Glycosphingolipid Biosynthesis Research Articles

P-257 Transcriptomic signature of live birth revealed in cumulus cells of women undergoing an ICSI cycle for infertility treatment

Abstract Study question Is the transcriptome of cumulus cells surrounding an oocyte that was fertilised and transferred predictive of live birth? Summary answer Live birth is associated with a distinct transcriptomic signature in cumulus cells isolated from an oocyte that was fertilised and transferred. What is known already The selection of the best embryo for transfer is currently based on standard morphological assessment, which is intrinsically subjective, and time-lapse monitoring, which fails to show benefit in predicting pregnancy. Alternative non-invasive selection methods are in development including analysis of cumulus cells (CCs) isolated from oocytes that resulted in an eligible embryo for transfer. However the majority of the existing studies were conducted using outdated methodologies such as transcriptomic analysis with PCR and microarrays. Moreover, the studies associating CCs’ transcriptome with clinical and embryological outcomes lack consensus due to non-homogenous cohorts while the outcome of live birth is inadequately explored. Study design, size, duration CC samples (n = 17), collected between 2018 and 2021 and biobanked, were retrospectively selected for RNA sequencing on the basis of their embryo transfer outcome. Thus, we analysed CCs associated with an oocyte that resulted in pregnancy (n = 6), no pregnancy (n = 7), live birth (n = 5) and pregnancy without live birth (n = 5). Differential gene expression analysis identified the differentially expressed genes (DEGs) between the groups in relation to pregnancy and live birth. Participants/materials, setting, methods The samples were retrieved from subfertile couples undergoing controlled ovarian stimulation and intracytoplasmic sperm injection with subsequent unbiopsied fresh or frozen embryo transfer at the IVF centre of University Hospital Zurich. Maternal age was below 43 years. Libraries from CCs were prepared using the Smart-seq2 pipeline and the RNA sequencing was performed on the NovaSeq 6000 system. The DESeq2 workflow was conducted and the threshold for the adjusted p-values (FDR) was set to 0.05. Main results and the role of chance No DEGs emerged when comparing the transcriptomic profiles of cumulus cells between the “no pregnancy” and “pregnancy” groups. However, 139 DEGs were identified in the comparison between the “pregnancy without live birth” and “live birth” groups. 28 DEGs were familiar and relevant to competent cumulus-oocyte-complexes: CTGF (fold change: 4.38, FDR: 0.005), SERPINE2 (fc: 3.08, FDR: 0.0002), PCK1 (fc:3.04, FDR: 0.0001), HHIP (fc:2.69, FDR: 0.006), HS3ST (fc:2.45, FDR: 0.009) and BIRC5 (fc: -2.69, FDR 0.0001) among others. Functional enrichment analysis of DEGs revealed that the incidence of live birth is associated with promotion of pathways such as runt-related transcription factor 1 (RUNX1/2) signalling, glycosaminoglycan biosynthesis and sonic hedgehog signalling (SHH), and reduction in pathways such as glycosphingolipid biosynthesis and pyrimidine metabolism in cumulus cells. Limitations, reasons for caution We reveal the transcriptome associated with live birth in CCs analysed with RNAseq on a limited number of samples. Consolidation of these findings via utilisation of a validation prospective cohort is pending. Wider implications of the findings CCs’ transcriptome could inform the development of non-invasive biomarkers to predict the chance of live birth across a group of embryos eligible for transfer. The transcriptomic findings inspire further explorations in the field of cumulus-oocyte bidirectional communication and establishment of oocyte competence. Trial registration number N/A

A BRAF mutation-associated gene risk model for predicting the prognosis of melanoma

BRAF mutation plays an important role in the pathogenesis and progression of melanoma and is correlated to the prognosis of melanoma patients. However, fewer studies have attempted to develop a BRAF mutation-associated gene risk model for predicting the prognosis of melanoma. The current research explores BRAF mutation-related biological features in melanoma and establishes a prognostic signature. First, we identified three significantly enriched KEGG pathways (glycosphingolipid biosynthesis - ganglio series, ether lipid metabolism, and glycosaminoglycan biosynthesis - keratan sulfate) and corresponding genes in the BRAF mutant group by gene set enrichment analysis. We then developed a prognostic signature based on 7 BRAF-associated genes (PLA2G2D, FUT8, PLA2G4E, PLA2G5, PLA2G1B, B3GNT2, and ST3GAL5) and assessed its prediction accuracy using ROC curve analysis. Finally, the nomogram was established according to the prognostic signature and independent clinical characteristics to predict the survival of melanoma patients. Furthermore, we found higher proportions of naive B cells, plasma cells, CD8 T cells, CD4 memory-activated T cells, and regulatory T cells in the low-risk group. Whereas lower proportions of M0, M1, and M2 macrophages and resting NK cells were observed in the high-risk group. The analysis also showed a significantly higher expression of immune checkpoint molecules (PD-1, PD-L1, CTLA4, BTLA, CD28, CD80, CD86, HAVCR2, ICOS, LAG3, and TIGIT) in the low-risk group. Our results provide novel insights into the effect of BRAF mutation on melanoma growth and indicate a promising direction toward immunotherapy and precision medicine in melanoma patients.

