Glycolysis Research Articles

The TRIM-NHL RNA-binding protein Brain Tumor coordinately regulates expression of the glycolytic pathway and vacuolar ATPase complex.

The essential Drosophila RNA-binding protein Brain Tumor (Brat) represses specific genes to control embryogenesis and differentiation of stem cells. In the brain, Brat functions as a tumor suppressor that diminishes neural stem cell proliferation while promoting differentiation. Though important Brat-regulated target mRNAs have been identified in these contexts, the full impact of Brat on gene expression remains to be discovered. Here, we identify the network of Brat-regulated mRNAs by performing RNAsequencing (RNA-seq) following depletion of Brat from cultured cells. We identify 158 mRNAs, with high confidence, that are repressed by Brat. De novo motif analysis identified a functionally enriched RNA motif in the 3' untranslated regions (UTRs) of Brat-repressed mRNAs that matches the biochemically defined Brat binding site. Integrative data analysis revealed a high-confidence list of Brat-repressed and Brat-bound mRNAs containing 3'UTR Brat binding motifs. Our RNA-seq and reporter assays show that multiple 3'UTR motifs promote the strength of Brat repression, whereas motifs in the 5'UTR are not functional. Strikingly, we find that Brat regulates expression of glycolytic enzymes and the vacuolar ATPase complex, providing new insight into its role as a tumor suppressor and the coordination of metabolism and intracellular pH.

Trained immunity of synovial macrophages is associated with exacerbated joint inflammation and damage after Staphylococcus aureus infection.

Investigate whether and which synoviocytes would acquire trained immunity characteristics that could exacerbate joint inflammation following a secondary Staphylococcus aureus infection. Lipopolysaccharide (LPS) and S. aureus were separately or double injected (21 days of interval) into the tibiofemoral joint cavity of male C57BL/6 mice. At different time points after these stimulations, mechanical nociception was analyzed followed by the analysis of signs of inflammation and damage in the affected joints. The trained immunity markers, including the glycolytic and mTOR pathway, were analyzed in whole tissue or isolated synoviocytes. A group of mice was treated with Rapamycin, an mTOR inhibitor before LPS or S. aureus stimulation. The double LPS - S. aureus hit promoted intense joint inflammation and damage compared to single joint stimulation, including markers in synoviocyte activation, production of proinflammatory cytokines, persistent nociception, and bone damage, despite not reducing the bacterial clearance. The double LPS - S. aureus hit joints increased the synovial macrophage population expressing CX3CR1 alongside triggering established epigenetic modifications associated with trained immunity events in these cells, such as the upregulation of the mTOR signaling pathway (p-mTOR and HIF1α) and the trimethylation of histone H3. Mice treated with Rapamycin presented reduced CX3CR1+ macrophage activation, joint inflammation, and bone damage. There is a trained immunity phenotype in CX3CR1+ synovial macrophages that contributes to the exacerbation of joint inflammation and damage during septic arthritis caused by S. aureus.

Claudin-9 (CLDN9) promotes gastric cancer progression by enhancing the glycolysis pathway and facilitating PD-L1 lactylation to suppress CD8+ T cell anti-tumor immunity

BackgroundGastric cancer (GC) is a common malignancy characterized by the absence of reliable prognostic indicators and effective therapeutic targets. Claudin-9 (CLDN9) has been demonstrated to be upregulated in various cancers. However, its prognostic value, biological function, and regulatory mechanisms in GC remain unclear. Therefore, this study aimed to elucidate the role of CLDN9 in GC progression and its underlying mechanisms. MethodsWe utilized consensus cluster, random survival forest, and multivariate Cox regression analyses to identify CLDN9 in GC. Subsequently, we evaluated the mRNA and protein levels of CLDN9 in GC using quantitative real-time polymerase chain reaction (PCR) (qRT-PCR), western blotting (WB), and immunohistochemistry (IHC). Furthermore, the role of CLDN9 in GC progression was investigated using a series of functional in vivo and in vitro experiments. Finally, we elucidated the molecular mechanisms of CLDN9 using bioinformatics, molecular biology, animal models, and patient tissue specimens. ResultsTwo GC subtypes with survival and functional differences were identified based on glycolytic metabolic genes in the Cancer Genome Atlas (TCGA)- Stomach adenocarcinoma (STAD) dataset. A prognostic risk score was calculated using seven genes to assess the overall survival (OS) in GC. Using random survival forest and multivariate Cox analyses, we identified CLDN9 as the key gene linked to the glycolytic subtype and prognosis of GC. CLDN9 expression was significantly upregulated in patients with GC as well as in GC cells. CLDN9 knockdown inhibited tumor proliferation, invasion, and metastasis both in vivo and in vitro. Mechanistically, CLDN9 was found to regulate lactate dehydrogenase A (LDHA) expression and promote glycolytic metabolism by activating the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/hypoxia-inducible factor 1-alpha (HIF1α) signaling pathway. Additionally, lactate, a glycolytic metabolite, enhanced programmed cell death ligand 1 (PD-L1) lactylation and stability, which suppressed anti-tumor immunity in CD8+ T cells, thereby contributing to GC progression. ConclusionsCLDN9 expression is associated with GC development and progression. Mechanistically, CLDN9 enhances the glycolysis pathway and facilitates PD-L1 lactylation through the PI3K/AKT/HIF1α signaling pathway, thereby suppressing anti-tumor immunity in CD8+ T cells. CLDN9 has the potential to serve as a novel prognostic marker and therapeutic target for GC.

