Journal: Cells

Article Title: Probiotics in the Prevention of the Calcium Oxalate Urolithiasis

doi: 10.3390/cells11020284

Figure Lengend Snippet: A summary of previous studies on the use of Lactobacillus spp. for the prevention of urolithiasis.

Article Snippet: Turroni et al. [ ] , In vitro , The oxalate-degrading activities of 14 Bifidobacterium strains ( B. adolescentis ATCC 15703, B. animalis subsp. Lactis DSM 10140, BA30, Bb12, BI07, and L15, B. bifidum S16, B. breve ATCC 15700 and BBSF, B. catenulatum B665, B. longum biotype longum S123, ATCC 15707, and W11, and B. longum biotype suis ATCC 27533), cultured in oxalate-rich media, was measured by a capillary electrophoresis technique. The identification of the oxc gene was performed with PCR , Only the five B. animalis subsp. Lactis strains (DSM 10140, BA30, Bb12, BI07, and L15) showed oxalate-degrading activity with 100% of oxalate consumption after 5 days of incubation, whereas no degrading activity was exhibited by all the other bifidobacterial strains tested..

Techniques: In Vitro, Bacteria, Activity Assay, Cell Culture, Electrophoresis, Incubation, Expressing, Isolation, Extraction, In Vivo, Clinical Proteomics, Concentration Assay, Animal Model, Microscopy, Control

Journal: Cells

Article Title: Probiotics in the Prevention of the Calcium Oxalate Urolithiasis

doi: 10.3390/cells11020284

Figure Lengend Snippet: Characteristics of Bifidobacterium spp.

Article Snippet: Turroni et al. [ ] , In vitro , The oxalate-degrading activities of 14 Bifidobacterium strains ( B. adolescentis ATCC 15703, B. animalis subsp. Lactis DSM 10140, BA30, Bb12, BI07, and L15, B. bifidum S16, B. breve ATCC 15700 and BBSF, B. catenulatum B665, B. longum biotype longum S123, ATCC 15707, and W11, and B. longum biotype suis ATCC 27533), cultured in oxalate-rich media, was measured by a capillary electrophoresis technique. The identification of the oxc gene was performed with PCR , Only the five B. animalis subsp. Lactis strains (DSM 10140, BA30, Bb12, BI07, and L15) showed oxalate-degrading activity with 100% of oxalate consumption after 5 days of incubation, whereas no degrading activity was exhibited by all the other bifidobacterial strains tested..

Techniques: Staining

Journal: Cells

Article Title: Probiotics in the Prevention of the Calcium Oxalate Urolithiasis

doi: 10.3390/cells11020284

Figure Lengend Snippet: A summary of previous studies on the use of Bifdobacterum spp. for the prevention of urolithiasis.

Article Snippet: Turroni et al. [ ] , In vitro , The oxalate-degrading activities of 14 Bifidobacterium strains ( B. adolescentis ATCC 15703, B. animalis subsp. Lactis DSM 10140, BA30, Bb12, BI07, and L15, B. bifidum S16, B. breve ATCC 15700 and BBSF, B. catenulatum B665, B. longum biotype longum S123, ATCC 15707, and W11, and B. longum biotype suis ATCC 27533), cultured in oxalate-rich media, was measured by a capillary electrophoresis technique. The identification of the oxc gene was performed with PCR , Only the five B. animalis subsp. Lactis strains (DSM 10140, BA30, Bb12, BI07, and L15) showed oxalate-degrading activity with 100% of oxalate consumption after 5 days of incubation, whereas no degrading activity was exhibited by all the other bifidobacterial strains tested..

Techniques: In Vitro, Bacteria, Western Blot, Activity Assay, Electrophoresis, Extraction, Probiotics, Cell Culture, Knock-Out

Journal: Cells

Article Title: Probiotics in the Prevention of the Calcium Oxalate Urolithiasis

doi: 10.3390/cells11020284

Figure Lengend Snippet: Characteristics of O. formigenes , Lactobacillus and Bifidobacterium in the prevention of urolithiasis.

