Journal: Scientific Reports

Article Title: A tool kit for rapid cloning and expression of recombinant antibodies

doi: 10.1038/srep05885

Figure Lengend Snippet: (A–B) Flow cytometric histograms showing fluorescent intensity of cells incubated with purified IgG/IgE antibodies plus secondary FITC-labelled goat anti-human IgG/IgE (open) and cells with secondary antibody only (filled) against the number of cells. (A) Flow cytometric assessment of anti-CSPG4 IgG 1 /IgE antibodies shows specific binding to native CSPG4 antigen present on the cell surface of A375 melanoma cells and no binding above background to primary melanocytes. (B) Fc regions of anti-CSPG4 IgG 1 and 102.1F10 IgG 4 isotypes demonstrate effector-binding to U937 monocytic cell line, expressing human Fcγ receptors. The IgE antibody isotypes also bind similarly to RBL SX38 mast cells, expressing human FcεR1 receptor. (C) Immunofluorescence staining of A375 cells confirms specific binding of anti-CSPG4 IgG 1 /IgE and no background binding with isotype control hapten specific NIP-IgG 1 /IgE detected by goat anti-human IgG/IgE-FITC. (D) Grass pollen allergen specificity is confirmed by anti-human sandwich ELISA, showing specific binding of recombinant 102.1F10 IgG 4 /IgE and original patient serum to the ELISA plate-bound Phl p 7 allergen and no binding above background with unspecific human myeloma IgG 4 and MOv18 IgE isotype controls.

Article Snippet: Primary human epidermal melanocytes (PCS-200-012, ATCC) were grownin Dermal Cell Basal Medium (PCS-200-030, ATCC) and supplemented with the Melanocyte Growth Kit (PCS-200-041, ATCC).

Techniques: Incubation, Purification, Binding Assay, Expressing, Immunofluorescence, Staining, Control, Sandwich ELISA, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: Scientific reports

Article Title: ETV1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes.

doi: 10.1038/s41598-018-28239-7

Figure Lengend Snippet: Figure 2. Cardiomyocyte-specific knockout of ETV1 slows atrial and His-Purkinje system conduction. Etv1flox/

Article Snippet: P1 NRVM heart lysates were purified using Miltenyi Biotec rat neonatal cardiomyocyte isolation kit (Miltenyi Biotec, 130–105–420) according to the manufacture’s protocol.

Techniques: Knock-Out

Journal: Scientific reports

Article Title: ETV1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes.

doi: 10.1038/s41598-018-28239-7

Figure Lengend Snippet: Figure 3. Cardiomyocyte deletion of ETV1 resulted in decreased expression of fast conduction genes in atrial and His-Purkinje system (HPS) myocytes. (A) Quantitative RT-PCR of fast conduction gene RNA levels (normalized to Gapdh) comparing 10–12-week-old Etv1 WT (Etv1flox/flox) and Etv1 cKO (Etv1flox/flox, Myh6- Cre) FACS-purified ventricular, atrial, and Purkinje myocytes. Relative Nkx2–5, Gja5, and Scn5a expression displayed versus control, Etv1 WT (n = 4). (B) Immunoblot assessment of Etv1 WT and Etv1 cKO atrial tissue lysates detecting NKX2–5, Cx40, NaV1.5, and Vinculin (loading control). (C) Protein level densitometric quantification (normalized to vinculin), displayed relative to Etv1 WT (n = 5). (D) Immunofluorescence evaluation of NKX2–5, Cx40, and NaV1.5 expression in 10-week-old Etv1 WT and Etv1 cKO atria/ventricular sections. (E) Immunofluorescence evaluation of NKX2–5, Cx40, and NaV1.5 expression in 10-week-old Etv1 WT and Etv1 cKO HPS sections. Positive CNTN2 expression identified HPS cells. Nuclei were identified by DAPI (blue). LA, left atria; LV, left ventricle. Data represent mean ± SEM. *P < 0.05, 2-tailed Student’s t test. Scale bars: 50 um.

Article Snippet: P1 NRVM heart lysates were purified using Miltenyi Biotec rat neonatal cardiomyocyte isolation kit (Miltenyi Biotec, 130–105–420) according to the manufacture’s protocol.

