Journal: Health Science Reports

Article Title: Increased Expression of miR‐326 Mediates Chemosensitivity in Pediatric Acute Lymphoblastic Leukemia Through ABCA2 and YY1 Downregulation: An Observational and Experimental Study

doi: 10.1002/hsr2.72720

Figure Lengend Snippet: Effect of miR‐326 overexpression on chemosensitivity in RN95 B‐ALL cells. (A) RT‐qPCR analysis confirming increased miR‐326 expression in cells transfected with miR‐326 mimic compared with scrambled miRNA and mock control cells. (B) Cell viability assessed by MTT assay 72 h after treatment with methotrexate (88 nM), dexamethasone (1.65 mM), doxorubicin (0.67 µM), or cytarabine (100 mM), administered 24 h after transfection. miR‐326 overexpression significantly reduced cell viability compared with controls. Data are mean ± SEM from experiments performed in quintuplicate. Statistical analysis used one‐way ANOVA after confirmation of normality with the Shapiro–Wilk test. The gray, hatched, and solid black bars represent mock control cells, RN95‐electroporated cells transfected with scrambled miRNA, and RN95‐electroporated cells transfected with miR‐326 mimic, respectively. * p < 0.05; ** p < 0.005; *** p < 0.0005.

Article Snippet: Cells were treated for 72 h with methotrexate (88 nM; Gibco, Milan, Italy), doxorubicin (0.67 μM; Pfizer, New York, USA), and dexamethasone (1.65 mM; Caspian Tamin, Rasht, Iran).

Techniques: Over Expression, Quantitative RT-PCR, Expressing, Transfection, Control, MTT Assay

Journal: bioRxiv

Article Title: Revealing the spatiotemporal dynamics of methionine metabolism with a genetically encoded single-fluorophore biosensor

doi: 10.64898/2026.06.05.730515

Figure Lengend Snippet: a, Diagram depicting the methionine cycle and interconnection with the methionine salvage pathway and folate cycle. Homocysteine (hcy) and 5’-methylthioadenosine (MTA) are highlighted in blue and were the metabolites used to study methionine synthesis. Created in BioRender, Cohen, K.A. (2026). b, Normalized response of localized Meteor (dark purple) and MeteorITE (light purple) to 150 μM hcy addition in HeLa cells at the (left) cytoplasm and (right) nucleus. c, Diagram depicting the impact of methotrexate (mtx) on the synthesis of methionine from homocysteine. Mtx inhibits the enzyme dihydrofolate reductase (DHFR), depleting 5-methyltetrahydrofolate (5-mTHF) and disrupting the conversion of homocysteine to methionine. Created in BioRender, Cohen, K.A. (2026). d, Normalized response of cyto-Meteor when incubated in normal conditions (light purple) or with 100 μM mtx (dark purple) for 2 hours prior to imaging with 150 μM hcy addition in HeLa cells. e, Maximum ratio change of cyto-Meteor in response to 150 μM hcy in HeLa cells incubated with (dark purple) or without (light purple) 100 μM mtx. * P = 0.0163 by unpaired two-tailed t-test. With mtx n = 63 cells from 4 independent experiments. Without mtx n = 50 cells from 4 independent experiments. f, Representative images of immunostaining for methionine synthase. Scale bar represents 20 μm. g, Nuclear to cytoplasmic ratio of methionine synthase intensity in starved HeLa cells. h, Normalized response of localized Meteor (dark purple) and MeteorITE (light purple) to 30 μM MTA addition in HeLa cells at the (left) cytoplasm and (right) nucleus. i, Maximum ratio change of Meteor and MeteorITE in response to either 150 μM hcy or 30 μM MTA at the (left) cytoplasm and (right) nucleus. Cyto hcy ** P = 0.0024; Cyto MTA ** P = 0.0035; Cyto hcy vs. MTA n.s. P = 0.8030; NLS hcy ** P = 0.0018; NLS MTA *** P = 0.0002; NLS hcy vs. MTA n.s. P = 0.3179 by unpaired two-tailed t-test. Cyto-Meteor hcy n = 97 from 3 independent experiments. Cyto-MeteorITE hcy n = 146 cells from 3 independent experiments. NLS-Meteor hcy n = 110 cells from 3 independent experiments. NLS-MeteorITE hcy n = 124 cells from 3 independent experiments. Cyto-Meteor MTA n = 86 from 3 independent experiments. Cyto-MeteorITE MTA n = 62 cells from 3 independent experiments. NLS-Meteor MTA n = 159 cells from 3 independent experiments. NLS-MeteorITE MTA n = 123 cells from 3 independent experiments. j, Time-to-half-maximum (t1/2) of subcellular targeted Meteor response to 30 μM methionine at the (left) cytoplasm and (right) nucleus in live HeLa cells from experiments shown in . Cyto * P = 0.0152; Nucleus n.s P = 0.3347 by unpaired two-tailed t-test.

