r 4 3 1  (STATA Corporation)

Journal: bioRxiv

Article Title: Auxin is metabolized through kynurenine in Hypericum perforatum L

doi: 10.64898/2026.05.18.726114

Figure Lengend Snippet: (A) Schematic representation of major tryptophan (Trp)-derived metabolic pathways, including the kynurenine pathway (center), the indole-3-pyruvic acid (IPA)–indole-3-acetic acid (IAA) pathway, and the tryptamine– serotonin–melatonin branch (top). Solid, dashed, and double boxes indicate metabolites reported in animals, plants, or both, respectively. Enzymes are indicated at each step: IDO1/IDO2 (indoleamine 2,3-dioxygenase), TDO (tryptophan 2,3-dioxygenase), AFMID (arylformamidase), KAT (kynurenine aminotransferase), TDC (tryptophan decarboxylase), TAA1/TAR (tryptophan aminotransferase), KYNU (kynureninase), KMO (kynurenine 3-monooxygenase), HAAO (3-hydroxyanthranilate 3,4-dioxygenase), ACMSD (α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase), and QPRT (quinolinate phosphoribosyltransferase). Inhibitor targets are indicated at the corresponding steps: JM6 and RO 61-8048 inhibit KMO, and PF-04859989 inhibits KAT. (B) Chemical structures of the kynurenine pathway metabolites quantified in this study: kynurenine, kynurenic acid (KYNA), and 3-hydroxyanthranilic acid (3-HAA). (C) Chemical structures of the inhibitors used in this study. Core structural differences between JM6 and RO 61-8048 are highlighted in red.

Article Snippet: The compound (R)-3-amino-1-hydroxy-3,4-dihydroquinolin-2(1H)-one (PF-04859989) was identified by a high-throughput screen of the Pfizer compound library as a high-affinity inhibitor of human kynurenine aminotransferase (KAT; ).

Techniques: Derivative Assay

Journal: bioRxiv

Article Title: Auxin is metabolized through kynurenine in Hypericum perforatum L

doi: 10.64898/2026.05.18.726114

Figure Lengend Snippet: ( A) Representative images of explants cultured on MSO (control), kynurenine (KYN), indole-3-acetic acid (IAA), and IAA combined with inhibitors (IAA + JM6, IAA + PF-04859989 [PF], and IAA + RO 61-8048 [RO]). Scale bar = 1 cm (B) Rooting frequency, (C) internodal length (cm per node), (D) root number, and (E) maximum root length (cm) of explants under each treatment. For rooting frequency (B), bars represent mean proportion rooted ± SE. For (C–E), boxplots represent median (center line), interquartile range (box), and range (whiskers). Differences relative to the MSO control were evaluated using Dunnett-adjusted contrasts (p < 0.05; n = 12–18 per treatment).

Article Snippet: The compound (R)-3-amino-1-hydroxy-3,4-dihydroquinolin-2(1H)-one (PF-04859989) was identified by a high-throughput screen of the Pfizer compound library as a high-affinity inhibitor of human kynurenine aminotransferase (KAT; ).

Techniques: Cell Culture, Control

Journal: bioRxiv

Article Title: Auxin is metabolized through kynurenine in Hypericum perforatum L

doi: 10.64898/2026.05.18.726114

Figure Lengend Snippet: (A–C) Representative extracted ion chromatograms (EICs) of PF-04859989 (A), RO 61-8048 (B), and JM6 (KMO inhibitor II) (C) detected in plant tissue by LC–HRMS. Each panel shows the precursor ion trace at the expected m/z and retention time. (D–F) Relative abundance of PF (D), RO (E), and JM6 (F) in roots and shoots following treatment with MSO (control), inhibitor alone, or IAA + inhibitor. Peak areas are shown as log□□-transformed values. Boxplots represent median (center line), interquartile range (box), and range (whiskers). Signals corresponding to each inhibitor were observed in treated tissues and were not detected in MSO controls. Detection was also observed in IAA co-application treatments.

