Germline C1GALT1C1 mutation causes a multisystem chaperonopathy.

Mutations in genes encoding molecular chaperones can lead to chaperonopathies, but none have so far been identified causing congenital disorders of glycosylation. Here we identified two maternal half-brothers with a novel chaperonopathy, causing impaired protein O-glycosylation. The patients have a decreased activity of T-synthase (C1GALT1), an enzyme that exclusively synthesizes the T-antigen, a ubiquitous O-glycan core structure and precursor for all extended O-glycans. The T-synthase function is dependent on its specific molecular chaperone Cosmc, which is encoded by X-chromosomal C1GALT1C1. Both patients carry the hemizygous variant c.59C>A (p.Ala20Asp; A20D-Cosmc) in C1GALT1C1. They exhibit developmental delay, immunodeficiency, short stature, thrombocytopenia, and acute kidney injury (AKI) resembling atypical hemolytic uremic syndrome. Their heterozygous mother and maternal grandmother show an attenuated phenotype with skewed X-inactivation in blood. AKI in the male patients proved fully responsive to treatment with the complement inhibitor Eculizumab. This germline variant occurs within the transmembrane domain of Cosmc, resulting in dramatically reduced expression of the Cosmc protein. Although A20D-Cosmc is functional, its decreased expression, though in a cell or tissue-specific manner, causes a large reduction of T-synthase protein and activity, which accordingly leads to expression of varied amounts of pathological Tn-antigen (GalNAcα1-O-Ser/Thr/Tyr) on multiple glycoproteins. Transient transfection of patient lymphoblastoid cells with wild-type C1GALT1C1 partially rescued the T-synthase and glycosylation defect. Interestingly, all four affected individuals have high levels of galactose-deficient IgA1 in sera. These results demonstrate that the A20D-Cosmc mutation defines a novel O-glycan chaperonopathy and causes the altered O-glycosylation status in these patients.

A unique miR775-GALT9 module regulates leaf senescence in Arabidopsis during post-submergence recovery by modulating ethylene and the abscisic acid pathway.

The submergence-induced hypoxic condition negatively affects the plant growth and development, and causes early onset of senescence. Hypoxia alters the expression of a number of microRNAs (miRNAs). However, the molecular function of submergence stress-induced miRNAs in physiological or developmental changes and recovery remains poorly understood. Here, we show that miR775 is an Arabidopsis thaliana-specific young and unique miRNA that possibly evolved non-canonically. miR775 post-transcriptionally regulates GALACTOSYLTRANSFERASE 9 (GALT9) and their expression is inversely affected at 24 h of complete submergence stress. The overexpression of miR775 (miR775-Oe) confers enhanced recovery from submergence stress and reduced accumulation of RBOHD and ROS, in contrast to wild-type and MIM775 Arabidopsis shoot. A similar recovery phenotype in the galt9 mutant indicates the role of the miR775-GALT9 module in post-submergence recovery. We predicted that Golgi-localized GALT9 is potentially involved in protein glycosylation. The altered expression of senescence-associated genes (SAG12, SAG29 and ORE1), ethylene signalling (EIN2 and EIN3) and abscisic acid (ABA) biosynthesis (NCED3) pathway genes occurs in miR775-Oe, galt9 and MIM775 plants. Thus, our results indicate the role for the miR775-GALT9 module in post-submergence recovery through a crosstalk between the ethylene signalling and ABA biosynthesis pathways.

Highly expressed B3GALT5-AS1 contributes to gastric cancer progression by recruiting WDR5 to mediate B3GALT5 and regulating ß-catenin/ZEB1 axis.

