Journal: International Journal of Molecular Sciences

Article Title: NGF Enhances CGRP Release Evoked by Capsaicin from Rat Trigeminal Neurons: Differential Inhibition by SNAP-25-Cleaving Proteases

doi: 10.3390/ijms23020892

Figure Lengend Snippet: BoNT/A blocked NGF-induced CGRP release and -enhancement of 20 nM CAP-evoked CGRP release. ( A ) After 2 days in the presence of 50 ng/mL NGF, TGNs were starved of the neurotrophin as detailed before, without or with the inclusion of 100 nM BoNT/A during this latter step. The release experiment was performed as described in A. ( B ) At the end of the protocol, one well each of BoNT/A-treated and non-treated cells were solubilised in 1× LDS and subjected to Western blotting. PVDF membranes were cut horizontally midway between the 25 k and 37 k molecular weight markers. The upper portion was exposed to antibodies reactive with syntaxin-1 (mouse monoclonal, 1:2000) and the lower piece probed with an antibody recognising both intact and BoNT/A-truncated SNAP-25 (mouse monoclonal, 1:3000). ( C ) The amount of cleaved SNAP-25 in BoNT/A-treated cells was calculated as a % (mean + s.e.m., N = 3) of the total SNAP-25 (intact + BoNT/A product). ( D ) Total CGRP (pg/well). ( E – G ) Histograms showing: ( E ) spontaneous CGRP release during the second 30 min. incubation into HBS without (grey bars) or induced by 100 ng/mL NGF (blue bars), ( F ) during the third period evoked by 20 nM CAP (minus the spontaneous release), and ( G ) during the third incubation with 1 µM CAP minus the spontaneous release. In all cases, CGRP release is expressed as a % of total CGRP (mean + s.e.m., N = 3, n = 9). Asterisks summarise the results of unpaired one-tailed Welch tests applied to the data plotted in panels E, F, and G, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: NGF 2.5S and antibodies to NGF (AN-240) and TRPV1 (ACC-030; used to determine TRPV1 expression by Western blotting) were supplied by Alomone Labs (Jerusalem, Israel).

Techniques: Western Blot, Molecular Weight, Incubation, One-tailed Test

Journal: International Journal of Molecular Sciences

Article Title: NGF Enhances CGRP Release Evoked by Capsaicin from Rat Trigeminal Neurons: Differential Inhibition by SNAP-25-Cleaving Proteases

doi: 10.3390/ijms23020892

Figure Lengend Snippet: Chimera/EA effectively inhibits 1 µM CAP-evoked CGRP release from starved TGNs, and its enhancement by NGF. TGNs were starved and incubated with 100 nM/EA using a protocol identical to that described previously for BoNT/A ( A). ( A ) Western blotting with anti-SNAP-25 antibodies confirms the disappearance of intact SNAP-25 and the appearance of a much faster-migrating product in cells exposed to/EA (+) but not control (−). ( B ) Histogram displaying the % of SNAP-25 cleaved, which was calculated as in C. ( C ) Total amounts (pg/well) of CGRP, determined as before, in control and/EA-treated cells. ( D ) Amounts of spontaneous CGRP release (% total) into HBS only, (grey bars), and that during incubation with 100 ng/mL NGF (blue bars), and ( E ) upon stimulation with 1 µM CAP (minus the spontaneous release) (mean + s.e.m. N = 3, n = 9). Unpaired one-tailed Welch test was applied to the data plotted in panels ( C – E ), * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: NGF 2.5S and antibodies to NGF (AN-240) and TRPV1 (ACC-030; used to determine TRPV1 expression by Western blotting) were supplied by Alomone Labs (Jerusalem, Israel).