Article Snippet: The compound (R)-3-amino-1-hydroxy-3,4-dihydroquinolin-2(1H)-one (PF-04859989) was identified by a high-throughput screen of the Pfizer compound library as a high-affinity inhibitor of human kynurenine aminotransferase (KAT; ).

Techniques: Control, Transformation Assay

Journal: bioRxiv

Article Title: Auxin is metabolized through kynurenine in Hypericum perforatum L

doi: 10.64898/2026.05.18.726114

Figure Lengend Snippet: Concentrations of (A, D) kynurenic acid (KYNA), (B, E) kynurenine (KYN), and (C, F) 3-hydroxyanthranilic acid (3-HAA) in shoots (A–C) and roots (D–F) of explants cultured on MSO (control), IAA, or IAA combined with kynurenine pathway inhibitors (IAA + JM6, IAA + PF-04859989, and IAA + RO 61-8048). Concentrations are shown as log□□ (ng g −1 FW). Boxplots represent median (center line), interquartile range (box), and range (whiskers). For shoots (A–C), different letters indicate significant differences among treatments (one-way ANOVA followed by Tukey’s HSD, p < 0.05; n = 3). For roots (D–F), differences relative to the MSO control were evaluated using Dunnett-adjusted contrasts (p < 0.05; n = 3).

Article Snippet: The compound (R)-3-amino-1-hydroxy-3,4-dihydroquinolin-2(1H)-one (PF-04859989) was identified by a high-throughput screen of the Pfizer compound library as a high-affinity inhibitor of human kynurenine aminotransferase (KAT; ).

Techniques: Cell Culture, Control

Journal: bioRxiv

Article Title: Auxin is metabolized through kynurenine in Hypericum perforatum L

doi: 10.64898/2026.05.18.726114

Figure Lengend Snippet: Indole-3-acetic acid (IAA) is primarily synthesized from tryptophan through the indole-3-pyruvate (IPyA) pathway via tryptophan aminotransferase (TAA) and YUCCA flavin monooxygenase (YUC). Free IAA may be regulated through conjugation, catabolism, oxidative transformation and through feedback effects on tryptophan-derived metabolism. Kynurenine pathway metabolism proceeds through N-formyl-kynurenine and kynurenine, which occupies a central branch point between kynurenic acid formation via kynurenine aminotransferase (KAT) and downstream oxidative metabolism toward 3-hydroxyanthranilic acid (3-HAA) via kynurenine monooxygenase (KMO). Reactive oxygen species (ROS), temperature, drought, iron, and Fe 2+ are shown as potential stress and redox inputs that may influence auxin and kynurenine-associated metabolism. The pharmacological inhibitors used in this study are shown at their proposed targets: PF-04859989 at KAT, and RO-61-8048 and JM6 at kynurenine monooxygenase (KMO). Dashed arrows indicate proposed interactions linking auxin catabolism or oxidative transformation with kynurenine-associated metabolite accumulation and potential feedback on tryptophan-dependent auxin biosynthesis.

Article Snippet: The compound (R)-3-amino-1-hydroxy-3,4-dihydroquinolin-2(1H)-one (PF-04859989) was identified by a high-throughput screen of the Pfizer compound library as a high-affinity inhibitor of human kynurenine aminotransferase (KAT; ).

Techniques: Synthesized, Conjugation Assay, Transformation Assay, Derivative Assay

Journal: bioRxiv

Article Title: Toxin-triggered activation of regulated exocytosis enhances bacterial egress from the intestinal layer

