Journal: bioRxiv

Article Title: Using Extracellular Vesicles Released by GDNF-transfected Macrophages for Therapy of Parkinson’s Disease

doi: 10.1101/2022.05.25.493424

Figure Lengend Snippet: Plasmid map for GDNF production

Article Snippet: Human GDNF cDNA (NM_199234) was provided by OriGene (Rockville, MD, USA) that was propagated in DH5α E.coli, followed by purification Giga-prep kits (Qiagen, Valencia, CA, USA).

Techniques: Plasmid Preparation

Journal: bioRxiv

Article Title: Using Extracellular Vesicles Released by GDNF-transfected Macrophages for Therapy of Parkinson’s Disease

doi: 10.1101/2022.05.25.493424

Figure Lengend Snippet: Bone-marrow derived macrophages were transfected by electroporation using four different conditions described in Experimental section. Then, cells were washed and cultured in complete media for up to 6 days. The GDNF expression levels in cells (solid bars), and EVs collected from conditioned media (stripped bars) was assessed by ELISA on day 1 (white bars), day 4 (grey bars), and day 6 (black bars). Successful transfection was accomplished with three conditions (#2 - #4). N = 4, *p < 0.05, compared to sham-transfected macrophages (dashed line, condition #1).

Article Snippet: Human GDNF cDNA (NM_199234) was provided by OriGene (Rockville, MD, USA) that was propagated in DH5α E.coli, followed by purification Giga-prep kits (Qiagen, Valencia, CA, USA).

Techniques: Derivative Assay, Transfection, Electroporation, Cell Culture, Expressing, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Using Extracellular Vesicles Released by GDNF-transfected Macrophages for Therapy of Parkinson’s Disease

doi: 10.1101/2022.05.25.493424

Figure Lengend Snippet: Primary macrophages were transfected with GDNF-encoding p DNA by electroporation (condition #4), and EV-GDNF were collected from conditioned media on day 6. EV-GDNF were characterized for size, zeta potential, and morphology by ZetaView QUATT Nanoparticle Tracking Microscope PMX-420 ( A ), and AFM ( B ). The presence of EV-specific membrane proteins was EV-GDNF was confirmed by Wes ( C ) and quantified using Compass SW software ( D ). The bar: 1 µm.

Article Snippet: Human GDNF cDNA (NM_199234) was provided by OriGene (Rockville, MD, USA) that was propagated in DH5α E.coli, followed by purification Giga-prep kits (Qiagen, Valencia, CA, USA).

Techniques: Transfection, Electroporation, Zeta Potential Analyzer, Microscopy, Membrane, Software

Journal: bioRxiv

Article Title: Using Extracellular Vesicles Released by GDNF-transfected Macrophages for Therapy of Parkinson’s Disease

doi: 10.1101/2022.05.25.493424

Figure Lengend Snippet: EVs samples from sham-transfected (white bars), and GDNF-transfected (black bars) macrophages were digested ( N =3) with trypsin and examined by nano-liquid chromatography tandem MS (nanoLC–MS/MS) with multiple reaction monitoring (MRM). Samples of 20 µg total protein were used, and 0.08 µg (0.4 % of the sample) was injected. No significant differences in specific proteins expression were found between sham EVs and EV-GDNF (t-tests, p < 0.05). Peptide identification is shown in . A CD81 peptide employed in other studies was not detected in these analyses. Values are means ± SD.

Article Snippet: Human GDNF cDNA (NM_199234) was provided by OriGene (Rockville, MD, USA) that was propagated in DH5α E.coli, followed by purification Giga-prep kits (Qiagen, Valencia, CA, USA).

Techniques: Transfection, Liquid Chromatography, Tandem Mass Spectroscopy, Targeted Proteomics, Injection, Expressing

Journal: bioRxiv

Article Title: Using Extracellular Vesicles Released by GDNF-transfected Macrophages for Therapy of Parkinson’s Disease

doi: 10.1101/2022.05.25.493424

Figure Lengend Snippet: Macrophages were transfected with GDNF-encoding p DNA by electroporation, and the levels of GDNF-DNA in the cells ( A ) and EVs released by these cells ( B ) were assessed. A significant amount of GDNF-DNA was detected in parent cells, as well as in the EVs. Statistical significance was assessed by One Way ANOVA corrected for multiple comparisons using the FDR. ** p < 0.01, or **** p < 0.0001.

Article Snippet: Human GDNF cDNA (NM_199234) was provided by OriGene (Rockville, MD, USA) that was propagated in DH5α E.coli, followed by purification Giga-prep kits (Qiagen, Valencia, CA, USA).

