Catabolite Repression Research Articles

Combined Transcriptome and Metabolome Analysis Reveals That Carbon Catabolite Repression Governs Growth and Pathogenicity in Verticillium dahliae

Carbon catabolite repression (CCR) is a common transcriptional regulatory mechanism that microorganisms use to efficiently utilize carbon nutrients, which is critical for the fitness of microorganisms and for pathogenic species to cause infection. Here, we characterized two CCR genes, VdCreA and VdCreC, in Verticillium dahliae that cause cotton Verticillium wilt disease. The VdCreA and VdCreC knockout mutants displayed slow growth with decreased conidiation and microsclerotium production and reduced virulence to cotton, suggesting that VdCreA and VdCreC are involved in growth and pathogenicity in V. dahliae. We further generated 36 highly reliable and stable ΔVdCreA and ΔVdCreC libraries to comprehensively explore the dynamic expression of genes and metabolites when grown under different carbon sources and CCR conditions. Based on the weighted gene co-expression network analysis (WGCNA) and correlation networks, VdCreA is co-expressed with a multitude of downregulated genes. These gene networks span multiple functional pathways, among which seven genes, including PYCR (pyrroline-5-carboxylate reductase), are potential target genes of VdCreA. Different carbon source conditions triggered entirely distinct gene regulatory networks, yet they exhibited similar changes in metabolic pathways. Six genes, including 6-phosphogluconolactonase and 2-ODGH (2-oxoglutarate dehydrogenase E1), may serve as hub genes in this process. Both VdCreA and VdCreC could comprehensively influence the expression of plant cell wall-degrading enzyme (PCWDE) genes, suggesting that they have a role in pathogenicity in V. dahliae. The integrated expression profiles of the genes and metabolites involved in the glycolysis/gluconeogenesis and pentose phosphate pathways showed that the two major sugar metabolism-related pathways were completely changed, and GADP (glyceraldehyde-3-phosphate) may be a pivotal factor for CCR under different carbon sources. All these results provide a more comprehensive perspective for further analyzing the role of Cre in CCR.

Ambrisentan reverses the prosenescent and anti-autophagic effect of high glucose on human pulmonary artery endothelial cells exposed to hypoxia

Abstract Background Cardiovascular (CV) comorbidities (systemic hypertension, hyperlipidemia, obesity, diabetes mellitus, peripheral arterial disease, coronary artery disease) are associated with reduced treatment response to specific drugs for group 1 pulmonary arterial hypertension (PAH). Often this epidemiological evidence is the result of misdiagnosis of group 2 PH associated with left heart disease, but it can also be explained as the intrinsic loss of the ability of pulmonary endothelial cells to respond to specific antiremodeling drugs. Aims To evaluate the impact of high glucose and hyperosmolar stress on the responsiveness of human pulmonary artery endothelial cells (hPAECs) to the endothelin receptor antagonist ambrisentan in terms of senescence, autophagic activity, viability and expression of some microRNAs involved in pulmonary arterial remodeling. Methods We incubated hPAECs with high glucose (HG, 30.5 mM) or high mannitol (HM, 25 mM), with/without ambrisentan (0.02 nM) in normoxia (N) or hypoxia (H) for 24 h and tested them for cell viability (MTT assay), protein and miRNA levels, autophagy and senescence (β-Gal assay) in vitro. Results H induced cell death as compared to N (752 ± 44 vs 701 ± 45 Abs units, p = 0.03). H reduced autophagy (Figure 1A-C) and induced senescence (Figure 1D), an effect further potentiated by HG and HM. Treatment with A in N and H significantly reversed the effect of HG but not HM on autophagy (Figure 1A-C) and senescence (Figure 1D). H increased the abundance of antiapoptotic miR124-3, compared to N in vehicle (V)-treated hPAEC (p=0.008) (Figure 2A), and induced a similar effect on antiapoptotic and proliferative miR191-3p, although not statistically significant (Figure 2B). In N, A potentiated the expression of both miR124-3p (p=0.007) and miR191-3p (p=0.02) in HG-treated hPAEC, and only miR191-3p in HM-treated hPAEC (p=0.01). A induced a similar effect on miR191-3p in hPAEC exposed to H+HG (p=0.02), with a non-statistically significant trend on miR124-3p. Conclusions In hPAEC exposed to H, A retains its pro-autophagic and antisenescent effects in an in vitro model mimicking diabetes. miR124-3p and miR191-3p may act as biomarkers of disease and treatment response to specific drugs in patients with PAH.Figure 1:autophagy and senescenceFigure 2:miRNA expression

