PFKFB4 Research Articles

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.

ROCK1 regulates glycolysis in pancreatic cancer via the c-MYC/PFKFB3 pathway

BackgroundDysregulation of Rho-associated coiled coil-containing protein kinases (ROCKs) is involved in the metastasis and progression of various malignant tumors. However, how one of the isomers, ROCK1, regulates glycolysis in tumor cells is incompletely understood. Here, we attempted to elucidate how ROCK1 influences pancreatic cancer (PC) progression by regulating glycolytic activity. MethodsThe biological function of ROCK1 was analyzed in vitro by establishing a silenced cell model. Coimmunoprecipitation confirmed the direct binding between ROCK1 and c-MYC, and a luciferase reporter assay revealed the binding of c-MYC to the promoter of the PFKFB3 gene. These results were verified in animal experiments. ResultsROCK1 was highly expressed in PC tissues and enriched in the cytoplasm, and its high expression was associated with a poor prognosis. Silencing ROCK1 inhibited the proliferation and migration of PC cells and promoted their apoptosis. Mechanistically, ROCK1 directly interacted with c-MYC, promoted its phosphorylation (Ser 62) and suppressed its degradation, thereby increasing the transcription of the key glycolysis regulatory factor PFKFB3, enhancing glycolytic activity and promoting PC growth. Silencing ROCK1 increased gemcitabine (GEM) sensitivity in vivo and in vitro. ConclusionsROCK1 promotes glycolytic activity in PC cells and promotes PC tumor growth through the c-MYC/PFKFB3 signaling pathway. ROCK1 knockdown can inhibit PC tumor growth in vivo and increase the GEM sensitivity of PC tumors, providing a crucial clinical therapeutic strategy for PC.

Genomic Deletion of PFKFB3 Decreases In Vivo Tumorigenesis.

Rapidly proliferative processes in mammalian tissues including tumorigenesis and embryogenesis rely on the glycolytic pathway for energy and biosynthetic precursors. The enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) plays an important regulatory role in glycolysis by activating the key rate-limiting glycolytic enzyme, 6-phosphofructo-1-kinase (PFK-1). We have previously determined that decreased PFKFB3 expression reduced glycolysis and growth in transformed cells in vitro and suppressed xenograft growth in vivo. In earlier studies, we created a constitutive knockout mouse to interrogate the function of PFKFB3 in vivo but failed to generate homozygous offspring due to the requirement for PFKFB3 for embryogenesis. We have now developed a novel transgenic mouse model that exhibits inducible homozygous pan-tissue Pfkfb3 gene deletion (Pfkfb3fl/fl). We have induced Pfkfb3 genomic deletion in these mice and found that it effectively decreased PFKFB3 expression and activity. To evaluate the functional consequences of Pfkfb3 deletion in vivo, we crossed Cre-bearing Pfkfb3fl/fl mice with oncogene-driven tumor models and found that Pfkfb3 deletion markedly decreased their glucose uptake and growth. In summary, our studies reveal a critical regulatory function for PFKFB3 in glycolysis and tumorigenesis in vivo and characterize an effective and powerful model for further investigation of its role in multiple biological processes.

Combined analysis of the effects of hypoxia and oxidative stress on DNA methylation and the transcriptome in HTR-8/SVneo trophoblast cells.