Techniques: Incubation, Western Blot, Control, One-tailed Test

Journal: International Journal of Molecular Sciences

Article Title: NGF Enhances CGRP Release Evoked by Capsaicin from Rat Trigeminal Neurons: Differential Inhibition by SNAP-25-Cleaving Proteases

doi: 10.3390/ijms23020892

Figure Lengend Snippet: NGF induces a minor increase in Ca 2+ - and SNARE-dependent CGRP release, whereas it greatly enhances the CAP-evoked exocytosis which is blocked by BoNT/A and/EA at low [CAP] but only abolished by BoNT/EA at higher [CAP]. ( A ) Illustrates the effect of acute NGF on CGRP exocytosis from control neonatal rat TGNs starved of the neurotrophin for 2 days, and ( B ) in TGNs pre-treated with BoNT/A or /EA. ( A ) NGF binds to its receptor TrkA, activates the signalling cascades shown , and induces Ca 2+ influx by an unidentified mechanism ( ? ). Elevated intracellular Ca 2+ ([Ca 2+ ] i ) triggers the fusion of large dense core vesicles (LDCVs) via SNARE-complexes (VAMP, syntaxin-1 and SNAP-25), thereby, causing exocytotic release of CGRP and surface delivery of vesicle constituents. This acute potentiation by NGF can involve the phosphatidylinositol 3-kinase—Src (PI3K-Src) pathway, which promotes trafficking of LDCVs, and insertion of their TRPV1 channels into the plasmalemma by Ca 2+ -regulated exocytosis (blue arrows) c.f. [ , ]. Additionally, the phospholipase C γ (PLCγ) cascade leads to sensitisation of TRPV1 already on the plasmalemma (red dashed arrows) . The outcome of these composite influences of NGF on TRPV1 is that when the channel is activated by CAP [Ca 2+ ] I is raised even more than normally [ , , ] and this further enhances CGRP release . ( B ) The proteases of BoNT/A and/EA delete 9 (purple arrow) and 26 (green arrow) residues from SNAP-25 (Insert), respectively, preventing the fusion of LDCVs; this blocks the minimal CGRP exocytosis elicited by NGF (arrow with crosses, left) and its enhancement of the release evoked by 20 nM CAP ( ) (arrow with crosses, centre). Stronger stimulation of TRPV1 with 1 µM CAP ( ) induces a lot more Ca 2+ influx ( [Ca 2+ ] i , ( [Ca 2+ ] i , ( [Ca 2+ ] i ; ) which causes a moderate increase in CGRP release but overcomes the inhibition by BoNT/A (purple cross with broken lines, right) while/EA (green cross, right) remains effective in diminishing CGRP release. Acute sensitisation by NGF of TRPV1 selectively enhances neuropeptide exocytosis stimulated by low [CAP] (<100 nM) and only moderately affects responses to ≥100 nM CAP ( B). Despite being impotent against 1 µM CAP-evoked CGRP release in starved cells (( B ), , and G), BoNT/A partially inhibits the moderate NGF-enhancement of 1 µM CAP-evoked CGRP release ( G), implicating membrane trafficking in the sensitisation process (B, PI3K-Src stimulated pathway) in accordance with its inhibition of NGF-induced, Ca 2+ -dependent CGRP exocytosis ( E).

Article Snippet: NGF 2.5S and antibodies to NGF (AN-240) and TRPV1 (ACC-030; used to determine TRPV1 expression by Western blotting) were supplied by Alomone Labs (Jerusalem, Israel).

Techniques: Control, Inhibition, Membrane

Journal: EBioMedicine

Article Title: Molecular mechanisms underlying altered neurobehavioural development of female offspring of mothers with polycystic ovary syndrome: FOS-mediated regulation of neurotrophins in placenta

doi: 10.1016/j.ebiom.2020.102993

Figure Lengend Snippet: Clinical characteristics of subjects.

Article Snippet: Serum biochemical indexes including, total testosterone (TT) (80979, 0.05 ng/mL, Crystal Chem Inc, USA), sex hormone-binding globulin (SHBG) (RAB0734, 1.2 pmol/L, Sigma-Aldrich, USA), BDNF (RAB0026, 80 pg/mL, Sigma-Aldrich, USA), NGF (CSB-E04683h, 6.86pg/mL, Cusabio, USA), interleukin 6 (IL6) (BMS213-2, 0.92 pg/mL, Invitrogen, USA), and interleukin 15 (IL15) (BMS2106, 3.4 pg/mL, Invitrogen, USA) were measured with enzyme-linked immunosorbent assay (ELISA) in maternal peripheral blood and umbilical cord blood.

