Factors affecting N-acetyl-ß-D-glucosaminidase as an indicator for mastitis detection in dairy sheep.

The study of new indirect methods for mastitis detection is of great relevance both at the economic level of the farm and dairies, and in terms of consumer health, and animal welfare. These methods help us to monitor the disease and speed up the decision-making process on treatment of the affected animal and the destination of the milk. The main aim of this work was to study the effect of intramammary infection and other non-infectious factors on the activity of the enzyme N-acetyl-ß-D-glucosaminidase (NAGase) in milk, in order to evaluate its use as an indicator for the early diagnosis of mastitis in sheep that could be less expensive, easier to measure and a better marker of inflammation or complementary to existing methods such as somatic cell count (SCC). Seven biweekly samplings were carried out, in which NAGase activity, SCC and milk were analyzed. Glands were classified according to their sanitary status based on the results of the SCC and bacteriological analysis. Non-infectious factors such as lactation stage, parity number and milking session had a statistically significant effect on NAGase values, finding the highest NAGase values at the onset and end of the study, in infectious mastitic glands of multiparous females and at morning milking. However, among the NAGase variation factors studied, the health status of the gland was the factor that caused the highest variation in enzyme levels, with infectious mastitic glands showing higher values than healthy glands. The predictive ability of NAGase was also studied by means of several logistic regression models, with the one that included NAGase together with lactation stage and parity obtaining the best results if sensitivity is to be prioritized, or the model that included NAGase, lactation stage, parity, milking and production if specificity is to be prioritized. From the results obtained, it can be concluded that the use of NAGase as an intramammary infection detection method in sheep can be useful when non-infectious factors that cause changes in the concentration of the enzyme are also considered.

Genetic features of patients with MPS type IIIB: Description of five pathogenic gene variations.

BACKGROUND: There are four distinct forms of Sanfilippo syndrome (MPS type III), each of which is an autosomal lysosomal storage disorder. These forms are caused by abnormalities in one of four lysosomal enzymes. This study aimed to identify possible genetic variants that contribute to Sanfilippo IIIB in 14 independent families in Southwest Iran. METHODS: Patients were included if their clinical features and enzyme assay results were suggestive. The patients were subsequently subjected to Sanger Sequencing to screen for Sanfilippo-related genes. Additional investigations have been conducted using various computational analyses to determine the probable functional effects of diagnosed variants. RESULTS: Five distinct variations were identified in the NAGLU gene. This included two novel variants in two distinct families and three previously reported variants in 12 distinct families. All of these variations were recognized as pathogenic using the MutationTaster web server. In silico analysis showed that all detected variants affected protein structural stability; four destabilized protein structures, and the fifth variation had the opposite effect. CONCLUSION: In this study, two novel variations in the NAGLU gene were identified. The results of this study positively contribute to the mutation diversity of the NAGLU gene. To identify new disease biomarkers and therapeutic targets, precision medicine must precisely characterize and account for genetic variations. New harmful gene variants are valuable for updating gene databases concerning Sanfilippo disease variations and NGS gene panels. This may also improve genetic counselling for rapid risk examinations and disease surveillance.

Local thiamet-G delivery by a thermosensitive hydrogel confers ischemic cardiac repair via myeloid M2-like activation in a STAT6 O-GlcNAcylation-dependent manner.

Infarct healing requires a dynamic and orchestrated inflammatory reaction following myocardial infarction (MI). While an uncontrolled excessive inflammatory response exaggerates ischemic injury post-MI, M2-like reparative macrophages may facilitate inflammation regression and promote myocardial healing. However, how protein post-translational modification regulates post-MI cardiac repair and dynamic myeloid activation remains unknown. Here we show that M2-like reparative, but not M1-like inflammatory activation, is enhanced by pharmacologically-induced hyper-O-GlcNAcylation. Mechanistically, myeloid knockdown of O-GlcNAc hydrolase O-GlcNAcase (Oga), which also results in hyper-O-GlcNAcylation, positively regulates M2-like activation in a STAT6-dependent fashion, which is controlled by O-GlcNAcylation of STAT6. Of note, both systemic and local supplementation of thiamet-G (TMG), an Oga inhibitor, effectively facilitates cardiac recovery in mice by elevating the accumulation of M2-like macrophages in infarcted hearts. Our study provides a novel clue for monocyte/macrophage modulating therapies aimed at reducing post-MI hyperinflammation in ischemic myocardium.

