Journal: The Journal of biological chemistry

Article Title: The arginine and nitric oxide metabolic pathway regulate the gut colonization and expansion of Ruminococcous gnavus.

doi: 10.1016/j.jbc.2024.107614

Figure Lengend Snippet: Figure 5. NO suppresses Ruminococcus gnavus growth, which is expanded in Nos2−/−mice. A–D, growth curves of four R. gnavus strains (ATCC 29149, ATCC 35913, AGR 2154, H2-28) in the presence of varying concentrations of the NO donor, DNNate (Veh, 0.05 mM, 0.5 mM, 5 mM) over 24 h. Insets show cultures turbidity at the end of the experiment with the indicated NO donor concentrations. E, relative abundance of R. gnavus in fecal samples from WT and Nos2 KO mice, showing a significant increase in R. gnavus abundance in Nos2 KO mice, **p < 0.01. n = 5 to 6 per genotype. F, fecal R. gnavus abundance was determined by quatitative polymerase chain reaction. Note that feeding NO donor to Nos2−/−mice reduced R. gnavus abundance (day 0–4), which recovered during a washout phase of NO donor (day 4–15). G, Alcian blue staining of goblet cells for WT and Nos2−/−mouse ileum. Quantification of Alcian blue positive cells per individual crypt-villus units of WT and Nos2−/−mouse ileum. The scale bars represent 100 mm. H, Mptx2 staining for WT and Nos2−/−mouse ileum. Quantification of Mptx2 abundance per crypt unit of WT and Nos2−/−mouse ileum. The scale bars represents 100 mm. I, Dclk1 staining for WT and Nos2−/−mouse ileum. Quantification of Dclk1 positive cells along individual crypt-villus units of WT and Nos2−/− mouse ileum. The scale bars represents 100 mm. J, lysozyme staining for WT and Nos2−/−mouse ileum. Quantification of lysozyme abundance per crypt unit of WT and Nos2−/−mouse ileum. The scale bars represents 100 mm. Data are expressed as mean ± SD (n = 3 per group). Statistical significance was determined by one-way ANOVA with post hoc testing (****p < 0.0001). ATCC, American Type Culture Collection; DNNoate, Deta-NONOate; NO, nitric oxide.

Article Snippet: Cultures were grown in 14 ml tubes containing 10 ml of AnaeroGRO Chopped Meat Glucose Broth (MB) (Hardy Diagnostics, AG19H) at 37 C. NO stress was induced using DNNoate (Thermo Fisher Scientific, 328651000) at concentrations of 0.05, 0.5, and 5 mM, and a DNNoate-free control.

Techniques: Polymerase Chain Reaction, Staining

Journal: Cell Cycle

Article Title: Mechanisms of nitric oxide-mediated inhibition of EMT in cancer

doi: 10.4161/cc.9.24.14229

Figure Lengend Snippet: DETANONOate inhibits NFκB and reverses the mesenchymal and invasive cell phenotypes. (A) Reversal of the mesenchymal cell phenotype by DETANONOate. Protein lysates derived from DU-145 and PC-3 cells before and after treatment with 1,000 µM DETANONOate for 4 and 12 h were subjected to western blot analysis for the determination of E-cadherin, vimentin and fibronectin expression. Actin was used as an internal control for loading. (B) DETANONOate inhibits the invasive properties of DU145 cells. 2.5 × 104 DU-145 cells were seeded in the upper chamber of the in vitro invasion assay system described in methods, while RPMI 1640 containing 10% FBS was added in the lower chamber. The cells were treated or left untreated with 200, 500 or 1,000 µM DETANONOate and incubated for 22 h. Invaded cells were counted under the microscope after matrigel membrane fixation and staining with crystal violet. Data are expressed as the percent invasion through the matrigel matrix and membrane relative to the migration through the control insert membrane. p < 0.03 treated vs. untreated cells (Mann-Whitney U test). (C) Inhibition of NFκB DNA binding activity by DETANONOate. DU145 cells were treated with 500 or 1,000 uM DETANONOate for 4 h and the derived nuclear extracts were subjected to EMSA. β-actin levels were used as loading control.

