Journal: Scientific Reports

Article Title: Molecular characterization of root-knot nematodes ( Meloidogyne spp.) from Arkansas, USA

doi: 10.1038/s41598-019-52118-4

Figure Lengend Snippet: Primers used for polymerase chain reaction and DNA sequencing.

Article Snippet: The 25-μl PCR was performed using 12.5-μl 2X Apex Taq red master mix DNA polymerase (Genesee Scientific Corporation, San Diego, CA, USA), 9.5-μl water, 1-μl each of 10-μM forward and reverse primers, and 1 μl of DNA template according to the manufacturer’s protocol in a Veriti® thermocycler (Life Technologies, Carlsbad, CA, USA).

Techniques: Polymerase Chain Reaction, Sequencing

Journal: Cell

Article Title: Therapeutic potential of co-signaling receptor modulation in hepatitis B

doi: 10.1016/j.cell.2024.05.038

Figure Lengend Snippet:

Article Snippet: PE anti human CD4 , Miltenyi Biotec , Cat# 130-113-225; RRID: AB_2726036.

Techniques: Purification, Virus, Recombinant, Staining, Saline, Fluorsave, Sequencing, DNA Labeling, Cell Isolation, Sample Prep, Reverse Transcription, Software, Microscopy

Journal: Cell Genomics

Article Title: Proteomics of immune cells from liver tumors reveals immunotherapy targets

doi: 10.1016/j.xgen.2023.100331

Figure Lengend Snippet:

Article Snippet: Human CD45 PerCP-Vio700 (clone REA747) , Miltenyi Biotec , Cat#130-110-774; RRID: AB_2658252.

Techniques: Recombinant, Sequencing, Modification, Transfection, DNA Extraction, Enzyme-linked Immunosorbent Assay, Cell Isolation, Gene Expression, Mouse Assay, Software

Journal: Frontiers in Immunology

Article Title: Multivalent MVA-vectored vaccine elicits EBV neutralizing antibodies in rhesus macaques that reduce EBV infection in humanized mice

doi: 10.3389/fimmu.2024.1445209

Figure Lengend Snippet: Generation and characterization of MVA-EBV5-2. (A) Schematic representation of MVA-EBV5-2 virus, with multivalent glycoprotein expression cassettes incorporated in the 69R/70L (G1L, gp350-gB) and 64L/65L (IGR3, gp42-gL-gH) MVA genomic sites. (B) Diagram depicting the reconstitution of recombinant MVA in BHK-21 cells, followed by viral serial passaging to evaluate transgene stability. (C) PCR amplification of the two glycoprotein expression cassettes in MVA-EBV5-2, illustrated in (A) , using DNA from uninfected BHK-21 cells (BHK), BHK-21 cells infected with empty MVA (BHK-MVA), or BHK-21 cells infected with serially passaged MVA-EBV5-2 virus (p0, p4, p7, and p10). NC denotes negative control, no DNA. Expected band sizes are listed above each gel and are indicated with arrows. (D) Binding of anti-gp350 antibody HB5, anti-gp42 antibody 15C8, and anti-B5R hybridoma supernatant to total protein from uninfected BHK-21 cells, BHK-21 cells infected with empty MVA, or BHK-21 cells infected with serially passaged MVA-EBV5-2 virus (p0–p10) was assessed using immunoblot. Bands corresponding to gp350, gp42, and B5R are indicated with arrows. B5R is a vaccinia antigen, and is used as a loading control. (E) Surface binding of the anti-gp350 monoclonal antibody (mAb) HB5, anti-gB mAb AMMO5, anti-gp42 mAb F-2-1, anti-gL mAb E1D1, and anti-gH mAb CL40 to BHK-21 cells infected with serially passaged MVA-EBV5-2 virus (p0, p4, p7, and p10) was measured using flow cytometry. Each bar represents the mean percent (%) + SD of infected cells from triplicate infections with positive signal for binding. See also Supplementary Figures S2–S4 .