Characterization of a GH20 ß-N-Acetylhexosaminidase from Flavobacterium algicola Suitable to Synthesize Lacto-N-triose II.

ß-N-Acetylhexosaminidases have attracted much attention in the enzymatic synthesis of lacto-N-triose II (LNT2) as a backbone precursor of human milk oligosaccharides (HMOs). In this study, a novel glycoside hydrolase (GH) 20 family ß-N-acetylhexosaminidase, FlaNag2353, from Flavobacterium algicola was biochemically characterized and applied to synthesize LNT2. FlaNag2353 displayed optimal activity to p-nitrophenyl N-acetyl-ß-d-glucosaminide (pNP-GlcNAc) at 40 °C and pH 8.0. In addition to its excellent hydrolysis activity toward pNP-GlcNAc and chitooligosaccharides, FlaNag2353 showed trans-glycosylation activity. Under conditions of pH 9.0 and 55 °C for 2 h and utilizing 200 mM lactose and 10 mM pNP-GlcNAc, FlaNag2353 synthesized LNT2 with a conversion ratio of 4.15% calculated from pNP-GlcNAc. Moreover, when applied to LNT2 synthesis with 10 mM pNP-GlcNAc and 9.7% (w/v) industrial waste whey powder, FlaNag2353 achieved a conversion ratio of 2.39%. This study has significant implications for broadening the applications of GH20 ß-N-acetylhexosaminidases and promoting the high-value utilization of whey powder.

Investigation of the relationship between the changes in vaginal microecological enzymes and human papillomavirus (HPV) infection.

This study aims to investigate the relationship between the human papillomavirus (HPV) infection and the altered vaginal microecological environment of patients. Initially, HPV genotyping and microecological detection were performed in 1281 subjects in the Department of Obstetrics and Gynecology of The First Hospital of Qinhuangdao (Qinhuangdao, China). The relationship between the enzymes of vaginal microecology, that is, proline aminopeptidase and acetylglucosaminidase, and vaginal inflammatory diseases, as well as the prognosis of HPV infection, was analyzed. The experimental findings indicated a close relationship between the expression of positive prolyl aminopeptidase and trichomonas vaginitis, as well as bacterial vaginitis. In addition, the expression of acetylglucosaminidase is closely associated with trichomonas vaginitis and vulvovaginal candidiasis. Furthermore, the observations indicated that positive prolyl aminopeptidase and acetylglucosaminidase could increase the risk of various subtypes of HPV infection in patients. The receiver operating characteristic curve analysis presented that the expression of prolyl aminopeptidase and acetylglucosaminidase could offer exceptional diagnostic efficacy, indicating their association with persistent HPV infection. In summary, our results highlighted that the expression of positive prolyl aminopeptidase and acetylglucosaminidase in the vaginal microecology could be substantially correlated to the occurrence and the development of vaginal inflammatory diseases, as well as the outcome and the risk of persistent HPV infection.

L-Glycosidase-Cleavable Natural Glycans Facilitate the Chemical Synthesis of Correctly Folded Disulfide-Bonded D-Proteins.

D-peptide ligands can be screened for therapeutic potency and enzymatic stability using synthetic mirror-image proteins (D-proteins), but efficient acquisition of these D-proteins can be hampered by the need to accomplish their in vitro folding, which often requires the formation of correctly linked disulfide bonds. Here, we report the finding that temporary installation of natural O-linked-ß-N-acetyl-D-glucosamine (O-GlcNAc) groups onto selected D-serine or D-threonine residues of the synthetic disulfide-bonded D-proteins can facilitate their folding in vitro, and that the natural glycosyl groups can be completely removed from the folded D-proteins to afford the desired chirally inverted D-protein targets using naturally occurring O-GlcNAcase. This approach enabled the efficient chemical syntheses of several important but difficult-to-fold D-proteins incorporating disulfide bonds including the mirror-image tumor necrosis factor alpha (D-TNFα) homotrimer and the mirror-image receptor-binding domain of the Omicron spike protein (D-RBD). Our work establishes the use of O-GlcNAc to facilitate D-protein synthesis and folding and proves that D-proteins bearing O-GlcNAc can be good substrates for naturally occurring O-GlcNAcase.

Assessment of human exposure to cadmium and its nephrotoxicity in the Chinese population.

