Journal: Communications Biology

Article Title: Toxoplasma gondii infection induces early host cell cycle arrest and DNA damage in primary human host cells by a MYR1-dependent mechanism

doi: 10.1038/s42003-024-07374-0

Figure Lengend Snippet: A , B HUVEC ( n = 3) were infected with T. gondii tachyzoites (MOI 0.5:1) of Tg Δhce1, Tg Δmyr1 mutants and wt parasites ( Tg Δku80) and fixed at 15 min, 1 h and 3 h p. i. Cells were stained in parallel for geminin (G2-phase marker, magenta), PCNA (S-phase marker, green) and DAPI (DNA maker, blue) ( B ). The total number of cells in G2-phase (geminin-positive/PCNA negative), S-phase (PCNA-positive/geminin negative) and G1-phase (geminin- and PCNA-negative) were determined. The data show that both, T. gondii wt and HCE1-deficient strain infection arrest the host cells in S-phase whilst the MYR1-deficient strain fails to do so. The scale bar represents 5 µm. Graph bars represent the median ± SD of three biological donors. The p values were calculated using a one-way ANOVA followed by a Kruskal Wallis multiple comparison test of variance, using the non-infected condition (n. i.) as control. p < 0.05 was considered as significant.

Article Snippet: T. gondii tachyzoites (RH, Δ ku 80, Δ myr 1 and Δ hce 1 strain) were maintained by serial passages in primary HFF cells (ATCC, maximum passage: 10).

Techniques: Infection, Staining, Marker, Comparison, Control

Journal: Communications Biology

Article Title: Toxoplasma gondii infection induces early host cell cycle arrest and DNA damage in primary human host cells by a MYR1-dependent mechanism

doi: 10.1038/s42003-024-07374-0

Figure Lengend Snippet: HUVEC ( n = 3) were infected with tachyzoites (MOI 0.5:1) of Tg Δ hce 1, Tg Δ myr 1 mutants and control parasites ( Tg Δ ku 80) ( A ) Comet assay was used to detect parasite-driven general DNA strand breaks induction (at 12 h p. i.) and showed that T. gondii infection induced DNA damage in a MYR1- and HCE1-independent fashion. Scale bar: 100 µm. B , C Host cell genome instability was evaluated by cell binucleation (indicating cytokinesis failure) and micronuclei formation. Current data show an increased percentage of binucleated cells and micronuclei in HUVEC infected with all, Tg Δ hce 1, Tg Δ myr 1 mutants and control parasites ( Tg Δ ku 80). D Exemplary illustrations of a T. gondii -infected host cell experiencing binucleated phenotype, and cells with micronuclei and simultaneous staining of γH2AX-based DNA damage foci. The results show that most of the binucleated cells were positive for DNA damage in contrast to micronuclei. Micronuclei are indicated by white arrowheads. On average, 1400 cells were counted in each non-infected and T. gondii strain infection in ( A − C ). The scale bar represents 5 µm. Graph bars represent the median ± SD of three biological donors. The p values were calculated using a one-way ANOVA followed by a Kruskal Wallis multiple comparison test of variance, using the non-infected condition (n. i.) as control. A p < 0.05 was considered as significant.

Article Snippet: T. gondii tachyzoites (RH, Δ ku 80, Δ myr 1 and Δ hce 1 strain) were maintained by serial passages in primary HFF cells (ATCC, maximum passage: 10).

Techniques: Infection, Control, Single Cell Gel Electrophoresis, Staining, Comparison

Journal: Communications Biology

Article Title: Toxoplasma gondii infection induces early host cell cycle arrest and DNA damage in primary human host cells by a MYR1-dependent mechanism

doi: 10.1038/s42003-024-07374-0

Figure Lengend Snippet: A , B HUVEC ( n = 3) were infected with tachyzoites (MOI 0.5:1) of Tg Δ hce 1, Tg Δ myr 1 mutants and control parasites ( Tg Δ ku 80), fixed at 15 min, 1 and 3 h p. i. and stained for γH2AX (cyan) to quantify DNA damage foci. Tachyzoites were detected by specific antibodies (red) and the nuclei were stained by DAPI (blue). B The percentage of DNA damage-positive cells was quantified in comparison to the total number of cells in the field of view at 15 min, 1 h and 3 h p. i. Data show that T. gondii infection-driven double-strand DNA breaks occur already at 15 min p. i. in a MYR1-dependent manner. The p-values were calculated using a one-way ANOVA followed by a Kruskal Wallis multiple comparison test of variance, using the non-infected condition (n. i.) as control. A p < 0.05 was considered as significant.

