Journal: Cell metabolism

Article Title: Heterogenous impairment of α cell function in type 2 diabetes is linked to cell maturation state.

doi: 10.1016/j.cmet.2021.12.021

Figure Lengend Snippet: Figure 1. Glucose suppresses human a cell exocytosis in concert with P/Q-channel activity (A) Schematic diagram illustrating that human islets were isolated, dispersed, and cultured for 1–2 days prior to whole-cell patch-clamp and subsequent a cell identification by glucagon immunostaining. (B) Total exocytosis upon a series of ten membrane depolarizations with increasing glucose in ND a cells (gray; n = 42, 28, 24, 20 cells from 9 donors) and T2D a cells (pink; n = 29, 16, 12, 27 cells from 6 donors). (C) Voltage-dependent Ca2+ channel activity with Ba2+ as a charge carrier in ND a cells with increasing glucose (n = 31, 24, 27 cells from 7 donors). (D) Effect of the P/Q-type Ca2+ channel activator GV-58 (10 mM) on Ca2+ current inactivation (left; n = 24, 28, 22, 21 cells) and charge entry (middle; n = 22, 20, 20, 23 cells) during a 500 ms depolarization from 70 to 0 mV and total exocytosis (right; n = 23, 22, 28, 34 cells) at 1 and 10 mM glucose (3 donors). (E) Effect of the P/Q-type Ca2+ channel blocker agatoxin (100 nM) and the L-type Ca2+ channel blocker isradipine (10 mM) on exocytosis at 1 mM glucose in ND a cells (n = 34, 30, 21 cells from 5 donors). (F) Voltage-dependent Ca2+ channel activity at 0 mV (Ba2+ as a charge carrier) with increasing glucose in ND a cells, with agatoxin (100 nM), and in T2D a cells (n = 31, 16, 24, 15, 27, 18 cells from 7 ND donors and 34, 31, 27 cells from 5 T2D donors). (G) Effect of agatoxin (100 nM) and the L-type Ca2+ channel blocker isradipine (10 mM) on total exocytosis in T2D a cells at 1 and 10 mM glucose (n = 30, 31, 29, 32, 31, 29 cells from 5 T2D donors). (H) Putative scheme for glucose regulation of depolarization-induced exocytosis in a cells and the dysfunction seen in T2D. *p < 0.05; **p < 0.01; ***p < 0.001 by one-way ANOVA (D, E, and G) or two-way ANOVA (B, C, and F) and Tukey post test compared with 1 mM glucose control, or as indicated.

Article Snippet: Cells were identified by post-hoc immunostaining for insulin with a rabbit anti-insulin primary antibody (Santa Cruz; #SC-9168; RRID: AB_2126540) and goat anti-rabbit Alexa Fluor 488 secondary (ThermoFisher, #A-11076; RRID: AB_141930), and with a guinea pig anti-glucagon primary antibody (Sigma-Aldrich, #G2654; RRID: AB_259852) and goat anti-guinea pig Alexa Fluor 594 secondary (ThermoFisher, #A-11076; RRID: AB_141930); or following collection for single-cell RNA sequencing analysis (Camunas-Soler et al., 2020).

Techniques: Activity Assay, Isolation, Cell Culture, Patch Clamp, Immunostaining, Membrane, Control

Journal: Cell metabolism

Article Title: Heterogenous impairment of α cell function in type 2 diabetes is linked to cell maturation state.

doi: 10.1016/j.cmet.2021.12.021

Figure Lengend Snippet: Figure 6. Electrophysiological fingerprints define a loss of ‘‘functional identity’’ in T2D a cells (A) Classifier models trained on islet cell electrical properties of a and b cells from donors with no diabetes (ND) using optimizable ensemble or extreme gradient boosting (XGBoost) approaches identify cell types with high accuracy regardless of the inclusion or exclusion of cell size from training data; 80% of data was used for training; 20% of data was reserved for validation and generation of confusion matrices. tSNE plots show cell types determined by immunostaining or sequencing (left) and assigned aprobability scores (right). (B) Ordinary least-squares multiple regression of electrophysiological properties of a cells from ND donors, model scoring, and donor/isolation variables. (C) Calculated aprobability (and bprobability) values from Model 3 applied to cells collected for patch-seq, without a priori knowledge of cell type and correlation with canonical b cell (light blue) and a cell (pink) markers. (D) bprobability and aprobability values derived from all three models, of all b and a cells from ND or T2D donors. (E) Volcano plot of transcript correlations with Model 3 aprobability values (slope/standard deviation) in a cells from donors with T2D. A negative correlation indicates transcripts associated with reduced aprobability. (F) Significantly enriched GO biological pathways from gene set enrichment analysis (GSEA) performed using aprobability - transcript correlation slopes of a cells from donors with T2D in all three Models as weighting. ***p < 0.001 and ****p < 0.0001 as indicated within models using non-parametric Kruskal-Wallis test (D) followed by Dunn’s post-test to correct for multiple comparisons. Pink/red points in (E) indicate significance at p < 0.05. FDRs for pathways identified by GSEA in (F) were <0.05 unless indicated otherwise.

