Journal: mSystems

Article Title: Analysis of co-occurrence of type II toxin–antitoxin systems and antibiotic resistance determinants in Staphylococcus aureus

doi: 10.1128/msystems.00957-24

Figure Lengend Snippet: Assessment of RNase activity of MazF-Sa, PemK-Sa1, PemK-Sa6, and mutants against different substrates. ( A ) PemK-Sa6 and its E19R and F42T mutants do not exhibit detectable activity against phage MS2 RNA. Very low activity is observed for the double mutant. PemK-Sa1 displays a strong RNase activity. ( B ) RNase activity of PemK-Sa6 toxin and its mutants against fluorescently labeled oligo RNAs. PemK-Sa6 single mutants as well as PemK-Shae are enzymatically inactive. Double-mutant PemK-Sa6 (E19R + F42T) shows activity comparable to PemK-Sa1. ( C ) RNase activity of PemK-Sa1 and MazF-Sa against cat194 transcripts containing indicated numbers of target sequences (UAUU and UACAU, respectively). MazF-Sa displays residual unspecific activity visible as digestion of transcripts devoid of target UACAU sites.

Article Snippet: Short fluorescently labeled RNA substrates (GenScript) contained 6-carboxyfluorescein (6-FAM) at the 5′-end and 5-carboxytetramethylrhodamine (5-TAMRA) at the 3′-end.

Techniques: Activity Assay, Mutagenesis, Labeling

Journal: Scientific Reports

Article Title: A Novel Small RNA-Cleaving Deoxyribozyme with a Short Binding Arm

doi: 10.1038/s41598-019-44750-x

Figure Lengend Snippet: Effect of binding arm length on catalysis and substrate sequence preference. ( A ) Effect of different lengths of left and right binding arms on RNA cleavage activity. Cleavage reactions catalyzed by different deoxyribozyme constructs were assayed on three separate gels and gel images were grouped together for comparison. ( B ) PAGE analysis of deoxyribozyme-catalyzed cleavage of RNA substrates containing 16 different cleavage site dinucleotide junctions. ( C ) PAGE analysis of deoxyribozyme-catalyzed cleavage of RNA substrates containing 16 different combinations of nucleotides at +2 and +3 positions. RNA substrates and deoxyribozyme variants with compensatory mutations were incubated in screen buffer for 3 h at 37 °C, followed by denaturing PAGE analysis. The error bars represent the SD calculated from three independent experiments.

Article Snippet: The fluorescently labeled RNA substrates were purchased from Takara (Dalian, China).

Techniques: Binding Assay, Sequencing, Activity Assay, Construct, Incubation

Journal: Journal of Muscle Research and Cell Motility

Article Title: Myosin VI in PC12 cells plays important roles in cell migration and proliferation but not in catecholamine secretion

doi: 10.1007/s10974-011-9279-0

Figure Lengend Snippet: MVI and the PC12 cell cycle. a Flow cytometric analysis of cell cycle of SA-MVI, mock transfected and sc 2 MVI cells. DNA content was estimated using propidium iodide ( left panels ) and cell cycle analysis ( right panels ) was made with Modtif software (Becton–Dickinson). These are representative histograms and analyses from three independent experiments. b SA β-galactosidase activity measurements of SA-MVI and mock transfected and sc 2 MVI cells. The bars represent the mean values (±SD) of fluorescence corresponding to SA β-galactosidase product. The values were obtained from three independent experiments. Other details as described under “ ” section

Article Snippet: Briefly, SA-MVI PC12 cells and the control ones (sc 2 MVI and mock control) were incubated with 33 mM non-fluorescent SA-β-gal substrate (C12FDG; Invitrogen, Paisley, UK) and after exhaustive wash the fluorescence corresponding to the product SA-β-galactosidase was immediately measured by flow cytometry using CellQuest Software; 10 000 events were counted for each sample (FACS Calibur, Becton–Dickinson).

