Journal: Journal of Diabetes Research

Article Title: Plasma BDNF Is Reduced in Acute Ischemic Stroke Patients With Type 2 Diabetes Mellitus and Associated With Fibrin-Rich Thrombi

doi: 10.1155/jdr/6693363

Figure Lengend Snippet: BDNF lower in AIS/DM patients. (a) Expression of plasma BDNF in healthy controls, AIS/non-DM patients, and AIS/DM patients. (b) Representative images of immunohistochemical detection of BDNF (brown) in clots from AIS/non-DM and AIS/DM patients. (c) The column graph represents mean ± SEM of staining scores in each group. (d) Representative clots from AIS/non-DM and AIS/DM patients were stained using MSB to visualize the fibrin (dark pink to red), RBCs (yellow), and platelets (gray). (e) Column graph displays the differences in fibrin content in clots. (f) Fibrin fluorescence of clots from AIS patients. (g) The bar graph represents mean ± SEM fluorescence values.

Article Snippet: Plasma BDNF concentrations were measured using the Human BDNF ELISA Kit (intra/intervariability < 10%) (SEKH-0101, Solarbio) following the manufacturer's instructions, with all assays performed by a research assistant blinded to the clinical status of the participants.

Techniques: Expressing, Clinical Proteomics, Immunohistochemical staining, Staining, Fluorescence

Journal: Journal of Diabetes Research

Article Title: Plasma BDNF Is Reduced in Acute Ischemic Stroke Patients With Type 2 Diabetes Mellitus and Associated With Fibrin-Rich Thrombi

doi: 10.1155/jdr/6693363

Figure Lengend Snippet: The clot permeability and ultrastructure. (a) Thrombus permeability was significantly lower in AIS/DM patients than AIS patients without DM. (b) Correlation between BDNF concentrations and thrombus permeability. (c) BDNF levels in patients based on the severity of stroke. (d) Scanning electron micrographs of thrombi. Arrows indicate fibrin; dotted lines indicate fibrin band. Bar = 20 μ m. (e) Transmission electron micrographs of thrombi from the AIS/non-DM and AIS/DM patients. Arrows indicate fibrin; dotted lines indicate fibrin band. Bar = 5 μ m.

Article Snippet: Plasma BDNF concentrations were measured using the Human BDNF ELISA Kit (intra/intervariability < 10%) (SEKH-0101, Solarbio) following the manufacturer's instructions, with all assays performed by a research assistant blinded to the clinical status of the participants.

Techniques: Permeability, Transmission Assay

Journal: Journal of Diabetes Research

Article Title: Plasma BDNF Is Reduced in Acute Ischemic Stroke Patients With Type 2 Diabetes Mellitus and Associated With Fibrin-Rich Thrombi

doi: 10.1155/jdr/6693363

Figure Lengend Snippet: BDNF influences fibrin density. (a) A ribbon diagram illustrates the binding of BDNF to the heparin-binding domain of fibrin(ogen), with the structure generated via DMFold. (b) rHu-BDNF was added to plasma pools from AIS/DM patients before induction of coagulation with thrombin; then, the fibrin density of the clot was analyzed. Visualization images of clots using rabbit polyclonal fibrinogen/FITC (bar = 100 μ m) and (c) the percentage of fibrin fibers versus control. Fibrin fibers were analyzed using ImageJ software. All samples were performed in triplicate. Data are expressed as mean ± SEM, ⁣ ∗ p < 0.05.

Article Snippet: Plasma BDNF concentrations were measured using the Human BDNF ELISA Kit (intra/intervariability < 10%) (SEKH-0101, Solarbio) following the manufacturer's instructions, with all assays performed by a research assistant blinded to the clinical status of the participants.

Techniques: Binding Assay, Generated, Clinical Proteomics, Coagulation, Control, Software

Journal: Journal of Diabetes Research

Article Title: Plasma BDNF Is Reduced in Acute Ischemic Stroke Patients With Type 2 Diabetes Mellitus and Associated With Fibrin-Rich Thrombi

doi: 10.1155/jdr/6693363

Figure Lengend Snippet: Observing the fibrin diameter by scanning electron microscopy. (a) Fibrin structures formed from venous blood from AIS/DM subjects were added with different concentrations of BDNF. The ultrastructure of fibrin was observed by a scanning electron microscope. (b) The column chart shows the difference in the diameter of fibrin fibers in clots of different groups. ⁣ ∗ p < 0.05 and ⁣ ∗∗∗ p < 0.001.

Article Snippet: Plasma BDNF concentrations were measured using the Human BDNF ELISA Kit (intra/intervariability < 10%) (SEKH-0101, Solarbio) following the manufacturer's instructions, with all assays performed by a research assistant blinded to the clinical status of the participants.

