Journal: Journal of Virology

Article Title: Monoclonal Antibodies Directed toward the Hepatitis C Virus Glycoprotein E2 Detect Antigenic Differences Modulated by the N-Terminal Hypervariable Region 1 (HVR1), HVR2, and Intergenotypic Variable Region

doi: 10.1128/JVI.02070-15

Figure Lengend Snippet: (A) Schematic representation of full-length E2, E2 661 , and E2 661 variants with deletions of HVR1 (Δ1), HVR2 (Δ2), the igVR (Δ3), or combinations thereof (Δ12, Δ13, Δ23, and Δ123). HVR2 and the igVR were replaced with a GSSG linker. Numbering is done according to the H77c prototype strain. Epitope I, II, and III regions are underlined on the E2 structure and overlap CD81 binding sites, shown in blue, orange, and green. A fourth region (yellow) is also implicated in CD81 interactions. Hypervariable region 1, HVR2, and the igVR are shown in red. The transmembrane domain and the C-terminal stem region are shown in black and gray, respectively, on the full-length E2 schematic. (B) Cartoon drawing of the E2 core domain with its surface overlaid (PDB accession number 4MWF ) . Coloring is according to that described above for panel A. The predicted location of the region spanning residues 411 to 420 (purple) that overlaps epitope I and precedes HVR1 is shown.

Article Snippet: The codon-optimized DNA sequence encoding H77c E2 661 was synthesized (GeneArt, Invitrogen, CA, USA) and contained an N-terminal human trypsin leader sequence and a C-terminal 6×His tag.

Techniques: Binding Assay

Journal: Journal of Virology

Article Title: Monoclonal Antibodies Directed toward the Hepatitis C Virus Glycoprotein E2 Detect Antigenic Differences Modulated by the N-Terminal Hypervariable Region 1 (HVR1), HVR2, and Intergenotypic Variable Region

doi: 10.1128/JVI.02070-15

Figure Lengend Snippet: Characterization of MAbs. (A) Immunoprecipitation of 35 S-labeled H77c E1E2 from lysates of transfected HEK293T cells using each MAb. Immunoprecipitates were run on reducing SDS-PAGE gels and phosphorimaged. Locations of E2 and multiple glycoforms of E1 are shown on the right. Positions of molecular weight markers (M) (in thousands) are shown on the left. Quantitation was performed by using imageQuant software, and results are the means ± standard deviations of data from three experiments (bottom). The antigens to which the MAbs were raised are indicated above the gel. (B) Analysis of the ability of MAbs to recognize denatured E2 661 . Nickel affinity-purified E2 661 was subjected to reducing SDS-PAGE and transferred onto nitrocellulose. Strips were probed with each MAb, followed by detection with anti-mouse Alexa 680-labeled antibody and infrared analysis (Li-COR Odyssey). The antigens to which the MAbs were raised are indicated above the gel. (C) Overlapping-peptide scan of antibodies reactive to denatured E2 661 . Synthetic 18-mers, overlapping the H77c E2 sequence by 11 amino acids, were used in a direct binding ELISA. Binding to a peptide is shown in gray and represents at least 10 times the background level. (D) Ability of MAbs raised to WT E2 661 to recognize an extended HVR1 peptide of strain H. Antibodies were applied to plates coated with peptide and titrated 0.5 log 10 .

Article Snippet: The codon-optimized DNA sequence encoding H77c E2 661 was synthesized (GeneArt, Invitrogen, CA, USA) and contained an N-terminal human trypsin leader sequence and a C-terminal 6×His tag.