Techniques: Transfection, Electroporation

Journal: bioRxiv

Article Title: Using Extracellular Vesicles Released by GDNF-transfected Macrophages for Therapy of Parkinson’s Disease

doi: 10.1101/2022.05.25.493424

Figure Lengend Snippet: The effect of EV-GDNF on motor functions and activity was assessed in Wire hanging test, and Rotarod test ( A, B ), as well as in OFA tests ( C, D ). ( A, B ) Transgenic mice were i.n . injected with EV-GDNF (triangles, 3×10 9 particles/10 µL/mouse), or sham EVs (empty circles, 3×10 9 particles/10 µL/mouse), or saline (filled circles, 10 µL/mouse). Wild type mice were i.n . injected with saline (filled squares, 10 µL/mouse) were used as controls. Wire hanging test ( A ), and Rotarod test ( B ) demonstrated significant improvements in motor functions upon treatment with EV-GDNF. ( C, D ) OFA tests at 12 mo. demonstrated improved behavior in EV-GDNF treated PD mice (striped bars) compared to PD mice treated with saline (white bars) that was similar as in healthy WT mice (black bars) including decreases in the hyperactivity and anxiety-like behavior. The differences between sham EVs and saline in PD mice were inconclusive. Values are means ± SEM ( N = 10), * p < 0.05, ** p < 0.005, and # p < 0.05, as compared to WT control.

Article Snippet: Human GDNF cDNA (NM_199234) was provided by OriGene (Rockville, MD, USA) that was propagated in DH5α E.coli, followed by purification Giga-prep kits (Qiagen, Valencia, CA, USA).

Techniques: Activity Assay, Transgenic Assay, Injection, Saline, Control

Journal: bioRxiv

Article Title: Using Extracellular Vesicles Released by GDNF-transfected Macrophages for Therapy of Parkinson’s Disease

doi: 10.1101/2022.05.25.493424

Figure Lengend Snippet: Transgenic mice (4 mo. old, N = 10) were i.n . injected with: saline (10 µL/mouse), or EV-GDNF (3×10 9 particles/10 µL/mouse), or sham EVs (3×10 9 particles/10 µL/mouse). Wild type control mice were intranasally injected with saline (10 µL/mouse). Animals were sacrificed at mo. 16, and brain slides were stained with TH, a marker for dopaminergic neurons ( A ); or Ab to CD11b for activated microglia ( B ). The images indicate significant preservation of TH-positive neurons and decrease in microglial activation in Parkin Q311(X)A mice upon EV-GDNF treatment compared to PD mice treated with saline. The administration of sham EVs did not cause significant therapeutic effects.

Article Snippet: Human GDNF cDNA (NM_199234) was provided by OriGene (Rockville, MD, USA) that was propagated in DH5α E.coli, followed by purification Giga-prep kits (Qiagen, Valencia, CA, USA).

Techniques: Transgenic Assay, Injection, Saline, Control, Staining, Marker, Preserving, Activation Assay

Journal: bioRxiv

Article Title: Using Extracellular Vesicles Released by GDNF-transfected Macrophages for Therapy of Parkinson’s Disease

doi: 10.1101/2022.05.25.493424

Figure Lengend Snippet: Transgenic mice (4 mo. old) were intranasally injected with: saline (10 µL/mouse), or EV-GDNF (3×10 9 particles/10 µL/mouse), or sham EVs (3×10 9 particles/10 µL/mouse). Wild type control mice were intranasally injected with saline (10 µL/mouse). Animals were sacrificed at mo. 16, brains were removed post-mortem, and homogenized in cell lysis buffer. Elevated cytokine levels in the brain, were recorded in PD mice treated with saline and Sham EVs. Administration of EV-GDNF significantly decreased pro-inflammatory molecules in the brain compared with PD mice treated with saline. N = 4, # p < 0.05 compared to healthy WT animals; * p < 0.05 compared to PD mice treated with saline, $ p <0.05 compared to PD mice treated with saline and sham EVs.

Article Snippet: Human GDNF cDNA (NM_199234) was provided by OriGene (Rockville, MD, USA) that was propagated in DH5α E.coli, followed by purification Giga-prep kits (Qiagen, Valencia, CA, USA).