Long non-coding RNAs (lncRNAs) play an important role in the progression of gastric cancer (GC), but its specific regulatory mechanism remains to be further studied. We previously identified that lncRNA B3GALT5-AS1 was upregulated in GC serum. Here, we investigated the functions and molecular mechanisms of B3GALT5-AS1 in GC tumorigenesis. qRT-PCR was used to detect B3GALT5-AS1 expression in GC. EdU, CCK-8, and colony assays were utilized to assess the proliferation ability of B3GAL5-AS1, and transwell, tube formation assay were used to assess the invasion and metastasis ability. Mechanically, FISH and nuclear plasmolysis PCR identified the subcellular localization of B3GALT5-AS1. RIP and CHIP assays were used to analyse the regulation of B3GALT5-AS1 and B3GALT5. We observed that B3GALT5-AS1 was highly expressed in GC, and silencing B3GALT5-AS1 could inhibit the proliferation, invasion, and migratory capacities of GC. Additionally, B3GALT5-AS1 was bound to WDR5 and modulated the expression of B3GALT5 via regulating the ZEB1/ß-catenin pathway. High-expressed B3AGLT5-AS1 promoted GC tumorigenesis and regulated B3GALT5 expression via recruiting WDR5. Our study is expected to provide a new idea for clinical diagnosis and treatment.

High expression of B3GALT5 suppresses the galectin-4-mediated peritoneal dissemination of poorly differentiated gastric cancer cells.

Peritoneal metastasis frequently accompanies metastatic and/or recurrent gastric cancer, leading to a poor prognosis owing to a lack of effective treatment. Hence, there is a pressing need to enhance our understanding of the mechanisms and molecules driving peritoneal metastasis. In a previous study, galectin-4 inhibition impeded peritoneal metastasis in a murine model. This study examined the glycan profiles of cell surface proteins and glycosphingolipids (GSLs) in cells with varying tumorigenic potentials to understand the intricate mechanisms underlying galectin-4-mediated regulation, particularly glycosylation. Detailed mass spectrometry analysis showed that galectin-4 knockout cells exhibit increased expression of lacto-series GSLs with ß1,3-linked galactose while showing no significant alterations in neolacto-series GSLs. We conducted real-time polymerase chain reaction (PCR) analysis to identify candidate glycosyltransferases that synthesize increased levels of GSLs. Subsequently, we introduced the candidate B3GALT5 gene and selected the clones with high expression levels. B3GALT5 gene-expressing clones showed GSL glycan profiles like those of knockout cells and significantly reduced tumorigenic ability in mouse models. These clones exhibited diminished proliferative capacity and showed reduced expression of galectin-4 and activated AKT. Moreover, co-localization of galectin-4 with flotillin-2 (a raft marker) decreased in B3GALT5-expressing cells, implicating GSLs in galectin-4 localization to lipid rafts. D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (a GSL synthase inhibitor) also affected galectin-4 localization in rafts, suggesting the involvement of GSL microdomains. We discovered that B3GALT5 plays a crucial role in regulating peritoneal metastasis of malignant gastric cancer cells by suppressing cell proliferation and modulating lipid rafts and galectin-4 via mechanisms that are yet to be elucidated.

Delving into human α1,4-galactosyltransferase acceptor specificity: The role of enzyme dimerization.

Human α1,4-galactosyltransferase (A4galt), a Golgi apparatus-resident GT, synthesizes Gb3 glycosphingolipid (GSL) and P1 glycotope on glycoproteins (GPs), which are receptors for Shiga toxin types 1 and 2. Despite the significant role of A4galt in glycosylation processes, the molecular mechanisms underlying its varied acceptor specificities remain poorly understood. Here, we attempted to elucidate A4galt specificity towards GSLs and GPs by exploring its interaction with GTs with various acceptor specificities, GP-specific ß1,4-galactosyltransferase 1 (B4galt1) and GSL-specific ß1,4-galactosyltransferase isoenzymes 5 and 6 (B4galt5 and B4galt6). Using a novel NanoBiT assay, we found that A4galt can form homodimers and heterodimers with B4galt1 and B4galt5 in two cell lines, human embryonic kidney cells (HEK293T) and Chinese hamster ovary cells (CHO-Lec2). We found that A4galt-B4galts heterodimers preferred N-terminally tagged interactions, while in A4galt homodimers, the favored localization of the fused tag depended on the cell line used. Furthermore, by employing AlphaFold for state-of-the-art structural prediction, we analyzed the interactions and structures of these enzyme complexes. Our analysis highlighted that the A4galt-B4galt5 heterodimer exhibited the highest prediction confidence, indicating a significant role of A4galt heterodimerization in determining enzyme specificity toward GSLs and GPs. These findings enhance our knowledge of A4galt acceptor specificity and may contribute to a better comprehension of pathomechanisms of the Shiga toxin-related diseases.