BACKGROUND: Cadmium (Cd) is a toxic heavy metal that widely detected in environment and accumulated in kidney, posing a great threat to human health. However, there is a lack of systematic investigation of exposure profile and association of Cd exposure with renal function in the Chinese population. METHODS: Related articles were searched from PubMed, Web of Science, China National Knowledge Internet, and Wanfang to construct an aggregate exposure pathway (AEP) framework for Cd and to explore the correlation between Cd and renal function using random effects models. RESULTS: A total of 220 articles were included in this study, among which 215 investigated human exposure and 12 investigated the association of Cd with renal outcomes. The AEP framework showed that 96.5 % and 62.5 % of total Cd intake were attributed to dietary intake in nonsmokers and smokers, respectively. And 35.2 % originated from cigarette smoke inhalation in smokers. In human body, Cd was detected in blood, urine, placenta, etc. Although the concentrations of Cd in blood and urine from subjects living in polluted areas showed a sharp downward trend since the early 21st century, higher concentration of Cd in the environment and human body in polluted areas was found. Kidney was the target organ. The level of blood Cd was positively associated with urinary ß2-microglobulin [ß2-MG, r (95 % CI) = 0.12 (0.05, 0.19)], albumin [0.13 (0.06, 0.20)], and retinol-binding protein [RBP, 0.14 (0.03, 0.24)]. Elevated urinary Cd was correlated with increases in ß2-MG [0.22 (0.15, 0.29)], albumin [0.23 (0.16, 0.29)], N-acetyl-ß-d-glucosaminidase [NAG, 0.33 (0.22, 0.44)], and RBP [0.22 (0.14, 0.30)]. CONCLUSIONS: Foods and cigarette smoke were two major ways for Cd intake, and Cd induced renal injury in the Chinese population. This study enhanced the understanding of human exposure and nephrotoxicity of Cd, and emphasized the need for controlling Cd level in polluted areas.

Synthesis of a fluorescent probe for measuring the activity of endo-ß-N-acetylglucosaminidases recognizing hybrid-type N-glycans.

A fluorescence-quenching-based assay system was constructed to determine the hydrolytic activity of endo-ß-N-acetylglucosaminidases (ENGases) interacting with hybrid-type N-glycans. This was achieved using a dual-labeled fluorescent probe with a nonasaccharide structure. We produced the nonasaccharide skeleton by the stepwise glycosylation of the galactose residue on a galactosyl chitobiose derivative. Next, we introduced azido and acetoxy groups into the nonasaccharide derivative in a stepwise manner, which led to stereochemistry inversion at both the C-4 and C-2 hydroxy groups on its galactose residue. The protecting groups of the resulting nonasaccharide derivative were removed, and the derivative was labeled with an N-methylanthraniloyl group to obtain a reporter dye and a 2,4-dinitrophenyl group as a quenching molecule to obtain target probe 1. The use of this probe along with a microplate reader enabled a facile evaluation of the hydrolytic activities of ENGases Endo-H, Endo-M, Endo-F3, Endo-S, and Endo-CC. Furthermore, this probe could also assist in the search for novel ENGases that are specific to hybrid-type N-glycans.

A fluorescent probe for selective detection of lysosomal ß-hexosaminidase in live cells.

Determining the activity of lysosomal ß-hexosaminidase in cells is of great importance for understanding the roles that these enzymes play in pathophysiological events. Herein, we designed the new fluorescent probe, ßGalNAc-Rhod-CM(NEt2), which consisted of a ßGalNAc-linked rhodol unit serving as a ß-hexosaminidase reactive fluorogenic moiety and a N,N'-diethylaminocoumarin (CM(NEt2)) group acting as a fluorescence marker for determining the degree of cell permeabilization. Treatment of ßGalNAc-Rhod-CM(NEt2) with ß-hexosaminidase promoted generation of Rhod-CM(NEt2), thereby leading to an increase in the intensity of fluorescence of Rhod. However, this probe did not respond to the functionally related glycosidase, O-GlcNAcase. The detection limit of ßGalNAc-Rhod-CM(NEt2) for ß-hexosaminidase was determined to be 0.52 nM, indicating that it has high sensitivity for this enzyme. Furthermore, the probe functioned as an excellent fluorogenic substrate for ß-hexosaminidase with kcat and Km values of 17 sec-1 and 22 µM, respectively. The results of cell studies using ßGalNAc-Rhod-CM(NEt2) showed that levels of ß-hexosaminidase activity in cells can be determined by measuring the intensity of fluorescence arising from Rhod and that the intensity of fluorescence of CM(NEt2) can be employed to determine the degree of cell permeabilization of the probe. Utilizing the new probe, we assessed ß-hexosaminidase activities in several types of cells and evaluated the effect of glucose concentrations in culture media on the activity of this enzyme.

