Journal: mBio

Article Title: Genome-Wide Association Study of Campylobacter - Positive Diarrhea Identifies Genes Involved in Toxin Processing and Inflammatory Response

doi: 10.1128/mbio.00556-22

Figure Lengend Snippet: Genomic context. Each dot represents a single variant, ordered by position on chromosome 8 along the x -axis. The y axis on the left shows the -log 10 P value from the meta-analysis, while the y axis on the right indicates the recombination rate in centimorgans per megabase. The colors indicate linkage disequilibrium (r 2 ) between each variant and the highest-scoring SNP (rs13281104, shown in purple). The zoomed-in portion shows exons 5 to 7 of ARHGEF10 and the 22 variants in the LD block with P < 5 × 10 −5 in the meta-analysis, colored by effect listed in GTEx. Red lines are CLN8 eQTLs in whole blood, blue lines are ARHGEF10 eQTLs in the brain, purple lines are eQTLs for both genes ( CLN8 in blood and ARHGEF10 in the brain), and gray lines are not identified as significant eQTLs in GTEx. The pink bars along the bottom indicate the approximate locations of enhancers listed in GeneCards.

Article Snippet: Samples were genotyped using TaqMan SNP Genotyping, Assay ID C__27061811_10 (Thermofisher) with CFX96 thermocycler (BioRAD).

Techniques: Variant Assay, Blocking Assay

Journal: mBio

Article Title: Genome-Wide Association Study of Campylobacter - Positive Diarrhea Identifies Genes Involved in Toxin Processing and Inflammatory Response

doi: 10.1128/mbio.00556-22

Figure Lengend Snippet: Assessment of interaction

Article Snippet: Samples were genotyped using TaqMan SNP Genotyping, Assay ID C__27061811_10 (Thermofisher) with CFX96 thermocycler (BioRAD).

Techniques:

Journal: bioRxiv

Article Title: A molecular clock controls periodically driven cell migration in confined spaces

doi: 10.1101/2020.12.29.424673

Figure Lengend Snippet: (A) (i) Representative time-lapse CFP images of RhoA2G – HUVECs superimposed on phase-contrast images and (ii) the corresponding pseudo-colored images of RhoA activity. The color bar indicates the ratio of the FRET signal over CFP. Scale bar: 50 μm. (iii) RhoA activity of the cell with the arrow in A (i). The numbers indicate peaks that were identified by the peak detection algorithm (Supplementary Figure 2A). (B) (i) Immunoblot of activation level and total abundance of GEF-H1, and of pMLC, MLC, acetylated a-tubulin and a-tubulin in HUVECs treated by ionomycin (3 μM). GAPDH was used as a loading control. (ii) Fold-change quantification of the active GEF-H1 level normalized by the total GEF-H1 abundance and (iii) total GEF-H1 level normalized to GAPDH. The dotted colored curves indicate results of independent experiments and the gray solid curves represent the averages of the dotted lines. The curve was created by the method of cubic spline interpolation using the function in Prism7. (C) A time-lapse kymograph tracking (i) A375 cell and (ii) shGEF-H1 A375 cell migrating in a spatially confining channel (version1). Asterisks indicate the nuclei of the cells. (iii) Migration speed dynamics of cells shown in (i) and (ii). (D) Average migration speed for shGEF-H1 A375 cells (n = 497) contrasted with that of un-infected A375 cells (n = 243). Data were analyzed by unpaired two-tailed t-test with error bar representing s.e.m. (**** P < 0.0001) (E) Immunoblot of pMLC and MLC in shGEF-H1 A375 and un-infected A375 cells in response to treatment with ionomycin (3 μM).

Article Snippet: The following Taqman primers were used and obtained from Applied Biosystems: ARHGEF2 (Hs01064532_m1), GAPDH (Hs02786624_g1).