Characterization of galactosyltransferase and sialyltransferase genes mediating the elongation of the extracellular O-GlcNAc glycans.

O-GlcNAc is a unique post-translational modification found in cytoplasmic, nuclear, and mitochondrial proteins. In a limited number of extracellular proteins, O-GlcNAc modifications occur through the action of EOGT, which specifically modifies subsets of epidermal growth factor-like (EGF) domain-containing proteins such as Notch receptors. The abnormalities due to EOGT mutations in mice and humans and the increased EOGT expression in several cancers signify the importance of EOGT pathophysiology and extracellular O-GlcNAc. Unlike intracellular O-GlcNAc monosaccharides, extracellular O-GlcNAc extends to form elongated glycan structures. However, the enzymes involved in the O-GlcNAc glycan extension have not yet been reported. In our study, we comprehensively screened potential galactosyltransferase and sialyltransferase genes related to the canonical O-GlcNAc glycan pathway and revealed the essential roles of B4GALT1 and ST3GAL4 in O-GlcNAc glycan elongation in human HEK293 cells. These findings were confirmed by sequential glycosylation of Drosophila EGF20 in vitro by EOGT, ß4GalT-1, and ST3Gal-IV. Thus, the findings from our study throw light on the specific glycosyltransferases that mediate O-GlcNAc glycan elongation in human HEK293 cells.

C1GALT1-mediated O-glycan T antigen increase enhances the migration and invasion ability of gastric cancer cells.

Gastric cancer (GC) is one of the most aggressive and lethal diseases in the world. Cancer metastasis is the mainly leading cause of death in GC patients. Aberrant Protein O-glycosylation is closely associated with tumor occurrence and metastasis. However, the effect of aberrant O-glycosylation on the progress of GC is not completely clear. This study aimed to investigate the biological function and its underlying effects mechanism of core 1 ß 1, 3-galactosyltransferase 1 (C1GALT1) C1GALT1-mediated O-glycan T antigen on GC progress. We conducted data mining analysis that C1GALT1 was obviously up-regulated in GC tissues than in para-carcinoma tissues. Elevated expression of C1GALT1 was closely associated with advanced TNM stage, lymph node metastasis, histological grade, and poor overall survival. In addition, C1GALT1 overexpression could promote GC cell proliferation, migration, and invasion, which was due to C1GALT1 overexpression-mediated O-glycan T antigen increase. Moreover, MUC1 was predicted to be a new downstream target of C1GALT1, which may be abnormally O-glycosylated by C1GALT1 thereby activating the cell adhesion signaling pathway. In conclusion, our studies proved that C1GALT1-mediated O-glycosylation increase could promote the metastasis of gastric cancer cells. These discoveries hint that C1GALT1 may serve as a novel therapeutic target for GC treatment.

Restoring galactose metabolism without restoring GALT rescues both compromised survival in larvae and an adult climbing deficit in a GALT-null D. Melanogaster model of classic galactosemia.