Techniques: Control

Journal: EBioMedicine

Article Title: Molecular mechanisms underlying altered neurobehavioural development of female offspring of mothers with polycystic ovary syndrome: FOS-mediated regulation of neurotrophins in placenta

doi: 10.1016/j.ebiom.2020.102993

Figure Lengend Snippet: Brain derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in placental villi. (a) Visualization of BDNF and NGF in placental villi of polycystic ovarian syndrome (PCOS) and control group by immunofluorescence with BDNF (green) and NGF (red). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue fluorescence). The syncytiotrophoblasts layers are labeled with white arrows, and the results of protein quantification are shown by bar plots (n = 5 in each group). All scale bars are 50 μm. BDNF and NGF mRNA expression level were quantified by real-time polymerase chain reaction (real-time PCR). PCOS: n = 20; control: n = 32. Data were presented as mean ± SEM. *P < 0.05, unpaired two-tailed Student's t tests. (b) The methylation status at the promoter region of BDNF (blue) and NGF (purple) genes in the placental villi from women with PCOS and controls using bisulfite genomic sequencing (BSP) (n = 3 in each group, significance was determined by Chi-square test).

Article Snippet: Serum biochemical indexes including, total testosterone (TT) (80979, 0.05 ng/mL, Crystal Chem Inc, USA), sex hormone-binding globulin (SHBG) (RAB0734, 1.2 pmol/L, Sigma-Aldrich, USA), BDNF (RAB0026, 80 pg/mL, Sigma-Aldrich, USA), NGF (CSB-E04683h, 6.86pg/mL, Cusabio, USA), interleukin 6 (IL6) (BMS213-2, 0.92 pg/mL, Invitrogen, USA), and interleukin 15 (IL15) (BMS2106, 3.4 pg/mL, Invitrogen, USA) were measured with enzyme-linked immunosorbent assay (ELISA) in maternal peripheral blood and umbilical cord blood.

Techniques: Derivative Assay, Control, Immunofluorescence, Fluorescence, Labeling, Expressing, Real-time Polymerase Chain Reaction, Two Tailed Test, Methylation, Genomic Sequencing

Journal: EBioMedicine

Article Title: Molecular mechanisms underlying altered neurobehavioural development of female offspring of mothers with polycystic ovary syndrome: FOS-mediated regulation of neurotrophins in placenta

doi: 10.1016/j.ebiom.2020.102993

Figure Lengend Snippet: Identification of FOS and its regulation of neurotrophins in placenta of polycystic ovary syndrome (PCOS). (a) Heatmaps of the global DNA methylation in the promoter and gene expression data for placentae from women with PCOS and controls. (n = 3 in each group) (b) Scatter plot of global DNA methylation levels in placentae in controls (x-axis) compared to women with PCOS (y-axis). Point density is shown as blue shading; Peacock blue and purple dots indicate significant down-regulated and up-regulated cytosine phosphate guanine (CpG) sites associated with a methylation difference > 0.20. (c) Volcano plot to inspect differentially expressed genes in placentae from women with PCOS and controls, with fold change as the abscissa and -log10 (P value) as the ordinate. Peacock blue and purple splashes represent genes that significantly up or down regulated respectively. Gray splashes mean genes without significantly different expression. FOS gene is marked as red. (d) Gene Ontology (GO) terms enriched in differentially expressed genes in placentae from women with PCOS. (e) Neural GO terms enriched in differentially expressed gene (top) and methylated DNA (bottom) in placentae from women with PCOS. (f) FOS gene mRNA and protein expression levels in human placentae, as assessed by real-time polymerase chain reaction (real-time PCR) (PCOS: n = 20; control: n = 32) and immunofluorescence with FOS (green), and nuclear DNA (blue) labeling (n = 5 in each group). All scale bars are 50 μm. (g) The summary data of methylation status at the promoter region of FOS gene in the placental villi using bisulfite genomic sequencing (BSP) (n = 3 in each group, significance was determined by Chi-square test). (h) In silico analysis of the genomic DNA 5 kb upstream and downstream of BDNF (pIV) and NGF (pI) transcription start site (TSS). Detection of FOS (i) protein expression levels, BDNF (j) and NGF (k) mRNA expression levels in overexpression group (n = 3) and control group (n = 3). Data were presented as mean ± SEM. *P < 0.05, unpaired two-tailed Student's t tests.

