iTRAQ-Based Quantitative Proteomic Analysis Reveals Toxicity Mechanisms in Chlamys farreri Exposed to Okadaic Acid

Okadaic acid (OA), produced by dinoflagellates during harmful algal blooms, is a principal diarrhetic shellfish poisoning toxin. This toxin poses a potential threat to bivalves with economic values. To better understand the toxicity mechanism of OA to bivalves, in this study, oxidative stress biomarkers (superoxide dismutase, SOD; catalase, CAT; glutathione S-transferase, GST; malondialdehyde, MDA) and the expression of detoxification genes (heat shock protein 70, HSP70; heat shock protein 90, HSP90; cytochrome P450, CYP450) were assessed in the gills of scallops Chlamys farreri after 24 h, 48 h and 96 h exposure to OA. In addition, the digestive glands of scallops exposed to OA for 96 h were dissected for an iTRAQ based quantitative proteomic analysis. The results of OA exposure experiments showed that OA induces oxidative stress and significant enhancement of the expression of detoxification genes in scallops. The proteomics analysis revealed that 159 proteins altered remarkably in OA-treated scallops, and these proteins were involved in phagosomes, regulation of actin cytoskeleton, adherens junction, tight junction, and focal adhesion. Amino acid biosynthesis, carbon metabolism, pentose phosphate pathway, fructose and mannose metabolism in the digestive glands were also significantly impacted. Our data shed new insights on the molecular responses and toxicity mechanisms of C. farreri to OA.

Ribosome Profiling and RNA Sequencing Reveal Genome-Wide Cellular Translation and Transcription Regulation Under Osmotic Stress in Lactobacillus rhamnosus ATCC 53103

To determine whether osmotic pressure affects the translation efficiency of Lactobacillus rhamnosus, the ribosome profiling assay was performed to analyze the changes in translation efficiency in L. rhamnosus ATCC 53103. Under osmotic stress, differentially expressed genes (DEGs) involved in fatty acid biosynthesis and metabolism, ribosome, and purine metabolism pathways were co-regulated with consistent expression direction at translation and transcription levels. DEGs involved in the biosynthesis of phenylalanine, tyrosine, and tryptophan, and the phosphotransferase system pathways also were co-regulated at translation and transcription levels, while they showed opposite expression direction at two levels. Moreover, DEGs involved in the two-component system, amino acid metabolism, and pyruvate metabolism pathways were only regulated at the transcription level. And DEGs involved in fructose and mannose metabolism were only regulated at the translation level. The translation efficiency of DEGs involved in the biosynthesis of amino acids was downregulated while in quorum sensing and PTS pathways was upregulated. In addition, the ribosome footprints accumulated in open reading frame regions resulted in impaired translation initiation and elongation under osmotic stress. In summary, L. rhamnosus ATCC 53103 could respond to osmotic stress by translation regulation and control the balance between survival and growth of cells by transcription and translation.

Discovery of metabolomic biomarkers for discriminating platinum-sensitive and platinum-resistant ovarian cancer by using GC-MS

This study aims to determine ovarian cancer (OC) patients with platinum resistance for alternative treatment protocols by using metabolomic methodologies. Urine and serum samples of platinum-resistant and platinum-sensitive OC were analyzed using GC-MS. After data processing of GC-MS raw data, multivariate analyses were performed to interpret complex data for biologically meaningful information and to identify the biomarkers that cause differences between two groups. The biomarkers were verified after univariate, multivariate, and ROC analysis. Finally, metabolomic pathways related to group separations were specified. The results of biomarker analysis showed that 3,4-dihydroxyphenylacetic acid, 4-hydroxybutyric acid, L-threonine, D- mannose, and sorbitol metabolites were potential biomarkers in urine samples. In serum samples, L-arginine, linoleic acid, L-glutamine, and hypoxanthine were identified as important biomarkers. R2Y, Q2, AUC, sensitivity and specificity values of platinum-resistant and sensitive OC patients’ urine and serum samples were 0.85, 0.545, 0.844, 91.30%, 81.08 and 0.570, 0.206, 0.743, 77.78%, 74.28%, respectively. In metabolic pathway analysis of urine samples, tyrosine metabolism and fructose and mannose metabolism were found to be statistically significant (p < 0.05) for the discrimination of the two groups. While 3,4-dihydroxyphenylacetic acid, L-tyrosine, and fumaric acid metabolites were effective in tyrosine metabolism. D-sorbitol and D-mannose metabolites were significantly important in fructose and mannose metabolism. However, seven metabolomic pathways were significant (p < 0.05) in serum samples. In terms of p-value, L-glutamine in the nitrogen metabolic pathway from the first three pathways; L-glutamine and pyroglutamic acid metabolites in D-glutamine and D-glutamate metabolism. In the arginine and proline metabolic pathway, L-arginine, L-proline, and L-ornithine metabolites differed significantly between the two groups.