Peculiarities of brain cell functioning during hyperglicemia and diabetes mellitus

Hyperglycemia is a symptom and damaging factor of diabetes mellitus (DM) that leads to systemic complications in the body, including macro- and microangiopathies of the brain, impaired blood supply, the appearance of foci of neurodegeneration and might be a trigger of neuroinflammation. Nervous tissue is characterized by a high level of energy consumption and is highly sensitive to fluctuations in the level of metabolic substrates. Therefore, it is extremely important to study the effect of high glucose levels on the functional state of the central nervous system. This review attempts to comprehensively assess the effects of hyperglycemia on brain cells. Analysis of experimental data obtained in in vivo and in vitro models of diabetes on the morphofunctional state of neurons, microglia and astrocytes showed that the direct and indirect effects of glucose in high concentrations depends on the cell type. Receptors and intracellular signaling cascades of astrocytes and microglia, that mediate the effects of hyperglycemia and the development of neuroinflammation, can act as therapeutic targets for the correction for the consequences of diabetes. Thus, finding ways to modulate the functional activity of glial cells may be an effective strategy to reduce the severity of the consequences of CNS damage.

Development of a xylose-inducible and glucose-insensitive expression system for Parageobacillus thermoglucosidasius

Inducible expression systems are pivotal for governing gene expression in strain engineering and synthetic biotechnological applications. Therefore, a critical need persists for the development of versatile and efficient inducible expression mechanisms. In this study, the xylose-responsive promoter xylA5p and its transcriptional regulator XylR were identified in Parageobacillus thermoglucosidasius DSM 2542. By combining promoter xylA5p with its regulator XylR, fine-tuning the expression strength of XylR, and reducing the glucose catabolite repression on xylose uptake, we successfully devised a xylose-inducible and glucose-insensitive expression system, denoted as IExyl*. This system exhibited diverse promoter strengths upon induction with xylose at varying concentrations and remained unhindered in the presence of glucose. Moreover, we showed the applicability of IExyl* in P. thermoglucosidasius by redirecting metabolic flux towards riboflavin biosynthesis, culminating in a 2.8-fold increase in riboflavin production compared to that of the starting strain. This glucose-insensitive and xylose-responsive expression system provides valuable tools for designing optimized biosynthetic pathways for high-value products and facilitates future synthetic biology investigations in Parageobacillus.Key points• A xylose-inducible and glucose-insensitive expression system IExyl* was developed.• IExyl* was applied to enhance the riboflavin production in P. thermoglucosidasius• A tool for metabolic engineering and synthetic biology research in Parageobacillus strains.

No Difference in Liver Damage Induced by Isocaloric Fructose or Glucose in Mice with a High-Fat Diet

Background/Objectives: The diverse effects of fructose and glucose on the progression of metabolic dysfunction-associated steatotic liver disease remain uncertain. This study investigated the effects, in animal models, of high-fat diets (HFDs) supplemented with either glucose or fructose. Methods: Six-week-old, male C57BL/6J mice were randomly allocated to four groups: normal diet (ND), HFD, HFD supplemented with fructose (30% w/v, HFD + Fru), and HFD supplemented with glucose (initially 30%, HFD + Glu). After 24 weeks, liver and plasma samples were gathered for analysis. In addition, 39 patients with obesity undergoing bariatric surgery with wedge liver biopsy were enrolled in the clinical study. Results: The HFD + Glu group consumed more water than did the HFD and HFD + Fru groups. Thus, we reduced the glucose concentration from 30% at baseline to 15% at week 2 and 10% starting from week 6. The HFD + Fru and HFD + Glu groups had a similar average caloric intake (p = 0.463). The HFD increased hepatic steatosis, plasma lipid levels, lipogenic enzymes, steatosis-related oxidative stress, hepatic inflammation, and early-stage liver fibrosis. Supplementation with fructose or glucose exacerbated liver damage, but no significant differences were identified between the two. The expression patterns of hepatic ceramides in HFD-fed mice (with or without supplemental fructose or glucose) were similar to those observed in patients with obesity and severe hepatic steatosis or metabolic dysfunction–associated steatohepatitis. Conclusions: Fructose and glucose similarly exacerbated liver damage when added to an HFD. Ceramides may be involved in the progression of hepatic lipotoxicity.

Fgk3, a Glycogen Synthase Kinase, Regulates Chitin Synthesis through the Carbon Catabolite Repressor FgCreA in Fusarium graminearum.