Classic galactosemia (CG) is an autosomal recessive disorder that results from profound deficiency of galactose-1-phosphate uridylyltransferase (GALT), the middle enzyme in the highly conserved Leloir pathway of galactose metabolism. That galactose metabolism is disrupted in patients with CG, and in GALT-null microbial, cell culture, and animal models of CG, has been known for many years. However, whether the long-term developmental complications of CG result from disrupted galactose metabolism alone, or from loss of some independent moonlighting function of GALT, in addition to disrupted galactose metabolism, has been posed but never resolved. Here, we addressed this question using a GALT-null Drosophila melanogaster model of CG engineered to express uridine diphosphate (UDP)-glucose/galactose pyrophosphorylase (UGGP), a plant enzyme that effectively bypasses GALT in the Leloir pathway by converting substrates uridine triphosphate (UTP) plus galactose-1-phosphate (gal-1P) into products UDP-galactose plus pyrophosphate (PPi). While GALT and UGGP share one substrate (gal-1P) and one product (UDP-galactose), they are structurally and evolutionarily unrelated enzymes. It is therefore extremely unlikely that they would also share a moonlighting function. We found that GALT-null flies expressing UGGP showed not only partial rescue of metabolic abnormalities and acute larval sensitivity to dietary galactose, as expected, but also full rescue of an adult motor deficit otherwise seen in this model. By extension, these results may offer insights to the underlying bases of at least some acute and long-term complications experienced by patients with CG.

Health and well-being of maturing adults with classic galactosemia.

Long-term outcomes in classic galactosemia (CG) have been studied previously, but all prior studies have relied on cohorts of patients that were small in number, or heavily skewed toward children and young adults, or both. Here, we extend what is known about the health and well-being of maturing adults with CG by analyzing the results of anonymous custom surveys completed by 92 affected individuals, ages 30-78, and 38 unaffected sibling controls, ages 30-79. The median age for patients was 38.5 years and for controls was 41 years. These study participants hailed from 12 different countries predominantly representing Europe and North America. Participants reported on their general life experiences and outcomes in seven different domains including: speech/voice/language, cognition, motor function, cataracts, bone health, psychosocial well-being, and gastrointestinal health. We also queried women about ovarian function. Our results indicated a prevalence of long-term complications across all outcome domains that aligned with levels previously reported in younger cohorts. Given the sample size and age range of participants in this study, these findings strongly suggest that the adverse developmental outcomes commonly linked to CG are not progressive with age for most patients. We also tested four candidate modifiers for possible association with each of the outcomes followed, including: days of neonatal milk exposure, rigor of dietary galactose restriction in early childhood, current age, and home continent. We observed no associations that reached even nominal significance, except for the following: cataracts with neonatal milk exposure (p = 2.347e-04), cataracts with age (p = 0.018), and bone health with home continent (p = 0.03).

The role of IL-1ß during human immunodeficiency virus type 1 infection.

Interleukin (IL)-1ß is a key innate cytokine that is essential for immune activation and promoting the inflammatory process. However, abnormal elevation in IL-1ß levels has been associated with unwanted clinical outcomes. IL-1ß is the most extensively studied cytokine among the IL-1 family of cytokines and its role in pathology is well established. During the course of human immunodeficiency virus type 1 (HIV-1) infection, the level of this proinflammatory cytokine is increased in different anatomical compartments, particularly in lymphatic tissues, and this elevation is associated with disease progression. The aim of this review is to address the pathological roles play by IL-1ß in the light of enhancing HIV-1 replication, driving immune cell depletion, and chronic immune activation. The role of IL-1ß in HIV-1 transmission (sexually or vertically 'from mother-to-child') will also be discussed. Additionally, the impact of the available antiretroviral therapy regimens on the levels of IL-1ß in HIV-1 treated patients is also discussed. Finally, we will provide a glance on how IL-1ß could be targeted as a therapeutic strategy.

A case report of classic galactosemia with a GALT gene variant and a literature review.

