhanced endoscopic imaging before resection, the endoscopic resectability of an HR-CAN lesion, and endoscopic assessment and standard report of CAN lesions. In addition, experts agreed on type of resections of HR-CAN (< 20 mm, >20 mm, with or without good lifting), endoscopic success (technical success and outcomes), histologic assessment, and follow-up in HR-CAN. CONCLUSIONS: This is the first step in developing international consensus-based recommendations for endoscopic management of CAN and HR-CAN. Although the quality of available evidence was considered low, consensus was reached on several aspects of the management of CAN and HR-CAN. The present work and proposed standardization might benefit future studies.

Evaluation of a real-time computer-aided polyp detection system during screening colonoscopy: AI-DETECT study.

BACKGROUND: Polyp detection and resection during colonoscopy significantly reduce long-term colorectal cancer risk. Computer-aided detection (CADe) may increase polyp identification but has undergone limited clinical evaluation. Our aim was to assess the effectiveness of CADe at colonoscopy within a bowel cancer screening program (BCSP). METHODS: This prospective, randomized controlled trial involved all eight screening-accredited colonoscopists at an English National Health Service (NHS) BCSP center (February 2020 to December 2021). Patients were randomized to CADe or standard colonoscopy. Patients meeting NHS criteria for bowel cancer screening were included. The primary outcome of interest was polyp detection rate (PDR). RESULTS: 658 patients were invited and 44 were excluded. A total of 614 patients were randomized to CADe (n = 308) or standard colonoscopy (n = 306); 35 cases were excluded from the per-protocol analysis due to poor bowel preparation (n = 10), an incomplete procedure (n = 24), or a data issue (n = 1). Endocuff Vision was frequently used and evenly distributed (71.7 % CADe and 69.2 % standard). On intention-to-treat (ITT) analysis, there was a borderline significant difference in PDR (85.7 % vs. 79.7 %; P = 0.05) but no significant difference in adenoma detection rate (ADR; 71.4 % vs. 65.0 %; P = 0.09) for CADe vs. standard groups, respectively. On per-protocol analysis, no significant difference was observed in these rates. There was no significant difference in procedure times. CONCLUSIONS: In high-performing colonoscopists in a BCSP who routinely used Endocuff Vision, CADe improved PDR but not ADR. CADe appeared to have limited benefit in a BCSP setting where procedures are performed by experienced colonoscopists.

Chelate-free "turn-on"-type fluorescence detection of trivalent metal ions.

Trivalent metal ions are essential elements in materials and life sciences. To date, fluorescence is the gold standard for the detection of trivalent metal ions, which form a complex with fluorescent chelate ligands (Lewis base type). In this study, we report pH-responsive fluorescent probes for the selective detection of trivalent metal ions that utilize a new chelate-free approach using Brønsted base-type probes. Moreover, an X-ray structure of the outer-sphere aluminum(III) aqua complex coordinated by the fluorescent probes through hydrogen bonds was discovered. The outer-sphere complex consisting of an Al(III) aqua complex and protonated cationic probe showed cation-cation coordination.

Intracellular ferritin heavy chain plays the key role in artesunate-induced ferroptosis in ovarian serous carcinoma cells.

Artesunate, an antimalarial drug, induces ferroptosis, but the mechanism is still unclear. In the present study, we investigated how Artesunate induces ferroptosis in ovarian serous carcinoma. Experiments were performed using the ovarian serous carcinoma cell lines CaOV3 and SKOV3ip1, and the sensitivity of CaOV3 to Artesunate was higher than that of SKOV3ip1. Ferroptosis inhibitors inhibited Artesunate-induced intracellular lipid peroxi-dation and cell death. However, unlike class 1 ferroptosis inducer erastin, Artesunate had no effect on intracellular glutathione-SH levels. We found that Artesunate-induced changes in lysosomal Fe|2+ were parallel to the induction of ferroptosis. Therefore, ferritin, which oxidizes and binds intracellular Fe|2+, may have an inhibitory effect on ferroptosis. Knockdown of nuclear coactivator 4, a key molecule of ferritinophagy (ferritin-specific autophagy), suppressed Artesunate-induced cell death. Knockdown of ferritin heavy chain by siRNA greatly enhanced the sensitivity to Artesunate, and overexpression of ferritin heavy chain greatly reduced the sensitivity of ovarian cancer cell lines to Artesunate. These results can explain the differential sensitivity of CaOV3 and SKOV3ip1 to Artesunate. In conclusion, enhancement of ferritinophagy is an important step involved in the mechanism of Artesunate-induced ferroptosis, and ferritin heavy chain levels may contribute to the regulation of sensitivity in Artesunate-induced ferroptosis in ovarian serous carcinoma cells.