The glycogen synthase kinase-3 (GSK3) orthologs are well-conserved in eukaryotic organisms. However, their functions remain poorly characterized in filamentous fungi. In our previous study, we unveiled the function of Fgk3, the GSK3 ortholog, in glycogen metabolism in Fusarium graminearum, the causal agent of Fusarium head blight. Interestingly, the fgk3 mutant was unstable and tended to produce fast-growing suppressors, including secondary suppressors. Using whole-genome sequencing, we identified suppressor mutations in FgCHS5, FgFKS1, FgCREA, FgSSN6, FgRGR1, and FgPP2A in nine primary and four secondary suppressors. Subsequently, we validated that deletion of FgCHS5 or FgCREAΔH253 mutation partially suppressed the defects of fgk3 in vegetative growth and cell wall integrity, suggesting that Fgk3 may regulate the chitin synthesis through FgCreA-mediated transcriptional regulation in F. graminearum. Accordingly, the FGK3 deletion led to hyphal swelling with abnormal chitin deposition, and deletion of FGK3 or FgCREA caused the upregulation of the expression of chitin synthases FgCHS5 and FgCHS6. The interaction between Fgk3 and FgCreA was verified by Yeast two-hybrid and Co-Immunoprecipitation assays. More importantly, we verified that the nuclear localization and protein stability of FgCreA relies on the Fgk3 kinase, while the H253 deletion facilitated the re-localization of FgCreA to the nucleus in the fgk3 mutant background, potentially contributing to the suppression of the fgk3 mutant's defects. Intriguingly, the ΔH253 mutation of FgCreA, identified in suppressor mutant S3, is adjacent to a conserved phosphorylation site, S254, suggesting that this mutation may inhibit the S254 phosphorylation and promote the nuclear localization of FgCreA. Collectively, our findings indicate that the glycogen synthase kinase Fgk3 regulates the chitin synthesis through the carbon catabolite repressor FgCreA in F. graminearum.

A conserved fungal morphogenetic kinase regulates pathogenic growth in response to carbon source diversity

Fungal pathogens must exhibit strong nutritional plasticity, effectively sensing and utilizing diverse nutrients to support virulence. How the signals generated by nutritional sensing are efficiently translated to the morphogenetic machinery for optimal growth and support of virulence remains incompletely understood. Here, we show that the conserved morphogenesis-related kinase, CotA, imparts isoform-specific control over Aspergillus fumigatus invasive growth in host-mimicking environments and during infection. CotA-mediated invasive growth is responsive to exogenous carbon source quality, with only preferred carbon sources supporting hyphal morphogenesis in a mutant lacking one of two identified protein isoforms. Strikingly, we find that the CotA protein does not regulate, nor is cotA gene expression regulated by, the carbon catabolite repression system. Instead, we show that CotA partially mediates invasive growth in specific carbon sources and virulence through the conserved downstream effector and translational repressor, SsdA. Therefore, A. fumigatus CotA accomplishes its conserved morphogenetic functions to drive pathogenic growth by translating host-relevant carbon source quality signals into morphogenetic outputs for efficient tissue invasive growth.

Novel motif associated with carbon catabolite repression in two major Gram-positive pathogen virulence regulatory proteins.

Carbon catabolite repression (CCR) is a widely conserved regulatory process that ensures enzymes and transporters of less-preferred carbohydrates are transcriptionally repressed in the presence of a preferred carbohydrate. This phenomenon can be regulated via a CcpA-dependent or CcpA-independent mechanism. The CcpA-independent mechanism typically requires a transcriptional regulator harboring a phosphotransferase regulatory domain (PRD) that interacts with phosphotransferase system (PTS) components. PRDs contain a conserved histidine residue that is phosphorylated by the PTS-associated HPr-His15~P protein. PRD-containing regulators often harbor additional domains that resemble PTS-associated EIIB protein domains with a conserved cysteine residue that can be phosphorylated by cognate PTS components. We noted that Mga, the PRD-containing central virulence regulator of Streptococcus pyogenes, has an EIIBGat domain containing a cysteine that, based on the presence of a similar motif in glycerol kinase, could be a target for phosphorylation. Using site-directed mutagenesis, we constructed phospho-ablative and phospho-mimetic substitutions of this cysteine and found that these substitutions modify the CCR of the Rgg2/3 quorum-sensing system. Moreover, we provide genetic evidence that the phospho-donor of this cysteine residue is likely to be ManL, the EIIA/B subunit of the mannose PTS system. Interestingly, a structurally distinct virulence gene regulator, PrfA of Listeria monocytogenes, harbors a similar cysteine-containing motif, and phospho-ablative and phospho-mimetic substitutions of the cysteine-altered CCR of PrfA-dependent virulence gene expression. Collectively, our data suggest that phosphorylation of a cysteine within the shared novel motif in Mga and PrfA may be a heretofore missing link between cellular metabolism and virulence.IMPORTANCEIn this study, we identified a novel cysteine-containing motif within the amino acid sequence of two structurally distinct transcriptional regulators of virulence in two Gram-positive pathogens that appears to link carbon metabolism with virulence gene expression. The results also highlight the potential post-translational modification of cysteine in bacterial species, a rare and understudied modification.