Long-Term Exposure of Cultured Astrocytes to High Glucose Impact on Their LPS-Induced Activation.

Diabetes mellitus is associated with various complications, mainly caused by the chronic exposure of the cells to high glucose (HG) concentrations. The effects of long-term HG exposure in vitro accompanied by lipopolysaccharide (LPS) application on astrocytes are relatively unknown. We used cell medium with normal (NG, 5.5 mM) or high glucose (HG, 25 mM) for rat astrocyte cultures and measured the release of NO, IL-6, ß-hexosaminidase and cell survival in response to LPS. We first demonstrated that HG long-term incubation of astrocytes increased the release of ß-hexosaminidase without decreasing MTT-detected cell survival, suggesting that there is no cell membrane damage or astrocyte death but could be lysosome exocytosis. Different from what was observed for NG, all LPS concentrations tested at HG resulted in an increase in IL-6, and this was detected for both 6 h and 48 h treatments. Interestingly, ß-hexosaminidase level increased after 48 h of LPS and only at HG. The NO release from astrocytes also increased with LPS application at HG but was less significant. These data endorsed the original hypothesis that long-term hyperglycemia increases proinflammatory activation of astrocytes, and ß-hexosaminidase could be a specific marker of excessive activation of astrocytes associated with exocytosis.

In vivo and in silico toxicity assessment of four common liquid crystal monomers to Daphnia magna: Novel endocrine disrupting chemicals in crustaceans?

Liquid crystal monomers (LCMs) are widely used in liquid crystal displays (LCDs) and are proposed to be a new generation of environmentally persistent, bioaccumulative and toxic (PBT) substances that are increasingly detected in rivers and seas. However, there is a lack of in vivo data that characterize adverse responses and toxic mechanisms of LCMs on aquatic organisms. The aim of this study was to comprehensively investigate the effect of four typical LCMs on the lethality, growth, molting, and reproductive capacity of Daphnia magna (D. magna), a highly studied aquatic species in environmental toxicology. Whole body and enzymatic biomarkers (i.e., body length, chitobiase, acetylcholinesterase, antioxidant defense) were measured to assess the toxicity of LCMs. The 48 h mortality rate and observations of disrupted thorax development and inhibition of ecdysis indicate that D. magna are sensitive to LCMs exposure. Oxidative stress, impaired neurotransmission, and disruptions in molting were observed in short-term biomarker tests using LCMs. A 21 day exposure of D. magna to LCMs resulted in reduced growth, reproduction, and population intrinsic growth rate. In addition, chitobiase and 20-hydroxyecdysone, enzymes important for the molting process, were altered at 7, 14 and 21 d. This is hypothesized to be related to endocrine imbalance resulting from LCM exposure. Based on molecular docking simulations, there is evidence that LCMs bind directly to ecdysteroid receptors; this may explain the observed endocrine disrupting effects of LCMs. These data support the hypothesis that LCMs are endocrine disrupting chemicals in aquatic species, impacting the process of molting. This may subsequently lead to lower reproduction and unbalanced population dynamics.

Prediction of key milk biomarkers in dairy cows through milk mid-infrared spectra and international collaborations.