Article Snippet: L-methionine (Sigma-Aldrich, Cat# M-9625), L-homocysteine (AA Blocks, Cat# AA0037CV), 5’-methylthioadenosine (MedChem Express, Cat# HY-16938-100mg), and sodium methotrexate (Thermo Scientific, Cat# J66364-MD) were dissolved in PBS.

Techniques: Incubation, Imaging, Two Tailed Test, Immunostaining

Journal: bioRxiv

Article Title: Metabolic glues as a means of purine sensing and chemotherapeutic response

doi: 10.64898/2026.05.05.723063

Figure Lengend Snippet: a. Simplified metabolism of 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG). Asterisk denotes ability of 6-TGMP to be transformed into 6-meTGMP that may inhibit de novo purine synthesis. b. FACS-based growth competition comparing ΔNUDT5 and mutants to wildtype HEK293T cells treated with 6-TG. Data are individual values from n=3 biological replicates from a representative experiment. Similar results were obtained in two independent experiments. c. Chemical structures of adenosine-5’-monophosphate (AMP) and 6-methylthioinosine-5’-monophosphate (6-meTIMP). d. Left – alignment of molecular glue interface of AMP and 6-meTIMP showing cryo-EM density for the nucleotides. Right – rearrangement of PPAT interface residues in the 6-meTIMP structure (dark sidechains) compared to the AMP-bounds structure (light sidechains) e. Hydrophobic pocket of PPAT engaged by 6-meTIMP. f. 2D-ligand diagram of the 6-meTIMP molecular glue interface. g. PPAT activity assay measuring nucleotide-dependent inhibition in the presence of NUDT5 with 0.25 mM PRPP. Data points are the mean and error bars are SEM from n=3 independent experiments. h. Left – Western blot of endogenous NUDT5 3xFLAG immunoprecipitations following 16-hour treatment with methotrexate (2 µM), 6-MP (50 µM), and MTX + 6-MP. Right – Quantification of PPAT immunoprecipitation normalized to NUDT5 3xFLAG bait and compared to a DMSO-treated control condition. Data are individual values from n=3 independent biological replicate experiments and error bars are SEM. i. Fractional enrichment of AMP (M+2) and GMP (M+3) isotopologs in [ 15 N-amide]-glutamine labeling experiments conducted in the presence of 6-MP. Data points are individual values of n=6 biological replicates from two independent experiments and error bars are SEM. Statistical comparisons were performed using Welch’s two-tailed t-test with Bonferroni correction between wildtype and each mutant. *** denotes a Bonferroni adjusted p-value < 0.001 and ** is p-value < 0.01. j. FACS-based growth competition experiment comparing growth of ΔNUDT5 and endogenous L217A/K218A (LKAA) NUDT5 mutants to wildtype HEK293T treated with 6-MP and 6-TG. Data show n=3 biological replicates from a representative experiment. Similar results were obtained in two independent experiments.

