Levels Of Galactose Research Articles

Abstract P1172: Shear-mediated Remodeling Of Platelet Surface Glycosylation: How Can Platelets Trim Their Sugar Coats?

Mechanical circulatory support (MCS) is associated with bleeding complications often requiring multiple transfusions or reoperation. Hypershear stress within device-supported circulation promotes platelet dysfunction associated with pro-apoptosis, impaired aggregation, and microvesiculation - all contributors to bleeding. Recent studies showed that glycosylation of surface receptors, i.e. “ sugar coat ,” plays a major role in the regulation of platelet function and lifespan. We tested the hypothesis that shear stress promotes platelet surface deglycosylation by increasing platelet glycosidase activity. Methods: Human platelets were obtained from ACD-anticoagulated blood via gel filtration on Sepharose 2B. Platelets were exposed to neuraminidase and continuous shear stress in a hemodynamic shearing device. Platelets were stained with fluorophore-conjugated lectins binding terminal sialic acids (SNA I, MAL II) and galactose (ECA), anti-CD41 and anti-CD42a a/b. Multi-colored flow cytometry was used to quantify platelet surface glycosylation. Neuraminidase and galactosidase activities of intact and lysed platelets were measured using enzyme-specific fluorogenic substrates. Results: Exposure to shear stress and neuraminidase induced a significant decrease in platelet 2,6-sialylation and galactosylation, while 2,3-sialylation remained unaltered. Neuraminidase reinforced shear-mediated platelet desialylation, causing a prominent decrease in SNA binding even at low shear, and increased platelet galactose levels by unmasking the galactose residues capped by sialic acids. Shear stress resulted in a 9.5-fold increase in platelet neuraminidase and a 1.4-fold increase in galactosidase activities vs their basal levels. Neutral neuraminidase and galactosidase were upregulated by shear, while acidic isoforms did not increase, suggesting that other than lysosomal glycosidase pools are involved. Shear stress promotes platelet deglycosylation via upregulation of platelet neuraminidase and galactosidase activities. Developing therapeutic strategies for the preservation of platelet glycosylation offers significant translational potential for the pharmacologic management of MCS platelet dysfunction and bleeding.

Artificial neural network model- and response surface methodology-based optimization of Atractylodis Macrocephalae Rhizoma polysaccharide extraction, kinetic modelling and structural characterization

Atractylodis Macrocephalae Rhizoma (AMR) is the dried rhizome of Atractylodes macrocephala Koidz, which is widely used in the development of health products. AMR contains a large number of polysaccharides, but at present there are fewer applications for these polysaccharides. In this study, the effects of different extraction methods on the Atractylodis Macrocephalae Rhizoma polysaccharide (AMRP) yield were investigated, and the conditions for ultrasound-assisted extraction were optimized by response surface methodology (RSM) and three neural network models (BP neural network, GA-BP neural network and ACO-GA-BP neural network). The best conditions were a liquid-to-solid ratio of 17 mL/g, ultrasonic power of 400 W, extraction temperature of 72 °C, and extraction time of 40 min, which yielded 31.31% AMRP. The kinetic equation of AMRP was determined and compared with the results predicted by three neural network models. It was finally determined that the extraction conditions, kinetic processes and kinetic equation predicted by the GA-ACO-BP neural network were optimal. In addition, AMRP was characterized using SEM, FTIR, HPLC, UV, XRD, and NMR, and the structural study revealed that AMRP has a rough exterior and a porous interior; moreover, it contains high levels of glucose (5.07%), arabinose (0.80%), and galactose (0.74%). AMRP has three crystal structures, consisting of two β-type monosaccharides and one α-type monosaccharide. Additionally, the effectiveness of AMRP as an antioxidant was demonstrated in an in vitro experiment.

The Morpho-Anatomy of Nectaries and Chemical Composition of Nectar in Pear Cultivars with Different Susceptibility to Erwinia amlylovora