BACKGROUND: Galactosemia is an autosomal recessive disorder resulting from an enzyme defect in the galactose metabolic pathway. The most severe manifestation of classic galactosemia is caused by galactose-1-phosphate uridylyltransferase (GALT) deficiency, and this condition can be fatal during infancy if left untreated. It also may result in long-term complications in affected individuals. CASE PRESENTATION: This report describes a patient whose initial clinical symptoms were jaundice and liver dysfunction. The patient's liver and coagulation functions did not improve after multiple admissions and treatment with antibiotics, hepatoprotective and choleretic agents and blood transfusion. Genetic analysis revealed the presence of two variants in the GALT gene in the compound heterozygous state: c.377 + 2dup and c.368G > C (p.Arg123Pro). Currently, the variant locus (c.377 + 2dup) in the GALT gene has not been reported in the Human Gene Mutation Database (HGMD), while c.368G > C (p.Arg123Pro) has not been reported in the Genome Aggregation Database (GnomAD) nor the HGMD in East Asian population. We postulated that the two variants may contribute to the development of classical galactosemia. CONCLUSIONS: Applications of whole-exome sequencing to detect the two variants can improve the detection and early diagnosis of classical galactosemia and, more specifically, may identify individuals who are compound heterozygous with variants in the GALT gene. Variants in the GALT gene have a potential therapeutic significance for classical galactosemia.

O-GlcNAcylation determines the function of the key O-GalNAc glycosyltransferase C1GalT1 in bladder cancer.

Protein glycosylation is a type of protein post-translational modification. One specific example is the modification of proteins with O-linked ß-N-acetylglucosamine (O-GlcNAc) and O-linked α-N-acetylgalactosamine (O-GalNAc). Enhanced levels of both O-GalNAc and O-GlcNAc in bladder cancer (BlCa) have been reported previously. However, the interplay between O-GalNAc and O-GlcNAc has yet to be explored. Herein, we find that the expression level of core1 ß-1,3-galactosyltransferase (C1GalT1), which is responsible for extending and maturing mucin-type O-glycans, is increased in BlCa. This increase is accompanied by O-GlcNAc modification of C1GalT1. This modification stabilizes C1GalT1 expression and strengthens its interaction with its chaperone Cosmc. Mutation at Thr229 or Thr233 attenuates C1GalT1 stability and facilitates its degradation via the proteasome pathway. Furthermore, a decrease in C1GalT1 inhibits the pro-tumorigenic effect on bladder cancer cells by suppressing glycolysis.

Hydroxychloroquine ameliorates immune functionality and intestinal flora disorders of IgA nephropathy by inhibition of C1GALT1/Cosmc pathway.

BACKGROUND: Hydroxychloroquine (HCQ) has emerged as a potential and secure antiproteinuric agent in IgA nephropathy (IgAN). This study endeavored to explore the impact of HCQ on the immune functionality and intestinal flora disorders in IgAN rats, as well as to elucidate the underlying mechanisms through in vivo and in vitro experiments. METHODS: IgAN model was established in Sprague-Dawley rats through the administration of BSA, LPS, and CCl4, and the IgAN rats received a continuous 8-week treatment with HCQ. Moreover, the human glomerular mesangial cells (HMCs) were incubated with IgA1 to establish an in vitro cellular model of IgAN. At the end of experimental period, samples were collected for further analysis. RESULTS: HCQ ameliorated the elevated levels of 24hUTP, SCr, BUN, the number of urinary RBC, and the activation of inflammation-related proteins within the TLR4/NF-κB signaling pathway. In the IgAN rat group, there was a pronounced escalation in IgA deposition, mesangial matrix hyperplasia, and glomerular inflammatory cell infiltration, while the administration of HCQ effectively mitigated these pathological changes. In addition, the reduced production of CD4+CD25+Foxp3+ Treg in the IgAN group was effectively reversed by HCQ. Furthermore, HCQ has the capacity to restore the compromised state of the intestinal mucosal barrier induced by IgAN and mitigate the circumstances of intestinal permeability and disruption in the intestinal flora. CONCLUSION: HCQ diminishes IgA aberrant glycosylation levels, ameliorates renal and intestinal histopathological damage, and attenuates intestinal flora disorders and immune dysfunction in IgAN rats by means of activating the C1GALT1/Cosmc pathway.

