Journal: Science immunology

Article Title: PDIA3 epitope-driven immune autoreactivity contributes to hepatic damage in type 2 diabetes

doi: 10.1126/sciimmunol.abl3795

Figure Lengend Snippet: Sera, collected from 25 healthy age- and sex-matched controls, 48 patients with a diagnosis of T2D, 28 subjects with a diagnosis of AIH, and 18 subjects with PBC, were tested in ELISA against the full-length human PDIA3 protein. Antibody titers were quantified in each subject, and each dot symbol represents the average of three technical replicates. Statistical analysis for each serum and the representative binding curves are presented in fig. S7 and table S7. The statistical significance was determined with one-way ANOVA including mixed-effects model restricted maximum likelihood (REML) followed by multiple comparisons performed with uncorrected Fisher’s LSD test (***P < 0.0001, **P < 0.001, and *P < 0.01). For specificity testing, commercially available polyclonal anti-PDIA3 antibody was used as positive control, whereas PBS buffer served as negative control.

Article Snippet: Human plasma experiments T2D plasma/sera were purchased from ProMedDx, Medical Laboratories (Norton, MA).

Techniques: Biomarker Discovery, Enzyme-linked Immunosorbent Assay, Binding Assay, Positive Control, Negative Control

Journal: Life Metabolism

Article Title: Apolipoprotein A-IV and its derived peptide, T55−121, improve glycemic control and increase energy expenditure

doi: 10.1093/lifemeta/loae010

Figure Lengend Snippet: The levels of apoA-IV increase in the sera of patients after bariatric surgeries. (a–d) Changes in serum biochemical parameters of patients with morbid obesity and T2D before and one year after LSG, including fasting blood glucose (a), fasting insulin (b), fasting C-peptide (c), and HbA1c (d). (e and f) Protein profiles in sera collected from patients before and after LSG (e) analyzed by SDS-PAGE (f). Protein bands 1 and 2 from post-surgery samples, indicated by red arrowheads, were subjected to MS-based proteomics. (g and h) Identification of proteins in bands 1 and 2 based on proteomics and immunoblot analysis. Proteomic analysis of bands 1 and 2 (g). Immunoblot analysis of apoA-IV and AGP-1 in sera of subjects before and after LSG (h). AGP-1 is a loading control. (i) The changes of plasma apoA-IV levels of diabetic obese patients ( n = 10) before and one year after RYGB were analyzed based on the reported proteomics data . Data are presented as mean ± SEM. Statistical significance was determined by the two-tailed Student’s t -test. *** P < 0.001.

Article Snippet: Spontaneous T2D KKAy mice were obtained from Beijing HFK Bioscience Co. Ltd (Beijing, China), and GK diabetic rats were generously supplied by Prof. Tao Xu’s Lab. All animals were maintained under a 12-h light/12-h dark cycle at 22 ± 1°C.

Techniques: SDS Page, Western Blot, Control, Clinical Proteomics, Two Tailed Test

Journal: Life Metabolism

Article Title: Apolipoprotein A-IV and its derived peptide, T55−121, improve glycemic control and increase energy expenditure

