Journal: Environmental Science and Pollution Research International

Article Title: Effective bioremediation of clarithromycin and diclofenac in wastewater by microbes and Arundo donax L

doi: 10.1007/s11356-023-27660-4

Figure Lengend Snippet: Streptomyces rochei DSMZ 41,732 ( a , b ) stock bacterium culture maintained in a GYM Streptomyces medium; Phanaerochete chrysosporium DSMZ 1556 ( c , d ) and Trametes versicolor DSMZ 11,309 ( e , f ) and stock fungal cultures maintained in malt extract peptone agar. Examples of the experimental set-up utilized for studying the Arundo donax growth and uptake of clarithromycin (CLA) and diclofenac (DCF) at different pharmaceutical doses and time of exposure ( g , h )

Article Snippet: Fig. 1 Streptomyces rochei DSMZ 41,732 ( a , b ) stock bacterium culture maintained in a GYM Streptomyces medium; Phanaerochete chrysosporium DSMZ 1556 ( c , d ) and Trametes versicolor DSMZ 11,309 ( e , f ) and stock fungal cultures maintained in malt extract peptone agar.

Techniques:

Journal: Environmental Science and Pollution Research International

Article Title: Effective bioremediation of clarithromycin and diclofenac in wastewater by microbes and Arundo donax L

doi: 10.1007/s11356-023-27660-4

Figure Lengend Snippet: Effect of clarithromycin (CLA) concentration (dose) and time of incubation ( T inc 0, 24, 48, 72, and 144 h) on the removal rate of CLA by Streptomyces rochei and Phanaerochete chrysosporium and of the interaction between dose and T inc on the removal rate of CLA by Trametes versicolor . Effect of the interaction between dose and T inc on the removal rate of diclofenac (DCF). Concentrations in the nutrient medium were for CLA 10 and 100 µg L −1 and for DCF 1 and 10 mg L. −1 . The T inc were 0, 24, 48, 72, and 144 h. Data are mean ± SE ( n = 4). Different letters indicate significant differences at P ≤ 0.05. Details about the two-way ANOVAs are given in Supplementary Table

Article Snippet: Fig. 1 Streptomyces rochei DSMZ 41,732 ( a , b ) stock bacterium culture maintained in a GYM Streptomyces medium; Phanaerochete chrysosporium DSMZ 1556 ( c , d ) and Trametes versicolor DSMZ 11,309 ( e , f ) and stock fungal cultures maintained in malt extract peptone agar.

Techniques: Concentration Assay, Incubation

Journal: Environmental Science and Pollution Research International

Article Title: Effective bioremediation of clarithromycin and diclofenac in wastewater by microbes and Arundo donax L

doi: 10.1007/s11356-023-27660-4

Figure Lengend Snippet: Streptomyces rochei DSMZ 41,732 ( a , b ) stock bacterium culture maintained in a GYM Streptomyces medium; Phanaerochete chrysosporium DSMZ 1556 ( c , d ) and Trametes versicolor DSMZ 11,309 ( e , f ) and stock fungal cultures maintained in malt extract peptone agar. Examples of the experimental set-up utilized for studying the Arundo donax growth and uptake of clarithromycin (CLA) and diclofenac (DCF) at different pharmaceutical doses and time of exposure ( g , h )

Article Snippet: Streptomyces rochei DSMZ 41,732 stock cultures were maintained at 28 °C in a GYM Streptomyces medium (DSMZ, medium 65), whereas P. chrysosporium DSMZ 1556 and T. versicolor DSMZ 11,309 and stock cultures were maintained at 25 °C and 35 °C, respectively, in malt extract peptone agar (medium 90) (DSMZ) (Fig. ) (DSMZ ).