Article Snippet: Additional recombinant gp42gHgL (gp42: a.a. 33–223, GenBank: AFY97939.1; gH: a.a. 19–679, GenBank: AFY97969.1; gL: a.a. 24–137, Genbank: AFY97944.1) proteins were produced and purified by GenScript, via co-transfection of EBV-gp42-His-Avi-PTT3, EBV-gH-His-Avi-PTT3, and EBV-gL-His-Avi-PTT3 plasmids, respectively, in ExpiCHO-S cells using proprietary methods.

Techniques: Virus, Expressing, Recombinant, Passaging, Amplification, Infection, Negative Control, Binding Assay, Western Blot, Control, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Multivalent MVA-vectored vaccine elicits EBV neutralizing antibodies in rhesus macaques that reduce EBV infection in humanized mice

doi: 10.3389/fimmu.2024.1445209

Figure Lengend Snippet: MVA-EBV5-2 is immunogenic in female BALB/c mice and elicits a neutralizing serum response against EBV infection in epithelial and Raji cells in vitro . (A) Female BALB/c mice were immunized with the indicated treatments (inset table, right) on Day 0, 28, and 56 (n=8/group). Blood was collected at the indicated timepoints (red droplets), and mice were terminally bled on Day 182. (B) IgG binding levels to gp350, gB, and gp42gHgL were measured using ELISA in pooled mouse serum (1/900 dilution). Each dot represents the mean of triplicate measurements, and black arrows represent immunization timepoints. (C, D) IgG1 (C) and IgG2a (D) binding levels to EBV gp350, gB, and gp42gHgL were measured using ELISA in individual mouse serum samples on Day 49 and 84 post-immunization (1/900 dilution; UV-EBV not shown). Each dot represents the mean of duplicate measurements for each animal, with the median and interquartile range shown for each group at each timepoint. Statistical differences were determined using Tukey’s multiple comparison test (** = p<0.01, *** = p<0.001, **** = p<0.0001). (E) The ability of Day 49 and 84 pooled sera to neutralize EBV infection in HEK-293 (left) and Raji (right) cells was measured via in vitro neutralization assays. Serum titration curves for each treatment group (% neutralization) are shown for each cell line, with the top dotted line representing 80% neutralization (IC80) and the bottom dotted line representing 50% neutralization (IC50) cut-offs.

Article Snippet: Additional recombinant gp42gHgL (gp42: a.a. 33–223, GenBank: AFY97939.1; gH: a.a. 19–679, GenBank: AFY97969.1; gL: a.a. 24–137, Genbank: AFY97944.1) proteins were produced and purified by GenScript, via co-transfection of EBV-gp42-His-Avi-PTT3, EBV-gH-His-Avi-PTT3, and EBV-gL-His-Avi-PTT3 plasmids, respectively, in ExpiCHO-S cells using proprietary methods.

Techniques: Infection, In Vitro, Binding Assay, Enzyme-linked Immunosorbent Assay, Comparison, Neutralization, Titration

Journal: Frontiers in Immunology

Article Title: Multivalent MVA-vectored vaccine elicits EBV neutralizing antibodies in rhesus macaques that reduce EBV infection in humanized mice