Knock-out of the major regulator Flo8 in Komagataella phaffii results in unique host strain performance for methanol-free recombinant protein production

Flo8 is a main transcriptional regulator of flocculation and pseudohyphal growth in yeast. Disruption of FLO8 in the popular recombinant protein production host Komagataella phaffii (Pichia pastoris) prevents pseudohyphal growth and reduces cell-to-surface adherence, making the mutant an interesting platform for research and industry. However, knowledge of the physiological impact of the mutation remained scarce. In-depth analysis of transcriptome data from FLO8-deficient K. phaffii revealed that Flo8 affects genes involved in cell cycle, mating, respiration, and catabolite repression additionally to flocculation targets. One gene with considerably increased expression in flo8 was GTH1, encoding a high-affinity glucose transporter in K. phaffii. Its promoter (PG1) was previously established as a strong, glucose-regulatable alternative to methanol-induced promoters. PG1 and its improved derivatives PG1-3, D-PGS4 and D-PGS5, proved to be promising candidates for controlling recombinant protein production in the FLO8-deficient background. In small-scale screenings, PG13-controlled intracellular EGFP levels were 2.8-fold higher, and yields of different secreted recombinant proteins were up to 4.8-fold increased. The enhanced productivity of the flo8 mutant in combination with the PG1 variants was transferrable to glucose-limited fed-batch processes and could largely be attributed to higher transcriptional activity of the promoter, leading to a much higher productivity per chromosomally integrated gene copy. K. phaffii flo8 has many advantageous characteristics, such as reduced surface growth and increased transcriptional strength of glucose-regulatable promoters. These features turn the flo8 strain into a valuable new base strain for various experimental designs and establish flo8 as an excellent strain background for methanol-free recombinant protein production processes.

Pyruvate stimulates transamination of leucine into α-ketoisocaproic acid and supports 3-methylbutanal production by Lactococcus lactis.

To investigate the effect of pyruvate and glucose on leucine transamination and 3-methylbutanal production by Lactococcus lactis, including the comparison with cells possessing glutamate dehydrogenase (GDH) activity. L. lactis cells were incubated in chemically defined medium (CDM) with the pH controlled at 5.2 to mimic cheese conditions. Pyruvate supplementation stimulated the production of the key flavour compound 3-methylbutanal by 3 to 4 times after 72 hours of incubation. Concurrently, alanine production increased, demonstrating the involvement of pyruvate in transamination reactions. Glucose-metabolising cells excreted α-ketoisocaproic acid and produced even 3 times more 3-methylbutanal after 24 hours than pyruvate-supplemented cells. Conjugal transfer technique was used to transfer the plasmid pGdh442 carrying the gdh gene encoding for GDH to L. lactis. Introducing GDH did not stimulate the excretion of α-ketoisocaproic acid and the production of 3-methylbutanal. These results demonstrate that Lactococcus uses pyruvate to transaminate leucine into α-ketoisocaproic acid which supports 3-methylbutanal production. Surprisingly, GDH activity did not stimulate leucine transamination and 3-methylbutanal production.

Genomic Characterization and Establishment of a Genetic Manipulation System for Trichoderma sp. (Harzianum Clade) LZ117.

Trichoderma species have been reported as masters in producing cellulolytic enzymes for the biodegradation of lignocellulolytic biomass and biocontrol agents against plant pathogens and pests. In our previous study, a novel Trichoderma strain LZ117, which shows potent capability in cellulase production, was isolated. Herein, we conducted multilocus phylogenetic analyses based on DNA barcodes and performed time-scaled phylogenomic analyses using the whole genome sequences of the strain, annotated by integrating transcriptome data. Our results suggest that this strain represents a new species closely related to T. atrobrunneum (Harzianum clade). Genes encoding carbohydrate-active enzymes (CAZymes), transporters, and secondary metabolites were annotated and predicted secretome in Trichoderma sp. LZ117 was also presented. Furthermore, genetic manipulation of this strain was successfully achieved using PEG-mediated protoplast transformation. A putative transporter gene encoding maltose permease (Mal1) was overexpressed, which proved that this transporter does not affect cellulase production. Moreover, overexpressing the native Cre1 homolog in LZ117 demonstrated a more pronounced impact of glucose-caused carbon catabolite repression (CCR), suggesting the importance of Cre1-mediated CCR in cellulase production of Trichoderma sp. LZ117. The results of this study will benefit further exploration of the strain LZ117 and related species for their applications in bioproduction.

