Journal: Journal of Virology

Article Title: Conserved Rotavirus NSP5 and VP2 Domains Interact and Affect Viroplasm

doi: 10.1128/JVI.01965-19

Figure Lengend Snippet: VP2 amino acid regions 103–134 and 840–880 are necessary for VLS formation. (A) Schematic representation of VP2 deletion mutants fused to an HA tag at the N terminus (not to scale). The dashed lines correspond to deleted regions. Positive (+) or negative (−) VLS phenotype is indicated at the right. N-terminal (NTD), central, apical, and dimerization (dim) VP2 tertiary domains are indicated. (B) Representative photomicrographs of MA104 cells expressing the indicated N and C termini of HA-VP2 deletion mutants alone (−NSP5 panel) or together with NSP5 (+NSP5 panel). At 16 hpt, cells were fixed and immunostained for detection of HA-VP2 deletion mutant (anti-HA, red) and NSP5 (anti-NSP5, green). A merged image is shown in the right column. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). White arrows point to VLS. Scale bar is 10 μm.

Article Snippet: MA104 cells (embryonic rhesus monkey kidney; ATCC CRL-2378) and HEK293T cells (human embryonic kidney; ATCC CRL-573) were cultured in complete Dulbecco ’ s modified Eagle ’ s medium (cDMEM; Gibco BRL) supplemented with 10% fetal calf serum (FCS) (AMIMED; BioConcept, Switzerland) and penicillin (100 U/ml)–streptomycin (100 μg/ml) (Gibco, Life Technologies).

Techniques: Expressing, Mutagenesis, Staining

Journal: Journal of Virology

Article Title: Conserved Rotavirus NSP5 and VP2 Domains Interact and Affect Viroplasm

doi: 10.1128/JVI.01965-19

Figure Lengend Snippet: VP2 residues L124, V865, and I878 are required for VLS formation. Alignment of rotavirus VP2 (strain SA11) amino acid region 103–135 (A) and region 840–880 (B). The VP2 amino acid sequences of several representative virus strains are shown. GenBank accession numbers are listed in Materials and Methods. Members of rotavirus families A to H are indicated. Highly conserved amino acids are shown in dark blue. Amino acid residues mutated to alanine are indicated at the top. A minus sign indicates deletion. (C) Immunofluorescence of MA104 cells expressing wt VP2 or point mutations alone (−NSP5 column) or together with NSP5 (+NSP5 panel). At 16 hpt, cells were fixed and VLS were detected by immunostaining of HA-VP2 (anti-HA, red) or VP2 (anti-VP2, green) with NSP5 (anti-NSP5, green or red). A merged image is shown in the right column. Nuclei were stained with DAPI (blue). Scale bar is 10 μm.

Article Snippet: MA104 cells (embryonic rhesus monkey kidney; ATCC CRL-2378) and HEK293T cells (human embryonic kidney; ATCC CRL-573) were cultured in complete Dulbecco ’ s modified Eagle ’ s medium (cDMEM; Gibco BRL) supplemented with 10% fetal calf serum (FCS) (AMIMED; BioConcept, Switzerland) and penicillin (100 U/ml)–streptomycin (100 μg/ml) (Gibco, Life Technologies).

Techniques: Virus, Immunofluorescence, Expressing, Immunostaining, Staining

Journal: Journal of Virology

Article Title: Conserved Rotavirus NSP5 and VP2 Domains Interact and Affect Viroplasm

doi: 10.1128/JVI.01965-19

Figure Lengend Snippet: VLS formation and triggering of NSP5 hyperphosphorylation from cognate and noncognate strains are sensitive to VP2 L124A. (A) Immunofluorescence of MA104 cells for detection of VLS composed of NSP5 from simian strain SA11 (top) or NSP5 from porcine strain OSU (bottom) in coexpression with VP2 from simian strain SA11 wt or L124A. At 16 hpt, cells were fixed and immunostained for detection of NSP5 (anti-NSP5, green) and VP2 (anti-VP2, red). A merged image is in the right column of each panel. Nuclei were stained with DAPI (blue). Scale bar is 10 μm. (B) Immunoblotting of lysates from MA104 cells coexpressing NSP5-OSU (lanes 1 to 3) or NSP5-SA11 (lanes 4 to 6) with VP2-SA11 wt (lanes 2 and 5) or L124A (lanes 4 and 6). The membranes were incubated with specific antibodies for the detection of NSP5. Alpha-tubulin was used as a loading control. The red brackets indicate NSP5 hyperphosphorylation. (C) Sequence alignment of NSP5 from simian strain SA11 and porcine strain OSU. The identity between the two proteins corresponds to 94.95%. Noncanonical casein kinase I alpha region and oligomerization tail are labeled in blue and red, respectively.