Circular Makerspaces as Alternative Employment Platforms for Circular Jobs

AbstractTransitioning towards a circular economy requires holistic consideration encompassing environmental, economic, and social dimensions. This perspective paper explores circular makerspaces as innovative platforms for fostering social integration and creating employment opportunities within the circular economy, as makerspaces can offer a more inclusive alternative to traditional employment platforms. They have the potential to unveil unrecognised talents, bridge access to under-utilised human capital, and act as pivotal conduits to a decent and inclusive circular labour force. Drawing insights from the European Horizon 2020 project: Pop-Machina, this perspective paper emphasizes the importance of collaborative efforts among policymakers, practitioners, and researchers to unlock the full transformative potential of circular makerspaces. By prioritizing the social aspect of sustainability and leveraging the network of circular makerspaces, circular makerspaces can unlock unexplored human capital, provide employment opportunities and cultivate inclusive, sustainable communities, while highlighting their potential for societal empowerment and innovation. Finally, this perspective paper underscores the need for ongoing research and collaboration to comprehensively understand and evaluate the role of circular makerspaces in the inclusive circular labour market, ensuring that the social dimension remains central to sustainable development endeavours and informing effective policy making.This project focused on the gene PFKFB3, a pivotal regulator of the Warburg effect, which facilitates enhanced glycolysis in cancer cells, including MCF7 human breast cancer lines. The objective was to investigate the effects of PFKFB3 knockout on the metabolic profile and proliferative capacity of MCF7 cells, hypothesizing that disruption of this gene would significantly impair cellular energy production and, consequently, cell growth. Through the application of CRISPR/Cas9 technology, PFKFB3 was specifically targeted and knocked out, followed by a meticulous process of selection to enrich for cells bearing the knockout. Growth assays, particularly MTT, were conducted to evaluate the impact of PFKFB3 deletion on cell proliferation. Contrary to expectations preliminary results indicated no significant difference in the growth rates between PFKFB3-knockout and control groups. This outcome suggests possible metabolic flexibility within MCF7 cells, allowing them to bypass the blockade of one glycolytic pathway—a concept supported by current understandings of cancer metabolism. Despite the lack of expected growth inhibition, this study provides critical insights into the adaptability of cancer cells to metabolic interventions and highlights the importance of targeting multiple metabolic pathways. Future directions will consider exploring the combined knockout of PFKFB3 and other glycolytic genes to overcome the metabolic resilience of cancer cells, thereby offering a more effective strategy for crippling cancer cell energy supplies.

The fate of pyruvate dictates cell growth by modulating cellular redox potential.

Pyruvate occupies a central node in carbohydrate metabolism such that how it is produced and consumed can optimize a cell for energy production or biosynthetic capacity. This has been primarily studied in proliferating cells, but observations from the post-mitotic Drosophila fat body led us to hypothesize that pyruvate fate might dictate the rapid cell growth observed in this organ during development. Indeed, we demonstrate that augmented mitochondrial pyruvate import prevented cell growth in fat body cells in vivo as well as in cultured mammalian hepatocytes and human hepatocyte-derived cells in vitro . This effect on cell size was caused by an increase in the NADH/NAD + ratio, which rewired metabolism toward gluconeogenesis and suppressed the biomass-supporting glycolytic pathway. Amino acid synthesis was decreased, and the resulting loss of protein synthesis prevented cell growth. Surprisingly, this all occurred in the face of activated pro-growth signaling pathways, including mTORC1, Myc, and PI3K/Akt. These observations highlight the evolutionarily conserved role of pyruvate metabolism in setting the balance between energy extraction and biomass production in specialized post-mitotic cells.