Plasma metabolites associated with clinically diagnosed Alzheimer’s Disease and the association is augmented using blood-based biomarkers. (P10-6.003)

<h3>Objective:</h3> Identify metabolites associated with clinical diagnosis and pathology of Alzheimer’s Disease (AD) <h3>Background:</h3> We investigated metabolites in plasma to capture systemic biochemical changes associated with AD diagnosed clinically and then using plasma biomarkers. <h3>Design/Methods:</h3> Exogenous and endogenous metabolites in plasma were measured in 300 clinical AD and 430 healthy non-demented individuals of Caribbean Hispanic ancestry using untargeted liquid-chromatography, performed on HILIC (+ ionization) and C18 (− ionization) columns, coupled to a Thermo Orbitrap HF-X mass spectrometer. Genome-wide SNP data and plasma biomarkers including Ab40, Ab42, P-tau181 and Neurofilament light chain (NfL) were obtained on all participants. Metabolite association with AD and P-tau181 levels were tested after adjustment for age and sex. Metabolites associated with P-tau181 levels (but not with NfL, a non-specific marker of neurodegeneration) were categorized as AD-specific. <h3>Results:</h3> Over 4000 metabolomic features were measured with high accuracy in the sample. 96 metabolites were associated with P-tau181 levels, but no metabolites survived multiple testing correction for association with clinical AD alone. Features putatively annotated as phosphatidylcholine (4.11E-07) and Carotamine (p=1.2e-05) were strongly associated P-tau181 levels. We observed similar results when using a diagnostic cutoff of P-tau181 levels for AD as the outcome. Metabolites associated with P-tau181 levels were enriched in amino acid metabolism, glutathione metabolism, tryrosine metabolism and hexose phosphorylation pathways. Ramipril (p=2.9e-04) and glycerophosphocholine (p=9.9e-04) were among the top associated with AD diagnosis. PLS-DA analysis identified biopterin metabolism, glycosphingolipid biosynthesis and tyrosine metabolism as the most discriminating pathways between AD and healthy controls when augmented by biomarkers. <h3>Conclusions:</h3> Metabolites specifically lipids and those involved in cardiovascular function were associated with AD and P-tau181 levels. Metabolite profiling can identify perturbed pathways in clinical and pre-clinical AD and integration with genome-wide SNP data will identify mechanisms underlying genetic association with disease. <b>Disclosure:</b> Dr. Kalia has nothing to disclose. Mrs. Reyes-Dumeyer has nothing to disclose. Mr. DUBEY has nothing to disclose. Dr. Nandakumar has nothing to disclose. Dr. Lee has nothing to disclose. Rafael Lantigua has nothing to disclose. Dr. Medrano has nothing to disclose. Dr. Rivera Mejia has nothing to disclose. Dr. Honig has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Eisai. Dr. Honig has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Biogen. Dr. Honig has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Medscape. Dr. Honig has received personal compensation in the range of $5,000-$9,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Prevail. Dr. Honig has received personal compensation in the range of $10,000-$49,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Cortexyme. Dr. Honig has received personal compensation in the range of $10,000-$49,999 for serving as an Expert Witness for Legal Firms . The institution of Dr. Honig has received research support from Roche. The institution of Dr. Honig has received research support from Eisau. Dr. Honig has received research support from Alector. The institution of Dr. Honig has received research support from Transposon. Dr. Mayeux has nothing to disclose. Badri Vardarajan has received personal compensation in the range of $10,000-$49,999 for serving as a Consultant for Kodikaz Therapeutics.

