Journal: International Journal of Molecular Sciences

Article Title: Characterization of the Activities of Vorinostat Against Toxoplasma gondii

doi: 10.3390/ijms26020795

Figure Lengend Snippet: Vorinostat promotes ROS production and induces apoptosis in T. gondii . ( A ) Assessment of ROS production. Tachyzoites of T. gondii RH strain were incubated with vorinostat or 0.1% DMSO (vehicle) for 24 h. Flow cytometry analysis was then performed using DCFH-DA staining to determine intracellular ROS levels. Data are presented as the means ± SD from at least three independent experiments. Statistical significance was determined by Student’s t -test; **** p < 0.0001. ( B ) Representative flow cytometric dot plots of Annexin V-FITC/PI staining are shown. ( C ) The percentage of apoptotic parasites following treatment with vorinostat or vehicle was quantified via flow cytometry. Mean percentages ± SD of Annexin V+, PI+, or PI− parasites were calculated from least three independent experiments. Statistical significance was determined by Student’s t -test; * p < 0.05, ns means not significant.

Article Snippet: T. gondii RH strain (type I) and ME49 strain (type II) were maintained in confluent monolayers of human foreskin fibroblast (HFF) cells (HS27; ATCC: CRL-1634, Manassas, VA, USA).

Techniques: Incubation, Flow Cytometry, Staining

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: VPS32, a member of the ESCRT complex, modulates adherence to host cells in the parasite Trichomonas vaginalis by affecting biogenesis and cargo sorting of released extracellular vesicles

doi: 10.1007/s00018-021-04083-3

Figure Lengend Snippet: Attachment of TvEpNEO and TvVPS32 parasites to NhPRE1 cells. a TvEpNEO and TvVPS32 parasites were labeled with Cell Tracker Blue CMAC (Invitrogen). Labeled parasites were then incubated for 30 min with NhPRE1 prostate cell monolayers grown on coverslips in 24-well plates at 37 °C and 5% CO2. Coverslips were washed to remove non-adherent parasites, mounted, and visualized by fluorescence microscopy. b Adherence of TvEpNEO (red bar) and TvVPS32 (green bar) parasites to NhPRE1 cells. Data are from three experiments performed in triplicate and show the fold increase in the number of parasites attached per coverslip standardized using TvEpNEO parasites with SD (p < 0.05). c Schematic representation of the experimental design. EVs from TvEpNEO (red) and TvVPS32 (green) were isolated. NhPRE1 cells were pre-incubated with increasing amount of EVs. Then, parasite attachment was analyzed by fluorescent microscopy. d Preincubation of NhPRE1 cells with VPS32-EVs or EpNEO-EVs increases adherence of a poorly adherent T. vaginalis strain (G3). Note that a stronger effect in increasing parasite adherence is observed when the cells are pre-incubated with EVs isolated from parasites transfected with VPS32. Statistical analyses were performed by comparing the adherence values of each treatment against the control treatment (*p < 0.05, **p < 0.01, ***p < 0.001). Besides, statistical analyses within each treatment were performed comparing between EVs concentration

Article Snippet: Parasites, cell cultures and media Trichomonas vaginalis strain G3 (ATCC PRA-98; Kent, UK) was cultured in TYM medium supplemented with 10% horse serum and 10 U/ml penicillin [ 27 ].

Techniques: Labeling, Incubation, Fluorescence, Microscopy, Isolation, Transfection, Control, Concentration Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: VPS32, a member of the ESCRT complex, modulates adherence to host cells in the parasite Trichomonas vaginalis by affecting biogenesis and cargo sorting of released extracellular vesicles

doi: 10.1007/s00018-021-04083-3

Figure Lengend Snippet: Analysis of EVs uptake by prostate cells. a T. vaginalis VPS32-EVs or EpNEO-EVs were labeled with PKH67 dye and incubated with NhPRE1cells for 3 h. EV binding was visualized using fluorescence microscopy by incubating NhPRE1 cells with PKH67-labeled EVs or PKH67-labeled PBS as control (green). The nucleus was stained with DAPI (blue). The images are representative of 20 images viewed under similar conditions. b Quantification of EVs binding. Data shown represent the mean ± SD from 3 independent experiments, each performed in duplicate. The maximal fluorescence after incubating with labeled EVs was arbitrarily set at 100%. c Effect on uptake by temperature that block endocytosis of fluorescently labeled VPS32-EVs or EpNEO-EVs detected using flow cytometry. T. vaginalis VPS32-EVs or EpNEO-EVs were labeled with PKH67 dye, incubated with NhPRE1 cells at 37 °C or 4 °C for 45 min and uptake was quantified by flow cytometry. d Quantification of VPS32-EVs or EpNEO-EVs uptake at 37 °C or 4 °C. The maximal fluorescence after incubating with labeled VPS32-EVs was arbitrarily set at 100%. e T. vaginalis VPS32-EVs or EpNEO-EVs were labeled with PKH67 dye, incubated with NhPRE1cells for 45 min at 37 °C and the binding quantified using flow cytometry. Three independent experiments were performed. A representative experiment is shown. f Quantification of PKH67-labeled EVs uptake to NhPRE1 cells using flow cytometry. The maximal fluorescence after incubating the NhPRE1 cells with labeled VPS32-EVs was set at 100%. Data are presented are the mean ± SD from three independent experiments

Article Snippet: Parasites, cell cultures and media Trichomonas vaginalis strain G3 (ATCC PRA-98; Kent, UK) was cultured in TYM medium supplemented with 10% horse serum and 10 U/ml penicillin [ 27 ].