Dynamic changes of protein lactylation and their correlations with the glycolytic process during the postmortem acidification of broiler breast

This experiment aimed to reveal the dynamic changes of protein post-translational lactylation modifications and their correlations with the glycolytic process in broiler breast muscle within 48 h of postmortem acidification. The experiment involved 12 male AA broilers, 42 days old, with similar body weights (2.8 ± 0.05 kg). The breast fillets (Pectoralis major) were collected after slaughter, and samples were taken at various time points: 0, 15 min, 30 min, 45 min, 60 min, 2 h, 4 h, 6 h, 8 h, 12 h, 18 h, 24 h, 36 h, and 48 h postmortem. The results showed that the rate of glycogen decline in the muscle was highest at 45 min postmortem, and glycogen levels tended to stabilize at 8 h postmortem. The lactate content in the breast reached its highest level at 4 h postmortem and began to decrease, stabilizing at 24 h postmortem. Additionally, the glycolytic potential increased gradually in the first 4 h postmortem, decreased rapidly from 4 to 8 h. Similarly, lactylation modification levels were highest at 8 h postmortem, but stabilized at 12 h postmortem. During this process, the protein expression of the enzymatic lactylation modifier p300 showed no significant difference, while the content of the nonenzymatic lactylation substrate lactoylglutathione significantly decreased at 8 h and 24 h postmortem. Correlation analysis found that lactylation levels were negatively correlated with glycogen content, glucose content, glycolytic potential, and pH value, while positively correlated with lactate content. Besides, there was a positive correlation between lactylation levels and the protein expression of hexokinase, phosphoglycerate kinase 2, phosphoglucomutase 1, and triosephosphate isomerase. Additionally, lactylation levels were positively correlated with the activities of lactate dehydrogenase and phosphofructokinase. In summary, our experiment elucidated the dynamic changes in the entire glycolytic pathway in broiler pectoral muscle during acidification. During this process, lactylation modifications may participate in the glycolysis process by regulating the protein expression and activity of glycolytic enzymes.

Transcriptomic and metabolomic investigations of methyl jasmonate-mediated enhancement of low temperature stress resistance in Cassia obtusifolia L.

Low temperature stress negatively impacts plant growth and development, reducing crop yields by disrupting metabolite production and accumulation. Cassia obtusifolia L., a widely cultivated medicinal plant in subtropical regions, is particularly vulnerable to such stress. Methyl jasmonate (MeJA) regulates plant growth, defense mechanisms, and secondary metabolite production. This study investigated how C. obtusifolia responds to low temperature stress under MeJA treatment using transcriptomic and metabolomic approaches. MeJA treatment upregulated several transcription factor families, including APE/ERF-ERF, WRKY, NAC, and MYB-related, which modulated the plant's response to cold stress. These transcription factors influenced the expression of genes involved in glycolysis, sugar metabolism, and amino acid pathways, contributing to the plant's adaptive responses. Differentially expressed genes were associated with key metabolic processes such as flavonoid biosynthesis, ribosome function, glycolysis/gluconeogenesis, and sugar metabolism. Using H-NMR and LC-MS techniques, we observed that MeJA induced the accumulation of sugars and amino acids in the leaves of C. obtusifolia seedlings under cold stress, while levels of three flavonoid compounds Isoliquiritigenin, Kaempferol-7-O-glucoside, and Phloretin significantly decreased. Enrichment analysis indicated that MeJA modulates the biosynthetic pathways of these compounds in a dose-dependent manner. Integrating metabolomic and transcriptomic data further elucidated alterations in the flavonoid biosynthetic network and other metabolic pathways under low temperature stress. These findings offer insights into the molecular mechanisms underlying flavonoid synthesis and other metabolic adjustments in C. obtusifolia under MeJA treatment.