Article Snippet: The NO donor, DETANONOate was purchased from Alexis (Biomol International LP).

Techniques: Derivative Assay, Western Blot, Expressing, Control, In Vitro, Invasion Assay, Incubation, Microscopy, Membrane, Staining, Migration, MANN-WHITNEY, Inhibition, Binding Assay, Activity Assay

Journal: Cell Cycle

Article Title: Mechanisms of nitric oxide-mediated inhibition of EMT in cancer

doi: 10.4161/cc.9.24.14229

Figure Lengend Snippet: DETANONOate inhibits Snail expression and activity. (A) DETANONOate inhibits Snail expression. Time kinetic analysis of Snail transcripts (upper part) and protein levels (lower part) after cell treatment with 1,000 uM DETANONOate, as assessed by real-time PCR and western blot analyses, respectively. The inhibition of Snail transcript levels are shown as increase in the normalized cycle threshold (Ct) values of the amplified product as function of time. The Ct values of GAPDH assessed in the same samples were used as normalizing controls. Data are expressed as the average normalized Ct values ± STDEV from one experiment performed in triplicates. *p < 0.05: treated vs. untreated cells. (B) Inhibition of Snail DNA binding activity by DETANONOate. DU145 cells were incubated in serum-free medium in the presence of absence of DETANONOate for 30 min or 4 h. The Snail's DNA-binding activity was evaluated by EMSA using a biotin-labeled oligonucleotide Snail probe. (C) DETANONOate induces S-nitrosylation of Snail protein. Immunoprecipitation of S-nitrosylated Snail after DETANONOate treatment (1,000 uM, 4 h). Total cell lysates were used in an immunoprecipitation assay using protein A beads as described in materials and methods. S-nitrosylated proteins were precipitated with an anti-S-nitroso-cysteine (SNO-Cys) antibody and the membranes were immunoblotted with Snail polyclonal antibody. Rabbit IgG was used as a negative control. (D) Direct role of Snail suppression in eMt inhibition. Total cell lysates were harvested from DU 145 cells after suppression of Snail for 72 h using Snail siRNA and subjected to western blot analysis for determination of the protein expression of the indicated gene products. A random nucleotide sequence (CNTR siRNA) was used as a control in the transfection assays. Actin was served as an internal control.

Article Snippet: The NO donor, DETANONOate was purchased from Alexis (Biomol International LP).

Techniques: Expressing, Activity Assay, Real-time Polymerase Chain Reaction, Western Blot, Inhibition, Amplification, Binding Assay, Incubation, Labeling, Immunoprecipitation, Negative Control, Sequencing, Control, Transfection

Journal: Cell Cycle

Article Title: Mechanisms of nitric oxide-mediated inhibition of EMT in cancer

doi: 10.4161/cc.9.24.14229

Figure Lengend Snippet: DETANONOate upregulates RKIP expression. (A) DETANONOate upregulates RKIP transcript levels in PC-3 cells as assessed by real-time PCR. The induction of RKIP transcript levels are shown as decrease in the normalized cycle threshold (Ct) values of the amplified product as a function of time. The Ct values of GAPDH assessed in the same samples were used as normalizing controls. Data are expressed as the average normalized Ct values ± STDEV from one experiment performed in triplicates. *p < 0.05: treated vs. untreated cells. (B) Induction of RKIP protein expression by DETANONOate. Protein lysates from DU145 and PC-3 cells were harvested at 4 and 12 h post-treatment with DETANONOate and subjected to western blot analysis for RKIP protein determination. (C) Regulation of RKIp promoter activity by Snail. DU145 cells were co-transfected with Snail siRNA or the relative control siRNA and the pRKIP-Luc wild type (pRKIP-Luc w/t), or the pRKIP-Luc mutant (pRKIP-Luc mut) promoter construct which has mutated 3 out of 5 E-boxes. *p = 0.013: transfected vs. transfected and treated with Snail siRNA cells. (D) Direct role of RKIP in EMT inhibition. Total cell lysates were harvested from DU145 cells after overexpression of RKIP for 48 h using a CMV-HA-RKIP vector and subjected to western blot analysis for determination of the protein expression of the indicated gene products. A CMV empty vector (CMV-HA-EV) was used as a negative transfection control. (E) Silencing of RKIP by siRNA reverses the DETANONOATE-mediated inhibition of the EMT phenotype in DU145 cells. Cells were pre-treated with RKIP siRNA or control siRNA for 70 h and then were exposed to DETANONOate (1,000 uM) for additional 12 h. A random nucleotide sequence (CNTR siRNA) was used as a negative silencing control. The expressions of RKIP, vimentin and fibronectin were assessed by western blot analysis. Actin was served as an internal control in all protein assays.