Article Snippet: T. gondii tachyzoites (RH, Δ ku 80, Δ myr 1 and Δ hce 1 strain) were maintained by serial passages in primary HFF cells (ATCC, maximum passage: 10).

Techniques: Infection, Control, Staining, Comparison

Journal: Communications Biology

Article Title: Toxoplasma gondii infection induces early host cell cycle arrest and DNA damage in primary human host cells by a MYR1-dependent mechanism

doi: 10.1038/s42003-024-07374-0

Figure Lengend Snippet: A , B HUVEC ( n = 3) were infected with tachyzoites (MOI 0.5:1) of Tg Δ hce 1, Tg Δ myr 1 mutants and control parasites ( Tg Δ ku 80). B T. gondii -infected cells (3 h p. i.) were stained in parallel for γH2AX (DNA damage foci marker), geminin and PCNA to study whether DNA damage foci formation was linked to a distinct cell cycle phase. γH2AX-positive cells were assigned to each cell cycle phase and plotted as the percentage of DNA damage-positive cells. The scale bar represents 5 µm. Graph bars represent the median ± SD of three biological donors. The p-values were calculated using a one-way ANOVA followed by a Kruskal Wallis multiple comparison test of variance, using the non-infected condition (n. i.) as control. A p < 0.05 was considered as significant.

Article Snippet: T. gondii tachyzoites (RH, Δ ku 80, Δ myr 1 and Δ hce 1 strain) were maintained by serial passages in primary HFF cells (ATCC, maximum passage: 10).

Techniques: Infection, Control, Staining, Marker, Comparison

Journal: Communications Biology

Article Title: Toxoplasma gondii infection induces early host cell cycle arrest and DNA damage in primary human host cells by a MYR1-dependent mechanism

doi: 10.1038/s42003-024-07374-0

Figure Lengend Snippet: HUVEC were infected with tachyzoites (MOI 0.5:1) of Tg Δ hce 1, Tg Δ myr 1 mutants and control parasites ( Tg Δ ku8 0) and analysed for key molecules of the ATM or ATR pathway by Western blotting-based protein quantification at 12 h p. i. Graph bars represent the median ± SD of four to six biological replicates. Vinculin quantification was used as a loading control of the assay. The results show that exclusively the ATM-dependent pathway was activated by T. gondii -infection in a MYR1- and HCE1-independent fashion. The p values were calculated using a one-way ANOVA followed by a Kruskal Wallis multiple comparison test of variance, using the non-infected condition (n. i.) as control. A p < 0.05 was considered as significant.

Article Snippet: T. gondii tachyzoites (RH, Δ ku 80, Δ myr 1 and Δ hce 1 strain) were maintained by serial passages in primary HFF cells (ATCC, maximum passage: 10).

Techniques: Infection, Control, Western Blot, Comparison

Journal: Communications Biology

Article Title: Toxoplasma gondii infection induces early host cell cycle arrest and DNA damage in primary human host cells by a MYR1-dependent mechanism

doi: 10.1038/s42003-024-07374-0

Figure Lengend Snippet: HUVEC ( n = 6) were infected with control tachyzoites (MOI 0.5:1) and both intracellular and extracellular ROS production was evaluated after 3 h p. i. A Exemplary histogram using flow cytometry-based quantification of intracellular ROS via the probe DCFH-DA. NAC treatment was used to block ROS production. DCFH-DA-based quantification of intracellular ROS production in T. gondii -infected HUVEC in the presence and absence of the ROS inhibitor NAC. A control of cells without DCFH-DA staining was included (WOS: without staining). Data showed that T. gondii- infected cells produced lower levels of ROS than uninfected controls. MFI: mean of fluorescence. FACS gating strategy in Supplementary Fig. . B Quantification of extracellular ROS (Amplex red) in T. gondii -infected HUVEC and control cells showed no infection-driven changes. RFI: relative fluorescence intensity. C Quantification of γH2AX-based DNA damage foci in NAC-treated and untreated T. gondii -infected HUVEC. No changes in DNA damage induction were detected when cells were pre-treated with the ROS inhibitor (NAC), suggesting that T. gondii -driven DNA damage occurs ROS-independently. The p values were calculated using a one-way ANOVA followed by a Kruskal Wallis multiple comparison test of variance, using the non-infected condition (n. i.) as control. A p < 0.05 was considered as significant.

