Advancements in protein glycosylation biomarkers for ovarian cancer through mass spectrometry-based approaches.

INTRODUCTION: Ovarian cancer, characterized by metastasis and reduced 5-year survival rates, stands as a substantial factor in the mortality of gynecological malignancies worldwide. The challenge of delayed diagnosis originates from vague early symptoms and the absence of efficient screening and diagnostic biomarkers for early cancer detection. Recent studies have explored the intricate interplay between ovarian cancer and protein glycosylation, unveiling the potential significance of glycosylation-oriented biomarkers. AREAS COVERED: This review examines the progress in glycosylation biomarker research, with particular emphasis on advances driven by mass spectrometry-based technologies. We document milestones achieved, discuss encountered limitations, and also highlight potential areas for future research and development of protein glycosylation biomarkers for ovarian cancer. EXPERT OPINION: The association of glycosylation in ovarian cancer is well known, but current research lacks desired sensitivity and specificity for early detection. Notably, investigations into protein-specific and site-specific glycoproteomics have the potential to significantly enhance our understanding of ovarian cancer and facilitate the identification of glycosylation-based biomarkers. Furthermore, the integration of advanced mass spectrometry techniques with AI-driven analysis and glycome databases holds the promise for revolutionizing biomarker discovery for ovarian cancer, ultimately transforming diagnosis and improving patient outcomes.

Effective decellularization of human skin tissue for regenerative medicine by supercritical carbon dioxide technique.

Allotransplantation, performed using an acellular dermal matrix (ADM), plays a significant role in the cultivation of constituted and damaged organs in clinical. Herein, we fabricated an innovative ADM for allografting derived from decellularized human skin by utilizing the supercritical fluid of carbon dioxide to eliminate immunogenic components. By using histological staining, the ADM product demonstrated the successful removal of cellular constituents without exerting any harmful influence on the extracellular matrix. The results from DNA electrophoresis also supported this phenomenon by showing the complete DNA removal in the product, accompanied by the absence of Major Histocompatibility Complex 1, which suggested the supercritical fluid is an effective method for cellular withdrawal. Moreover, the mechanical property of the ADM products, which showed similarity to that of native skin, displayed great compatibility for using our human-derived ADM as an allograft in clinical treatment. Specifically, the cell viability demonstrated the remarkable biocompatibility of the product to human bio-cellular environment which was noticeably higher than that of other products. Additionally, the significant increase in the level of growth factors such as vascular endothelial growth factor, urokinase-type plasminogen activator receptor, granulocyte-macrophage colony-stimulating factor suggested the ability to stimulate cellular processes, proving the products to be innovative in the field of regeneration when applied to clinical in the future. This study provides a thoroughly extensive analysis of the new ADM products, enabling them to be applied in industrial and clinical treatment.

Deuterium Oxide Labeling for Global Omics Relative Quantification (DOLGOReQ): Application to Glycomics.

A new relative quantification strategy for glycomics, named deuterium oxide (D2O) labeling for global omics relative quantification (DOLGOReQ), has been developed based on the partial metabolic D2O labeling, which induces a subtle change in the isotopic distribution of glycan ions. The relative abundance of unlabeled to D-labeled glycans was extracted from the overlapped isotopic envelope obtained from a mixture containing equal amounts of unlabeled and D-labeled glycans. The glycan quantification accuracy of DOLGOReQ was examined with mixtures of unlabeled and D-labeled HeLa glycans combined in varying ratios according to the number of cells present in the samples. The relative quantification of the glycans mixed in an equimolar ratio revealed that 92.4 and 97.8% of the DOLGOReQ results were within a 1.5- and 2-fold range of the predicted mixing ratio, respectively. Furthermore, the dynamic quantification range of DOLGOReQ was investigated with unlabeled and D-labeled HeLa glycans mixed in different ratios from 20:1 to 1:20. A good correlation (Pearson's r > 0.90) between the expected and measured quantification ratios over 2 orders of magnitude was observed for 87% of the quantified glycans. DOLGOReQ was also applied in the measurement of quantitative HeLa cell glycan changes that occur under normoxic and hypoxic conditions. Given that metabolic D2O labeling can incorporate D into all types of glycans, DOLGOReQ has the potential as a universal quantification platform for large-scale comparative glycomic experiments.

Spatial and temporal diversity of glycome expression in mammalian brain.