The topography and morpho-anatomical characteristics of floral nectaries and the chemical analysis of nectar have been studied in seven pear cultivars with different susceptibility to Erwinia amylovora. The susceptible cultivar Williams, the moderately resistant cultivars Bella di guigno, Poire de Cure and the low susceptible cultivar Alexander Lucas originated from Pyrus communis, while highly resistant cultivars Chojuro and Nijisseiki from P. pyraster and Kieffer as interspecies hybrid P. communis × P. pyraster were included in this experiment and studied for the first time. Large differences in size and structure of the nectaries were observed in these seven pear cultivars. The epidermal cells were with cuticle being more or less striated in Alexander Lucas, Kieffer and Williams. Resistant cultivars had a narrow, elongated cell shape of epidermal cells while those susceptible had an isodiametric. Stomata were mesomorphic in all cultivars except in Poire de Cure and Williams, being slightly xeromorphic since they were situated in deep hollows. Guard cells of the modified stomata were much larger in resistant cultivars. Hypanthium cells were larger in resistant compared to susceptible cultivars. The most abundant sugars were glucose, fructose, sorbitol and sucrose in nectar of all studied pear cultivars. The resistant cultivars (Chojuro, Kieffer and Nijisseiki) had a ~2-fold higher level of sorbitol and galactose, ~2.2-fold higher isomaltose, ~2.7-fold turanose, ~3.35-fold maltose, ~4.4-fold melibiose and ~12.7-fold higher melesitose compared to susceptible cultivars. The sum of quantified phenolic acids varied from 0.049 (Williams) up to 4.074 µg CAE/mL (Kieffer), while flavonoid glycosides levels ranged from 1.224 (Williams) up to 11.686 µg RE/mL (Nijisseiki). In the nectar of the resistant cultivars, rutin, apigetrin, together with patuletin and luteolin glycosides were detected but not in susceptible cultivars, which could be considered as the markers of resistance.

Association of the serum IgG glycosylation with disease activity of anti-transcription intermediary factor 1 gamma positive dermatomyositis.

Dermatomyositis (DM) is an idiopathic inflammatory myopathy characterised by the presence of a variety of myositis-specific autoantibodies (MSA) in the circulation. As one of the commonly-detected MSA, anti-transcription intermediary factor 1 gamma (TIF1γ) autoantibody is strongly associated with DM-related malignancy and disease activity. We investigated the glycosylation patterns of serum IgG and to determine the clinical significance of specific glycosylation patterns in patients with anti-TIF1γ positive DM. Lectin microarray was used to reveal the glycosylation patterns of serum IgG among 52 DM, 46 disease controls (DC) and 49 healthy controls (HC). Lectin blot was used to further validate the specific alteration of glycosylation. The correlation between glycan levels and clinical features was also evaluated. The results of lectin microarray showed that compared with the DC group, the DM group had significantly lower glycan levels of mannose and glucose. Compared with the HC group, the glycans levels of GalNAc, galactose, Galβ3GalNAc, sialic acid, and fucose were observed significantly higher in DM group. Lectin blot demonstrated that anti-TIF1γ positive DM had lower glycan level of GlcNAc (recognized by LEL) compared to patients with MSA negative DM, DC, and HC groups. Additionally, the glycan level of GlcNAc was positively associated with manual muscle test (r=0.547, p=0.028), or negatively associated with IL-6 level (r=-0.756, p=0.049) and disease activity score (r=-0.507, p=0.045). Anti-TIF1γ positive DM presents a unique glycosylation pattern in serum IgG. Considering that the glycan level of GlcNAc reflects the inflammatory state and disease activity, glycosylation has a role in clinical utility by monitoring disease in patients with anti-TIF1γ positive DM.

Treatable cause of leukodystrophy: Galactosaemia

Galactosaemia is a group of autosomal recessive metabolic disorders characterised by increased blood levels of galactose. It is characterised by cataracts, organomegaly, sepsis and developmental delay. We are reporting a case of galactosaemia presenting as a neurodegenerative disease with a leukodystrophy-like presentation. An 11-month-old boy born to a second-degree consanguineously married couple presented with developmental delay, vomiting, lethargy and refusal of feeds. Examination showed normocephaly, cataract, hepatomegaly and spasticity in all limbs with exaggerated deep tendon reflexes. Magnetic resonance imaging (MRI) of the brain was suggestive of diffuse hyperintensities in periventricular, subcortical and deep white matter on T2WI. Plasma galactose levels were high (3784.0 u M/L) and the RBC Galactose 1-Phosphate uridylyltransferase (GALT) enzyme was low (<0.04). Genetic testing showed homozygous missense mutation c.428C>T (p.Ser143Leu) in exon 5 of the GALT gene. For any child who presents with cataracts, hepatomegaly, developmental delay and leukodystrophy picture on an MRI of the brain, a treatable cause of galactosaemia should be considered.