doi: 10.64898/2026.02.06.704300

Figure Lengend Snippet: Caco-2 monolayer was infected with 2.5 x 10 6 bacteria of the Y. pseudotuberculosis wildtype strain YPIII (wt), the YP147 (YPIII Δ cnfY ) +/- the empty vector pJNS11 (pV), and the cnfY + complementation plasmid pJNS10 (p cnfY ). ( A , D , E ) 3 hours post-infection the medium was removed and replaced by medium containing gentamicin and inhibitors of the Cdc42/Rac1 (ML141) ( A ), phospholipase C (PLC γ-1) (U-73122) ( D ), and Ca 2+ channel IP3-R (2-APB) ( D ), or activators for Cdc42/Rac1 (Activator II), PLC γ-1 (m-3M3FBS), or IP3-R (Myo-Inositol) ( E ). After 30 minutes, the medium from the basolateral chamber was collected and plated onto LB agar to determine the CFUs of egressed bacteria. The mean +/- SEM of three independent biological replicates is shown. Significant differences were determined using a two-way ANOVA test with Tukey correction and indicated by asterisks (P-value: * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001). ( B , C ) Caco-2 monolayer was infected with 2.5 x 10 6 bacteria of the Y. pseudotuberculosis wildtype strain YPIII (wt), the Yersinia virulence-negative strain (ΔpYV), the cnfY -negative mutant strain YP147 (Δ cnfY ) without or with cnfY + complementation plasmid pJNS10 (p cnfY ). After 3.5 h post-infection, infected Caco-2 cells were lysed, cell extracts were prepared. Activation of the different Rho GTPases was tested by the isolation of the GTP-bound form by pull-downs with Rho-GTPase-binding agarose beads and Western blotting using Rho GTPase-specific antibodies, e.g., against Cdc42. M: Protein size marker. As negative and positive controls, high concentrations of GDP and GTP (GTP γS) were added to the uninfected whole-cell extract samples. Equal concentrations of extracts were used for pull-down assays, which were assessed by Western blotting with Actin antibodies. ( B ) shows a scheme of the procedure (Created in BioRender. Dersch, P. (2026)), and ( C ) the Western blots; ( F , G ) CNF Y -mediated induction of inositol triphosphate (IP 3 ) production. ( F ) Scheme of triggered Cdc42-mediated activation of phospholipase C (PLC γ-1), which leads to the formation of inositol monophosphate (IP 3 ) from PIP 2. IP 3 is rapidly metabolized to inositol monophosphate (IP 1 ), and IP 1 can thus be used as a proxy for IP 3 levels by adding LiCl, which blocks the metabolism of IP 1 . Created in BioRender. Dersch, P. (2026). ( G ) Caco-2 monolayer was infected with 2.5 x 10 6 bacteria of the Y. pseudotuberculosis wildtype strain YPIII (wt), YP147 (YPIII Δ cnfY ) +/- the empty vector pJNS11 (pV), and the cnfY + complementation plasmid pJNS10 (p cnfY ). After at least 2 h post-infection, the medium was removed and replaced with medium +/- gentamicin and/or 50 mM LiCl to block IP 1 metabolism, as indicated. After an additional 1.5 h, the Caco-2 cells were lysed, and the cellular concentration of IP 1 was determined by ELISA using an anti-IP 1 monoclonal antibody. The IP 3 concentrations were calculated based on the IP 1 amounts. The mean +/- SEM of four independent biological replicates is shown. Significant differences were determined using a two-way ANOVA test with Tukey correction and indicated by asterisks (P-value: **** < 0.0001).

Article Snippet: After 2-4 hours post-infection, the media in the upper chamber were removed and replaced with a medium containing gentamicin (to kill extracellular bacteria on the apical side of the Caco-2 monolayer), with or without inhibitors/activators of cell signaling pathways/molecules with the following concentration of the inhibitors for RhoA (30 μM, Rhosin, Sigma-Aldrich, #555460), Rac-1/Cdc42 (2.5 μM ML141, MedChemExpress, #HY-12755), PLCγ-1 (5 μM, U-73122, MedChemExpress, #HY-13419), IP 3 -R (40 μM 2-APB, MedChemExpress, #HY-W009724), and the activators of Cdc42/Rac1 (1 unit/ml, Rac1/Cdc42 activator II, Cytoskeleton, Inc., #CN02-A), PLC γ-1 (15 μM m-3M3FBS, MedChemExpress, #HY-19619), and IP 3 -R (30 μM D-myo-Inositol-1,2,4,5-tetraphosphate, sodium salt, Santa Cruz Biotechnology, #sc-362076).