Mannose Metabolism Is a Metabolic Vulnerability Unveiled By Standard and Novel Therapies in Acute Myeloid Leukemia

Introduction The development of resistance to standard and novel therapies remains the main obstacle to cure in acute myeloid leukemia (AML). Metabolic rewiring has emerged as a therapeutically actionable vulnerability in AML, where specific metabolic adaptations arising as a result of driver mutations or in response to therapy have been reported. Mannose phosphoisomerase (MPI) is the first enzyme in the mannose metabolism (MM) pathway, that leads to the production of GDP-Mannose, a key sugar donor for N-glycosylation reactions. MPI was amongst the top drop-out genes in our published CRISPR-Cas9 screen aiming to identify sensitizers to FLT3 tyrosine kinase inhibitors (TKI) in AML carrying activating FLT3 internal tandem duplication (ITD) mutations (Gallipoli et al., Blood 2018). Moreover, analysis of published genomic datasets indicates that MPI expression levels are higher in AML compared to normal samples, correlate with patient outcome (A) and further increase in paired relapsed to diagnosis samples. We therefore hypothesized that MM and MPI inhibition sensitize AML cells to both FLT3-TKI and standard chemotherapy and tested this in preclinical models. Methods Experiments were performed in human AML cell lines, primary AML mononuclear cells and normal CD34 + stem/progenitor cells. Liquid chromatography coupled to mass spectrometry and oxygen consumption/extracellular acidification rate as measured by a Seahorse analyser were employed to assess metabolic changes. Gene-expression was measured by RNA-sequencing and confirmed by RT-qPCR. Viability, surface protein expression and lipid peroxidation were assessed by flow-cytometry. Protein expression and localisation was measured by western blot and immunofluorescence. Gene silencing was performed using CRISPR-Cas9 gene editing and inducible short hairpin RNA interference. Results We show that genetic and chemical MPI inhibition sensitizes FLT3 WT and FLT3 ITD AML cell lines in vitro to standard cytarabine chemotherapy and FLT3-TKI respectively and these effects are rescued by the addition of MPI downstream product mannose. We validate these findings in vivo using MPI knock-out (KO) cell line xenografts and in vitro using primary AML samples following MPI knock-down (B). We also demonstrate a lack of toxicity to normal CD34 + cells. Surprisingly, global metabolomic analysis show that MPI KO cells accumulate fatty acids and particularly polyunsaturated fatty acids (PUFA) (C), due to both increased uptake and reduced fatty acid oxidation (FAO). MPI KO cells downregulate several FAO metabolism genes and this is corroborated by the strong positive correlation of MPI expression levels with multiple genes involved in FAO across multiple primary AML datasets. Metabolic profiling demonstrates that MPI KO cells have reduced oxidative phosphorylation metabolism and in particular are unable to oxidise palmitate, an effect rescued by mannose or the FAO activator fenofibrate (D). We link this metabolic defect to the specific activation of the ATF6 arm of the unfolded protein response (UPR) due to a reduction in protein glycosylation in MPI KO cells. We validate this by demonstrating that activating or inhibiting ATF6 respectively phenocopies or rescues the effects of MPI KO (E) and show this to be secondary to ATF6 driving FAO inhibition, via transcriptional downregulation of PPARα, a master regulator of lipid catabolism. Finally, we show that accumulation of PUFA in MPI KO cells is accompanied by reduced cysteine levels and increased markers of oxidative stress and lipid peroxidation, such as 4-hydroxynonenal (4-HNE), all features associated with ferroptosis (F). We validate this by showing that combining FLT3-TKI with MPI KO leads to ferroptotic cell death in AML cells, which can be rescued by the radical-trapping antioxidant ferrostatin. Conclusions We provide further evidence to support the role of metabolic rewiring in driving therapy resistance in AML and show for the first time that targeting MPI and MM sensitizes AML cells to cytarabine and FLT3-TKI. Mechanistically we unveil a novel connection between MM and fatty acid metabolism, via activation of the ATF6 arm of the UPR, leading to cellular PUFA accumulation, lipid peroxidation and ferroptotic cell death. Finally, our findings also suggest that triggering ferroptosis can be leveraged as a therapeutically actionable mechanism driving cell death in therapy-resistant AML cells. Figure 1 Figure 1. Disclosures Vassiliou: Kymab Ltd: Divested equity in a private or publicly-traded company in the past 24 months; STRM.BIO: Consultancy; Astrazeneca: Consultancy.