Abstract 2042: Graph-based pathway analysis of T cell populations in hepatocellular carcinoma reveals novel metabolic regulators of tumor-infiltration lymphocyte activity

Abstract With advances in single cell RNA sequencing (scRNAseq), accurate detection of perturbed pathways between conditions or cell types of interest becomes a critical analytical step. The ability to model alterations in a set of genes corresponding of a biological function is particularly useful when comparing cells between healthy and tumor tissues. Yet, few tools exist to detect changes in the multivariate distribution of genes corresponding to a given pathway. We developed a novel, graph-based statistical framework based on optimal matching for testing differential distribution of biological pathways in scRNAseq. We applied our method to data generated from &amp;gt;4,000 T cells isolated from six individuals with hepatocellular carcinoma (HCC). The T cell populations were purified from three tissue locations: peripheral blood, tumor-infiltrating immune cells (TIICs), and normal tissue adjacent to the tumor. We examined the distribution of gene sets that belong to a particular metabolic pathway across T cell subtypes to address the following questions: (1) Which pathways have a similar distribution across T cell subtypes in a given tissue? (2) Are there pathways that have a stable distribution across T cell subtypes in a normal/healthy tissue, but a perturbed distribution in HCC? (3) For pathways that have a disparate distribution across the T cell subtypes, which subtypes show the most distinct distribution? Of the 86 metabolic pathways compared, 41, 63 and 76 metabolic pathways were indistinguishable across T cell subtypes in the tumor, peripheral blood and adjacent normal tissues, respectively. We observed that most metabolic pathways do not show evidence for dissimilar distribution across cell types, suggesting that T cell subtypes might be more similar than previously appreciated in terms of how they regulate their basic metabolic machinery. Further, for each pair of tissue locations, we computed the overlap in perturbed pathways, and found that the concordance was substantially higher for blood and adjacent normal, than what either of these tissues had with the tumor tissue. Interestingly, five metabolic pathways were differentially distributed across the T cell subtypes in each tissue examined in HCC: glycolysis, purine metabolism, glycosphingolipid biosynthesis, pyruvate metabolism, and glycerophospholipid metabolism. Our model also found that CD4+ regulatory T cells were the strongest contributors, driving the differential distribution of these pathways between T cell subtypes in HCC. Altogether, our approach allows for a systems level characterization of pathway activity across multiple cell types with a variety of applications in single cell pathway analysis in oncology. Our work here also highlights unexpected regulatory mechanisms of regulatory T cells that might play in role in the immunobiology of HCC. Citation Format: Divyansh Agarwal, Tina Bharani, Somabha Mukherjee. Graph-based pathway analysis of T cell populations in hepatocellular carcinoma reveals novel metabolic regulators of tumor-infiltration lymphocyte activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2042.

Cell type- and region-specific translatomes in an MPTP mouse model of Parkinson's disease

Parkinson's disease (PD) is the most common neurodegenerative movement disorder, characterized by the progressive loss of nigrostriatal dopaminergic neurons (DANs), involving the dysregulation of both neurons and glial cells. Cell type- and region-specific gene expression profiles can provide an effective source for revealing the mechanisms of PD. In this study, we adopted the RiboTag approach to obtain cell type (DAN, microglia, astrocytes)- and brain region (substantia nigra, caudate-putamen)-specific translatomes at an early stage in an MPTP-induced mouse model of PD. Through DAN-specific translatome analysis, the glycosphingolipid biosynthetic process was identified as a significantly downregulated pathway in the MPTP-treated mice. ST8Sia6, a key downregulated gene related to glycosphingolipid biosynthesis, was confirmed to be downregulated in nigral DANs from postmortem brains of patients with PD. Specific expression of ST8Sia6 in DANs exerts anti-inflammatory and neuroprotective effects in MPTP-treated mice. Through cell type (microglia vs. astrocyte) and brain region (substantia nigra vs. caudate-putamen) comparisons, nigral microglia showed the most intense immune responses. Microglia and astrocytes in the substantia nigra showed similar levels of activation in interferon-related pathways and interferon gamma (IFNG) was identified as the top upstream regulator in both cell types. This work highlights that the glycosphingolipid metabolism pathway in the DAN is involved in neuroinflammation and neurodegeneration in an MPTP mouse model of PD and provides a new data source for elucidating the pathogenesis of PD.

Glucosylceramide is essential for Heartland and Dabie bandavirus glycoprotein-induced membrane fusion.