7578 SGLT2 Inhibitor Is Associated With A Decreased Risk Of Cerebrovascular Events And Dementia Compared To Thiazolidinedione In Subjects With Type 2 Diabetes

Abstract Disclosure: H. Park: None. S. Park: None. H. Kim: None. D. Byun: None. Subjects with type 2 diabetes (T2D) are at higher risk of developing cerebrovascular disease, cognitive decline, and dementia. Hyperinsulinemia and insulin resistance in the brain have been linked to amyloid beta metabolism, raising the risk of Alzheimer's dementia. Recently, several clinical studies have shown potential beneficial effects of sodium glucose co-transporter-2 (SGLT2) inhibitors, and thiazolidinediones, in the risk of cerebrovascular events and dementia in T2D. This study was aimed to investigate the comparative efficacy of SGLT2 inhibitors and thiazolidinediones in modifying the risk of cerebrovascular events, dementia, and Alzheimer's dementia. Using the common data model, we analyzed patients from December 31, 2011 to December 30, 2022 from 15 hospitals in South Korea. Clinical outcomes between SGLT2 inhibitors and thiazolidinediones were compared using hazard ratios (HRs), with large-scale propensity score matching and a random-effects model applied. Our analyses showed that subjects on SGLT2 inhibitors had a significantly reduced rate of cerebrovascular events (HR 0.2787, 95% CI 0.1882-0.4127, p<0.00001), dementia (HR 0.5395, 95% CI 0.3824-0.7614, p=0.0004), and Alzheimer's dementia (HR 0.586, 95% CI 0.4634-0.7411, p<0.00001), compared to those on thiazolidinediones. In summary, these results suggest that SGLT2 inhibitor administration is associated with a lower risk of cerebrovascular events, dementia, and Alzheimer's dementia compared to thiazolidinedione in subjects with T2D. Further studies are warranted to confirm our findings and elucidate the underlying mechanisms. Presentation: 6/3/2024

6692 Proarrhythmic Risk Of Hypoglycemia; A Case Of Insulin-Induced Hypoglycemia Provoking Torsade De Pointes-Cardiac Arrest

Abstract Disclosure: A.M. San Hernandez: None. A. Adelkhanova: None. H. Ashraf: None. J. Aguayo Herrera: None. M.F. Siddiqui: None. Introduction: Torsade de Pointes is a life-threatening polymorphic ventricular tachycardia which is associated with QTc prolongation caused by medications, metabolic and electrolyte abnormalities. Very few cases of hypoglycemia causing QTc prolongation and Torsades de Pointes have been reported in the literature. Although the mechanism is unclear, the effects of low blood glucose have been proposed to induce a sympathetic response, hypokalemia and calcium disturbances. We report a case of an insulin-induced hypoglycemia causing Torsades de Pointes cardiac arrest, highlighting the proarrhythmic risk of hypoglycemia. Case presentation: A 57-year-old female with a history of Insulin-dependent Type 2 Diabetes Mellitus (DM) was brought in by EMS due to severe symptomatic hypoglycemia (POC blood glucose of 30 mg/dL), requiring treatment with dextrose. Patient reported reduced oral intake due to vomiting for 1 week and continued insulin administration without self-monitoring of blood glucose. Laboratory work up on presentation revealed severe hypoglycemia VBG= 55 mg/dL and mild hypomagnesemia Mg=1.4mg/dL (Ref Range 1.7-2.2mg/dl) which was treated. There was no hypokalemia or hypocalcemia. ECG showed sinus rhythm with prolonged QTc interval of 573ms and non-specific ST changes.Few hours later, patient developed torsades de pointes, requiring ACLS resuscitation. ROSC was achieved after two rounds of CPR. Patient received dextrose infusion to maintain euglycemic state. She was not taking any QTc prolonging medication. There was no history of autoimmune disease. Except for hypoglycemia, no other metabolic abnormality could be identified. Cardiac and pulmonary etiologies of cardiac arrest were ruled out. Post-ROSC ECG showed sinus tachycardia with normalization of QTc to 454ms. Thus, Insulin-induced hypoglycemia was appointed as the main culprit for her cardiac arrest, in the setting of QTc prolongation. Patient received diabetes self-management and hypoglycemia education. Conclusion: Several studies support strong correlation between hypoglycemia and arrhythmic events during bedtime in people with diabetes “The dead in bed syndrome”. Hypoglycemia causes an acquired long QT syndrome independent of other risk factors such as cardiac autonomic neuropathy. Possible mechanisms include; sympathoadrenal activation, which regulates potassium, calcium and chloride channels or altered autonomic regulation caused by hypoglycemia itself regardless of the effect of insulin or hypokalemia. QT prolongation is a risk factor for sudden cardiac death by causing torsades de pointes ventricular tachycardia (VT).People with insulin dependent diabetes are at high risk of hypoglycemia and should be cautioned about the proarrhythmic risk of hypoglycemia. Education is crucial, and Continuous glucose monitor (CGM) is a valuable tool to be use in such a patient population. Presentation: 6/3/2024

