Polymorphisms in B3GAT1, SLC9A9 and MGAT5 are associated with variation within the human plasma N-glycome of 3533 European adults.

The majority of human proteins are post-translationally modified by covalent addition of one or more complex oligosaccharides (glycans). Alterations in glycosylation processing are associated with numerous diseases and glycans are attracting increasing attention both as disease biomarkers and as targets for novel therapeutic approaches. Using a recently developed high-throughput high-performance liquid chromatography (HPLC) analysis method, we have reported, in a pilot genome-wide association study of 13 glycan features in 2705 individuals from three European populations, that polymorphisms at three loci (FUT8, FUT6/FUT3 and HNF1A) affect plasma levels of N-glycans. Here, we extended the analysis to 33 directly measured and 13 derived glycosylation traits in 3533 individuals and identified three novel gene association (MGAT5, B3GAT1 and SLC9A9) as well as replicated the previous findings using an additional European cohort. MGAT5 (meta-analysis association P-value = 1.80 × 10(-10) for rs1257220) encodes a glycosyltransferase which is known to synthesize the associated glycans. In contrast, neither B3GAT1 (rs7928758, P = 1.66 × 10(-08)) nor SLC9A9 (rs4839604, P = 3.50 × 10(-13)) had previously been associated functionally with glycosylation of plasma proteins. Given the glucuronyl transferase activity of B3GAT1, we were able to show that glucuronic acid is present on antennae of plasma glycoproteins underlying the corresponding HPLC peak. SLC9A9 encodes a proton pump which affects pH in the endosomal compartment and it was recently reported that changes in Golgi pH can impair protein sialylation, giving a possible mechanism for the observed association.

Human plasma glycome in attention-deficit hyperactivity disorder and autism spectrum disorders.

Over a half of all proteins are glycosylated, and their proper glycosylation is essential for normal function. Unfortunately, because of structural complexity of nonlinear branched glycans and the absence of genetic template for their synthesis, the knowledge about glycans is lagging significantly behind the knowledge about proteins or DNA. Using a recently developed quantitative high throughput glycan analysis method we quantified components of the plasma N-glycome in 99 children with attention-deficit hyperactivity disorder (ADHD), 81 child and 5 adults with autism spectrum disorder, and a total of 340 matching healthy controls. No changes in plasma glycome were found to associate with autism spectrum disorder, but several highly significant associations were observed with ADHD. Further structural analysis of plasma glycans revealed that ADHD is associated with increased antennary fucosylation of biantennary glycans and decreased levels of some complex glycans with three or four antennas. The design of this study prevented any functional conclusions about the observed associations, but specific differences in glycosylation appears to be strongly associated with ADHD and warrants further studies in this direction.

High throughput isolation and glycosylation analysis of IgG-variability and heritability of the IgG glycome in three isolated human populations.

All immunoglobulin G molecules carry N-glycans, which modulate their biological activity. Changes in N-glycosylation of IgG associate with various diseases and affect the activity of therapeutic antibodies and intravenous immunoglobulins. We have developed a novel 96-well protein G monolithic plate and used it to rapidly isolate IgG from plasma of 2298 individuals from three isolated human populations. N-glycans were released by PNGase F, labeled with 2-aminobenzamide and analyzed by hydrophilic interaction chromatography with fluorescence detection. The majority of the structural features of the IgG glycome were consistent with previous studies, but sialylation was somewhat higher than reported previously. Sialylation was particularly prominent in core fucosylated glycans containing two galactose residues and bisecting GlcNAc where median sialylation level was nearly 80%. Very high variability between individuals was observed, approximately three times higher than in the total plasma glycome. For example, neutral IgG glycans without core fucose varied between 1.3 and 19%, a difference that significantly affects the effector functions of natural antibodies, predisposing or protecting individuals from particular diseases. Heritability of IgG glycans was generally between 30 and 50%. The individual's age was associated with a significant decrease in galactose and increase of bisecting GlcNAc, whereas other functional elements of IgG glycosylation did not change much with age. Gender was not an important predictor for any IgG glycan. An important observation is that competition between glycosyltransferases, which occurs in vitro, did not appear to be relevant in vivo, indicating that the final glycan structures are not a simple result of competing enzymatic activities, but a carefully regulated outcome designed to meet the prevailing physiological needs.