Techniques:

Journal: Environmental Science and Pollution Research International

Article Title: Effective bioremediation of clarithromycin and diclofenac in wastewater by microbes and Arundo donax L

doi: 10.1007/s11356-023-27660-4

Figure Lengend Snippet: Effect of clarithromycin (CLA) concentration (dose) and time of incubation ( T inc 0, 24, 48, 72, and 144 h) on the removal rate of CLA by Streptomyces rochei and Phanaerochete chrysosporium and of the interaction between dose and T inc on the removal rate of CLA by Trametes versicolor . Effect of the interaction between dose and T inc on the removal rate of diclofenac (DCF). Concentrations in the nutrient medium were for CLA 10 and 100 µg L −1 and for DCF 1 and 10 mg L. −1 . The T inc were 0, 24, 48, 72, and 144 h. Data are mean ± SE ( n = 4). Different letters indicate significant differences at P ≤ 0.05. Details about the two-way ANOVAs are given in Supplementary Table

Article Snippet: Streptomyces rochei DSMZ 41,732 stock cultures were maintained at 28 °C in a GYM Streptomyces medium (DSMZ, medium 65), whereas P. chrysosporium DSMZ 1556 and T. versicolor DSMZ 11,309 and stock cultures were maintained at 25 °C and 35 °C, respectively, in malt extract peptone agar (medium 90) (DSMZ) (Fig. ) (DSMZ ).

Techniques: Concentration Assay, Incubation

Journal: PLoS Pathogens

Article Title: ZBTB2 represses HIV-1 transcription and is regulated by HIV-1 Vpr and cellular DNA damage responses

doi: 10.1371/journal.ppat.1009364

Figure Lengend Snippet: ZASC1 and ZBTB2 interact. (A) Schematic of ZASC1 and ZBTB2 and relative location of N-terminal and internal deletion variants tested for interaction. Blue indicates interaction and red indicates a failure to interact. The location of zinc fingers in each protein are indicated in yellow. Sequences of the interaction domains in ZASC1 zinc finger 6 (ZF6) and ZBTB2 zinc finger 1 are shown below the respective protein maps, with Cys and His residues implicated in Zn 2+ coordination indicated in green. The ZASC1 mZF6 variant has C376, C379S,H392A, H397A mutations while the ZBTB2 mZF1 variant has C256S, C259S, H272A, H276A mutations. HEK293 cells (1X10 7 ) were transfected with expression plasmids encoding the epitope tagged forms of the indicated proteins. 48 h post-transfection, cells were lysed, and epitope tagged proteins were immunoprecipitated (IP). Starting material and IP material was separated by SDS-PAGE and analyzed by western blotting (WB) using the indicated antibodies as described in materials and methods. (B) Co-immunoprecipitation of ZASC1 variants by WT ZBTB2. (C) Co-immunoprecipitation of ZBTB2 variants by WT ZASC1. Numbers to the left of graphs indicate position of molecular weight markers in kDa. Numbers below the graph indicate relative band intensity relative to the well expressing WT ZASC1 and WT ZBTB2. In B and C, no appreciable signal from the HA-CD4 control protein IP was detected (less than 0.1% of input).

Article Snippet: CRISPR/Cas9 plasmids targeting ZASC1 (sc-41729) and ZBTB2 (sc-412860) and corresponding homologous directed repair plasmids (sc-41729-HDR, sc-412860-HDR) were obtained commercially (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Zinc-Fingers, Variant Assay, Transfection, Expressing, Immunoprecipitation, SDS Page, Western Blot, Molecular Weight, Control

Journal: PLoS Pathogens

Article Title: ZBTB2 represses HIV-1 transcription and is regulated by HIV-1 Vpr and cellular DNA damage responses

doi: 10.1371/journal.ppat.1009364

Figure Lengend Snippet: Fluorescent images of U2OS cells transfected with expression vectors encoding the indicated mCherry-fusion proteins (mCherry with a nuclear localization signal, SP1 and ZASC1 variants) and GFP- fusion proteins (firefly luciferase and ZBTB2 variants). Nuclei were stained with DAPI. Scale bar applies to all panels.