Techniques: Transfection, Cell Cycle Assay, Software, Activity Assay, Fluorescence

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Reduced potency of cytotoxic T lymphocytes from patients with high-risk myelodysplastic syndromes

doi: 10.1007/s00262-016-1865-y

Figure Lengend Snippet: Gating strategy. a For the bone marrow CX3CR1 screening assay, BMMCs were gated on the lymphocyte subset based on scatter properties, subsequently dead cells were excluded, CD3+ cells selected and CD8+, and CD8− cells were gated based on expression of CD8. Finally, expression of CX3CR1 was assessed for the CD3+, CD3+CD8+ and CD3+CD8− subsets. b In the cytotoxicity assay, assessment of peripheral blood lymphocyte effectors was performed by primarily gating on lymphocytes based on scatter properties (right-arrow), and then, unwanted events were excluded using a dump channel designed to remove CD19+ (B cells), CD14+ (monocytes) and dead cells. Subsequently, T cells and NK cells were gated based on expression of CD3 and CD56, (T cells CD3+ and NK cells CD3−CD56+). CD3+ cells were subdivided into CD57+ and CD57− cells. Degranulation (CD107a) and production of TNF and IFNγ were then assessed from PBLs cultured alone or in the presence of target cells (inside black frame). For the T cells, graphs from samples cultured alone (above dotted line) and in the presence of P815 target cells with anti-CD3 (below dotted line) are shown. Analysis of granule content (granzymes and perforin), co-stimulatory receptors and markers of activation were investigated for the same subsets, but only for unstimulated PBLs. Isotype controls were only used for granzyme A, granzyme B and perforin. Investigation of doublet formation between target cells and lymphocytes was performed by first gating on target cells based on scatter properties (down-arrow), then doublets were selected (high FSC-A/FSC-H), and conjugates were defined based on the expression of lymphocyte markers (CD3+, CD4+, CD8+ and CD3+CD57+). c In the cytotoxicity assay, P815 target cell cytotoxicity was assessed by first gating on the target cells based on scatter properties; subsequently, the target cells were selected based on the target cell tracer dye TFL4 (in the APC channel) at the same time gating away cells dead prior to co-culture (NFL1 detected in the Pacific Orange channel), before target cell cytotoxicity was assessed by PanToxiLux fluorescence (converted by granzyme B/caspase-3 present in the target cell from a non-fluorescent substrate into a fluorescent product detectable in the FITC channel). Target cell cytotoxicity; d MDS-L target cells were initially gated out based on light scatter properties; subsequently, CD34 was used to identify the cells and at the same time gating away cells dead prior to initiation of the co-culture (NFL1). CD34 was chosen to identify the target cells as staining by the target cell tracer TFL4 was poor (data not shown). Finally, the MDS-L-directed cytotoxicity was investigated using PanToxiLux, as described for (c). e BMMC CD34+ target cells were gated out as the other target cells based on light scatter properties, TFL4/NFL1, and subsequently cells positive for CD45 and CD33 were gated away (selection of double-negative cells), penultimately CD34+ cells were selected and finally cytotoxicity assessed by PanToxiLux fluorescence as previously described

Article Snippet: Effector and target cells were co-cultured for 1 h in media added non-fluorescent PanToxiLux substrate (PTL, OncoImmunin).

Techniques: Screening Assay, Expressing, Cytotoxicity Assay, Cell Culture, Activation Assay, Co-Culture Assay, Fluorescence, Staining, Selection

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Reduced potency of cytotoxic T lymphocytes from patients with high-risk myelodysplastic syndromes

