Journal: Pathogens

Article Title: Type II Restriction-Modification System from Gardnerella vaginalis ATCC 14018

doi: 10.3390/pathogens9090703

Figure Lengend Snippet: Susceptibility of E. coli gDNA to cleavage by REase in G. vaginalis ATCC 14018 culture supernatant. ( A ) gDNAs extracted from E. coli DH10B (lanes 2–4) and three individual DH10B-Me colonies (lanes 5–13) were incubated with fresh sBHI medium (negative control) (lanes 2, 5, 8, 11), G. vaginalis ATCC 14018 culture (OD 600 = 0.6) supernatant (lanes 3, 6, 9, 12), and BsuRI (lanes 4, 7, 10, 13). ( B ) gDNAs extracted from E. coli DH10B (lanes 2–3), three individual DH10B-RSFMe colonies (lanes 4–9), LMG194 (lanes 10–11), and three individual LMG194-RSFMe colonies (lanes 12–17) were incubated with fresh sBHI medium (negative control) (lanes 2, 4, 6, 8, 10, 12, 14, 16) and G. vaginalis ATCC 14018 culture (OD 600 = 0.6) supernatant (lanes 3, 5, 7, 9, 11, 13, 15, 17). Lanes 18, 19: untreated pBR322 and pBR322 incubated with ATCC 14018 culture supernatant, respectively. (A, B) Lane 1: Gene Ruler™ Ladder Mix.

Article Snippet: In addition to ATCC 14018, G. vaginalis ATCC 14019 and G. vaginalis DSM 4944 possess putative REase that recognize GGCC (rebase.neb.com/rebase/rebase.html).

Techniques: Incubation, Negative Control

Journal: Pathogens

Article Title: Type II Restriction-Modification System from Gardnerella vaginalis ATCC 14018

doi: 10.3390/pathogens9090703

Figure Lengend Snippet: REase activity of R.Gva14018I mutants. ( A ) pBR322 incubated with E. coli ArcticExpress (DE3) expressing R.Gva14018I-4: cell lysates (lanes 3, 4), clarified lysates (lanes 8, 9), and crude protein R.Gva14018I-4 preparations (lane 15). pBR322 incubated with R.Gva14018I-8-expressing cell lysates (lanes 5, 6), clarified lysates (lanes 10, 11), and crude protein R.Gva14018I-8 preparations (lane 16). Lanes 2, 7: pBR322 treated with cell lysate and clarified lysate of E. coli cells transformed with pET28(+) vector, respectively. Lanes 12, 13: pBR322 treated with undiluted (OD 600 = 0.6) and a 10-fold diluted G. vaginalis ATCC 14018 cell-free supernatant, respectively. Lane 14: untreated pBR322. ( B ) REase activity of the purified recombinant R.Gva14018I-4 and wild-type REase in cell-free supernatant of ATCC 14018. The substrates pUC57, pBR322, gDNAs from calf thymus, human cell line HCT116, E. coli LMG194, and E. coli LMG194-Me were incubated with the supernatant (lanes 3, 6, 9, 12, 15, 18) or R.Gva14018I-4 (lanes 4, 7, 10, 13, 16, 19). Lanes 2, 5, 8, 11, 14, 17: untreated respective DNA substrate. (A, B) Lane 1: Gene Ruler™ Ladder Mix.

Article Snippet: In addition to ATCC 14018, G. vaginalis ATCC 14019 and G. vaginalis DSM 4944 possess putative REase that recognize GGCC (rebase.neb.com/rebase/rebase.html).

Techniques: Activity Assay, Incubation, Expressing, Transformation Assay, Plasmid Preparation, Purification, Recombinant

Journal: Pathogens

Article Title: Type II Restriction-Modification System from Gardnerella vaginalis ATCC 14018

doi: 10.3390/pathogens9090703

Figure Lengend Snippet: Identification of wild-type and recombinant REases via immunoblot ( A ) and REase activity towards the DNA substrate ( B ). ( A ) Immunoblot with purified R.Gva14018I-4 pAbs. Lane 3: cell lysate of ATCC 14018 (approx. 0.3 mg of the total protein); lane 5: cell lysate of ATCC 49145 (approx. 0.4 mg of the total protein); lanes 7–9: 0.2, 0.5, and 1 µg of recombinant R.Gva14018I-4. Lanes 2, 4, 6: empty lanes; lane 1: PageRuler Prestained Protein Ladder (Thermo Fisher Scientific #26616). ( B ) Cell lysates subjected to immunoblot analysis ( A ) were tested for REase activity via a DNA agarose gel electrophoresis. pBR322 was incubated with 10-fold serially diluted cell lysates of strain ATCC 14018 (lanes 2–4) and ATCC 49145 (lanes 5–7), purified R.Gva14018I-4 (lane 8), PBS (lane 9), and cell-free culture supernatant of ATCC 14018 (lane 10). Lane 1: Gene Ruler™ Ladder Mix.