doi: 10.3389/fimmu.2024.1445209

Figure Lengend Snippet: MVA-EBV5-2 is immunogenic in male BALB/c mice and elicits a neutralizing serum response against EBV infection in epithelial and Raji cells in vitro . (A) Male BALB/c mice were immunized with the indicated treatments (inset table, right) on Day 0, 28, and 56 (n=8/group). Blood was collected at the indicated timepoints (red droplets), and mice were terminally bled on Day 119. (B) IgG binding levels to EBV gp350, gB, and gp42gHgL were measured using ELISA in pooled mouse serum (1/900 dilution). Each dot represents the mean of triplicate measurements, and black arrows represent immunization timepoints. (C, D) IgG1 (C) and IgG2a (D) binding levels to EBV gp350, gB, and gp42gHgL were measured using ELISA in individual mouse serum samples on Day 49 and 84 post-immunization (1/900 dilution; UV-EBV not shown). Each dot represents the mean of duplicate measurements for each animal, with the median and interquartile range shown for each group at each timepoint. Statistical differences were determined using Tukey’s multiple comparison test (** = p<0.01, *** = p<0.001, **** = p<0.0001). (E) The ability of Day 49 and 84 pooled sera to neutralize EBV infection in HEK-293 (left) and Raji (right) cells was measured via in vitro neutralization assays. Serum titration curves for each treatment group (% neutralization) are shown for each cell line, with the top dotted line representing 80% neutralization (IC80) and the bottom dotted line representing 50% neutralization (IC50) cut-offs.

Article Snippet: Additional recombinant gp42gHgL (gp42: a.a. 33–223, GenBank: AFY97939.1; gH: a.a. 19–679, GenBank: AFY97969.1; gL: a.a. 24–137, Genbank: AFY97944.1) proteins were produced and purified by GenScript, via co-transfection of EBV-gp42-His-Avi-PTT3, EBV-gH-His-Avi-PTT3, and EBV-gL-His-Avi-PTT3 plasmids, respectively, in ExpiCHO-S cells using proprietary methods.

Techniques: Infection, In Vitro, Binding Assay, Enzyme-linked Immunosorbent Assay, Comparison, Neutralization, Titration

Journal: Frontiers in Immunology

Article Title: Multivalent MVA-vectored vaccine elicits EBV neutralizing antibodies in rhesus macaques that reduce EBV infection in humanized mice

doi: 10.3389/fimmu.2024.1445209

Figure Lengend Snippet: MVA-EBV5-2 is immunogenic in rhLCV-negative rhesus macaques. (A) rhLCV-negative female and male rhesus macaques were immunized with the indicated treatments (inset table, right) on Day 0 and Day 28 (n=5/group). Blood (red droplets) and saliva (blue droplets) were collected at the indicated timepoints. (B, C) IgG binding levels to EBV gp350, gB, and gp42gHgL were measured using ELISA in individual rhesus macaque serum samples (1/100 dilution). In (B) , the median and interquartile range are shown for each group at each timepoint, each animal was tested in duplicate. Black arrows represent immunization timepoints. In (C) , the same data as in B is presented but shown for individual animals, each dot representing the mean of duplicate measurements for each animal, with the median and interquartile range shown for each group at each timepoint. Statistical differences were determined using Tukey’s multiple comparison test (* = p<0.05, ** = p<0.01). (D, E) IgG binding levels to EBV gp350, gB, and gp42gHgL were measured using ELISA in individual rhesus macaque saliva samples (undiluted). In (D) , the median and interquartile range are shown for each group at each timepoint, each animal was tested in duplicate. In (E) , the same data as in (B) is presented but shown for individual animals, each dot representing the mean of duplicate measurements for each animal, with the median and interquartile range shown for each group at each timepoint. Statistical differences were determined using Tukey’s multiple comparison test (* = p<0.05, ** = p<0.01). See also Supplementary Figure S5 .

Article Snippet: Additional recombinant gp42gHgL (gp42: a.a. 33–223, GenBank: AFY97939.1; gH: a.a. 19–679, GenBank: AFY97969.1; gL: a.a. 24–137, Genbank: AFY97944.1) proteins were produced and purified by GenScript, via co-transfection of EBV-gp42-His-Avi-PTT3, EBV-gH-His-Avi-PTT3, and EBV-gL-His-Avi-PTT3 plasmids, respectively, in ExpiCHO-S cells using proprietary methods.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: miR-137 is a tumor suppressor in endometrial cancer and is repressed by DNA hypermethylation

doi: 10.1038/s41374-018-0092-x

Figure Lengend Snippet: DNA methylation of miR-137 in primary endometrial tissues. ( a ) Outline for identifying hypermethylated miRNA in endometrial cancer. ( b ) Methylation profiles of 10 normal endometrial tissues (N) and 67 primary tumors (Ca) created after methyl-capture sequencing analysis. Red and white dashed-line squares represent methylated and unmethylated regions, respectively. Green bar: the nearby CpG island.