Glycoconjugate-Specific Developmental Changes in the Horse Vomeronasal Organ

The vomeronasal organ (VNO) is a tubular pheromone-sensing organ in which the lumen is covered with sensory and non-sensory epithelia. This study used immunohistochemistry and lectin histochemistry techniques to evaluate developmental changes, specifically of the glycoconjugate profile, in the horse VNO epithelium. Immunostaining analysis revealed PGP9.5 expression in some vomeronasal non-sensory epithelium (VNSE) cells and in the vomeronasal receptor cells of the vomeronasal sensory epithelium (VSE) in fetuses, young foals, and adult horses. Olfactory marker protein expression was exclusively localized in receptor cells of the VSE in fetuses, young foals, and adult horses and absent in VNSE. To identify the glycoconjugate type, lectin histochemistry was performed using 21 lectins. Semi-quantitative analysis revealed that the intensities of glycoconjugates labeled with WGA, DSL, LEL, and RCA₁₂₀ were significantly higher in adult horse VSE than those in foal VSE, whereas the intensities of glycoconjugates labeled with LCA and PSA were significantly lower in adult horse VSE. The intensities of glycoconjugates labeled with s-WGA, WGA, BSL-II, DSL, LEL, STL, ConA, LCA, PSA, DBA, SBA, SJA, RCA₁₂₀, jacalin, and ECL were significantly higher in adult horse VNSE than those in foal VNSE, whereas the intensity of glycoconjugates labeled with UEA-I was lower in adult horse VNSE. Histochemical analysis of each lectin revealed that various glycoconjugates in the VSE were present in the receptor, supporting, and basal cells of foals and adult horses. A similar pattern of lectin histochemistry was also observed in the VNSE of foals and adult horses. In conclusion, these results suggest that there is an increase in the level of N-acetylglucosamine (labeled by WGA, DSL, LEL) and galactose (labeled by RCA₁₂₀) in horse VSE during postnatal development, implying that they may influence the function of VNO in adult horses.

Comprehensive analysis of non-synonymous missense SNPs of human galactose mutarotase (GALM) gene: an integrated computational approach

Missense Non-synonymous single nucleotide polymorphisms (nsSNPs) of Galactose Mutarotase (GALM) are associated with the Novel type of Galactosemia (Galactosemia type 4) together with symptoms such as high blood galactose levels and eye cataracts. The objective of the present study was to identify deleterious nsSNPs of GALM recorded on the dbSNP database through comprehensive insilico analysis. Among the 319 missense nsSNPs reported, various insilco tools predicted R78S, R82G, A163E, P210S, Y281C, E307G and F339C as the most deleterious mutations. Structural analysis, PTM analysis and molecular dynamics simulations (MDS) were carried out to understand the effect of these mutations on the structural and physicochemical properties of the GALM protein. The residues R82G and E307G were found to be part of the binding site that resulted in decreased surface accessibility. Replacing the charged wild-type residue with a neutral mutant type affected its substrate binding. All 7 mutations were found to increase the rigidity of the protein structure, which is unfavorable during ligand binding. The mutation F339E made the protein structure more rigid than all the other mutations. Y281 is a phosphorylated site, and therefore, less significant structural changes were observed when compared to other mutations; however, it may have significant differences in the usual functioning of the protein. In summary, the structural and functional analysis of missense SNPs of GALM is important to reduce the number of potential mutations to be evaluated in vitro to understand the association with some genetic diseases. Communicated by Ramaswamy H. Sarma

Serum levels of degraded monosaccharides in children with Henoch-Schönlein purpura.

To examine the serum levels of degraded monosaccharides in children with Henoch-Schönlein purpura (HSP) and to study the clinical significance of degraded monosaccharides in HSP. A prospective analysis was performed on 132 children who were diagnosed with HSP from September 2019 to January 2022, and 132 healthy children were enrolled as the control group. High-performance liquid chromatography was used to determine the content of degraded monosaccharides in serum in both groups. The receiver operating characteristic (ROC) curve was used to evaluate the efficiency of degraded monosaccharides for the diagnosis of HSP. Compared with the control group, the HSP group had significantly higher serum levels of mannose, glucosamine, aminogalactose, and galactose (P<0.001). The four degraded monosaccharides had an area under the ROC curve of 0.919, 0.913, 0.832, and 0.932 respectively for the diagnosis of HSP (P<0.05). Children with HSP have higher serum levels of mannose, glucosamine, aminogalactose, and galactose than the healthy population. The levels of degraded monosaccharides may have an important value for the diagnosis of HSP.

