Journal: Viruses

Article Title: The AC2 Protein of a Bipartite Geminivirus Stimulates the Transcription of the BV1 Gene through Abscisic Acid Responsive Promoter Elements

doi: 10.3390/v12121403

Figure Lengend Snippet: The abscisic acid (ABA)-responsive elements (ABREs) in the MYMV P BV1 are needed for AC2-mediated activation. ( A ) Top: Schematic representation of the P BV1 -GFP construct. The boundaries of the Q1 section are denoted with two vertical bars, and the three regions (A, B, and C) subjected to deletions in are highlighted with shades of different colors. Bottom: The sequences of the A and B regions, along with that of seven mutants in the three ABREs, are variously mutated, are shown. The three ABRE motifs are underlined and highlighted in blue fonts. The conserved late element (CLE) motif is highlighted with a double underline. The mutated nt (GTGG → CCTT) are in red fonts. ( B ) Western blot detection of the GFP protein in the agro-infiltrated leaves harboring the various mutants. Each protein sample was extracted from four separate leaf sections infiltrated with the same Agrobacterium suspension, taken from four different plants. The relative ratios were averages of two independent sets of experiments, with the band intensity value of P BV1 + AC2 set at 100%.

Article Snippet: In experiments testing the promoter responsiveness to abscisic acid (ABA) treatment, a 50-mM ABA stock solution was prepared by dissolving ABA powder (GoldBio, St. Louis, MO, USA) in dimethyl sulfoxide (DMSO).

Techniques: Activation Assay, Construct, Western Blot

Journal: Glycobiology

Article Title: Contemporary human H3N2 influenza A viruses require a low threshold of suitable glycan receptors for efficient infection.

doi: 10.1093/glycob/cwad060

Figure Lengend Snippet: Fig. 1. Construction of MDCK and hCK cells that overexpress B3GNT2 and/or B4GALT1. a) MDCK and hCK cells were modified with recombinant lentiviruses containing transfer plasmids for the insertion of the B3GNT2 and/or B4GALT1 gene and the Hygromycin B resistance gene. The knock-in cells were selected with 300 μg/ml Hygromycin B. b) RT-qPCR was performed with primers that anneal to both the human and dog B3GNT2, B4GALT1, or ST6GAL1 genes. The values relative to the dog GAPDH gene were used, which were then normalized to the highest value of each gene. Mean and SD (n = 3) are shown. c) Peptide MS of the B3GNT2, B4GALT1, and ST6GAL1 proteins. Only peptides unique to human proteins were selected. All samples were normalized against tubulin-β and then normalized to the highest value of each protein. Mean and SD (n = 3) are shown.

Article Snippet: The B3GNT2 and B4GALT1 genes were always proceeded by the signal sequence of the human GalT, which we copied from the EGFP-GalT plasmid (gift from Jennifer LippincottSchwartz, Addgene plasmid # 11929; Cole et al. 1996).

Techniques: Recombinant, Knock-In, Quantitative RT-PCR

Journal: Glycobiology

Article Title: Contemporary human H3N2 influenza A viruses require a low threshold of suitable glycan receptors for efficient infection.

doi: 10.1093/glycob/cwad060

Figure Lengend Snippet: Fig. 2. Flow cytometric characterization of B3GNT2/B4GALT1 knock-in MDCK and hCK cells. a) The gating strategy that was used to select single, alive cells. b) Flow cytometry measurements with lectins SNA (recognizes α2,6-Sia), LEL (recognizes elongated glycans), and ECA (recognizes glycans without Sia) were performed. Furthermore, Gf-CoV-2014 NTD was used to detect elongated glycans, H5 HA of A/Vietnam/1203/2004 was used to detect α2,3-Sia, and H1 HA from A/Puerto-Rico/8/1934 was used as a standard influenza virus. Triplicate measurements were performed, of which the mean and all individual measurements are displayed. c) A diverse set of H3 HAs was used to characterize the cells. Triplicate measurements were performed, of which the mean and all individual measurements are displayed. Titration curves of A/Hong-Kong/1/1968, A/Netherlands/109/2003, and A/Singapore/INFH-16-0019/2016 are shown in Fig. S4. Flow cytometric experiments with neuraminidase treatment of the cells are shown in Fig. S3.