Techniques: Labeling, Incubation, Binding Assay, Fluorescence, Microscopy, Control, Staining, Blocking Assay, Flow Cytometry

Journal: PLoS Neglected Tropical Diseases

Article Title: Leukocyte Lysis and Cytokine Induction by the Human Sexually Transmitted Parasite Trichomonas vaginalis

doi: 10.1371/journal.pntd.0004913

Figure Lengend Snippet: (A) PBMC and Tv strains Tv G3 (laboratory adapted) or Tv MSA1132 (clinical isolate) were differentially labeled with CFSE or Cell Tracker Blue, respectively and co-cultured at an MOI of 0.5 for 4 hours. Cells were then stained with anti-CD3 and anti-CD19 to detect T-cells or B-cells, respectively, and lymphocyte death was assessed with flow cytometry analysis. (B) Killing of lymphocytes by Tv MSA1132 was assessed as in (A) at the indicated MOI for 4 hours. (C) Killing of lymphocytes by Tv MSA1132 was assessed as in (A) at an MOI 0.5 for the indicated period of time. All data in (A-C) are averages with standard deviation of triplicate wells and representative of at least 3 donors/ independent experiments.

Article Snippet: Tv strains G3 (Beckenham, UK 1973, ATCC-PRA-98), and MSA1132 (Mt.

Techniques: Labeling, Cell Culture, Staining, Flow Cytometry, Standard Deviation

Journal: PLoS Neglected Tropical Diseases

Article Title: Leukocyte Lysis and Cytokine Induction by the Human Sexually Transmitted Parasite Trichomonas vaginalis

doi: 10.1371/journal.pntd.0004913

Figure Lengend Snippet: Primary human monocytes were cultured with either dead intact Tv G3, Tv MSA1132, unstimulated, or treated with LPS ( A, B, D ) or poly (I:C) (C) or LPS and IFNγ (E) for 16 hours. Prior to killing, Tv strains were either untreated ( M . hominis +) or treated with additional antibiotic to clear the symbiont ( M . hominis -). Supernatants were collected and the indicated cytokines were measured using Cytometric Bead Array (CBA) or ELISA. Data shown are average of triplicate wells with standard deviation, and are representative of 3 donors/ independent experiments.

Article Snippet: Tv strains G3 (Beckenham, UK 1973, ATCC-PRA-98), and MSA1132 (Mt.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Cancer Communications

Article Title: Candidate therapeutic agents in a newly established triple wild‐type mucosal melanoma cell line

doi: 10.1002/cac2.12315

Figure Lengend Snippet: Establishment and characterization of MM9H‐1 cell line. (A) Clinical imaging of the patient. The patient was a 67‐year‐old woman who was diagnosed with mucosal melanoma at T4aN1M0 stage. CT axial view reveals a low‐density mass around the lower anterior teeth, and the boundary display is not clear. The arrows indicate the primary foci (red), the location of the cryobiopsy specimens, and the metastatic lymph node (yellow). (B) Histopathology of the patient. HE staining of the biopsy shows abundance of tumor cells with pigmentation. Immunohistochemistry analysis shows positive staining for HMB45 and Melan‐A and negative for AE1/AE3. (C) Phase contrast image of cultured mucosal melanoma MM9H‐1 cells. The cells exhibit a spindle cell‐type appearance, a typical fusiform morphology (the 5th passage). (D) MM9H‐1 cells are positive for staining of melanoma‐specific biomarkers Melan‐A, S100B HMB45, S100A6. (E) Cell suspension from the 10 th ‐passage cells formed a black clump at the bottom of the centrifuge tube after centrifugation. (F) Flow cytometry for the purity of MM9H‐1. MM9H‐1 cells were filtered with or without anti‐melanoma (MCSP) microbeads and then stained with or without MCSP antibody. The purity of MCSP‐positive cells is >97%. (G‐J) Establishment of PDX model. (G) Growth curves of different PDX passages, which start to increase in size at around 10 days after transplantation (P1, P2, P3 = PDX tumors in first, second and third passages). Tumor volume was analyzed with Student's t test. (H) Representative image of PDX‐P1 tumors subcutaneously grow in nude mice. (I) H&E staining of the subcutaneous PDX‐P3 tumor, with vessel formation indicated by the yellow box. (J) Immunohistochemistry analysis of PDX‐P3 shows positive staining for Melan‐A, HMB45, S100 and negative for AE1/AE3. * P < 0.05. Abbreviations: CT, computed tomography; HE, hematoxylin and eosin; HMB45, human melanoma black 45; MCSP, melanoma‐associated chondroitin sulfate proteoglycan; PDX, patient‐derived tumor xenografts

Article Snippet: When the cell confluence reached about 50%, cells were fixed with 4% paraformaldehyde for 20 min at 22‐25°C and permeabilized with 0.3% Triton X‐100 for 10 min. After blocking with 5% bovine serum albumin at 22‐25°C for 1 h, the cells were incubated with primary antibodies against S100A6 (1:100, 10245‐1‐AP, Proteintech, Wuhan, Hubei, China), S100 beta (1:100, 15146‐1‐AP, Proteintech), Melan‐A (1:100, 60348‐1‐Ig, Proteintech), human melanoma black 45 (HMB45) (1:1000, ab212829, Abcam, Cambridge, UK), and a triple anti‐melanoma antibody cocktail against HMB45, Melan‐A, and Tyrosinase (1:200, ab733, Abcam) overnight at 4°C.

Techniques: Imaging, Histopathology, Staining, Immunohistochemistry, Cell Culture, Suspension, Centrifugation, Flow Cytometry, Transplantation Assay, Computed Tomography, Derivative Assay