Genomics meets glycomics-the first GWAS study of human N-Glycome identifies HNF1α as a master regulator of plasma protein fucosylation.

Over half of all proteins are glycosylated, and alterations in glycosylation have been observed in numerous physiological and pathological processes. Attached glycans significantly affect protein function; but, contrary to polypeptides, they are not directly encoded by genes, and the complex processes that regulate their assembly are poorly understood. A novel approach combining genome-wide association and high-throughput glycomics analysis of 2,705 individuals in three population cohorts showed that common variants in the Hepatocyte Nuclear Factor 1α (HNF1α) and fucosyltransferase genes FUT6 and FUT8 influence N-glycan levels in human plasma. We show that HNF1α and its downstream target HNF4α regulate the expression of key fucosyltransferase and fucose biosynthesis genes. Moreover, we show that HNF1α is both necessary and sufficient to drive the expression of these genes in hepatic cells. These results reveal a new role for HNF1α as a master transcriptional regulator of multiple stages in the fucosylation process. This mechanism has implications for the regulation of immunity, embryonic development, and protein folding, as well as for our understanding of the molecular mechanisms underlying cancer, coronary heart disease, and metabolic and inflammatory disorders.

Extending the Protection Ability and Life Cycle of Medical Masks through the Washing Process.

The reuse of decontaminated disposable medical face masks can contribute to reducing the environmental burden of discarded masks. This research is focused on the effect of household and laboratory washing at 50 °C on the quality and functionality of the nonwoven structure of polypropylene medical masks by varying the washing procedure, bath composition, disinfectant agent, and number of washing cycles as a basis for reusability. The barrier properties of the medical mask were analyzed before and after the first and fifth washing cycle indirectly by measuring the contact angle of the liquid droplets with the front and back surface of the mask, further by measuring air permeability and determining antimicrobial resistance. Additional analysis included FTIR, pH of the material surface and aqueous extract, as well as the determination of residual substances-surfactants-in the aqueous extract of washed versus unwashed medical masks, while their aesthetic aspect was examined by measuring their spectral characteristics. The results showed that household washing had a stronger impact on the change of some functional properties, primarily air permeability, than laboratory washing. The addition of the disinfectant agent, didecyldimethylammonium chloride, contributes to the protective ability and supports the idea that washing of medical masks under controlled conditions can preserve barrier properties and enable reusability.

Screening novel biomarkers for metabolic syndrome by profiling human plasma N-glycans in Chinese Han and Croatian populations.

N-glycans play an essential role in biological process and are associated with age, gender, and body mass parameters in Caucasian populations, whereas no study has been reported in Chinese populations. To investigate the correlation between N-glycan structures and metabolic syndrome (MetS) components, we conducted a population-based study in 212 Chinese Han individuals. The replication study was performed on 520 unrelated individuals from a Croatian island Korcula. The most prominent observation was the consistent positive correlations between different forms of triantennary glycans and negative correlations between glycans containing core-fucose with MetS components including BMI, SBP, DBP, and fasting plasma glucose (FPG) simultaneously. Significant differences in a number of N-glycan traits were also detected between normal and abnormal groups of BMI, BP, and FPG, respectively. In the multivariate analysis, the level of monosialylated glycans (structure loadings = -0.776) was the most correlated with the MetS related risk factors, especially with SBP (structure loadings = 0.907). Results presented here are showing that variations in the composition of the N-glycome in human plasma could represent the alternations of human metabolism and could be potential biomarkers of MetS.

High throughput plasma N-glycome profiling using multiplexed labelling and UPLC with fluorescence detection.

A rapid glycomic profiling method is described wherein N-glycans from plasma samples individually labelled with aniline, 2-aminobenzamide and 2-aminoacridone are mixed, co-injected and separated in the same HILIC-fluorescence run. Transfer of the multiplexed method to UPLC-fluorescence permits an increase in sample throughput from 24 to 864 plasma samples per day.

Angiomyoma--angioleiomyoma of the cheek.