Article Snippet: In addition to ATCC 14018, G. vaginalis ATCC 14019 and G. vaginalis DSM 4944 possess putative REase that recognize GGCC (rebase.neb.com/rebase/rebase.html).

Techniques: Recombinant, Western Blot, Activity Assay, Purification, Agarose Gel Electrophoresis, Incubation

Journal: The Journal of Infectious Diseases

Article Title: Prevotella timonensis Bacteria Associated With Vaginal Dysbiosis Enhance Human Immunodeficiency Virus Type 1 Susceptibility Of Vaginal CD4 + T Cells

doi: 10.1093/infdis/jiae166

Figure Lengend Snippet: Prevotella timonensis –induced human immunodeficiency virus type 1 (HIV-1) uptake, fusion, and viral replication in vaginal CD4 + T cells. Vaginal epithelium explants ( A ) or CD4 + T cells isolated from phytohemagglutinin-stimulated peripheral blood mononuclear cells ( B–D ) were stimulated overnight by UV-inactivated bacteria ( Lactobacillus crispatus [LC], Lactobacillus iners [LI], Gardnerella vaginalis [GV], Fannyhessea vaginae [FV], Megasphaera elsdenii [ME], and Prevotella timonensis [PT], all on multiplicity of infection [MOI] 10) and subsequently exposed to HIV-1 (SF162; MOI 0.1) for 3 d unless stated differently. A , HIV-1 infection was measured by flow cytometry after intracellular staining for HIV-1 capsid p24 and depicted here as % p24 + cells of CD3 + cells of emigrated fraction (explant model, n = 4–8). B , HIV-1 uptake in CD4 + T cells after 4 h HIV-1 exposure as measured by p24 enzyme-linked immunosorbent assay after trypsin treatment and cell lysis (n = 3). C , Pooled data of β-lactamase activity measured by flow cytometry, representing viral fusion upon 4 h infection with NL4.3BaL-BlaM-Vpr (n = 4). D , De novo HIV-1 replication, determined by detecting green fluorescent protein, after HIV-1 NL4.3eGFP-BaL infection (n = 6–7). Symbols represent independent donors, bars represent mean ± standard deviation. * P < .05, ** P < .01, 2-tailed t test.

Article Snippet: Lactobacillus crispatus (German Collection of Microorganisms and Cell Cultures GmbH [DSMZ]-20584), Lactobacillus iners (DSMZ-13335), Gardnerella vaginalis (DSMZ-4944), Fannyhessea vaginae (DMSZ-15829), Megasphaera elsdenii (DSMZ-20460), Bacteroides fragilis (ATCC-25285), Bacteroides thetaiotaomicron (DSMZ-2079), Escherichia coli (NC0749147), Prevotella amnii (DSMZ-23384), Prevotella bivia (DSMZ-20514), Prevotella copri (or Segatella copri ; DSMZ-18205), Prevotella intermedia (DSMZ-20706), and Prevotella timonensis (or Hoylesella timonensis ; DSMZ-22865) were cultured as recommended by DSMZ.

Techniques: Virus, Isolation, Bacteria, Infection, Flow Cytometry, Staining, Enzyme-linked Immunosorbent Assay, Lysis, Activity Assay, Standard Deviation

Journal: The Journal of Infectious Diseases

Article Title: Prevotella timonensis Bacteria Associated With Vaginal Dysbiosis Enhance Human Immunodeficiency Virus Type 1 Susceptibility Of Vaginal CD4 + T Cells

