Journal: The Journal of Neuroscience

Article Title: Neuregulin Directly Decreases Voltage-Gated Sodium Current in Hippocampal ErbB4-Expressing Interneurons

doi: 10.1523/JNEUROSCI.1420-12.2012

Figure Lengend Snippet: NRG1 decreases ErbB4+ neuron excitability. A , Neurons live-labeled with mAb77 viewed in the recording chamber using phase contrast (left) or fluorescent (right) microscopy; ErbB4+ (arrow) and ErbB4− (arrowhead) neurons are marked (magnification

Article Snippet: ErbB4 in parvalbumin-positive interneurons is critical for neuregulin 1 regulation of long-term potentiation.

Techniques: Labeling, Microscopy

Journal: The Journal of Neuroscience

Article Title: Neuregulin Directly Decreases Voltage-Gated Sodium Current in Hippocampal ErbB4-Expressing Interneurons

doi: 10.1523/JNEUROSCI.1420-12.2012

Figure Lengend Snippet: NRG1 application dramatically reduces voltage-gated sodium channel currents. A , B , Representative traces of Na v currents from an ErbB4+ interneuron before (baseline) and after NRG1 ( A ) or vehicle ( B ) application for 10 and 20 min. Far right shows overlaid

Article Snippet: ErbB4 in parvalbumin-positive interneurons is critical for neuregulin 1 regulation of long-term potentiation.

Techniques:

Journal: The Journal of Neuroscience

Article Title: Neuregulin Directly Decreases Voltage-Gated Sodium Current in Hippocampal ErbB4-Expressing Interneurons

doi: 10.1523/JNEUROSCI.1420-12.2012

Figure Lengend Snippet: NRG1 does not affect potassium currents. A , B , Representative traces of K v channel currents from ErbB4+ interneurons with NRG1 ( A ) or vehicle application ( B ) for baseline, 10 min, and 20 min recordings. C , D , Averaged I / V relationship for K + current density

Article Snippet: ErbB4 in parvalbumin-positive interneurons is critical for neuregulin 1 regulation of long-term potentiation.

Techniques:

Journal: Frontiers in Cellular Neuroscience

Article Title: Connecting Immune Cell Infiltration to the Multitasking Microglia Response and TNF Receptor 2 Induction in the Multiple Sclerosis Brain

doi: 10.3389/fncel.2020.00190

Figure Lengend Snippet: TNFR2 protein expression in the normal-appearing white matter in the MS brain. Immunostaining for TNFR2 (A) and double immunofluorescence for CD68 (green) and TNFR2 (red) (B) reveal absence of TNFR2 immunoreactivity in the non-pathological subcortical WM of two control cases (C25 and C30, respectively). Immunostainings for MOG (C) and MHC class II antigen (D) show intact myelin and widespread microglial activation in the NAWM surrounding an inflamed blood vessel (MS100 case); the inset in (D) shows a ramified MHC class II+ microglial cell at high magnification. Staining of an adjacent section for TNFR2 (E,F) shows immunoreactivity in numerous cells with microglial morphology throughout the same area. Absence of staining after incubation of an adjacent brain section with normal serum is shown in the inset in (E) . The inset in (F) shows a TNFR2+ cell with a ramified morphology at high magnification. Double immunofluorescence staining for CD68 (green) and TNFR2 (red) reveals presence of many CD68+ microglial cells co-expressing TNFR2 in the NAWM (G) . In the inset in (G) , two CD68+ TNFR2+ microglial cells are shown at high magnification. Double immunofluorescence stainings for TNFR2 and CNPase (H) or GFAP (I) reveal absence of TNFR2 immunoreactivity in oligodendrocytes and astrocytes in the NAWM. Sections in (A,C–F) are counterstained with hematoxylin. Nuclei are stained with DAPI (blue) in (B,G–I) . Bars: 500 μm in the inset in (E) ; 200 μm in (A,C,D) ; 100 μm in (B,E,G) ; 50 μm in (F) ; 20 μm in (I) and insets in (D,F,G) ; 10 μm in (H) .