Article Snippet: The following drugs and chemicals were used in this study at amounts specified in figures and legends: Pevonedistat; MLN4924 (MedChemExpress, HY-70062), methotrexate; MTX (MedChemExpress, HY-14519), lometrexol; LMX (MedChemExpress, HY-14521), brequinar (MedChemExpress, HY-108325) , rapamycin (Adooq Biosciences, A10782), 5-Phospho-D-ribose 1-diphosphate; PRPP (Sigma-Aldrich, P8296) , L-glutamine (Sigma-Aldrich, G8540); adenosine-5’-monophosphate; AMP (Sigma-Aldrich 01930), inosine-5’-monophosphate; IMP (MedChemExpress, HY-W010759), guanosine-5’-monophosphate; GMP (Sigma-Aldrich, G8377), AICA-ribonucleotide (Cayman Chemicals 33907), adenine (Thermo Scientific, A17622.14), hypoxanthine (MedChemExpress, HY-N0091), 6-thioguanine; 6-TG (Thermo Scientific, B21280.03), 6-mercaptopurine; 6-MP (Adooq Biosciences, A15898), 6-thioinosine-5’-monophosphate; 6-TIMP (Jena Biosciences, NU-1148), 6-methylthioinosine-5’-monophosphate; 6-meTIMP (Jena Biosciences, NU-1226), 6-methylthioguanosine-5’-monophosphate; 6-meTGMP (Jena Biosciences, NU-1128), 6-benzylthioinosine-5’-monophosphate; 6-benzylTIMP (WuXi, custom synthesis), 6-ethylthioinosine-5’-monophosphate 6-etTIMP (WuXi, custom synthesis), 6-ethylmercaptopurine riboside; 6-EMPR (WuXi, custom synthesis).

Techniques: Transformation Assay, Cryo-EM Sample Prep, Activity Assay, Inhibition, Western Blot, Immunoprecipitation, Control, Labeling, Two Tailed Test, Mutagenesis

Journal: bioRxiv

Article Title: Metabolic glues as a means of purine sensing and chemotherapeutic response

doi: 10.64898/2026.05.05.723063

Figure Lengend Snippet: a. Example cryo-EM density of the PPAT-NUDT5 6-meTIMP molecular glue interface with model fit. b,c. PPAT activity assay measuring inhibitory effects of 6-meTIMP in the presence and absence of wildtype NUDT5 and indicated mutants and c. compared to AMP only. d. Left – Representative Western blot of immunoprecipitations from endogenous NUDT5 3xFLAG HEK293T cells treated with indicated drugs for 16 hours: methotrexate (MTX; 2 µM), lometrexol (LMX; 10 µM), 6-mercaptopurine (6-MP; 50 µM), MLN4924 (1 µM), brequinar (2 µM), and rapamycin (1 µM). Right – quantification of PPAT immunoprecipitation relative to NUDT5 3xFLAG bait and normalized to a DMSO-treated control condition. Data are individual values from n=3 biological replicates from independent experiments and error bars are SEM. e. Western blot of immunoprecipitations from endogenous NUDT5 3xFLAG HEK293T cells treated with MTX (2 µM) for the indicated amounts of time. Similar results were obtained in two independent experiments. f. Western blot of endogenous PPAT 3xFLAG immunoprecipitations following 16-hour treatment with MTX (2 µM), 6-MP (50 µM), and MTX + 6-MP g. Time-resolved microscopy (incucyte) growth assays of wildtype and mutant HEK293T cells treated with the indicated drugs. Data are the mean and error bars are SEM of n=6 biological replicates. h. Levels of intracellular 6-TIMP and 6-meTIMP metabolites following 16-hour treatment with 6-MP (20 µM). Data are individual values and error bars are SEM from n=3 biological replicates. i. PPAT activity assay measuring inhibitory effects of 6-meTGMP in the presence and absence of wildtype NUDT5 and indicated mutants. Activity data shown in panels b, c and i are the mean and error bars are SEM of n=3 independent experiments.