Article Snippet: MA104 cells (embryonic rhesus monkey kidney; ATCC CRL-2378) and HEK293T cells (human embryonic kidney; ATCC CRL-573) were cultured in complete Dulbecco ’ s modified Eagle ’ s medium (cDMEM; Gibco BRL) supplemented with 10% fetal calf serum (FCS) (AMIMED; BioConcept, Switzerland) and penicillin (100 U/ml)–streptomycin (100 μg/ml) (Gibco, Life Technologies).

Techniques: Immunofluorescence, Staining, Western Blot, Incubation, Control, Sequencing, Labeling

Journal: Journal of Virology

Article Title: Conserved Rotavirus NSP5 and VP2 Domains Interact and Affect Viroplasm

doi: 10.1128/JVI.01965-19

Figure Lengend Snippet: VP2 residues L124, V865, and I878 are necessary to trigger NSP5 hyperphosphorylation. Coexpression of VP2 N terminus (lanes 3 to 7) (A) or C terminus (lanes 3 to 7) (B) deletion mutants with NSP5. NSP5 alone (lane 1) or in coexpression with wt VP2 (fl; lane 2) is shown. NSP5 in cellular extracts was detected by immunoblotting. Alpha-tubulin was used as a loading control. (C) Immunoblotting of total cellular lysates expressing NSP5 alone (lane 1) or in coexpression with VP2 wt (lane 2) or point mutations (lanes 3 to 5). NSP5 hyperphosphorylation was detected with anti-NSP5, and VP2 was detected with anti-VP2. Alpha-tubulin was used as a loading control. (D) λ-Phosphatase treatment of NSP5 coexpressed with empty vector (−), wt VP2, or VP2 L124A. NSP5 is visualized by immunoblotting. Untreated (−) and treated (+) samples are indicated. Red brackets indicate the NSP5 mobility shift. The red dots show the NSP5 unphosphorylated isoform. (E) Schematic representation of point mutations in wt NSP5 and NSP5 S67D. S to D modification is indicated in red (44). (F) Immunofluorescence of MA104 cells for detection of VLSs formed with wt or mutant NSP5 with wt VP2 (upper) or VP2 L124A (lower). At 16 hpt, cells were fixed and immunostained for detection of VLS with anti-NSP5 (green) and anti-VP2 (red). Nuclei were stained with DAPI (blue). Scale bar is 10 μm. (G) Immunoblotting of total cellular lysates expressing wt NSP5 (lanes 1 to 3) or NSP5 S67D (lanes 4 to 6) with wt VP2 or VP2 L124A. Where indicated, the membranes were incubated with anti-VP2 or anti-NSP5. The brackets indicate the NSP5 hyperphosphorylation smear. (H) Pulldown assay of cell extracts coexpressing H6-NSP5 (lanes 1 and 2 and lanes 5 and 6) or H6-NSP5 S67D (lanes 3 and 4 and lanes 7 and 8) with wt VP2 (lanes 1, 3, 5, and 7) or VP2 L124A (lanes 2, 4, 6, and 8). Samples were detected by immunoblotting using anti-NSP5 and anti-VP2 antibodies. Input (lanes 1 to 4) and elution fractions are indicated (lanes 5 to 8).

Article Snippet: MA104 cells (embryonic rhesus monkey kidney; ATCC CRL-2378) and HEK293T cells (human embryonic kidney; ATCC CRL-573) were cultured in complete Dulbecco ’ s modified Eagle ’ s medium (cDMEM; Gibco BRL) supplemented with 10% fetal calf serum (FCS) (AMIMED; BioConcept, Switzerland) and penicillin (100 U/ml)–streptomycin (100 μg/ml) (Gibco, Life Technologies).