Article Snippet: Furthermore, only TNFR2, but not TNFR1, correlated strongly with genes involved in macrophage effector functions (Factor 2) ( ).

Techniques: Expressing, Immunostaining, Immunofluorescence, Activation Assay, Staining, Incubation, Double Immunofluorescence Staining

Journal: Frontiers in Cellular Neuroscience

Article Title: Connecting Immune Cell Infiltration to the Multitasking Microglia Response and TNF Receptor 2 Induction in the Multiple Sclerosis Brain

doi: 10.3389/fncel.2020.00190

Figure Lengend Snippet: TNFR2 protein expression in subpial gray matter lesions. Rare TNFR2+ CD68+ cells with macrophage ( A , arrow) or microglia morphology ( B , arrow) are present at the edge of two different chronic active subpial GM lesions (MS160 and MS180 case, respectively). TNFR2 immunoreactivity is observed in cells resembling reactive astrocytes in a chronic active subpial GM lesion (MS79 case) (C) ; a TNFR2+ GFAP+ astrocyte present in the same area is shown in (D) . Double immunofluorescence staining for CD68 (green) and TNFR2 (red) shows presence of many CD68+ TNFR2+ cells with a macrophage morphology in an active subpial GM lesion (MS79 case) (E) ; a TNFR2+ CD68+ macrophage is shown at high magnification in the inset. Bars: 50 μm in (A,C,E) ; 20 μm in (B,D) ; 10 μm in the inset in (E) .

Article Snippet: Furthermore, only TNFR2, but not TNFR1, correlated strongly with genes involved in macrophage effector functions (Factor 2) ( ).

Techniques: Expressing, Double Immunofluorescence Staining

Journal: Frontiers in Cellular Neuroscience

Article Title: Connecting Immune Cell Infiltration to the Multitasking Microglia Response and TNF Receptor 2 Induction in the Multiple Sclerosis Brain

doi: 10.3389/fncel.2020.00190

Figure Lengend Snippet: Regulation of TNF receptors and microglia/macrophage markers during demyelination and remyelination in mouse cerebellar slices. Mouse cerebellar slices maintained in vitro for 7 days were grown in the absence or presence of lysolecithin (0.5 mg/ml) for 16 h and then cultured in normal medium. (A,B) Slices were collected immediately (0 days), at 2 days and 4 days after toxin removal. RNA was extracted and reverse transcribed and the expression of the indicated genes was investigated using real time RT-PCR. Data are expressed as 2 −ΔCt relative to GAPDH. (C) Culture supernatants were collected at 8, 24, and 48 h after toxin removal and the amount of soluble TNFR1 and TNFR2 was measured using specific ELISA. Means ± SEM of 3 experiments are shown. * p

Article Snippet: Furthermore, only TNFR2, but not TNFR1, correlated strongly with genes involved in macrophage effector functions (Factor 2) ( ).

Techniques: In Vitro, Cell Culture, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Cellular Neuroscience

Article Title: Connecting Immune Cell Infiltration to the Multitasking Microglia Response and TNF Receptor 2 Induction in the Multiple Sclerosis Brain

doi: 10.3389/fncel.2020.00190

Figure Lengend Snippet: TNFR1 and TNFR2 gene expression in microdissected white matter and gray matter from control and MS brains. The graphs depict the differences in TNFR1 and TNFR2 gene expression between the indicated control and MS brain parenchymal areas. Data are expressed as 2 −ΔCt relative to the housekeeping gene GAPDH. Comparisons between control and MS WM and GM areas were performed using the Mann-Whitney test; statistically significant differences ( p

Article Snippet: Furthermore, only TNFR2, but not TNFR1, correlated strongly with genes involved in macrophage effector functions (Factor 2) ( ).

Techniques: Expressing, MANN-WHITNEY

Journal: Frontiers in Cellular Neuroscience

Article Title: Connecting Immune Cell Infiltration to the Multitasking Microglia Response and TNF Receptor 2 Induction in the Multiple Sclerosis Brain

doi: 10.3389/fncel.2020.00190

Figure Lengend Snippet: TNFR2 protein expression in actively demyelinating and chronic active white matter lesions in the MS brain. Immunostainings for MOG (A) , MHC class II antigen (B) , and TNFR2 (C) in serial MS brain sections (MS180 case) show an actively demyelinating WM lesion containing numerous TNFR2+ cells with a macrophage-like morphology; the asterisk marks the perivascular immune infiltrate in the center of the lesion (barely visible in the section stained for TNFR2). The frame in (C) marks the area shown at higher magnification in (D) ; the inset in (D) shows two macrophage-like TNFR2+ cells. Immunostainings for CD68 (E) and MOG (F) in serial brain sections of case MS121 show the presence of foamy macrophages throughout a subcortical actively demyelinating WM lesion; the frame in (E) marks the area shown at high magnification in the inset. Double immunofluorescence staining for MOG (green, G ) and the macrophage marker Iba-1 (red, H ) shows Iba-1+ macrophages containing MOG (merge, I ). Double immunofluorescence staining for CD68 (green) and TNFR2 (red) (J,K) reveals the presence of numerous CD68+ macrophages expressing TNFR2 in the same area shown in (E,F) ; TNFR2+ CD68+ foamy macrophages are shown at high power magnification in (L,M) . Inset in (J) shows absence of staining in an adjacent brain section after replacement of primary antibodies with a mixture of preimmune rabbit serum and unconjugated mouse IgG1. MOG (N) , MHC class II antigen (O) , and CD68 (P) immunostainings in brain sections of MS79 case identify a subcortical chronic WM lesion with an active border (rim) and an inactive, demyelinated core (center). CD68 immunoreactivity is present throughout the lesion whereas MHC class II+ cells localize in the lesion border. Immunostaining for TNFR2 (Q) reveals that this receptor is expressed in many cells with microglia and macrophage morphologies in the lesion border; the inset in (Q) shows one TNFR2+ cell displaying the typical microglial morphology at high magnification. Double immunofluorescence staining for CD68 (green, R ) and TNFR2 (red, S ) in a serial section confirms the expression of TNFR2 in numerous CD68+ microglial cells and macrophages in the active border of the chronic WM lesion (merge, T ); one CD68+ TNFR2+ cell with a bipolar morphology is shown at high magnification in the inset in (T) . Double immunofluorescence for GFAP (green) and TNFR2 (red) (U) reveals the presence of an occasional TNFR2+ astrocyte (arrow) in the rim of the same chronic active WM lesion. Sections in (A–F) and (N–Q) are counterstained with hematoxylin; nuclei are stained with DAPI (blue) in (G–M) and (R–U) . Bars: 200 μm in (A–C,E,F,N–P) and inset in (J) ; 100 μm in (Q,R–T) ; 50 μm in (D,J) ; 20 μm in (G–I,K–M,U) and insets in (D,Q) ; 10 μm in the inset in (T) .

Article Snippet: Furthermore, only TNFR2, but not TNFR1, correlated strongly with genes involved in macrophage effector functions (Factor 2) ( ).

Techniques: Expressing, Staining, Double Immunofluorescence Staining, Marker, Immunostaining, Immunofluorescence

Journal: Frontiers in Cellular Neuroscience

Article Title: Connecting Immune Cell Infiltration to the Multitasking Microglia Response and TNF Receptor 2 Induction in the Multiple Sclerosis Brain

doi: 10.3389/fncel.2020.00190

Figure Lengend Snippet: Effect of selective TNFR2 activation on MBP expression in remyelinating cerebellar slices. Cerebellar slices from P10 mice were grown for 7 days in vitro and then treated with lysolecithin (0.5 mg/ml) for 16 h. Medium was then replaced with fresh medium containing TNFR2 mAb (2 μg/ml) or rat IgG2a (2 μg/ml) as isotype control. (A) After 4 days slices were collected, RNA was extracted and reverse transcribed and MBP RNA expression was assessed by real time RT-PCR. Data are expressed as fold increase of each RNA (normalized to GAPDH) in TNFR2 mAb-treated slices relatively to isotype control slices (2 −ΔΔCt ). (B) After 7 days in vitro cerebellar slices were immunostained for MBP and NFH and the index of MBP protein expression was calculated as the ratio between the immunofluorescence intensity of MBP and NFH; one representative experiment of 3 performed is shown. Means ± SEM of three independent experiments are shown; ** p

Article Snippet: Furthermore, only TNFR2, but not TNFR1, correlated strongly with genes involved in macrophage effector functions (Factor 2) ( ).

Techniques: Activation Assay, Expressing, Mouse Assay, In Vitro, RNA Expression, Quantitative RT-PCR, Immunofluorescence

Journal: AMB Express

Article Title: Improving the temperature characteristics and catalytic efficiency of a mesophilic xylanase from Aspergillus oryzae, AoXyn11A, by iterative mutagenesis based on in silico design

doi: 10.1186/s13568-017-0399-9

Figure Lengend Snippet: The temperature characteristics of four recombinant xylanases. a The T opt of xylanase was measured, at pH optimum, at temperatures ranging from 40 to 70 °C. b The t 1/2 50 of xylanase was assayed by incubated it at 50 °C for different times

Article Snippet: Enzyme activity and protein assays Xylanase activity was assayed by measuring the amount of reducing sugars from birchwood xylan (Sigma, St. Louis, MO, USA) using the 3,5-dinitrosalicylic acid (DNS) method (Gao et al. ).

Techniques: Recombinant, Incubation

Journal: bioRxiv

Article Title: Recombinant SARS-CoV-2 spike S1-Fc fusion protein induced high levels of neutralizing responses in nonhuman primates

doi: 10.1101/2020.04.21.052209

Figure Lengend Snippet: Virus neutralizing activities of antisera from either S1-Fc-immunized animals or COVID-19 convalescence patient A) The neutralizing activity of rabbit sera was evaluated with SARS-CoV-2 pseudo-virus. The horizontal coordinate is Log10 serum dilution and the vertical coordinate is the percentage of the neutralization. B) The neutralizing titer of monkey and COVID-19 Convalescent patient’s sera was examined using live SARS-CoV-2. The vertical coordinate is the neutralizing titer.

Article Snippet: We thank Dr. Qiang Gao and his team from SinoVac for performing the live SARS-CoV-2 neutralizing assay, Dr. Jianqing Xu at Fudan Univ. for providing the ACE2-transfected HEK 293T cells, Dr. Pinliang Hu at Beijing Beyond Biotech. for purification of S1-Fc protein.

Techniques: Activity Assay, Neutralization

Journal: bioRxiv

Article Title: Recombinant SARS-CoV-2 spike S1-Fc fusion protein induced high levels of neutralizing responses in nonhuman primates

doi: 10.1101/2020.04.21.052209

Figure Lengend Snippet: Anti-SARS-CoV-2 S1 antibody levels in S1-Fc immunized mice and rabbits A) Serum samples collected from different mice on different days were evaluated with S1-His coated plates by ELISA using mouse IgG-specific secondary antibodies. The vertical coordinate is IgG titer. B) Serum samples collected from different rabbits on different days were evaluated with S1-His coated plates by ELISA using rabbit IgG-specific secondary antibodies. The horizontal coordinate is dilution and the vertical coordinate is absorbance value at 450nm.

Article Snippet: We thank Dr. Qiang Gao and his team from SinoVac for performing the live SARS-CoV-2 neutralizing assay, Dr. Jianqing Xu at Fudan Univ. for providing the ACE2-transfected HEK 293T cells, Dr. Pinliang Hu at Beijing Beyond Biotech. for purification of S1-Fc protein.

Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Recombinant SARS-CoV-2 spike S1-Fc fusion protein induced high levels of neutralizing responses in nonhuman primates

doi: 10.1101/2020.04.21.052209

Figure Lengend Snippet: Anti-SARS-CoV-2 S1 antibody levels in S1-Fc immunized monkeys A) The titers of total anti-S1 antibodies in monkey serum. Sera were evaluated by ELISA using HRP-conjugated goat-anti-human IgG (H+L) secondary antibodies. The horizontal coordinate is time and the vertical coordinate is the titer of anti-S1 antibodies in the sera. B) The levels of IgG and IgM in monkey sera. Sera were examined with either HRP-conjugated goat anti human IgG Fc-specific secondary antibodies (1:20000) or HRP-conjugated mouse monoclonal anti-μ-Chain of human IgM (1:5000). The horizontal coordinate is time and the vertical coordinate is absorbance value at 450nm.

Article Snippet: We thank Dr. Qiang Gao and his team from SinoVac for performing the live SARS-CoV-2 neutralizing assay, Dr. Jianqing Xu at Fudan Univ. for providing the ACE2-transfected HEK 293T cells, Dr. Pinliang Hu at Beijing Beyond Biotech. for purification of S1-Fc protein.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Small Rho GTPases and the Effector VipA Mediate the Invasion of Epithelial Cells by Filamentous Legionella pneumophila

doi: 10.3389/fcimb.2018.00133

Figure Lengend Snippet: Rho family small GTPases are needed for the attachment of filamentous Lp to LECs. Recruitment of Cdc42 (A) , Rac1 (B) , and RhoA (C) at the membrane wraps formed by Lp attachment. Cells transiently expressing Cdc42-GFP, Rac1-GFP or RhoA-GFP were infected for 2 h, fixed and external bacteria were immunolabeled (blue). Panels to the right show higher magnifications of framed areas. (D) Quantification of Rho GTPase recruitment at membrane wraps from (A–C) . Means ± SEM from 3 independent experiments are shown (n > 25 bacteria per experiment). (E,F) Confocal micrographs showing the recruitment of PAK-PBD-YFP and rGBD-GFP at the membrane wraps formed by Lp 2h p.i. (G) Quantification from (E,F) . Means ± SEM from 3 independent experiments are shown ( n > 25 bacteria in each experiment). (H–J) Lp attachment to NCI-H292 cells following inhibition of Cdc42, Rac1, and Rho by ML141, Nsc23766 (NSC) and C3 transferase (C3) respectively. Cells were treated with the indicated inhibitors for 1 h followed by infection with Lp for 1 h in the presence of the inhibitors. Quantification of bacterial attachment, normalized to vehicle treated cells is shown. (K–M) Attachment of Lp to cells expressing wild-type or dominant-negative (DN) forms of Rho GTPases. Attachment of bacteria to cells pre-treated for 1 h with inhibitor for mDia (N) or cells expressing dominant negative form of mDia (mDia-DN) (O) . Attachment of bacteria to cells pre-treated with CK666, Y27632 or blebbistatin to inhibit Arp2/3, ROCK or myosin II respectively (P–R) . Data shown are means ± SEM where bacterial attachment to at least 200 cells was analyzed. The number of independent experiments analyzed for each treatment were as follows: (H,I,K–P,R) n = 3; (J,Q) n = 4. Vehicle controls for each condition were as follows: (H,N–R) DMSO; (I) water; (J) glycerol. For all fluorescence micrographs the main panels are merged z-stacks and the higher magnifications are single z-planes. All scale bars shown, 12 μm. Statistical tests were performed using Student's t -test, *** p