Article Snippet: The B3GNT2 and B4GALT1 genes were always proceeded by the signal sequence of the human GalT, which we copied from the EGFP-GalT plasmid (gift from Jennifer LippincottSchwartz, Addgene plasmid # 11929; Cole et al. 1996).

Techniques: Knock-In, Flow Cytometry, Virus, Titration

Journal: Glycobiology

Article Title: Contemporary human H3N2 influenza A viruses require a low threshold of suitable glycan receptors for efficient infection.

doi: 10.1093/glycob/cwad060

Figure Lengend Snippet: Fig. 3. N-glycan analysis of WT and B3GNT2/B4GALT1 knock-in MDCK and hCK cells using MS. The N-glycans from WT and B3GNT2/B4GALT1 knock-in MDCK and hCK cells were measured using HILIC-IMS-QTOF positive mode MS. a) Chromatograms of hCK WT and hCK-B3GNT2 cells were constructed for the glycans with at least 2 and at most 7 LacNAc repeating units. The extracted-ion-counts for the 10 most abundant glycan features per LacNAc repeating unit group were summed to yield a chromatogram. b) The N-glycans found in HILIC-IMS-QTOF positive mode MS with at least 1 LacNAc repeating unit were analyzed for the number of LacNAc repeating units present and the relative abundance was calculated. Further analysis is presented in Fig. S5. Full glycan feature lists for each cell line are presented in Tables SII–SIX. c) Analysis of the N-glycans was additionally performed by LC–MS/MS, followed by analysis of the glycan oxonium ions (Table SX). Sia capped (repeating) LacNAc oxonium ions with masses (from left to right) 657.2349, 1,022.3671, 1,387.4993, and 1,752.6315 were identified and the amounts detected were normalized to the core fragments. Mean and standard errors (n = 3) are shown. Further analysis is shown in Fig. S6 and annotated spectra are present in Fig. S7.

Article Snippet: The B3GNT2 and B4GALT1 genes were always proceeded by the signal sequence of the human GalT, which we copied from the EGFP-GalT plasmid (gift from Jennifer LippincottSchwartz, Addgene plasmid # 11929; Cole et al. 1996).

Techniques: Glycoproteomics, Knock-In, Hydrophilic Interaction Liquid Chromatography, Construct, Liquid Chromatography with Mass Spectroscopy

Journal: Glycobiology

Article Title: Contemporary human H3N2 influenza A viruses require a low threshold of suitable glycan receptors for efficient infection.

doi: 10.1093/glycob/cwad060

Figure Lengend Snippet: Fig. 5. Influenza virus inoculation of B3GNT2 and B4GALT1 knock-in MDCK and hCK cells. End-point titrations with 4 control viruses and 8 recent H3N2 IAVs (details in Table 1) were performed, of which a) 4 control viruses, b) 4 3C.2a viruses, and c) 4 3C.3a viruses. Infectious titers were determined either using a hemagglutination assay a) or a NP staining b, c) when a hemagglutination assay was not possible. d) An infection study using hCK and hCK-B3GNT2 cells with a 2-fold dilution of 12 H3N2 IAVs from the 3C.2a clade (details in Table 2) that could previously either not be isolated in hCK cells (#1–8) or could be isolated in hCK cells (#9–12) was performed. Infection was assessed by the presence of cytopathic effects. Individual and mean values are shown.

Article Snippet: The B3GNT2 and B4GALT1 genes were always proceeded by the signal sequence of the human GalT, which we copied from the EGFP-GalT plasmid (gift from Jennifer LippincottSchwartz, Addgene plasmid # 11929; Cole et al. 1996).

Techniques: Virus, Knock-In, Control, Hemagglutination Assay, Staining, Infection, Isolation

Journal: PLoS ONE

Article Title: An Integrated Physical, Genetic and Cytogenetic Map of Brachypodium distachyon , a Model System for Grass Research

doi: 10.1371/journal.pone.0013461

Figure Lengend Snippet: Characteristics of the two BAC libraries used to construct the Brachypodium physical map.

Article Snippet: The Hin dIII library (BD_ABa) consisted of 36,864 clones with an average insert size of 128 Kb , and the Eco RI library (BD_CBa) consisted of 36,864 clones with an average insert size of 124 Kb ( ).

Techniques: Construct