Demonstrating the utility of sugar-phosphate phosphatases in coupled enzyme assays: galactose-1-phosphate uridylyltransferase as proof-of-concept.

During our biochemical characterization of select bacterial phosphatases belonging to the haloacid dehalogenase superfamily of hydrolases, we discovered a strong bias of Salmonella YidA for glucose-1-phosphate (Glc-1-P) over galactose-1-phosphate (Gal-1-P). We sought to exploit this ability of YidA to discriminate these two sugar-phosphate epimers in a simple coupled assay that could be a substitute for current cumbersome alternatives. To this end, we focused on Gal-1-P uridylyltransferase (GalT) that is defective in individuals with classical galactosemia, an inborn disorder. GalT catalyzes the conversion of Gal-1-P and UDP-glucose to Glc-1-P and UDP-galactose. When recombinant YidA was coupled to GalT, the final orthophosphate product (generated from selective hydrolysis of Glc-1-P by YidA) could be easily measured using the inexpensive malachite green reagent. When this new YidA-based colorimetric assay was benchmarked using a recombinant Duarte GalT variant, it yielded kcat/Km values that are ~2.5-fold higher than the standard coupled assay that employs phosphoglucomutase and glucose-6-phosphate dehydrogenase. Although the simpler design of our new GalT coupled assay might find appeal in diagnostics, a testable expectation, we spotlight the GalT example to showcase the untapped potential of sugar-phosphate phosphatases with distinctive substrate-recognition properties for measuring the activity of various metabolic enzymes (e.g. trehalose-6-phosphate synthase, N-acetyl-glucosamine-6-phosphate deacetylase, phosphofructokinase).

Novel GALT variations and genetic spectrum in Turkish population with the correlation of genotype and phenotype.

Classic galactosemia (OMIM#230400) is an autosomal recessive inborn error of carbohydrate metabolism caused by a deficiency of the galactose-1-phosphate-uridyl-transferase enzyme encoded by the GALT gene. Even though a galactose-restricted diet efficiently resolves the acute complications, it is insufficient to prevent long-term complications regarding speech defects, intellectual functioning, premature ovarian failure, cataract, hepatomegaly, dysarthria, ataxia, and tremor. Seventy-seven patients who were genetically diagnosed with classic galactosemia were included in this cohort. Identified novel variants were classified based on their predicted effect on the GALT function. Further, potential genotype-phenotype correlations were investigated via statistical analysis. In total, 18 different sequence variants were identified, including four novels (c.200delG/p.(Arg67Profs* 19), c.533T>G/ p.(Met178Arg), c.708_709delGT/p.(Ser236Argfs* 30), c.467C>A/p.(Ser156* )). Jaundice was the most common short-term finding with 80% (61/77). Even with early diagnosis, intellectual disability is encountered with 36% (27/74) of the long-term complications. Patients with biallelic missense variants have a significantly higher prevalence of cataracts (OR: 17.9). Longitudinal observations showed attenuation of cataracts and hepatomegaly. This study has shown the GALT variation spectrum of the Turkish population with a 30-year retrospective cohort, submitting a significant contribution to the genotype/phenotype correlation in galactosemia. This study also highlights the cost-effective importance of Sanger sequencing in the diagnosis of single-gene metabolic diseases.

Impact of Early ARV Initiation on Relative Proportions of Effector and Regulatory CD8 T Cell in Mesenteric Lymph Nodes and Peripheral Blood During Acute SIV Infection of Rhesus Macaques.