Techniques: Activity Assay, Western Blot, Activation Assay, Control, Migration, Infection, Two Tailed Test

Journal: bioRxiv

Article Title: A molecular clock controls periodically driven cell migration in confined spaces

doi: 10.1101/2020.12.29.424673

Figure Lengend Snippet: (A) (i) Representative fluorescent images of EB1-GFP-expressing HUVECs cultured on a nano-grooved patterned substratum following treatment by DMSO, BAPTA (1 μM), ionomycin (3 μM) and thapsigargin (1 μM) for 15 hours. The white arrow indicates the direction of the grooves and ridges in the pattern. Scale bar: 40 μm. Results of an automated tracking analysis representing (ii) speed and (iii) angle of trajectories of EB1-GFP clusters from images taken every 2 seconds for 2 minutes. (B) Comparison of the length of persistent MT growth over 2 minutes for the conditions of treatment by DMSO, BAPTA (1 μM) (* P = 0.0401), ionomycin (3 μM) (* P = 0.0113) and thapsigargin (1 μM) (* P = 0.0447). Data were analyzed by unpaired two-tailed t test with error bar representing s.e.m. (C) Comparison of axial ratios of HUVECs under the conditions of treatment with DMSO (n = 106 cells), paclitaxel (0.5 μM) (n = 140 cells), colchicine (0.5 μM) (n = 132 cells), BAPTA (1 μM) (n = 135 cells), ionomycin (3 μM) (n = 130 cells) and thapsigargin (1 μM) (n = 113 cells). Data were analyzed by one-way ANOVA with error bar representing s.e.m. (* P = 0.0116, **** P < 0.0001). (D) Immunoblot of GEF-H1 and a-tubulin abundance in HUVECs following treatment with paclitaxel (0.05, 0.5, 5 μM) and nocodazole (0.05, 0.5, 5 μM) for 15 hours. GAPDH was used as a loading control. Analysis is based on 3 independent experiments. (E) Immunoblot of GEF-H1 and a-tubulin abundance in HUVECs following treatment with MG-132 (10 μM), chloroquine (50 μM) and nocodazole (0.5 μM) for 15 hours. GAPDH was used as a loading control. Analysis is based on 3 independent experiments.

Article Snippet: The following Taqman primers were used and obtained from Applied Biosystems: ARHGEF2 (Hs01064532_m1), GAPDH (Hs02786624_g1).

Techniques: Expressing, Cell Culture, Comparison, Two Tailed Test, Western Blot, Control

Journal: bioRxiv

Article Title: A molecular clock controls periodically driven cell migration in confined spaces

doi: 10.1101/2020.12.29.424673

Figure Lengend Snippet: (A) (i) Immunoblot of GEF-H1 abundance in HUVECs following treatment with DMSO, BAPTA (1 μM), ionomycin (3 μM) and thapsigardin (1 μM) for 15 hours. GAPDH was used as a loading control. Analysis is based on 3 independent experiments. (ii) Fold-change quantification of GEF-H1 abundance normalized over GAPDH (** P = 0.0034, **** P < 0.0001). Data were analyzed by unpaired two-tailed t-test with the error bar representing s.e.m. (B-D) (i) Immunoblot analysis (ii) and fold-change quantification of GEF-H1 in HUVECs following treatment of DMSO, thapsigargin (1 μM) and/or (B) 666-15 (100 nM) (** P = 0.005, * P = 0.0208), (C) VIVIT (1, 10 μM) (** P = 0.0021) and (D) BML (10, 50 μM) (*** P = 0.0001) for 15 hours. a-tubulin was used as a loading control. Data were analyzed by unpaired two-tailed t test with error bar representing s.e.m. Analysis is based on 3 independent experiments. (E) GEF-H1 mRNA expression level change vs. GAPDH in HUVECs following treatment with BAPTA (1 μM), thapsigargin (1 μM) and/or 666-15 (100 nM) for 15 hours. Data were analyzed by unpaired two-tailed t test with error bar representing s.e.m. (**** P < 0.0001, *** P = 0.0001) (F) Schematic diagram summarizing the effect of intracellular Ca 2+ on microtubules and GEF-H1 abundance.