The authors present the case of an angiomyoma--angioleiomyoma of the cheek in a 58-year-old man. The tumour was palpable, although clinically not visible, and the only case of a tumour of smooth muscle treated in the Clinical Department of Oral Surgery over the last 40 years. The operation was performed in the Outpatient Department by intraoral procedure. The postoperative course passed without complications. Current literature on leiomyomas is cited in the Introduction, followed by presentation of the case and histological characteristics of the tumour The example is presented as a rarity and one of the differential diagnostic possibilities in the diagnostics of soft tissue tumours in the oral cavity.

Effects of aging, body mass index, plasma lipid profiles, and smoking on human plasma N-glycans.

Protein glycosylation affects nearly all molecular interactions at the cell surface and in the intercellular space. Many of the physiological variations which are part of homeostatic mechanisms influence glycosylation. However, a comprehensive overview of changes in glycosylation caused by aging and common lifestyle parameters is still lacking. After analyzing N-glycans in the plasma of 1914 individuals from the Croatian islands of Vis and Korcula, we performed a comprehensive analysis of the dependence of different glycosylation features (position of fucose, level of galactosylation, sialylation and branching) on aging, smoking, body fat and plasma lipid status. A number of statistically significant associations were observed. Glycosylation changes with aging were especially evident in females, mostly in association with the transition from pre-menopausal to post-menopausal age. Levels of core-fucosylated, non-galactosylated, digalactosylated and disialylated biantennary glycans were shown to be mainly age dependent, but the level of branching and higher levels of galactosylation were found to correlate with lipid status. For the majority of glycans which we analyzed, all examined parameters explained up to 5% of the variance. The only notable exception were non-galactosylated glycans where 20% of the variance was explained mostly by age and blood pressure. In general, only a small fraction of the variability in glycan levels observed in a population was explained by age and other measured parameters, indicating that even in the absence of a genetic template, glycan levels are mostly determined by genetic background and/or specific pathophysiological processes.

Common aberrations from the normal human plasma N-glycan profile.

After performing hydrophilic interaction and weak anion exchange high-performance liquid chromatography to analyze N-glycans in the plasma of 1991 people, we identified several individuals that differed significantly from the "normal" profile of N-glycans. By performing consensus scoring of pairwise distances between vectors containing measured glycan values, we formed six groups of individuals with specific glyco-phenotypes. Some aberrations from the normal plasma protein patterns were found to be associated with clinical conditions (such as renal problems in people with increased monosialylated biantennary glycans, A2G2S1), while other substantial changes in N-glycan structure, such as the near complete absence of neutral glycans or antennary fucosylated tri- and tetraantennary glycans, were not associated with any observed adverse health outcomes. These results demonstrate the existence of specific altered glyco-phenotypes in some individuals and indicate that in some cases they might represent risk factors for the development of specific diseases.

High-throughput glycoanalytical technology for systems glycobiology.

The development of glycoanalytical HPLC-based high-throughput technology has greatly enhanced the study of glycobiology, facilitating the discovery of disease-related solutions and providing an informative view of glycosylation and its relationship with other biological disciplines in a systems biology approach.

Health condition of first permanent molars in year 1977 and 2007 in children in Istria (Croatia).

The aim of this study was to examine differences between health condition of the first permanent molar (M1) in children in 1977 and 2007. The materials for the study consisted of data on the health condition of M1 determined in a study in 1977 (Group I) for children from the district of Buje in Istria. The health condition of M1 was examined again in the same area in 2007 (Group II). The first permanent molar is most frequently affected by caries and represent a good indicator for general caries incidence of children. Study included 709 subjects in Group I (363 boys, 346 girls) and 460 subjects in Group II (242 boys, 218 girls), aged from 6 years and 0 months to 12 years and 5 months. The difference in the frequency of intact, decayed, filled and missingd M1 was examined in both groups. Chi2 test was used to determine the differences between the number of I (intact), D (decayed), F (filled) and M (missing) teeth for each age group in Group I and Group II. In Group I there were 29.3% intact, 48.9% decayed, 17.4% filled and 4.3% missing M1, and in Group II there were 53.0% intact, 22.6% decayed, 22.1% filled and 2.1% missing M1. During the period of 30 years, a significant increase of number of dental surgeries, and thus better preventive and health education, resulted in the significant increase in the number of intact (24.0%) and filled (4.7%) M1, and decrease in the number of decayed (26.3%) and missing (2.2%) M1. From 1977 to 2007, the number of intact M1 in group II increased considerably according to group I, while the number of decayed M1 in group II significant decreased according to group I. These changes were the result of a considerably increased number of dental surgeries.