Novel mechanisms of intestinal flora regulation in high-altitude hypoxia

BackgroundThis study investigates the molecular mechanisms behind firmicutes-mediated macrophage (Mψ) polarization and glycolytic metabolic reprogramming through HIF-1α in response to intrinsic mucosal barrier injury induced by high-altitude hypoxia. MethodsEstablishing a hypoxia mouse model of high altitude, we utilized single-cell transcriptome sequencing to identify key cell types involved in regulating intestinal mucosal barrier damage caused by high-altitude hypoxia. Through proteomic analysis of colonic tissue Mψ and metabolomic analysis of Mψ metabolites, we determined crucial proteins and metabolic pathways influencing intestinal mucosal barrier damage induced by high-altitude hypoxia. Mechanistic validation was conducted using RAW264.7 Mψ in vitro by assessing cell viability with CCK-8 assay following treatment with different metabolites. The hypoxia mouse model was further validated in vivo by transplanting gut microbiota of Firmicutes. Histological examinations through H&E staining assessed colonic cell morphology and structure, while the FITC-dextran assay evaluated intestinal tissue permeability. Hypoxia probe signal intensity in mouse colonic tissue was assessed via metronidazole staining. Various experimental techniques, including flow cytometry, immunofluorescence, ELISA, Western blot, and RT-qPCR, were employed to study the impact of HIF-1α/glycolysis pathway and different gut microbiota metabolites on Mψ polarization. ResultsBioinformatics analysis revealed that single-cell transcriptomics identified Mψ as a key cell type, with their polarization pattern playing a crucial role in the intestinal mucosal barrier damage induced by high-altitude hypoxia. Proteomics combined with metabolomics analysis indicated that HIF-1α and the glycolytic pathway are pivotal proteins and signaling pathways in the intestinal mucosal barrier damage caused by high-altitude hypoxia. In vitro cell experiments demonstrated that activation of the glycolytic pathway by HIF-1α led to a significant upregulation of mRNA levels of IL-1β, IL-6, and TNFα while downregulating mRNA levels of IL-10 and TGFβ, thereby promoting M1 Mψ activation and inhibiting M2 Mψ polarization. Further mechanistic validation experiments revealed that the metabolite butyric acid from Firmicutes bacteria significantly downregulated the protein expression of HIF-1α, GCK, PFK, PKM, and LDH, thus inhibiting the HIF-1α/glycolytic pathway that suppresses M1 Mψ and activates M2 Mψ, consequently alleviating the hypoxic symptoms in RAW264.7 cells. Subsequent animal experiments confirmed that Firmicutes bacteria inhibited the HIF-1α/glycolytic pathway to modulate Mψ polarization, thereby mitigating intestinal mucosal barrier damage in high-altitude hypoxic mice. ConclusionThe study reveals that firmicutes, through the inhibition of the HIF-1α/glycolysis pathway, mitigate Mψ polarization, thereby alleviating intrinsic mucosal barrier injury in high-altitude hypoxia.

PKM1 Regulates the Expression of Autophagy and Neuroendocrine Markers in Small Cell Lung Cancer

Small cell lung cancer (SCLC) is known as recalcitrant cancer with high malignancy and heterogeneity. Immunotherapy has changed the treatment pattern of extensive-disease SCLC (ED-SCLC), but the beneficiary population is limited. Therefore, exploring new therapeutic strategies is an urgent clinical problem to be solved for SCLC. SCLC is characterized by highly active glycolytic metabolism and pyruvate kinase M1 (PKM1) is one of the isozymes of PK, an important rate-limiting enzyme in glycolysis pathway. Previous studies have shown that PKM1 is related to autophagy and drug sensitivity, however, how PKM1 regulates drug sensitivity in SCLC and its mechanism remain unclear. The aim of this study was to investigate the biological functions of PKM1 in SCLC, including its effects on proliferation, migration, autophagy, drug sensitivity, and expression of neuroendocrine (NE)-related markers in SCLC. Western blot was used to detect the expression level of PKM1 in SCLC cells. PKM1 gene-overexpressed SCLC cell lines were constructed by stable lentivirus transfection. Proliferation of cells and drug sensitivity were detected by MTT, and migration ability of cells was determined by Transwell. The level of autophagy was detected by flow cytometry. Western blot was used to determine the expression levels of NE-related proteins. PKM1 was differentially expressed among various SCLC cell lines, and was lower in H1092 cells (P<0.01). Compared with the control group, there was no significant difference in proliferation level of PKM1 overexpressing H1092 cell, but the migration ability was significantly increased (P<0.001), the drug sensitivity was reduced, and the level of autophagy was inhibited (P<0.001). Additionally, overexpression of PKM1 could upregulate the expression of non-neuroendocrine (non-NE)-related proteins (P<0.01) and decrease the expression of NE-related proteins (P<0.01). PKM1 was differentially expressed in SCLC cell lines, and high expression of PKM1 did not affect the proliferation, but affected the migration of SCLC cells. PKM1 might affect drug sensitivity by inhibiting autophagy and regulating the expression of NE markers. These results provide a theoretical basis for exploring the role of PKM1 in SCLC.

