Journal: Virulence

Article Title: Dynamic Dissection of Dynein and Kinesin-1 Cooperatively Mediated Intercellular Transport of Porcine Epidemic Diarrhea Coronavirus along Microtubule Using Single Virus Tracking

doi: 10.1080/21505594.2021.1878748

Figure Lengend Snippet: Microtubule, dynein and kinesin-1 are involved in PEDV infection in Vero cells. (a)/(b) Fluorescence images of DiD-labeled PEDV particles in Vero cells respectively transfected with EGFP-microtubule and mKO2-dynein/mKO2-KIF5B. Scale bars indicate 5 μm in the whole fields of view (FoVs) and 1 μm in the zoomed-in FoVs. (c) Titers of PEDV particles from control DMSO, nocodazole (Noco), ciliobrevin D (Cilio D), control siRNA and KIF5B siRNA pretreated Vero cells. The KIF5B level was determined by Western blot using the KIF5B antibody, and equal loading was verified with the anti-GAPDH antibody. Each data point represents mean ± standard deviation from three independent experiments. Statistical analysis on all data was performed using one-way ANOVA (***, P < 0.001)

Article Snippet: The siRNA sequences targeting kinesin-1 heavy chain (KIF5B) were prepared by Sangon Biotech (China).

Techniques: Infection, Fluorescence, Labeling, Transfection, Control, Western Blot, Standard Deviation

Journal: Virulence

Article Title: Dynamic Dissection of Dynein and Kinesin-1 Cooperatively Mediated Intercellular Transport of Porcine Epidemic Diarrhea Coronavirus along Microtubule Using Single Virus Tracking

doi: 10.1080/21505594.2021.1878748

Figure Lengend Snippet: Microtubule, dynein and kinesin-1 affect PEDV fusion and accumulation in the perinuclear region. (a) Fluorescence images of Vero cells first respectively treated with control DMSO, Noco, Cilio D, control siRNA and KIF5B siRNA, then infected with DiD-labeled PEDV particles for 1 h and finally fixed. Actin was stained with AbFluor 488-conjugated phalloidin. Scale bar, 5 μm. (b) Statistical analysis on the DiD fluorescence intensity from (a) (n = 20 cells). a.u.: arbitrary units. (c) Fluorescence images of Vero cells first transfected with EGFP-MT and respectively treated with control DMSO, Noco, Cilio D, control siRNA and KIF5B siRNA and then infected with DiD-labeled PEDV particles. Nuclei were stained with DAPI. Scale bar, 5 μm. (d) Quantification on the percentage virions within 2 μm of the nuclei in 15 infected cells respectively treated with control DMSO, Cilio D, Noco, control siRNA and KIF5B siRNA. The KIF5B level was determined using Western blot with the KIF5B antibody, and equal loading was verified using the anti-GAPDH antibody. Each data point represents mean ± standard deviation from three independent experiments. Statistical analysis of all data was performed using one-way ANOVA (***, P < 0.001)

Article Snippet: The siRNA sequences targeting kinesin-1 heavy chain (KIF5B) were prepared by Sangon Biotech (China).

Techniques: Fluorescence, Control, Infection, Labeling, Staining, Transfection, Western Blot, Standard Deviation

Journal: Virulence

Article Title: Dynamic Dissection of Dynein and Kinesin-1 Cooperatively Mediated Intercellular Transport of Porcine Epidemic Diarrhea Coronavirus along Microtubule Using Single Virus Tracking

doi: 10.1080/21505594.2021.1878748

Figure Lengend Snippet: Microtubule, dynein and kinesin-1 are not required for PEDV attachment or internalization. (a) Detection on PEDV N protein and GAPDH using Western blot from the infected PEDV particles in the control DMSO, Noco and Cilio D pretreated Vero cells at 0 h and 1 h PEDV post-infection (p.i.). (b) Quantification on the amount of PEDV N protein according to the amount of GAPDH. (c) Copy numbers of PEDV RNA from the infected PEDV particles in the control DMSO, Noco and Cilio D pretreated Vero cells at 0 h and 1 h PEDV post-infection using RT-PCR. (d) Detection on the proteins of KIF5B, PEDV N protein and GAPDH using Western blot from the infected PEDV particles in the control siRNA and KIF5B siRNA pretreated Vero cells at 0 h and 1 h PEDV post-infection (p.i.). (e) and (f) Quantification on the amount of KIF5B and PEDV N protein according to the amount of GAPDH. (g) Copy numbers of PEDV RNA from infected PEDV particles in the control siRNA and KIF5B siRNA pretreated Vero cells at 0 h and 1 h PEDV post-infection using RT-PCR. Each data point represents mean ± standard deviation from three independent experiments. Statistical analysis of all data was performed using one-way ANOVA (***, P < 0.001). ns: nonsignificant

Article Snippet: The siRNA sequences targeting kinesin-1 heavy chain (KIF5B) were prepared by Sangon Biotech (China).