At the individual cow level, suboptimum fertility, mastitis, negative energy balance, and ketosis are major issues in dairy farming. These problems are widespread on dairy farms and have an important economic impact. The objectives of this study were (1) to assess the potential of milk mid-infrared (MIR) spectra to predict key biomarkers of energy deficit (citrate, isocitrate, glucose-6 phosphate [glucose-6P], free glucose), ketosis (ß-hydroxybutyrate [BHB] and acetone), mastitis (N-acetyl-ß-d-glucosaminidase activity [NAGase] and lactate dehydrogenase), and fertility (progesterone); (2) to test alternative methodologies to partial least squares (PLS) regression to better account for the specific asymmetric distribution of the biomarkers; and (3) to create robust models by merging large datasets from 5 international or national projects. Benefiting from this international collaboration, the dataset comprised a total of 9,143 milk samples from 3,758 cows located in 589 herds across 10 countries and represented 7 breeds. The samples were analyzed by reference chemistry for biomarker contents, whereas the MIR analyses were performed on 30 instruments from different models and brands, with spectra harmonized into a common format. Four quantitative methodologies were evaluated to address the strongly skewed distribution of some biomarkers. Partial least squares regression was used as the reference basis, and compared with a random modification of distribution associated with PLS (random-downsampling-PLS), an optimized modification of distribution associated with PLS (KennardStone-downsampling-PLS), and support vector machine (SVM). When the ability of MIR to predict biomarkers was too low for quantification, different qualitative methodologies were tested to discriminate low versus high values of biomarkers. For each biomarker, 20% of the herds were randomly removed within all countries to be used as the validation dataset. The remaining 80% of herds were used as the calibration dataset. In calibration, the 3 alternative methodologies outperform the PLS performances for the majority of biomarkers. However, in the external herd validation, PLS provided the best results for isocitrate, glucose-6P, free glucose, and lactate dehydrogenase (coefficient of determination in external herd validation [R2v] = 0.48, 0.58, 0.28, and 0.24, respectively). For other molecules, PLS-random-downsampling and PLS-KennardStone-downsampling outperformed PLS in the majority of cases, but the best results were provided by SVM for citrate, BHB, acetone, NAGase, and progesterone (R2v = 0.94, 0.58, 0.76, 0.68, and 0.15, respectively). Hence, PLS and SVM based on the entire dataset provided the best results for normal and skewed distributions, respectively. Complementary to the quantitative methods, the qualitative discriminant models enabled the discrimination of high and low values for BHB, acetone, and NAGase with a global accuracy around 90%, and glucose-6P with an accuracy of 83%. In conclusion, MIR spectra of milk can enable quantitative screening of citrate as a biomarker of energy deficit and discrimination of low and high values of BHB, acetone, and NAGase, as biomarkers of ketosis and mastitis. Finally, progesterone could not be predicted with sufficient accuracy from milk MIR spectra to be further considered. Consequently, MIR spectrometry can bring valuable information regarding the occurrence of energy deficit, ketosis, and mastitis in dairy cows, which in turn have major influences on their fertility and survival.

Characterization of novel endo-ß-N-acetylglucosaminidases from intestinal Barnesiella intestinihominis that hydrolyze multi-branched complex-type N-glycans.

Endo-ß-N-acetylglucosaminidases (ENGases) are enzymes that hydrolyze N-linked glycans. Many ENGases have been characterized, but few have been identified with hydrolytic activity towards multi-branched complex-type N-glycans. In this study, three candidate ENGases were identified from Barnesiella intestinihominis based on database searches and phylogenetic analysis. A domain search identified the N x E motif in all three candidates, suggesting that they were members of glycosyl hydrolase family 85 (GH85). The three candidate ENGases, named Endo-BIN1, Endo-BIN2, and Endo-BIN3, were expressed in Escherichia coli cells, and their hydrolytic activity towards N-glycans and glycoproteins was measured by high performance liquid chromatography analysis and SDS-PAGE analysis. All ENGases showed hydrolytic activity towards glycoproteins, but only Endo-BIN2 and Endo-BIN3 showed hydrolytic activity towards pyridylaminated N-glycans. The optimum pH of Endo-BIN1, Endo-BIN2, and End-BIN3 was pH 6.5, 4.0, and 7.0, respectively. We measured substrate specificities of Endo-BIN2 and Endo-BIN3 towards pyridylaminated N-glycans, and found that the two Endo-BIN enzymes showed similar substrate specificity, preferring bi-antennary complex-type N-glycans with galactose or α2,6-linked sialic acid residues at the non-reducing ends. Endo-BIN2 and Endo-BIN3 were also able to hydrolyze multi-branched complex-type N-glycans. SDS-PAGE analysis revealed that all Endo-BIN enzymes were capable of releasing complex-type N-glycans from glycoproteins such as rituximab, transferrin, and fetuin. We expect that B. intestinihominis possesses ENGases to facilitate the utilization of complex-type N-glycans from host cells. These findings will have applications in N-glycan remodeling of glycoproteins and the development of pharmaceuticals.

Cryo-EM structure of human O-GlcNAcylation enzyme pair OGT-OGA complex.