Reduction in mitochondrial ATP synthesis mimics the effect of low glucose in depolarizing neurons from the subpostremal nucleus of the solitary tract of rats.

Neurons of the subpostremal nucleus of the solitary tract (NTS) respond to changes in extracellular glucose with alterations in membrane potential with both depolarization and hyperpolarization. From 5 mM glucose, a rapid shift to 0.5 mM glucose produces a membrane depolarization by an unknown mechanism in most neurons. However, the mechanism involved in this response needs to be known. Here, we investigated if the low glucose-induced depolarization could be mimicked by reducing ATP synthesis and possible mediators of this effect. We showed that applying the mitochondrial uncoupler CCCP (1 µM) reproduced the effects of low glucose depolarizing the membrane, generating an inward current, and decreasing membrane resistance. On the other hand, activation of AMPK did not alter these parameters. To test if low glucose and CCCP could depolarize the membrane by affecting the ionic gradient, we inhibited the electrogenic Na/K pump with 10 µM of ouabain. We observed a similar membrane depolarization but not a decrease in membrane resistance. We conclude that perfusion of neurons of the subpostremal NTS with a low glucose solution depolarizes the membrane by probably reducing intracellular ATP, but not by activating AMPK or decreasing the ionic gradient across the membrane.

Dynamic glycolytic reprogramming effects on dendritic cells in pancreatic ductal adenocarcinoma

BackgroundPancreatic ductal adenocarcinoma tumors exhibit resistance to chemotherapy, targeted therapies, and even immunotherapy. Dendritic cells use glucose to support their effector functions and play a key role in anti-tumor immunity by promoting cytotoxic CD8+ T cell activity. However, the effects of glucose and lactate levels on dendritic cells in pancreatic ductal adenocarcinoma are unclear. In this study, we aimed to clarify how glucose and lactate can impact the dendritic cell antigen-presenting function and elucidate the relevant mechanisms.MethodsGlycolytic activity and immune cell infiltration in pancreatic ductal adenocarcinoma were evaluated using patient-derived organoids and resected specimens. Cell lines with increased or decreased glycolysis were established from KPC mice. Flow cytometry and single-cell RNA sequencing were used to evaluate the impacts on the tumor microenvironment. The effects of glucose and lactate on the bone marrow-derived dendritic cell antigen-presenting function were detected by flow cytometry.ResultsThe pancreatic ductal adenocarcinoma tumor microenvironment exhibited low glucose and high lactate concentrations from varying levels of glycolytic activity in cancer cells. In mouse transplantation models, tumors with increased glycolysis showed enhanced myeloid-derived suppressor cell infiltration and reduced dendritic cell and CD8+ T cell infiltration, whereas tumors with decreased glycolysis displayed the opposite trends. In three-dimensional co-culture, increased glycolysis in cancer cells suppressed the antigen-presenting function of bone marrow-derived dendritic cells. In addition, low-glucose and high-lactate media inhibited the antigen-presenting and mitochondrial functions of bone marrow-derived dendritic cells.ConclusionsOur study demonstrates the impact of dynamic glycolytic reprogramming on the composition of immune cells in the tumor microenvironment of pancreatic ductal adenocarcinoma, especially on the antigen-presenting function of dendritic cells.

The potato RNA metabolism machinery is targeted by the cyst nematode effector RHA1B for successful parasitism.