Article Snippet: Cells were identified by post-hoc immunostaining for insulin with a rabbit anti-insulin primary antibody (Santa Cruz; #SC-9168; RRID: AB_2126540) and goat anti-rabbit Alexa Fluor 488 secondary (ThermoFisher, #A-11076; RRID: AB_141930), and with a guinea pig anti-glucagon primary antibody (Sigma-Aldrich, #G2654; RRID: AB_259852) and goat anti-guinea pig Alexa Fluor 594 secondary (ThermoFisher, #A-11076; RRID: AB_141930); or following collection for single-cell RNA sequencing analysis (Camunas-Soler et al., 2020).

Techniques: Functional Assay, Biomarker Discovery, Immunostaining, Sequencing, Isolation, Derivative Assay, Standard Deviation

Journal: Cell metabolism

Article Title: Heterogenous impairment of α cell function in type 2 diabetes is linked to cell maturation state.

doi: 10.1016/j.cmet.2021.12.021

Figure Lengend Snippet: Figure 7. A role for maturation state in a cell dysfunction in T2D (A) Heterogenous expression of transcript makers for islet cell lineage and a cell maturity in 980 patch-seq a cells. (B) ARX and MAFB protein expression in a cells at the protein level in situ by immunostaining. Violin plots show the relative levels of GCG, ARX, and MAFB expressed in ARXlow and ARXhi a cells. (C) aprobability values from the three separate classifier models in ND and T2D a cells separated by high and low expression of ARX (see also Figure S6). (D) Correlation of exocytosis in ND and T2D a cells with a cell lineage and identity markers. Bars to the left of the centerline indicate correlation with low exocytosis at the given glucose concentration. (E) In a separate human islet single-cell dataset (Avrahami et al., 2020), expression of a juvenile a cell gene set in ND and T2D a cells separated by low and high ARX expression. The heatmap displays relative expression levels as median log2 FPKM values. (F) Total exocytosis in ND and T2D a cells at 1 mM glucose separated by low and high expression of ISL1, NEUROD1, NKX2-2, and ARX. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 by two-way ANOVA followed by two-stage step-up method for estimation of FDR (C, D, and F) or Tukey post- test (B).

Article Snippet: Cells were identified by post-hoc immunostaining for insulin with a rabbit anti-insulin primary antibody (Santa Cruz; #SC-9168; RRID: AB_2126540) and goat anti-rabbit Alexa Fluor 488 secondary (ThermoFisher, #A-11076; RRID: AB_141930), and with a guinea pig anti-glucagon primary antibody (Sigma-Aldrich, #G2654; RRID: AB_259852) and goat anti-guinea pig Alexa Fluor 594 secondary (ThermoFisher, #A-11076; RRID: AB_141930); or following collection for single-cell RNA sequencing analysis (Camunas-Soler et al., 2020).

Techniques: Expressing, In Situ, Immunostaining, Concentration Assay

Journal: Psychopharmacology

Article Title: Antidepressant-like activity of the neuropeptide Y Y5 receptor antagonist Lu AA33810: behavioral, molecular, and immunohistochemical evidence

doi: 10.1007/s00213-016-4495-3

Figure Lengend Snippet: The effect of L-AAA (100 μg/2 μl, twice) and Lu AA33810 (10 mg/kg, i.p.) administered alone or in combination on the protein level of GFAP ( a ), BDNF ( b ), TrkB ( c ), and Y5 receptor (Y5R) ( d ) in the rat PFC determined by Western blot analysis 144 h after the second toxin injection. Data are expressed as % changes vs. control. Immunoblot bands corresponding to GFAP, BDNF, TrkB, Y5R, and GAPDH are seen ( e ). Values represent the mean ± SEM ( n = 6–10 rats per group) and were evaluated by two-way ANOVA, followed by the Bonferroni multiple comparison test. * p < 0.05, ** p < 0.01 vs. control; # p < 0.05, ## p < 0.01 vs. L-AAA-treated group

Article Snippet: Behavioral data, immunoblots, and immunohistochemical analysis were evaluated using the two-way ANOVA (analysis of variance), followed by the Newman-Keuls (behavioral results) or Bonferroni (biochemical and immunohistochemical results) multiple comparison post hoc tests (GraphPad Software, San Diego CA, USA).