doi: 10.1093/lifemeta/loae010

Figure Lengend Snippet: ApoA-IV promotes GSIS in human primary islets, partially through Gαs-coupled GPCR/cAMP signaling. (a) GSIS in human primary islets treated with vehicle or 100 μg/mL sh-apoA-IV for 3 h. (b) GSIS in spontaneous T2D KKAy mouse primary islets treated with vehicle or 100 μg/mL sm-apoA-IV for 3 h. (c) GSIS assay in MIN6 cells treated with vehicle or 100 μg/mL sm-apoA-IV for 3 h. (d) Immunoblot analysis of apoA-IV-Flag in pancreas (top) and brain (bottom) at indicated times after intraperitoneal administration of recombinant apoA-IV-Flag protein. GAPDH is a loading control. (e) Fluorescent sections of pancreas and brain after 2 h incubation with 10 μg/mL apoA-IV-GFP or GFP. Scale bar = 20 μm. (f) Blood glucose levels in mice with vehicle or STZ treatment. (g) The IPGTT in STZ-induced T1D mice ( n = 10) with the administration of saline or sm-apoA-IV (6 mg/kg body weight). (h) The cAMP sensor fluorescence changes in MIN6 cells with 0, 100, or 200 μg/mL sm-apoA-IV. (i) Fluorescent DAG sensor changes in MIN6 cells treated with 0, 100, or 200 μg/mL sm-apoA-IV. (j) Fluorescent Ca 2+ sensor changes in MIN6 cells treated with 0, 100, or 200 μg/mL sm-apoA-IV. (k) GSIS in MIN6 cells treated with 100 μg/mL sm-apoA-IV alone, 100 μg/mL sm-apoA-IV together with 20 μmol/L SQ22536 (AC inhibitor), or 100 μg/mL sm-apoA-IV together with 10 μmol/L NF449 (Gαs-selective antagonist). (l) Immunoblot analysis of phosphorylated CREB (p-CREB) and CREB in MIN6 cells treated with 100 μg/mL sm-apoA-IV alone, or 100 μg/mL sm-apoA-IV together with 20 μmol/L of SQ22536. HSP90 is a loading control. (m) Ratio of p-CREB/HSP90 calculated from l. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. ns, no significance. GSIS, glucose-stimulated insulin secretion. STZ, streptozocin. sh-apoA-IV, signal peptide-removed human apoA-IV. sm-apoA-IV, signal peptide-removed mouse apoA-IV.

Article Snippet: Spontaneous T2D KKAy mice were obtained from Beijing HFK Bioscience Co. Ltd (Beijing, China), and GK diabetic rats were generously supplied by Prof. Tao Xu’s Lab. All animals were maintained under a 12-h light/12-h dark cycle at 22 ± 1°C.

Techniques: Western Blot, Recombinant, Control, Incubation, Saline, Fluorescence

Journal: Advanced Science

Article Title: Human Islet Amyloid Polypeptide (hIAPP) Protofibril‐Specific Antibodies for Detection and Treatment of Type 2 Diabetes

doi: 10.1002/advs.202202342

Figure Lengend Snippet: The mAbs engage hIAPP protofibrils in vivo. Immunofluorescence shows increased hIAPP protofibril staining with T2D disease progression with a) 07G10 and b) 10H04. Insulin (magenta), mAb (yellow), nuclei (cyan). c) Magnified image of 07G10 and 10H04 staining in WT and severe T2D. Protofibril staining is indicated by white arrowheads. d) Quantification of candidate mAb signal as the disease progresses compared to healthy controls. n = 3 per group; error bars represent S.E.M, ordinary one‐way ANOVA. **** p < 0.0001, *** p < 0.001, and ** p < 0.01.

Article Snippet: To confirm that our mAbs can also detect protofibrils in human T2D, we obtained pancreatic sections from healthy and T2D donors (Novus, Biochain) and performed immunofluorescence with our mAbs and Thioflavin S (ThS) staining to mark hIAPP aggregates.

Techniques: In Vivo, Immunofluorescence, Staining, Biomarker Discovery

Journal: Advanced Science

Article Title: Human Islet Amyloid Polypeptide (hIAPP) Protofibril‐Specific Antibodies for Detection and Treatment of Type 2 Diabetes

doi: 10.1002/advs.202202342

Figure Lengend Snippet: The mAbs engage hIAPP protofibrils in human T2D. Immunofluorescence shows increased hIAPP protofibril staining in pancreatic islet tissue from a T2D donor compared to nondiabetic control with a) 07G10 and b) 10H04. c) Magnified inset shows co‐localization of protofibril signal and hIAPP aggregate signal detected by ThS staining with protofibril signal localized at the core of larger aggregates. mAb (magenta), ThS (yellow), nuclei (cyan). Co‐localization of protofibril signal and ThS signal is indicated by white arrowheads.

Article Snippet: To confirm that our mAbs can also detect protofibrils in human T2D, we obtained pancreatic sections from healthy and T2D donors (Novus, Biochain) and performed immunofluorescence with our mAbs and Thioflavin S (ThS) staining to mark hIAPP aggregates.