Due to climate changes, there has been a large expansion of emerging tick-borne zoonotic viruses, including Heartland bandavirus (HRTV) and Dabie bandavirus (DBV). As etiologic agents of hemorrhagic fever with high fatality, HRTV and DBV have been recognized as dangerous viral pathogens that likely cause future wide epidemics. Despite serious health concerns, the mechanisms underlying viral infection are largely unknown. HRTV and DBV Gn and Gc are viral surface glycoproteins required for early entry events during infection. Glycosphingolipids, including galactosylceramide (GalCer), glucosylceramide (GlcCer) and lactosylceramide (LacCer), are a class of membrane lipids that play essential roles in membrane structure and viral lifecycle. Here, our genome-wide CRISPR/Cas9 knockout screen identifies that glycosphingolipid biosynthesis pathway is essential for HRTV and DBV infection. The deficiency of UDP-glucose ceramide glucosyltransferase (UGCG) that produces GlcCer resulted in the loss of infectivity of recombinant viruses pseudotyped with HRTV or DBV Gn/Gc glycoproteins. Conversely, exogenous supplement of GlcCer, but not GalCer or LacCer, recovered viral entry of UGCG-deficient cells in a dose-dependent manner. Biophysical analyses showed that GlcCer targeted the lipid-head-group binding pocket of Gc to form a stable protein-lipid complex, which allowed the insertion of Gc protein into host lysosomal membrane lipid bilayers for viral fusion. Mutagenesis showed that D841 residue at the Gc lipid binding pocket was critical for GlcCer interaction and thereby, viral entry. These findings reveal detailed mechanism of GlcCer glycosphingolipid in HRTV and DBV Gc-mediated membrane fusion and provide a potential therapeutic target for tickborne virus infection.

Composition of the intestinal microbiota of infant rhesus macaques at different ages before and after weaning

BackgroundRhesus macaques and humans are closely related genetically and share similar physiological and pathological characteristics. Exploring the impact of diet on the early establishment of gut microbiota in non-human primates can provide relevant clinical models for healthy infant growth and development. At present, few writers have focused on the composition and changes of the intestinal microbes of infant rhesus macaques throughout their progression from birth to formula feeding after weaning. In this study, we used 16S rRNA sequencing technology to explore the composition of the intestinal flora of rhesus macaques at different ages and analyzed the trends in the microbial changes. ResultsThe results showed that the relative abundance of Bifidobacterium and Lactobacillus in the intestinal flora of infant rhesus macaques significantly decreased, and Prevotella increased with age. Bifidobacterium longum and Bifidobacterium breve are effective biomarkers to predict grouping. The metabolic pathways enriched in early life mainly concentrated in glycosphingolipid biosynthesis (lacto and neolacto series) and the degradation and metabolism of alcohols and esters. ConclusionsWe found that age was an important factor that affected the changes in the intestinal flora. This study revealed the change trend of flora in breastfed and formula-fed infant rhesus monkeys in different growth months, and found that the dominant flora changed greatly. This research provides a medically relevant theoretical basis for understanding the healthy development of infants.

Glycolysis regulates KRAS plasma membrane localization and function through defined glycosphingolipids

Oncogenic KRAS expression generates a metabolic dependency on aerobic glycolysis, known as the Warburg effect. We report an effect of increased glycolytic flux that feeds into glycosphingolipid biosynthesis and is directly linked to KRAS oncogenic function. High resolution imaging and genetic approaches show that a defined subset of outer leaflet glycosphingolipids, including GM3 and SM4, is required to maintain KRAS plasma membrane localization, with GM3 engaging in cross-bilayer coupling to maintain inner leaflet phosphatidylserine content. Thus, glycolysis is critical for KRAS plasma membrane localization and nanoscale spatial organization. Reciprocally oncogenic KRAS selectively upregulates cellular content of these same glycosphingolipids, whose depletion in turn abrogates KRAS oncogenesis in pancreatic cancer models. Our findings expand the role of the Warburg effect beyond ATP generation and biomass building to high-level regulation of KRAS function. The positive feedforward loop between oncogenic KRAS signaling and glycosphingolipid synthesis represents a vulnerability with therapeutic potential.

Analysis of the mechanism of Buyang Huanwu Decoction against cerebral ischemia-reperfusion by multi-omics