Galactosemia: Biochemistry, Molecular Genetics, Newborn Screening, and Treatment.

Galactosemia is an inborn disorder of carbohydrate metabolism characterized by the inability to metabolize galactose, a sugar contained in milk (the main source of nourishment for infants), and convert it into glucose, the sugar used by the body as the primary source of energy. Galactosemia is an autosomal recessive genetic disease that can be diagnosed at birth, even in the absence of symptoms, with newborn screening by assessing the level of galactose and the GALT enzyme activity, as GALT defect constitutes the most frequent cause of galactosemia. Currently, galactosemia cannot be cured, but only treated by means of a diet with a reduced content of galactose and lactose. Although the diet is able to reverse the neonatal clinical picture, it does not prevent the development of long-term complications. This review provides an overview of galactose metabolism, molecular genetics, newborn screening and therapy of galactosemia. Novel treatments for galactosemia currently being investigated in (pre)clinical studies and potentially able to prevent long-term complications are also presented.

OsPP65 Negatively Regulates Osmotic and Salt Stress Responses Through Regulating Phytohormone and Raffinose Family Oligosaccharide Metabolic Pathways in Rice

Although type 2C protein phosphatases (PP2Cs) have been demonstrated to play important roles in regulating plant development and various stress responses, their specific roles in rice abiotic stress tolerance are still largely unknown. In this study, the functions of OsPP65 in rice osmotic and salt stress tolerance were investigated. Here, we report that OsPP65 is responsive to multiple stresses and is remarkably induced by osmotic and salt stress treatments. OsPP65 was highly expressed in rice seedlings and leaves and localized in the nucleus and cytoplasm. OsPP65 knockout rice plants showed enhanced tolerance to osmotic and salt stresses. Significantly higher induction of genes involved in jasmonic acid (JA) and abscisic acid (ABA) biosynthesis or signaling, as well as higher contents of endogenous JA and ABA, were observed in the OsPP65 knockout plants compared with the wild-type plants after osmotic stress treatment. Further analysis indicated that JA and ABA function independently in osmotic stress tolerance conferred by loss of OsPP65. Moreover, metabolomics analysis revealed higher endogenous levels of galactose and galactinol but a lower content of raffinose in the OsPP65 knockout plants than in the wild-type plants after osmotic stress treatment. These results together suggest that OsPP65 negatively regulates osmotic and salt stress tolerance through regulation of the JA and ABA signaling pathways and modulation of the raffinose family oligosaccharide metabolism pathway in rice. OsPP65 is a promising target for improvement of rice stress tolerance using gene editing.

Congenital portosystemic venous shunt associated with 22q11.2 deletion syndrome: a case report

Background22q11.2 deletion syndrome (22qDS) is the most common chromosomal microdeletion syndrome and is associated with a high rate of congenital heart disease (CHD) and neurodevelopmental abnormalities. Congenital portosystemic venous shunts (CPSS) are rare developmental abnormalities of the portal venous system. The clinical manifestations of CPSS are varied, and some patients have CHD or genetic chromosomal abnormalities, but their relationship remains unknown. We report the first case of CPSS associated with 22qDS.Case presentationA newborn boy referred to our institution was diagnosed with 22qDS due to characteristic facial features and complications of tetralogy of Fallot. A subsequent newborn screening test indicated hypergalactosemia and high blood levels of ammonia and bile acids. Upon closer examination, these abnormalities were found to be caused by the CPSS. Abdominal contrast-enhanced computed tomography and angiography confirmed that abnormal blood vessels ascended from the splenic vein and short-circuited to the left renal vein. Intracardiac repair for CHD was performed at 1 year of age, followed by transcatheter occlusion of the CPSS using a multilayer device (vascular plug) and detachable coil at 2 years of age. After treatment, the abnormal blood parameters promptly normalized.ConclusionsAs the blood flow of CPSS bypasses the liver, the levels of galactose, bile acids, and ammonia in the systemic veins can increase. Some patients with CPSS have CHD, and these toxic substances may cause liver and lung lesions as well as portosystemic encephalopathy (PSE). Several genetic chromosomal abnormalities, including 22qDS, and CPSS have similar symptoms, and neurodevelopmental abnormalities, particularly those caused by PSE, may be difficult to diagnose. Blood tests, such as newborn screening, and abdominal imaging are useful in the early diagnosis of CPSS.