Article Snippet: HEC1A and Ishikawa H cells (2×10 5 ) were seeded into 6-well plates with transfected plasmids of human miR-137 or empty vector (2 μg/well; Origene, Rockville MD), using the TurboFectin Transfection Reagent (Origene) according to the manufacturer’s instructions.

Techniques: DNA Methylation Assay, Methylation, Sequencing

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: miR-137 is a tumor suppressor in endometrial cancer and is repressed by DNA hypermethylation

doi: 10.1038/s41374-018-0092-x

Figure Lengend Snippet: miR-137 is hypermethylated and shows loss of expression in human primary endometrial tumors. ( a ) Genomic map of the CpG island nearby miR-137 (black bar), COBRA and bisulfite pyrosequencing (Pyro) amplicons and TCGA methylation probes. CpG sites are lines under bars. ( b ) Dot plot showing miR-137 methylation levels in 10 pairs of endometrioid endometrial cancer (EEC) and normal adjacent tissues (NAT) quantified by bisulfite pyrosequencing. ( c ) DNA methylation was determined by bisulfite pyrosequencing in human endometrial tissues. NAT: normal adjacent tissue; Serous: serous endometrial tumor. Each dot represents one specimen. Each horizontal line indicates the mean of methylation within each group. DNA methylation of miR-137 by probes in paired samples ( d ) and subtypes of endometrial tumors ( e ) in TCGA cohort. Pair (n=33); N: normal endometrium (n=13); EEC (n=312); serous (n=98). ( f ) Expression of miR-137 in TCGA endometrial cohort. Gray portions: miR-137 not expressed; white portions: miR-137 expressed. Number in each bar indicates sample size. *: P <0.05; **: P <0.01; ***: P <0.001.

Article Snippet: HEC1A and Ishikawa H cells (2×10 5 ) were seeded into 6-well plates with transfected plasmids of human miR-137 or empty vector (2 μg/well; Origene, Rockville MD), using the TurboFectin Transfection Reagent (Origene) according to the manufacturer’s instructions.

Techniques: Expressing, Combined Bisulfite Restriction Analysis Assay, Methylation, DNA Methylation Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: miR-137 is a tumor suppressor in endometrial cancer and is repressed by DNA hypermethylation

doi: 10.1038/s41374-018-0092-x

Figure Lengend Snippet: miR-137 is hypermethylated and reactivated by epigenetic inhibitors in endometrial cancer cells. ( a ) DNA methylation levels determined by bisulfite pyrosequencing in a normal endometrial cell line (EM-E6/E7/TERT) and 11 endometrial cancer cell lines. ( b–c ) Reactivation of pre-/pri- and mature miR-137 after epigenetic inhibitors in endometrial cancer cells after endometrial cancer cells were treated with epigenetic inhibitors. Cancer cells (HEC1A, Ishikawa H, and Hec50co) were treated with 5-aza-2′-deoxycytidine (DAC) and/or trichostatin A (TSA). Gene expression was determined by RT-qPCR and compared to untreated controls (CTR). U6 served as an internal control. Bars: means±SD; *: P <0.05.

Article Snippet: HEC1A and Ishikawa H cells (2×10 5 ) were seeded into 6-well plates with transfected plasmids of human miR-137 or empty vector (2 μg/well; Origene, Rockville MD), using the TurboFectin Transfection Reagent (Origene) according to the manufacturer’s instructions.