Ethnopharmacological relevanceBuyang Huanwu Decoction (BYHW) is a classic representative formula for treating qi deficiency and the blood stasis syndrome of stroke in the Qing Dynasty physician Wang Qingren's Correction on the Errors of Medical Works. However, the research on the mechanism of BYHW in the treatment of stroke is not systematic and comprehensive. Aim of the studyCombined with multi-omics analysis methods to explore the potential targets of BYHW in the treatment of cerebral ischemia-reperfusion (I/R). Materials and methodsThe rat middle cerebral artery occlusion (MCAO) model was established to study the effect of BYHW on cerebral I/R injury in rats. Then, the potential targets and pathways of BYHW in the treatment of cerebral I/R injury were analyzed by proteomic, transcriptomic, and metabolomic methods. Finally, 4D-PRM was used to validate potential targets. ResultsBYHW effectively improved the neurological function scores of MCAO rats and significantly reduced the rate of cerebral infarction in MCAO rats. Multi-omics analysis had identified 15 potential targets and 4 potential signaling pathways. The results of 4D-PRM targeted proteomics verification showed that Pde1b was reversed up-regulated, and Aprt, Gpd1, Glb1, HEXA, and HEXB were reversed down-regulated. ConclusionBYHW may improve cerebral I/R through Aprt, Pde1b, Gpd1, Glb1, HEXA and HEXB targets, and Glycerophospholipid metabolism, Purine metabolism and Glycosphingolipid biosynthesis - globoseries pathway.

A sensitive method for determining UDP-glucose: ceramide glucosyltransferase (UGCG) activity in biological samples using deuterated glucosylceramide as acceptor substrate.

Glucosylceramide synthase (UGCG) is a key enzyme in the biosynthesis of glycosphingolipids and its activity is related to the resistance to anticancer drugs and is involved in the derangement of metabolism in various diseases. Moreover, UGCG acts as a major controller of the balanced levels of individual brain sphingolipids that may trigger neurodegeneration in Gaucher disease and in Parkinson disease associated to pathogenic variants in the glucocerebrosidase-encoding gene GBA. We have developed an effective method for determining UGCG activity in vitro using deuterated ceramide as an acceptor, and quantitation of the formed deuterated glucosylceramide by liquid chromatography coupled with tandem mass spectrometry. The method enabled us to determine the kinetic parameters of UGGC and the effect of the inhibitor GZ667161 on the enzyme activity expressed in model cells, as well as to measure UGCG specific activity in human fibroblasts using a simple crude cell homogenate. This novel approach may be useful in determining the actual UGCG activity levels in patient cells and tissues of animal models of diseases, and to study novel drugs targeting glycosphingolipid metabolism.

ST3GAL5‐catalyzed gangliosides inhibit TGF‐β‐induced epithelial‐mesenchymal transition via TβRI degradation

Epithelial‐mesenchymal transition (EMT) is pivotal in the initiation and development of cancer cell metastasis. We observed that the abundance of glycosphingolipids (GSLs), especially ganglioside subtypes, decreased significantly during TGF‐β‐induced EMT in NMuMG mouse mammary epithelial cells and A549 human lung adenocarcinoma cells. Transcriptional profiling showed that TGF‐β/SMAD response genes and EMT signatures were strongly enriched in NMuMG cells, along with depletion of UDP‐glucose ceramide glucosyltransferase (UGCG), the enzyme that catalyzes the initial step in GSL biosynthesis. Consistent with this finding, genetic or pharmacological inhibition of UGCG promoted TGF‐β signaling and TGF‐β‐induced EMT. UGCG inhibition promoted A549 cell migration, extravasation in the zebrafish xenograft model, and metastasis in mice. Mechanistically, GSLs inhibited TGF‐β signaling by promoting lipid raft localization of the TGF‐β type I receptor (TβRI) and by increasing TβRI ubiquitination and degradation. Importantly, we identified ST3GAL5‐synthesized a‐series gangliosides as the main GSL subtype involved in inhibition of TGF‐β signaling and TGF‐β‐induced EMT in A549 cells. Notably, ST3GAL5 is weakly expressed in lung cancer tissues compared to adjacent nonmalignant tissues, and its expression correlates with good prognosis.

Proteomic profiling of cerebrospinal fluid in pediatric myelin oligodendrocyte glycoprotein antibody-associated disease