Techniques: Transgenic Assay, Injection, Saline, Control, Lysis

Journal: bioRxiv

Article Title: Using Extracellular Vesicles Released by GDNF-transfected Macrophages for Therapy of Parkinson’s Disease

doi: 10.1101/2022.05.25.493424

Figure Lengend Snippet: Transgenic mice (4 mo. old) were intranasally injected with: saline (10 µL/mouse), or ( 3 ) EV-GDNF (3×10 9 particles/10 µL/mouse), or sham EVs (3×10 9 particles/10 µL/mouse). Wild type control mice were intranasally injected with saline (10 µL/mouse). Animals were sacrificed at mo. 16, brain slides were stained with Nissl staining ( A – D ) and H&E staining ( E – H ). The obtained bright light images show lower number of Nissl bodies with neuronal shrinkage ( B ) and damages tissues with degeneration in the neurons ( F ) in PD mice treated with saline when compared to WT mice ( A, E ). Histological analysis indicate neuroprotective effects in the brain of PD mice treated with EV-GDNF with healthy morphology in tissue structure and high integrity of neurons ( C, G ) when comparted to PD mice treated with saline ( B, F ). The administration of sham EVs did not have significant therapeutic effect in PD mice ( D, H ). Black arrows, degenerated neurons; blue arrows, elongated irregular nuclear morphology.

Article Snippet: Human GDNF cDNA (NM_199234) was provided by OriGene (Rockville, MD, USA) that was propagated in DH5α E.coli, followed by purification Giga-prep kits (Qiagen, Valencia, CA, USA).

Techniques: Transgenic Assay, Injection, Saline, Control, Staining

Journal: bioRxiv

Article Title: Using Extracellular Vesicles Released by GDNF-transfected Macrophages for Therapy of Parkinson’s Disease

doi: 10.1101/2022.05.25.493424

Figure Lengend Snippet: Transgenic mice (4 mo. old) were intranasally injected with: saline (10 µL/mouse), or EV-GDNF (3×10 9 particles/10 µL/mouse), or sham EVs (3×10 9 particles/10 µL/mouse) weekly three times. Wild type control mice were intranasally injected with saline (10 µL/mouse). Animals were sacrificed at mo. 16, brain slides were stained with Nissl staining. The obtained bright light images show lower number of Nissl bodies with neuronal shrinkage and damages tissues with degeneration in the neurons in PD mice treated with saline when compared to WT mice. Histological analysis indicates neuroprotective effects in the brain of PD mice treated with GDNF-EVs with healthy morphology in tissue structure and high integrity of neurons when comparted to PD mice treated with saline. The administration of sham EVs did not have significant therapeutic effect in PD mice.

Article Snippet: Human GDNF cDNA (NM_199234) was provided by OriGene (Rockville, MD, USA) that was propagated in DH5α E.coli, followed by purification Giga-prep kits (Qiagen, Valencia, CA, USA).

Techniques: Transgenic Assay, Injection, Saline, Control, Staining

Journal: bioRxiv

Article Title: Using Extracellular Vesicles Released by GDNF-transfected Macrophages for Therapy of Parkinson’s Disease

doi: 10.1101/2022.05.25.493424

Figure Lengend Snippet: Transgenic mice (4 mo. old) were intranasally injected with: saline (10 µL/mouse), or EV-GDNF (3×10 9 particles/10 µL/mouse), or sham EVs (3×10 9 particles/10 µL/mouse) weekly three times. Wild type control mice were intranasally injected with saline (10 µL/mouse). Animals were sacrificed at mo. 16, brain slides were stained with Nissl staining. The obtained bright light images show damaged tissues with degeneration in the neurons in PD mice treated with saline when compared to WT mice. Histological analysis indicates neuroprotective effects in the brain of PD mice treated with GDNF-EVs with healthy morphology in tissue structure when comparted to PD mice treated with saline. The administration of sham EVs did not have significant therapeutic effect in PD mice.

Article Snippet: Human GDNF cDNA (NM_199234) was provided by OriGene (Rockville, MD, USA) that was propagated in DH5α E.coli, followed by purification Giga-prep kits (Qiagen, Valencia, CA, USA).

Techniques: Transgenic Assay, Injection, Saline, Control, Staining

Journal: bioRxiv

Article Title: Using Extracellular Vesicles Released by GDNF-transfected Macrophages for Therapy of Parkinson’s Disease

doi: 10.1101/2022.05.25.493424

Figure Lengend Snippet: Transgenic mice (4 mo. of age) were i.n . injected with saline, or EV-GDNF, or sham EVs (3×10 9 particles/10 µL/mouse, once a week, 3x weeks). At 16 mo. of age total weigh of the animals was recorded. No gross toxicity manifested in the losing weight was detected in mice injected with EV-GDNF and well as sham EVs.

Article Snippet: Human GDNF cDNA (NM_199234) was provided by OriGene (Rockville, MD, USA) that was propagated in DH5α E.coli, followed by purification Giga-prep kits (Qiagen, Valencia, CA, USA).