Techniques: DNA Methylation Assay, Expressing, Quantitative RT-PCR

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: miR-137 is a tumor suppressor in endometrial cancer and is repressed by DNA hypermethylation

doi: 10.1038/s41374-018-0092-x

Figure Lengend Snippet: Functional analysis of miR-137 in endometrial cancer cells. ( a ) Relative expression of miR-137 in stably transfected clones of HEC1A and Ishikawa H cancer cells by RT-qPCR. pCMV-MIR: mock transfection with vector; miR-137: miR-137 transfection. U6 served as an internal control. Bars: means±SD. ( b ) Cellular proliferation in transfected HEC1A and Ishikawa H cells at different time points, as determined by MTS assays. ( c–d ) Colony number and size in miR-137 -transfected HEC1A and Ishikawa H endometrial cancer cells. ( e–f ) Gene expression by RT-qPCR and Western blotting in transfected cells. Bars: means±SD. *: P <0.05; **: P <0.01; ***: P <0.001.

Article Snippet: HEC1A and Ishikawa H cells (2×10 5 ) were seeded into 6-well plates with transfected plasmids of human miR-137 or empty vector (2 μg/well; Origene, Rockville MD), using the TurboFectin Transfection Reagent (Origene) according to the manufacturer’s instructions.

Techniques: Functional Assay, Expressing, Stable Transfection, Transfection, Clone Assay, Quantitative RT-PCR, Plasmid Preparation, Western Blot

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: miR-137 is a tumor suppressor in endometrial cancer and is repressed by DNA hypermethylation

doi: 10.1038/s41374-018-0092-x

Figure Lengend Snippet: miR-137 impairs xenograft tumor formation. ( a ) Xenograft tumor growth curves in miR-137 stably transfected clones of HEC1A cells. pCMV-MIR: mock transfection with vector; miR-137: miR-137 transfection. ( b ) Tumor weights of miR-137 - or mock-transfected HEC1A cells. ( c ) Protein expression of EZH2 and LSD1 in transfected cells by Western blotting. β-actin serves as a loading control. ( d–e ) Representative sections and summary of H&E and IHC staining of xenograft tumors. Bars: means±SE. *: P <0.05; **: P <0.01.

Article Snippet: HEC1A and Ishikawa H cells (2×10 5 ) were seeded into 6-well plates with transfected plasmids of human miR-137 or empty vector (2 μg/well; Origene, Rockville MD), using the TurboFectin Transfection Reagent (Origene) according to the manufacturer’s instructions.

Techniques: Stable Transfection, Transfection, Clone Assay, Plasmid Preparation, Expressing, Western Blot, Immunohistochemistry

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: miR-137 is a tumor suppressor in endometrial cancer and is repressed by DNA hypermethylation

doi: 10.1038/s41374-018-0092-x

Figure Lengend Snippet: miR-137 targets EZH2 amd LSD1. ( a ) 3′-UTR reporter assays were conducted in cells co-transfected with reporter plasmids, negative contol miRNA, miR-137, or anti-miR-137. Fold changes were normalized to plamsid and negative control miRNA transfections. Gene expression of EZH2 ( b ) and LSD1 ( c ) in miR-137 -transfected HEC1A cancer cells by RT-qPCR. The stably transfected cells were subsequently transfected with EZH2 and control vector pCMV-HA (HA), or with LSD1 and its empty vector pCMV6 (PS). GAPDH served as an internal control. ( d ) Cellular proliferation in transfected cells at different time points was determined by MTS assays. *: P <0.05; ***: P <0.001.

Article Snippet: HEC1A and Ishikawa H cells (2×10 5 ) were seeded into 6-well plates with transfected plasmids of human miR-137 or empty vector (2 μg/well; Origene, Rockville MD), using the TurboFectin Transfection Reagent (Origene) according to the manufacturer’s instructions.

Techniques: Transfection, Negative Control, Expressing, Quantitative RT-PCR, Stable Transfection, Plasmid Preparation