BackgroundMyelin oligodendrocyte glycoprotein (MOG) antibody-associated disease (MOGAD) is an autoimmune demyelinating disorder of the central nervous system.MethodsExtracted proteins from 34 cerebrospinal fluid (CSF) samples [patients with MOGAD (MOG group, n = 12); healthy controls (HC group, n = 12); patients with MOG seronegative and metagenomics next-generation sequencing-negative inflammatory neurological diseases (IND group, n = 10)] were processed and subjected to label-free quantitative proteomics. Supervised partial least squares-discriminant analysis (PLS-DA) and orthogonal PLS-DA (O-PLS-DA) models were also performed based on proteomics data. Functional analysis of differentially expressed proteins (DEPs) was performed using Gene Ontology, InterPro, and Kyoto Encyclopedia Genes and Genomes. An enzyme-linked immunosorbent assay was used to determine the complement levels in serum from patients with MOGAD.ResultsFour hundred and twenty-nine DEPs (149 upregulated and 280 downregulated proteins) were identified in the MOG group compared to the HC group according to the P value and fold change (FC). Using the O-PLS-DA model, 872 differentially abundant proteins were identified with variable importance projection (VIP) scores > 1. Five proteins (gamma-glutamyl hydrolase, cathepsin F, interalpha-trypsin inhibitor heavy chain 5, latent transforming growth factor beta-binding protein 4 and leukocyte-associated immunoglobulin-like receptor 1) overlapping between the top 30 DEPs with top-ranked P value and FC and top 30 proteins in PLS-DA VIP lists were acquired. Functional analysis revealed that the dysregulated proteins in the MOG group were primarily involved in complement and coagulation cascades, cell adhesion, axon guidance, and glycosphingolipid biosynthesis compared to the HC group.ConclusionThe proteomic alterations in CSF samples from children with MOGAD identified in the current study might provide opportunities for developing novel biomarker candidates.

Analyzing the Therapeutic Mechanism of Mongolian Medicine Zhonglun-5 in Rheumatoid Arthritis Using a Bagging Algorithm with Serum Metabonomics.

Rheumatoid arthritis (RA) is a complex autoimmune disorder. Zhonglun-5 (ZL), a traditional Mongolian medicine, exhibits an excellent clinical effect on RA; however, its molecular mechanism remains unclear. In this study, rat serum metabolomic analysis was performed to identify potential biomarkers for RA and investigate its treatment mechanism. A Dionex Ultimate 3000 ultrahigh-performance liquid chromatography system coupled with a Q-Exactive Focus Orbitrap mass spectrometer was used for metabonomics analysis. Bootstrap aggregation (bagging) classification algorithm was applied to process data from control (CG), model (MG), and treatment administration groups. The classification accuracy was 100.00% (6/6) in the decision tree model and 83.33% (5/6) in the K-nearest neighbor (KNN) model, accompanied by 18 training samples and 6 testing samples. Using volcanic map analysis, 24 biomarkers were identified between CG and MG, including those related to glycosphingolipid biosynthesis, arachidonic acid, fatty acids, amino acids, bile acids, vitamins, and sphingolipids. A set diagram of the heatmap and drug-biomarker network of potential biomarkers was constructed. After ZL administration, the levels of these biomarkers returned to normal, indicating that ZL had a therapeutic effect in rats with RA. This study established a solid theoretical foundation to promote further research on the clinical applicability of ZL.

Effect and Mechanism Analysis of Pig FUT8 Gene on Resistance to Escherichia coli F18 Infection.

Post-weaning diarrhea caused by enterotoxigenic Escherichia coli F18 (E. coli F18) causes significant economic losses for pig producers. Fucosyltransferase 8 (FUT8) is a glycosyltransferase that catalyzes core fucosylation; however, its role in mediating the resistance to E. coli F18 infection in pigs remains unknown. In this study, we systematically verified the relationship between FUT8 expression and E. coli resistance. The results showed that FUT8 was expressed in all detected tissues of Meishan piglets and that its expression was significantly increased in the duodenum and jejunum of E. coli F18-sensitive individuals when compared to E. coli F18-resistant individuals. FUT8 expression increased after exposure to E. coli F18 (p < 0.05) and decreased significantly after LPS induction for 6 h (p < 0.01). Then, the IPEC-J2 stable cell line with FUT8 interference was constructed, and FUT8 knockdown decreased the adhesion of E. coli F18ac to IPEC-J2 cells (p < 0.05). Moreover, we performed a comparative transcriptome study of IPEC-J2 cells after FUT8 knockdown via RNA-seq. In addition, further expression verification demonstrated the significant effect of FUT8 on the glycosphingolipid biosynthesis and Toll-like signaling pathways. Moreover, the core promoter of FUT8, which was located at −1213 bp to −673 bp, was identified via luciferase assay. Interestingly, we found a 1 bp C base insertion mutation at the −774 bp region, which could clearly inhibit the transcriptional binding activity of C/EBPα to an FUT8 promoter. Therefore, it is speculated that FUT8 acts in a critical role in the process of E. coli infection; furthermore, the low expression of FUT8 is conducive to the enhancement of E. coli resistance in piglets. Our findings revealed the mechanism of pig FUT8 in regulating E. coli resistance, which provided a theoretical basis for the screening of E. coli resistance in Chinese local pig breeds.