Techniques: Expressing, Quantitative RT-PCR, Purification, Control, Western Blot, Immunofluorescence

Journal: Scientific reports

Article Title: ETV1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes.

doi: 10.1038/s41598-018-28239-7

Figure Lengend Snippet: Figure 5. ETV1 regulates the diversity of sodium channel biophysical properties between ventricular, atrial, and Purkinje myocytes. Whole-cell patch clamp data from dissociated cardiomyocytes (ventricular, right atrial, Purkinje myocytes) using 10–12 week-old Etv1 WT (Etv1flox/flox) and Etv1 cKO (Etv1flox/flox, Myh6-Cre) mice in a Cntn2-EGFP background (n = 4). (A) Comparison of sodium current–voltage (I–V) relationship. Maximum conductance was calculated to assess significant differences among experimental groups. (B) Voltage dependence of steady-state activation. Voltage at half activation (V0.5, activation) was calculated to assess significant differences among experimental groups. (C) Voltage dependence of steady-state inactivation. Voltage at half inactivation (V0.5, inactivation) was calculated to assess significant differences among experimental groups. (D) Time course of recovery from inactivation. Tau of recovery (τrecovery) was calculated to assess significant differences among experimental groups. Number of cells analyzed per cell type (ventricle, right atria, Purkinje) included in each graph legend. Patch clamp protocol diagrams are included for each endpoint. Data represent mean ± SEM. *P < 0.05, 1-way ANOVA.

Article Snippet: P1 NRVM heart lysates were purified using Miltenyi Biotec rat neonatal cardiomyocyte isolation kit (Miltenyi Biotec, 130–105–420) according to the manufacture’s protocol.

Techniques: Patch Clamp, Comparison, Activation Assay

Journal: Scientific reports

Article Title: ETV1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes.

doi: 10.1038/s41598-018-28239-7

Figure Lengend Snippet: Figure 6. ETV1-transduced neonatal rat ventricular myocytes (NRVMs) upregulates a His-Purkinje system gene signature. (A) Volcano plot of relative transcript expression from NRVMs transduced with either Ad-Etv1- EGFP or Ad-EGFP. RNA-sequencing (RNA-seq) comparison revealed a total of 9,236 differentially expressed genes (normalized counts ≥ 5, padj < 0.05). All significantly different genes (padj < 0.05) are labeled blue (downregulated) or red (enriched) and all nonsignificantly different transcripts labeled in gray. Of these there were 4,696 upregulated and 4,540 downregulated genes in Ad-Etv1-EGFP versus Ad-EGFP transduced NRVMs. (B) Functional clustering of upregulated genes in Ad-Etv1 transduced NRVMs highlighted significantly enriched ETV1-dependent cellular processes (top 20 non-redundant categories are shown). Pathways are color coded to represent genes clustered into functional classes for heat maps in C. (C) Comparative RNA-seq between 21-day-old (P21) wild-type mouse FACS-purified Purkinje cell (PC)/ventricular myocytes (VM) and Ad-Etv1-EGFP/Ad-EGFP transduced NRVMs. Heat map representation of 88 genes differentially expressed in Ad-Etv1-EGFP versus Ad-EGFP transduced NRVMs (n = 3) plotted adjacent to average fold change expression in PCs and VMs. Genes clustered into functional groups demonstrate that ETV1 regulates a PC transcriptome in neonatal cardiomyocytes.

Article Snippet: P1 NRVM heart lysates were purified using Miltenyi Biotec rat neonatal cardiomyocyte isolation kit (Miltenyi Biotec, 130–105–420) according to the manufacture’s protocol.

Techniques: Expressing, Transduction, RNA Sequencing, Comparison, Labeling, Functional Assay, Purification

Journal: Scientific reports

Article Title: ETV1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes.

doi: 10.1038/s41598-018-28239-7

Figure Lengend Snippet: Figure 8. Activation of ETV1 in human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) leads to increased expression of rapid conduction genes and sodium current. (A) Schematic representation of hiPSC-CM generation and maturation (day 0–21), transduction of Ad-Etv1-EGFP or Ad-EGFP (day 24), and timepoint for experimentation (day 38–40). (B) Quantitative RT-PCR analysis of Etv1, NKX2–5, GJA5, SCN5A, and MYL2 in hiPSC-CM transduced with either Ad-Etv1-EGFP or Ad-EGFP (n = 4). (C) Whole-cell patch clamp was performed on Ad-Etv1-EGFP (n = 12) or Ad-EGFP (n = 9) transduced hiPSC-CMs. Sodium current–voltage (I–V) relationship comparison. (D) hiPSC-CM NaV peak conductance (gNaV-peak). gNaV-peak following −120 mV to −35 mV depolarization step was measured for Ad-Etv1-EGFP (n = 12) or Ad-EGFP (n = 9) transduced hiPSC-CMs. Data represent mean ± SEM. *P < 0.05, 2-tailed Student’s t test.