Article Snippet: Serum biochemical indexes including, total testosterone (TT) (80979, 0.05 ng/mL, Crystal Chem Inc, USA), sex hormone-binding globulin (SHBG) (RAB0734, 1.2 pmol/L, Sigma-Aldrich, USA), BDNF (RAB0026, 80 pg/mL, Sigma-Aldrich, USA), NGF (CSB-E04683h, 6.86pg/mL, Cusabio, USA), interleukin 6 (IL6) (BMS213-2, 0.92 pg/mL, Invitrogen, USA), and interleukin 15 (IL15) (BMS2106, 3.4 pg/mL, Invitrogen, USA) were measured with enzyme-linked immunosorbent assay (ELISA) in maternal peripheral blood and umbilical cord blood.

Techniques: DNA Methylation Assay, Gene Expression, Methylation, Expressing, Real-time Polymerase Chain Reaction, Control, Immunofluorescence, Labeling, Genomic Sequencing, In Silico, Over Expression, Two Tailed Test

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Carotid Baroreceptor Stimulation Ameliorates Pulmonary Arterial Remodeling in Rats With Hypoxia‐Induced Pulmonary Hypertension

doi: 10.1161/JAHA.124.035868

Figure Lengend Snippet: A , Venn map showed DEGs among Con‐sham, Hypoxia‐sham and Hypoxia‐CBS group. B , CBS made a change in DEGs altered by hypoxia, which was shown in GO enrichment analysis. C , PPI network showed that the top hub genes of DEGs. D , Heat map showed the 21 hypoxia‐upregulated genes reversed by CBS treatment. E and F , The relative mRNA expression levels of POSTN ( E ) and BDNF ( F ) Con‐sham, n=3; Con‐CBS, n=3; Hypoxia‐sham, n=4; Hypoxia‐CBS, n=4 (normalized to GAPDH mRNA). Values were mean±SEM. * P <0.05, ** P <0.01, *** P <0.001. BDNF indicates brain‐derived neurotrophic factor; BP, biological process; CBS, carotid baroreceptor stimulation; CC, cellular component; Con, Control; DEGs, differentially expressed genes; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; GO, Gene Ontology; MF, molecular function; POSTN, periostin; and PPI, protein–protein interaction network.

Article Snippet: Norepinephrine (HY‐13715), prazosin (HY‐B0193A), U0126 (HY‐12031), recombinant BDNF (brain‐derived neurotrophic factor) protein (HY‐P74383), recombinant POSTN (periostin protein; HY‐P71196) were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Expressing, Derivative Assay, Control

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Carotid Baroreceptor Stimulation Ameliorates Pulmonary Arterial Remodeling in Rats With Hypoxia‐Induced Pulmonary Hypertension

doi: 10.1161/JAHA.124.035868

Figure Lengend Snippet: A , Immunofluorescence staining with α‐SMA for PASMC identification, scale bar=50 μm. B , PASMCs were treated with different concentrations of norepinephrine (0.01, 0.1, 1 μmol/L) for 48 hours. n=6. C and D , Different concentrations of Pra (0.1, 1, 10 μmol/L) or U0126 (40, 80, 160 μmol/L) inhibited norepinephrine‐induced cell viability. n=6. E and F , CCK8 and cell count assay were used to evaluate cell proliferation under different conditions. E , N=6; F , N=3. G through K , Representative immunoblot bands and the protein expression levels of p‐ERK1/2/ERK1/2 ratio ( H ), PCNA ( I ), POSTN ( J ), BMPR2 ( K ) in primary cultured PASMCs. n=3. L through N , Representative immunoblot bands ( G ) and the protein expression levels of BDNF , P‐TrkB , and TrkB under normoxic ( M ) or hypoxic conditions ( N ). Values were mean±SEM. * P <0.05, ** P <0.01, *** P <0.001. α‐SMA indicates a‐smooth muscle actin; BDNF, brain‐derived neurotrophic factor; BMPR2, bone morphogenetic protein receptor, type II; CCK8, cell counting kit‐8; DAPI, 4′,6‐diamidino‐2‐phenylindole; ERK1/2, extracellular signal regulated kinase 1/2; HPH, hypoxia‐induced pulmonary hypertension; NE, norepinephrine; PA, pulmonary artery; PASMCs, pulmonary artery smooth muscle cells; PCNA, proliferating cell nuclear antigen; P‐ERK1/2, phosphorylated‐extracellular signal regulated kinase 1/2; POSTN, periostin; Pra, prazosin; P‐TrkB, phosphorylated‐tyrosine kinase receptor B; and TrkB, tyrosine kinase receptor B.

