Journal: eLife

Article Title: Sensorimotor pathway controlling stopping behavior during chemotaxis in the Drosophila melanogaster larva

doi: 10.7554/eLife.38740

Figure Lengend Snippet:

Article Snippet: Antibody , Rabbit anti-HA-Tag (C29F4) , Cell Signaling Technology, MA, USA , RRID: AB_1549585 , 1:500 dilution.

Techniques: Plasmid Preparation, Software

Journal: Lipids in Health and Disease

Article Title: Palmitic acid causes increased dihydroceramide levels when desaturase expression is directly silenced or indirectly lowered by silencing AdipoR2

doi: 10.1186/s12944-021-01600-y

Figure Lengend Snippet: AdipoR2 overexpression causes decreased dihydroceramide levels (data from the R2OE experiment). Western blots detection of the HA-tagged over-expressed AdipoR2 using an anti-HA antibody ( A ) or an anti-AdipoR2 antibody ( B ); anti-GAPDH was used as a loading control. (D-F) Relative abundance of SFA, MUFA and PUFA in phosphatidylcholines (PC) and phosphatidylethanolamines (PE) of control (WT) and AdipoR2 over-expressing cells (R2OE) treated with 400 μM PA for 6 h. The sums of five sphingolipid classes are shown as box plots ( J-N ) while their fatty acid composition is shown as a heat map ( O ). In the box plots, boxes indicate the 25th to 75th percentile while the whiskers indicate the data points still within 1.5 of the box range. For D-N, significant differences from the WT control were determined using Student’s t-tests, with * P < 0.05, ** P < 0.01 and *** P < 0.001. For the heat map, the amount of each lipid species was normalized to the average of the WT + 400 μM PA treatment and the heat maps show fold differences from the mean across all treatments (each column is a replicate); only lipid species with significantly different levels among siRNAs in a given culture condition are included (ANOVA, q < 0.05)

Article Snippet: The blots were then incubated overnight at 4 °C with primary antibodies, as follows: rabbit monoclonal anti-HA antibody (C29F4; Cell signaling) 1:5000 dilution, rabbit anti-GAPDH antibody (14C10; Cell Signaling, Danvers, USA) or rabbit polyclonal anti-AdipoR2 (1:1000, described in [ ]).

Techniques: Over Expression, Western Blot, Control, Expressing

Journal: PLOS Pathogens

Article Title: Species-specific emergence of H7 highly pathogenic avian influenza virus is driven by intrahost selection differences between chickens and ducks

doi: 10.1371/journal.ppat.1011942

Figure Lengend Snippet: (A) Schematic representation of the modified HA proteins of H7N7 LPAIVs and HPAIVs that were used in this study. The FLAGtag and HAtag are located downstream of the signal peptide (SP) and are connected to the mature HA1 via a linker (L) sequence. The viruses differ only at the N-terminus and at the cleavage site at the border of HA1 and HA2. (B) Chicken fibroblasts (DF-1 cells) were inoculated with H7N7-LP FLAGtag (in blue), H7N7-HP HAtag (in red), or mock-inoculated (in black) at an MOI of 0.1. Expression of the influenza virus nucleoprotein (NP), FLAGtag, and HAtag was assessed at 24 hpi by flow cytometry. (C, D) Replication kinetics in primary chicken and duck embryonic fibroblasts (CEF; DEF) following inoculation with H7N7-LP, H7N7-LP FLAGtag , H7N7-HP, or H7N7-HP HAtag at an MOI of 0.001. Infectious titers in supernatants were determined by endpoint titration in MDCK cells and expressed as log 10 TCID 50 /mL. Data are presented as arithmetic mean ± SD of log 10 transformed values from two independent experiments. The dotted lines indicate the limit of detection of the endpoint titration assay.

Article Snippet: The plates were washed with PBS, and stained overnight at 4°C with a mixture of primary antibodies diluted in PBS with 0.1% bovine serum albumin (BSA): 1 μg/mL anti-influenza virus NP antibody (mouse IgG2α; ATCC HB-65) and 44 ng/mL anti-HAtag antibody (rabbit IgG; C29F4; Cell Signaling).

Techniques: Modification, Sequencing, Expressing, Virus, Flow Cytometry, Titration, Transformation Assay

Journal: PLOS Pathogens

Article Title: Species-specific emergence of H7 highly pathogenic avian influenza virus is driven by intrahost selection differences between chickens and ducks

doi: 10.1371/journal.ppat.1011942

Figure Lengend Snippet: (A-D) Viral RNA quantification in oropharyngeal (OP) and cloacal (CL) swabs of chickens and ducks mono-inoculated with ~10 4 EID 50 of H7N7-HP HAtag or ~10 6 EID 50 of H7N7-LP FLAGtag . Viral RNA levels were determined by RT-qPCR targeting the influenza virus matrix segment and expressed as 40-cycle threshold (Ct). Data are depicted as arithmetic mean ± SD calculated from the number of animals indicated above the graphs (#: the number of alive animals/the total number of animals in the experiment). (E-H) Infectious virus titers in swabs from panels (A-D). Infectious titers were determined by endpoint titration in MDCK cells and expressed as log 10 TCID 50 /mL. Data are depicted as arithmetic mean ± SD calculated from log 10 transformed values from the number of animals indicated above panels (A-D). The horizontal dotted lines indicate the limit of detection of the endpoint titration assay.

Article Snippet: The plates were washed with PBS, and stained overnight at 4°C with a mixture of primary antibodies diluted in PBS with 0.1% bovine serum albumin (BSA): 1 μg/mL anti-influenza virus NP antibody (mouse IgG2α; ATCC HB-65) and 44 ng/mL anti-HAtag antibody (rabbit IgG; C29F4; Cell Signaling).

