Journal: Frontiers in Systems Neuroscience

Article Title: Open Source Tools for Temporally Controlled Rodent Behavior Suitable for Electrophysiology and Optogenetic Manipulations

doi: 10.3389/fnsys.2018.00018

Figure Lengend Snippet: Head-fixed setup. (A) The animal is held by an implanted head bar with a pair of metal holders (1), facing a custom-made lick port hosting an IR emitter and an IR receiver (2, 3) for lick detection and a plastic water spout (4). Air-puff is delivered via a cannula placed near the animal’s face (5). Visual and auditory cues are delivered by a central LED (6) and lateral speakers (7). (B) Schematic diagram of the behavior setup. Cue and reinforcement delivery are controlled by Bpod. Motion is monitored with a camera using Bonsai open software.

Article Snippet: The tones were uploaded as .wav files to a USB-based microcontroller development system Teensy 3.2 and its audio adaptor board (TEENSY32 and TEENSY3_AUDIO, PJRC) using the Bpod r0.5 behavior control system (Sanworks LLC ).

Techniques: Software

Journal: Frontiers in Systems Neuroscience

Article Title: Open Source Tools for Temporally Controlled Rodent Behavior Suitable for Electrophysiology and Optogenetic Manipulations

doi: 10.3389/fnsys.2018.00018

Figure Lengend Snippet: Sound calibration and delivery. (A) Components: computer (1), miniUSB-USB A cable (2), Bpod (3), RJ45 cable (4), miniUSB-USB A cable (5), Audio Adaptor Board for Teensy + Teensy USB Development Board + SD card (6), 3.5 mm stereo jack to jack cable (7), Adafruit Audio Amplifier (8), 12V power supply (9), Digikey 8 Ohm Magnetic Speakers (10), EMM-6 Electret Measurement Microphone (11), Male–Female three-pin XLR cable (12), AudioBox iOne (13), USB B -USB A cable (14). (B) Schematic of the setup. A sine wave is generated in Matlab and sent to Bpod, which loads it to the Teensy apparatus as a. wav file. When played by the speakers, the sound is detected by the microphone, delivered to the computer and the dB SPL is read using the TrueRTA software. (C) The dB SPL levels at each frequency before (blue) and after (red) the calibration process. Solid black line indicates the calibration target volume (60 dB SPL).

Article Snippet: The tones were uploaded as .wav files to a USB-based microcontroller development system Teensy 3.2 and its audio adaptor board (TEENSY32 and TEENSY3_AUDIO, PJRC) using the Bpod r0.5 behavior control system (Sanworks LLC ).

Techniques: Generated, Software

Journal: Frontiers in Systems Neuroscience

Article Title: Open Source Tools for Temporally Controlled Rodent Behavior Suitable for Electrophysiology and Optogenetic Manipulations

doi: 10.3389/fnsys.2018.00018

Figure Lengend Snippet: Delay measurements. (A) Internal delay. Left, signals were sent from the BNC output port (blue) and the RJ45 output connector for communication with the port interface board (red) directly to the oscilloscope. Right, example signals detected by the oscilloscope. Arrow, measured delay. (B) Delay of visual cue. Left, signals were sent from Bpod to the oscilloscope both directly (blue) and via the port interface board (red). Right, example of the signals detected by the oscilloscope. (C) Delay of sound delivery. Left, signals were sent directly (blue) or via the Teensy board (red). The oscilloscope receives the latter signal from the line out pins of the Teensy slave board. Right, example of the signals detected by the oscilloscope. (D) Delay of reinforcement delivery. Left, signals were sent from the BNC output port (blue) directly to the oscilloscope and to two port interface boards. One port was receiving commands to open and close the water valve (red), while the other was receiving similar input for controlling the air valve (yellow) along with a constant PWM signal (orange). The latter was sent to the oscilloscope throughout a circuit that water or air could close or break, respectively, changing the oscilloscope voltage input. Top right, example of the signals detected by the oscilloscope for water delay measurement. Bottom right, example of the signals detected by the oscilloscope for air delay measurement.