Article Snippet: Turroni et al. [ ] , In vitro , The oxalate-degrading activities of 14 Bifidobacterium strains ( B. adolescentis ATCC 15703, B. animalis subsp. Lactis DSM 10140, BA30, Bb12, BI07, and L15, B. bifidum S16, B. breve ATCC 15700 and BBSF, B. catenulatum B665, B. longum biotype longum S123, ATCC 15707, and W11, and B. longum biotype suis ATCC 27533), cultured in oxalate-rich media, was measured by a capillary electrophoresis technique. The identification of the oxc gene was performed with PCR , Only the five B. animalis subsp. Lactis strains (DSM 10140, BA30, Bb12, BI07, and L15) showed oxalate-degrading activity with 100% of oxalate consumption after 5 days of incubation, whereas no degrading activity was exhibited by all the other bifidobacterial strains tested..

Techniques: Bacteria, Membrane, Probiotics, Activity Assay, Lyophilization, Formulation, Capsules, Clinical Proteomics

Journal: Cell chemical biology

Article Title: MYC regulation of D2HGDH and L2HGDH influences the epigenome and epitranscriptome

doi: 10.1016/j.chembiol.2020.02.002

Figure Lengend Snippet: a) D2HGDH and b) L2HGDH. Luciferase activity of promoter regions – arrows indicate transcriptional start sites; blue squares are putative E-boxes. c) D2HGDH and d) L2HGDH. Left panels: ChIP-qPCR of MYC binding to E-boxes in D2HGDH or L2HGDH promoters; +ctrl is the LIN28B promoter, −ctrl is a promoter region lacking a predicted E-box. Right panels: Luciferase activity of D2HGDH and L2HGDH promoters in cells co-transfected with MYC. e) D2HGDH and f) L2HGDH. Left panels: Q-RT-PCR of D2HGDH and L2HGDH mRNA in P493-6 cells; Center panel: Immunoblot of tetracycline-regulated MYC expression in P493-6 cells; Right panels: Q-RT-PCR of D2HGDH and L2HGDH mRNA in mature B-cells from Eμ-Myc mice and WT mice. Data shown are mean −/+ SD.

Article Snippet: The following antibodies were used: c-Myc (clone 9E10; #sc-40, Santa Cruz Biotechnology), D2HGDH (#13895-1-AP, Proteintech), L2HGDH (#15707-1-AP, Proteintech), ALKBH5 (# ab69325, Abcam), FTO (c-3, #sc-271713, Santa Cruz Biotechnology), m6A (#202003, Synaptic System), TET1 (#SAB2700730, Sigma-Aldrich), TET2 (#18950, Cell Signaling Technology), TET3 (#: ABS463, EMD Millipore Corporation), METTL3, METTL14, WTAP (# 69391, # 51104 and #56501, all from Cell Signaling Technology), 5-Methylcytosine and 5-hydroxymethylcytosine (#28692 and #51660, Cell Signaling Technology), β-actin (# A2228, Sigma-Aldrich), Lamin A (#A303-433A-M, Bethyl Laboratories), Tubulin (#62204 Invitrogen).

Techniques: Luciferase, Activity Assay, ChIP-qPCR, Binding Assay, Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing

Journal: Cell chemical biology

Article Title: MYC regulation of D2HGDH and L2HGDH influences the epigenome and epitranscriptome

doi: 10.1016/j.chembiol.2020.02.002

Figure Lengend Snippet: a) Left to right panels – MYC-induced modulation of 5hmC and 5mC levels in WT and D2HGDH or L2HGDH KO P493-6 cells, quantification of MYC-induced TET activity in WT and D2HGDH or L2HGDH KO P493-6 cells, and WBs of D2HGDH, L2HGDH, TET1, TET2, TET3 and MYC. b) Left to right panels – MYC-induced modulation of m6A levels in WT and D2HGDH or L2HGDH KO P493-6 cells, quantification of MYC-induced RNA demethylase activity in WT and D2HGDH or L2HGDH KO P493-6 cells, and WBs of FTO, ALKBH5, METL3, WTAP and METTL14 in WT and D2HGDH or L2HGDH KO P493-6 cells. c) Left to right panels – 5hmC and m6A levels in D2HGDH/L2HGDH WT or KO clones in DLBCL cell lines OCI-Ly1 and OCI-Ly8; WBs for D2HGDH and L2HGDH are shown at the top. The percentage of MYC-induced changes in a) and b) represent the difference between the measures in MYC OFF and ON cells (full data in Figure S3). Data shown are mean −/+ SD. In a) and b), TET1/ALKBH5 and TET3/FTO were detected in the same gel and share the β-actin loading control.