Techniques: Quantitative RT-PCR, Virus, Titration, Transformation Assay

Journal: PLOS Pathogens

Article Title: Species-specific emergence of H7 highly pathogenic avian influenza virus is driven by intrahost selection differences between chickens and ducks

doi: 10.1371/journal.ppat.1011942

Figure Lengend Snippet: (A, B) Viral RNA quantification in tissues harvested at 3 dpi from chickens (A) and ducks (B) inoculated with ~10 4 EID 50 of H7N7-HP HAtag or ~10 6 EID 50 of H7N7-LP FLAGtag . Viral RNA amounts were determined by RT-qPCR targeting the influenza virus matrix segment and expressed as 40-cycle threshold (Ct) normalized/gram tissue. Bars represent the arithmetic mean. The dotted lines distinguish tissues from the respiratory, digestive, and miscellaneous systems. (C, D) Infectious virus titers in tissues from panels (A, B). Infectious titers were determined by endpoint titration in MDCK cells and expressed as log 10 TCID 50 /gram tissue. Bars represent the arithmetic mean of log 10 transformed values. Dotted lines similar to (A, B). The horizontal solid lines indicate the limit of detection of the endpoint titration assay per tissue.

Article Snippet: The plates were washed with PBS, and stained overnight at 4°C with a mixture of primary antibodies diluted in PBS with 0.1% bovine serum albumin (BSA): 1 μg/mL anti-influenza virus NP antibody (mouse IgG2α; ATCC HB-65) and 44 ng/mL anti-HAtag antibody (rabbit IgG; C29F4; Cell Signaling).

Techniques: Quantitative RT-PCR, Virus, Titration, Transformation Assay

Journal: PLOS Pathogens

Article Title: Species-specific emergence of H7 highly pathogenic avian influenza virus is driven by intrahost selection differences between chickens and ducks

doi: 10.1371/journal.ppat.1011942

Figure Lengend Snippet: (A) Method for distinguishing H7N7-HP HAtag and H7N7-LP FLAGtag plaques based on epitope tag staining. The number of plaque forming units (PFU) per virus per sample was determined by co-staining plaques with antibodies targeting influenza virus nucleoprotein (NP) and HAtag. Plaques formed by H7N7-LP FLAGtag stain positive for NP (green). Plaques formed by H7N7-HP HAtag stain positive for both NP (green) and HAtag (red). (B) Severity of disease in six chickens co-inoculated with H7N7-HP HAtag and H7N7-LP FLAGtag . Disease severity scores (0–5) were determined thrice daily, each box representing one time point. Dotted lines indicate planned time points for euthanasia and dissection. (C) H7N7-HP HAtag and H7N7-LP FLAGtag RNA quantification in oropharyngeal (OP) and cloacal (CL) swabs from co-inoculated chickens. Viral RNA amounts were determined by HPAIV/LPAIV differentiating RT-qPCR targeting the HA cleavage site and expressed as 40-cycle threshold (Ct). (D) Infectious virus titers in the swabs from panel (C). Oropharyngeal and CL swab titers are depicted as PFU/mL as measured by the HPAIV/LPAIV differentiating plaque assay. (E) H7N7-HP HAtag and H7N7-LP FLAGtag RNA quantification in tissues harvested at 3 dpi as determined by HPAIV/LPAIV differentiating RT-qPCR targeting the HA cleavage site and expressed as 40-Ct normalized/gram tissue. The dotted lines distinguish tissues from the respiratory, digestive, and miscellaneous systems. (F) Infectious virus titers of tissues from panel (E), depicted as PFU/gram tissue as determined by the HPAIV/LPAIV differentiating plaque assay. Dotted lines are similar to (E).

Article Snippet: The plates were washed with PBS, and stained overnight at 4°C with a mixture of primary antibodies diluted in PBS with 0.1% bovine serum albumin (BSA): 1 μg/mL anti-influenza virus NP antibody (mouse IgG2α; ATCC HB-65) and 44 ng/mL anti-HAtag antibody (rabbit IgG; C29F4; Cell Signaling).

Techniques: Staining, Virus, Dissection, Quantitative RT-PCR, Plaque Assay

Journal: PLOS Pathogens

Article Title: Species-specific emergence of H7 highly pathogenic avian influenza virus is driven by intrahost selection differences between chickens and ducks

doi: 10.1371/journal.ppat.1011942

Figure Lengend Snippet: (A) Degree of viral antigen expression in tissues of chickens and ducks mono-inoculated with ~10 4 EID 50 H7N7-HP HAtag or ~10 6 EID 50 H7N7-LP FLAGtag and euthanized at 3 dpi. Viral antigen expression was determined by immunohistochemical detection of the influenza virus nucleoprotein. Tissues showing no positivity were scored as (0), sporadic single positive cells as (1), multiple sites of positive cells as (2), and diffuse positivity as (3). N.P. = not performed. (B) Degree of viral antigen expression in tissues from H7N7-LP FLAGtag /H7N7-HP HAtag co-inoculated chickens and ducks euthanized at 3 dpi. Staining and scoring were performed as in (A).

Article Snippet: The plates were washed with PBS, and stained overnight at 4°C with a mixture of primary antibodies diluted in PBS with 0.1% bovine serum albumin (BSA): 1 μg/mL anti-influenza virus NP antibody (mouse IgG2α; ATCC HB-65) and 44 ng/mL anti-HAtag antibody (rabbit IgG; C29F4; Cell Signaling).

Techniques: Expressing, Immunohistochemical staining, Virus, Staining