Techniques: Electron Microscopy, Microscopy

Journal: Journal of Diabetes Research

Article Title: Plasma BDNF Is Reduced in Acute Ischemic Stroke Patients With Type 2 Diabetes Mellitus and Associated With Fibrin-Rich Thrombi

doi: 10.1155/jdr/6693363

Figure Lengend Snippet: rHu-BDNF influences fibrin polymerization, coagulation, and lysis. (a) Representative turbidity curves were recorded spectrophotometrically at 350 nm (37°C) with 30-s intervals. (b) Maximum turbidity values were determined under identical monitoring conditions. (c) Maximum clot firmness was assessed by thromboelastographic analyses (MA). Plasma samples supplemented with recombinant BDNF prior to thrombin/tPA-induced coagulation underwent subsequent polymerization analysis. (d) Thrombin clotting time (TT) and (e) lysis time were measured. All samples were performed in triplicate. Data are expressed as mean ± SEM, n = 3 different pools. ⁣ ∗ p < 0.05, ⁣ ∗∗ p < 0.01, and ⁣ ∗∗∗ p < 0.001.

Article Snippet: Plasma BDNF concentrations were measured using the Human BDNF ELISA Kit (intra/intervariability < 10%) (SEKH-0101, Solarbio) following the manufacturer's instructions, with all assays performed by a research assistant blinded to the clinical status of the participants.

Techniques: Coagulation, Lysis, Clinical Proteomics, Recombinant

Journal: bioRxiv

Article Title: LRRK2 Phosphorylates Neuronal Elav RNA-Binding Proteins to Regulate Phenotypes Relevant to Parkinson’s Disease

doi: 10.1101/2022.04.24.489327

Figure Lengend Snippet: (a) Western blot of immunoprecipitates of anti-Flag antibody or an isotype control antibody (IgG) from cells transfected with plasmid expressing Flag-HuD. (b) Log10 enrichment of the indicated mRNAs (x-axis) in immunoprecipitates of Flag-HuD from SH-SY5Y cells normalized to levels in IgG control immunoprecipitates measured by RT-qPCR in 4-6 independent experiments. * p<0.05, ** p<0.01, *** p<0.001 ANOVA with Tukey’s correction where multiple groups were compared to IgG control. (c) Western blot of LRRK2 phosphoserine-935 in cell lysates after treatment with control vehicle or GSK2578215A LRRK2 kinase inhibitor. (d) Western blot of Flag in immunoprecipitates of Flag-HuD from cells treated with control vehicle or GSK2578215A LRRK2 kinase inhibitor. (e) Quantification of RNAs by RT-qPCR in Flag-HuD immunoprecipitates from SH-SY5Y cells over four independent experiments from cells treated with control vehicle or GSK2578215A LRRK2 kinase inhibitor. (f) Western blot of LRRK2 in cell lysates after treatment with control siRNA or LRRK2 siRNA. (g) Western blot of Flag in immunoprecipitates of Flag-HuD from cells treated with control siRNA or LRRK2 siRNA. (h) Quantification of RNAs by RT-qPCR in Flag-HuD immunoprecipitates from SH-SY5Y cells treated with control siRNA or LRRK2 siRNA over 3-4 independent experiments. (i,j) Quantification of RNAs by RT-qPCR in immunoprecipitates of Flag-HuB (i) or Flag-HuC (j) from SH-SY5Y cells treated with control siRNA or LRRK2 siRNA over 2-4 independent experiments. (k) Quantification of RNAs by RT-qPCR in neuronal ELAV immunoprecipitates from midbrain of 3-4 wild-type and LRRK2 G2019S mice. (l) Quantification of gel-shift assay of HuD binding to γ -32P labeled U-rich RNA containing neuronal ELAV binding sites from BDNF 3’UTR or a control RNA from another segment of the BDNF 3’UTR. The effect of the presence of LRRK2 variants and LRRK2 kinase inhibitors (GSK2578215A [GSK], or HG-10-102-01 [HG]) was tested. * p<0.05, ** p<0.01, *** p<0.001 ANOVA with Tukey’s correction where multiple groups were compared, one-sample t-test (in e,h,I,j where one group was compared.

Article Snippet: Seventy-two hours after transfection cell culture media was centrifuged at 1500 x g for 10 min and supernatants were immediately used in the Quantikine Human BDNF Immunoassay (Catalogue DBD00, R&D Systems).