Stability of N-glycan profiles in human plasma.

Glycan heterogeneity was shown to be associated with numerous diseases and glycan analysis has a great diagnostic potential. Recently, we reported high biological variability of human plasma N-glycome at the level of population. The observed variations were larger than changes reported to be associated with some diseases; thus, it was of great importance to examine the temporal constancy of human N-glycome before glycosylation changes could be routinely analyzed in diagnostic laboratories. Plasma samples were taken from 12 healthy individuals. The blood was drawn on seven occasions during 5 days. N-Linked glycans, released from plasma proteins, were separated using hydrophilic interaction high-performance liquid chromatography into 16 groups (GP1-GP16) and quantified. The results showed very small variation in all glycan groups, indicating very good temporal stability of N-glycome in a single individual. Coefficients of variation from 1.6% for GP8 to 11.4% for GP1 were observed. The average coefficient of variation was 5.6%. These variations were comparable to those observed when analytical procedure was tested for its precision. Good stability of plasma N-glycome in healthy individuals implies that glycosylation is under significant genetic control. Changes observed in glycan profiles are consequence of environmental influences and physiologic responses and therefore have a significant diagnostic potential.

Comparison of Er:YAG and Er,Cr:YSGG Laser in the Treatment of Oral Leukoplakia Lesions Refractory to the Local Retinoid Therapy.

Objective: The aim of this study was to compare the efficacy of Er:YAG and Er,Cr:YSGG laser in the treatment of oral leukoplakia refractory to conventional retinoid therapy. Materials and methods: The study sample consisted of 54 patients (16 men and 38 women) who were histopathologically diagnosed with oral leukoplakia that was refractory to conventional retinoid therapy. Patients were randomly allocated into two groups according to the type of the laser used for treatment of oral leukoplakia: Group 1. Er:YAG laser; Group 2. Er,Cr:YSGG laser. Patients were recalled at 6 months and 1 year after treatment to evaluate possible recurrence and assess the patients' postoperative quality of life. Results: After initial ablation, the degree of residual lesion was significantly greater in the Er:YAG laser group (74.1%), compared with the Er,Cr:YSGG group (18.5%) (p = 0.0001). Six months and 1 year after the second ablation, there was no lesion recurrence in either laser group. Fourteen days after the initial ablation, the visual analog scale (VAS) pain rating and the total oral health impact profile score fell significantly in both groups (p < 0.0001). However, in the Er,Cr:YSGG laser group, the average value of the VAS rating was significantly lower than in the Er:YAG laser group (p = 0.039). Conclusions: The Er:YAG and Er,Cr:YSGG lasers showed similar efficacy in the treatment of oral leukoplakia and resulted in full postoperative recovery without recurrence after 1 year of follow-up.

Changes of mandibular dental arch shape during adolescence and its influence on late mandibular incisor crowding.

The aim was to analyze the changes in mandibular dental arch shape during adolescence and assess its relation to late mandibular incisor crowding. Longitudinal study included 68 orthodontically untreated subjects (49% female) and analyzed their data for the ages of 12, 15, 18 and 21 years. Measurements included anterior arch depth, intercanine, interpremolar/anterior and intermolar/posterior width, Little's Irregularity and Bolton's index and the ratio between anterior arch depth and width. Males had significantly greater posterior widths than females at any age (p < 0.05). The anterior arch depth continuously decreased (p < 0.05), while width increased after the age of 18 years. Mandibular incisor crowding increased during all investigated periods (p < 0.05). The increase of intercanine width at 12-21 years of age reduced the risk for mandibular incisor crowding in the same period by 74% (OR: 0.265: 95% CI 0.076-0.931; p = 0.045). The shape of mandibular dental arch continues to change during adolescence becoming more squared while mandibular incisor crowding increases. The increase in mandibular intercanine width reduces the risk of crowding.

Idiopathic Exposed Bone Lesions of the Jaw.