Techniques: Immunoprecipitation, Labeling, Transfection, SDS Page, Molecular Weight, Quantitation Assay, Software, Affinity Purification, Sequencing, Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Virology

Article Title: Monoclonal Antibodies Directed toward the Hepatitis C Virus Glycoprotein E2 Detect Antigenic Differences Modulated by the N-Terminal Hypervariable Region 1 (HVR1), HVR2, and Intergenotypic Variable Region

doi: 10.1128/JVI.02070-15

Figure Lengend Snippet: Direct binding of MAbs to E2 661 with single and multiple variable region deletions. Data shown are the means ± standard deviations of data from at least two independent experiments. Percent binding was calculated relative to the binding observed for WT E2 661 . Nonlinear regression analysis was performed with Prism v 6.0f. Data from two independent analyses of MAb24 reactivity are shown for reproducibility. The amounts of E2 661 containing single and multiple deletions of the variable regions added to plates were equivalent, as indicated by GNA-lectin capture of proteins followed by detection with an antibody to the 6×His epitope tag (anti-His) from two independent analyses performed three times (means ± standard deviations).

Article Snippet: The codon-optimized DNA sequence encoding H77c E2 661 was synthesized (GeneArt, Invitrogen, CA, USA) and contained an N-terminal human trypsin leader sequence and a C-terminal 6×His tag.

Techniques: Binding Assay

Journal: Journal of Virology

Article Title: Monoclonal Antibodies Directed toward the Hepatitis C Virus Glycoprotein E2 Detect Antigenic Differences Modulated by the N-Terminal Hypervariable Region 1 (HVR1), HVR2, and Intergenotypic Variable Region

doi: 10.1128/JVI.02070-15

Figure Lengend Snippet: Ability of MAbs to inhibit binding between E2 661 or Δ123 and recombinant MBP-LEL 113–201 . (A) Serial dilutions of antibody were mixed with 50 ng E2 E2 661 or Δ123 and applied to plates coated with purified dimeric MBP-LEL 113–201 . Bound E2 was detected with rabbit anti-His and horseradish peroxidase-labeled goat anti-rabbit IgG. Results shown are the means ± standard deviations of data from at least 2 independent experiments. Data were normalized to the percentage of E2 binding to CD81 in the absence of MAb. Curves were fitted with one-site-specific binding with the Hill slope equation in Prism v 6.0f. (B) Binding of E2 661 proteins containing one or more variable region deletions to solid-phase MBP-LEL 113–201 . The inset graph shows the capture of E2 proteins with GNA-lectin and detection with anti-His antibody and confirms that similar amounts of E2 protein were present in every well. (C) Biosensor analysis of binding of E2 661 and variants containing one or more variable region deletions to dimeric MBP-LEL 113–201 . Four concentrations of each E2 661 protein were flowed over biosensor chips coated with MBP-LEL 113–201 , and the curves generated with 100 μg/ml E2 protein are shown.

Article Snippet: The codon-optimized DNA sequence encoding H77c E2 661 was synthesized (GeneArt, Invitrogen, CA, USA) and contained an N-terminal human trypsin leader sequence and a C-terminal 6×His tag.

Techniques: Binding Assay, Recombinant, Purification, Labeling, Generated

Journal: Journal of Virology

Article Title: Monoclonal Antibodies Directed toward the Hepatitis C Virus Glycoprotein E2 Detect Antigenic Differences Modulated by the N-Terminal Hypervariable Region 1 (HVR1), HVR2, and Intergenotypic Variable Region

doi: 10.1128/JVI.02070-15

Figure Lengend Snippet: Summary of characteristics of the 18 MAbs raised against E2 661 and Δ123

Article Snippet: The codon-optimized DNA sequence encoding H77c E2 661 was synthesized (GeneArt, Invitrogen, CA, USA) and contained an N-terminal human trypsin leader sequence and a C-terminal 6×His tag.

Techniques: Enzyme-linked Immunosorbent Assay, Inhibition, Binding Assay, Neutralization

Journal: Journal of Virology

Article Title: Monoclonal Antibodies Directed toward the Hepatitis C Virus Glycoprotein E2 Detect Antigenic Differences Modulated by the N-Terminal Hypervariable Region 1 (HVR1), HVR2, and Intergenotypic Variable Region

doi: 10.1128/JVI.02070-15

Figure Lengend Snippet: Comparative analysis of the abilities of MAbs to inhibit E2 binding to CD81 and dissociation of E2 from CD81

Article Snippet: The codon-optimized DNA sequence encoding H77c E2 661 was synthesized (GeneArt, Invitrogen, CA, USA) and contained an N-terminal human trypsin leader sequence and a C-terminal 6×His tag.