Article Snippet: T. gondii tachyzoites (RH, Δ ku 80, Δ myr 1 and Δ hce 1 strain) were maintained by serial passages in primary HFF cells (ATCC, maximum passage: 10).

Techniques: Infection, Control, Flow Cytometry, Blocking Assay, Staining, Produced, Fluorescence, Comparison

Journal: Communications Biology

Article Title: Toxoplasma gondii infection induces early host cell cycle arrest and DNA damage in primary human host cells by a MYR1-dependent mechanism

doi: 10.1038/s42003-024-07374-0

Figure Lengend Snippet: Hypothetic illustration of T. gondii -driven effects on host cellular genome integrity.

Article Snippet: T. gondii tachyzoites (RH, Δ ku 80, Δ myr 1 and Δ hce 1 strain) were maintained by serial passages in primary HFF cells (ATCC, maximum passage: 10).

Techniques:

Journal: Cell host & microbe

Article Title: HIV-1 Balances the Fitness Costs and Benefits of Disrupting the Host Cell Actin Cytoskeleton Early after Mucosal Transmission

doi: 10.1016/j.chom.2018.12.008

Figure Lengend Snippet: (A) Cell surface expression of CD4 and MHC I on uninfected TCM (black histograms) or TCM infected with HIV-GFP encoding either wildtype Nef, ΔNef, NefF191A or NefLLAA (green histograms).

Article Snippet: To generate a nef -deleted strain, HIV-GFP was digested with MluI and XhoI, and re-ligated using T4 DNA polymerase and quick ligase (NEB #E0542L).

Techniques: Expressing, Infection

Journal: Cell host & microbe

Article Title: HIV-1 Balances the Fitness Costs and Benefits of Disrupting the Host Cell Actin Cytoskeleton Early after Mucosal Transmission

doi: 10.1016/j.chom.2018.12.008

Figure Lengend Snippet: (A) ORF diagram of HIV-Lifeact-GFP.

Article Snippet: To generate a nef -deleted strain, HIV-GFP was digested with MluI and XhoI, and re-ligated using T4 DNA polymerase and quick ligase (NEB #E0542L).

Techniques:

Journal: Cell host & microbe

Article Title: HIV-1 Balances the Fitness Costs and Benefits of Disrupting the Host Cell Actin Cytoskeleton Early after Mucosal Transmission

doi: 10.1016/j.chom.2018.12.008

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: To generate a nef -deleted strain, HIV-GFP was digested with MluI and XhoI, and re-ligated using T4 DNA polymerase and quick ligase (NEB #E0542L).

Techniques: Recombinant, Purification, SYBR Green Assay, Plasmid Preparation, Software

Journal: Standards in Genomic Sciences

Article Title: High-quality draft genome sequences of five anaerobic oral bacteria and description of Peptoanaerobacter stomatis gen. nov., sp. nov., a new member of the family Peptostreptococcaceae

doi: 10.1186/s40793-015-0027-8

Figure Lengend Snippet: Maximum-Likelihood phylogenetic tree based on 16S rRNA gene sequence comparisons of strains ACC19a, CM2, CM5, OBRC8, and AS15 (shown in bold) together with other representatives of the Peptostreptococcaceae family and other related human bacteria. The tree was derived based on Tamura-Nei model using MEGA 5 . Bootstrap values > 50 % calculated for 1000 subsets are shown at branch-points. Bar 0.02 substitutions per position. Strains whose genomes have been sequenced are marked with an asterisk

Article Snippet: The type strain ACC19a (=DSM 28705 T ; =HM-483 T ; =ATCC BAA-2665 T ) was isolated from the human subgingival dental plaque.

Techniques: Sequencing, Derivative Assay

Journal: Standards in Genomic Sciences

Article Title: High-quality draft genome sequences of five anaerobic oral bacteria and description of Peptoanaerobacter stomatis gen. nov., sp. nov., a new member of the family Peptostreptococcaceae

doi: 10.1186/s40793-015-0027-8

Figure Lengend Snippet: Classification and general features of the five oral isolates according to the MIGS recommendation [ 34 ]

Article Snippet: The type strain ACC19a (=DSM 28705 T ; =HM-483 T ; =ATCC BAA-2665 T ) was isolated from the human subgingival dental plaque.