Terminal glycosylation pattern as a determinant of human iPSC-derived cardiomyocytes transduction with AAV vectors

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Young scientists grant MNS9/2021 Polish National Science Centre PRELUDIUM grant 2019/33/N/NZ1/03066 Introduction Although gene therapy has become a feasible alternative to regular treatment in numerous disorders, efficient targeting of human heart still seems out of reach. So far, adeno-associated viral vectors (AAVs) have been the most promising transgene carriers in in vivo applications. Transduction with AAVs relies on serotype-specific binding to the glycan residues on the cell surface. While AAV serotype 9 (AAV9) - binding galactose - was successfully used for delivery of therapeutic genes to cardiac muscle in murine models, its effect was underwhelming in large animals. Switching the serotype to AAV1 or AAV6 – binding sialic acids – enabled transduction of porcine hearts, and cardiomyocytes derived from human induced pluripotent stem cells (hiPSC) in 2D in vitro culture. But, as evidenced in clinical trials where less than 1% of heart cells contained delivered transgene, such strategy was not suitable for in vivo application in humans. Purpose Taking into account differences in glycosylation patterns between humans and other mammals, we aimed to investigate the role of surface glycans in AAV6 and AAV9 transduction mechanism in 2D and 3D cultures of human cardiomyocytes. Methods We generated hiPSC- derived cardiomyocytes (hiPSC-CMs) and epicardial fibroblasts (hiPSC-CFs). Cells were cultured in regular 2D conditions or 3D co-cultures (as spheroids), since direct cells interaction may influence the availability of terminal sugar residues. Surface glycans were stained with lectins and imaged using confocal microscope. Results While in 2D conditions AAV6 transduces hiPSC-CMs very efficiently, it is significantly less potent in 3D culture. Conversely, AAV9 maintains most of its functionality in 3D model. Our data collected from spheroids, indicate that cells in such 3D cultures undergo significant changes in terminal glycosylation pattern. While the level of galactose (AAV9 receptor) increased from day 1 to day 7, the signal from sialic acids attached to galactose (AAV6 receptor) decreased. This effect was accompanied by the upregulation of sialidase-3 expression, that regulates availability of galactose residues on the cell surface. As a result, transduction efficiency was improved in spheroids exposed to AAV9 on day 7 of 3D culture, in comparison to those transduced on day 1. Inhibition of sialidases activity with NADNA reduced the level of terminal galactose in both, hiPSC-CMs and hiPSC-CFs, and was associated with more efficient transduction of cardiomyocytes with AAV6, and less efficient transduction with AAV9. Additionally, we observed that galactosylation of collagen can be another factor that influences cell-vector interaction in 2D and 3D culture. Conclusions Our results demonstrate that the availability of terminal sugar residues is a key factor regulating transduction of hiPSC-CMs with AAV vectors, and underline the need for development of appropriate models for testing of AAV functionality in preclinical studies.

Physiological insights into enhanced lipid accumulation and temperature tolerance by Tetraselmis suecica ultraviolet mutants

High outdoor temperatures significantly inhibit the growth and lipid production of the industrially promising marine microalga Tetraselmis suecica, which is viewed as a potential feedstock for high-value bioproducts and biofuels. To overcome this limitation, T. suecica was subjected to ultraviolet irradiation to generate mutants capable of being productive at higher temperatures. The top two high-lipid mutants UV-25 and UV-31 isolated at 25 °C and 31 °C, respectively, were compared to the wild type (WT) to delineate physiological alterations and shed light on the mutants' increased biomass and lipid productivity. At 25 °C, UV-25 and UV-31 exhibited lipid productivity of 36.12 and 31.33 mg/L day, which were 1.4- and 1.2-fold higher than WT, respectively. This increase in lipid biosynthesis correlated well with increased carotenoid content in UV-25 (2.2-fold) and UV-31 (3.6-fold), indicating an improved capacity to quench reactive oxygen species. At 31 °C, the growth and lipid accumulation of UV-31 remained high, signifying adaptation to higher temperatures. This is attributed to a well-coordinated modulation of the mutant's cellular metabolism through an increase in galactose and phosphatidylglycerol levels, as well as in protein, all of which contributed to its performance at elevated temperatures. The study successfully established a UV mutagenesis strategy for producing superior- performing microalgae strains with industrially desired traits, paving the way for future outdoor cultivation deployment.