Article Snippet: The following drugs and chemicals were used in this study at amounts specified in figures and legends: Pevonedistat; MLN4924 (MedChemExpress, HY-70062), methotrexate; MTX (MedChemExpress, HY-14519), lometrexol; LMX (MedChemExpress, HY-14521), brequinar (MedChemExpress, HY-108325) , rapamycin (Adooq Biosciences, A10782), 5-Phospho-D-ribose 1-diphosphate; PRPP (Sigma-Aldrich, P8296) , L-glutamine (Sigma-Aldrich, G8540); adenosine-5’-monophosphate; AMP (Sigma-Aldrich 01930), inosine-5’-monophosphate; IMP (MedChemExpress, HY-W010759), guanosine-5’-monophosphate; GMP (Sigma-Aldrich, G8377), AICA-ribonucleotide (Cayman Chemicals 33907), adenine (Thermo Scientific, A17622.14), hypoxanthine (MedChemExpress, HY-N0091), 6-thioguanine; 6-TG (Thermo Scientific, B21280.03), 6-mercaptopurine; 6-MP (Adooq Biosciences, A15898), 6-thioinosine-5’-monophosphate; 6-TIMP (Jena Biosciences, NU-1148), 6-methylthioinosine-5’-monophosphate; 6-meTIMP (Jena Biosciences, NU-1226), 6-methylthioguanosine-5’-monophosphate; 6-meTGMP (Jena Biosciences, NU-1128), 6-benzylthioinosine-5’-monophosphate; 6-benzylTIMP (WuXi, custom synthesis), 6-ethylthioinosine-5’-monophosphate 6-etTIMP (WuXi, custom synthesis), 6-ethylmercaptopurine riboside; 6-EMPR (WuXi, custom synthesis).

Techniques: Cryo-EM Sample Prep, Activity Assay, Western Blot, Immunoprecipitation, Control, Microscopy, Mutagenesis

Journal: bioRxiv

Article Title: Mitochondrion-IMC contact sites are critical for cofactor biosynthesis and egress signaling in Toxoplasma gondii

doi: 10.64898/2026.04.08.717193

Figure Lengend Snippet: A) Schematic representation of folate metabolism in Toxoplasma . PM = plasma membrane. IMC = inner membrane complex. OMM = outer mitochondrial membrane. IMM = inner mitochondrial membrane. DHF = dihydrofolate. DHFR-TS = dihydrofolate reductase. THF = tetrahydrofolate. B) Total activity of DHFR-TS in crude extracts of intracellular tachyzoites. Enzymatic activity was performed in n=4. Data are means ± s.d. Statistical analysis is a Two-tailed unpaired t-test, ** P<0.01. C). Total activity of DHFR-TS in crude extracts of intracellular parasites TATi-IMC10. Parasites were maintained in the presence or absence (control) of anhydrotetracycline (ATc) for 72 hours. Enzymatic activity was performed in n=4. Data are means ± s.d. Statistical analysis is a Two-tailed unpaired t-test, ** P<0.01. D) Untargeted metabolomics results for key metabolites in parasites’ folate pathway. Metabolite quantification was performed in three technical replicates and three biological replicates (n=9). Data are means ± s.d.. Statistical analysis is a Two-tailed unpaired t-test, ** P<0.01; * P<0.05. E) Representative images of parental and knockout parasites after methotrexate-fluorescein (MTX-488) incorporation. Parasites were stained with TMRE to track mitochondria. White arrows indicate a region of the cell that resembles the nucleus. F) Total fluorescence of MTX-488 parasites. Fluorescence intensity was measured in 30 individual parasites (n = 3). G) Quantification of the differential accumulation of MTX-488 in the mitochondrion and the nucleus in parental (ku80) and knockout parasites. A total of 30 individual parasites were compared in each replicate (n=3). Data are means ± s.d. Statistical analysis is a Two-tailed unpaired t-test; ns, not significant (P > 0.05). H) IFA of intracellular parental (Δku80) and knockout (Δlmf1) parasites expressing DHFR-TS with an HA epitope tag. Parasites were stained with DAPI (nuclear marker) and TOM40 (mitochondrial marker).

Article Snippet: Fluorescein methotrexate (Molecular Probes) was resuspended to a concentration of 1 mM in alkaline water.

Techniques: Clinical Proteomics, Membrane, Activity Assay, Two Tailed Test, Control, Knock-Out, Staining, Fluorescence, Expressing, Marker