The potato (Solanum tuberosum) cyst nematode Globodera pallida induces a multinucleate feeding site (syncytium) in potato roots as its sole source of nutrition. Here, we demonstrate that the G. pallida effector RING-H2 finger A1b (RHA1B), which is a functional ubiquitin ligase, interferes with the carbon catabolite repression 4 (CCR4)-negative on TATA-less (NOT) deadenylase-based RNA metabolism machinery that regulates syncytium development in G. pallida-infected potato. Specifically, RHA1B targets the CCR4-associated factor 1 (CAF1) and StNOT10 subunits of the CCR4-NOT complex for proteasome-mediated degradation, leading to upregulation of the cyclin gene StCycA2 involved in syncytium formation. The StCAF1 subunit of CCR4-NOT recruits the RNA binding protein StPUM5 to deadenylate StCycA2 mRNA, resulting in shortened poly-A tails of StCycA2 mRNA and subsequently reduced transcript levels. Knockdown of either subunit (StCAF1 or StNOT10) of the CCR4-NOT complex or StPUM5 in transgenic potato plants resulted in enlarged syncytia and enhanced susceptibility to G. pallida infection, which resembles the phenotypes of StCycA2 overexpression transgenic potato plants. Genetic analyses indicate that transgenic potato plants overexpressing RHA1B exhibit similar phenotypes as transgenic potato plants with knockdown of StNOT10, StCAF1, or StPUM5. Thus, our data suggest that G. pallida utilizes the RHA1B effector to manipulate RNA metabolism in host plants, thereby promoting syncytium development for parasitic success.

Are1-mediated nitrogen metabolism is associated with iron regulation in the mycoparasite Trichoderma atroviride

Trichoderma atroviride is a mycoparasitic fungus with antagonistic activity against fungal pathogens and is used as a pathogen control agent alternative to synthetic fungicides. Sensing nutrient availability in the environment and adjusting metabolism for optimal growth, development and reproduction is essential for adaptability and is relevant to its mycoparasitic activity. During mycoparasitism, secondary metabolites are produced to weaken the fungal prey and support the attack. Are1-like proteins act as major GATA-type transcription factors in the activation of genes subject to nitrogen catabolite repression. Since the quality and quantity of nitrogen has been proven particularly relevant in remodeling the biosynthesis of secondary metabolites in fungi, we decided to functionally characterize Are1, the ortholog of Aspergillus nidulans AreA, in T. atroviride. We show that the growth of the T. atroviride ∆are1 mutant is impaired in comparison to the wild type on several nitrogen sources. Deletion of are1 enhanced sensitivity to oxidative and cell-wall stressors and altered the mycoparasitic activity. We were able to identify for the first time a link between Are1 and iron homeostasis via a regulatory mechanism that does not appear to be strictly linked to the nitrogen source, but rather to an independent role of the transcription factor.

Elevated blood glucose levels are associated with the progression of brain hypometabolism, and HDL-C and APOE4 add to this association.

Brain glucose hypometabolism has consistently been found in neurodegenerative disorders, including Alzheimer's disease (AD). High blood glucose and HDL cholesterol (HDL-C) levels have also been linked to neurodegeneration and AD. However, there is limited understanding of the relationships between dementia-related risk factors in the brain and blood. A linear mixed model was used to examine the relationship between blood glucose and HDL-C levels and the progression of brain hypometabolism, adjusting for APOE4 and other clinical covariates. The hypometabolic convergence index (HCI) was measured by fluorodeoxyglucose-18 (FDG) positron emission tomography (PET) in participants from the Alzheimer's Disease Neuroimaging Initiative (ADNI). Data visualizations were generated to understand the joint effects of plasma glucose, HDL-C, and APOE4 on HCI. There were 336 individuals (781 observations), of whom 22.62% had AD. The majority were male (63.98%) and of white race, and 48.51% were carriers of APOE4. Over time, high blood glucose level was associated with the progression of brain glucose hypometabolism (β=0.33, 95% CI: 0.02, 0.64, p<0.05). A high plasma HDL-C level (β=1.22, 95% CI: 0.09, 2.35, p<0.05), more study visits (β=1.67, 95% CI: 1.37, 1.98, p<0.001), and being an APOE4 allele carrier (β=1.29, 95% CI: 0.15, 2.42, p<0.05) were also significant predictors of brain hypometabolism progression. APOE4 carrier status and number of visits account for the largest proportion of the variance from the fixed effects model. Random effects due to participant characteristics and fixed effects together accounted for 95.2% of the model variance. Subgroup analysis revealed that these effects were observed only in those without AD. High plasma glucose levels facilitated the progression of brain hypometabolism. The effect was more prominent in the APOE4 double-carriers with elevated HDL-C. Elevated blood glucose may reflect systemic insulin resistance, which could impair brain glucose uptake, resulting in brain hypometabolism. Controlling blood glucose and HDL-C levels in APOE4 carriers may improve brain metabolism, potentially delaying the onset of dementia.

