Journal: Scientific Reports

Article Title: The prognostic significance of VISTA and CD33-positive myeloid cells in cutaneous melanoma and their relationship with PD-1 expression

doi: 10.1038/s41598-020-71216-2

Figure Lengend Snippet: Clinico-histopathological characteristics of the 136 cutaneous melanomas according to CD33, VISTA and PD-1 expression.

Article Snippet: Paraffin-embedded sections were immunostained with anti-VISTA (1:200, ProSci Incorporation, CA, USA), anti-CD33 (1:100, Leica Biosystems, Newcastle, UK), or anti-PD-1 (1:100, Ventana, Tucson, AZ, USA) antibodies.

Techniques: Expressing

Journal: Scientific Reports

Article Title: The prognostic significance of VISTA and CD33-positive myeloid cells in cutaneous melanoma and their relationship with PD-1 expression

doi: 10.1038/s41598-020-71216-2

Figure Lengend Snippet: Correlations among CD33, VISTA, and PD-1 expression.

Article Snippet: Paraffin-embedded sections were immunostained with anti-VISTA (1:200, ProSci Incorporation, CA, USA), anti-CD33 (1:100, Leica Biosystems, Newcastle, UK), or anti-PD-1 (1:100, Ventana, Tucson, AZ, USA) antibodies.

Techniques: Expressing

Journal: Scientific Reports

Article Title: The prognostic significance of VISTA and CD33-positive myeloid cells in cutaneous melanoma and their relationship with PD-1 expression

doi: 10.1038/s41598-020-71216-2

Figure Lengend Snippet: Expression of CD33 and VISTA in cutaneous melanoma. ( A ) High intratumoral expression (immunohistochemical score of 3, red color, cytoplasmic) of CD33 in acral melanoma (× 100). ( B ) High intratumoral expression (score 3, red color, cytoplasmic) of VISTA in nodular melanoma (× 100). ( C ) High peritumoral expression (score 3, red color, cytoplasmic) of CD33 in acral melanoma (× 100). ( D ) High peritumoral expression (score 3, red color, cytoplasmic) of VISTA in nodular melanoma (× 200). ( E ) Dual immunofluorescence staining of ( A ) CD33 and VISTA (CD33, red, cytoplasmic; VISTA, green, cytoplasmic) and ( F ) PD-1 and VISTA (PD-1, red, cytoplasmic; VISTA, green, cytoplasmic). Double-positive cells are shown in yellow (white arrow). VISTA, V-domain Ig suppressor of T-cell activation; PD-1, programmed cell death protein-1.

Article Snippet: Paraffin-embedded sections were immunostained with anti-VISTA (1:200, ProSci Incorporation, CA, USA), anti-CD33 (1:100, Leica Biosystems, Newcastle, UK), or anti-PD-1 (1:100, Ventana, Tucson, AZ, USA) antibodies.

Techniques: Expressing, Immunohistochemical staining, Immunofluorescence, Staining, Activation Assay

Journal: Scientific Reports

Article Title: The prognostic significance of VISTA and CD33-positive myeloid cells in cutaneous melanoma and their relationship with PD-1 expression

doi: 10.1038/s41598-020-71216-2

Figure Lengend Snippet: Patient survival according to CD33 and VISTA expression. ( A ) OS according to CD33 expression. ( B ) OS according to VISTA expression. ( C ) Worse OS in patients with high expression of both CD33 and VISTA. ( D ) OS according to the degree of CD33 and VISTA expression ( E ) Worse OS in cases with dual expression of VISTA and PD-1 among patients with high expression of PD-1. VISTA, V-domain Ig suppressor of T-cell activation; PD-1, programmed cell death protein-1.

Article Snippet: Paraffin-embedded sections were immunostained with anti-VISTA (1:200, ProSci Incorporation, CA, USA), anti-CD33 (1:100, Leica Biosystems, Newcastle, UK), or anti-PD-1 (1:100, Ventana, Tucson, AZ, USA) antibodies.

Techniques: Expressing, Activation Assay

Journal: Scientific Reports

Article Title: The prognostic significance of VISTA and CD33-positive myeloid cells in cutaneous melanoma and their relationship with PD-1 expression

doi: 10.1038/s41598-020-71216-2

Figure Lengend Snippet: Univariate and multivariate analyses of OS and PFS.

Article Snippet: Paraffin-embedded sections were immunostained with anti-VISTA (1:200, ProSci Incorporation, CA, USA), anti-CD33 (1:100, Leica Biosystems, Newcastle, UK), or anti-PD-1 (1:100, Ventana, Tucson, AZ, USA) antibodies.