Serum Metabolomics of Benign Essential Blepharospasm Using Liquid Chromatography and Orbitrap Mass Spectrometry.

Benign essential blepharospasm (BEB) is a form of focal dystonia that causes excessive involuntary spasms of the eyelids. Currently, the pathogenesis of BEB remains unclear. This study is aimed at investigating the serum metabolites profiles in patients with BEB and healthy control and to identify the mechanism and biomarkers of this disease. 30 patients with BEB and 33 healthy controls were recruited for this study. We conducted the quantitative and nontargeted metabolomics analysis of the serum samples from 63 subjects by using liquid chromatography and Orbitrap mass spectrometry (LC-Orbitrap MS). Multivariate statistical analysis was performed to detect and identify different metabolites between the two groups. The Kyoto Encyclopedia of Genes and Genomes (KEGG) and receiver operating characteristic (ROC) curve analysis of the altered metabolites were performed. A total of 134 metabolites were found and identified. The metabolites belonged to several metabolic pathways including phenylalanine metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, arginine biosynthesis, linoleic acid metabolism, tryptophan metabolism, aminoacyl-tRNA biosynthesis, sphingolipid metabolism, glycosphingolipid biosynthesis, leucine and isoleucine biosynthesis, and vitamin B6 metabolism. Eight metabolites were identified as the potential biomarkers. These results demonstrated that serum metabolic profiling of BEB patients was significantly different from healthy controls based on LC-Orbitrap MS. Besides, metabolomics might provide useful information for a better understanding of BEB.

Comprehensive Analysis Revealed the Potential Roles of N6-Methyladenosine (m6A) Mediating E. coli F18 Susceptibility in IPEC-J2 Cells.

Post-weaning diarrhea caused by enterotoxigenic Escherichia coli F18 (E. coli F18) causes significant economic losses for pig producers. N6-methyladenosine (m6A) is a highly abundant epitranscriptomic marker that has been found to be involved in regulating the resistance of host cells to pathogenic infection, but its potential role in E. coli F18-exposed intestinal porcine epithelial cells (IPEC-J2) remains undetermined. Here, we demonstrated that m6A and its regulators modulate E. coli F18 susceptibility. Briefly, we revealed that the Wilms' tumor 1-associating protein (WTAP) expressions were markedly elevated in IPEC-J2 cells upon E. coli F18 exposure. WTAP are required for the regulation of E. coli F18 adhesion in IPEC-J2 cells. Additionally, WTAP knockdown significantly suppressed m6A level at N-acetyllactosaminide beta-1,6-N-acetylglucosaminyl-transferase (GCNT2) 3'UTR, resulting in the enhancement of TH N6-methyladenosine RNA binding protein 2 (YTHDF2)-mediated GCNT2 mRNA stability. Subsequently, the altered GCNT2 expressions could inhibit the glycosphingolipid biosynthesis, thus improving resistance to E. coli F18 infection in IPEC-J2. Collectively, our analyses highlighted the mechanism behind the m6A-mediated management of E. coli F18 susceptibility, which will aid in the development of novel approaches that protect against bacterial diarrhea in piglets.

MiRNA Expression Associated with HbF in Saudi Sickle Cell Anemia.

Background and Objectives: Sickle cell anemia (SCA) is a hereditary monogenic disease due to a single β-globin gene mutation that codes for the production of sickle hemoglobin. Its phenotype is modulated by fetal hemoglobin (HbF), a product of γ-globin genes. Exploring the molecules that regulate γ-globin genes at both transcriptional and translational levels, including microRNA (miRNA), might help identify alternative therapeutic targets. Materials and Methods: Using next-generation sequencing we identified pre-miRNAs and mature miRNA expression signatures associated with different HbF levels in patients homozygous for the sickle hemoglobin gene. The involvement of identified miRNAs in potential SCD-related pathways was investigated with the DIANA TOOL and miRWalk 2.0 database. Results: miR-184 were most highly upregulated in reticulocytes. miR-3609 and miR-483-5p were most highly downregulated in sickle cell anemia with high HbF. miR-370-3p that regulates LIN28A, and miR-451a which is effective in modulating α- and β- globin levels were also significantly upregulated. miRNA targeted gene pathway interaction identified BCL7A, BCL2L1, LIN28A, KLF6, GATA6, solute carrier family genes and ZNF genes associated with erythropoiesis, cell cycle regulation, glycosphingolipid biosynthesis, cAMP, cGMP-PKG, mTOR, MAPK and PI3K-AKT signaling pathways and cancer pathways. Conclusions: miRNA signatures and their target genes identified novel miRNAs that could regulate fetal hemoglobin production and might be exploited therapeutically.