Article Snippet: P1 NRVM heart lysates were purified using Miltenyi Biotec rat neonatal cardiomyocyte isolation kit (Miltenyi Biotec, 130–105–420) according to the manufacture’s protocol.

Techniques: Activation Assay, Derivative Assay, Expressing, Transduction, Quantitative RT-PCR, Patch Clamp, Comparison

Journal: Toxicology and applied pharmacology

Article Title: Monoubiquitinated γ-H2AX: abundant product and specific biomarker for non-apoptotic DNA double-strand breaks

doi: 10.1016/j.taap.2018.07.007

Figure Lengend Snippet: Panels A-C used antibodies #1. (A) Impact of gel polyacrylamide percentage and transfer conditions to PVDF membranes. Treatments of H460 cells: 6 h with 3 mM hydroxyurea (HU), 6 or 16 h with 5 μM aphidicolin (Aphi) or 1 h with 5 μM bleomycin (Bleo). All γ-H2AX images had 5 s film exposures. (B) Ethanol effect in semi-dry transfer. IMR90 cells were treated with 40 μM etoposide (Eto) for 1 h. (C) Comparison of wet (pH 8.2 and pH 8.8) and semi-dry transfers using 12% ethanol-containing buffers (10 s film exposures for all γ-H2AX images). IMR90 cells were treated with 40 μM etoposide for 1 h. (D) Effectiveness of different antibodies using semi-dry transfer with 12% ethanol. Normal human keratinocytes were treated for 1 h with 30 μM etoposide or 0.5 μM bleomycin. Antibodies #1–3: 1:1000 dilutions and 5 s exposures, antibody #4: 1:1000 dilution and 60 s exposure. (E) Comparison of antibodies #1 and #5 (both at 1:1000 dilutions) in H460 cells treated with bleomycin for 1 h. (F) γ-H2AX blot (Ab#1) in control and H2AX-depleted IMR90 cells treated with 100 μM etoposide for 1h.

Article Snippet: IMR90, WI38, normal human neonatal keratinocytes (PCS-200–010), H460, A549, U2OS, mouse embryonic fibroblasts (MEFs, SCRC-1040), telomerase-immortalized human bronchial epithelial HBEC3 (CRL-4051), Daudi, HCT116, Ramos, Raji and CA46 were obtained from ATCC.

Techniques: Comparison, Control

Journal: International Journal of Environmental Research and Public Health

Article Title: High Incidence of Partial Biotinidase Deficiency in the First 3 Years of a Regional Newborn Screening Program in Italy

doi: 10.3390/ijerph19138141

Figure Lengend Snippet: DBS biotinidase activity distribution in all the newborns screened. The dotted line represents the 85 U/dL cutoff.

Article Snippet: Screening test for BD was performed by measuring BTD activity in DBS samples by a semi-quantitative time-resolved immunofluorescence method on a fully automated integrated screening plate processor (GSP ® Neonatal Biotinidase kit, PerkinElmer, Wallac Oy, Turku, Finland).

Techniques: Activity Assay

Journal: International Journal of Environmental Research and Public Health

Article Title: High Incidence of Partial Biotinidase Deficiency in the First 3 Years of a Regional Newborn Screening Program in Italy

doi: 10.3390/ijerph19138141

Figure Lengend Snippet: DBS biotinidase activity in false positives (gray boxes) and true positives (white boxes) at the initial and repeat DBS screening test.

Article Snippet: Screening test for BD was performed by measuring BTD activity in DBS samples by a semi-quantitative time-resolved immunofluorescence method on a fully automated integrated screening plate processor (GSP ® Neonatal Biotinidase kit, PerkinElmer, Wallac Oy, Turku, Finland).

Techniques: Activity Assay

Journal: International Journal of Environmental Research and Public Health

Article Title: High Incidence of Partial Biotinidase Deficiency in the First 3 Years of a Regional Newborn Screening Program in Italy

doi: 10.3390/ijerph19138141

Figure Lengend Snippet: Biotinidase activity at the DBS screening test and variants in carriers and in affected infants.