Article Snippet: CRISPR/Cas9 plasmids targeting ZASC1 (sc-41729) and ZBTB2 (sc-412860) and corresponding homologous directed repair plasmids (sc-41729-HDR, sc-412860-HDR) were obtained commercially (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Transfection, Expressing, Luciferase, Staining

Journal: PLoS Pathogens

Article Title: ZBTB2 represses HIV-1 transcription and is regulated by HIV-1 Vpr and cellular DNA damage responses

doi: 10.1371/journal.ppat.1009364

Figure Lengend Snippet: (A) Jurkat cells were transfected with a HIV-1 promoter-driven reporter plasmid upstream of gaussian luciferase and the herpes simplex virus tk promoter upstream of cypridinia luciferase and the protein encoding plasmids indicated at the bottom. The effects on the basal and TAT activated expression of the WT HIV-1 LTR promoter and the control tk promoter in the presence or absence of ZBTB2 or a ZBTB2 variant with a deletion in the POZ domain (a.a. 1–79, ΔPOZ) are shown). The data shown are the average mean chemiluminescent reporter values obtained in an experiment performed with quadruplicate samples and are representative of three independent experiments. (B) SUPT1 cells transfected with siRNAs targeting ZBTB2 or non-targeting siRNA. Two days post transfection, cells were challenged with VSV-G pseudotyped HIV-1 vectors with non-sense mutations in envelope, and Vpr with firefly luciferase in the NEF locus. Infections were done with the Vpr deletion virus or virions transcomplemented with a Vpr expression plasmid in the producer cells. A sample of cells were harvested for total protein (see C) at this time. 48 hours post infection HIV-1 transcription was assayed for by measuring firefly luciferase and cell viability was monitored by the ATP assay CellTiter-glo (Promega). The data shown are the average mean values obtained in an experiment performed with quadruplicate samples and are representative of three independent experiments. (C and D) Western blots demonstrating loss of ZASC1 or ZBTB2 protein accumulation in (C) SUPT1 cells transfected with siRNAs targeting ZBTB2 or non-targeting (NT) siRNA or (D) ZASC1 and ZBTB2 knockout cells generated by CRISPR/CAS9 in SUPT1 and Jurkat T cell lines. (E) Co-immunoprecipitation assays of endogenous proteins. WT Jurkat, ZASC1 or ZBTB2 knockout cells (1X10 7 ) were lysed and incubated with ZASC1, ZBTB2 or non-specific (NS) rabbit IgG. Co-immunoprecipitating complexes were detected by western blotting. (F) Cells (5X10 4 /well) were seeded in quadruplicate a 96 well plate, incubated for 48 hours and assayed for cell viability using the ATP assay CellTiter-glo (Promega). (G) Cells were passaged for the indicated time, counted, plotted and fitted to a growth curve using and the doubling time and 95% confidence interval was calculated using Prism graphing software. Error bars indicate the standard deviation of the data in all panels. ANOVA analysis was performed and for P-values < 0.05 a Tukey’s HSD was performed and relevant P-values reported.

Article Snippet: CRISPR/Cas9 plasmids targeting ZASC1 (sc-41729) and ZBTB2 (sc-412860) and corresponding homologous directed repair plasmids (sc-41729-HDR, sc-412860-HDR) were obtained commercially (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Transfection, Plasmid Preparation, Luciferase, Virus, Expressing, Control, Variant Assay, Infection, ATP Assay, Western Blot, Knock-Out, Generated, CRISPR, Immunoprecipitation, Incubation, Software, Standard Deviation