Association of galactose and insulin resistance in polycystic ovary syndrome: A case-control study

SummaryBackgroundPolycystic ovary syndrome (PCOS) is closely linked to metabolic disorders. Recent reports have identified galactose as having strong associations with metabolic disorders, however, the correlation between galactose and PCOS remains largely unknown.MethodsThe serum galactose levels of 104 patients with PCOS and 98 controls were measured, and their relationships with several metabolic parameters were analyzed. The study took place at the Center for Reproductive Medicine at Shengjing Hospital of China Medical University, Shenyang, China from July 13 to Oct 20, 2020. The relationships between serum galactose and PCOS as well as PCOS-related insulin resistance were investigated via logistic regression analyses, and the performance of serum galactose as a potential biomarker for PCOS was evaluated using receiver operating characteristic curve analysis.FindingsHigher serum galactose levels were observed in the patients with PCOS than in the controls (p = 0.001). There was still a correlation between circulating galactose levels and PCOS after adjusting for covariates (p = 0.002; odds ratio (OR), 1.133; 95% confidence interval (CI) 1.047–1.227). Serum galactose levels were shown to be most closely related to the fasting serum insulin level (r = 0.370, p = 0.001) and were higher in the insulin-resistant subgroup than in the non-insulin-resistant subgroup of patients with PCOS (p = 0.001). There was no difference in serum galactose levels between the insulin-resistant and non-insulin-resistant subgroups of women in the control group (p > 0.05). Furthermore, higher serum galactose levels were shown to be associated with insulin resistance in PCOS (p = 0.004; OR, 26.017; 95% CI, 2.907–232.810). The area under the curve for galactose-mediated prediction of PCOS was 80.0%, with a sensitivity of 71.0% and a specificity of 86.4%.InterpretationHigher circulating galactose levels correlate with PCOS and PCOS-related insulin resistance; therefore, it may serve as a potential biomarker for patients with PCOS. These findings require further validation in a study with a larger sample size.FundingNational Natural Science Foundation of China (No. 82,071,607 and 32,100,691); LiaoNing Revitalization Talents Program (No. XLYC1907071); Fok Ying Tung Education Foundation (No. 151,039); Key Research and Development Program of Liaoning Province (NO. 2,018,225,062); Outstanding Scientific Fund of Shengjing Hospital (No. 202,003).

Altered IgG glycosylation at COVID-19 diagnosis predicts disease severity.

The nature of the immune responses associated with COVID‐19 pathogenesis and disease severity, as well as the breadth of vaccine coverage and duration of immunity, is still unclear. Given the unpredictability for developing a severe/complicated disease, there is an urgent need in the field for predictive biomarkers of COVID‐19. We have analyzed IgG Fc N‐glycan traits of 82 SARS‐CoV‐2+ unvaccinated patients, at diagnosis, by nano‐LC‐ESI‐MS. We determined the impact of IgG Fc glyco‐variations in the induction of NK cells activation, further evaluating the association between IgG Fc N‐glycans and disease severity/prognosis. We found that SARS‐CoV‐2+ individuals display, at diagnosis, variations in the glycans composition of circulating IgGs. Importantly, levels of galactose and sialic acid structures on IgGs are able to predict the development of a poor COVID‐19 disease. Mechanistically, we demonstrated that a deficiency on galactose structures on IgG Fc in COVID‐19 patients appears to induce NK cells activation associated with increased release of IFN‐γ and TNF‐α, which indicates the presence of pro‐inflammatory immunoglobulins and higher immune activation, associated with a poor disease course. This study brings to light a novel blood biomarker based on IgG Fc glycome composition with capacity to stratify patients at diagnosis.

NMR metabolic composition profiling of high pressure pasteurized milk preserved by hyperbaric storage at room temperature

1 H NMR spectroscopy was used to study the effect of hyperbaric storage (HS) on the chemical composition of high pressure pasteurized milk (HPP milk). A subsequent multivariate analysis (MVA) was applied to distinguish samples stored under different conditions. Three different pressure levels (50/75/100 MPa), at naturally variable uncontrolled room temperature (≈ 20 °C), and up to 40 days of storage were compared with controls at atmospheric pressure and room temperature (AP/RT) or under refrigeration (RF). Only AP/RT samples presented spoilage compounds as lactate, methylamine and trimethylamine derived from microbial deterioration of milk. However, RF spectral profiles were closer to initial milk samples than the HS profiles, mainly in the aliphatic region which corresponds to an increase in soluble proteins for the HS samples. Additionally, MVA revealed higher levels of citrate, carnitine, lactose, and orotate in initial and RF samples, while HS samples had higher levels of acetate, N-acetylglucosamine, choline and galactose. In conclusion, although HS and RF samples were separated by MVA, there was no presence of microbial spoilage compounds in the spectra, being HS at RT an eco-friendlier alternative to preserve foods than traditional RF. • High-pressure pasteurized milk (HPP milk) presented a typical 1D 1 H NMR spectrum. • Refrigerated (RF) samples were closer to the initial samples of HPP milk. • Hyperbaric storage (HS) showed higher levels of acetate, choline and galactose. • RF presented higher levels of citrate, carnitine, lactose and orotate. • HS raised soluble protein, potentially with higher digestibility/nutritional value.