Nobiletin, an activator of the pyruvate kinase isozyme M1/M2 protein, upregulated the glycolytic signalling pathway and alleviated depressive-like behaviour caused by artificial light exposure at night in zebrafish

We established a zebrafish model of depression-like behaviour induced by exposure to artificial light at night (ALAN) and found that nobiletin (NOB) alleviated depression-like behaviour. Subsequently, based on the results of a 24-h free movement assay, clock gene expression and brain tissue transcriptome sequencing, the glycolysis signalling pathway was identified as a potential target through which NOB exerted antidepressant effects. Using the ALAN zebrafish model, we found that supplementation with exogenous L-lactic acid alleviated depressive-like behaviour. Molecular docking and molecular dynamics simulations revealed an inter-molecular interaction between NOB and the pyruvate kinase isozyme M1/M2 (PKM2) protein. We then used compound 3 k to construct a zebrafish model in which PKM2 was inhibited. Our analysis of this model suggested that NOB alleviated depression-like behaviour via inhibition of PKM2. In summary, NOB alleviated depressive-like behaviour induced by ALAN in zebrafish via targeting of PKM2 and activation of the glycolytic signalling pathway.

G6pdN126D Variant Increases the Risk of Developing VEGFR (Vascular Endothelial Growth Factor Receptor) Blocker-Induced Pulmonary Vascular Disease.

G6PD (glucose-6-phosphate-dehydrogenase) is a key enzyme in the glycolytic pathway and has been implicated in the pathogenesis of cancer and pulmonary hypertension-associated vascular remodeling. Here, we investigated the role of an X-linked G6pd mutation (N126D polymorphism), which is known to increase the risk of cardiovascular disease in individuals from sub-Saharan Africa and many others with African ancestry, in the pathogenesis of pulmonary hypertension induced by a vascular endothelial cell growth factor receptor blocker used for treating cancer. CRISPR-Cas9 genome editing was used to generate the G6pd variant (N126D; G6pdN126D) in rats. A single dose of the vascular endothelial cell growth factor receptor blocker sugen-5416 (SU; 20 mg/kg in DMSO), which is currently in a Phase 2/3 clinical trial for cancer treatment, was subcutaneously injected into G6pdN126D rats and their wild-type littermates. After 8 weeks of normoxic conditions, right ventricular pressure and hypertrophy, pulmonary artery remodeling, the metabolic profile, and cytokine expression were assessed. Right ventricular pressure and pulmonary arterial wall thickness were increased in G6PDN126D+SU/normoxic rats. Simultaneously, levels of oxidized glutathione, inositol triphosphate, and intracellular Ca2+ were increased in the lungs of G6PDN126D+SU/normoxic rats, whereas nitric oxide was decreased. Also increased in G6PDN126D+SU/normoxic rats were pulmonary levels of plasminogen activator inhibitor-1, thrombin-antithrombin complex, and expression of proinflammatory cytokines CCL3 (chemokine [C-C motif] ligand), CCL5, and CCL7. Our results suggest G6PDN126D increases inositol triphosphate-Ca2+ signaling, inflammation, thrombosis, and hypertrophic pulmonary artery remodeling in SU-treated rats. This suggests an increased risk of vascular endothelial cell growth factor receptor blocker-induced pulmonary hypertension in those carrying this G6PD variant.

ZFP64 drives glycolysis-mediated stem cell-like properties and tumorigenesis in breast cancer

BackgroundBreast cancer (BC) is a great clinical challenge because of its aggressiveness and poor prognosis. Zinc Finger Protein 64 (ZFP64), as a transcriptional factor, is responsible for the development and progression of cancers. This study aims to investigate whether ZFP64 regulates stem cell-like properties and tumorigenesis in BC by the glycolytic pathway.ResultsIt was demonstrated that ZFP64 was overexpressed in BC specimens compared to adjacent normal tissues, and patients with high ZFP64 expression had shorter overall survival and disease-free survival. The analysis of the association of ZFP64 expression with clinicopathological characteristics showed that high ZFP64 expression is closely associated with N stage, TNM stage, and progesterone receptor status. Knockdown of ZFP64 suppressed the viability and colony formation capacity of BC cells by CCK8 and colony formation assays. The subcutaneous xenograft models revealed that ZFP64 knockdown reduced the volume of formatted tumors, and decreased Ki67 expression in tumors. The opposite effects on cell proliferation and tumorigenesis were demonstrated by ZFP64 overexpression. Furthermore, we suggested that the stem cell-like properties of BC cells were inhibited by ZFP64 depletion, as evidenced by the decreased size and number of formatted mammospheres, the downregulated expressions of OCT4, Nanog, and SOX2 proteins, as well as the reduced proportion of CD44+/CD24− subpopulations. Mechanistically, glycolysis was revealed to mediate the effect of ZFP64 using mRNA-seq analysis. Results showed that ZFP64 knockdown blocked the glycolytic process, as indicated by decreasing glycolytic metabolites, inhibiting glucose consumption, and reducing lactate and ATP production. As a transcription factor, we identified that ZFP64 was directly bound to the promoters of glycolysis-related genes (ALDOC, ENO2, HK2, and SPAG4), and induced the transcription of these genes by ChIP and dual-luciferase reporter assays. Blocking the glycolytic pathway by the inhibition of glycolytic enzymes ENO2/HK2 suppressed the high proliferation and stem cell-like properties of BC cells induced by ZFP64 overexpression.ConclusionsThese data support that ZFP64 promotes stem cell-like properties and tumorigenesis of BC by activating glycolysis in a transcriptional mechanism.