Techniques: Binding Assay

Journal: Journal of Virology

Article Title: Monoclonal Antibodies Directed toward the Hepatitis C Virus Glycoprotein E2 Detect Antigenic Differences Modulated by the N-Terminal Hypervariable Region 1 (HVR1), HVR2, and Intergenotypic Variable Region

doi: 10.1128/JVI.02070-15

Figure Lengend Snippet: Ability of MAbs to bind their epitopes on the surface of VLPs. (A) VLPs containing genotype 1a H77c E1E2 glycoproteins were pelleted through a sucrose cushion, subjected to reducing SDS-PAGE, and transferred onto nitrocellulose. Membranes were probed with a mixture of H52 (anti-E2) and A4 (anti-E1) or with IgG obtained from an HIV-positive individual. (B) Binding of anticapsid antibody to VLPs is enhanced by permeabilization with Triton X-100. Capsid protein (anti-CA) was detected with MAb183. No binding was observed by using an irrelevant MAb to a Myc epitope tag (anti-myc) or in the absence of primary antibody (No primary). (C) Ability of MAbs to bind VLPs in a direct binding ELISA. Data shown are the means ± standard deviations of data from two independent experiments. OD, optical density.

Article Snippet: The codon-optimized DNA sequence encoding H77c E2 661 was synthesized (GeneArt, Invitrogen, CA, USA) and contained an N-terminal human trypsin leader sequence and a C-terminal 6×His tag.

Techniques: SDS Page, Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: Genes & development

Article Title: CDK4 loss-of-function mutations cause microcephaly and short stature.

doi: 10.1101/gad.352311.124

Figure Lengend Snippet: Figure 1. Individuals with biallelic CDK4 variants display microcephaly and short stature. (A) Family pedigrees with segregation of CDK4 variants. (Square) Male, (circle) female, (filled symbols) individuals with microcephaly, (strikethrough) deceased. WT Reference (+), variants v1 and v2, and zygosity are indicated for each studied individual. (B) Diagram of CDK4 transcript (top) and protein (bottom); coding exons are depicted as black rectangles. Red lines indicate variant location. (SS) Splice site disrupted. (C) Altered splicing predictions for the c.218G > A substitution generated using Alamut. (Blue rectangles) Strength of splice donor predictions for individual splice algo- rithms, (blue triangle) predicted donor splice site. (D) Growth parameters at birth and at last assessment (postnatal). (W) Weight, (OFC) orbito–frontal circumference. Z-scores show standard deviations from population mean for age and sex. Dashed lines indicate a 95% con- fidence interval for the general population. Individual subject data points from families A (circles) and B (squares) are graphed, and mean values are plotted. (E) MRI scan of age-matched control (4 years 8 months) and affected individuals with a CDK4 variant. Coronal FLAIR projection shows simplified parietal and temporal gyri, reduced white matter volume, and the absence of brain malformations. Scale bars, 10 cm. (See also Supplemental Figure S1C for additional MRI projections.) (F) Photographs of all affected individuals.

Article Snippet: Patient fibroblasts were transduced with lentiviral particles containing pLIX_403-CDK4 and/or pLIX_403CDK6, a construct where full-length codon-optimized CDK4 or CDK6 was gateway-cloned into pLIX_403 (Addgene 41395 and 158560).