Article Snippet: The NO donor, DETANONOate was purchased from Alexis (Biomol International LP).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Amplification, Western Blot, Activity Assay, Transfection, Control, Mutagenesis, Construct, Inhibition, Over Expression, Plasmid Preparation, Sequencing

Journal: Cell Cycle

Article Title: Mechanisms of nitric oxide-mediated inhibition of EMT in cancer

doi: 10.4161/cc.9.24.14229

Figure Lengend Snippet: Validation of the NO-mediated reversal of mesenchymal cell phenotype in mice bearing PC-3 xenografts. (A) Immunohistochemical analysis of fibronectin, vimentin and E-cadherin expression in PC-3 cells following treatment with 500 and 1,000 uM of DETANONOate. Rabbit IgG control was used as a negative control. (B) Representative immunohistochemical staining for RKIP, Snail, Fibronectin, and E-cadherin expression in biopsies derived from mice bearing PC-3 tumors. As above, rabbit IgG control was used as negative control.

Article Snippet: The NO donor, DETANONOate was purchased from Alexis (Biomol International LP).

Techniques: Biomarker Discovery, Immunohistochemical staining, Expressing, Control, Negative Control, Staining, Derivative Assay

Journal: Cell Cycle

Article Title: Mechanisms of nitric oxide-mediated inhibition of EMT in cancer

doi: 10.4161/cc.9.24.14229

Figure Lengend Snippet: the NFκB/Snail/RKIP crosstalk in DETANONOate-mediated inhibition of EMT. We propose that the NO-mediated inhibition of NFκB activity serves as a modulator of EMT responses not only by itself but also via regulation of downstream EMt-related targets such as Snail. The NO-mediated suppression of Snail expression and activity in turn results in the derepression of its transcriptional targets RKIP and E-cadherin and the inhibition of Snail-regulated mesenchymal markers. RKIP induction attenuates directly a feedback loop of Snail suppression via NFκB inhibition in addition to suppression mediated directly by NO. The reported modifications in the expressions and activities of the above gene products constitute a suggested mechanism by which DETANONOate inhibits the mesenchymal cell phenotype and the migratory and invasive properties of metastatic prostate cells in vitro and in vivo.

Article Snippet: The NO donor, DETANONOate was purchased from Alexis (Biomol International LP).

Techniques: Inhibition, Activity Assay, Expressing, In Vitro, In Vivo

Journal: Advanced Healthcare Materials

Article Title: Immunometabolic Effect of Nitric Oxide on Human Macrophages Challenged With the SARS‐CoV2‐Induced Cytokine Storm. A Fluxomic Approach