Techniques: Infection, Bacteria, Plasmid Preparation, Mutagenesis, Activation Assay, Isolation, Binding Assay, Western Blot, Marker, Blocking Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Toxin-triggered activation of regulated exocytosis enhances bacterial egress from the intestinal layer

doi: 10.64898/2026.02.06.704300

Figure Lengend Snippet: ( A-D ) Caco-2, and the isogenic cell lines Caco-2ΔStx3, Caco-2ΔStx4, Caco-2ΔSNAP23, and were seeded into transwells and cultivated until the TEER had reached > 400 Ω•cm². Whole-cell extracts were prepared and analyzed by Western blotting using antibodies specific to Stx3 ( A ), Stx4 ( B ), and SNAP23 ( C ), and actin as a loading control ( A-C ). M: protein marker. ( D ) Monolayers of wildtype Caco-2 cells (wt), and ΔStx3, ΔStx4, and ΔSNAP23 mutant derivatives were infected with Y. pseudotuberculosis wildtype strain YPIII ( cnfY + ) or YP147 (Δ cnfY ). The CFUs of egressed bacteria were determined 3.5 h after infection. The mean +/- SEM of three independent biological replicates is shown. Significant differences were determined using a two-way ANOVA test with Tukey correction and indicated by asterisks (P-value: **** < 0.0001). ( E ) Illustration of the CNF Y -triggered activation of Yersinia egress through the induction of the Cdc42-dependent Ca 2+ -regulated exocytosis machinery. Created in BioRender. Dersch, P. (2026). Upon uptake of Y. pseudotuberculosis, the bacteria reside in a membrane-bound vacuole, to which the v-SNARE protein VAMP-3 is initially recruited. Later, during transcytosis, VAMP3 is replaced by VAMP7. The CNF Y toxin is secreted by the intracellular bacteria (indicated by red arrows), leading to the activation of Cdc42 at the cell membrane by the deamidation of Gln61 of the Rho GTPase. This activates PLC-γ1, leading to the cleavage of phosphatidylinositol-4,5-bisphosphate (PIP 2 ), producing inositol triphosphate (IP 3 ) and diacylglycerol (DAG). IP 3 interacts with the IP 3 receptor (IP 3 -R) in the endoplasmic reticulum (ER), which triggers the activation of adjacent Ca 2+ channels and the subsequent formation of a functional SNARE complex of VAMP7, Stx4, and SNAP25, and allows egress of Yersinia by exocytosis.

Article Snippet: After 2-4 hours post-infection, the media in the upper chamber were removed and replaced with a medium containing gentamicin (to kill extracellular bacteria on the apical side of the Caco-2 monolayer), with or without inhibitors/activators of cell signaling pathways/molecules with the following concentration of the inhibitors for RhoA (30 μM, Rhosin, Sigma-Aldrich, #555460), Rac-1/Cdc42 (2.5 μM ML141, MedChemExpress, #HY-12755), PLCγ-1 (5 μM, U-73122, MedChemExpress, #HY-13419), IP 3 -R (40 μM 2-APB, MedChemExpress, #HY-W009724), and the activators of Cdc42/Rac1 (1 unit/ml, Rac1/Cdc42 activator II, Cytoskeleton, Inc., #CN02-A), PLC γ-1 (15 μM m-3M3FBS, MedChemExpress, #HY-19619), and IP 3 -R (30 μM D-myo-Inositol-1,2,4,5-tetraphosphate, sodium salt, Santa Cruz Biotechnology, #sc-362076).

Techniques: Western Blot, Control, Marker, Mutagenesis, Infection, Bacteria, Activation Assay, Membrane, Functional Assay

Journal: Molecules

Article Title: Polyphenol-Enriched Fraction from Chestnut Shells as a Source of Bioactive Compounds for Friedreich Ataxia

doi: 10.3390/molecules31010070

Figure Lengend Snippet: Modulation of ferroptosis schematic. Accumulation of lipid hydroperoxides drives cells to death. The glutathione-dependent enzyme GPX4 reduces toxic lipid hydroperoxides to lipid alcohols. Erastin, BSO, and RSL3 impair GPX4 activity, sensitizing cells to ferroptosis. Iron overload and possibly ALOX activity also sensitize cells to ferroptosis. Iron chelators, lipid peroxide scavengers, and possibly ALOX inhibitors render cells ferroptosis-resistant. Blue arrows, inhibition of ferroptosis; green arrows, triggers of ferroptosis.