Article Snippet: The following antibodies were used: c-Myc (clone 9E10; #sc-40, Santa Cruz Biotechnology), D2HGDH (#13895-1-AP, Proteintech), L2HGDH (#15707-1-AP, Proteintech), ALKBH5 (# ab69325, Abcam), FTO (c-3, #sc-271713, Santa Cruz Biotechnology), m6A (#202003, Synaptic System), TET1 (#SAB2700730, Sigma-Aldrich), TET2 (#18950, Cell Signaling Technology), TET3 (#: ABS463, EMD Millipore Corporation), METTL3, METTL14, WTAP (# 69391, # 51104 and #56501, all from Cell Signaling Technology), 5-Methylcytosine and 5-hydroxymethylcytosine (#28692 and #51660, Cell Signaling Technology), β-actin (# A2228, Sigma-Aldrich), Lamin A (#A303-433A-M, Bethyl Laboratories), Tubulin (#62204 Invitrogen).

Techniques: Activity Assay, Clone Assay, Control

Journal: Cell chemical biology

Article Title: MYC regulation of D2HGDH and L2HGDH influences the epigenome and epitranscriptome

doi: 10.1016/j.chembiol.2020.02.002

Figure Lengend Snippet: a) Quantification of αKG in P493-6 cells (MYC ON or OFF) with WT or KO of D2HGDH and L2HGDH; MYC immunoblots are on the left, ***p<0.001, two-way ANOVA, Bonferroni's post-test. b) 5hmC and m6A levels in P493-6 cells (MYC OFF or ON; D2HGDH and L2HGDH WT or KO), with or without DMαKG rescue (5mM); WBs are on the right; p values are from two-tailed Student’s t-test c) 5hmC and 5mC (left panels) and m6A (right panel) levels in P493-6 cells (MYC OFF or ON) exposed to DMOG (1mM), octyl-D-2-HG or L-2-HG (100μM); p values are from one-way ANOVA. WBs of relevant proteins are also shown. d) Left panel: TET activity in DLBCL cell lines exposed to DMαKG (5mM) ,**p<0.01, ***p<0.001, Student’s t-test; center-panel: RNA demethylase activity of HEK-293T cells exposed to DMαKG, one-way ANOVA; right panel: RNA demethylase activity in DLBCL cell lines exposed to octyl-D-2-HG (100μM), **p<0.01, ***p<0.001, Student’s t-test. e) Fold increase in 5hmC and decrease of m6A (left and right panels) upon MYC induction in cells grown in full media (Glut+), in glutamine-free media (Glut−) or in the presence of CB-839 (1μM) (3 biological replicates for both assays, 5 and 9 technical replicates for 5hmC and m6A, respectively); p values are from two-tailed Student’s t-test. Data are mean −/+ SD. In b), the sets of proteins MYC/TET2/FTO/METLL14, TET3/METLL3/WTAP and TET1/ALKBH5 were detected in the same filter and share the β-actin loading control. In c), TET3/METLL3/METLL14, and FTO/ALKBH5 share the β-actin loading control.

Article Snippet: The following antibodies were used: c-Myc (clone 9E10; #sc-40, Santa Cruz Biotechnology), D2HGDH (#13895-1-AP, Proteintech), L2HGDH (#15707-1-AP, Proteintech), ALKBH5 (# ab69325, Abcam), FTO (c-3, #sc-271713, Santa Cruz Biotechnology), m6A (#202003, Synaptic System), TET1 (#SAB2700730, Sigma-Aldrich), TET2 (#18950, Cell Signaling Technology), TET3 (#: ABS463, EMD Millipore Corporation), METTL3, METTL14, WTAP (# 69391, # 51104 and #56501, all from Cell Signaling Technology), 5-Methylcytosine and 5-hydroxymethylcytosine (#28692 and #51660, Cell Signaling Technology), β-actin (# A2228, Sigma-Aldrich), Lamin A (#A303-433A-M, Bethyl Laboratories), Tubulin (#62204 Invitrogen).