doi: 10.1007/s00262-016-1865-y

Figure Lengend Snippet: Analysis of TCR-induced redirected cytotoxicity and CD3+CD57+ T cell characteristics. Peripheral blood lymphocytes and P815 target cells with added anti-CD3 were co-cultured for 4 h, and target cell cytotoxicity was measured indirectly by flow cytometry using the PanToxiLux assay (OncoImmunin) for the assessment of intracellular granzyme B and caspase 3 activation in target cells. a Scatter plot showing P815 cytotoxicity following co-culture with PBMCs from healthy controls, low- and high-risk MDS patients (white, gray and black circles, respectively) (Kruskal–Wallis with Dunn’s posttest, N = 27, p < 0.05). b Scatter plot showing doublets of P815 cells and CD3+ cells as a percentage of all P815 cells following co-culture with PBMCs from healthy controls, low- and high-risk MDS patients (white, gray and black circles, respectively). (Kruskal–Wallis with Dunn’s post test. N = 27 *p < 0.05, **p < 0.01). c Effector cell degranulation and cytokine production by CD3+CD57+ T cells were measured in parallel co-cultures. Unstimulated CD3+CD57+ T cells were investigated for cytotoxic granule content (granzyme A, granzyme B and perforin), co-stimulatory receptors (DNAM-1, NKG2D and 2B4), co-inhibitory receptors (PD-1 and TIM3), adhesion molecules (CD11a, CD18 and ICAM) and extracellular markers of activation (HLA-DR, CD38, and CD45RA). Bars represent median and interquartile range (K–W with Dunn’s posttest, N = 24, *p < 0.05)

Article Snippet: Effector and target cells were co-cultured for 1 h in media added non-fluorescent PanToxiLux substrate (PTL, OncoImmunin).

Techniques: Cell Culture, Flow Cytometry, Activation Assay, Co-Culture Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Reduced potency of cytotoxic T lymphocytes from patients with high-risk myelodysplastic syndromes

doi: 10.1007/s00262-016-1865-y

Figure Lengend Snippet: Reactivity of PBLs toward autologous lymphocyte-depleted bone marrow. a Comparison of degranulation and cytokine production of CD57− and CD57+ T cells as well as NK cells following co-culture of PBMCs with autologous lymphocyte-depleted BMMCs. b Degranulation and production of TNF and IFNγ by CD57+ T cells from low-risk and high-risk MDS patients (gray and black bars, respectively). Bars represent median and interquartile range. c CD34+ BMMC-directed cytotoxicity was measured in co-culture of PBMCs and autologous lymphocyte-depleted BMMCs using the PanToxiLux assay (intracellular granzyme B and caspase 3 activation). No significant difference was observed when comparing PanToxiLux levels in CD34+ cells from low- and high-risk MDS patients (N = 11, M–W) (*p < 0.05, **p < 0.01, ***p < 0.001)

Article Snippet: Effector and target cells were co-cultured for 1 h in media added non-fluorescent PanToxiLux substrate (PTL, OncoImmunin).

Techniques: Comparison, Co-Culture Assay, Activation Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Reduced potency of cytotoxic T lymphocytes from patients with high-risk myelodysplastic syndromes

doi: 10.1007/s00262-016-1865-y

Figure Lengend Snippet: Levels of bone marrow CD57+ T cells, CD57+ T cell degranulation and CD34+ BMMC-directed cytotoxicity following initiation of hypomethylating therapy. Assay of serial bone marrow samples taken prior to and following initiation of azacytidine therapy (samples 2 and 3 taken approximately 3–6 months after initiation of treatment). a Frequency of CD57+ T cells in bone marrow samples from the three time points. b Degranulation of CD57+ peripheral blood T cells collected prior to treatment with azacytidine when co-cultured with autologous T and NK cell-depleted BMMC samples from the given time points. c Expression of PD-1 on CD57+ T cells from bone marrow samples collected prior to and following treatment with azacytidine. d CD34+ BMMC-directed cytotoxicity using the PanToxiLux assay (granzyme B and caspase-3 activation) for co-cultures PBMCs collected prior to azacytidine treatment with autologous lymphocyte-depleted BMMCs from the given time points. Symbols used (diamond, square, circle, up- and down-facing triangles) correspond to individual patients in figures a–c

Article Snippet: Effector and target cells were co-cultured for 1 h in media added non-fluorescent PanToxiLux substrate (PTL, OncoImmunin).

Techniques: Cell Culture, Expressing, Activation Assay