doi: 10.1002/adhm.202401688

Figure Lengend Snippet: Cytokine (CK) administration to hMφ fails to induce a significant NOS2 expression but increases ROS production and mitochondrial inner membrane potential. Effect of incubation with the NO‐donor DETA‐NO. Human and murine Mφ (2×10 6 cells) were treated as indicated in the corresponding panels. A) Results show the RT‐qPCR cycle thresholds of NOS2 and the normalization gene RPLP0 in human and murine Mφ after 24 h of incubation with the indicated stimuli; B) representative Western blot analysis of NOS‐2 in human and murine Mφ after incubation for 24 h with 500 µ m DETA‐NO and/or CK. LPS and IFN‐γ were used at 1 µg mL −1 and 20 ng mL −1 , respectively, as a positive control; the levels of β‐actin were used as a normalization for lane charge. C) Nitrite accumulation in the culture medium of human and murine Mφ corresponding to panel B. D) Cell viability; E) ROS production; F) mitochondrial membrane potential (determined by CMXRos fluorescence intensity, in %) of hMφ after 24 h of treatment with the indicated stimuli. Results show the mean ± SD of at least 10 distinct donors, or a representative blot (B). CK represents 20 ng mL −1 each of IL‐1β, IL‐6, IL‐8, TNFα, GM‐CSF, and IFNγ. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 versus untreated cells or the 500 µ m DETA‐NO condition (CK samples); ns, not statistically significant.

Article Snippet: After that, the NO donor DETA NONOate (DETA‐NO; ALX‐430‐014, Enzo) was added, and the cells were incubated for another 24h.

Techniques: Expressing, Membrane, Incubation, Quantitative RT-PCR, Western Blot, Positive Control, Fluorescence

Journal: Advanced Healthcare Materials

Article Title: Immunometabolic Effect of Nitric Oxide on Human Macrophages Challenged With the SARS‐CoV2‐Induced Cytokine Storm. A Fluxomic Approach

doi: 10.1002/adhm.202401688

Figure Lengend Snippet: Effect of DETA‐NO administration on glucose and oxygen consumption in hMφ upon cytokine storm (CK) treatment. A) Cells (2×10 6 ) were cultured in RPMI1640 medium and 2% FCS, and incubated with 500 µ m DETA‐NO and CK. The glucose consumption and lactate accumulation were determined in the culture medium at the indicated times. B) The ratio between glucose consumption and lactate accumulation was determined for the indicated periods as a measure of the glycolytic flux from glucose to lactate (left panel), while the metabolic rate was calculated by subtracting the concentrations of each metabolite at the indicated times. Metabolic rate is shown as absolute values (right panel) C) The oxygen consumption rate (OCR) after 24 h of treatment of hMφ (2×10 4 cells well −1 ) with the indicated stimuli was assayed in a Seahorse XF platform. At the indicated times oligomycin (OL), 2′,4′‐dinitrophenol (DNP), and rotenone plus antimycin (R+A) were added. The basal and maximal respiration rates were quantified (right panels). Results show the means ± SD from 7 different healthy donors assayed by triplicate. * p < 0.05; ** p < 0.01; *** p < 0.001 versus the corresponding untreated condition.

Article Snippet: After that, the NO donor DETA NONOate (DETA‐NO; ALX‐430‐014, Enzo) was added, and the cells were incubated for another 24h.

Techniques: Cell Culture, Incubation

Journal: Advanced Healthcare Materials

Article Title: Immunometabolic Effect of Nitric Oxide on Human Macrophages Challenged With the SARS‐CoV2‐Induced Cytokine Storm. A Fluxomic Approach

doi: 10.1002/adhm.202401688

Figure Lengend Snippet: Regulatory effects of DETA‐NO and CK on the expression of inflammation‐related genes in hMφ. hMφ (10 7 cells per condition) from healthy donors were incubated for 24 h with the indicated stimuli and the RNA was isolated and analyzed by RT‐qPCR. Results show the mean ± SD of fold induction (F.I.) from 10 different donors assayed per triplicate. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 versus the untreated condition, or the DETA‐NO condition (upper values). ns , not statistically significant.

Article Snippet: After that, the NO donor DETA NONOate (DETA‐NO; ALX‐430‐014, Enzo) was added, and the cells were incubated for another 24h.