Journal: PLoS Pathogens

Article Title: ZBTB2 represses HIV-1 transcription and is regulated by HIV-1 Vpr and cellular DNA damage responses

doi: 10.1371/journal.ppat.1009364

Figure Lengend Snippet: (A) WT or ΔZASC1 Jurkat and SupT1 cells were challenged with equivalent amounts (based on p24 levels) of either VSV-G pseudotyped HIV-1 vector with nonsense mutations in envelope and Vpr, luciferase in the NEF locus, and either a WT LTR or an LTR with all 4 ZASC1 binding sites mutated (mZBS) . (B) Replication of WT HIV-1 or HIV-1 with a Vpr nonsense mutation (ΔVpr) in WT or ΔZASC1 Jurkat cells. Cells (1X10 6 ) were infected with the indicated virus at an moi of 0.005. Supernatant was titered in quadruplicate on TZMBL reporter cells at the indicated time points and infectious units counted by X-gal staining. The data shown are the average mean values obtained in an experiment performed with quadruplicate samples and are representative of three independent experiments. The bar graph is a focus on the day 10 time point. Error bars indicate the standard deviation of the data in all panels. ANOVA analysis was performed and for P-values < 0.05 a Tukey’s HSD was performed and relevant P-values reported.

Article Snippet: CRISPR/Cas9 plasmids targeting ZASC1 (sc-41729) and ZBTB2 (sc-412860) and corresponding homologous directed repair plasmids (sc-41729-HDR, sc-412860-HDR) were obtained commercially (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Plasmid Preparation, Luciferase, Binding Assay, Mutagenesis, Infection, Virus, Staining, Standard Deviation

Journal: PLoS Pathogens

Article Title: ZBTB2 represses HIV-1 transcription and is regulated by HIV-1 Vpr and cellular DNA damage responses

doi: 10.1371/journal.ppat.1009364

Figure Lengend Snippet: (A) WT Jurkat and ΔZASC1 Jurkat cells transfected with reporter plasmids expressing Gaussia luciferase from the HIV-1 promoter and with the ZASC1 and/or TAT expression plasmids indicated in the table at the bottom. Raw relative light units of HIV-1 LTR promoter-driven reporter gene expression for each cell line was reported in the presence and absence of ZASC1 and TAT. (B) WT and ΔZBTB2 Jurkat cells were transfected with reporter plasmids containing HIV-1 promoter-driven Gaussia luciferase and HSV TK-promoter driving Cypridinia luciferase expression. Raw luciferase activity for both the HIV-1 LTR and HSV TK promoters is plotted. (C) WT and ΔZASC1 or (D) ΔZBTB2 Jurkat cells were transfected with the above luciferase reporter plasmids, TAT, and plasmids expressing either WT Vpr or the cell-cycle deficient Vpr R80A mutant. Raw Gaussia luciferase activity for the HIV-1 LTR promoter divided by the raw Cypridinia luciferase activity of the TK promoter is plotted as relative light units. The data shown in each of panels are the average mean values obtained in an experiment performed with quadruplicate samples and are representative of three independent experiments. (E) Western blots showing expression of WT and R80A Vpr mutants in each cell line. Numbers indicate band intensity relative to WT Vpr lanes. Error bars indicate the standard deviation of the data in all panels. ANOVA analysis was performed and for P-values < 0.05 a Tukey’s HSD was performed and relevant P-values reported.

Article Snippet: CRISPR/Cas9 plasmids targeting ZASC1 (sc-41729) and ZBTB2 (sc-412860) and corresponding homologous directed repair plasmids (sc-41729-HDR, sc-412860-HDR) were obtained commercially (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Transfection, Expressing, Luciferase, Gene Expression, Activity Assay, Mutagenesis, Western Blot, Standard Deviation

Journal: PLoS Pathogens

Article Title: ZBTB2 represses HIV-1 transcription and is regulated by HIV-1 Vpr and cellular DNA damage responses