The alterations in DNA methylation and transcriptome in trophoblast cells under conditions of low oxygen and oxidative stress have major implications for pregnancy-related disorders. However, the exact mechanism is still not fully understood. In this study, we established models of hypoxia (H group) and oxidative stress (HR group) using HTR-8/SVneo trophoblast cells and performed combined analysis of genome-wide DNA methylation changes using reduced representation bisulphite sequencing and transcriptome expression changes using RNA sequencing. Our findings revealed that the H group exhibited a higher number of differentially methylated genes and differentially expressed genes than the HR group. In the H group, only 0.90% of all differentially expressed genes displayed simultaneous changes in DNA methylation and transcriptome expression. After the threshold was expanded, this number increased to 6.29% in the HR group. Notably, both the H group and HR group exhibited concurrent alterations in DNA methylation and transcriptome expression within Axon guidance and MAPK signalling pathway. Among the top 25 differentially methylated KEGG pathways in the promoter region, 11 pathways were commonly enriched in H group and HR group, accounting for 44.00%. Among the top 25 KEGG pathways in transcriptome with significant differences between the H group and HR group, 10 pathways were consistent, accounting for 40.00%. By integrating our previous data on DNA methylation from preeclamptic placental tissues, we identified that the ANKRD37 and PFKFB3 genes may contribute to the pathogenesis of preeclampsia through DNA methylation-mediated transcriptome expression under hypoxic conditions.

DNA methylation signatures of youth-onset type 2 diabetes and exposure to maternal diabetes

ObjectiveYouth-onset type 2 diabetes (T2D) is physiologically distinct from adult-onset, but it is not clear how the two diseases differ at a molecular level. In utero exposure to maternal type 2 diabetes (T2D) is known to be a specific risk factor for youth-onset T2D. DNA methylation (DNAm) changes associated with T2D but which differ between youth- and adult-onset might delineate the impacts of T2D development at different ages and could also determine the contribution of exposure to in utero diabetes.MethodsWe performed an epigenome-wide analysis of DNAm on whole blood from 218 youth with T2D and 77 normoglycemic controls from the iCARE (improving renal Complications in Adolescents with type 2 diabetes through REsearch) cohort. Associations were tested using multiple linear regression models while adjusting for maternal diabetes, sex, age, BMI, smoking status, second-hand smoking exposure, cell-type proportions and genetic ancestry.ResultsWe identified 3830 differentially methylated sites associated with youth T2D onset, of which 3794 were moderately (adjusted p-value < 0.05 and effect size estimate > 0.01) associated and 36 were strongly (adjusted p-value < 0.05 and effect size estimate > 0.05) associated. A total of 3725 of these sites were not previously reported in the EWAS Atlas as associated with T2D, adult obesity or youth obesity. Moreover, three CpGs associated with youth-onset T2D in the PFKFB3 gene were also associated with maternal T2D exposure (FDR < 0.05 and effect size > 0.01). This is the first study to link PFKFB3 and T2D in youth.ConclusionOur findings support that T2D in youth has different impacts on DNAm than adult-onset, and suggests that changes in DNAm could provide an important link between in utero exposure to maternal diabetes and the onset of T2D.

M6A methylation of FKFB3 reduced pyroptosis of gastric cancer by NLRP3.

Gastric cancer is a kind of malignant tumor that seriously endangers human life and health. Its incidence rate and mortality rate are among the highest in the global malignant tumors. Therefore, this study explored the role of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) in the progression of gastric cancer and its underlying mechanism. Patients with gastric cancer were collected, and human GC cell lines (stomach gastric carcinoma 7901, stomach gastric carcinoma 823 , human gastric carcinoma cell line 803 and adenocarcinoma gastric stomach) were used in this study. We utilized glucose consumption, cell migration, and ELISA assay kits to investigate the function of GC. To understand its mechanism, we employed quantitative PCR (qPCR), western blot, and m6A methylated RNA immunoprecipitation assay. FKFB3 protein expression levels in patients with gastric cancer were increased. The induction of PFKFB3 mRNA expression levels in patients with gastric cancer or gastric cancer cell lines. Gastric cancer patients with high PFKFB3 expression had a lower survival rate. PFKFB3 high expression possessed the probability of pathological stage, lymph node metastasis or distant metastasis in patients with gastric cancer. PFKFB3 upregulation promoted cancer progression and Warburg effect progression of gastric cancer. PFKFB3 upregulation reduced pyroptosis and suppressed nucleotidebinding domain, leucinerich repeat containing protein 3-induced pyroptosis of gastric cancer. M6A-forming enzyme methyltransferase-like 3 increased PFKFB3 stability. Taken together, the M6A-forming enzyme methyltransferase-like 3 increased PFKFB3 stability and reduced pyroptosis in the model of gastric cancer through the Warburg effect. The PFKFB3 gene represents a potential therapeutic strategy for the treatment of gastric cancer.