O-GlcNAcylation is a conserved post-translational modification that attaches N-acetyl glucosamine (GlcNAc) to myriad cellular proteins. In response to nutritional and hormonal signals, O-GlcNAcylation regulates diverse cellular processes by modulating the stability, structure, and function of target proteins. Dysregulation of O-GlcNAcylation has been implicated in the pathogenesis of cancer, diabetes, and neurodegeneration. A single pair of enzymes, the O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), catalyzes the addition and removal of O-GlcNAc on over 3,000 proteins in the human proteome. However, how OGT selects its native substrates and maintains the homeostatic control of O-GlcNAcylation of so many substrates against OGA is not fully understood. Here, we present the cryo-electron microscopy (cryo-EM) structures of human OGT and the OGT-OGA complex. Our studies reveal that OGT forms a functionally important scissor-shaped dimer. Within the OGT-OGA complex structure, a long flexible OGA segment occupies the extended substrate-binding groove of OGT and positions a serine for O-GlcNAcylation, thus preventing OGT from modifying other substrates. Conversely, OGT disrupts the functional dimerization of OGA and occludes its active site, resulting in the blocking of access by other substrates. This mutual inhibition between OGT and OGA may limit the futile O-GlcNAcylation cycles and help to maintain O-GlcNAc homeostasis.

The O-GlcNAc dichotomy: when does adaptation become pathological?

O-Linked attachment of ß-N-acetylglucosamine (O-GlcNAc) on serine and threonine residues of nuclear, cytoplasmic, and mitochondrial proteins is a highly dynamic and ubiquitous post-translational modification that impacts the function, activity, subcellular localization, and stability of target proteins. Physiologically, acute O-GlcNAcylation serves primarily to modulate cellular signaling and transcription regulatory pathways in response to nutrients and stress. To date, thousands of proteins have been revealed to be O-GlcNAcylated and this number continues to grow as the technology for the detection of O-GlcNAc improves. The attachment of a single O-GlcNAc is catalyzed by the enzyme O-GlcNAc transferase (OGT), and their removal is catalyzed by O-GlcNAcase (OGA). O-GlcNAcylation is regulated by the metabolism of glucose via the hexosamine biosynthesis pathway, and the metabolic abnormalities associated with pathophysiological conditions are all associated with increased flux through this pathway and elevate O-GlcNAc levels. While chronic O-GlcNAcylation is well associated with cardiovascular dysfunction, only until recently, and with genetically modified animals, has O-GlcNAcylation as a contributing mechanism of cardiovascular disease emerged. This review will address and critically evaluate the current literature on the role of O-GlcNAcylation in vascular physiology, with a view that this pathway can offer novel targets for the treatment and prevention of cardiovascular diseases.

Growing and dividing: how O-GlcNAcylation leads the way.

Cell cycle errors can lead to mutations, chromosomal instability, or death; thus, the precise control of cell cycle progression is essential for viability. The nutrient-sensing posttranslational modification, O-GlcNAc, regulates the cell cycle allowing one central control point directing progression of the cell cycle. O-GlcNAc is a single N-acetylglucosamine sugar modification to intracellular proteins that is dynamically added and removed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. These enzymes act as a rheostat to fine-tune protein function in response to a plethora of stimuli from nutrients to hormones. O-GlcNAc modulates mitogenic growth signaling, senses nutrient flux through the hexosamine biosynthetic pathway, and coordinates with other nutrient-sensing enzymes to progress cells through Gap phase 1 (G1). At the G1/S transition, O-GlcNAc modulates checkpoint control, while in S Phase, O-GlcNAcylation coordinates the replication fork. DNA replication errors activate O-GlcNAcylation to control the function of the tumor-suppressor p53 at Gap Phase 2 (G2). Finally, in mitosis (M phase), O-GlcNAc controls M phase progression and the organization of the mitotic spindle and midbody. Critical for M phase control is the interplay between OGT and OGA with mitotic kinases. Importantly, disruptions in OGT and OGA activity induce M phase defects and aneuploidy. These data point to an essential role for the O-GlcNAc rheostat in regulating cell division. In this review, we highlight O-GlcNAc nutrient sensing regulating G1, O-GlcNAc control of DNA replication and repair, and finally, O-GlcNAc organization of mitotic progression and spindle dynamics.

Exploiting O-GlcNAc transferase promiscuity to dissect site-specific O-GlcNAcylation.