Basement-Membrane-Related Gene Signature Predicts Prognosis in WHO Grade II/III Gliomas.

Gliomas that are classified as grade II or grade III lesions by the World Health Organization (WHO) are highly aggressive, and some may develop into glioblastomas within a short period, thus portending the conferral of a poor prognosis for patients. Previous studies have implicated basement membrane (BM)-related genes in glioma development. In this study, we constructed a prognostic model for WHO grade II/III gliomas in accordance with the risk scores of BM-related genes. Differentially expressed genes (DEGs) in the glioma samples relative to normal samples were screened from the GEO database, and five prognostically relevant BM-related genes, including NELL2, UNC5A, TNC, CSPG4, and SMOC1, were selected using Cox regression analyses for the risk score model. The median risk score was calculated, based on which high- and low-risk groups of patients were generated. The clinical information, pathological information, and risk group were combined to establish a prognostic nomogram. Both the nomogram and risk score model performed well in the independent CGGA cohort. Gene set enrichment analysis (GSEA) and immune profile, drug sensitivity, and tumor mutation burden (TMB) analyses were performed in the two risk groups. A significant enrichment of ‘Autophagy–other’, ‘Collecting duct acid secretion’, ‘Glycosphingolipid biosynthesis–lacto and neolacto series’, ‘Valine, leucine, and isoleucine degradation’, ‘Vibrio cholerae infection’, and other pathways were observed for patients with high risk. In addition, higher proportions of monocytes and resting CD4 memory T cells were observed in the low- and high-risk groups, respectively. In conclusion, the BM-related gene risk score model can guide the clinical management of WHO grade II and III gliomas.

Oxidative stress, apoptosis, and transcriptional responses in Acropora microphthalma under simulated diving activities.

This study simulated the effects of diving activities on the physiology, enzymatic, and transcriptional responses of Acropora microphthalma. Touching had less impact on Fv/Fm, but a few zooxanthellae were decreased and minor MDA was elevated. Caspase 3 was activated to remove damaged cells, and SOD was increased to alleviate oxidative damage. Under double or triple diving stress, we observed mass loss of zooxanthellae and Fv/Fm, a significant increase in MDA, and SOD, CAT was activated in response to oxidative stress. Transcriptome analyses showed that corals activated immune signaling pathways, anti-oxidation pathways, lysosomal, phagosomal, and cellular autophagy pathways to manage oxidation stress. Moreover, it up-regulated carbohydrate metabolisms, as well as lipopolysaccharide metabolism, glycosphingolipid biosynthesis, photorespiration, amino acid metabolism, and fatty acid beta-oxidation, but down-regulated fatty acid biosynthesis to answer energy insufficiency. This research supported that even in a short time, improper diving activities could have a serious impact on coral health.

An Integrated Analysis of Lactation-Related miRNA and mRNA Expression Profiles in Donkey Mammary Glands.

Donkey milk is consumed by humans for its nutritional and therapeutic properties. MicroRNAs (miRNAs) and messenger RNAs (mRNAs) have been implicated in the regulation of milk component synthesis and mammary gland development. However, the regulatory profile of the miRNAs and mRNAs involved in lactation in donkeys is unclear. We performed mRNA-seq and miRNA-seq and constructed coexpression regulatory networks for the mammary glands during the lactating and nonlactating period of jennies. We identified 3144 differentially expressed (DE) mRNAs (987 upregulated mRNAs and 2157 downregulated mRNAs) and 293 DE miRNAs (231 upregulated miRNAs and 62 downregulated miRNAs) in the lactating group compared to the nonlactating group. The DE miRNA target mRNA were significantly associated with pathways related to RNA polymerase, glycosphingolipid biosynthesis, mRNA surveillance, ribosome biogenesis in eukaryotes, glycerophospholipid metabolism, Ras signaling, and the fly hippo signaling pathway. The mRNA–miRNA coregulation analysis showed that novel-m0032-3p, miR-195, miR-26-5p, miR-23-3p, miR-674-3p, and miR-874-3p are key miRNAs that target mRNAs involved in immunity and milk lipid, protein, and vitamin metabolism in the jenny mammary gland. Our results improve the current knowledge of the molecular mechanisms regulating bioactive milk component metabolism in the mammary glands and could be used to improve milk production in donkeys.