The Functionally Characterization of Putative Genes Involved in the Formation of Mannose in the Aplanospore Cell Wall of Haematococcus pluvialis (Volvocales, Chlorophyta)

Unicellular volvocalean green algal Haematococcus pluvialis, known as astaxanthin rich microalgae, transforms into aplanospore stage from the flagellate stage when exposed to the stress environments. However, the mechanism of the formation of aplanospore cell wall, which hinders the extraction of astaxanthin and the genetic manipulation is still unclear. In this study, the cell wall components under salicylic acid and high light stresses were explored, and cellulose was considered the main component in the flagellates, which changed gradually into mannose in the aplanospore stages. During the period, the genes related to the cellulose and mannose metabolisms were identified based on the RNA-seq data, which presented a similar expression pattern. The positive correlations were observed among these studied genes by Pearson Correlation (PC) analysis, indicating the coordination between pathways of cellulose and mannose metabolism. The study firstly explored the formation mechanism of aplanospore cell wall, which might be of scientific significance in the study of H. pluvialis.

Glutathione peroxidase 3 (GPX3) expression predicts the prognosis of numerous malignant tumors: A pan-cancer Analysis

Background Malignant tumor is a general term for uncontrollable growth of cells. Several studies have investigated that role of GPX3 in tumors. However, no pan-cancer analysis has be conducted to assess the diagnostic and prognostic potential of GPX3. Methods GPX3 mRNA and protein expression profile was analyzed in the TCGA + GTEx database and CPTAC database. Kaplan-Meier survival curves and forest plots were constructed to help evaluate the impact of GPX3 on the survival and prognosis of cancer patients. Gene mutations of GPX3 were analyzed based on TMB/MSI. The correlation between GPX3 and tumor immune infiltration was assessed using TIMER2. Enrichment analysis was performed to determine tumor-related signaling pathways associated with GPX3. A prediction model for STAD was established. Results GPX3 was downregulated in most malignant tumors, and was significantly associated with the survival and prognosis of malignant tumors, such as STAD, PAAD, COAD, etc. Moreover, GPX3 expression was positively correlated and negatively with MSI in 13 and 20 categories of tumor respectively after GPX3 expression was positively correlated and negatively with TMB in 9 and 24 tumors separately (P < 0.05). A positive correlation was found between GPX3 and the infiltration level of major immune cells and Cancer-associated fibroblasts (P < 0.05). The effects of GPX3 were mediated by the AMPK signaling pathway, fructose and mannose metabolism. Conclusions This is a novel pan-cancer analysis on the relationship between GPX3 and human tumors. Findings of this research will deepen our understanding on the role of GPX3 in the development, regulation and prognosis of malignant tumors.

Glutathione Peroxidase 3 (GPX3) Expression Predicts the Prognosis of Numerous Malignant Tumors: A Pan-Cancer Analysis

Background: Malignant tumor is a general term for uncontrollable growth of cells. Several studies have investigated that role of GPX3 in tumors. However, no pan-cancer analysis has be conducted to assess the diagnostic and prognostic potential of GPX3. Methods: GPX3 mRNA and protein expression profile was analyzed in the TCGA+GTEx database and CPTAC database. Kaplan-Meier survival curves and forest plots were constructed to help evaluate the impact of GPX3 on the survival and prognosis of cancer patients. Gene mutations of GPX3 were analyzed based on TMB/MSI. The correlation between GPX3 and tumor immune infiltration was assessed using TIMER2. Enrichment analysis was performed to determine tumor-related signaling pathways associated with GPX3. A prediction model for STAD was established. Results: GPX3 was downregulated in most malignant tumors, and was significantly associated with the survival and prognosis of malignant tumors, such as STAD, PAAD, COAD, etc. Moreover, GPX3 expression was positively correlated and negatively with MSI in 13 and 20 categories of tumor respectively after GPX3 expression was positively correlated and negatively with TMB in 9 and 24 tumors separately (P<0.05). A positive correlation was found between GPX3 and the infiltration level of major immune cells and Cancer-associated fibroblasts (P<0.05). The effects of GPX3 were mediated by the AMPK signaling pathway, fructose and mannose metabolism. Conclusions: This is a novel pan-cancer analysis on the relationship between GPX3 and human tumors. Findings of this research will deepen our understanding on the role of GPX3 in the development, regulation and prognosis of malignant tumors.