Techniques: Expressing

Journal: Pathogens and global health

Article Title: Elucidating the survival and response of carbapenem resistant Klebsiella pneumoniae after exposure to imipenem at sub-lethal concentrations.

doi: 10.1080/20477724.2018.1538281

Figure Lengend Snippet: Figure 1. Similarity between groups of replicates of imipenem-treated and untreated K/1309–39. (a) Dendogram of sample distances and clustering matrix profiles between biological replicates of imipenem-treated and untreated K/1309–39 (b) PCA plot showing similarity among biological replicates and differences between imipenem-treated and untreated K/1309–39.

Article Snippet: Briefly, the strains were grown overnight in Luria Bertani (LB) plates with and without sub- lethal concentrations of imipenem (Gold Biotechnology, USA) (K/1310–33, 32μg/ml; K/1309–39, 12μg/ml; K/1309– 38, 4μg/ml).

Techniques:

Journal: Pathogens and global health

Article Title: Elucidating the survival and response of carbapenem resistant Klebsiella pneumoniae after exposure to imipenem at sub-lethal concentrations.

doi: 10.1080/20477724.2018.1538281

Figure Lengend Snippet: Figure 3. Comparison of 1H NMR spectra of imipenem-treated and untreated K/1309–39. 1, branched chain amino acid (BCAA); 2, ethanol; 3, threonine; 4, alanine; 5, acetate; 6, methionine; 7, pyruvate; 8, succinate; 9, trimethylamine-N-oxide (TMAO); 10, glycerophosphocholine; 11, phenylacetate; 12, glycine; 13, valine; 14, anserine; 15, uracil; 16, fumarate; 17, tyrosine; 18, xanthine; 19, hypoxanthine; 20, methyladenine; 21, FAPγ-adenine; 22, formate.

Article Snippet: Briefly, the strains were grown overnight in Luria Bertani (LB) plates with and without sub- lethal concentrations of imipenem (Gold Biotechnology, USA) (K/1310–33, 32μg/ml; K/1309–39, 12μg/ml; K/1309– 38, 4μg/ml).

Techniques: Comparison

Journal: bioRxiv

Article Title: Somatic Mutations in MCOLN3 in Aldosterone-Producing Adenomas cause Primary Aldosteronism

doi: 10.1101/2024.10.20.619295

Figure Lengend Snippet: Real-time quantitative PCR (qRT-PCR) analyses of ( A ) MCOLN3 mRNA in multiple snap frozen human tissues showed highest expression in the adrenal cortex. MCOLN3 expression is expressed and compared relative to liver MCOLN3 expression ( B ) MCOLN3 and CYP11B2 mRNA analysis of adrenocortical-derived tissues expressed and compared relative to the whole adrenal. Aldosterone-producing adenomas (APAs), but not cortisol-producing adenomas (CPAs), exhibited significantly higher MCOLN3 expression compared to the adrenal. As opposed to the whole adrenal, CYP11B2 transcript levels in APAs, but not CPAs, were increased. Data is presented as the ( A ) mean ±SD or ( B ) median with 95% confidence interval of at least four tissue samples normalized to PPIA . Significance was analyzed using a Kruskal-Wallis with a Dunn’s multiple comparison test. * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: GenScript supplied the cDNA open reading frames (ORF) of human wild type MCOLN3 gene (OHu23350) and MCOLN3 p.Y391D variant (obtained through site-directed mutagenesis of the wild type clone) in the pcDNA3.1-C-(k)DYK backbone.

Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Derivative Assay, Comparison

Journal: bioRxiv

Article Title: Somatic Mutations in MCOLN3 in Aldosterone-Producing Adenomas cause Primary Aldosteronism

doi: 10.1101/2024.10.20.619295

Figure Lengend Snippet: Hematoxylin and eosin (H&E) staining, aldosterone synthase (CYP11B2) and mucolipin- 3 (MCOLN3) immunohistochemistry staining of 5 µm FFPE sections representative of ( A ) an adrenal with classical adrenal architecture and ( B ) an adrenal with disrupted zona glomerulosa CYP11B2 expression with aldosterone-producing micronodules (APMs). Scale bar, 2 mm.

Article Snippet: GenScript supplied the cDNA open reading frames (ORF) of human wild type MCOLN3 gene (OHu23350) and MCOLN3 p.Y391D variant (obtained through site-directed mutagenesis of the wild type clone) in the pcDNA3.1-C-(k)DYK backbone.