INTRODUCTION: Osteonecrosis of the jaw is defined as exposed bone in the oral cavity that does not heal longer than eight weeks after identification. The two most common predisposing factors for osteonecrosis of the jaw are medication-related and radiotherapy. Rarely, exposed bone in the maxillofacial region can occur due to other causes and represents a clinical and therapeutic challenge for the dentist because there is no universally accepted treatment protocol. CASE PRESENTATION: We report a case of a patient with two idiopathic lesions of exposed bone which have healed after systemic antibiotic therapy, seven weeks after the first examination. CONCLUSION: Exposed bone lesions of the jaw are a rare entity and are poorly documented in the literature. It is necessary to exclude possible local or systemic contributing factors. Surgical and conservative therapy (antibiotics) are the treatment of choice.

Clinical and Radiographic Assessment of Cases Referred to Endodontic Surgery.

OBJECTIVE: The objective of the study was to assess the quality of root canal fillings of cases referred to endodontic surgery using preoperative radiographs and correlate it with endodontic surgery treatment decision. The objective was also to analyse clinical symptoms and size of periapical lesions on radiographs and correlate them with treatment decisions including non-surgical retreatment, endodontic surgery and extraction. MATERIALS AND METHODS: A questionnaire was composed to record the data. Eighty-six patients with 109 teeth, who were referred to endodontic surgery, participated in the research. The quality of root canal filling was assessed according to its homogeneity and filling length on digital radiographs. The data were analyzed using χ2-test and t-test. RESULTS: Of the teeth referred to endodontic surgery, 97.2% were treated by a general practice dentist, endodontic retreatment was attempted in 20.6%, and root canal filling was homogeneous and within 1 mm from the apex in 21.6%. Endodontic surgery, retreatment, extraction and no treatment were selected in 90.1%, 5.4%, 1.8% and 2.7% of the cases, respectively. CONCLUSIONS: Low percentage of adequate root canal fillings and high percentage of endodontic surgery decisions suggest that there is a need to increase awareness of non-surgical retreatment options.

Glycomics meets lipidomics--associations of N-glycans with classical lipids, glycerophospholipids, and sphingolipids in three European populations.

Recently, high-throughput technologies have been made available which allow the measurement of a broad spectrum of glycomics and lipidomics parameters in many samples. The aim of this study was to apply these methods and investigate associations between 46 glycan and 183 lipid traits measured in blood of 2041 Europeans from three different local populations (Croatia - VIS cohort; Sweden - NSPHS cohort; Great Britain - ORCADES cohort). N-glycans have been analyzed with High Performance Liquid Chromatography (HPLC) and lipids with Electrospray Ionization Tandem Mass Spectrometry (ESI-MS/MS) covering sterol lipids, glycerolipids, glycerophospholipids and sphingolipids in eight subclasses. Overall, 8418 associations were calculated using linear mixed effect models adjusted for pedigree, sex, age and multiple testing. We found 330 significant correlations in VIS. Pearson's correlation coefficient r ranged from -0.27 to 0.34 with corresponding p-values between 1.45 × 10(-19) and 4.83 × 10(-6), indicating statistical significance. A total of 71 correlations in VIS could be replicated in NSPHS (r = [-0.19; 0.35], p = [4.16 × 10(-18); 9.38 × 10(-5)]) and 31 correlations in VIS were also found in ORCADES (r = [-0.20; 0.24], p = [2.69 × 10(-10); 7.55 × 10(-5)]). However, in total only 10 correlations between a subset of triantennary glycans and unsaturated phosphatidylcholine, saturated ceramide, and sphingomyelin lipids in VIS (r = [0.18; 0.34], p = [2.98 × 10(-21); 1.69 × 10(-06)]) could be replicated in both NSPHS and ORCADES. In summary, the results show strong and consistent associations between certain glycans and lipids in all populations, but also population-specific correlations which may be caused by environmental and genetic differences. These associations point towards potential interactive metabolic pathways.

Variability, heritability and environmental determinants of human plasma N-glycome.

Plasma glycans were analyzed in 1008 individuals to evaluate variability and heritability, as well as the main environmental determinants that affect glycan structures. By combining HPLC analysis of fluorescently labeled glycans with sialidase digestion, glycans were separated into 33 chromatographic peaks and quantified. A high level of variability was observed with the median ratio of minimal to maximal values of 6.17 and significant age- and gender-specific differences. Heritability estimates for individual glycans varied widely, ranging from very low to very high. Glycome-wide environmental determinants were also detected with statistically significant effects of different variables including diet, smoking and cholesterol levels.