CD8 T cells are key players in the clearance of human immunodeficiency virus (HIV)-infected cells, such that CD8 T-cell dysfunction contributes to viral persistence despite antiretroviral (ARV) therapy. Mesenteric lymph nodes (MLNs) are major sites of gut mucosal immunity. While different CD8 T cell subsets such as CD8 alpha-alpha (CD8αα), CD8 alpha-beta (CD8αß), CD8 regulatory T cells (Treg), and mucosa-associated invariant T cells (MAIT) are present in the gut and exhibit distinct functions, their dynamics remain poorly understood due to the lack of accessibility to these tissues in humans. We thus assessed CD8 T cells in MLNs versus peripheral blood in simian immunodeficiency virus (SIV)-infected rhesus macaques (RMs) following early ARV therapy initiation. SIV infection was associated with an increase over time of both CD8αß and CD8αα T cells in the blood and MLNs, whereas early ARV initiation significantly decreased the frequencies of CD8αα but not CD8αß T cells in MLNs. A significant decrease in the expression of chemokine receptors CCR6 and CXCR3 by CD8 T cells, which are essential for T-cell trafficking to the inflammatory sites, was observed in chronically SIV-infected RMs. Surprisingly, while MAIT cells are increased in ARV-treated RMs, their frequencies in MLN are extremely low and were not impacted by ARV. The acute infection resulted in an early CD39+FoxP3+ CD8 Tregs increase in both compartments, which was normalized after early ARV. Frequencies of CD8 Treg cells were positively correlated with frequencies of CD4 Tregs and accordingly negatively correlated with the Th17/Treg ratio in the blood but not in MLNs. Overall, our results underscore the difference in CD8 T-cell subset dynamics in the blood and MLNs. IMPORTANCE Changes in CD8 T-cell subsets during acute SIV/HIV infections and following early ARV initiation in gut lymphoid tissues are poorly understood. Using an acute SIV infection model in rhesus macaques, we assessed the impact of early ARV, initiated 4 days postinfection, on relative proportions of CD8 T-cell subsets in MLNs compared to blood. We found that acute SIV infection and early ARV initiation differentially affect the distribution of effector CD8 T cells, CD8 MAIT cells, and CD8 Tregs in MLNs compared to blood. Overall, early ARV initiation maintains the frequency of effector CD8 T cells while reducing immunosuppressive CD39+ CD8 Tregs. Our study provides deeper insight into the dynamics of the CD8 T-cell compartment in gut mucosal immune surveillance during acute SIV infection and following early ARV initiation.

Development, optimization and validation of LC-MS/MS method for the determination of DBS GALT enzyme activity.

Galactosemia is a carbohydrate metabolism disorder often caused by galactose-1-phosphate uridyl transferase (GALT) deficiency. Detecting GALT deficiency involves measuring intra-erythrocyte enzyme activity. We aimed to create a robust liquid chromatography-mass spectrometry (LC-MS/MS) method to assess GALT activity in dried blood spot (DBS) samples. We validated this method and compared it to the fluorometric approach. We investigated the impact of K2EDTA and lithium heparin tubes on enzyme activity to identify the best sample collection tube. We also assessed the reaction-stopping method. The developed approach employed [13C6]-galactose-1-phosphate as a substrate and UDP-N-acetylglycosamine as an internal standard (IS). The mean ± SD value for GALT activity of DBS samples was determined as 6.37 ± 1.96 µmol/gHb/hour. The linear range was 0.4-50 µM (2.4-310% of normal) in the DBS method. The % coefficient of variation (%CV) values were less than 15 for intra-day and inter-day repeatability studies. Over 90% recovery was achieved in recovery studies, and no ion suppression from matrix was detected. DBS samples were quite stable for 31 days under different storage conditions. Enzyme activity results reported as <3.5 U/g Hb by fluorometric method, were quantitatively determined for even very low concentrations by LC-MS/MS method.

Maternal Leukocytes and Infant Immune Programming during Breastfeeding.