Implementation of optical diagnosis with a "resect and discard" strategy in clinical practice: DISCARD3 study.

BACKGROUND AND AIMS: Optical diagnosis (OD) of polyps can be performed with advanced endoscopic imaging. For high-confidence diagnoses, a "resect and discard" strategy could offer significant histopathology time and cost savings. The implementation threshold is a ≥90% OD-histology surveillance interval concordance. Here we assessed the OD learning curve and feasibility of a resect and discard strategy for ≤5-mm and <10-mm polyps in a bowel cancer screening setting. METHODS: In this prospective feasibility study, 8 bowel cancer screening endoscopists completed a validated OD training module and performed procedures. All <10-mm consecutive polyps had white-light and narrow-band images taken and were given high- or low-confidence diagnoses until 120 high-confidence ≤5-mm polyp diagnoses had been performed. All polyps had standard histology. High-confidence OD errors underwent root-cause analysis. Histology and OD-derived surveillance intervals were calculated. RESULTS: Of 565 invited patients, 525 patients were included. A total of 1560 <10-mm polyps underwent OD and were resected and retrieved (1329 ≤5 mm and 231 6-9 mm). There were no <10-mm polyp cancers. High-confidence OD was accurate in 81.5% of ≤5-mm and 92.8% of 6-9-mm polyps. Sensitivity for OD of a ≤5-mm adenoma was 93.0% with a positive predictive value of 90.8%. OD-histology surveillance interval concordance for ≤5-mm OD was 91.3% (209/229) for U.S. Multi-Society Task Force, 98.3% (225/229) for European Society of Gastrointestinal Endoscopy, and 98.7% (226/229) for British Society of Gastroenterology guidelines, respectively. CONCLUSIONS: A resect and discard strategy for high-confidence ≤5-mm polyp OD in a group of bowel cancer screening colonoscopists is feasible and safe, with performance exceeding the 90% surveillance interval concordance required for implementation in clinical practice. (Clinical trial registration number: NCT04710693.).

State of the Art in Endoscopic Therapy for the Management of Gastroenteropancreatic Neuroendocrine Tumors.

OPINION STATEMENT: Gastroenteropancreatic neuroendocrine neoplasms (GEP NENs) comprise a heterogeneous group of slow growing tumors arising from the neuroendocrine cells of the gastrointestinal (GI) tract. Although they are considered relatively rare, their incidence is rising and it is believed that the more frequent use of endoscopy and imaging studies have at least in part contributed to the increased diagnosis especially of localized neoplasms. The management of these neoplasms should be guided by a multidisciplinary NEN team following appropriate staging investigations. Localized neoplasms of the GI tract may be suitable for endoscopic therapy, while patients with pancreatic NENs, unsuitable for surgery, should be considered for endoscopic ultrasound (EUS)-guided ablation. In this review, we discuss the evidence regarding endoscopic resection of luminal NENs and EUS-guided therapy of pancreatic NENs. The efficacy, safety, and other longer-term outcomes of these techniques are summarized. In conclusion, this review of endoscopic therapies for localized NENs may be a useful guide for NEN clinicians and endoscopists who are considering these therapeutic options for the management of focal GEP NENs.

Acid responsiveness of emissive morpholinyl aminoquinolines and their use for cell fluorescence imaging.

Herein, we report emissive aminoquinoline derivatives (TFMAQ) containing alkylmorpholine and arylmorpholine groups and their photophysical properties, acid-responsiveness, and organelle targeting. The alkylmorpholine group is well-known to favour accumulation in lysosomes and be acid-responsive, but, counterintuitively, the TFMAQ derivatives containing ethylmorpholine groups showed limited accumulation in lysosomes and, instead, preferential accumulation in lipid droplets. The findings reported here will aid the development of organelle/tissue specific dyes for cell imaging and diagnosis.

Increased levels of acidic free-N-glycans, including multi-antennary and fucosylated structures, in the urine of cancer patients.