Techniques: Immunofluorescence, Staining, Control

Journal: PLoS ONE

Article Title: Inhibition of β-catenin dependent WNT signalling upregulates the transcriptional repressor NR0B1 and downregulates markers of an A9 phenotype in human embryonic stem cell-derived dopaminergic neurons: Implications for Parkinson’s disease

doi: 10.1371/journal.pone.0261730

Figure Lengend Snippet: Cultures showed increased NEUROD1 along with reduced ALDH1A1 , TH , KCNJ6 and LMX1A , but no change in NR4A2 , SLC6A3 SLC18A2 or TUBB3 . Control wells underwent transfection with an empty plasmid. Results are expressed as mean ± SEM of three (biological) replicate experiments. Analysis was performed with t-tests where each post transfection timepoint represented a paired measure (t = 0 was not included in analyses). * and ** indicate P<0.05 and P<0.01, respectively. shows NEUROD1 immunolabelling following transfection.

Article Snippet: At this time, cultures were transfected with NEUROD1 (SC118625, Origene), NR0B1 (SC319624, Origene), NR0B2 (SC122920, Origene) or blank plasmids (PCMV6XL5, Origene) according to manufacturer’s instructions.

Techniques: Control, Transfection, Plasmid Preparation

Journal: PLoS ONE

Article Title: Inhibition of β-catenin dependent WNT signalling upregulates the transcriptional repressor NR0B1 and downregulates markers of an A9 phenotype in human embryonic stem cell-derived dopaminergic neurons: Implications for Parkinson’s disease

doi: 10.1371/journal.pone.0261730

Figure Lengend Snippet: Panels (a) and (b) respectively show the effects of the WNT modulators and NEUROD1 overexpression upon NR0B1 . Panels (c) and (d) show effects upon NR0B2 expression. Results are expressed as mean ± SEM of three (biological) replicate experiments. Analysis of panel (a) was via one-way ANOVA with post-hoc Dunnett’s test. For panels (b) and (d) analysis was via Student’s t-tests (at each time point as in ). For panel (c) analysis was via a non-parametric Kruskal-Wallis test with post-hoc Dunn’s test to accommodate undetectable levels of NR0B2 in two out of three samples, both undetectable values were assigned to 0.0028 (equal to the lowest value detected).

Article Snippet: At this time, cultures were transfected with NEUROD1 (SC118625, Origene), NR0B1 (SC319624, Origene), NR0B2 (SC122920, Origene) or blank plasmids (PCMV6XL5, Origene) according to manufacturer’s instructions.

Techniques: Over Expression, Expressing

Journal: PLoS ONE

Article Title: Inhibition of β-catenin dependent WNT signalling upregulates the transcriptional repressor NR0B1 and downregulates markers of an A9 phenotype in human embryonic stem cell-derived dopaminergic neurons: Implications for Parkinson’s disease

doi: 10.1371/journal.pone.0261730

Figure Lengend Snippet: Expression patterns following transfection with NR0B1 (green), NR0B2 (red) or blank (blue) plasmids show that NR0B1 transfection increases both NR0B1 and NR0B2 and impacts expression of NEUROD1 , TH , KCNJ6 , LMX1A and TUBB3 . Control wells underwent transfection with an empty plasmid. Results are expressed as mean ± SEM of three (biological) replicate experiments. Analysis was performed with One-way ANOVA followed by post-hoc Dunnett’s test where each post transfection timepoint represented a repeated measure (t = 0 was not included in analyses). * and ** indicate P<0.05 and P<0.01, respectively. shows increased NR0B1 and NR0B2 immunolabelling up to six days after transfection.

Article Snippet: At this time, cultures were transfected with NEUROD1 (SC118625, Origene), NR0B1 (SC319624, Origene), NR0B2 (SC122920, Origene) or blank plasmids (PCMV6XL5, Origene) according to manufacturer’s instructions.

Techniques: Expressing, Transfection, Control, Plasmid Preparation