Article Snippet: The tones were uploaded as .wav files to a USB-based microcontroller development system Teensy 3.2 and its audio adaptor board (TEENSY32 and TEENSY3_AUDIO, PJRC) using the Bpod r0.5 behavior control system (Sanworks LLC ).

Techniques:

Journal: Frontiers in Systems Neuroscience

Article Title: Open Source Tools for Temporally Controlled Rodent Behavior Suitable for Electrophysiology and Optogenetic Manipulations

doi: 10.3389/fnsys.2018.00018

Figure Lengend Snippet: Temporally precise delivery of stimuli and feedback. (A) Distribution of minimal elapsed time between sending signals to the BNC and RJ45 output of Bpod (mean ± SD, 0.045 ± 0.001 ms). (B) Board delay: distribution of delays between the signals from the BNC output port and the LED output wire terminal of the port interface board (mean ± SD, 0.047 ± 0.003 ms) (C) Delay distribution of sound delivery, between the signals from the BNC output port and the Teensy board (mean ± SD, 6.59 ± 0.9 ms). (D) Delay distribution of air puff delivery (mean ± SD, 3.48 ± 0.02 ms). (E) Delay distribution of water delivery (mean ± SD, 8.61 ± 0.81 ms).

Article Snippet: The tones were uploaded as .wav files to a USB-based microcontroller development system Teensy 3.2 and its audio adaptor board (TEENSY32 and TEENSY3_AUDIO, PJRC) using the Bpod r0.5 behavior control system (Sanworks LLC ).

Techniques:

Journal: Emerging microbes & infections

Article Title: ABTB1 facilitates the replication of influenza A virus by counteracting TRIM4-mediated degradation of viral NP protein.

doi: 10.1080/22221751.2023.2270073

Figure Lengend Snippet: Figure 1. ABTB1 positively regulates the replication of IAV. (A, B) siRNA knockdown of ABTB1 in A549 cells. A549 cells were trans- fected with siRNA targeting ABTB1 or with scrambled siRNA for 48 h. Knockdown of ABTB1 expression was detected by RT-qPCR (A) and western blotting (B) with a mouse anti-ABTB1 mAb. ****, P < 0.0001. (C) Viability of A549 cells treated with ABTB1-specific or scrambled siRNA. A549 cells were treated with siRNA targeting ABTB1 or with scrambled siRNA for 48 h. Cell viability was deter- mined by using a CellTiter-Glo assay. (D-F) Replication of IAVs in A549 cells treated with ABTB1-specific or scrambled siRNA. A549 cells were transfected with siRNA targeting ABTB1 or with scrambled siRNA for 48 h and infected with WSN (H1N1) (MOI = 0.01) (D), AH05 (H5N1) (MOI = 0.1) (E), or SH13 (H9N2) virus (MOI = 0.1) (F). Supernatants were collected at 24 and 48 h p.i., and virus titres were determined by performing plaque assays on MDCK cells. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (G, H) Generation of the ABTB1_KO A549 cell line by using the CRISPR/Cas9 system. The knockout of ABTB1 was validated by sequencing analysis (G) and western blotting (H). (I) Viability of ABTB1_KO and control A549 cells determined by using the CellTiter-Glo assay. (J) Replica- tion of WSN (H1N1) virus in ABTB1_KO or control A549 cells. ABTB1_KO or control A549 cells were infected with WSN (H1N1) virus (MOI = 0.01). Supernatants were collected at 24 and 48 h p.i., and virus titres were determined by using plaque assays on MDCK cells. ***, P < 0.001; ****, P < 0.0001. (K, L) Establishment of a stable A549 cell line overexpressing ABTB1. The stable overexpres- sion of ABTB1 was confirmed by RT-qPCR (K) and western blotting with a mouse anti-ABTB1 mAb (L) in comparison to the A549 control cell line transduced with an empty retrovirus. ***, P < 0.001. (M) Replication of WSN (H1N1) virus in ABTB1-overexpressing or control A549 cells. The ABTB1-overexpressing or empty retrovirus-transduced control A549 cells were infected with WSN (H1N1) virus (MOI = 0.01). Supernatants were collected at 24 and 48 h p.i., and virus titres were determined by performing plaque assays on MDCK cells. **, P < 0.01. (N) Expression of ABTB1 in A549 cells infected with WSN (H1N1) virus. A549 cells were infected with WSN (H1N1) virus (MOI = 5). Whole cell lysates were analyzed by western blotting with a mouse anti-ABTB1 mAb and a mouse anti-NP mAb at the indicated timepoints p.i. Data are representative of at least three independent experiments. Means ± SD are shown in (A, C-F, I-K, M) (n = 3).