PFKFB3 Regulates the Growth and Migration of Ovarian Cancer Cells through Pyroptosis and Warburg Effect Progression.

Ovarian cancer is one of the most common malignant tumors in female reproductive organs. Its incidence rate is second only to uterine body cancer and cervical cancer, posing a serious threat to women's health. Herein, we explored that PFKFB3 in cancer progression of ovarian cancer and its underlying mechanism. All the serum samples from ovarian cancer were collected by our hospital. PFKFB3 mRNA expressions in patients with ovarian cancer and ovarian cancer cell lines were up-regulated. PFKFB3 protein expressions in ovarian cancer cells were induced. ovarian cancer patients with high PFKFB3expression had lower survival rate. The PFKFB3gene promoted cell proliferation and EDU cells, and increased cell metastasis of ovarian cancer. Si-PFKFB3 reduced cell proliferation and EDU cells, and decreased cell metastasis of ovarian cancer. PFKFB3 gene up-regulation reduced caspase-3/9 activity levels of ovarian cancer. Si-PFKFB3 also promoted caspase-3/9 activity levels of ovarian cancer. PFKFB3 gene promoted Warburg effect progression of ovarian cancer. PFKFB3 gene reduced NLRP3-induced pyroptosis of ovarian cancer. PFKFB3 suppressed NLRP3 expression. NLRP3 was one target spot for PFKFB3 on pyroptosis of ovarian cancer. Taken together, we conclude that PFKFB3 suppressed NLRP3 axis to reduce pyroptosis and increase Warburg effect progression of ovarian cancer, and provide molecular insight into the mechanisms by which the PFKFB3 regulates pyroptosis of ovarian cancer.

Garlic-derived exosomes regulate PFKFB3 expression to relieve liver dysfunction in high-fat diet-fed mice via macrophage-hepatocyte crosstalk

BackgroundAlthough macrophage-mediated low-grade chronic inflammation and liver dysfunction have been found to be associated with the development of non-alcoholic fatty (NAFLD) and widely reported, but strategies and drugs targeting macrophages for the treatment of NAFLD are limited. Hypothesis/purposeGarlic-derived exosomes (GDE) can be useful for NAFLD due to its anti-inflammatory activity. Clarify whether GDE improves liver dysfunction through macrophage-hepatocyte crosstalk. MethodsGDE was isolated with PEG precipitation and ultracentrifuge. Inflammatory cytokines were detected by qRT-PCR and ELISA. Expression of 6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase 3 (PFKFB3) was determined using qRT-PCR and western blot. Crosstalk between macrophages and hepatocytes was identified through a co-culture experiment. Small RNA sequencing and bioinformatic analysis were used to identify the key element of GDE regulating the expression of PFKFB3 gene. ResultsGDE regulated the expression of PFKFB3 to reduce the inflammatory response in LPS-treated differentiated THP-1 macrophages. Data from small RNA sequencing and bioinformatics analysis reveal that miR-396e, one of the most abundant miRNAs of GDE, is the key component to regulate PFKFB3 expression. Mechanistically, miR-396e-mediating PFKFB3 expression plays a crucial role in GDE inhibiting inflammatory response and enhancing lipid metabolism in hepatocytes via the macrophage-hepatocyte crosstalk. Notably, GDE supplementation reduced the inflammatory response and improved liver dysfunction in high-fat diet-fed mice. ConclusionGDE may be useful for improving the symptoms of NAFLD via macrophage-hepatocyte crosstalk and its role in PFKFB3 expression.

Bariatric surgery-induced weight loss and associated genome-wide DNA-methylation alterations in obese individuals

BackgroundObesity is a multifactorial and chronic condition of growing universal concern. It has recently been reported that bariatric surgery is a more successful treatment for severe obesity than other noninvasive interventions, resulting in rapid significant weight loss and associated chronic disease remission. The identification of distinct epigenetic patterns in patients who are obese or have metabolic imbalances has suggested a potential role for epigenetic alterations in causal or mediating pathways in the development of obesity-related pathologies. Specific changes in the epigenome (DNA methylome), associated with metabolic disorders, can be detected in the blood. We investigated whether such epigenetic changes are reversible after weight loss using genome-wide DNA methylome analysis of blood samples from individuals with severe obesity (mean BMI ~ 45) undergoing bariatric surgery.ResultsOur analysis revealed 41 significant (Bonferroni p < 0.05) and 1169 (false discovery rate p < 0.05) suggestive differentially methylated positions (DMPs) associated with weight loss due to bariatric surgery. Among the 41 significant DMPs, 5 CpGs were replicated in an independent cohort of BMI-discordant monozygotic twins (the heavier twin underwent diet-induced weight loss). The effect sizes of these 5 CpGs were consistent across discovery and replication sets (p < 0.05). We also identified 192 differentially methylated regions (DMRs) among which SMAD6 and PFKFB3 genes were the top hypermethylated and hypomethylated regions, respectively. Pathway enrichment analysis of the DMR-associated genes showed that functional pathways related to immune function and type 1 diabetes were significant. Weight loss due to bariatric surgery also significantly decelerated epigenetic age 12 months after the intervention (mean = − 4.29; p = 0.02).ConclusionsWe identified weight loss-associated DNA-methylation alterations targeting immune and inflammatory gene pathways in blood samples from bariatric-surgery patients. The top hits were replicated in samples from an independent cohort of BMI-discordant monozygotic twins following a hypocaloric diet. Energy restriction and bariatric surgery thus share CpGs that may represent early indicators of response to the metabolic effects of weight loss. The analysis of bariatric surgery-associated DMRs suggests that epigenetic regulation of genes involved in endothelial and adipose tissue function is key in the pathophysiology of obesity.

Recessive Dystrophic Epidermolysis bullosa due to Hemizygous 40 kb Deletion of COL7A1 and the Proximate PFKFB4 Gene Focusing on the Mutation c.425A>G Mimicking Homozygous Status

Background: Dystrophic Epidermolysis bullosa (DEB) is a rare inherited mechanobullous disease characterised by the hyperfragility of the skin and mucous membranes. It is (typically) caused by (loss-of-function) mutations in the COL7A1 gene that impair the formation of collagen type VII, which represents the major constituent of anchoring fibrils within the basement membrane zone of epithelialised tissues. In a 4-year-old patient diagnosed with the clinical features of recessive DEB, genotyping via Next-Generation EB Panel Sequencing initially revealed the homozygosity of the maternal c.425A>G mutation, while the paternal heterozygosity in exon 3 was lacking. This genetic profile suggested incongruent gene transmission due to uniparental isodisomy (UPD) or the occurrence of a hemizygous deletion of unknown size. Methods: Thus, the EB panel sequencing of genomic DNA, followed by a paternity test and analysis of microsatellite markers, as well as multiplex ligation-dependent probe amplification (MLPA) copy number analysis using patient and parental DNA, were performed. Results: This approach revealed a paternally derived hemizygous deletion spanning from exon 3 to exon 118. Linear amplification-mediated PCR (LAM-PCR) determined the breaking points within intron 2 of the COL7A1 gene, comprising a 40kb segment within intron 1 of the adjacent PFKFB4 gene. Conclusion: This report highlights the relevance of advanced molecular profiling to determine new/exceptional/unusual genotypes and the accurate mode of genetic transmission in DEB.

Identification of genomic determinants contributing to cytokine release in immunotherapies and human diseases

BackgroundCytokine release syndrome (CRS) is a strong immune system response that can occur as a result of the reaction of a cellular immunotherapy with malignant cells. While the frequency and management of CRS in CAR T-cell therapy has been well documented, there is emerging interest in pre-emptive treatment to reduce CRS severity and improve overall outcomes. Accordingly, identification of genomic determinants that contribute to cytokine release may lead to the development of targeted therapies to prevent or abrogate the severity of CRS.MethodsForty three clinical CD22 CAR T-cell products were collected for RNA extraction. 100 ng of mRNA was used for Nanostring assay analysis which is based on the nCounter platform. Several public datasets were used for validation purposes.ResultsWe found the expression of the PFKFB4 gene and glycolytic pathway activity were upregulated in CD22 CAR T-cells given to patients who developed CRS compared to those who did not experience CRS. Moreover, these results were further validated in cohorts with COVID-19, influenza infections and autoimmune diseases, and in tumor tissues. The findings were similar, except that glycolytic pathway activity was not increased in patients with influenza infections and systemic lupus erythematosus (SLE).ConclusionOur data strongly suggests that PFKFB4 acts as a driving factor in mediating cytokine release in vivo by regulating glycolytic activity. Our results suggest that it would beneficial to develop drugs targeting PFKFB4 and the glycolytic pathway for the treatment of CRS.

Dimethyl Fumarate Blocks Tumor Necrosis Factor-Alpha-Driven Inflammation and Metabolic Rewiring in the Retinal Pigment Epithelium.

The retinal pigment epithelium (RPE) acts as a metabolic gatekeeper between photoreceptors and the choroidal vasculature to maintain retinal function. RPE dysfunction is a key feature of age-related macular degeneration (AMD), the leading cause of blindness in developed countries. Inflammation is a key pathogenic mechanism in AMD and tumor necrosis factor-alpha (TNFα) has been implicated as a pro-inflammatory cytokine involved in AMD. While mitochondrial dysfunction has been implicated in AMD pathogenesis, the interplay between inflammation and cellular metabolism remains elusive. The present study explores how the pro-inflammatory cytokine, TNFα, impacts mitochondrial morphology and metabolic function in RPE. Matured human primary RPE (H-RPE) were treated with TNFα (10 ng/ml) for up to 5 days. TNFα-induced upregulation of IL-6 secretion and inflammatory genes (IL-6, IL-8, MCP-1) was accompanied by increased oxidative phosphorylation (OXPHOS) and reduced glycolysis, leading to an increase in cellular adenosine triphosphate (ATP) content. Transmission electron microscopy (TEM) revealed defects in mitochondrial morphology with engorged mitochondria and loss of cristae integrity following TNFα treatment. Pre-treatment with the anti-inflammatory drug, 80 μM dimethyl fumarate (DMFu), blocked TNFα-induced inflammatory activation of RPE (IL-6, IL-8, MCP-1, CFH, CFB, C3) and normalized their bioenergetic profile to control levels by regulating PFKFB3 and PKM2 gene expression. Furthermore, DMFu prevented TNFα-induced mitochondrial dysfunction and morphological anomalies. Thus, our results indicate that DMFu serves as a novel therapeutic avenue for combating inflammatory activation and metabolic dysfunction of RPE in AMD.

MOLECULAR BASIS OF THE DEVELOPMENT OF INSULIN RESISTANCE IN OBESE ADOLESCENT AND ADULT MEN

The aim of this work was to study the association between the expression of glucose metabolism related genes and insulin resistance, which expression is changed in obese adolescents and adult men with and without insulin resistance, for better understanding the molecular basis of the development of obesity complications and evaluation of possible contribution of these genes in development of insulin resistance. Methods. The expression level of genes related to glucose metabolism and their regulations was studied by real-time qPCR in adipose tissue and blood cells using SYBRGreen Mix and specific for each mRNA forward and reverse primers. Total RNA was extracted using TRIZOL reagent. For reverse transcription of mRNAs we used Thermo Scientific Verso cDNA Synthesis Kit (Germany). The values of mRNA expressions were normalized to the level of ACTB mRNA and represented as percent of control (100 %). Results. It was shown that in obese patients with insulin resistance the expression level of IRS1 (insulin receptor substrate 1), HK2 (hexokinase 2), PFKFB2 (6-phosphofructokinase/fructose-2,6-bisphosphatase 2) and PFKFB3 as well as circadian factors CLOCK and ARNTL genes in subcutaneous adipose tissue is significantly decreased as compared to obese men with normal sensitivity to insulin. At the same time, the development of insulin resistance in obese patients leads to up-regulation of PFKFB4, PER1, HSPA6, ALDH1A3, COL5A1, TIMP1, TIMP2, SPARC, and VCAN gene expressions in subcutaneous adipose tissue. The expression level of IGF1 (insulin-like growth factor 1) and IGFBP5 (IGF binding protein 5) as well as ENO1 (enolase 1) and ENO2 is down-regulated in the blood of obese adolescent with insulin resistance, but IGFBP2 and IGFBP7 gene expressions are significantly increased in these patients. Conclusions. The results of this investigation provide evidence that the development of insulin resistance in obese patients is associated with gene specific changes in the expression of many very important regulatory genes, which are endoplasmic reticulum stress responsible.

\u03b2-cell-specific deletion of PFKFB3 restores cell fitness competition and physiological replication under diabetogenic stress

HIF1α and PFKFB3 play a critical role in the survival of damaged β-cells in type–2 diabetes while rendering β-cells non-responsive to glucose stimulation. To discriminate the role of PFKFB3 from HIF1α in vivo, we generated mice with conditional β-cell specific disruption of the Pfkfb3 gene on a human islet pancreatic polypeptide (hIAPP+/−) background and a high-fat diet (HFD) [PFKFB3βKO + diabetogenic stress (DS)]. PFKFB3 disruption in β-cells under DS led to selective purging of hIAPP-damaged β-cells and the disappearance of insulin- and glucagon positive bihormonal cells. PFKFB3 disruption induced a three-fold increase in β-cell replication as evidenced by minichromosome maintenance 2 protein (MCM2) expression. Unlike high-, lower DS or switch to restricted chow diet abolished HIF1α levels and reversed glucose intolerance of PFKFB3βKO DS mice. Our data suggest that replication and functional recovery of β-cells under DS depend on β-cell competitive and selective purification of HIF1α and PFKFB3-positive β-cells.

The PPAR-γ agonist pioglitazone exerts proinflammatory effects in bronchial epithelial cells during acute Pseudomonas aeruginosa pneumonia.

Pseudomonas aeruginosa is a common respiratory pathogen that causes injurious airway inflammation during acute pneumonia. Peroxisome proliferator-activated receptor (PPAR)-γ is involved in the regulation of metabolic and inflammatory responses in different cell types and synthetic agonists of PPAR-γ exert anti-inflammatory effects on myeloid cells in vitro and in models of inflammation in vivo. We sought to determine the effect of the PPAR-γ agonist pioglitazone on airway inflammation induced by acute P. aeruginosa pneumonia, focusing on bronchial epithelial cells. Mice pretreated with pioglitazone or vehicle (24 and 1h) were infected with P. aeruginosa via the airways. Pioglitazone treatment was associated with increased expression of chemokine (Cxcl1, Cxcl2, and Ccl20) and cytokine genes (Tnfa, Il6, and Cfs3) in bronchial brushes obtained 6h after infection. This pro-inflammatory effect was accompanied by increased expression of Hk2 and Pfkfb3 genes encoding rate-limiting enzymes of glycolysis; concurrently, the expression of Sdha, important for maintaining metabolite flux in the tricarboxylic acid cycle, was reduced in bronchial epithelial cells of pioglitazone treated-mice. Pioglitazone inhibited bronchoalveolar inflammatory responses measured in lavage fluid. These results suggest that pioglitazone exerts a selective proinflammatory effect on bronchial epithelial cells during acute P. aeruginosa pneumonia, possibly by enhancing intracellular glycolysis.

Hypoxia-inducible CircPFKFB4 Promotes Breast Cancer Progression by Facilitating the CRL4DDB2 E3 Ubiquitin Ligase-mediated p27 Degradation

Hypoxic microenvironment and circular RNAs (circRNAs) have shown critical implications in breast cancer (BC) progression. However, the specific functions and underlying mechanisms of circRNAs in BC under hypoxia remain largely unknown. We first screened for differentially expressed circRNAs in normoxic and hypoxic MCF-7 cells using circRNA microarray. A novel hypoxia-induced circRNA, circPFKFB4, was identified. Clinical investigation showed that circPFKFB4 was highly expressed in BC tissues and cell lines, and its overexpression was positively correlated with the advanced clinical stage and poor prognosis of BC patients. Functionally, circPFKFB4 promoted the proliferation of BC cells both in vitro and in vivo. Mechanistically, HIF1α bound to hypoxia response elements in the promoter region of the PFKFB4 gene to facilitate the biogenesis of circPFKFB4 under hypoxia. Hypoxia-induced circPFKFB4 directly bound to both DDB1 and DDB2 and promoted the CRL4DDB2 E3 ubiquitin ligase assembly, resulting in p27 ubiquitination and BC progression under hypoxia. Our findings revealed a novel interaction between circPFKFB4 and the CRL4DDB2 E3 ubiquitin ligase, suggesting that circPFKFB4 might serve as a promising biomarker and therapeutic target for BC.

IRF8 and BATF3 interaction enhances the cDC1 specific Pfkfb3 gene expression

Dendritic cells (DCs) play central role in innate as well as adaptive immune responses regulated by diverse DC subtypes that vary in terms of surface markers, transcriptional profile and functional responses. Generation of DC diversity from progenitor stage is tightly regulated by complex molecular inter-play between transcription factors. We earlier demonstrated that Batf3 and Id2 expression have a synergistic effect on the Irf8 directed classical cDC1 development. In present study, Bi-molecular fluorescence complementation assay suggested that IRF8 interacts with BATF3, and ID2 may aid cDC1 development independently. Genome wide recruitment analysis of IRF8 and BATF3 from different DC subtypes led to identification of the overlapping regions of occupancy by these two transcription factors. Further analysis of overlapping peaks of IRF8 and BATF3 occupancy in promoter region within the cDC1 subtype specific transcriptional pattern identified a metabolically important Pfkfb3 gene. Among various immune cell types; splenic cDC1 subtype displayed enhanced expression of Pfkfb3. Analysis of Irf8-/-, Irf8R294C and Batf3DCKO DC confirmed direct regulation of Pfkfb3 enhanced expression specifically in cDC1 subtype. Further we show that inhibition of PFKFB3 enzymatic activity by a chemical agent PFK15 led to reduction in cDC1 subtype in both in vitro FLDC cultures as well as in vivo mouse spleens. Together, our study identified the direct regulation of cDC1 specific enhanced expression of Pfkfb3 in glycolysis and cDC1 biology.