Mammalian brain glycome remains a relatively poorly understood area compared to other large-scale "omics" studies, such as genomics and transcriptomics due to the inherent complexity and heterogeneity of glycan structure and properties. Here, we first performed spatial and temporal analysis of glycome expression patterns in the mammalian brain using a cutting-edge experimental tool based on liquid chromatography-mass spectrometry, with the ultimate aim to yield valuable implications on molecular events regarding brain functions and development. We observed an apparent diversity in the glycome expression patterns, which is spatially well-preserved among nine different brain regions in mouse. Next, we explored whether the glycome expression pattern changes temporally during postnatal brain development by examining the prefrontal cortex (PFC) at different time point across six postnatal stages in mouse. We found that glycan expression profiles were dynamically regulated during postnatal developments. A similar result was obtained in PFC samples from humans ranging in age from 39 d to 49 y. Novel glycans unique to the brain were also identified. Interestingly, changes primarily attributed to sialylated and fucosylated glycans were extensively observed during PFC development. Finally, based on the vast heterogeneity of glycans, we constructed a core glyco-synthesis map to delineate the glycosylation pathway responsible for the glycan diversity during the PFC development. Our findings reveal high levels of diversity in a glycosylation program underlying brain region specificity and age dependency, and may lead to new studies exploring the role of glycans in spatiotemporally diverse brain functions.

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.

Comprehensive Profiling of Surface Gangliosides Extracted from Various Cell Lines by LC-MS/MS.

Gangliosides act as a surface marker at the outer cellular membrane and play key roles in cancer cell invasion and metastasis. Despite the biological importance of gangliosides, they have been still poorly characterized due to the lack of effective analytical tools. Herein, we performed molecular profiling and structural elucidation of intact gangliosides in various cell lines including CFPAC1, A549, NCI-H358, MCF7, and Caski. We identified and quantified a total of 76 gangliosides on cell membrane using C18 LC-MS/MS. Gangliosides found in each cell line exhibited high complexity and diversity both qualitatively and quantitatively. The most abundant species was GM3(d34:1) in CFPAC1, NCI-H358, and MCF7, while GM2(d34:1) and GM1(d34:1) were major components in A549 and Caski, respectively. Notably, glycan moieties showed more diversity between cancer cell lines than ceramide moieties. In addition, noncancerous pancreatic cell line (hTERT/HPNE) could be distinguished by gangliosides containing different levels of sialic acid compared with cancerous pancreatic cell line (CFPAC1). These results clearly demonstrated the feasibility of our analytical platform to comprehensive profile of cell surface gangliosides for identifying cell types and subgrouping cancer cell types.

Positive Effect of Incubated Adipose-Derived Mesenchymal Stem Cells on Microfat Graft Survival.

Autologous fat grafting is commonly used for soft tissue augmentation, but its unpredictably high resorption rate remains a major limitation. Although adipose-derived mesenchymal stem cells (ASCs) are an attractive candidate for enhancing graft retention, their poor posttransplantation viability limits their application. The authors aimed to evaluate the effect of incubated ASCs on microfat graft survival in an immunocompromised mouse model. Lipoaspirates for microfat injection were collected from the wasted lower abdominal adipose tissues of 5 patients who had undergone breast reconstructive surgery with an abdominal flap. Adipose-derived mesenchymal stem cells were also isolated and proliferated from these fat tissues. Sixty athymic mice were randomly allocated to a control group (microfat grafting alone; n = 30) or ASCs group (microfat grafting plus simultaneous human ASCs injection; n = 30). The volume and weight of survived fat were measured at 8 and 16 weeks, and histopathological and immunologic staining was performed at 16 weeks. The survived fat volume of the ASCs group was significantly greater than that of the control group at 8 and 16 weeks, whereas the weight of survived fat tissues did not significantly differ. Histologic evaluation of the harvested fat indicated significantly higher levels of adipocytes, and fewer cysts and fibrosis in the tissues in the ASCs group than in the control group. The ASCs group also exhibited a significantly higher number of capillary vessels than the control group on CD31 and alpha-smooth muscle actin staining. In conclusion, transplanted fat survival is markedly higher when simultaneous microfat graft and ASCs injection were performed, as compared with that in the classical microfat graft alone method in mice; this improvement was primarily attributed to the increased ability to produce blood vessels.

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.

Shaofu Zhuyu decoction ameliorates obesity-mediated hepatic steatosis and systemic inflammation by regulating metabolic pathways.