Techniques: Western Blot, Two Tailed Test, Activity Assay, Control

Journal: Cell chemical biology

Article Title: MYC regulation of D2HGDH and L2HGDH influences the epigenome and epitranscriptome

doi: 10.1016/j.chembiol.2020.02.002

Figure Lengend Snippet: a) WBs of TET1-3, FTO and ALKBH5 in nuclear and cytoplasmic fractions, and whole cell lysate (WCL) of P493-6 cells upon MYC suppression and its re-expression. Two exposures (short and long) are shown for FTO. b) WBs of TET1-3, FTO and ALKBH5 in the subcellular fractions and WCL of DLBCL cell lines exposed to DMαKG (5mM), octyl-D-2-HG or octyl-L-2-HG (100μM) for 8h. c) WBs of TET1-3, FTO and ALKBH5 in subcellular fractions and WCL of P493-6 cells (D2HGDH and L2HGDH WT or KO) upon MYC suppression and its re-expression. d) WBs of TET1-3, FTO and ALKBH5 in HEK-293 cells expressing an empty vector (MSCV), D2HGDH WT or the G131X and A208T mutants. Densitometric measurements of protein levels are shown at the bottom of WB displays – all values are relative to controls and already corrected by WCL abundance, which is shown for reference. e) immunofluorescence of HEK-293T cells transiently transfected with a FLAG-TET3 plasmid and exposed to DMαKG (5mM), octyl-D-2-HG and L-2-HG (100μM/each) or DMSO for 6h; scale bar is 10 μm. Scoring of nuclear vs. nuclear and cytoplasmic expression in the three conditions is shown on the right; p<0.001, Chi-square.

Article Snippet: The following antibodies were used: c-Myc (clone 9E10; #sc-40, Santa Cruz Biotechnology), D2HGDH (#13895-1-AP, Proteintech), L2HGDH (#15707-1-AP, Proteintech), ALKBH5 (# ab69325, Abcam), FTO (c-3, #sc-271713, Santa Cruz Biotechnology), m6A (#202003, Synaptic System), TET1 (#SAB2700730, Sigma-Aldrich), TET2 (#18950, Cell Signaling Technology), TET3 (#: ABS463, EMD Millipore Corporation), METTL3, METTL14, WTAP (# 69391, # 51104 and #56501, all from Cell Signaling Technology), 5-Methylcytosine and 5-hydroxymethylcytosine (#28692 and #51660, Cell Signaling Technology), β-actin (# A2228, Sigma-Aldrich), Lamin A (#A303-433A-M, Bethyl Laboratories), Tubulin (#62204 Invitrogen).

Techniques: Expressing, Plasmid Preparation, Immunofluorescence, Transfection

Journal: Cell chemical biology

Article Title: MYC regulation of D2HGDH and L2HGDH influences the epigenome and epitranscriptome

doi: 10.1016/j.chembiol.2020.02.002

Figure Lengend Snippet: KEY RESOURCE TABLE

Article Snippet: The following antibodies were used: c-Myc (clone 9E10; #sc-40, Santa Cruz Biotechnology), D2HGDH (#13895-1-AP, Proteintech), L2HGDH (#15707-1-AP, Proteintech), ALKBH5 (# ab69325, Abcam), FTO (c-3, #sc-271713, Santa Cruz Biotechnology), m6A (#202003, Synaptic System), TET1 (#SAB2700730, Sigma-Aldrich), TET2 (#18950, Cell Signaling Technology), TET3 (#: ABS463, EMD Millipore Corporation), METTL3, METTL14, WTAP (# 69391, # 51104 and #56501, all from Cell Signaling Technology), 5-Methylcytosine and 5-hydroxymethylcytosine (#28692 and #51660, Cell Signaling Technology), β-actin (# A2228, Sigma-Aldrich), Lamin A (#A303-433A-M, Bethyl Laboratories), Tubulin (#62204 Invitrogen).

Techniques: Recombinant, Western Blot, Stripping, Activity Assay, Methylation, Extraction, DNA Purification, Luciferase, Enzymatic Assay, Cell Isolation, Reverse Transcription, shRNA, Plasmid Preparation, Software, Membrane