Proteoglycan 4 (Lubricin) and Regulation of Xanthine Oxidase in Synovial Macrophage as A Mechanism of Controlling Synovitis.

Synovial macrophages (SMs) are important effectors of joint health and disease. A novel Cx3CR1 + TREM2 + SM population expressing the tight junction protein claudin-5, was recently discovered in synovial lining. Ablation of these SMs was associated with onset of arthritis. Proteoglycan 4 (PRG4) is a mucinous glycoprotein that fulfills lubricating and homeostatic roles in the joint. The aim of this work is to study the role of PRG4 in modulating synovitis in the context of SM homeostasis and assess the contribution of xanthine oxidase (XO)-hypoxia inducible factor alpha (HIF-1α) axis to this regulation. We used Prg4 FrlioxP/FrtloxP ;R26 FlPoER/+ , a novel transgenic mouse, where the Prg4 Frt allele normally expresses the PRG4 protein and was designed to flank the first two exons of Prg4 with a flippase recognition target and "LOXP" sites. Inducing flippase activity with tamoxifen (TAM) inactivates the Frt allele and thus creates a conditional knockout state. We studied anti-inflammatory SMs and XO by quantitative immunohistochemistry, isolated RNA and studied immune pathway activations by multiplexed assays and isolated SMs and studied PRG4 signaling dysfunction in relation to glycolytic switching due to pro-inflammatory activation. Prg4 inactivated mice were treated with oral febuxostat, a specific XO inhibitor, and quantification of Cx3CR1 + TREM2 + SMs, XO immunostaining and synovitis assessment were conducted. Prg4 inactivation induced Cx3CR1 + TREM2 + SM loss (p < 0.001) and upregulated glycolysis and innate immune pathways in the synovium. In isolated SMs, Xdh (p < 0.01) and Hif1a (p < 0.05) were upregulated. Pro-inflammatory activation of SMs was evident by enhanced glycolytic flux and XO-generated reactive oxygen species (ROS). Febuxostat reduced glycolytic flux (p < 0.001) and HIF-1α levels (p < 0.0001) in SMs. Febuxostat also reduced systemic inflammation (p < 0.001), synovial hyperplasia (p < 0.001) and preserved Cx3CR1 + TREM2 + SMs (p < 0.0001) in synovia of Prg4 inactivated mice. PRG4 is a biologically significant modulator of synovial homeostasis via inhibition of XO expression and downstream HIF-1a activation. PRG4 signaling is anti-inflammatory and promotes synovial homeostasis in chronic synovitis, where direct XO inhibition is potentially therapeutic in chronic synovitis.

The tumor suppressor Parkin exerts anticancer effects through regulating mitochondrial GAPDH activity.

Cancer cells preferentially utilize glycolysis for energy production, and GAPDH is a critical enzyme in glycolysis. Parkin is a tumor suppressor and a key protein involved in mitophagy regulation. However, the tumor suppression mechanism of Parkin has still not been elucidated. In this study, we identified mitochondrial GAPDH as a new substrate of the E3 ubiquitin ligase Parkin, which mediated GAPDH ubiquitination in human cervical cancer. The translocation of GAPDH into mitochondria was driven by the PINK1 kinase, and either PINK1 or GAPDH mutation prevented the accumulation of GAPDH in mitochondria. Parkin caused the ubiquitination of GAPDH at multiple sites (K186, K215, and K219) located within the enzyme-catalyzed binding domain of the GAPDH protein. GAPDH ubiquitination was required for mitophagy, and stimulation of mitophagy suppressed cervical cancer cell growth, indicating that mitophagy serves as a type of cell death. Mechanistically, PHB2 served as a key mediator in GAPDH ubiquitination-induced mitophagy through stabilizing PINK1 protein and GAPDH mutation resulted in the reduced distribution of PHB2 in mitophagic vacuole. In addition, ubiquitination of GAPDH decreased its phosphorylation level and enzyme activity and inhibited the glycolytic pathway in cervical cancer cells. The results of in vivo experiments also showed that the GAPDH mutation increased glycolysis in cervical cancer cells and accelerated tumorigenesis. Thus, we concluded that Parkin may exert its anticancer function by ubiquitinating GAPDH in mitochondria. Taken together, our study further clarified the molecular mechanism of tumor suppression by Parkin through the regulation of energy metabolism, which provides an experimental basis for the development of new drugs for the treatment of human cervical cancer.