Article Snippet: The following Taqman primers were used and obtained from Applied Biosystems: ARHGEF2 (Hs01064532_m1), GAPDH (Hs02786624_g1).

Techniques: Western Blot, Control, Two Tailed Test, Expressing

Journal: bioRxiv

Article Title: A molecular clock controls periodically driven cell migration in confined spaces

doi: 10.1101/2020.12.29.424673

Figure Lengend Snippet: (A) Representative fluorescent images of EB1-GFP and shGEF-H1 EB1-GFP expressing HUVECs on a nanogrooved patterned substratum. The white arrow indicates the direction of the grooves and ridges in the pattern, analyzed as in . Scale bar: 40 μm. Results of an automated tracking analysis representing (ii) speed and (iii) angle of trajectories of EB1-GFP clusters from images taken every 2 seconds for 2 minutes. (B) Comparison of length of persistent growth of microtubules over 2 minutes determined using EB1-GFP in un-infected (n = 13 cells) and shGEF-H1 expressing (n = 23 cells) HUVECs. Data were analyzed by unpaired two-tailed t test with error bar representing s.e.m. (** P = 0.0036) (C) (i) Immunoblot analysis of GEF-H1 and mDia1 abundances in cells infected with scrambled shRNA, shGEF- Hl (HUVECs, HEK293 cells) or a construct encoding the GEF-H1-eGFP (HEK293 cells). a-tubulin was used as a loading control. Analysis is based on 3 independent experiments. (ii) Fold-change quantification of mDia1 expression relative to a-tubulin. Data were analyzed by unpaired two-tailed t test with error bar representing s.e.m. (shGEF-H1 HUVECs **** P < 0.0001, shGEF-H1 HEK293 cells * P = 0.0460, GEF-H1- eGFP HEK293 cells * P = 0.0499) (D) (i) Immunoblot analysis of abundances of GEF-H1, mDia1, acetylated a-tubulin and a-tubulin in MS1 cells following treatments with DMSO, thapsigargin (1, 10 μM) and BAPTA (1, 10 μM) for 15 hours. (ii) Fold-change quantification of expression relative to a-tubulin of GEF- H1 (BAPTA 1 μM: * P = 0.0353, thapsigargin 1 μM: * P = 0.0167) and mDia1 (thapsigargin 1 μM: * P = 0.028, thapsigargin 10 μM: ** P = 0.0031) for the experiments shown in (i). Images are representative of 3 independent experiments. (E) Immunoblot analysis of the abundances of mDia1, acetylated a-tubulin, a-tubulin and GEF-H1 in HUVECs following treatment with DMSO and SMIFH2 (1, 10, 20, 50 μM) for 15 hours. GAPDH was used as a loading control. Analysis is based on 3 independent experiments.

Article Snippet: The following Taqman primers were used and obtained from Applied Biosystems: ARHGEF2 (Hs01064532_m1), GAPDH (Hs02786624_g1).

Techniques: Expressing, Comparison, Infection, Two Tailed Test, Western Blot, shRNA, Construct, Control

Journal: bioRxiv

Article Title: A molecular clock controls periodically driven cell migration in confined spaces