Alteration of Gut Microbiota Relates to Metabolic Disorders in Primary Aldosteronism Patients

PurposeThis study aimed to determine the relationships among gut microbiota, primary aldosteronism (PA), and related metabolic disorders.MethodsThe study enrolled 13 PA patients, 26 sex-matched primary hypertension patients, and 26 sex-matched healthy controls. Demographic and clinical characteristics such as age, body mass index (BMI), blood aldosterone–renin ratio, blood potassium, blood glucose, blood lipid parameters, and history of diabetes mellitus (DM) were compared between the three groups. The gut microbiota of each participant was examined by 16S rRNA gene sequencing. Spearman correlation analysis was performed to demonstrate the relationship between gut microbiota and clinical characteristics.ResultsBMI and the percentage of DM in PA patients were higher than those in healthy controls (p &lt; 0.05), but not higher than those in primary hypertension patients (p &gt; 0.05). The gut microbiota of healthy controls and primary hypertension patients had a higher alpha diversity level than that of PA patients. PA patients had fewer short-chain fatty acid (SCFA)-producing genera (Prevotella, Blautia, Coprococcus, Anaerostipes, and Ruminococcus) and more inflammation-associated genera (Megamonas, Sutterella, and Streptococcus) than healthy controls (p &lt; 0.05). The gut microbiota of PA patients was more inclined to encode microbial pathways involved in sugar metabolism, such as starch and sucrose metabolism and fructose and mannose metabolism. Blood potassium was negatively correlated with the relative abundance of Romboutsia (R = −0.364, q = 0.023). Diastolic blood pressure (DBP) was positively correlated with Romboutsia (R = 0.386, q = 0.015). Systolic blood pressure (SBP) was negatively correlated with Blautia (R = −0.349, q = 0.030).ConclusionsThe alteration of gut microbiota in PA patients, especially bacteria and pathways involved in inflammation, SCFAs, and sugar metabolism, may be associated with chronic metabolic disorders.

Metagenomic analysis revealed the potential role of gut microbiome in gout

Emerging evidence indicates an association between gut microbiome and arthritis diseases including gout. However, how and which gut bacteria affect host urate degradation and inflammation in gout remains unclear. Here we performed a metagenome analysis on 307 fecal samples from 102 gout patients and 86 healthy controls. Gout metagenomes significantly differed from those of healthy controls. The relative abundances of Prevotella, Fusobacterium, and Bacteroides were increased in gout, whereas those of Enterobacteriaceae and butyrate-producing species were decreased. Functionally, gout patients had greater abundances for genes in fructose, mannose metabolism and lipid A biosynthesis, and lower for genes in urate degradation and short chain fatty acid production. A three-pronged association between metagenomic species, functions and clinical parameters revealed that decreased abundances of species in Enterobacteriaceae were associated with reduced amino acid metabolism and environmental sensing, which together contribute to increased serum uric acid and C-reactive protein levels in gout. A random forest classifier based on three gut microbial genes showed high predictivity for gout in both discovery and validation cohorts (0.91 and 0.80 accuracy), with high specificity in the context of other chronic disorders. Longitudinal analysis showed that uric-acid-lowering and anti-inflammatory drugs partially restored gut microbiota after 24-week treatment. Comparative analysis with obesity, type 2 diabetes, ankylosing spondylitis and rheumatoid arthritis indicated that gout metagenomes were more similar to those of autoimmune than metabolic diseases. Our results suggest that gut dysbiosis was associated with dysregulated host urate degradation and systemic inflammation and may be used as non-invasive diagnostic markers for gout.

Fructose and Mannose in Inborn Errors of Metabolism and Cancer

History suggests that tasteful properties of sugar have been domesticated as far back as 8000 BCE. With origins in New Guinea, the cultivation of sugar quickly spread over centuries of conquest and trade. The product, which quickly integrated into common foods and onto kitchen tables, is sucrose, which is made up of glucose and fructose dimers. While sugar is commonly associated with flavor, there is a myriad of biochemical properties that explain how sugars as biological molecules function in physiological contexts. Substantial research and reviews have been done on the role of glucose in disease. This review aims to describe the role of its isomers, fructose and mannose, in the context of inborn errors of metabolism and other metabolic diseases, such as cancer. While structurally similar, fructose and mannose give rise to very differing biochemical properties and understanding these differences will guide the development of more effective therapies for metabolic disease. We will discuss pathophysiology linked to perturbations in fructose and mannose metabolism, diagnostic tools, and treatment options of the diseases.