Techniques: Staining

Journal: Standards in Genomic Sciences

Article Title: High-quality draft genome sequences of five anaerobic oral bacteria and description of Peptoanaerobacter stomatis gen. nov., sp. nov., a new member of the family Peptostreptococcaceae

doi: 10.1186/s40793-015-0027-8

Figure Lengend Snippet: Transmission and scanning electron micrographs of anaerobic oral bacteria from the family Peptostreptococcaceae. General morphology and Gram-positive cell wall structure of strains CM5 ( a ) and ACC19a ( b ), peritrichous flagella of strain CM2 ( c ), rosettes or brush-like structures formed by strain AS15 ( d ). Bars, 500 nm ( a, b ), 1 μm (c) and 5 μm ( d )

Article Snippet: The type strain ACC19a (=DSM 28705 T ; =HM-483 T ; =ATCC BAA-2665 T ) was isolated from the human subgingival dental plaque.

Techniques: Transmission Assay

Journal: Standards in Genomic Sciences

Article Title: High-quality draft genome sequences of five anaerobic oral bacteria and description of Peptoanaerobacter stomatis gen. nov., sp. nov., a new member of the family Peptostreptococcaceae

doi: 10.1186/s40793-015-0027-8

Figure Lengend Snippet: Genomes statistics

Article Snippet: The type strain ACC19a (=DSM 28705 T ; =HM-483 T ; =ATCC BAA-2665 T ) was isolated from the human subgingival dental plaque.

Techniques: CRISPR

Journal: Standards in Genomic Sciences

Article Title: High-quality draft genome sequences of five anaerobic oral bacteria and description of Peptoanaerobacter stomatis gen. nov., sp. nov., a new member of the family Peptostreptococcaceae

doi: 10.1186/s40793-015-0027-8

Figure Lengend Snippet: Project information

Article Snippet: The type strain ACC19a (=DSM 28705 T ; =HM-483 T ; =ATCC BAA-2665 T ) was isolated from the human subgingival dental plaque.

Techniques: Sequencing

Journal: Standards in Genomic Sciences

Article Title: High-quality draft genome sequences of five anaerobic oral bacteria and description of Peptoanaerobacter stomatis gen. nov., sp. nov., a new member of the family Peptostreptococcaceae

doi: 10.1186/s40793-015-0027-8

Figure Lengend Snippet: Summary of the genomes: one chromosome each and no plasmids

Article Snippet: The type strain ACC19a (=DSM 28705 T ; =HM-483 T ; =ATCC BAA-2665 T ) was isolated from the human subgingival dental plaque.

Techniques:

Journal: Standards in Genomic Sciences

Article Title: High-quality draft genome sequences of five anaerobic oral bacteria and description of Peptoanaerobacter stomatis gen. nov., sp. nov., a new member of the family Peptostreptococcaceae

doi: 10.1186/s40793-015-0027-8

Figure Lengend Snippet: Number of genes associated with general COG functional categories obtained from BROAD or JCVI pipelines

Article Snippet: The type strain ACC19a (=DSM 28705 T ; =HM-483 T ; =ATCC BAA-2665 T ) was isolated from the human subgingival dental plaque.

Techniques: Functional Assay, Modification, Transduction

Journal: Standards in Genomic Sciences

Article Title: High-quality draft genome sequences of five anaerobic oral bacteria and description of Peptoanaerobacter stomatis gen. nov., sp. nov., a new member of the family Peptostreptococcaceae

doi: 10.1186/s40793-015-0027-8

Figure Lengend Snippet: Predicted values of DNA-DNA hybridization a between strains ACC19a, CM2, CM5, OBRC8, AS15 and related members of the family Peptostreptococcaceae

Article Snippet: The type strain ACC19a (=DSM 28705 T ; =HM-483 T ; =ATCC BAA-2665 T ) was isolated from the human subgingival dental plaque.

Techniques: DNA-DNA Hybridization, Staining

Journal: Standards in Genomic Sciences

Article Title: High-quality draft genome sequences of five anaerobic oral bacteria and description of Peptoanaerobacter stomatis gen. nov., sp. nov., a new member of the family Peptostreptococcaceae

doi: 10.1186/s40793-015-0027-8

Figure Lengend Snippet: Number of genes identified in biosynthetic pathway a from whole genome sequences of strains ACC19a, CM2, CM5, OBRC8, AS15 and related organisms from the family Peptostreptococcaceae

Article Snippet: The type strain ACC19a (=DSM 28705 T ; =HM-483 T ; =ATCC BAA-2665 T ) was isolated from the human subgingival dental plaque.

Techniques: