Journal: Acta Neuropathologica Communications

Article Title: Upregulation of carbonic anhydrase 1 beneficial for depressive disorder

doi: 10.1186/s40478-023-01545-6

Figure Lengend Snippet: Decreased expression of CAR1 in MDD patients and depression-like model rodents. (A) The protein level of CAR1 in healthy controls (n = 29) and MDD patients (n = 36) revealed by ELISA assay. (B) Western blot analysis for the expression level of CAR1 in the serum of mice after CSDS exposure. (C) Western blot analysis showed the changes of CAR1 in the hippocampus and prefrontal cortex of mice after CSDS or CRS (n = 6 for each group) treatment. (D) Western blot analysis of CA family isoforms in the hippocampi between CSDS mice model and control mice group. (E) Western blot analysis showed the changes of CAR1 in the hippocampus among CSDS susceptible group (n = 6), resilient group (n = 7) and control group (n = 6). (F) Q-PCR identification of car1 mRNA in the hippocampi among among CSDS susceptible group (n = 7), resilient group (n = 8) and control group (n = 7). (G) Western blot analysis showed the changes in the cerebellum and entorhinal cortex of CSDS mice. (H) The expression of CAR1 in the hippocampus and prefrontal cortex from the CUMS-treated rats. β-actin or GAPDH was used as loading controls. Unpaired t -test analysis, *p < 0.05, **p < 0.01

Article Snippet: Primary antibodies used were rabbit anti-Car1 (1:2000, Abcam, Cat: ab108367), mouse anti-β-actin (1:10000, Proteintech, Cat: 66009-1-Ig), mouse anti-GAPDH (1:10000, Proteintech, Cat: 60004-1-Ig); secondary antibodies used in experiments were (goat Anti-Mouse IgG (H + L)-HRP Conjugate (1:10000, Bio-Rad, Cat:1,706,516), goat Anti-Rabbit IgG (H + L)-HRP Conjugate (1:8000, Bio-Rad, Cat:1,706,515).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Acta Neuropathologica Communications

Article Title: Upregulation of carbonic anhydrase 1 beneficial for depressive disorder

doi: 10.1186/s40478-023-01545-6

Figure Lengend Snippet: Specific expression of CAR1 in astrocytes. (A, B) A sample confocal image of a hippocampal slice stained with antibody against CAR1 (green) and DAPI (blue). Boxed area in A was zoomed in B. (C) Co-immunostaining with antibodies against CAR1 (green) and different cell type markers (red). Solid arrowheads: representative double-labeled cells. (D) The quantification of CAR1 + /GFAP + cells in CAR1 + cells or GFAP + cells. (E) RNAscope signals of CAR1 (white) in astrocytes labeled by anti-GFAP immunostaining (red) of the ventral hippocampus. (F, G) Zoomed in figures from E. F for the CA1 region and G for the DG region

Article Snippet: Primary antibodies used were rabbit anti-Car1 (1:2000, Abcam, Cat: ab108367), mouse anti-β-actin (1:10000, Proteintech, Cat: 66009-1-Ig), mouse anti-GAPDH (1:10000, Proteintech, Cat: 60004-1-Ig); secondary antibodies used in experiments were (goat Anti-Mouse IgG (H + L)-HRP Conjugate (1:10000, Bio-Rad, Cat:1,706,516), goat Anti-Rabbit IgG (H + L)-HRP Conjugate (1:8000, Bio-Rad, Cat:1,706,515).

Techniques: Expressing, Staining, Immunostaining, Labeling

Journal: Acta Neuropathologica Communications

Article Title: Upregulation of carbonic anhydrase 1 beneficial for depressive disorder

doi: 10.1186/s40478-023-01545-6

Figure Lengend Snippet: A causal role of CAR1 in depression-like behaviors and the activity changes in granule cells. (A) Western blotting of hippocampal sample to validate the absence of CAR1 in CAR1 −/− mice(top); depression-like behavioral tests including FST and TST for CAR1 −/− mice and WT mice (bottom). (B, C) Example traces and their average amplitude and frequency of mEPSC (D) or mIPSC (E) in DG granule cells. (D) Representative responses of granule cells (black, WT; red, CAR1 −/− ) to perforant pathway stimulations (top); the peak response and the area under the curve quantification (bottom) of evoked responses (WT, n = 14; CAR1 −/− , n = 17). (E) The representative mIPSC traces of DG granule cells from WT mice injected with AAV-control, CAR1 −/− mice with AAV-control, or AAV-CAR1 (left), and their average amplitude and frequency of mIPSC of DG granule cells (right). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Student’s t-test and one-way ANOVA)

Article Snippet: Primary antibodies used were rabbit anti-Car1 (1:2000, Abcam, Cat: ab108367), mouse anti-β-actin (1:10000, Proteintech, Cat: 66009-1-Ig), mouse anti-GAPDH (1:10000, Proteintech, Cat: 60004-1-Ig); secondary antibodies used in experiments were (goat Anti-Mouse IgG (H + L)-HRP Conjugate (1:10000, Bio-Rad, Cat:1,706,516), goat Anti-Rabbit IgG (H + L)-HRP Conjugate (1:8000, Bio-Rad, Cat:1,706,515).

Techniques: Activity Assay, Western Blot, Injection

Journal: Acta Neuropathologica Communications

Article Title: Upregulation of carbonic anhydrase 1 beneficial for depressive disorder

doi: 10.1186/s40478-023-01545-6

Figure Lengend Snippet: Decreased the concentration of bicarbonate and proton in the extracellular space of the hilus of CAR1 −/− mice. (A) Example mIPSC traces (top) and their average amplitude and frequency (bottom) of DG granule cells from WT mice underHCO 3 − -free ACS for the normal ACSF condition. (B) Example mIPSC traces (top) and their average amplitude and frequency (bottom) of DG granule cells from WT mice and CAR1 −/− mice under HCO 3 − -free ACSF condition. (C) Schematic representation of a construct (top) and the timeline of the experimental procedure (middle). A representative image of the vHPC after bilateral infusions of AAV (bottom left). The infection of astrocytes was confirmed by co-immunostaining with EGFP (green) and GFAP (red) (bottom right). (D) The immobility time in the FST test after AAV-CAR1 infection. *p < 0.05, **p < 0.01 (Student’s t-test ). (E) Representative mIPSC traces of DG granule cells from WT mice injected with AAV-control, CAR1 −/− mice with AAV-control or AAV-CAR1 underHCO 3 − -free ACSF condition (top) and their average amplitude and frequency analysis (bottom). (F) mIPSC traces of DG granule cells after CAR1 overexpression in CAR1 −/− astrocytes with control, acetazolamide, and amiloride treatment as well as their average amplitude and frequency quantifications (bottom). (G) Representative images in the DG hilus after bilateral infusions of pHLIP at the vHPC (n = 4) (left); fluorescence density quantification of pHLIP labeled hilus interneurons from WT and CAR1 −/− mice (right). (H) Representative images in the DG hilus after bilateral infusions of pHLIP at vHPC into WT mice infected with AAV-GFP, CAR1 −/− mice with AAV-GFP or AAV-CAR1 and their fluorescence density quantification; boxed regions in the top panels were zoomed in the bottom panels. (I) The validation of pHLIP marked hilus interneuron identities by co-immunostaining with neuropeptide Y (NPY), Somatostatin (SST), and parvalbumin (PV). *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t-test and one-way ANOVA)

Article Snippet: Primary antibodies used were rabbit anti-Car1 (1:2000, Abcam, Cat: ab108367), mouse anti-β-actin (1:10000, Proteintech, Cat: 66009-1-Ig), mouse anti-GAPDH (1:10000, Proteintech, Cat: 60004-1-Ig); secondary antibodies used in experiments were (goat Anti-Mouse IgG (H + L)-HRP Conjugate (1:10000, Bio-Rad, Cat:1,706,516), goat Anti-Rabbit IgG (H + L)-HRP Conjugate (1:8000, Bio-Rad, Cat:1,706,515).

Techniques: Concentration Assay, Construct, Infection, Immunostaining, Injection, Over Expression, Fluorescence, Labeling

Journal: Acta Neuropathologica Communications

Article Title: Upregulation of carbonic anhydrase 1 beneficial for depressive disorder

doi: 10.1186/s40478-023-01545-6

Figure Lengend Snippet: Pharmacological activation of CAR or overexpression of CAR1 in vHPC astrocytes promoted anti-depression-like behavior. (A) The immobility time of FST after intraperitoneal injection with saline (n = 9) or L-Phe (10 mg/kg, n = 10; 15 mg/kg, n = 9). (B) Enzyme activity of CAR in the hippocampus after injecting saline (n = 5) and L-Phe (15 mg/kg, n = 5). (C, E) The images to show the cannula traces into the lateral ventricle (C) or into the vHPC (E). (D, F) The immobility time in FST 30 min after injection into the lateral ventricle (L-Phe2.5ug/ul, n = 4; saline, n = 6, D) or into the vHPC(L-Phe25 µg/side, n = 10; saline, n = 9, F). (G) Schematic representation of a construct andthe timeline of the experimental procedure; the bottom panel shows the overexpression with the Western blotting. (H) A representative confocal image after lenti-viral injection in the vHPC. (I) Behavior tests after CAR1 overexpression. (J) Schematic representation of an AAV construct with GFAP promoter and the timeline of experimental procedure; the bottom panel shows the overexpression with the Western blotting. (K) A representative confocal image of the vHPC after AAV injection. (L) The infection of astrocytes was confirmed by co-immunostaining with EGFP (green) and GFAP (red). (M) The immobility time in FST (GFAP-CAR1, n = 11; GFAP-Control, n = 7) after CSDS treatment

Article Snippet: Primary antibodies used were rabbit anti-Car1 (1:2000, Abcam, Cat: ab108367), mouse anti-β-actin (1:10000, Proteintech, Cat: 66009-1-Ig), mouse anti-GAPDH (1:10000, Proteintech, Cat: 60004-1-Ig); secondary antibodies used in experiments were (goat Anti-Mouse IgG (H + L)-HRP Conjugate (1:10000, Bio-Rad, Cat:1,706,516), goat Anti-Rabbit IgG (H + L)-HRP Conjugate (1:8000, Bio-Rad, Cat:1,706,515).

Techniques: Activation Assay, Over Expression, Injection, Activity Assay, Construct, Western Blot, Infection, Immunostaining

Journal: bioRxiv

Article Title: A novel Gorilla-derived oncolytic Adenovirus with natural selective replication in cancer cells

doi: 10.64898/2026.02.26.708271

Figure Lengend Snippet: (A) Schematic representation of the genetic structure of the GRAd32Fk25 EV20 SC vector. (B) A549, NCI-H727 or NCI-H1975 were infected with GRAd32Fk25 EV20 SC expressing a single-chain antibody targeting HER3, based on the EV20 sequence. Infection was performed at MOI 1-10-50-100, and supernatant-containing antibodies were separated on SDS-PAGE and blotted with an anti-human antibody. (C) MRC5 cells were infected either with GRAd32Fk25 expressing the EV20 SC, or with GRAd25 expressing the same EV20 SC at an MOI of 10 or 100. The supernatant-containing antibody were separated on SDS-PAGE and blotted with an anti-human antibody. (D) Affinity for HER3 (ECD) of supernatants containing GRAd-produced EV20 SC (1:2 or 1:10 diluted) derived from A549, NCI-H727, or NCI-H1975 infected with 100 MOI of GRAd32Fk25 EV20 SC or mock control. Affinity was evaluated through direct ELISA and absorbance values are expressed in OD (450nm) are reported. (E) Affinity for cell surface HER3 on A375m exposed to supernatants containing GRAd-produced EV20 SC derived from A549, NCI-H727, or NCI-H1975 infected with 100 MOI of GRAd32Fk25 EV20 SC or mock control. Affinity was evaluated by FACS and levels are expressed in arbitrary units (A.U.) of mean fluorescence intensity (M.F.I.). (F) Western blotting images for the evaluation of p-HER3, total HER3, p-Akt, total Akt, and actin (loading control) protein levels in A375m exposed to supernatants containing GRAd-produced EV20 SC (1:2 or 1:10 diluted) derived from A549, NCI-H727, or NCI-H1975 infected with 100 MOI of GRAd32Fk25 EV20 SC and treated or not with neuregulin-1β (NRG-1β). Equal amounts of protein samples were loaded per lane. All adenoviruses contained in the supernatants were heat-inactivated for 30 minutes at 56°C; EV20 is the positive control, EV20 heated is the positive control treated for 30 minutes at 56°C.

Article Snippet: For the evaluation of CD46 and CAR cell surface levels, 2-3 x 10 5 cells (MRC5, HUVEC, A549, NCI-H1299, NCI-H1975, NCI-H727) were collected and incubated with anti-human CD46 (1:50; mouse monoclonal; #12239-MM05, Sino Biological) or with anti-human CAR (1:50; rabbit monoclonal; #10799-R271, Sino Biological) for 30 min at 4°C.

Techniques: Plasmid Preparation, Infection, Expressing, Sequencing, SDS Page, Produced, Derivative Assay, Control, Direct ELISA, Fluorescence, Western Blot, Positive Control