Article Snippet: Screening test for BD was performed by measuring BTD activity in DBS samples by a semi-quantitative time-resolved immunofluorescence method on a fully automated integrated screening plate processor (GSP ® Neonatal Biotinidase kit, PerkinElmer, Wallac Oy, Turku, Finland).

Techniques: Activity Assay

Journal: International Journal of Environmental Research and Public Health

Article Title: High Incidence of Partial Biotinidase Deficiency in the First 3 Years of a Regional Newborn Screening Program in Italy

doi: 10.3390/ijerph19138141

Figure Lengend Snippet: DBS biotinidase activity in relation to the birth weight ( A ) and to the pregnancy term ( B ).

Article Snippet: Screening test for BD was performed by measuring BTD activity in DBS samples by a semi-quantitative time-resolved immunofluorescence method on a fully automated integrated screening plate processor (GSP ® Neonatal Biotinidase kit, PerkinElmer, Wallac Oy, Turku, Finland).

Techniques: Activity Assay

Journal: International Journal of Environmental Research and Public Health

Article Title: High Incidence of Partial Biotinidase Deficiency in the First 3 Years of a Regional Newborn Screening Program in Italy

doi: 10.3390/ijerph19138141

Figure Lengend Snippet: Mean DBS biotinidase activity in relation to the birth season.

Article Snippet: Screening test for BD was performed by measuring BTD activity in DBS samples by a semi-quantitative time-resolved immunofluorescence method on a fully automated integrated screening plate processor (GSP ® Neonatal Biotinidase kit, PerkinElmer, Wallac Oy, Turku, Finland).

Techniques: Activity Assay

Journal: International Journal of Environmental Research and Public Health

Article Title: High Incidence of Partial Biotinidase Deficiency in the First 3 Years of a Regional Newborn Screening Program in Italy

doi: 10.3390/ijerph19138141

Figure Lengend Snippet: Mean biotinidase activity in DBS vs. the age at specimen collection.

Article Snippet: Screening test for BD was performed by measuring BTD activity in DBS samples by a semi-quantitative time-resolved immunofluorescence method on a fully automated integrated screening plate processor (GSP ® Neonatal Biotinidase kit, PerkinElmer, Wallac Oy, Turku, Finland).

Techniques: Activity Assay

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Tylosema esculentum (Marama) Tuber and Bean Extracts Are Strong Antiviral Agents against Rotavirus Infection

doi: 10.1155/2011/284795

Figure Lengend Snippet: Dose-dependent cytopathic effect of rotavirus on bovine intestinal epithelial cells (CIEB), pig intestinal epithelial cells (CLAB), and human intestinal epithelial cells (H4). The graphs for cytopathic effect of rotavirus on the different cells are shown in terms of mean absorbance at each concentration ± standard deviation at 595 nm.

Article Snippet: NO 2 release was significant from human intestinal epithelial cells (H4) exposed to all extracts used {seed coat water and ethanolic extracts (MSCW and MSCE, resp.), cotyledon water and ethanolic extracts (MCW and MCE, resp.), and tuber water extract (MTW)}, while only MCE and MTW significantly increased NO in CLAB cells ( ).

Techniques: Concentration Assay, Standard Deviation

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Tylosema esculentum (Marama) Tuber and Bean Extracts Are Strong Antiviral Agents against Rotavirus Infection

doi: 10.1155/2011/284795

Figure Lengend Snippet: Survival of bovine intestinal epithelial cells (CIEB), pig intestinal epithelial cells (CLAB), and human intestinal epithelial cells (H4) from cytopathic effects of rotavirus in the presence of T. esculentum extracts. Shown above are bars of % survival of epithelial cells {% change in cell viability after coincubation of rotavirus with marama extracts compared to rotavirus only wells at OD 595 nm ± % standard deviation}, respective levels of significance in terms of T values (*). T values were determined using Statsoft Statistica software ( P < .05). Tests were done in triplicate wells. * T value between 0 and 5 (low level significance), ** T value greater than 5, but less than 10 (middle level significance), and *** T value greater than 10 (high level significance). MCE: T. esculentum bean cotyledon ethanolic extract, MSCE: T. esculentum bean husk ethanolic extract, MTW: T. esculentum tuber water extract, MSCW: T. esculentum bean husk water extract, MCW: T. esculentum bean cotyledon water extract, and Combivir: (lamivudine and zidovudine).