DNA Methylation Age Mediates Effect of Metabolic Profile on Cardiovascular and General Aging.

Alterations in lipid metabolism and DNA methylation are 2 hallmarks of aging. Connecting metabolomic, epigenomic, and aging outcomes help unravel the complex mechanisms underlying aging. We aimed to assess whether DNA methylation clocks mediate the association of circulating metabolites with incident atherosclerotic cardiovascular disease (ASCVD) and frailty. The China Kadoorie Biobank is a prospective cohort study with a baseline survey from 2004 to 2008 and a follow-up period until December 31, 2018. We used the Infinium Methylation EPIC BeadChip to measure the methylation levels of 988 participants' baseline blood leukocyte DNA. Metabolite profiles, including lipoprotein particles, lipid constituents, and various circulating metabolites, were measured using quantitative nuclear magnetic resonance. The pace of DNA methylation age acceleration (AA) was calculated using 5 widely used epigenetic clocks (the first generation: Horvath, Hannum, and Li; the second generation: Grim and Pheno). Incident ASCVD was ascertained through linkage with local death and disease registries and national health insurance databases, supplemented by active follow-up. The frailty index was constructed using medical conditions, symptoms, signs, and physical measurements collected at baseline. A total of 508 incident cases of ASCVD were documented during a median follow-up of 9.5 years. The first generation of epigenetic clocks was associated with the risk of ASCVD (P<0.05). For each SD increment in LiAA, HorvathAA, and HannumAA, the corresponding hazard ratios for ASCVD risk were 1.16 (1.05-1.28), 1.10 (1.00-1.22), and 1.17 (1.04-1.31), respectively. Only LiAA mediated the association of various metabolites (lipids, fatty acids, histidine, and inflammatory biomarkers) with ASCVD, with the mediating proportion reaching up to 15% for the diameter of low-density lipoprotein (P=1.2×10-2). Regarding general aging, a 1-SD increase in GrimAA was associated with an average increase of 0.10 in the frailty index (P=2.0×10-3), and a 33% and 63% increased risk of prefrailty and frailty at baseline (P=1.5×10-2 and 5.8×10-2), respectively; this association was not observed with other clocks. GrimAA mediated the effect of various lipids, fatty acids, glucose, lactate, and inflammatory biomarkers on the frailty index, with the mediating proportion reaching up to 22% for triglycerides in very small-sized very low-density lipoprotein (P=6.0×10-3). These findings suggest that epigenomic mechanisms may play a role in the associations between circulating metabolites and the aging process. Different mechanisms underlie the first and second generations of DNA methylation age in cardiovascular and general aging.

Regulatory insight for a Zn2Cys6 transcription factor controlling effector-mediated virulence in a fungal pathogen of wheat.

The regulation of virulence in plant-pathogenic fungi has emerged as a key area of importance underlying host infections. Recent work has highlighted individual transcription factors (TFs) that serve important roles. A prominent example is PnPf2, a member of the Zn2Cys6 family of fungal TFs, which controls the expression of effectors and other virulence-associated genes in Parastagonospora nodorum during infection of wheat. PnPf2 orthologues are similarly important for other major fungal pathogens during infection of their respective host plants, and have also been shown to control polysaccharide metabolism in model saprophytes. In each case, the direct genomic targets and associated regulatory mechanisms were unknown. Significant insight was made here by investigating PnPf2 through chromatin-immunoprecipitation (ChIP) and mutagenesis approaches in P. nodorum. Two distinct binding motifs were characterised as positive regulatory elements and direct PnPf2 targets identified. These encompass known effectors and other components associated with the P. nodorum pathogenic lifestyle, such as carbohydrate-active enzymes and nutrient assimilators. The results support a direct involvement of PnPf2 in coordinating virulence on wheat. Other prominent PnPf2 targets included TF-encoding genes. While novel functions were observed for the TFs PnPro1, PnAda1, PnEbr1 and the carbon-catabolite repressor PnCreA, our investigation upheld PnPf2 as the predominant transcriptional regulator characterised in terms of direct and specific coordination of virulence on wheat, and provides important mechanistic insights that may be conserved for homologous TFs in other fungi.