Article Snippet: The following primary antibodies were purchased from commercial sources: rabbit anti-NP polyclonal antibody (pAb) (PA5-32242, Invitrogen, Carlsbad, CA, USA), rabbit anti-M1 pAb (GTX125928, GeneTex, Irvine, CA, USA), rabbit anti-HA pAb (GTX127357, GeneTex), rabbit antiHA tag pAb (51064-2-AP, Proteintech, Wuhan, China), rabbit anti-V5 pAb (AB3792, Merck Millipore, Darmstadt, Germany), rabbit anti-GAPDH pAb (10494-1-AP, Proteintech, Wuhan, China), mouse anti-actin mAb (sc-47778, Santa Cruz, Dallas, TX, USA), mouse anti-Flag mAb (F3165, SigmaAldrich), mouse anti-Myc mAb (M4439, SigmaAldrich), mouse anti-V5 mAb (V8012, SigmaAldrich), rabbit anti-Flag pAb (F7425, SigmaAldrich), rabbit anti-Myc pAb (C3956, SigmaAldrich), mouse anti-ABTB1 mAb (CF811778, OriGene, Rockville, MD, USA), and rabbit anti-TRIM4 pAb (PAB32752, Bioswamp, Wuhan, China).

Techniques: Knockdown, Expressing, Quantitative RT-PCR, Western Blot, Glo Assay, Transfection, Infection, Virus, CRISPR, Knock-Out, Sequencing, Control, Comparison, Transduction

Journal: Emerging microbes & infections

Article Title: ABTB1 facilitates the replication of influenza A virus by counteracting TRIM4-mediated degradation of viral NP protein.

doi: 10.1080/22221751.2023.2270073

Figure Lengend Snippet: Figure 2. ABTB1 is involved in the early stage of the IAV life cycle. (A) Cellular localization of NP in ABTB1_KO or control A549 cells infected with WSN (H1N1) virus (MOI = 5). At 2, 3, 4, and 7 h p.i., the infected cells were fixed and stained with a mouse anti-NP mAb, followed by incubation with Alexa Fluor 633 goat anti-mouse IgG (H + L) (red). The nuclei were stained with DAPI. The dis- tribution pattern of viral NP was statistically shown in the right panel. (B) Viral NP expression in ABTB1_KO or control A549 cells infected with WSN (H1N1) virus (MOI = 5). At the indicated timepoints p.i., the whole cell lysates were western blotted with a mouse anti-NP mAb. The band intensities of NP, quantified by using ImageJ software, were normalized to GAPDH and were expressed as relative ratios compared with A549 control cells (right panel). (C) Cellular localization of NP in ABTB1-overexpressing or empty retrovirus-transduced control A549 cells infected with WSN (H1N1) virus (MOI = 5). At 2, 3, 4 and 7 h p.i., the infected cells were fixed and stained with a mouse anti-NP mAb, followed by incubation with Alexa Fluor 633 goat anti-mouse IgG (H + L) (red). The nuclei were stained with DAPI. (D) Viral NP expression in ABTB1-overexpressing or empty retrovirus-transduced control A549 cells infected with WSN (H1N1) virus (MOI = 5). At 2, 3, 4 and 7 h p.i., the whole cell lysates were western blotted with a mouse anti-NP mAb. Data are representative of at least three independent experiments.