Long noncoding RNA GSEC promotes neutrophil inflammatory activation by supporting PFKFB3-involved glycolytic metabolism in sepsis

Metabolic reprogramming is a hallmark of neutrophil activation in sepsis. LncRNAs play important roles in manipulating cell metabolism; however, their specific involvement in neutrophil activation in sepsis remains unclear. Here we found that 11 lncRNAs and 105 mRNAs were differentially expressed in three transcriptome datasets (GSE13904, GSE28750, and GSE64457) of gene expression in blood leukocytes and neutrophils of septic patients and healthy volunteers. After Gene Ontology biological process analysis and lncRNA–mRNA pathway network construction, we noticed that GSEC lncRNA and PFKFB3 were co-expressed and associated with enhanced glycolytic metabolism. Our clinical observations confirmed the expression patterns of GSEC lncRNA and PFKFB3 genes in neutrophils in septic patients. Performing in vitro experiments, we found that the expression of GSEC lncRNA and PFKFB3 was increased when neutrophils were treated with inflammatory stimuli. Knockdown and overexpression experiments showed that GSEC lncRNA was essential for mediating PFKFB3 mRNA expression and stability in neutrophil-like dHL-60 cells. In addition, we found that GSEC lncRNA-induced PFKFB3 expression was essential for mediating dHL-60 cell inflammatory cytokine expression. Performing mechanistic experiments, we found that glycolytic metabolism with PFKFB3 involvement supported inflammatory cytokine expression. In summary, our study uncovers a mechanism by which GSEC lncRNA promotes neutrophil inflammatory activation in sepsis by supporting glycolytic metabolism with PFKFB3.

Cardiac developmental toxicity and transcriptome analyses of zebrafish (Danio rerio) embryos exposed to Mancozeb

Mancozeb (MZ), an antibacterial pesticide, has been linked to reproductive toxicity, neurotoxicity, and endocrine disruption. However, whether MZ has cardiactoxicity is unclear. In this study, the cardiotoxic effects of exposure to environment-related MZ concentrations ranging from 1.88 μM to 7.52 μM were evaluated at the larval stage of zebrafish. Transcriptome sequencing predicted the mechanism of MZ-induced cardiac developmental toxicity in zebrafish by enrichment analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO). Consistent with morphological changes, the osm, pfkfb3, foxh1, stc1, and nrarpb genes may effect normal development of zebrafish heart by activating NOTCH signaling pathways, resulting in pericardial edema, myocardial fibrosis, and congestion in the heart area. Moreover, differential gene expression analysis indicated that cyp-related genes (cyp1c2 and cyp3c3) were significantly upregulated after MZ treatment, which may be related to apoptosis of myocardial cells. These results were verified by real-time quantitative RT-qPCR and acridine orange staining. Our findings suggest that MZ-mediated cardiotoxic development of zebrafish larvae may be related to the activation of Notch and apoptosis-related signaling pathways.

Expression of Alternative Splice Variants of 6-Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase-4 in Normoxic and Hypoxic Melanoma Cells.

Cancer-specific isoenzyme of phosphofructokinase II (PFKFB4), as our previous research has shown, may be one of the most important enzymes contributing to the intensification of glycolysis in hypoxic malignant melanoma cells. Although the PFKFB4 gene seems to play a crucial role in the progression of melanoma, so far there are no complete data on the expression of PFKFB4 at the isoform level and the influence of hypoxia on alternative splicing. Using RT-qPCR and semi-quantitative RT-PCR, we presented the PFKFB4 gene expression profile at the level of six isoforms described in the OMIM NCBI database in normoxic and hypoxic melanoma cells. Additionally, using VMD software, we analyzed the structure of isoforms at the protein level, concluding about the catalytic activity of individual isoforms. Our research has shown that five isoforms of PFKFB4 are expressed in melanoma cells, of which the D and F isoforms are highly constitutive, while the canonical B isoform seems to be the main isoform induced in hypoxia. Our results also indicate that the expression profile at the level of the PFKFB4 gene does not reflect the expression at the level of individual isoforms. Our work clearly indicates that the PFKFB4 gene expression profile should be definitely analyzed at the level of individual isoforms. Moreover, the analysis at the protein level allowed the selection of those isoforms whose functional validation should be performed to fully understand the importance of PFKFB4 expression in the metabolic adaptation of malignant melanoma cells.