Shaofu Zhuyu decoction (SFZYD, also known as Sobokchugeo-tang), a classical prescription drug in traditional East Asian medicine, has been used to treat blood stasis syndrome (BSS). Hepatic steatosis is the result of excess caloric intake, and its pathogenesis involves internal retention of phlegm and dampness, blood stasis, and liver Qi stagnation. To evaluate the effects of treatment with SFZYD on obesity-induced inflammation and hepatic steatosis, we fed male C57BL/6N mice a high fat diet (HFD) for 8 weeks and then treated them with SFZYD by oral gavage for an additional 4 weeks. The results of histological and biochemical examinations indicated that SFZYD treatment ameliorates systemic inflammation and hepatic steatosis. A partial least squares-discriminant analysis (PLS-DA) scores plot of serum metabolites showed that HFD mice began to produce metabolites similar to those of normal chow (NC) mice after SFZYD administration. We noted significant alterations in the levels of twenty-seven metabolites, alterations indicating that SFZYD regulates the TCA cycle, the pentose phosphate pathway and aromatic amino acid metabolism. Increases in the levels of TCA cycle intermediate metabolites, such as 2-oxoglutaric acid, isocitric acid, and malic acid, in the serum of obese mice were significantly reversed after SFZYD treatment. In addition to inducing changes in the above metabolites, treatment with SFZYD also recovered the expression of genes related to hepatic mitochondrial dysfunction, including Ucp2, Cpt1α, and Ppargc1α, as well as the expression of genes involved in lipid metabolism and inflammation, without affecting glucose uptake or insulin signaling. Taken together, these findings suggest that treatment with SFZYD ameliorated obesity-induced systemic inflammation and hepatic steatosis by regulating inflammatory cytokine and adipokine levels in the circulation and various tissues. Moreover, treatment with SFZYD also reversed alterations in the levels of metabolites of the TCA cycle, the pentose phosphate pathway and aromatic amino acid metabolism.

Direct analysis of aberrant glycosylation on haptoglobin in patients with gastric cancer.

Based on our previous studies, differential analysis of N-glycan expression bound on serum haptoglobin reveals the quantitative variation on gastric cancer patients. In this prospective case-control study, we explore the clinically relevant glycan markers for gastric cancer diagnosis. Serum samples were collected from patients with gastric cancer (n = 44) and healthy control (n = 44). N-glycans alteration was monitored by intact analysis of Hp using liquid chromatography-mass spectrometry followed by immunoaffinity purification with the serum samples. Intensity and frequency markers were defined depending on the mass spectrometry data analysis. Multiple markers were found with high diagnostic efficacy. As intensity markers (I-marker), six markers were discovered with the AUC > 0.8. The high efficiency markers exhibited AUC of 0.93 with a specificity of 86% when the sensitivity was set to 95%. We additionally established frequency marker (f-marker) panels based on the tendency of high N-glycan expression. The AUC to conclude patients and control group were 0.82 and 0.79, respectively. This study suggested that N-glycan variation of serum haptoglobin were associated with patients with gastric cancer and might be a promising marker for the cancer screening.

Glycomic profiling of targeted serum haptoglobin for gastric cancer using nano LC/MS and LC/MS/MS.

Gastric cancer has one of the highest cancer mortality rates worldwide, largely because of difficulties in early-stage detection. Aberrant glycosylation in serum proteins is associated with many human diseases including inflammation and various types of cancer. Serum-based global glycan profiling using mass spectrometry has been explored and has already led to several potential glycan markers for several disease states. However, localization of the aberrant glycosylation is desirable in order to improve the specificity and sensitivity for clinical use. Here, we combined protein-specific immunoaffinity purification, glycan release, and MS analysis to examine haptoglobin glycosylation of gastric cancer patients for glyco-markers. Age- and sex-matched 60 serum samples (30 cancer patients and 30 healthy controls) were used to profile and quantify haptoglobin N-glycans. A T-test based statistical analysis was performed to identify potential glyco-markers for gastric cancer. Interestingly, abundances of several tri- and tetra-antennary fucosylated N-glycans were increased in gastric cancer patients. Additionally, structural analysis via LC/MS/MS indicated that the fucosylated complex type N-glycans were primarily decorated with antenna fucose, which can be categorized as sialyl-Lea or sialyl-Lex type structures. This platform demonstrates quantitative, structure-specific profiling of haptoglobin glycosylation for the purposes of biomarker discovery for gastric cancer.