We recently reported increased levels of urinary free-glycans in some cancer patients. Here, we focused on cancer related alterations in the levels of high molecular weight free-glycans. The rationale for this study was that branching, elongation, fucosylation and sialylation, which lead to increases in the molecular weight of glycans, are known to be up-regulated in cancer. Urine samples from patients with gastric cancer, pancreatic cancer, cholangiocarcinoma and colorectal cancer and normal controls were analyzed. The extracted free-glycans were fluorescently labeled with 2-aminopyridine and analyzed by multi-step liquid chromatography. Comparison of the glycan profiles revealed increased levels of glycans in some cancer patients. Structural analysis of the glycans was carried out by performing chromatography and mass spectrometry together with enzymatic or chemical treatments. To compare glycan levels between samples with high sensitivity and selectivity, simultaneous measurements by reversed-phase liquid chromatography-selected ion monitoring of mass spectrometry were also performed. As a result, three lactose-core glycans and 78 free-N-glycans (one phosphorylated oligomannose-type, four sialylated hybrid-type and 73 bi-, tri- and tetra-antennary complex-type structures) were identified. Among them, glycans with α1,3-fucosylation ((+/- sialyl) Lewis X), triply α2,6-sialylated tri-antennary structures and/or a (Man3)GlcNAc1-core displayed elevated levels in cancer patients. However, simple α2,3-sialylation and α1,6-core-fucosylation did not appear to contribute to the observed increase in the level of glycans. Interestingly, one tri-antennary free-N-glycan that showed remarkable elevation in some cancer patients contained a unique Glcß1-4GlcNAc-core instead of the common GlcNAc2-core at the reducing end. This study provides further insights into free-glycans as potential tumor markers and their processing pathways in cancer.

Dynamics of salivary human herpesvirus-6 and -7 shedding in pregnant women.

Reactivation of Betaherpesvirinae (Human herpesvirus 6A: HHV-6A, -6B, HHV-7) may be associated with mental illness and host fatigue. This study aimed to determine whether viral reactivation, measured by monitoring salivary viral DNA load, can be used to monitor depression in pregnant and postpartum women. Saliva samples were collected from 64 pregnant women at five points of observation periods. The HHV-6- and HHV-7-specific qPCRs were carried out to measure viral DNA load. When HHV-6 DNA was detected in saliva, nested PCR was used to discriminate between HHV-6A and -6B. In both viruses, a significant correlation was observed between detection frequency and viral DNA load in saliva. In the low-shedding group, HHV-6 DNA was significantly higher in the third trimester (p < 0.0001), the time of delivery (p = 0.0003), 1 month after birth (p = 0.0023) compared with the first trimester, and HHV-7 was at the time of delivery (p = 0.0277) and 1 month after birth (p = 0.0235). Most of the detected HHV-6 DNAs in saliva were HHV-6B. Both viral DNA loads were significantly lower (HHV-6: p = 0.0101, HHV-7: p = 0.0044) in the subjects with abnormal Edinburgh Postnatal Depression Scale (EPDS) scores. The detection rate and viral DNA load of both viruses in saliva increased after the third trimester. Salivary virus DNA shedding was significantly lower in subjects with an abnormal EPDS score.

Structural analysis of N-glycans in chicken trachea and lung reveals potential receptors of chicken influenza viruses.

Although avian influenza A viruses (avian IAVs) bind preferentially to terminal sialic acids (Sia) on glycans that possess Siaα2-3Gal, the actual glycan structures found in chicken respiratory tracts have not been reported. Herein, we analyzed N-glycan structures in chicken trachea and lung, the main target tissues of low pathogenic avian IAVs. 2-Aminopyridine (PA)-labeled N-glycans from chicken tissues were analyzed by combined methods using reversed-phase liquid chromatography (LC), electrospray ionization (ESI)-mass spectrometry (MS), MS/MS, and multistage MS (MSn), with or without modifications using exoglycosidases, sialic acid linkage-specific alkylamidation (SALSA), and/or permethylation. The results of SALSA indicated that PA-N-glycans in both chicken trachea and lung harbored slightly more α2,6-Sia than α2,3-Sia. Most α2,3-Sia on N-glycans in chicken trachea was a fucosylated form (sialyl Lewis X, sLex), whereas no sLex was detected in lung. By contrast, small amounts of N-glycans with 6-sulfo sialyl LacNAc were detected in lung but not in trachea. Considering previous reports that hemagglutinins (HAs) of avian IAVs originally isolated from chicken bind preferentially to α2,3-Sia with or without fucosylation and/or 6-sulfation but not to α2,6-Sia, our results imply that avian IAVs do not evolve to possess HAs that bind preferentially to α2,6-Sia, regardless of the abundance of α2,6-Sia.