Article Snippet: The following primary antibodies were purchased from commercial sources: rabbit anti-NP polyclonal antibody (pAb) (PA5-32242, Invitrogen, Carlsbad, CA, USA), rabbit anti-M1 pAb (GTX125928, GeneTex, Irvine, CA, USA), rabbit anti-HA pAb (GTX127357, GeneTex), rabbit antiHA tag pAb (51064-2-AP, Proteintech, Wuhan, China), rabbit anti-V5 pAb (AB3792, Merck Millipore, Darmstadt, Germany), rabbit anti-GAPDH pAb (10494-1-AP, Proteintech, Wuhan, China), mouse anti-actin mAb (sc-47778, Santa Cruz, Dallas, TX, USA), mouse anti-Flag mAb (F3165, SigmaAldrich), mouse anti-Myc mAb (M4439, SigmaAldrich), mouse anti-V5 mAb (V8012, SigmaAldrich), rabbit anti-Flag pAb (F7425, SigmaAldrich), rabbit anti-Myc pAb (C3956, SigmaAldrich), mouse anti-ABTB1 mAb (CF811778, OriGene, Rockville, MD, USA), and rabbit anti-TRIM4 pAb (PAB32752, Bioswamp, Wuhan, China).

Techniques: Control, Infection, Virus, Staining, Incubation, Expressing, Western Blot, Software

Journal: Emerging microbes & infections

Article Title: ABTB1 facilitates the replication of influenza A virus by counteracting TRIM4-mediated degradation of viral NP protein.

doi: 10.1080/22221751.2023.2270073

Figure Lengend Snippet: Figure 3. ABTB1 promotes the nuclear import of the vRNP complex of IAV. (A) Binding and endocytosis assay of WSN (H1N1) virus in A549 cells transfected with ABTB1-specific or scrambled siRNA. At 48 h post-transfection, the siRNA treated cells were pre-incu- bated on ice for 30 min, followed by infection with WSN (H1N1) virus (MOI = 20) on ice for 1 h. For the binding assay, the bound virus particles on the cell surface were stained with a rabbit anti-HA pAb and Alexa Fluor 488 goat anti-rabbit IgG (H + L), and visualized by confocal microscopy. For the endocytosis assay, the infected cells were further incubated at 37°C for 30 min, stained with a rabbit anti-HA pAb and Alexa Fluor 488 goat anti-rabbit IgG (H + L), and visualized by confocal microscopy. (B) Fusion assay of WSN (H1N1) virus in A549 cells transfected with the indicated siRNA. At 48 h post-transfection, the siRNA-treated cells were infected with WSN (H1N1) virus labeled with R18 and SP-DiOC18 on ice for 30 min, incubated at 37°C for 3 h, and visualized by confocal microscopy. (C) Uncoating assay of WSN (H1N1) virus in A549 cells transfected with the indicated siRNA. At 48 h post-transfection, the siRNA-treated cells were infected with WSN (H1N1) virus (MOI = 100) on ice for 1 h, followed by incubation at 37°C for 3 h in the presence of CHX. The cells were then stained with a rabbit anti-M1 pAb and Alexa Fluor 488 goat anti-rabbit IgG (H + L), and visualized by confocal microscopy. (D) Visualization of the nuclear import of the incoming vRNP complex of WSN (H1N1) virus in siRNA-treated A549 cells in the presence of CHX. At 48 h post-transfection, the siRNA-treated cells were infected with WSN (H1N1) virus (MOI = 50) on ice for 1 h, followed by incubation at 37°C for 3, 4, or 5 h in the presence of CHX. The cells were stained with a mouse anti-NP mAb and Alexa Fluor 633 goat anti-mouse IgG (H + L), and visualized by confocal microscopy. (E) Visualization of the nuclear import of the incoming vRNP complex of WSN (H1N1) virus in ABTB1-overexpressing A549 cells in the presence of CHX. ABTB1-overexpressing or empty retrovirus-transduced control A549 cells were infected with WSN (H1N1) virus (MOI = 50) on ice for 1 h, followed by the procedures described in (D). Data are representative of at least three independent experiments.