Article Snippet: Norepinephrine (HY‐13715), prazosin (HY‐B0193A), U0126 (HY‐12031), recombinant BDNF (brain‐derived neurotrophic factor) protein (HY‐P74383), recombinant POSTN (periostin protein; HY‐P71196) were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Immunofluorescence, Staining, Cell Characterization, Western Blot, Expressing, Cell Culture, Derivative Assay, Cell Counting

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Carotid Baroreceptor Stimulation Ameliorates Pulmonary Arterial Remodeling in Rats With Hypoxia‐Induced Pulmonary Hypertension

doi: 10.1161/JAHA.124.035868

Figure Lengend Snippet: A , After the cells were transfected with Ad‐shPOSTN or Ad‐shNC for 96 hours, POSTN protein expression level was detected using Western blot to verify the silence efficiency. n=3. B and C , CCK8 and cell count assay were used to evaluate cell proliferation. B , n=6; C , n=3. D through F , Representative immunoblot bands ( D ) and the protein expression levels of PCNA ( E ) and BMPR2 ( F ). n=3. G , Representative immunoblot bands of BDNF , P‐TrkB , and TrkB in PASMCs. H and I , Bar graph showed the protein expression levels of BDNF , P‐TrkB , and TrkB under normoxic ( H ) or hypoxic ( I ) conditions. n=3. J through L , Representative immunoblot bands of BDNF , POSTN , and bar diagram showed the result of quantitative analysis after cell transfection with siBDNF or siNC under normoxic or hypoxic conditions. n=3. M and N , Effect of BDNF on POSTN expression as showed by western blot. O and P , CCK8 and cell count assay were performed to measure cell proliferation after stimulated by BDNF at 24 hours and 48 hours. n=3. Values were mean±SEM. * P <0.05, ** P <0.01, *** P <0.001. Ad‐shNC indicates adenovirus mediated negative control; Ad‐shPOSTN, adenovirus mediated POSTN silencing; BDNF, brain‐derived neurotrophic factor; BMPR2, bone morphogenetic protein receptor, type II; CCK8, cell counting kit‐8; NE, norepinephrine; PASMCs, pulmonary artery smooth muscle cells; PCNA, proliferating cell nuclear antigen; POSTN, periostin; P‐TrkB, phosphorylated‐tyrosine kinase receptor B; and TrkB, tyrosine kinase receptor B.

Article Snippet: Norepinephrine (HY‐13715), prazosin (HY‐B0193A), U0126 (HY‐12031), recombinant BDNF (brain‐derived neurotrophic factor) protein (HY‐P74383), recombinant POSTN (periostin protein; HY‐P71196) were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Transfection, Expressing, Western Blot, Cell Characterization, Negative Control, Derivative Assay, Cell Counting

Journal: Investigative Ophthalmology & Visual Science

Article Title: Crystallins Play a Crucial Role in Glaucoma and Promote Neuronal Cell Survival in an In Vitro Model Through Modulating Müller Cell Secretion

doi: 10.1167/iovs.63.8.3

Figure Lengend Snippet: Antibodies Used in Microarray Analysis

Article Snippet: Anti-nerve growth factor beta ( NGFb ) , ORB11126, Biorybt, Cambridge, United Kingdom.

Techniques: Microarray