doi: 10.1371/journal.ppat.1009364

Figure Lengend Snippet: (A) ChIP results from Jurkat cells transduced with NL43E-R-Luc using an antibody against ZBTB2 or non-specific IgG control antibody and primer sets targeting the HIV-1 promoter (-54 to +32) and downstream sequences in the Vif gene (+4,599 to +4,619) or a region on chromosome 12 that lacks active genes (gene desert). ChIP analysis of (B) ZASC1 and (C) ZBTB2 recruitment to the HIV-1 promoter in infected WT Jurkat and Jurkat ZASC1 knockout cells. (D-E) ChIP analysis of (D) ZASC1 and (E) ZBTB2 recruitment to the HIV-1 promoter in infected WT Jurkat and Jurkat ZBTB2 knockout cells. (F) ChIP analysis of ZBTB2 recruitment to the HIV-1 promoter in WT Jurkat cells infected with an HIV-1 vector with either a WT LTR or an LTR variant with all four ZASC1 binding sites mutated. Immunoprecipitation and Real-time PCR analysis were performed and normalized to input controls as a percentage of starting material for immunoprecipitation and fold enrichment of experimental IPs relative to IgG controls reported. For each panel, the data shown are the average mean values obtained in an experiment performed with quadruplicate samples and are representative of three or more independent experiments. For (A), ANOVA analysis was performed and for P-values < 0.05 a Tukey’s HSD was performed and relevant P-values reported. For (B to F) P-values were calculated using a standard Student’s t-test and significant changes are indicated.

Article Snippet: CRISPR/Cas9 plasmids targeting ZASC1 (sc-41729) and ZBTB2 (sc-412860) and corresponding homologous directed repair plasmids (sc-41729-HDR, sc-412860-HDR) were obtained commercially (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Transduction, Control, Infection, Knock-Out, Plasmid Preparation, Variant Assay, Binding Assay, Immunoprecipitation, Real-time Polymerase Chain Reaction

Journal: PLoS Pathogens

Article Title: ZBTB2 represses HIV-1 transcription and is regulated by HIV-1 Vpr and cellular DNA damage responses

doi: 10.1371/journal.ppat.1009364

Figure Lengend Snippet: ChIP analysis of (A) histone H3 acetylation for the HIV-1 promoter in WT Jurkat cells infected with an HIV-1 vector with either a WT LTR or an LTR variant with all four ZASC1 binding sites mutated. ChIP analysis of (B) histone H3 acetylation and (C) total histone H3 on the HIV-1 promoter in HIV-1 vector infected WT Jurkat, Jurkat ZASC1 knockout cells and Jurkat ZBTB2 knockout cells. Immunoprecipitation and Real-time PCR analysis were performed and normalized to input controls as a percentage of starting material for immunoprecipitation and fold enrichment of experimental IPs relative to IgG controls reported. For each panel, the data shown are the average mean values obtained in an experiment performed with quadruplicate samples and are representative of three or more independent experiments. Error bars indicate the standard deviation of the data in all panels. For (A), P-values were calculated using a standard Student’s t-test and significant changes are indicated, for (B & C) ANOVA analysis was performed and for P-values < 0.05 a Tukey’s HSD was performed and relevant P-values reported.

Article Snippet: CRISPR/Cas9 plasmids targeting ZASC1 (sc-41729) and ZBTB2 (sc-412860) and corresponding homologous directed repair plasmids (sc-41729-HDR, sc-412860-HDR) were obtained commercially (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Infection, Plasmid Preparation, Variant Assay, Binding Assay, Knock-Out, Immunoprecipitation, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: PLoS Pathogens

Article Title: ZBTB2 represses HIV-1 transcription and is regulated by HIV-1 Vpr and cellular DNA damage responses

doi: 10.1371/journal.ppat.1009364

Figure Lengend Snippet: U2OS cells were transfected with GFP-ZBTB2 and either mCherry with a nuclear localization signal (A) or mCherry-ZASC1 (B-F) and imaged 24 hours post transfection. ATR kinase was activated by 320 nm UV light (C & D), 8 mM of the DNA damaging agent methyl methanesulfonate (MMS) ATR activation was inhibited by treating with 10 μM of ATR inhibitor (CAS 905973-89-9) (D, F). (G and H) To quantify the effects observed in (A-F), independent transfections were performed with the indicated conditions and the relative ZBTB2 fluorescence signals in the nucleus and a nucleus-adjacent cytoplasmic ring were measured in ≥100 cells, and the fold change in nuclear to cytoplasmic signal reported. (G) presents the effects of DNA damaging UV treatment and ATR inhibitor, and (H) presents the effects of 8 mM of the DNA damaging agent MMS and 10 μM ATR inhibitor. Error bars indicate the standard deviation of the data in all panels. ANOVA analysis was performed and for P-values < 0.05 a Tukey’s HSD was performed and relevant P-values reported. Western blots showing the effects of (I) UV or (J) MMS treatment on the levels of ZASC1, ZBTB2 and a HA-tagged CD4 control protein. Numbers indicate band intensity relative to lane expressing both ZASC1 and ZBTB2. Results are representative of three independent experiments.