Gas production by Paucilactobacillus wasatchensis WDCO4 is increased in Cheddar cheese containing sodium gluconate

Paucilactobacillus wasatchensis can use gluconate (GLCN) as well as galactose as an energy source and because sodium GLCN can be added during salting of Cheddar cheese to reduce calcium lactate crystal formation, our primary objective was to determine if the presence of GLCN in cheese is another risk factor for unwanted gas production leading to slits in cheese. A secondary objective was to calculate the amount of CO2 produced during storage and to relate this to the amount of gas-forming substrate that was utilized. Ribose was added to promote growth of Pa. wasatchensis WDC04 (P.waWDC04) to high numbers during storage. Cheddar cheese was made with lactococcal starter culture with addition of P.waWDC04 on 3 separate occasions. After milling, the curd was divided into six 10-kg portions. To the curd was added (A) salt, or salt plus (B) 0.5% galactose + 0.5% ribose (similar to previous studies), (C) 1% sodium GLCN, (D) 1% sodium GLCN + 0.5% ribose, (E) 2% sodium GLCN, (F) 2% sodium GLCN + 0.5% ribose. A vat of cheese without added P.waWDC04 was made using the same milk and a block of cheese used as an additional control. Cheeses were cut into 900-g pieces, vacuum packaged and stored at 12°C for 16 wk. Each month the bags were examined for gas production and cheese sampled and tested for lactose, galactose and GLCN content, and microbial numbers. In the control cheese, P.waWDC04 remained undetected (i.e., <104 cfu/g), whereas in cheeses A, C, and E it increased to 107 cfu/g, and when ribose was included with salting (cheeses B, D, and F) increased to 108 cfu/g. The amount of gas (measured as headspace height or calculated as mmoles of CO2) during 16 wk storage was increased by adding P.waWDC04 into the milk, and by adding galactose or GLCN to the curd. Galactose levels in cheese B were depleted by 12 wk while no other cheeses had residual galactose. Except for cheese D, the other cheeses with GLCN added (C, E and F) showed little decline in GLCN levels until wk 12, even though gas was being produced starting at wk 4. Based on calculations of CO2 in headspace plus CO2 dissolved in cheese, galactose and GLCN added to cheese curd only accounted for about half of total gas production. It is proposed that CO2 was also produced by decarboxylation of amino acids. Although P.waWDC04 does not have all the genes for complete conversion and decarboxylation of the amino acids in cheese, this can be achieved in conjunction with starter culture lactococcal. Adding GLCN to curd can now be considered another confirmed risk factor for unwanted gas production during storage of Cheddar cheese that can lead to slits and cracks in cheese. Putative risk factors now include having a community of bacteria in cheese leading to decarboxylation of amino acids and release of CO2 as well autolysis of the starter culture that would provide a supply of ribose that can promote growth of Pa. wasatchensis.

Metabolic profiling of serum and urine in lactating dairy cows affected by subclinical ketosis using proton nuclear magnetic resonance spectroscopy.