Ethnic comparison in takotsubo syndrome: novel insights from the International Takotsubo Registry.

BACKGROUND: Ethnic disparities have been reported in cardiovascular disease. However, ethnic disparities in takotsubo syndrome (TTS) remain elusive. This study assessed differences in clinical characteristics between Japanese and European TTS patients and determined the impact of ethnicity on in-hospital outcomes. METHODS: TTS patients in Japan were enrolled from 10 hospitals and TTS patients in Europe were enrolled from 32 hospitals participating in the International Takotsubo Registry. Clinical characteristics and in-hospital outcomes were compared between Japanese and European patients. RESULTS: A total of 503 Japanese and 1670 European patients were included. Japanese patients were older (72.6 ± 11.4 years vs. 68.0 ± 12.0 years; p < 0.001) and more likely to be male (18.5 vs. 8.4%; p < 0.001) than European TTS patients. Physical triggering factors were more common (45.5 vs. 32.0%; p < 0.001), and emotional triggers less common (17.5 vs. 31.5%; p < 0.001), in Japanese patients than in European patients. Japanese patients were more likely to experience cardiogenic shock during the acute phase (15.5 vs. 9.0%; p < 0.001) and had a higher in-hospital mortality (8.2 vs. 3.2%; p < 0.001). However, ethnicity itself did not appear to have an impact on in-hospital mortality. Machine learning approach revealed that the presence of physical stressors was the most important prognostic factor in both Japanese and European TTS patients. CONCLUSION: Differences in clinical characteristics and in-hospital outcomes between Japanese and European TTS patients exist. Ethnicity does not impact the outcome in TTS patients. The worse in-hospital outcome in Japanese patients, is mainly driven by the higher prevalence of physical triggers. TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov ; Unique Identifier: NCT01947621.

Electromyographic evaluation of perioral muscle activities during facial expression and button-pull exercise.

BACKGROUND: Muscle weakness negatively affects perioral muscles and quality of life. The button-pull exercise is used to teach lip closure (LC) and to strengthen muscles. However, how the muscles accomplish LC during button-pull and its training effect on each muscle are unknown. OBJECTIVES: This study aimed to investigate the LC mechanism and the efficacy of perioral muscle training. METHODS: Electromyographic (EMG) activities were obtained from perioral muscles along with the lip closing force (LCF) and were normalised to the maximum LC activities. Correlations between muscle activities and LCF were assessed during LCF increment/decrement task. The effectiveness of training methods was evaluated during functional face tasks (FFT). The effects of button-pull on muscles were evaluated during static loadings with two sized buttons. RESULTS: The muscles were active during LC, and the amplitudes changed with the LCF. In FFT, the muscles were simultaneously active and the total activity was highest during the blowing task. In button-pull, maximum button-pulling forces (BPFs) were significantly larger with the large button (p = .0001). In the static loading task, muscle activities increased with increasing button load. However, the small button produced significantly greater EMG activity than the large button in most of the load (p < .005). CONCLUSION: LC is accomplished by the cooperation of perioral muscles. In button-pull, a larger button requires a larger BPF, but a smaller button shows higher muscle activities. Face expression exercises compare favourably with button-pull. Forceful LC and blowing tasks may be effective and balanced training of the perioral muscles.

Strictly regulated agonist-dependent activation of AMPA-R is the key characteristic of TAK-653 for robust synaptic responses and cognitive improvement.