Protein O-GlcNAcylation is an evolutionary conserved post-translational modification catalysed by the nucleocytoplasmic O-GlcNAc transferase (OGT) and reversed by O-GlcNAcase (OGA). How site-specific O-GlcNAcylation modulates a diverse range of cellular processes is largely unknown. A limiting factor in studying this is the lack of accessible techniques capable of producing homogeneously O-GlcNAcylated proteins, in high yield, for in vitro studies. Here, we exploit the tolerance of OGT for cysteine instead of serine, combined with a co-expressed OGA to achieve site-specific, highly homogeneous mono-glycosylation. Applying this to DDX3X, TAB1, and CK2α, we demonstrate that near-homogeneous mono-S-GlcNAcylation of these proteins promotes DDX3X and CK2α solubility and enables production of mono-S-GlcNAcylated TAB1 crystals, albeit with limited diffraction. Taken together, this work provides a new approach for functional dissection of protein O-GlcNAcylation.

Disease pathology signatures in a mouse model of Mucopolysaccharidosis type IIIB.

Mucopolysaccharidosis type IIIB (MPS IIIB) is a rare and devastating childhood-onset lysosomal storage disease caused by complete loss of function of the lysosomal hydrolase α-N-acetylglucosaminidase. The lack of functional enzyme in MPS IIIB patients leads to the progressive accumulation of heparan sulfate throughout the body and triggers a cascade of neuroinflammatory and other biochemical processes ultimately resulting in severe mental impairment and early death in adolescence or young adulthood. The low prevalence and severity of the disease has necessitated the use of animal models to improve our knowledge of the pathophysiology and for the development of therapeutic treatments. In this study, we took a systematic approach to characterizing a classical mouse model of MPS IIIB. Using a series of histological, biochemical, proteomic and behavioral assays, we tested MPS IIIB mice at two stages: during the pre-symptomatic and early symptomatic phases of disease development, in order to validate previously described phenotypes, explore new mechanisms of disease pathology and uncover biomarkers for MPS IIIB. Along with previous findings, this study helps provide a deeper understanding of the pathology landscape of this rare disease with high unmet medical need and serves as an important resource to the scientific community.

Impact of multi-heavy metal exposure on renal damage indicators in Korea: An analysis using Bayesian Kernel Machine Regression.

Exposure to cadmium (Cd), arsenic (As), and mercury (Hg) is associated with renal tubular damage. People living near refineries are often exposed to multiple heavy metals at high concentrations. This cross-sectional study investigated the association between combined urinary Cd, As, and Hg levels and renal damage markers in 871 residents living near the Janghang refinery plant and in a control area. Urinary Cd, As, Hg, N-acetyl-ß-D-glucosaminidase (NAG), and ß2-microglobulin (ß2-MG) levels were measured. The combined effects of Cd, As, and Hg on renal tubular damage markers were assessed using linear regression and a Bayesian Kernel Machine Regression (BKMR) model. The results of the BKMR model were compared using a stratified analysis of the exposure and control groups. While the linear regression showed that only Cd concentration was significantly associated with urinary NAG levels (ß = 0.447, P value < .05), the BKMR model showed that Cd and Hg levels were also significantly associated with urinary NAG levels. The combined effect of the 3 heavy metals on urinary NAG levels was significant and stronger in the exposure group than in the control group. However, no relationship was observed between the exposure concentrations of the 3 heavy metals and urinary ß2-MG levels. The results suggest that the BKMR model can be used to assess the health effects of heavy-metal exposure on vulnerable residents.

Protein O-GlcNAcylation in multiple immune cells and its therapeutic potential.

O-GlcNAcylation is a post-translational modification of proteins that involves the addition of O-GlcNAc to serine or threonine residues of nuclear or cytoplasmic proteins, catalyzed by O-GlcNAc transferase (OGT). This modification is highly dynamic and can be reversed by O-GlcNAcase (OGA). O-GlcNAcylation is widespread in the immune system, which engages in multiple physiologic and pathophysiologic processes. There is substantial evidence indicating that both the hexosamine biosynthesis pathway (HBP) and O-GlcNAcylation are critically involved in regulating immune cell function. However, the precise role of O-GlcNAcylation in the immune system needs to be adequately elucidated. This review offers a thorough synopsis of the present research on protein O-GlcNAcylation, accentuating the molecular mechanisms that control immune cells' growth, maturation, and performance via this PTM.