doi: 10.1101/2020.12.29.424673

Figure Lengend Snippet: (A) Schematic diagram of the putative molecular network driving the molecular clock. (B) Experimental results (left) of the temporal dynamics of GEF-H1 activity in response to treatment with ionomycin (from (ii)) and model prediction (right) of temporal dynamics of the GEF-H1 activity (blue, left y-axis), RhoA-GTP abundance (red, left y-axis) and GEF-H1 abundance (green, right y-axis) (C) Model prediction (left) and experimental results (right) of a time delay between oscillatory RhoA activation (assayed as the migration speed) and mDia1 activity (assayed as actin polymerization). Time lapse images of F- TRActin-eGFP A375 cells migrating under confinements in narrow channels (version1) (top right). Horizontal bars indicate the timing of the migration speed (orange) and GFP intensity (purple) peaks corresponding to a portion of the graph below (bottom right). White arrows indicate blebs at the lateral cell periphery. Scale bar: 10 μm. Quantification of the migration speed and the intensity of actin filamentation over time for this experiment is shown in bottom right. (D) The effects of pharmacological perturbations of the molecular clock analyzed by mathematical model prediction (left) and experimental validation (right). Gray lines indicate simulation results for various model parameters (left). The dotted color lines indicate independent experimental results and the dark gray line represents averages of these experimental data (right). (E) (i) Time-lapse kymograph tracking A375 cells under confinements (version1) following treatments with C646 (10 μM) and SMIFH2 (50 μM) for 15 hours. Scale bar: 1 hour. (ii) Migration speed dynamics of the cells in (i). (F) Migration speed peak frequency over 1-hour (*P = 0.0202, **P = 0.0064) (G) Average migration speed for A375 cells treated as in (E) and untreated control cells (*P = 0.0214, **P = 0.0067). Data were analyzed by unpaired two-tailed t test with error bar representing s.e.m. (n=10 each)

Article Snippet: The following Taqman primers were used and obtained from Applied Biosystems: ARHGEF2 (Hs01064532_m1), GAPDH (Hs02786624_g1).

Techniques: Activity Assay, Activation Assay, Migration, Biomarker Discovery, Control, Two Tailed Test

Journal: Stroke

Article Title: Increased blood-brain barrier permeability and brain edema after focal cerebral ischemia induced by hyperlipidemia: role of lipid peroxidation and calpain-1/2, matrix metalloproteinase-2/9, and RhoA overactivation.

doi: 10.1161/STROKEAHA.111.615559

Figure Lengend Snippet: Figure 4. The RhoGTPase RhoA is overactivated and the tight junction protein occludin is downregulated in ischemic microvessels of hyperlipidemic mice. Western blot analysis and pull-down assay for RhoA (A), Western blot analysis for the guanine nucleotide exchange factor (GEF) of RhoA, leukemia-associated guanine exchange factor (LARG) (B), coprecipitation study for RhoA and LARG (C), and Western blot analysis for occludin (D) using cerebral microvessel extracts of normolipidemic and hyperlipidemic mice 24 hours after middle cerebral artery (MCA) occlusion. Note that LARG is overexpressed in ischemic microvessels of hyperlipidemic mice (B) and directly interacts with RhoA (C), which explains the overactivation of RhoA that is known to destabilize tight junctions. Data are meansstandard deviation (SD) (n4 Western blots, pull-down assays, or coprecipitation studies/group). C, contralateral nonischemic; I, ischemic. *P0.05/**P0.01 compared with corresponding nonischemic/†P0.05 compared with ischemic normal diet (1-way analy- sis of variance [ANOVA] followed by least significant difference [LSD] tests).

Article Snippet: Antibodies and histochemicals for Western blotting, pulldown and immunoprecipitation studies The following antibodies and chemicals were used: anti-calpastatin (sc-20779), anti- leukemia-associated Rho guanine nucleotide exchange factor (LARG) (sc-25638), anti-RhoA (sc-418), anti-occludin (sc-27151) and protein A/G plus-agarose (sc-2003) were purchased from Santa Cruz Biotechnology.