Article Snippet: The following inhibitors were used in this study: PP2 (25 μM, Tocris) (Hanke et al., ), (100 μM, Sigma) (Vlahos et al., ), membrane permeable C3 transferase (0.5 μg/mL, Cytoskeleton Inc.) (Ridley and Hall, ), ML141 (20 μM, Tocris) (Surviladze et al., ), Blebbistatin (200 μM, Sigma) (Straight et al., ), Nsc23766 (50 μM, Tocris) (Gao et al., ), ROCK (1 μM, Millipore) (Narumiya et al., ), SMIFH2 (25 μM, Millipore) (Rizvi et al., ), CK-666 (80 μM, Sigma) (Nolen et al., ).

Techniques: Expressing, Infection, Immunolabeling, Inhibition, Dominant Negative Mutation, Fluorescence

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Small Rho GTPases and the Effector VipA Mediate the Invasion of Epithelial Cells by Filamentous Legionella pneumophila

doi: 10.3389/fcimb.2018.00133

Figure Lengend Snippet: Rho family small GTPases are needed for the attachment of filamentous Lp to LECs. Recruitment of Cdc42 (A) , Rac1 (B) , and RhoA (C) at the membrane wraps formed by Lp attachment. Cells transiently expressing Cdc42-GFP, Rac1-GFP or RhoA-GFP were infected for 2 h, fixed and external bacteria were immunolabeled (blue). Panels to the right show higher magnifications of framed areas. (D) Quantification of Rho GTPase recruitment at membrane wraps from (A–C) . Means ± SEM from 3 independent experiments are shown (n > 25 bacteria per experiment). (E,F) Confocal micrographs showing the recruitment of PAK-PBD-YFP and rGBD-GFP at the membrane wraps formed by Lp 2h p.i. (G) Quantification from (E,F) . Means ± SEM from 3 independent experiments are shown ( n > 25 bacteria in each experiment). (H–J) Lp attachment to NCI-H292 cells following inhibition of Cdc42, Rac1, and Rho by ML141, Nsc23766 (NSC) and C3 transferase (C3) respectively. Cells were treated with the indicated inhibitors for 1 h followed by infection with Lp for 1 h in the presence of the inhibitors. Quantification of bacterial attachment, normalized to vehicle treated cells is shown. (K–M) Attachment of Lp to cells expressing wild-type or dominant-negative (DN) forms of Rho GTPases. Attachment of bacteria to cells pre-treated for 1 h with inhibitor for mDia (N) or cells expressing dominant negative form of mDia (mDia-DN) (O) . Attachment of bacteria to cells pre-treated with CK666, Y27632 or blebbistatin to inhibit Arp2/3, ROCK or myosin II respectively (P–R) . Data shown are means ± SEM where bacterial attachment to at least 200 cells was analyzed. The number of independent experiments analyzed for each treatment were as follows: (H,I,K–P,R) n = 3; (J,Q) n = 4. Vehicle controls for each condition were as follows: (H,N–R) DMSO; (I) water; (J) glycerol. For all fluorescence micrographs the main panels are merged z-stacks and the higher magnifications are single z-planes. All scale bars shown, 12 μm. Statistical tests were performed using Student's t -test, *** p

Article Snippet: The following inhibitors were used in this study: PP2 (25 μM, Tocris) (Hanke et al., ), (100 μM, Sigma) (Vlahos et al., ), membrane permeable C3 transferase (0.5 μg/mL, Cytoskeleton Inc.) (Ridley and Hall, ), ML141 (20 μM, Tocris) (Surviladze et al., ), Blebbistatin (200 μM, Sigma) (Straight et al., ), Nsc23766 (50 μM, Tocris) (Gao et al., ), ROCK (1 μM, Millipore) (Narumiya et al., ), SMIFH2 (25 μM, Millipore) (Rizvi et al., ), CK-666 (80 μM, Sigma) (Nolen et al., ).

Techniques: Expressing, Infection, Immunolabeling, Inhibition, Dominant Negative Mutation, Fluorescence