Article Snippet: Erastin (2-(1-(4-(2-(4-Chlorophenoxy)acetyl)-piperazin-1-yl)-ethyl)-3-(2-ethoxyphenyl)-3H-quinazolin-4-one), RSL3 ((1S,3R)-2-(2-Chloroacetyl)-2,3,4,9-tetrahydro-1-[4-(methoxycarbonyl)phenyl]-1H-pyrido [3,4-b]indole-3-carboxylic acid methyl ester), ethyl3-(benzylamino)-4-(cyclohexylamino) benzoate (SRS11-92), and OMAV (Omaveloxolone RTA 408- N -(2-Cyano-3,12-dioxo-28-noroleana-1,9(11)-dien-17-yl)-2,2-difluoropropanamide) were obtained from Selleckchem (Houston, TX, USA).

Techniques: Activity Assay, Inhibition

Journal: Molecules

Article Title: Polyphenol-Enriched Fraction from Chestnut Shells as a Source of Bioactive Compounds for Friedreich Ataxia

doi: 10.3390/molecules31010070

Figure Lengend Snippet: Comparative anti-ferroptotic activity in CSDE fractions. ( A ) Primary, patient-derived FRDA fibroblasts Coriell 3816 were treated with the ferroptosis inducer erastin at 7 µM for 24 h. Addition of CSDE and fractions A to D at 20 µg/mL 2 h after erastin treatment increased cell survival. ( B ) Primary, patient-derived FRDA fibroblasts Coriell 3816 were treated with 7 µM erastin or 20 nM RSL3 for 24 h. Treatment with fraction A and D at 20 µg/mL and 10 µg/mL increased cell survival. Survival is calculated relative to cells not treated with erastin or RSL3 (NT); SR11-92 1 µM was used as positive control for both assays and the statistical significance of the data with SR11-92 is not shown. CC = carrier control; *** = p < 0.005; dotted line = p < 0.01; * = p < 0.05 by one-way ( A ) or two-way ANOVA ( B ) with Bonferroni post hoc test. The experiment shows the average and SD of five ( A ) or four ( B ) technical replicates and it is representative of at least two independent experiments.

Article Snippet: Erastin (2-(1-(4-(2-(4-Chlorophenoxy)acetyl)-piperazin-1-yl)-ethyl)-3-(2-ethoxyphenyl)-3H-quinazolin-4-one), RSL3 ((1S,3R)-2-(2-Chloroacetyl)-2,3,4,9-tetrahydro-1-[4-(methoxycarbonyl)phenyl]-1H-pyrido [3,4-b]indole-3-carboxylic acid methyl ester), ethyl3-(benzylamino)-4-(cyclohexylamino) benzoate (SRS11-92), and OMAV (Omaveloxolone RTA 408- N -(2-Cyano-3,12-dioxo-28-noroleana-1,9(11)-dien-17-yl)-2,2-difluoropropanamide) were obtained from Selleckchem (Houston, TX, USA).

Techniques: Activity Assay, Derivative Assay, Positive Control, Control

Journal: Molecules

Article Title: Polyphenol-Enriched Fraction from Chestnut Shells as a Source of Bioactive Compounds for Friedreich Ataxia