Ketosis is associated with high milk yield during lactating or insufficient feed intake in lactating dairy cows. However, few studies have been conducted on the metabolomics of ketosis in Korean lactating dairy cows. The present study aimed to investigate the serum and urine metabolites profiling of lactating dairy cows through proton nuclear magnetic resonance (1H-NMR) spectroscopy and comparing those between healthy (CON) and subclinical ketosis (SCK) groups. Six lactating dairy cows were categorized into CON and SCK groups. All experimental Holstein cows were fed total mixed ration. Serum and urine samples were collected from the jugular vein of the neck and by hand sweeping the perineum, respectively. The metabolites in the serum and urine were determined using 1H-NMR spectroscopy. Identification and quantification of metabolites was performed by Chenomx NMR Suite 8.4 software. Metabolites statistical analysis was performed by Metaboanalyst version 5.0 program. In the serum, the acetoacetate level was significantly (p < 0.05) higher in the SCK group than in the CON group, and whereas acetate, galactose and pyruvate levels tended to be higher. CON group had significantly (p < 0.05) higher levels of 5-aminolevulinate and betaine. Indole-3-acetate, theophylline, p-cresol, 3-hydroxymandelate, gentisate, N-acetylglucosamine, N-nitrosodimethylamine, xanthine and pyridoxine levels were significantly (p < 0.05) higher in the urine of the SCK group than that in the CON group, which had higher levels of homogentisate, ribose, gluconate, ethylene glycol, maltose, 3-methyl-2-oxovalerate and glycocholate. Some significantly (p < 0.05) different metabolites in the serum and urine were associated with ketosis diseases, inflammation, energy balance and body weight. This study will be contributed useful a future ketosis metabolomics studies in Korea.

Hexose transporter SWEET5 confers galactose sensitivity to Arabidopsis pollen germination via a galactokinase.

Galactose is an abundant and essential sugar used for the biosynthesis of many macromolecules in different organisms, including plants. Galactose metabolism is tightly and finely controlled, since excess galactose and its derivatives are inhibitory to plant growth. In Arabidopsis (Arabidopsis thaliana), root growth and pollen germination are strongly inhibited by excess galactose. However, the mechanism of galactose-induced inhibition during pollen germination remains obscure. In this study, we characterized a plasma membrane-localized transporter, Arabidopsis Sugars Will Eventually be Exported Transporter 5, that transports glucose and galactose. SWEET5 protein levels started to accumulate at the tricellular stage of pollen development and peaked in mature pollen, before rapidly declining after pollen germinated. SWEET5 levels are responsible for the dosage-dependent sensitivity to galactose, and galactokinase is essential for these inhibitory effects during pollen germination. However, sugar measurement results indicate that galactose flux dynamics and sugar metabolism, rather than the steady-state galactose level, may explain phenotypic differences between sweet5 and Col-0 in galactose inhibition of pollen germination.

Cytokines in the Immune Microenvironment Change the Glycosylation of IgG by Regulating Intracellular Glycosyltransferases.

BackgroundChanges in IgG glycosylation, as a novel pathological feature, are observed in various autoimmune diseases (AIDs). The glycosylation patterns of IgG play a critical role in regulating the biological function and stability of IgG involved in the pathophysiology of many AIDs. However, the intracellular regulatory mechanisms underlying the effects of disturbances in various cytokines on IgG glycosylation are poorly understood. Thus, we investigated the regulatory effects of elevated cytokines in AIDs on intracellular IgG glycosylation within B cells.MethodsFirst, we established a controlled primary culture system in vitro to differentiate human CD19+ B cells into antibody-secreting cells (ASCs). Then, the IgG concentrations in the supernatants were measured by enzyme-linked immunoassay (ELISA) under IFN-γ, TNF-α, IL-21, IL-17A, BAFF, or APRIL stimulation. Next, the glycosylation levels of IgG under different stimuli were compared via a lectin microarray. The fine carbohydrate structures of IgG were confirmed by matrix-assisted laser desorption/ionization-quadrupole ion trap-time of flight-mass spectrometry (MALDI-TOF-MS). Finally, the expression of glycosyltransferases and glycosidases in B cells under stimulation with several cytokines was detected by real-time PCR and western blotting.ResultsWe found that cytokines significantly promoted IgG production in vitro and led to considerably different IgG glycan patterns. Specifically, the results of lectin microarray showed the galactose level of IgG was increased by IFN-γ stimulation (p<0.05), and the sialylation of IgG was increased by IL-21 and IL-17A (p<0.05). The MALDI-TOF-MS data showed that the frequency of agalactosylation was decreased by IFN-γ with the increased frequency of mono-galactosylation and decreased frequency of digalactosylation, accompanied by upregulation of β-1,4-galactosyltransferase 1. Both frequencies of mono-sialylated and disialylated N-glycans were increased by IL-21 and IL-17A with decreased frequency of asialylation, and the expression of β-galactoside α-2,6-sialyltransferase 1 was upregulated by IL-21 and IL-17A.ConclusionAbnormally elevated cytokines in the microenvironment regulates IgG glycan patterns by regulating intracellular glycosyltransferases in human B cells.