The fetal immune system develops in a rather sterile environment relative to the outside world and, therefore, lacks antigenic education. Soon after birth, the newborn is exposed to the hostile environment of pathogens. Recently, animal- and limited human-based studies have indicated that help from the mother, upon transfer of leukocytes and their products via breast milk feeding, greatly assists the newborn's immune system. Here, I discuss the newest advances on how milk leukocytes impact early life immunity, with an emphasis on the development of the infant T cell repertoire and early immune responses in the periphery and gut-associated lymphoid tissue. A deeper understanding of these novel mechanistic insights may inform potential translational approaches to improving immunity in infants.

Intranasal administration of ß-1, 3-galactosyltransferase 2 confers neuroprotection against ischemic stroke by likely inhibiting oxidative stress and NLRP3 inflammasome activation.

Ischemic stroke is one of the major causes of morbidity and mortality. The ß-1, 3-galactosyltransferase 2 (B3galt2), a member of ß-1, 3-galactosyltransferase family, is playing a vital role in the pathological process of cerebral ischemic injury, but its underlying mechanisms remain unclear. In the present study, we examined the involvement of oxidative stress and NLRP3 inflammasome activation in the neuroprotective effect of B3galt2. Cerebral ischemia/reperfusion (I/R) injury was simulated in a mouse middle cerebral artery occlusion (MCAO) model. Recombinant human B3galt2 (rh-B3galt2) was administered intranasally 1 h post MCAO, and TGF-ß1-siRNA was administered intracerebroventricularly 24 h before MCAO. Outcome measures included brain infarct volume, neurological function, blood-brain barrier (BBB) permeability, neuronal apoptosis, oxidative stress, and the inflammatory response. First, we found that rh-B3galt2 significantly alleviated brain infarct volume and BBB permeability, improved neurological function, and attenuated I/R-induced neuron apoptosis and oxidative stress. Furthermore, rh-B3galt2 attenuated pro-inflammatory cytokines, NF-κB, IL-6, TNF-α, and IL-1ß, and inhibited NLRP3 inflammasome activation. Finally, inhibition of TGF-ß1 by TGF-ß1-siRNA abolished the anti-oxidative and anti-inflammatory effects of rh-B3galt2 in mice after I/R. Collectively, our study demonstrated that rh-B3galt2 exerts neuroprotective effects by regulating cerebral ischemia-induced oxidative stress and NLRP3 inflammasome, which is mainly dependent on the heightening of the TGF-ß1 pathway. Thus, B3galt2 might be considered a new therapeutic target for ischemic stroke treatment.

UGCG modulates heart hypertrophy through B4GalT5-mediated mitochondrial oxidative stress and the ERK signaling pathway.

Mechanical pressure overload and other stimuli often contribute to heart hypertrophy, a significant factor in the induction of heart failure. The UDP-glucose ceramide glycosyltransferase (UGCG) enzyme plays a crucial role in the metabolism of sphingolipids through the production of glucosylceramide. However, its role in heart hypertrophy remains unknown. In this study, UGCG was induced in response to pressure overload in vivo and phenylephrine stimulation in vitro. Additionally, UGCG downregulation ameliorated cardiomyocyte hypertrophy, improved cardiomyocyte mitochondrial oxidative stress, and reduced the ERK signaling pathway. Conversely, UGCG overexpression in cardiomyocytes promoted heart hypertrophy development, aggravated mitochondrial oxidative stress, and stimulated ERK signaling. Furthermore, the interaction between beta-1,4-galactosyltransferase 5 (B4GalT5), which catalyses the synthesis of lactosylceramide, and UGCG was identified, which also functions as a synergistic molecule of UGCG. Notably, limiting the expression of B4GalT5 impaired the capacity of UGCG to promote myocardial hypertrophy, suggesting that B4GalT5 acts as an intermediary for UGCG. Overall, this study highlights the potential of UGCG as a modulator of heart hypertrophy, rendering it a potential target for combating heart hypertrophy.