A Widespread Radical-Mediated Glycolysis Pathway.

Glycyl radical enzymes (GREs) catalyze mechanistically diverse radical-mediated reactions, playing important roles in the metabolism of anaerobic bacteria. The model bacterium Escherichia coli MG1655 contains two GREs of unknown function, YbiW and PflD, which are widespread among human intestinal bacteria. Here, we report that YbiW and PflD catalyze ring-opening C-O cleavage of 1,5-anhydroglucitol-6-phosphate (AG6P) and 1,5-anhydromannitol-6-phosphate (AM6P), respectively. The product of both enzymes, 1-deoxy-fructose-6-phosphate (DF6P), is then cleaved by the aldolases FsaA or FsaB to form glyceraldehyde-3-phosphate (G3P) and hydroxyacetone (HA), which are then reduced by the NADH-dependent dehydrogenase GldA to form 1,2-propanediol (1,2-PDO). Crystal structures of YbiW and PflD in complex with their substrates provided insights into the mechanism of radical-mediated C-O cleavage. This "anhydroglycolysis" pathway enables anaerobic growth of E. coli on 1,5-anhydroglucitol (AG) and 1,5-anhydromannitol (AM), and we probe the feasibility of harnessing this pathway for the production of 1,2-PDO, a highly demanded chiral chemical feedstock, from inexpensive starch. Discovery of the anhydroglycolysis pathway expands the known catalytic repertoire of GREs, clarifies the hitherto unknown physiological functions of the well-studied enzymes FsaA, FsaB, and GldA, and demonstrates how enzyme discovery efforts can cast light on prevalent yet overlooked metabolites in the microbiome.

The antagonistic activity of Streptomyces spiroverticillatus (No. HS1) against of poplar canker pathogen Botryosphaeria dothidea

BackgroundPoplar canker caused by Botryosphaeria dothidea is one of the most severe plant disease of poplars worldwide. In our study, we aimed to investigate the modes of antagonism by fermentation broth supernatant (FBS) of Streptomyces spiroverticillatus HS1 against B. dothidea.ResultsIn vitro, the strain and FBS of S. spiroverticillatus HS1 significantly inhibited mycelial growth and biomass accumulation, and also disrupted the mycelium morphology of B. dothidea. On the 3rd day after treatment, the inhibition rates of colony growth and dry weight were 80.72% and 52.53%, respectively. In addition, FBS treatment damaged the plasma membrane of B. dothidea based on increased electrical conductivity in the culture medium, and malondialdehyde content of B. dothidea mycelia. Notably, the analysis of key enzymes in glycolysis pathway showed that the activity of hexokinase (HK), phosphofructokinase (PFK), and pyruvate kinase (PK), Ca2+Mg2+-ATPase were significantly increased after FBS treatment. But the glucose contents were significantly reduced, and pyruvate contents were significantly increased in B. dothidea after treatment with FBS.ConclusionsThe inhibitory mechanism of S. spiroverticillatus HS1 against B. dothidea was a complex process, which was associated with multiple levels of mycelial growth, cell membrane structure, material and energy metabolism. The FBS of S. spiroverticillatus HS1 could provide an alternative approach to biological control strategies against B. dothidea.

Limosilactobacillus Regulating Microbial Communities to Overcome the Hydrolysis Bottleneck with Efficient One-Step Co-Production of H2 and CH4.