Analytical detection and characterization of biopharmaceutical glycosylation by MS.

Glycosylation plays an important role in ensuring the proper structure and function of most biotherapeutic proteins. Even small changes in glycan composition, structure, or location can have a drastic impact on drug safety and efficacy. Recently, glycosylation has become the subject of increased focus as biopharmaceutical companies rush to create not only biosimilars, but also biobetters based on existing biotherapeutic proteins. Against this backdrop of ongoing biopharmaceutical innovation, updated methods for accurate and detailed analysis of protein glycosylation are critical for biopharmaceutical companies and government regulatory agencies alike. This review summarizes current methods of characterizing biopharmaceutical glycosylation, including compositional mass profiling, isomer-specific profiling and structural elucidation by MS and hyphenated techniques.

Effects of fibrin-binding oligopeptide on osteopromotion in rabbit calvarial defects.

PURPOSE: Fibronectin (FN) has been shown to stimulate bone regeneration in animal models. The aim of this study was to evaluate the capacity of bovine bone mineral coated with synthetic oligopeptides to enhance bone regeneration in rabbit calvarial defects. METHODS: Oligopeptides including fibrin-binding sequences of FN repeats were synthesized on the basis of primary and tertiary human plasma FN structures. Peptide coated and uncoated bone minerals were implanted into 10 mm calvarial defects in New Zealand white rabbits, and the animals were sacrificed at 4 or 8 weeks after surgery. After specimens were prepared, histologic examination and histomorphometric analysis were performed. RESULTS: At 4 weeks after surgery, the uncoated groups showed a limited amount of osteoid formation at the periphery of the defect and the oligopeptide coated groups showed more osteoid formation and new bone formation in the center of the defect as well as at the periphery. At 8 weeks, both sites showed increased new bone formation. However, the difference between the two sites had reduced. CONCLUSIONS: Fibrin-binding synthetic oligopeptide derived from FN on deproteinized bovine bone enhanced new bone formation in rabbit calvarial defects at the early healing stage. This result suggests that these oligopeptides can be beneficial in reconstructing oral and maxillofacial deformities or in regenerating osseous bone defects.

Luteolin suppresses cisplatin-induced apoptosis in auditory cells: possible mediation through induction of heme oxygenase-1 expression.

Luteolin has been shown to possess antitumorigenic, antioxidant, and anti-inflammatory properties. In the present study, we investigated the protective mechanism of luteolin against cisplatin-induced apoptosis in auditory (House Ear Institute-Organ of Corti 1 [HEI-OC1]) cells. Luteolin was found to induce the expression of heme oxygenase-1 (HO-1) in a dose- and time-dependent manner. Luteolin also activated the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase pathway, which plays an important role in the expression of HO-1. Luteolin protected the cells against cisplatin-induced apoptotic cell death. The protective effect of luteolin was abrogated by zinc protoporphyrin IX (ZnPP IX), an HO inhibitor, and antisense oligodeoxynucleotides against the HO-1 gene. Furthermore, pretreatment with luteolin inhibited the activation of caspase-3 and the mitochondrial dysfunction, and the effect of luteolin on the activation of caspase-3 disappeared in the presence of ZnPP IX or PD098059. These results demonstrate that the expression of HO-1 by luteolin is mediated by the ERK pathway, and also that the activating of HO-1 inhibits cisplatin-induced apoptosis in HEI-OC1 1 cells.

Brazilin and the extract from Caesalpinia sappan L. protect oxidative injury through the expression of heme oxygenase-1.

In this study, we examined the protective effects of Caesalpinia sappan L. and its major component, brazilin, against tert-butylhydroperoxide (t-BHP)-induced cell death in House Ear Institute-Organ of Corti 1 (HEI-OC1) cells. We found that the extract of C. sappan L. and brazilin induced antioxidant response element (ARE)-luciferase activity and heme oxygenase-1 (HO-1) expression in a concentration-dependent manner. The inductive effect of brazilin was more potent than the extract of C. sappan L. and the expression of HO-1 reached a peak at 12 h after brazilin treatment. The extract and brazilin protected the cells against t-BHP-induced cell death. Their protective effects were abrogated by zinc protoporphyrin IX (ZnPP IX), a HO inhibitor. These results demonstrate that the extract of C. sappan L. and brazilin induce the expression of HO-1 and the enzyme diminishes t-BHP-induced cell death in HEI-OC1 cells.