Article Snippet: The following primary antibodies were purchased from commercial sources: rabbit anti-NP polyclonal antibody (pAb) (PA5-32242, Invitrogen, Carlsbad, CA, USA), rabbit anti-M1 pAb (GTX125928, GeneTex, Irvine, CA, USA), rabbit anti-HA pAb (GTX127357, GeneTex), rabbit antiHA tag pAb (51064-2-AP, Proteintech, Wuhan, China), rabbit anti-V5 pAb (AB3792, Merck Millipore, Darmstadt, Germany), rabbit anti-GAPDH pAb (10494-1-AP, Proteintech, Wuhan, China), mouse anti-actin mAb (sc-47778, Santa Cruz, Dallas, TX, USA), mouse anti-Flag mAb (F3165, SigmaAldrich), mouse anti-Myc mAb (M4439, SigmaAldrich), mouse anti-V5 mAb (V8012, SigmaAldrich), rabbit anti-Flag pAb (F7425, SigmaAldrich), rabbit anti-Myc pAb (C3956, SigmaAldrich), mouse anti-ABTB1 mAb (CF811778, OriGene, Rockville, MD, USA), and rabbit anti-TRIM4 pAb (PAB32752, Bioswamp, Wuhan, China).

Techniques: Binding Assay, Endocytosis Assay, Virus, Transfection, Infection, Staining, Confocal Microscopy, Incubation, Single Vesicle Fusion Assay, Labeling, Control

Journal: Emerging microbes & infections

Article Title: ABTB1 facilitates the replication of influenza A virus by counteracting TRIM4-mediated degradation of viral NP protein.

doi: 10.1080/22221751.2023.2270073

Figure Lengend Snippet: Figure 7. ABTB1 promotes the replication of IAV by degrading TRIM4β. (A) Confocal microscopy to visualize the effect of ABTB1 on the expression of TRIM4. HEK293T cells were transfected individually with plasmids expressing Flag-ABTB1 and Myc-TRIM4β or in combination. At 48 h post-transfection, the cells were incubated with a rabbit anti-Flag pAb and a mouse anti-Myc mAb, followed by staining with Alexa Fluor 488 goat anti-rabbit IgG (H + L) (green) and Alexa Fluor 633 goat anti-mouse IgG (H + L) (red). Nuclei were stained with DAPI. (B) ABTB1 reduces the expression of TRIM4β as determined by western blotting. HEK293T cells were trans- fected with plasmids expressing Myc-TRIM4β and gradually increasing amounts of Flag-ABTB1; the expression of TRIM4β and ABTB1 was determined by western blotting. (C) ABTB1 does not interact with TRIM35 as assessed in a co-IP assay. HEK293T cells were transfected individually or in combination with plasmids expressing Flag-ABTB1 and V5-TRIM35. Cell lysates were immunoprecipitated with a mouse anti-Flag mAb and western blotted with a rabbit anti-Flag pAb or a rabbit anti-V5 pAb to reveal the presence of ABTB1 and TRIM35, respectively. (D) ABTB1 has no effect on the stability of TRIM35. HEK293T cells were trans- fected with plasmids expressing V5-TRIM35 and gradually increasing amounts of Flag-ABTB1, and the expression of TRIM35 and ABTB1 was determined by western blotting. (E) ABTB1 mediates the degradation of TRIM4β via the ubiquitin-proteasome system. HEK293T cells were transfected with plasmids expressing ABTB1 and TRIM4β in the presence of DMSO, MG132, 3-MA, or CQ. The level of ABTB1 and TRIM4β was determined by western blotting. (F) ABTB1 suppresses the degradative effect of TRIM4β on IAV NP. HEK293T cells were transfected with plasmids expressing WSN NP alone or in combination with TRIM4β and ABTB1. The levels of WSN NP, TRIM4β, and ABTB1 were determined by western blotting. (G, H) ABTB1 partially counteracts the inhibitory effect of TRIM4β on the replication of IAV. HEK293T cells were transfected with empty vector, or plasmids expressing TRIM4β alone or in combination with ABTB1. The expression of TRIM4β and ABTB1 was confirmed by western blotting (G). At 48 h post-transfection, the cells were infected with WSN (H1N1) virus (MOI = 0.01). Supernatants were collected at 24 and 48 h p.i., and virus titres were determined by performing plaque assays on MDCK cells (H). *, P < 0.05; **, P < 0.01; ***, P < 0.001. Data are repre- sentative of at least three independent experiments. Means ± SD are shown in (H) (n = 3).