Techniques: Western Blot, Control, Transfection, Plasmid Preparation, Expressing, Quantitative RT-PCR, Gel Shift, Binding Assay, Labeling

Journal: bioRxiv

Article Title: LRRK2 Phosphorylates Neuronal Elav RNA-Binding Proteins to Regulate Phenotypes Relevant to Parkinson’s Disease

doi: 10.1101/2022.04.24.489327

Figure Lengend Snippet: (a) Coomassie total protein stain of recombinant GST, GST-HuD, LRRK2 WT, LRRK2 G2019S and LRRK2 D1994A. (b) Model of synthetic RNAs used in experiments and Supplementary Figure 1d. RNA derived from a neuronal ELAV binding segment of the BDNF 3’UTR which contains U-rich motifs or an independent segment of the BDNF 3’UTR that is not regulated by neuronal ELAVs . (c) Representative autoradiographic images of gel-shift assay using RNA probes as in (b) labeled with γ -32P and incubated with GST, GST-HuD, and LRRK2 variants +/- LRRK2 kinase inhibitors.

Article Snippet: Seventy-two hours after transfection cell culture media was centrifuged at 1500 x g for 10 min and supernatants were immediately used in the Quantikine Human BDNF Immunoassay (Catalogue DBD00, R&D Systems).

Techniques: Staining, Recombinant, Derivative Assay, Binding Assay, Gel Shift, Labeling, Incubation

Journal: bioRxiv

Article Title: LRRK2 Phosphorylates Neuronal Elav RNA-Binding Proteins to Regulate Phenotypes Relevant to Parkinson’s Disease

doi: 10.1101/2022.04.24.489327

Figure Lengend Snippet: (a) Western blot of levels of LRRK2 and HuD in SH-SY5Y cells after transfection with Flag-HuD or control plasmid, and control siRNA or LRRK2 siRNA (b) RT-qPCR for BDNF, α-synuclein (SNCA) and p21 mRNAs in SH-SY5Y cells after transfection with Flag-HuD or control plasmid, and control siRNA or LRRK2 siRNA (c) Western blot of levels of α -synuclein and BDNF in SH-SY5Y cells after transfection with Flag-HuD or control plasmid, and control siRNA or LRRK2 siRNA. Note that Westerns in (a) and (c) were performed on the same membrane and therefore use the same Tubulin control. (d) Quantification of the levels of α -synuclein and p21 as in (c). (e) ELISA of BDNF in cell supernatants of SH-SY5Y cells after transfection with Flag-HuD or control plasmid, and control siRNA or LRRK2 siRNA (f) Luciferase assays of neuronal ELAV binding sites in the BDNF 3’UTR in U2OS cells transfected with Flag-HuD and treated with control siRNA or LRRK2 siRNA. The control is an independent section of the BDNF 3’UTR that does not contain neuronal ELAV binding sites. * p<0.05, ** p<0.01, *** p<0.001 ANOVA with Tukey’s correction.

Article Snippet: Seventy-two hours after transfection cell culture media was centrifuged at 1500 x g for 10 min and supernatants were immediately used in the Quantikine Human BDNF Immunoassay (Catalogue DBD00, R&D Systems).

Techniques: Western Blot, Transfection, Control, Plasmid Preparation, Quantitative RT-PCR, Membrane, Enzyme-linked Immunosorbent Assay, Luciferase, Binding Assay

Journal: bioRxiv

Article Title: LRRK2 Phosphorylates Neuronal Elav RNA-Binding Proteins to Regulate Phenotypes Relevant to Parkinson’s Disease

doi: 10.1101/2022.04.24.489327

Figure Lengend Snippet: (a,b) Western blot (top) and quantification (bottom) of α -synuclein (a) and BDNF (b) in wild-type, HuD -/- , LRRK2 G2019S, and LRRK2 G2019S x HuD -/- mice of 4 weeks of age with dorsal midbrain (DM), ventral midbrain (VM) and striatum (STR) samples normalized to Tubulin (loading control). n=5-6 mice. (c) Western blot (left) and quantification (right) of LRRK2 in wild-type, HuD -/- , LRRK2 G2019S, and LRRK2 G2019S x HuD -/- mice of 4 weeks of age with dorsal midbrain (DM), and striatum (STR) samples normalized to Tubulin (loading control). n=5-6 mice. Note that blots for Lrrk2 and alpha-synuclein were performed on the same membranes and therefore use the same Tubulin control. * p<0.05, ** p<0.01, *** p<0.001 ANOVA with Tukey’s correction where multiple groups were compared, t-test (two tailed assuming unequal variance) where one group was compared.

Article Snippet: Seventy-two hours after transfection cell culture media was centrifuged at 1500 x g for 10 min and supernatants were immediately used in the Quantikine Human BDNF Immunoassay (Catalogue DBD00, R&D Systems).

Techniques: Western Blot, Control, Two Tailed Test