Techniques: Transgenic Assay, Injection, Saline

Journal: Pharmaceuticals

Article Title: Transcriptomic Analysis of Fumarate Compounds Identifies Unique Effects of Isosorbide Di-(Methyl Fumarate) on NRF2, NF-kappaB and IRF1 Pathway Genes

doi: 10.3390/ph15040461

Figure Lengend Snippet: RT-PCR analyses and NF-κB luciferase reporter assay. ( A – G ) RT-PCR analyses ( OSGIN1 , IL6 , ICAM1 , MALT1 , TNFAIP3 , IRF1, and CXCL8 ). Average relative expression is shown for each gene (±1 standard error; n = 3 replicates per treatment). Relative expression was calculated using the 2 −∆∆Ct method and ∆Ct values were further normalized to the CTL treatment. Average relative expression is shown for each treatment (bottom margin). Treatments with different letters have significantly different average expression ( p < 0.05, Fisher’s least significant difference). ( H ) NF-κB reporter assay. Human fetal astrocytes were transfected with firefly/Renilla luciferase constructs and treated with TNF-α (20 ng/ml) alone ( n = 6) or TNF-α (20 ng/mL) + IDMF (2.5 µM) ( n = 3) for 6 h. The ratio of luciferase and Renilla (internal control) signals was used to monitor NF-κB induction. The average ratio value is shown (±1 standard error) for each treatment ( p -value: two-sample Wilcoxon rank sum test).

Article Snippet: Experiments were performed using human fetal astrocytes (HA) derived from cerebral cortex (cat no. 882A-05f; Cell Applications, San Diego, CA, USA) grown to subconfluence for 48 h in HA growth medium (cat. no. 820-500, Cell Applications) in a 96-well black wall tissue culture plate.

Techniques: Reverse Transcription Polymerase Chain Reaction, Luciferase, Reporter Assay, Expressing, Transfection, Construct, Control

Journal: Oncotarget

Article Title: Melatonin promotes goat spermatogonia stem cells (SSCs) proliferation by stimulating glial cell line-derived neurotrophic factor (GDNF) production in Sertoli cells

doi: 10.18632/oncotarget.12720

Figure Lengend Snippet: A. Light field of SSCs cultured with melatonin and GDNF, bar=50 μm. B. Cell density after being cultured with different cell mediums at 48 h. The initial number was 5*10 4 . C. QRT-PCR and western blot analysis of proliferation, self-renewal and Sertoli cell markers. D. Western blot analysis of proliferation and Sertoli cell markers. *, P<0.05,**, P<0.01.

Article Snippet: GDNF levels were determined by using a Human glial cell line-derived neurotrophic factor (GDNF) ELISA Kit (BOSTER).

Techniques: Cell Culture, Quantitative RT-PCR, Western Blot

Journal: Oncotarget

Article Title: Melatonin promotes goat spermatogonia stem cells (SSCs) proliferation by stimulating glial cell line-derived neurotrophic factor (GDNF) production in Sertoli cells

doi: 10.18632/oncotarget.12720

Figure Lengend Snippet: A. ELISA analysis of GDNF levels in the SSCs medium. B. Western Blot analysis of phosphorylation levels of AKT and ERK. *, P<0.05,**, P<0.01.

Article Snippet: GDNF levels were determined by using a Human glial cell line-derived neurotrophic factor (GDNF) ELISA Kit (BOSTER).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Phospho-proteomics

Journal: ACS Biomaterials Science & Engineering

Article Title: Promoting and Orienting Axon Extension Using Scaffold-Free Dental Pulp Stem Cell Sheets

doi: 10.1021/acsbiomaterials.1c01517

Figure Lengend Snippet: NTF expression by DPC sheets. (A) Analysis of the NTF mRNA expression using qPCR. Biological variability was evaluated by isolating mRNA from cell sheets formed using different human donor cells, denoting different human samples with HS. (B) Total BDNF, GDNF, and NT3 protein produced by DPC sheets, quantified by performing ELISA on the conditioned media of the sheets. Total protein was also normalized to average number of cells per cell sheet. The data represent an average of sheets formed using four different donor cells, and error bars indicate the standard deviation across biological replicates (*: p -value < 0.05, **: p -value < 0.005).

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) kits for human BDNF (PicoKine ELISA Kit, Boster Biological Technology), GDNF (PicoKine ELISA Kit, Boster Biological Technology), and NT-3 (RayBiotech) were used to measure the protein concentration of the DPC sheet CM.

Techniques: Expressing, Produced, Enzyme-linked Immunosorbent Assay, Standard Deviation