Techniques: Variant Assay, Generated, Control

Journal: Genes & development

Article Title: CDK4 loss-of-function mutations cause microcephaly and short stature.

doi: 10.1101/gad.352311.124

Figure Lengend Snippet: Figure 3. Full-length CDK4 protein is undetectable in patient fibroblasts. (A,B) Immunoblots of total cell extracts obtained from expo- nentially growing control (C1 and C2) and patient (P1 and P2) fibroblasts without (A) and with (B) CDK4 complementation. α-Tubulin was used as the loading control. A rabbit monoclonal antibody to C-terminal CDK4 was used; a different mouse CDK4 antibody raised against full-length CDK4 was used in Figure 5A. A smaller ∼12 kDa molecular weight band was variably detected in P1 with this antibody (Sup- plemental Fig. S2D) that might correspond to the 46 amino acid truncated nonfunctional protein predicted from RNA studies. (C) CDK6 and Cyclin D1 levels were unchanged in patient fibroblasts compared with wild-type controls.

Article Snippet: Patient fibroblasts were transduced with lentiviral particles containing pLIX_403-CDK4 and/or pLIX_403CDK6, a construct where full-length codon-optimized CDK4 or CDK6 was gateway-cloned into pLIX_403 (Addgene 41395 and 158560).

Techniques: Western Blot, Control, Molecular Weight

Journal: Genes & development

Article Title: CDK4 loss-of-function mutations cause microcephaly and short stature.

doi: 10.1101/gad.352311.124

Figure Lengend Snippet: Figure 4. CDK4 mutations do not alter mitosis. (A) Percentage of mitotic cells (p-Histone H3 ser10-positive) in control (C1 and C2) and patient (P1 and P2) fibroblasts as measured by flow cy- tometry. Data points are from three independent experiments (two for C1); one-way ANOVA with Tukey post test; mean ± SEM. (B) Quantification of metaphase cells with more than two centrosomes, expressed as percentage. Numbers of cells analyzed were as follows: C1, 79; C2, 94; P1, 150; and P2, 101. Two-tailed t- test; mean ± SEM; measurements were pooled from two indepen- dent experiments. (C) Representative confocal images of control (C1 and C2) and patient (P1 and P2) fibroblasts fixed and stained for DAPI (gray), α-tubulin (green), and pericentrin (magenta). Scale bars, 5 µm.

Article Snippet: Patient fibroblasts were transduced with lentiviral particles containing pLIX_403-CDK4 and/or pLIX_403CDK6, a construct where full-length codon-optimized CDK4 or CDK6 was gateway-cloned into pLIX_403 (Addgene 41395 and 158560).

Techniques: Control, Two Tailed Test, Staining

Journal: Genes & development

Article Title: CDK4 loss-of-function mutations cause microcephaly and short stature.

doi: 10.1101/gad.352311.124

Figure Lengend Snippet: Figure 5. CDK4 mutations impair G1-to-S progression and lead to reduced cell proliferation. (A) Western blot of control and patient-de- rived fibroblasts with and without WT CDK4 complementation. (B, left) Growth curves of control and patient-derived fibroblasts with and without WT CDK4 complementation. (Right) Bar graph showing quantification of doubling times; one-way ANOVA with Tukey post test. P-values are indicated; mean ± SEM. (C) Cell cycle distribution (G0/G1, S, and G2/M) derived from BrdU and DNA (DAPI) flow cytometry scatter plots show fewer cells in S phase (BrdU+) in patient-derived fibroblasts compared with controls. n = 3 independent experiments; mean ± SEM. Gates are shown on representative plots at the right. (D) Cell cycle distribution after complementation of patient-derived fibroblasts with CDK4. Reduced G0/G1 and increased S-phase populations consistent with rescue of a G1/S progression defect. n = 3 in- dependent experiments; mean ± SEM. (See also Supplemental Fig. S4A.) (E) Quantification of DNA synthesis rate (BrdU mean fluorescence intensity [MFI] of gated population in the red rectangle) from experiments depicted in C.

Article Snippet: Patient fibroblasts were transduced with lentiviral particles containing pLIX_403-CDK4 and/or pLIX_403CDK6, a construct where full-length codon-optimized CDK4 or CDK6 was gateway-cloned into pLIX_403 (Addgene 41395 and 158560).

Techniques: Western Blot, Control, Derivative Assay, Flow Cytometry, DNA Synthesis, Fluorescence