Techniques: Staining, Immunohistochemistry, Expressing

Journal: bioRxiv

Article Title: Somatic Mutations in MCOLN3 in Aldosterone-Producing Adenomas cause Primary Aldosteronism

doi: 10.1101/2024.10.20.619295

Figure Lengend Snippet: Sanger sequence chromatograms of tumor and adjacent adrenal tissue DNA for validation of the MCOLN3 variants. A) Translational consequence of a point mutation in the MCOLN3 Y391D variant resulting in the single residue change, Tyr391Asp (blue). B) MCOLN3 N411_V412deIinsI variant presents with the deletion of two amino acids, Asn411 and Val412 (red), and the insertion of Ile411(yellow) causing a single residue frame shift (green). Mutations are absent in the tumor adjacent adrenal tissue (top sequence). C) Simplified schematic representation of the MCOLN3 transmembrane adapted from the work done by Zhou et al. . The location of MCOLN3 Y391D (cyan star) and MCOLN3 N411_V412deIinsI (yellow star) in segment 4 (S4) and segment 5 (S5), respectively, is shown in close proximity to the ion selectivity filter. The disease-causing Mcoln3 A419P variant identified in mice is shown in S5 by the white star. Neon green circles mark the amino acids involved in binding of MCOLN3 agonist, ML-SA1. D) Alignment of orthologs among various species shows the conservation of Tyr391 and Val412 in the mucolipin-3 protein. Affected amino acid residue associated with the novel variants are highlighted in blue ( MCOLN3 Y391D ) and red and green ( MCOLN3 N411_V412deIinsI ). Highlighted in grey are residues conserved in orthologs.

Article Snippet: GenScript supplied the cDNA open reading frames (ORF) of human wild type MCOLN3 gene (OHu23350) and MCOLN3 p.Y391D variant (obtained through site-directed mutagenesis of the wild type clone) in the pcDNA3.1-C-(k)DYK backbone.

Techniques: Sequencing, Mutagenesis, Variant Assay, Residue, Binding Assay

Journal: bioRxiv

Article Title: Somatic Mutations in MCOLN3 in Aldosterone-Producing Adenomas cause Primary Aldosteronism

doi: 10.1101/2024.10.20.619295

Figure Lengend Snippet: Hematoxylin and eosin (H&E) staining, and MCOLN3, CYP11B2 and CYP17A1 immunohistochemistry staining of the resected APAs identified with the MCOLN3 Y391D (UM102 and ELA25) and MCOLN3 N411_V412delinsI (AA30) variants. Scale bar, 2 mm.

Article Snippet: GenScript supplied the cDNA open reading frames (ORF) of human wild type MCOLN3 gene (OHu23350) and MCOLN3 p.Y391D variant (obtained through site-directed mutagenesis of the wild type clone) in the pcDNA3.1-C-(k)DYK backbone.

Techniques: Staining, Immunohistochemistry

Journal: bioRxiv

Article Title: Somatic Mutations in MCOLN3 in Aldosterone-Producing Adenomas cause Primary Aldosteronism

doi: 10.1101/2024.10.20.619295

Figure Lengend Snippet: HAC15 cells were transfected with 1 µg of empty pIRES-CD8 plasmid (empty) or with pIRES-CD8 plasmids containing MCOLN3 WT (WT) or MCOLN3 Y391D (Y391D) via electroporation. 48h post-transfection, the relative mRNA expression levels of ( A ) MCOLN3 , ( B ) NR4A2 , ( C ) CYP11B2 , ( D ) CYP17A1 , and ( E ) CYP11B1 were quantified by qRT-PCR. ( F ) Aldosterone content in cell medium was quantified using an ELISA and normalized to total protein content. Data are presented as the mean ±SEM of at least four independent experiments. Each data point represents the mean of the experimental triplicates. Statistical analyses were performed using an ordinary one-way ANOVA with a Tukey’s multiple comparison test. * P <0.05; *** P <0.001; **** P <0.0001

Article Snippet: GenScript supplied the cDNA open reading frames (ORF) of human wild type MCOLN3 gene (OHu23350) and MCOLN3 p.Y391D variant (obtained through site-directed mutagenesis of the wild type clone) in the pcDNA3.1-C-(k)DYK backbone.

Techniques: Transfection, Plasmid Preparation, Electroporation, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Comparison

Journal: bioRxiv

Article Title: Somatic Mutations in MCOLN3 in Aldosterone-Producing Adenomas cause Primary Aldosteronism