doi: 10.3390/molecules31010070

Figure Lengend Snippet: Polyphenol-rich fraction decreases lipid peroxidation. ( A ) Primary, patient-derived FRDA fibroblasts Coriell 3816 were treated with increasing doses of RSL3 for 24 h in the presence of carrier control or fraction D at 20 µg/mL. The following day, C-11 Bodipy™ dye was added at 10 µM for 30 min. Fluorescence was measured on a plate reader using 485/520 (ex/em) and 485/590 (ex/em) filters. An increase in the 520/590 ratio is indicative of lipid peroxidation. ( B ) FRDA fibroblasts Coriell 3816 were incubated overnight with 10 nM RSL3 and treated with CSDE, fraction D, F2–3, or F7 all at concentrations of 20 µg/mL. Lipid peroxidation was measured as described above. Data are represented as fold increase in lipid peroxidation relative to cells not treated with RSL3 (NT). SR11-92 1 µM was used as a positive control for both assays and the statistical significance of the SR11-92 data is not shown. CC = carrier control; CSDE = chestnut shell dry extract; *** = p < 0.005; ** = p < 0.01 by one-way ANOVA with Bonferroni post hoc test. The experiment shows the average and SD of four independent technical replicates, and it is representative of at least two independent experiments.

Article Snippet: Erastin (2-(1-(4-(2-(4-Chlorophenoxy)acetyl)-piperazin-1-yl)-ethyl)-3-(2-ethoxyphenyl)-3H-quinazolin-4-one), RSL3 ((1S,3R)-2-(2-Chloroacetyl)-2,3,4,9-tetrahydro-1-[4-(methoxycarbonyl)phenyl]-1H-pyrido [3,4-b]indole-3-carboxylic acid methyl ester), ethyl3-(benzylamino)-4-(cyclohexylamino) benzoate (SRS11-92), and OMAV (Omaveloxolone RTA 408- N -(2-Cyano-3,12-dioxo-28-noroleana-1,9(11)-dien-17-yl)-2,2-difluoropropanamide) were obtained from Selleckchem (Houston, TX, USA).

Techniques: Derivative Assay, Control, Fluorescence, Incubation, Positive Control

Journal: Molecules

Article Title: Polyphenol-Enriched Fraction from Chestnut Shells as a Source of Bioactive Compounds for Friedreich Ataxia

doi: 10.3390/molecules31010070

Figure Lengend Snippet: PCA decreases lipid peroxidation and increases survival. ( A ) Primary, patient-derived FRDA fibroblasts Coriell 3816 were treated with 10 nM RSL3 for 24 h in the presence of carrier control (CC), 2.5 µM GA, 0.5 µM PCA, or 0.82 µM PHBA. The following day, C-11 Bodipy™ dye was added at 10 µM for 30 min. Fluorescence was measured on a plate reader using 485/520 (ex/em) and 485/590 (ex/em) filters. An increase in the 520/590 ratio is indicative of lipid peroxidation. Data are represented as the fold increase in lipid peroxidation relative to cells not treated with RSL3 (NT). ( B ) Primary, patient-derived FRDA fibroblasts Coriell 3816 were treated with 7 µM erastin for 24 h. Addition of 0.5 µM PCA or 0.82 µM PHBA increased survival whereas 2.5 µM GA caused toxicity. SR 11-92 1 µM was used as a positive control for both assays and the statistical significance of the SR 11-92 data is not shown. CC = carrier control; CSDE = chestnut shell dry extract; *** = p < 0.005; ** = p < 0.01; * = p < 0.05 by one-way ANOVA with Bonferroni post hoc test. The data shown are the mean and the standard deviation calculated from three independent technical replicates, and they are representative of at least two independent experiments.

Article Snippet: Erastin (2-(1-(4-(2-(4-Chlorophenoxy)acetyl)-piperazin-1-yl)-ethyl)-3-(2-ethoxyphenyl)-3H-quinazolin-4-one), RSL3 ((1S,3R)-2-(2-Chloroacetyl)-2,3,4,9-tetrahydro-1-[4-(methoxycarbonyl)phenyl]-1H-pyrido [3,4-b]indole-3-carboxylic acid methyl ester), ethyl3-(benzylamino)-4-(cyclohexylamino) benzoate (SRS11-92), and OMAV (Omaveloxolone RTA 408- N -(2-Cyano-3,12-dioxo-28-noroleana-1,9(11)-dien-17-yl)-2,2-difluoropropanamide) were obtained from Selleckchem (Houston, TX, USA).

Techniques: Derivative Assay, Control, Fluorescence, Positive Control, Standard Deviation