Article Snippet: CRISPR/Cas9 plasmids targeting ZASC1 (sc-41729) and ZBTB2 (sc-412860) and corresponding homologous directed repair plasmids (sc-41729-HDR, sc-412860-HDR) were obtained commercially (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Transfection, Activation Assay, Fluorescence, Standard Deviation, Western Blot, Control, Expressing

Journal: PLoS Pathogens

Article Title: ZBTB2 represses HIV-1 transcription and is regulated by HIV-1 Vpr and cellular DNA damage responses

doi: 10.1371/journal.ppat.1009364

Figure Lengend Snippet: U2OS cells were transfected with GFP-ZBTB2 and mCherry-ZASC1 and imaged 24 hours post transfection. Co-expression of WT Vpr (A) resulted in a primarily cytoplasmic ZBTB2 localization which was reversed (B) by treating with 10 μM of ATR inhibitor (CAS 905973-89-9). (C) Co-expression of the cell-cycle arrest-deficient Vpr R80A mutant failed to relocalize ZBTB2 to the nucleus. (D and E) To quantify the effects observed in (A-C), independent transfections were performed with the indicated conditions and the relative ZBTB2 fluorescence signals in the nucleus and a nucleus-adjacent cytoplasmic ring were measured in ≥100 cells and the fold change in nuclear to cytoplasmic signal reported. See for representative results of cells expressing ZBTB2 alone or in combination with ZASC1. (D) presents the effects of WT Vpr expression and the effect of 10 μM ATR inhibitor, and (E) presents the effects expressing the Vpr R80A cell cycle arrest deficient mutant. Error bars indicate the standard deviation of the data in all panels. ANOVA analysis was performed and for P-values < 0.05 a Tukey’s HSD was performed and relevant P-values reported. Western blots showing the effects of (F) Vpr and ATR inhibitor or (G) WT and R80A Vpr on the levels of ZASC1, ZBTB2 and a HA-tagged CD4 control protein. Numbers indicate band intensity relative to lane expressing both ZASC1 and ZBTB2. Results are representative of three independent experiments.

Article Snippet: CRISPR/Cas9 plasmids targeting ZASC1 (sc-41729) and ZBTB2 (sc-412860) and corresponding homologous directed repair plasmids (sc-41729-HDR, sc-412860-HDR) were obtained commercially (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Transfection, Expressing, Mutagenesis, Fluorescence, Standard Deviation, Western Blot, Control

Journal: PLoS Pathogens

Article Title: ZBTB2 represses HIV-1 transcription and is regulated by HIV-1 Vpr and cellular DNA damage responses

doi: 10.1371/journal.ppat.1009364

Figure Lengend Snippet: In the presence of Vpr or other activation of the DNA damage response, ZBTB2 is exported from the nucleus, ZASC1 binds the HIV-1 LTR promoter and facilitates the assembly of the TAT P-TEFb elongation complex. Under conditions of low Vpr, ZBTB2 is recruited to the HIV-1 promoter by ZASC1, binds cellular HDAC complexes and represses HIV-1 transcription.

Article Snippet: CRISPR/Cas9 plasmids targeting ZASC1 (sc-41729) and ZBTB2 (sc-412860) and corresponding homologous directed repair plasmids (sc-41729-HDR, sc-412860-HDR) were obtained commercially (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Activation Assay