doi: 10.1101/2024.10.20.619295

Figure Lengend Snippet: HAC15 cells were transfected with 1 µg empty vector (empty) or with 0.5 µg of wild type (WT) or MCOLN3 Y391D (Y391D)-containing plasmids (adjusted to 1 µg plasmid concentration using empty vector). 24-30 h after transfection, cells were used for whole-cell patch-clamp experiments to measure the membrane potential ( A ) and for measurements of the cytosolic calcium activity with fura-2 ( B, C ). ( A ) Membrane potential of cells with mutant MCOLN3 Y391D was depolarized compared to cells with empty vector or with MCOLN3 WT (empty, n=7; MCOLN3 WT , n=8; MCOLN3 Y391D , n=17). ( B ) For calcium measurements, basal calcium activities were calculated from the first control phase of the experiments shown in C . Basal calcium activity of cells with MCOLN3 Y391D was increased compared to cells with empty vector or with MCOLN3 WT . Depending on the transfection rate, the signals from 2-6 cells with bound CD8 beads per dish were averaged. Shown here is the mean ±SEM of the mean Fura-2 ratio from n dishes: empty, n=10; MCOLN3 WT , n=11; MCOLN3 Y391D , n=10. Figure C summarizes changes of fura-2 ratio over time upon application of the indicated extracellular solutions. Cells with MCOLN3 Y391D showed a greater dependence of cytosolic calcium on extracellular calcium changes. This was characterized by a significant decrease in calcium activity when extracellular calcium was removed and an increase when extracellular calcium was increased. *, P <0.05, MCOLN3 WT vs. MCOLN3 Y391D cells or empty CD8. Statistical significance (*) was tested with the GraphPad Prism 10 software using one-way ANOVA analysis with Tukey’s multiple comparison.

Article Snippet: GenScript supplied the cDNA open reading frames (ORF) of human wild type MCOLN3 gene (OHu23350) and MCOLN3 p.Y391D variant (obtained through site-directed mutagenesis of the wild type clone) in the pcDNA3.1-C-(k)DYK backbone.

Techniques: Transfection, Plasmid Preparation, Concentration Assay, Patch Clamp, Membrane, Activity Assay, Mutagenesis, Control, Software, Comparison

Journal: bioRxiv

Article Title: Somatic Mutations in MCOLN3 in Aldosterone-Producing Adenomas cause Primary Aldosteronism

doi: 10.1101/2024.10.20.619295

Figure Lengend Snippet: Cell currents at different voltage-clamp steps ( A-C ) and cell membrane potentials ( D ) were measured using the whole-cell patch-clamp technique. ( A-C ) All cells showed outward currents typically present in adrenal cell lines and native adrenal glomerulosa cells. Overexpression of MCOLN3 Y391D led to additional large inward currents at negative voltage-clamp steps (-100 mV up to -40 mV), which were absent in cells with empty vector and MCOLN3 WT cells. Replacement of extracellular Na + by NMDG + (Na + free, red triangles) led to a significant reduction of these inward currents in mutant cell, albeit not to the level of control cells. ( D ) Membrane voltage of cells with MCOLN3 Y391D was depolarized compared to cells with empty vector or with MCOLN3 WT , both under control and Na + free conditions. Removal of Na + led to a more hyperpolarized membrane potential in HAC15- MCOLN3 WT and HAC15- MCOLN3 Y391D cells, but to a higher extent in cells with HAC15- MCOLN3 Y391D cells. Statistical significance (* and # ) was tested with the GraphPad Prism 10 software using a two-way ANOVA analysis with Tukey’s multiple comparison. * P < 0.05 for comparison of ctrl versus Na + free solution in (A-D). # P < 0.05 for comparison of HAC15- MCOLN3 Y391D cells vs HAC15- MCOLN3 WT and empty control cells in ( D ); Data are presented as mean ±SEM (empty CD8-vector n=7, MCOLN3 WT n=8, MCOLN3 Y391D n=17). The number of experiments in ( A-C ) is equal to the number in ( D ) because the membrane voltage was measured alternately with the current measurements.

Article Snippet: GenScript supplied the cDNA open reading frames (ORF) of human wild type MCOLN3 gene (OHu23350) and MCOLN3 p.Y391D variant (obtained through site-directed mutagenesis of the wild type clone) in the pcDNA3.1-C-(k)DYK backbone.

Techniques: Membrane, Patch Clamp, Over Expression, Plasmid Preparation, Mutagenesis, Control, Software, Comparison

Journal: bioRxiv

Article Title: Somatic Mutations in MCOLN3 in Aldosterone-Producing Adenomas cause Primary Aldosteronism

doi: 10.1101/2024.10.20.619295

Figure Lengend Snippet: Mechanism 1 proposes that MCOLN3 Y391D resulted in a gain-of-function calcium permeable channel which directly increases cytosolic calcium levels. In mechanism 2, MCOLN3 Y391D altered the ion selectivity of MCOLN3 resulting in the influx of sodium ions (Na + ) which causes cell membrane depolarization and the activation of voltage-gated calcium channels. Elevated cytosolic calcium levels induce the transcription of aldosterone synthase (CYP11B2) and hyperaldosteronism. Image created with BioRender.com

Article Snippet: GenScript supplied the cDNA open reading frames (ORF) of human wild type MCOLN3 gene (OHu23350) and MCOLN3 p.Y391D variant (obtained through site-directed mutagenesis of the wild type clone) in the pcDNA3.1-C-(k)DYK backbone.

Techniques: Membrane, Activation Assay