Techniques: Western Blot, Infection, Control, Reverse Transcription Polymerase Chain Reaction, Standard Deviation

Journal: Virulence

Article Title: Dynamic Dissection of Dynein and Kinesin-1 Cooperatively Mediated Intercellular Transport of Porcine Epidemic Diarrhea Coronavirus along Microtubule Using Single Virus Tracking

doi: 10.1080/21505594.2021.1878748

Figure Lengend Snippet: Five types of PEDV intercellular transport observed by single-virus tracking in live Vero cells. (a)-(b) Representative time-lapse fluorescence images of PEDV particles during intercellular transport in live Vero cells transfected with (a) EGFP-MT and mKO2-dynein and (b) EGFP-MT and mKO2-KIF5B in different types: (1) unidirectional movement toward microtubule plus ends, (2) unidirectional movement toward microtubule minus ends, (3) bidirectional movement along the same microtubule, (4) bidirectional movement along different microtubules and (5) motionless state. White arrows indicate PEDV particles. Scale bar, 1 μm. (c) Average velocities corresponding to motion states of PEDV intercellular transport according to (a). (d) Average velocities corresponding to motion states of PEDV intercellular transport according to (b). (e) Proportions corresponding to five types of PEDV intercellular transport according to (a). (f) Proportions corresponding to five types of PEDV intercellular transport according to (b)

Article Snippet: The siRNA sequences targeting kinesin-1 heavy chain (KIF5B) were prepared by Sangon Biotech (China).

Techniques: Virus, Fluorescence, Transfection

Journal: Virulence

Article Title: Dynamic Dissection of Dynein and Kinesin-1 Cooperatively Mediated Intercellular Transport of Porcine Epidemic Diarrhea Coronavirus along Microtubule Using Single Virus Tracking

doi: 10.1080/21505594.2021.1878748

Figure Lengend Snippet: Cooperation between dynein and kinesin-1 during PEDV intercellular transport. (a)-(b) Representative time-lapse fluorescence images of PEDV particles during intercellular transport in live Vero cells transfected with EGFP-MT and mKO2- KIF5B corresponding to the (a) control DMSO treated and (b) dynein inhibited conditions. White arrows indicate PEDV particles. Scale bar, 1 μm. (c) Proportions corresponding to five types of PEDV intercellular transport according to (a) and (b). (d) Average velocities corresponding to motion states of PEDV intercellular transport according to (a) and (b). (e)-(f) Representative time-lapse fluorescence images of PEDV particles during intercellular transport in live Vero cells transfected with EGFP-MT and mKO2-dynein corresponding to (e) control siRNA and (f) KIF5B knockdown conditions. White arrows indicate PEDV particles. Scale bar, 1 μm. (g) Proportions corresponding to five types of PEDV intercellular transport according to (e) and (f). (h) Average velocities corresponding to motion states of PEDV intercellular transport according to (e) and (f). (i) Models corresponding to motion states of PEDV intercellular transport

Article Snippet: The siRNA sequences targeting kinesin-1 heavy chain (KIF5B) were prepared by Sangon Biotech (China).

Techniques: Fluorescence, Transfection, Control, Knockdown

Journal: Nature Communications

Article Title: Structural basis of epitope selectivity and potent protection from malaria by PfCSP antibody L9

doi: 10.1038/s41467-023-38509-2

Figure Lengend Snippet: a Buried surface area (BSA) contributions of individual residues to rsCSP binding in the L9 heavy chain. Sequence alignment to the IGHV3-33 germline gene shown below. b Same as in a, for the L9 light chain. c , d Structural details of NPNV binding. e NPNA 2 epitope structure in the NPNA-specific mAb 2243 (PBD 6O23), highlighting the two key CH-π interactions of germline-encoded aromatic residues (W52 H and Y94 L ) with the repeat prolines. f Same as in e, with X-ray structures of six NPNA-specific mAbs superimposed to highlight structural conservation. These six mAbs are shown in g . g Electrostatic surface potentials from L9 cryo-EM structure +/− peptide (upper left two panels) and X-ray structures of six other NPNA-specific mAbs bound to peptide; electrostatic potentials were calculated in PyMol . The PDB accession codes are in parentheses. K b : Boltzmann constant; T : temperature in kelvin; c e : electron charge in coulombs. Source data are provided as a Source Data file.

Article Snippet: L9 heavy and light chain sequences were synthesized and codon-optimized for mammalian expression and cloned into pHCMV3 by Genscript Inc.

Techniques: Binding Assay, Sequencing, Cryo-EM Sample Prep

Journal: Nature Communications

Article Title: Structural basis of epitope selectivity and potent protection from malaria by PfCSP antibody L9

doi: 10.1038/s41467-023-38509-2

Figure Lengend Snippet: a Ribbon diagram of Fab B (cyan) and C (maroon); side chains of interacting residues are shown. b – d Structural details of key homotypic interactions. Dashed lines indicate specific contacts. e Buried surface area (BSA) contributions of individual residues to the homotypic interface in L9 light chain. Sequence alignment with F10 K and germline IGKV1-5 gene is shown below. f Same as in e , for L9 heavy chain, with sequence alignment to F10 H and germline IGHV3-33 gene. Source data are provided as a Source Data file.

Article Snippet: L9 heavy and light chain sequences were synthesized and codon-optimized for mammalian expression and cloned into pHCMV3 by Genscript Inc.

Techniques: Sequencing