The efficient co-production of H2 and CH4 via anaerobic digestion (AD) requires separate stages, as it cannot yet be achieved in one step. Lactic acid bacteria (LAB) (Limosilactobacillus) release H2 and acetate by enhancing hydrolysis, potentially increasing CH4 production with simultaneous H2 accumulation. This study investigated the enhanced effect of one-step co-production of H2 and CH4 in AD by LAB and elucidated its enhancement mechanisms. The results showed that 236.3 times increase in H2 production and 7.1 times increase in CH4 production are achieved, resulting in profits of 469.39 USD. Model substrates lignocellulosic straw, sodium acetate, and H2 confirmes LAB work on the hydrolysis stage and subsequent sustainable volatile fatty acid production during the first 6 days of AD. In this stage, the enrichment of Limosilactobacillus carrying bglB and xynB, the glycolysis pathway, and the high activity of protease, acetate kinase, and [FeFe] hydrogenase, jointly achieved rapid acetate and H2 accumulation, driving hydrogenotrophic methanogenesis dominated. From day 7 to 24, with enriched Methanosarcina, and increased methenyltetrahydromethanopterin hydrogenase activity, continuously produced acetate led to the mainly acetoclastic methanogenesis shift from hydrogenotrophic methanogenesis. The power generation capacity of LAB-enhanced AD is 333.33 times that of China's 24,000m3 biogas plant.

Chemiosmotic nutrient transport in synthetic cells powered by electrogenic antiport coupled to decarboxylation

Cellular homeostasis depends on the supply of metabolic energy in the form of ATP and electrochemical ion gradients. The construction of synthetic cells requires a constant supply of energy to drive membrane transport and metabolism. Here, we provide synthetic cells with long-lasting metabolic energy in the form of an electrochemical proton gradient. Leveraging the L-malate decarboxylation pathway we generate a stable proton gradient and electrical potential in lipid vesicles by electrogenic L-malate/L-lactate exchange coupled to L-malate decarboxylation. By co-reconstitution with the transporters GltP and LacY, the synthetic cells maintain accumulation of L-glutamate and lactose over periods of hours, mimicking nutrient feeding in living cells. We couple the accumulation of lactose to a metabolic network for the generation of intermediates of the glycolytic and pentose phosphate pathways. This study underscores the potential of harnessing a proton motive force via a simple metabolic network, paving the way for the development of more complex synthetic systems.

Comparative Proteomic Analysis Reveals the Effect Mechanisms of Glucose on the Biomass and Phenolic Glycoside Esters Synthesis Activity of Candida Parapsilosis ACCC 20221 Whole-Cell Catalyst.

A new Candida parapsilosis ACCC 20221 (C. parapsilosis ACCC 20221) whole-cell catalyst with a high phenolic glycoside esters synthesis activity and large biomass was obtained after culture with glucose. The possible mechanisms were revealed by using comparative proteomics. It found the expression of proteins involved in post-translational modification, protein turnover, and chaperone, and RNA processing and modification was upregulated, which ensured the metabolic balance and accurate translation, correct folding, and post-translational modification of proteins, thus enhancing the production of lipases in C. parapsilosis ACCC 20221 cultured with glucose. Moreover, the glycolysis pathway, tricarboxylic acid cycle, and fatty acids synthesis were enhanced, while the β-oxidation of fatty acids was weakened in C. parapsilosis ACCC 20221 cells cultured with glucose, which led to an increase in energy generation and cell membrane synthesis; thus, large biomass was obtained. In addition, CCE40476.1 and CAC86400.1, which were likely to exert arbutin esters synthesis activity in C. parapsilosis ACCC 20221, were screened, and it was found that vinyl propionate could easily enter the catalytic pocket of CCE40476.1 and form hydrogen bonding interactions with Leu191 and Ser266.

Identification of TPI1 As a potential therapeutic target in pancreatic cancer with dependency of TP53 mutation using multi-omics analysis.

Mutations of KRAS, CDKN2A, TP53, and SMAD4 are the four major driver genes for pancreatic ductal adenocarcinoma (PDAC), of which mutations of KRAS and TP53 are the most frequently recognized. However, molecular-targeted therapies for mutations of KRAS and TP53 have not yet been developed. To identify novel molecular targets, we newly established organoids with the Kras mutation (KrasmuOR) and Trp53 loss of function using Cre transduction and CRISPR/Cas9 (Krasmu/p53muOR) from murine epithelia of the pancreatic duct in KrasLSL-G12D mice, and then analyzed the proteomic and metabolomic profiles in both organoids by mass spectrometry. Hyperfunction of the glycolysis pathway was recognized in Krasmu/p53muOR compared with KrasmuOR. Loss of function of triosephosphate isomerase (TPI1), which is involved in glycolysis, induced a reduction of cell proliferation in human PDAC cell lines with the TP53 mutation, but not in PDAC or in human fibroblasts without TP53 mutation. The TP53 mutation is clinically recognized in 70% of patients with PDAC. In the present study, protein expression of TPI1 and nuclear accumulation of p53 were recognized in the same patients with PDAC. TPI1 is a potential candidate therapeutic target for PDAC with the TP53 mutation.