Agonistic profiles of AMPA receptor (AMPA-R) potentiators may be associated with seizure risk and bell-shaped dose-response effects. Here, we report the pharmacological characteristics of a novel AMPA-R potentiator, TAK-653, which exhibits minimal agonistic properties. TAK-653 bound to the ligand binding domain of recombinant AMPA-R in a glutamate-dependent manner. TAK-653 strictly potentiated a glutamate-induced Ca2+ influx in hGluA1i-expressing CHO cells through structural interference at Ser743 in GluA1. In primary neurons, TAK-653 augmented AMPA-induced Ca2+ influx and AMPA-elicited currents via physiological AMPA-R with little agonistic effects. Interestingly, TAK-653 enhanced electrically evoked AMPA-R-mediated EPSPs more potently than AMPA (agonist) or LY451646 (AMPA-R potentiator with a prominent agonistic effect) in brain slices. Moreover, TAK-653 improved cognition for both working memory and recognition memory, while LY451646 did so only for recognition memory, and AMPA did not improve either. These data suggest that the facilitation of phasic AMPA-R activation by physiologically-released glutamate is the key to enhancing synaptic and cognitive functions, and nonselective activation of resting AMPA-Rs may negatively affect this process. Importantly, TAK-653 had a wide safety margin against convulsion; TAK-653 showed a 419-fold (plasma Cmax) and 1017-fold (AUC plasma) margin in rats. These findings provide insight into a therapeutically important aspect of AMPA-R potentiation.

Tissue N-Glycan Analysis Using LC-MS, MS/MS, and MSn.

Tissue glycans usually contain various structures, from simple to highly complicated, in different quantities. N-Glycans are particularly heterogeneous, with up to pentaantennary structures, different branch sequences, and several isomeric structures. 2-Aminopyridine (PA) tagging on released N-glycans is useful for separating isomers and to quantitatively analyze both the major and minor glycan structures in tissues using reversed-phase liquid chromatography (LC)-mass spectrometry (MS) and MS/MS analysis. Because the structural differences of PA-N-glycans influence their retention on a reversed-phase C18 column, it is easy to deduce the core structure, including core Fuc and bisecting GlcNAc as well as the branching pattern of each PA-N-glycan, based on the results of elution position, full MS, and MS/MS analysis. If more detailed structural analysis is required, combining sequential exoglycosidase digestions, sialic acid linkage-specific alkylamidation (SALSA), and/or SALSA/permethylation is useful for determining glycosidic linkages of branches. This article includes detailed protocols for the preparation of N-glycans released from glycoproteins/glycopeptides by glycoamidase F or hydrazinolysis, PA-tagging of N-glycans, fractionation with anion-exchange chromatography, and chemical or enzymatic modifications of PA-N-glycans, as well as reversed-phase LC-MS, MS/MS, and MSn analysis of PA-N-glycans from tissues. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Preparation of released N-glycans from tissue samples using glycoamidase F Alternate Protocol: Preparation of released N-glycans from tissue samples by hydrazinolysis Basic Protocol 2: PA-tagging of N-glycans and sample cleanup Support Protocol 1: Monitoring of PA-N-glycans using normal-phase HPLC Basic Protocol 3: Anion-exchange chromatography of PA-N-glycans Basic Protocol 4: Sequential exoglycosidase digestions Basic Protocol 5: Determination of Sia-linkages by SALSA Support Protocol 2: Cotton-HILIC solid-phase extraction to remove reagents for alkylamidation Basic Protocol 6: Sequential modifications of glycans with SALSA and permethylation Basic Protocol 7: LC-MS and MS/MS analysis of PA-N-glycans (before permethylation) Basic Protocol 8: LC-MS, MS/MS, and MSn analysis of PA-N-glycans (after permethylation).

Toward robust N-glycomics of various tissue samples that may contain glycans with unknown or unexpected structures.

Glycans in tissues are structurally diverse and usually include a large number of isomers that cannot be easily distinguished by mass spectrometry (MS). To address this issue, we developed a combined method that can efficiently separate and identify glycan isomers. First, we separated 2-aminopyridine (PA)-derivatized N-glycans from chicken colon by reversed-phase liquid chromatography (LC) and directly analyzed them by electrospray ionization (ESI)-MS and MS/MS to obtain an overview of the structural features of tissue glycans. Next, we deduced the structures of isomers based on their elution positions, full MS, and MS/MS data, before or after digestions with several exoglycosidases. In this method, the elution position differed greatly depending on the core structure and branching pattern, allowing multiantennary N-glycan structures to be easily distinguished. To further determine linkages of branch sequences, we modified PA-N-glycans with sialic acid linkage-specific alkylamidation and/or permethylation, and analyzed the products by LC-MS and multistage MS. We determined the relative abundances of core structures, branching patterns, and branch sequences of N-glycans from chicken colon, and confirmed presence of characteristic branch sequences such as Lex, sialyl Lex, sulfated LacNAc, LacNAc repeat, and LacdiNAc. The results demonstrated that our method is useful for comparing N-glycomes among various tissue samples.