doi: 10.1093/infdis/jiae166

Figure Lengend Snippet: Antiretroviral drugs used in pre-exposure prophylaxis (PrEP) and combination antiretroviral therapy (cART) block Prevotella timonensis –enhanced human immunodeficiency virus type 1 (HIV-1) infection in vaginal CD4 + T cells. A–C , CD4 + T cells isolated from phytohemagglutinin (PHA)–stimulated peripheral blood mononuclear cells (PBMCs) ( A and B ) or vaginal epithelium explants ( C ) were stimulated overnight by UV-inactivated bacteria ( Lactobacillus crispatus [LC], Lactobacillus iners [LI], Gardnerella vaginalis [GV], Fannyhessea vaginae [FV], Megasphaera elsdenii [ME], and Prevotella timonensis [PT], all on multiplicity of infection [MOI] 10) and subsequently exposed to HIV-1 (SF162; MOI 0.1) for 3 d. HIV-1 infection was measured by flow cytometry after intracellular staining for HIV-1 capsid p24 and depicted here as % p24 + cells of total cells (CD4 + T cells isolated from PHA-stimulated PBMCs ( A and B ) or CD3 + cells of emigrated fraction (explant model, C ). HIV-1 infection in the presence or absence of the PrEP drug tenofovir (TFV, reverse-transcriptase inhibitor, 50 µM, A , n = 4) or replication inhibitors used in cART ( B , n = 3; C , n = 5); maraviroc (Mar, CCR5 blockage, 30 µM); reverse transcriptase inhibitors zidovudine (ZDV, 20 µM) and lamivudine (3TC, 50 µM); raltegravir (Ral, integrase inhibitor, 100 nM); and indinavir (IDV, protease inhibitor, 5 µM). Symbols represent independent donors, bars represent mean ± standard deviation. * P < .05, ** P < .01, *** P < .001, 2-tailed t test.

Article Snippet: Lactobacillus crispatus (German Collection of Microorganisms and Cell Cultures GmbH [DSMZ]-20584), Lactobacillus iners (DSMZ-13335), Gardnerella vaginalis (DSMZ-4944), Fannyhessea vaginae (DMSZ-15829), Megasphaera elsdenii (DSMZ-20460), Bacteroides fragilis (ATCC-25285), Bacteroides thetaiotaomicron (DSMZ-2079), Escherichia coli (NC0749147), Prevotella amnii (DSMZ-23384), Prevotella bivia (DSMZ-20514), Prevotella copri (or Segatella copri ; DSMZ-18205), Prevotella intermedia (DSMZ-20706), and Prevotella timonensis (or Hoylesella timonensis ; DSMZ-22865) were cultured as recommended by DSMZ.

Techniques: Blocking Assay, Virus, Infection, Isolation, Bacteria, Flow Cytometry, Staining, Reverse Transcription, Protease Inhibitor, Standard Deviation

Journal: Cell Death and Differentiation

Article Title: Epigenetic regulation of the Warburg effect by H2B monoubiquitination

doi: 10.1038/s41418-019-0450-2

Figure Lengend Snippet: PKM2 disrupts the access of the RNF20-RAD6 ubiquitination machinery to H2B. a GFP-tagged H2B transfected H1299 cells were co-transfected with or without (−) Flag-tagged PKM2 (left and middle) or Flag-tagged KD mutant of PKM2 (K367M) (right) at an increasing dosage for 48 h. The cells were then lysed and subjected to Co-IP analysis. Normal IgG (NIgG) was used as a negative control. b H1299 and A549 cells were transfected with control or PKM2-specific shRNA or siRNA as indicated for 48 h. The cells were then lysed and subjected to Co-IP analysis with an antibody against H2B followed by western blot analysis with antibodies as indicated. Normal IgG (NIgG) was used as a negative control. c H1299 cells were co-transfected with Flag-tagged PKM2 and GFP-tagged H2B for 48 h. The cells were then treated with 100 ng/mL EGF for the indicated times. Co-IP analysis was performed with anti-GFP antibody followed by western blot analysis with antibodies as indicated. d H1299 cells were treated with 100 ng/mL EGF for the indicated times. The cells were then harvested, and western blot analysis was performed with antibodies as indicated. e H1299 cells transfected with control siRNA (siCont.) or PKM2-specific siRNA (siPKM2) were treated with 100 ng/mL EGF for the indicated times. The cells were then harvested, and western blot analysis was performed with antibodies as indicated. f GFP-H2B transfected H1299 cells were treated with 100 ng/mL EGF for the indicated times. The cells were then lysed and subjected to Co-IP analysis with anti-GFP antibody, and western blot analysis was performed with antibodies as indicated. Normal IgG (NIgG) was used as a negative control

Article Snippet: The antibodies against PKM2 (#4053), RAD6 (#4944), RNF20 (#11974), RNF40 (#12187), H2B (#2934), and Pol-II (#14958) were purchased from Cell Signaling Technology.

Techniques: Transfection, Mutagenesis, Co-Immunoprecipitation Assay, Negative Control, shRNA, Western Blot