Techniques: Western Blot, Pull Down Assay

Journal: Viruses

Article Title: A Human and Rhesus Macaque Interferon-Stimulated Gene Screen Shows That Over-Expression of ARHGEF3/XPLN Inhibits Replication of Hepatitis C Virus and Other Flavivirids

doi: 10.3390/v14081655

Figure Lengend Snippet: Validation of the antiviral activity of ARHGEF3/XPLN on HCV RNA replication. ( A ) Relative HCV RNA abundance from HCVcc infection of Huh7 cells expressing ARHGEF3 from Human (Hs) or rhesus macaque (Mm) as compared to negative control at 72 hpi. ( B ) Relative RLU (%) following HCV SGR RNA transfection of Huh7 cells expressing ARHGEF3 from Human (Hs) or rhesus macaque (Mm) as compared to negative control at 72 hpt. ( C ) Effect of ARHGEF3/XPLN -specific mutations (Myc-tagged WT, N-terminus truncation, N-terminus only and the GEF-inactivation mutant W440L) on HCV RNA replication following SGR RNA transfection. ( D ) Schematic of ARHGEF3 showing the N (amino acids 1–125), diffuse B cell lymphoma homology (DH) and plekstrin homology (PH) domains, alongside the location of the W440 residue. RLU activity was measured and compared to controls at 72 hpt. Values from combined three technical replicates from two independent experiments were used and variation is shown as standard error of the mean.

Article Snippet: RT-qPCR was carried out using Taqman protocols for primers and probes against HCV (in-house [ ]) as well as human Mx1 (Hs00895608), ARHGEF3 (Hs04241552), and the control GAPDH (Hs402869); assays were performed with TaqMan Fast Universal PCR Master Mix (Applied Biosystems, Waltham, MA, USA).

Techniques: Biomarker Discovery, Activity Assay, Infection, Expressing, Negative Control, Transfection, Mutagenesis, Residue

Journal: Viruses

Article Title: A Human and Rhesus Macaque Interferon-Stimulated Gene Screen Shows That Over-Expression of ARHGEF3/XPLN Inhibits Replication of Hepatitis C Virus and Other Flavivirids

doi: 10.3390/v14081655

Figure Lengend Snippet: Antiviral activity of ARHGEF3 against flaviviruses. RLU activity in Huh7 cells stably expressing ARHGEF3/XPLN or IRF1 following infection with recombinant luciferase-expressing YFV ( A ) and ZIKV ( B ) as measured at 48 hpi and 72 hpi, respectively. Values from 3 technical replicates for YFV and 9 technical replicates for ZIKV were used and variation is shown as standard deviation of the mean alongside p values for Student’s T test between EMPTY and ARHGEF3 WT results.

Article Snippet: RT-qPCR was carried out using Taqman protocols for primers and probes against HCV (in-house [ ]) as well as human Mx1 (Hs00895608), ARHGEF3 (Hs04241552), and the control GAPDH (Hs402869); assays were performed with TaqMan Fast Universal PCR Master Mix (Applied Biosystems, Waltham, MA, USA).

Techniques: Activity Assay, Stable Transfection, Expressing, Infection, Recombinant, Luciferase, Standard Deviation

Journal: Viruses

Article Title: A Human and Rhesus Macaque Interferon-Stimulated Gene Screen Shows That Over-Expression of ARHGEF3/XPLN Inhibits Replication of Hepatitis C Virus and Other Flavivirids

doi: 10.3390/v14081655

Figure Lengend Snippet: Abundance of ARHGEF3/XPLN RNA in vivo in HCV-infected human liver biopsies. RNA levels (FKPM) of known anti-HCV ISGs in liver biopsies from uninfected individuals ( n = 4) ( A ). Fold-change in RNA (FPKM) levels of known ISGs in liver biopsies from infected individuals (HCV gt1: pink; gt3: green (each n = 5)) compared to uninfected controls ( B ).

Article Snippet: RT-qPCR was carried out using Taqman protocols for primers and probes against HCV (in-house [ ]) as well as human Mx1 (Hs00895608), ARHGEF3 (Hs04241552), and the control GAPDH (Hs402869); assays were performed with TaqMan Fast Universal PCR Master Mix (Applied Biosystems, Waltham, MA, USA).

Techniques: In Vivo, Infection