Techniques: Expressing, Incubation, Isolation, Quantitative RT-PCR

Journal: Advanced Healthcare Materials

Article Title: Immunometabolic Effect of Nitric Oxide on Human Macrophages Challenged With the SARS‐CoV2‐Induced Cytokine Storm. A Fluxomic Approach

doi: 10.1002/adhm.202401688

Figure Lengend Snippet: RNAseq gene expression analysis of hMφ after treatment with DETA‐NO and CK. Results show the Volcano plots of all quantified genes in the transcriptome analysis of 10 7 hMφ per condition (24 h of treatment), and the corresponding Venn diagrams (up‐ and down‐regulated genes) to define the specificities of each treatment (untreated control, 500 µ m DETA‐NO and CK). Statistically significant differentially expressed genes (up‐ or down‐regulated; red and blue colors, respectively) are defined as those with at least ± 30% change (vertical lines in the Volcano plots) and p < 0.05. Volcano plots were generated as the mean log 2 of fold change versus ‐log 10 of hMφ p‐values from 10 distinct healthy donors.

Article Snippet: After that, the NO donor DETA NONOate (DETA‐NO; ALX‐430‐014, Enzo) was added, and the cells were incubated for another 24h.

Techniques: Gene Expression, Control, Generated

Journal: Advanced Healthcare Materials

Article Title: Immunometabolic Effect of Nitric Oxide on Human Macrophages Challenged With the SARS‐CoV2‐Induced Cytokine Storm. A Fluxomic Approach

doi: 10.1002/adhm.202401688

Figure Lengend Snippet: Top canonical pathways modified in hMφ treated with DETA‐NO plus CK versus CK. Upregulated and downregulated canonical pathways with a higher normalized enrichment score (NES), in absolute value.

Article Snippet: After that, the NO donor DETA NONOate (DETA‐NO; ALX‐430‐014, Enzo) was added, and the cells were incubated for another 24h.

Techniques: Modification

Journal: Advanced Healthcare Materials

Article Title: Immunometabolic Effect of Nitric Oxide on Human Macrophages Challenged With the SARS‐CoV2‐Induced Cytokine Storm. A Fluxomic Approach

doi: 10.1002/adhm.202401688

Figure Lengend Snippet: Modulation of metabolic fluxes predicted by human Genome‐Scale Metabolic Model (GSMM) in hMφ treated with DETA‐NO and CK. A) Distribution of metabolic fluxes across several pathways, as determined by the GIM3E algorithm and sampling procedure. B) Relative log2 fold change of pathways among conditions determined from qMTA procedure. * p < 0.05; ** p < 0.01; *** p < 0.001 (panel A) versus the control value or the CK condition (bar values). ns , not statistically significant.

Article Snippet: After that, the NO donor DETA NONOate (DETA‐NO; ALX‐430‐014, Enzo) was added, and the cells were incubated for another 24h.

Techniques: Sampling, Control

Journal: Advanced Healthcare Materials

Article Title: Immunometabolic Effect of Nitric Oxide on Human Macrophages Challenged With the SARS‐CoV2‐Induced Cytokine Storm. A Fluxomic Approach