Techniques: Western Blot, Control, Expressing, Plasmid Preparation, Mobility Shift, Modification, Immunofluorescence, Mutagenesis, Staining, Incubation

Journal: Journal of Virology

Article Title: Conserved Rotavirus NSP5 and VP2 Domains Interact and Affect Viroplasm

doi: 10.1128/JVI.01965-19

Figure Lengend Snippet: VP2 L124A disrupts VLS formation. For immunofluorescence of VLS formation, MA104 cells expressed NSP5 with HA-VP2 plus increasing amounts (0.25, 0.5, and 1 μg) of either wt VP2 (A) or VP2 L124A (B). At 16 hpt, cells were fixed and immunostained for VLS by detecting NSP5 (anti-NSP5, red) and HA-VP2 (anti-HA, green). Nuclei were stained with DAPI (blue). White arrowheads denote impaired VLS. Scale bar is 10 μm. (C) Immunofluorescence of VLS(NSP5+NSP2) alone (upper), with wt VP2 (middle), or with VP2 L124A (lower). At 16 hpt, MA104 cells were fixed and immunostained for the VLS with anti-NSP5 (red) and anti-NSP2 or anti-VP2 (green). Nuclei were stained with DAPI (blue). A merged image is shown at the right of each panel. The white arrowheads point to misshaped VLS. Scale bar is 10 μm.

Article Snippet: MA104 cells (embryonic rhesus monkey kidney; ATCC CRL-2378) and HEK293T cells (human embryonic kidney; ATCC CRL-573) were cultured in complete Dulbecco ’ s modified Eagle ’ s medium (cDMEM; Gibco BRL) supplemented with 10% fetal calf serum (FCS) (AMIMED; BioConcept, Switzerland) and penicillin (100 U/ml)–streptomycin (100 μg/ml) (Gibco, Life Technologies).

Techniques: Immunofluorescence, Staining

Journal: PLoS ONE

Article Title: Synthesis and Evaluation of the 2-Aminothiazoles as Anti-Tubercular Agents

doi: 10.1371/journal.pone.0155209

Figure Lengend Snippet: a Compounds were tested for activity against M . tuberculosis . Minimum inhibitory concentration (MIC, in μM) is the minimum concentration required to inhibit the growth of M . tuberculosis in liquid culture. MICs of active compounds are the average of two independent experiments. b Toxic concentration (TC50, in μM) is the concentration required to inhibit growth of Vero cells by 50%. Selectivity index (SI) is the ratio of TC50 to MIC. Cpd = compound; Rif = rifampicin; ND = not determined.

Article Snippet: The Vero cell line (African green monkey kidney epithelial cells: ATCC CRL-1587) was grown in DMEM, High Glucose, GlutaMAXTM (Invitrogen), 10% fetal bovine serum (FBS), and 1X penicillin-streptomycin solution (100 U/mL).

Techniques: Activity Assay, Concentration Assay

Journal: PLoS ONE

Article Title: Synthesis and Evaluation of the 2-Aminothiazoles as Anti-Tubercular Agents

doi: 10.1371/journal.pone.0155209

Figure Lengend Snippet: a MIC (μM) is the minimum concentration required to inhibit the growth of M . tuberculosis in liquid culture. MICs of active compounds are the average of two independent experiments. b Toxic concentration (TC 50 , in μM) is the concentration required to inhibit growth of Vero cells by 50%. Selectivity index (SI) is the ratio of TC 50 to MIC. Cpd = compound; Rif = rifampicin; ND = not determined.

Article Snippet: The Vero cell line (African green monkey kidney epithelial cells: ATCC CRL-1587) was grown in DMEM, High Glucose, GlutaMAXTM (Invitrogen), 10% fetal bovine serum (FBS), and 1X penicillin-streptomycin solution (100 U/mL).

Techniques: Concentration Assay

Journal: PLoS ONE

Article Title: Synthesis and Evaluation of the 2-Aminothiazoles as Anti-Tubercular Agents

doi: 10.1371/journal.pone.0155209

Figure Lengend Snippet: a Compounds were tested for inhibition of M . tuberculosis . Minimum inhibitory concentration (MIC, in μM) is the minimum concentration required to completely inhibit the growth of M . tuberculosis in liquid culture. MICs of active compounds are the average of two independent experiments. b Toxic concentration (TC 50 , in μM) is the concentration required to inhibit growth of Vero cells by 50%. Selectivity index (SI) is the ratio of TC50 to MIC. Cpd = compound; Rif = rifampicin; ND = not determined.

Article Snippet: The Vero cell line (African green monkey kidney epithelial cells: ATCC CRL-1587) was grown in DMEM, High Glucose, GlutaMAXTM (Invitrogen), 10% fetal bovine serum (FBS), and 1X penicillin-streptomycin solution (100 U/mL).

Techniques: Inhibition, Concentration Assay