Techniques: Western Blot, Injection, Control, Comparison

Journal: Psychopharmacology

Article Title: Antidepressant-like activity of the neuropeptide Y Y5 receptor antagonist Lu AA33810: behavioral, molecular, and immunohistochemical evidence

doi: 10.1007/s00213-016-4495-3

Figure Lengend Snippet: ( Upper panel ) Representative microphotographs of coronal sections of the rat brain, illustrating the effect of L-AAA (100 μg/2 μl, twice) and Lu AA33810 (10 mg/kg, i.p.) administered alone or in combination on the number of GFAP-positive cells in the mPFC. Numerous GFAP-positive astrocytes are seen in the section from a control rat in contrast to few astrocytes in the section from L-AAA injected rat. Moreover, the shrinkage of astrocyte cells and processes is also seen after the gliotoxin. LuAA33810 prevented the decrease in the number of astrocytes and their pathological changes. Calibration bars 50 μm. ( Bottom panel ) A histogram showing the number of GFAP-positive cells counted by stereological method. Data are expressed as % changes vs. control. Values represent the mean ± SEM ( n = 5–6 rats per group) and were evaluated by two-way ANOVA, followed by the Bonferroni multiple comparison test. *** p < 0.001 vs. control; # p < 0.05 vs. L-AAA-treated group

Article Snippet: Behavioral data, immunoblots, and immunohistochemical analysis were evaluated using the two-way ANOVA (analysis of variance), followed by the Newman-Keuls (behavioral results) or Bonferroni (biochemical and immunohistochemical results) multiple comparison post hoc tests (GraphPad Software, San Diego CA, USA).

Techniques: Control, Injection, Comparison

Journal: Psychopharmacology

Article Title: Antidepressant-like activity of the neuropeptide Y Y5 receptor antagonist Lu AA33810: behavioral, molecular, and immunohistochemical evidence

doi: 10.1007/s00213-016-4495-3

Figure Lengend Snippet: ( Upper panel ) Representative microphotographs of coronal sections of the rat brain showing the expression of BDNF-positive cells in the mPFC of the experimental groups. Many BDNF-positive cells are seen in the control rat. Gliotoxin L-AAA (100 μg/2 μl, twice) induced a strong decrease in the number of BDNF-ir cells, while Lu AA33810 (10 mg/kg, i.p.) reversed this effect. Arrows point to some of BDNF-positive cells. Calibration bars 50 μm. ( Bottom panel ) A histogram showing the effect of L-AAA and Lu AA33810 administered alone or in combination on the number of BDNF-ir cells in the rat PFC. Data are expressed as % changes vs. control. Values represent the mean ± SEM ( n = 5–6 rats per group) and were evaluated by two-way ANOVA, followed by the Bonferroni multiple comparison test. * p < 0.05 vs. control; ## p < 0.001 vs. L-AAA-treated group

Article Snippet: Behavioral data, immunoblots, and immunohistochemical analysis were evaluated using the two-way ANOVA (analysis of variance), followed by the Newman-Keuls (behavioral results) or Bonferroni (biochemical and immunohistochemical results) multiple comparison post hoc tests (GraphPad Software, San Diego CA, USA).

Techniques: Expressing, Control, Comparison

Journal: Journal of Human Genetics

Article Title: Concurrent MCL1 and JUN amplification in pseudomyxoma peritonei: a comprehensive genetic profiling and survival analysis

doi: 10.1038/jhg.2013.132

Figure Lengend Snippet: Immunohistochemical staining for MCL1 in two patient samples demonstrated moderate (left, patient 2) and moderate-to-strong (right, patient 6) cytoplasmic staining. Both patients had unique MCL1 and JUN coamplification in samples of pseudomyxoma peritonei tested by next-generation genetic sequencing (original magnification × 200, left; × 100, right).

Article Snippet: Additionally, we performed a post hoc immunohistochemical analysis (Histoserv, Inc., Germantown, MD, USA), using MCL1 (myeloid cell leukemia sequence 1) primary antibody staining on FFPE slides that were sectioned into 3-μm slides and stained with MCL1 monoclonal antibody (ab31948; Abcam, Cambridge, MA, USA).

Techniques: Immunohistochemical staining, Staining, Sequencing