Article Snippet: NO 2 release was significant from human intestinal epithelial cells (H4) exposed to all extracts used {seed coat water and ethanolic extracts (MSCW and MSCE, resp.), cotyledon water and ethanolic extracts (MCW and MCE, resp.), and tuber water extract (MTW)}, while only MCE and MTW significantly increased NO in CLAB cells ( ).

Techniques: Standard Deviation, Software

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Tylosema esculentum (Marama) Tuber and Bean Extracts Are Strong Antiviral Agents against Rotavirus Infection

doi: 10.1155/2011/284795

Figure Lengend Snippet: T. esculentum extracts show direct inhibition of rotavirus infectivity on bovine intestinal epithelial cells (CIEB), pig intestinal epithelial cells (CLAB), and human intestinal epithelial cells (H4). Shown above are bars of % survival of epithelial cells {% change in cell viability after pre-exposure of rotavirus to T. esculentum extracts compared to rotavirus only wells at OD 595 nm ± % standard deviation}, respective levels of significance in terms of T values (*). T values were determined using Statsoft Statistica software ( P < .05). Tests were done in triplicate wells. * T value between 0 and 5 (low level significance), ** T value greater than 5, but less than 10 (middle level significance), and *** T value greater than 10 (high level significance). MCE: T. esculentum bean cotyledon ethanolic extract, MSCE: T. esculentum bean husk ethanolic extract, MTW: T. esculentum tuber water extract, MSCW: T. esculentum bean husk water extract, MCW: T. esculentum bean cotyledon water extract, and AZT: Combivir (lamivudine and zidovudine).

Article Snippet: NO 2 release was significant from human intestinal epithelial cells (H4) exposed to all extracts used {seed coat water and ethanolic extracts (MSCW and MSCE, resp.), cotyledon water and ethanolic extracts (MCW and MCE, resp.), and tuber water extract (MTW)}, while only MCE and MTW significantly increased NO in CLAB cells ( ).

Techniques: Inhibition, Infection, Standard Deviation, Software

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Tylosema esculentum (Marama) Tuber and Bean Extracts Are Strong Antiviral Agents against Rotavirus Infection

doi: 10.1155/2011/284795

Figure Lengend Snippet: Effect of coincubation of rotavirus with T. esculentum extracts on monolayer polarity (TER) (Ω/cm 2 ) of H4 cells over time. Shown graphs of change in epithelial cell monolayer polarity exposed to extracts or AZT and rotavirus (treatment wells), extracts or AZT alone (control), or rotavirus alone (negative control) over time compared to respective controls. Tests were done in triplicate wells (12-well Transwell plates). MCE: T. esculentum bean cotyledon ethanolic extract (1 mg/mL), MSCE: T. esculentum bean husk ethanolic extract (0.1 mg/mL), MTW: T. esculentum tuber water extract (0.1 mg/mL), MSCW: T. esculentum bean husk water extract (0.01 mg/mL), MCW: T. esculentum bean cotyledon water extract (2 mg/mL), and AZT: Combivir (lamivudine and zidovudine) (0.75 mg/mL).

Article Snippet: NO 2 release was significant from human intestinal epithelial cells (H4) exposed to all extracts used {seed coat water and ethanolic extracts (MSCW and MSCE, resp.), cotyledon water and ethanolic extracts (MCW and MCE, resp.), and tuber water extract (MTW)}, while only MCE and MTW significantly increased NO in CLAB cells ( ).

Techniques: Control, Negative Control

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Tylosema esculentum (Marama) Tuber and Bean Extracts Are Strong Antiviral Agents against Rotavirus Infection

doi: 10.1155/2011/284795

Figure Lengend Snippet: Effect of T. esculentum extracts on release of nitric oxide (NO) (% release compared to control) in human small intestine epithelial cells (H4) and pig small intestine epithelial cells (CLAB). MCE: T. esculentum bean cotyledon ethanolic extract (1 mg/mL), MSCE: T. esculentum bean husk ethanolic extract (0.1 mg/mL), MTW: T. esculentum tuber water extract (0.1 mg/mL), MSCW: T. esculentum bean husk water extract (0.01 mg/mL), and MCW: T. esculentum bean cotyledon water extract (2 mg/mL).

Article Snippet: NO 2 release was significant from human intestinal epithelial cells (H4) exposed to all extracts used {seed coat water and ethanolic extracts (MSCW and MSCE, resp.), cotyledon water and ethanolic extracts (MCW and MCE, resp.), and tuber water extract (MTW)}, while only MCE and MTW significantly increased NO in CLAB cells ( ).

Techniques: Control