Article Snippet: The following primary antibodies were purchased from commercial sources: rabbit anti-NP polyclonal antibody (pAb) (PA5-32242, Invitrogen, Carlsbad, CA, USA), rabbit anti-M1 pAb (GTX125928, GeneTex, Irvine, CA, USA), rabbit anti-HA pAb (GTX127357, GeneTex), rabbit antiHA tag pAb (51064-2-AP, Proteintech, Wuhan, China), rabbit anti-V5 pAb (AB3792, Merck Millipore, Darmstadt, Germany), rabbit anti-GAPDH pAb (10494-1-AP, Proteintech, Wuhan, China), mouse anti-actin mAb (sc-47778, Santa Cruz, Dallas, TX, USA), mouse anti-Flag mAb (F3165, SigmaAldrich), mouse anti-Myc mAb (M4439, SigmaAldrich), mouse anti-V5 mAb (V8012, SigmaAldrich), rabbit anti-Flag pAb (F7425, SigmaAldrich), rabbit anti-Myc pAb (C3956, SigmaAldrich), mouse anti-ABTB1 mAb (CF811778, OriGene, Rockville, MD, USA), and rabbit anti-TRIM4 pAb (PAB32752, Bioswamp, Wuhan, China).

Techniques: Confocal Microscopy, Expressing, Transfection, Incubation, Staining, Western Blot, Co-Immunoprecipitation Assay, Immunoprecipitation, Ubiquitin Proteomics, Plasmid Preparation, Infection, Virus

Journal: Emerging microbes & infections

Article Title: ABTB1 facilitates the replication of influenza A virus by counteracting TRIM4-mediated degradation of viral NP protein.

doi: 10.1080/22221751.2023.2270073

Figure Lengend Snippet: Figure 8. Proposed model for ABTB1-mediated regulation of the nuclear import of IAV vRNP by targeting and degrading TRIM4. In the cytoplasm, TRIM4 inhibits the nuclear import of vRNP by interacting with and targeting NP into the ubiquitin-proteasome degradation pathway. TRIM4 is also an interacting partner and target of ABTB1. The presence of ABTB1 leads to the proteasomal degradation of TRIM4. Consequently, the degradation of NP, impairment of the nuclear import of the vRNP complex, and reduction of virus replication mediated by TRIM4 are largely counteracted by ABTB1.

Article Snippet: The following primary antibodies were purchased from commercial sources: rabbit anti-NP polyclonal antibody (pAb) (PA5-32242, Invitrogen, Carlsbad, CA, USA), rabbit anti-M1 pAb (GTX125928, GeneTex, Irvine, CA, USA), rabbit anti-HA pAb (GTX127357, GeneTex), rabbit antiHA tag pAb (51064-2-AP, Proteintech, Wuhan, China), rabbit anti-V5 pAb (AB3792, Merck Millipore, Darmstadt, Germany), rabbit anti-GAPDH pAb (10494-1-AP, Proteintech, Wuhan, China), mouse anti-actin mAb (sc-47778, Santa Cruz, Dallas, TX, USA), mouse anti-Flag mAb (F3165, SigmaAldrich), mouse anti-Myc mAb (M4439, SigmaAldrich), mouse anti-V5 mAb (V8012, SigmaAldrich), rabbit anti-Flag pAb (F7425, SigmaAldrich), rabbit anti-Myc pAb (C3956, SigmaAldrich), mouse anti-ABTB1 mAb (CF811778, OriGene, Rockville, MD, USA), and rabbit anti-TRIM4 pAb (PAB32752, Bioswamp, Wuhan, China).

Techniques: Ubiquitin Proteomics, Virus