doi: 10.1002/adhm.202401688

Figure Lengend Snippet: Fluxes of glycolysis, pentose phosphate, and TCA cycle pathways in hMφ treated with DETA‐NO and CK. Upregulated and downregulated reactions are indicated for Glycolysis, Pentose phosphate, and TCA cycle pathways. Reaction colors correspond to the average flux values obtained from the sampling procedure (units: 1884 pmol/(h·cell)). The thickness and color of the arrows are based on the flux values. Fluxes with values higher than 1.5 are assigned the maximum scale color. In the diagrams, the arrow indicates the direction of the flux. Glycolysis : HEX1: hexokinase 1; PGI: glucose‐6‐phosphate isomerase; PFK: phosphofructokinase; FBA: fructose‐1,6‐bisphosphate aldolase; TPI: triosephosphate isomerase; GAPD: glyceraldehyde 3‐phosphate dehydrogenase; PGK: phosphoglycerate kinase; PGM: phosphoglycerate mutase; ENO: enolase; PYRt2m: mitochondrial pyruvate carrier; PDH: pyruvate dehydrogenase; LDH_L: L‐lactate dehydrogenase; glc: glucose; g6p: glucose‐6‐phosphate; f6p: fructose‐6‐phosphate; fdp: fructose 1,6‐bisphosphate; dhap: dihydroxyacetone phosphate; g3p: glyceraldehyde‐3‐phosphate; 13dpg: 1,3‐bisphosphoglycerate; 3pg: 3‐phosphoglycerate; 2pg: 2‐phosphoglycerate; pep: phosphoenolpyruvate; pyr: pyruvate; lac_L: L‐lactate; accoa: acetyl‐coenzyme A; atp: adenosine triphosphate; adp: adenosine diphosphate; h: proton; nadh: nicotinamide adenine dinucleotide; nadph: nicotinamide adenine dinucleotide phosphate; pi: inorganic phosphate; coa: coenzyme‐A; _c: cytosolic; _m: mitochondrial. Pentose phosphate pathway (PPP) : G6PDH2r: glucose‐6‐phosphate dehydrogenase; PGL: 6‐phosphogluconolactonase; GND: 6‐phosphogluconate dehydrogenase; RPE: ribulose‐phosphate 3‐epimerase; RPI: ribose‐5‐phosphate isomerase; PPM: phosphopentomutase; TKT1: transketolase; TALA: transaldolase; glc: glucose; g6p: glucose‐6‐phosphate; f6p: fructose‐6‐phosphate; 6pgl: 6‐phosphogluconate; ru5p: ribulose‐5‐phosphate; r5p: ribose‐5‐phosphate; r1p: ribose‐1‐phosphate; xu5p: xilulose‐5‐phosphate; s7p: sedoheptulose‐7‐phosphate; e4p: erythrose‐4‐phosphate; g3p: glyceraldehyde‐3‐phosphate; h: proton; nadh: nicotinamide adenine dinucleotide; nadph: nicotinamide adenine dinucleotide; _c: cytosolic. TCA : CS: citrate synthase; r0317 and r0426: aconitase 1 or 2; MAR13087: aconitate descarboxylase; ICDH: isocitrate dehydrogenase; AKGD: α‐ketoglutarate dehydrogenase; SUCOAS1: succinate synthase; DIC: dicarboxylate carrier; SUCD1: succinate dehydrogenase; FUM: fumarase; MDH: malate dehydrogenase; accoa: acetyl‐coenzyme A; coa: coenzyme A; cit: citrate; HC00342: cis‐aconitate; itacon: itaconate; icit: isocitrate; akg: α‐ketoglutarate; succoa: succinyl‐coenzyme A; succ: succinate; so3: sulfite; gtp: guanosine trisphosphate; gdp: guanosine diphosphate; pi: phosphate inorganic; fum: fumarate; mal: malate; oaa: oxaloacetate; h: proton; nadh: nicotinamide adenine dinucleotide; fadh2: flavin adenine dinucleotide; _c: cytosolic; _m: mitochondrial.

Article Snippet: After that, the NO donor DETA NONOate (DETA‐NO; ALX‐430‐014, Enzo) was added, and the cells were incubated for another 24h.

Techniques: Sampling

Journal: Advanced Healthcare Materials

Article Title: Immunometabolic Effect of Nitric Oxide on Human Macrophages Challenged With the SARS‐CoV2‐Induced Cytokine Storm. A Fluxomic Approach

doi: 10.1002/adhm.202401688

Figure Lengend Snippet: Analysis of the modulation of itaconate synthesis pathway by human Genome‐Scale Metabolic Model (GSMM) in hMφ treated with DETA‐NO and CK . (A) Schematic representation of itaconate synthesis. The effect of DETA‐NO on the aconitase activity was calculated using the GSMM ReconD3 algorithm and expressed as average flux values obtained from the sampling procedure. (B) Description of the individual reactions used to establish the GSMM analysis. (A) Results show the mean ± SD of the corresponding values expressed as “relative flux”. * p < 0.05; ** p < 0.01; *** p < 0.001 versus the control value or the CK condition (bar values). ns, not statistically significant.

Article Snippet: After that, the NO donor DETA NONOate (DETA‐NO; ALX‐430‐014, Enzo) was added, and the cells were incubated for another 24h.

Techniques: Activity Assay, Sampling, Control