Journal: Diabetes

Article Title: Seroreactivity Against Tyrosine Phosphatase PTPRN Links Type 2 Diabetes and Colorectal Cancer and Identifies a Potential Diagnostic and Therapeutic Target.

doi: 10.2337/db20-1206

Figure Lengend Snippet: Figure 1—Assessment of PTPRN dysregulation in CRC. A: Evaluation of mRNA levels in CRC tissue compared with normal adjacent tis- sue using Oncomine. Higher PTPRN mRNA levels were found in CRC tissue in comparison with control tissue (39,40). B: The cBioPortal database retrieved data about the presence of PTPRN mutations at a genetic level in 3% of patients with CRC (TCGA, COAD). C: Sur- vival analysis retrieved from the GEPIA2 web-based tool revealed that a higher PTPRN expression in patients with CRC (READ and COAD) and patients with COAD (TCGA data sets) was related with a large decrease in their overall and disease-free survival (P values < 0.05). CNA, copy number alteration; HR, hazard ratio.

Article Snippet: After blocking, membranes were incubated overnight at 4 C with an anti-HaloTag monoclonal antibody (#6921A; Promega) diluted 1:1,000, an anti-PTPRN monoclonal antibody (sc-130570; Santa Cruz Biotechnology) diluted 1:200, an anti–phosphorylated (p-)insulin Rb polyclonal antibody (sc-25103-R; Santa Cruz Biotechnology) diluted 1:1,000, an anti-IRS1 polyclonal antibody (sc-559; Santa Cruz Biotechnology) diluted 1:1,000, an anti–pFOXO1/3 polyclonal antibody (#9464; Cell Signaling Technology), an anti-AKT polyclonal antibody (#4691; Cell Signaling Technology,), an anti–p-AKT polyclonal antibody (#9275; Cell Signaling Technology), an anti-ERK polyclonal antibody (#4695; Cell Signaling Technology), an anti–p-ERK polyclonal antibody (#9101; Cell Signaling Technology), an anti-GSK3b monoclonal antibody (#61021; BD Transduction Laboratories), an anti-GAPDH (800–656–7625; Rockland Immunochemicals) diluted 1:2,000, or an anti-RhoGDi (sc-360; Santa Cruz Biotechnology) diluted 1:1,000.

Techniques: Comparison, Control, Expressing

Journal: Diabetes

Article Title: Seroreactivity Against Tyrosine Phosphatase PTPRN Links Type 2 Diabetes and Colorectal Cancer and Identifies a Potential Diagnostic and Therapeutic Target.

doi: 10.2337/db20-1206

Figure Lengend Snippet: Figure 2—In vitro PTPRN expression and seroreactivity analysis of PTPRN. A: PTPRN (979 aa in length) is composed of a signal peptide (SP), an ECD, a transmembrane domain (TM), and an ICD. The amino acids composing each domain are highlighted. B: Immunostaining using a monoclonal antibody against the HaloTag of the indicated fusion proteins in vitro expressed. C: Covalent immobilization of the proteins into MBs by HaloTag was verified through luminescence using a monoclonal antibody against the tag. D: Seroreactivity analysis of autoantibodies against ECD, ICD, and PTPRN in plasma samples from healthy individuals and those with diabetes, CRC, and CRC with T2D. E: PTPRN and ECD seroreactivity was significantly higher in the CRC group than in the control group (P value <0.05). F: When com- paring the control group with the patients with CRC and the CRC with patients with T2D separately, PTPRN could significantly discrimi- nate between control subjects and patients with T2D with CRC (P value <0.05), whereas ECD could significantly discriminate between the control group and the patients with CRC (P value <0.05). G: Not statistically significant differences were found when comparing EBNA1 seroreactivity among groups. EBNA1 seroreactivity was used as control because >90% of the human population has antibodies against EBNA1. H: Both PTPRN and ECD autoantibodies could discriminate between individuals with diabetes and patients with CRC and patients with CRC with T2D separately (P values <0.05). Measurements were performed in triplicate. Bar graphs represent the mean ± SD (E, F, and H). *P < 0.05.

Article Snippet: After blocking, membranes were incubated overnight at 4 C with an anti-HaloTag monoclonal antibody (#6921A; Promega) diluted 1:1,000, an anti-PTPRN monoclonal antibody (sc-130570; Santa Cruz Biotechnology) diluted 1:200, an anti–phosphorylated (p-)insulin Rb polyclonal antibody (sc-25103-R; Santa Cruz Biotechnology) diluted 1:1,000, an anti-IRS1 polyclonal antibody (sc-559; Santa Cruz Biotechnology) diluted 1:1,000, an anti–pFOXO1/3 polyclonal antibody (#9464; Cell Signaling Technology), an anti-AKT polyclonal antibody (#4691; Cell Signaling Technology,), an anti–p-AKT polyclonal antibody (#9275; Cell Signaling Technology), an anti-ERK polyclonal antibody (#4695; Cell Signaling Technology), an anti–p-ERK polyclonal antibody (#9101; Cell Signaling Technology), an anti-GSK3b monoclonal antibody (#61021; BD Transduction Laboratories), an anti-GAPDH (800–656–7625; Rockland Immunochemicals) diluted 1:2,000, or an anti-RhoGDi (sc-360; Santa Cruz Biotechnology) diluted 1:1,000.

Techniques: In Vitro, Expressing, Immunostaining, Clinical Proteomics, Control

Journal: Diabetes

Article Title: Seroreactivity Against Tyrosine Phosphatase PTPRN Links Type 2 Diabetes and Colorectal Cancer and Identifies a Potential Diagnostic and Therapeutic Target.

doi: 10.2337/db20-1206

Figure Lengend Snippet: Figure 3—Diagnostic potential of the detection of PTPRN autoantibodies. The diagnostic potential for the detection of autoantibodies against PTPRN and ECD was evaluated using ROC curves. A: Autoantibody detection could discriminate between control and CRC group with an AUC of 75.5%. B: Autoantibody detection could discriminate between patients with T2D and CRC with patients with T2D with an AUC of 90.0%. Sens, sensitivy; Spec, specificity.

Article Snippet: After blocking, membranes were incubated overnight at 4 C with an anti-HaloTag monoclonal antibody (#6921A; Promega) diluted 1:1,000, an anti-PTPRN monoclonal antibody (sc-130570; Santa Cruz Biotechnology) diluted 1:200, an anti–phosphorylated (p-)insulin Rb polyclonal antibody (sc-25103-R; Santa Cruz Biotechnology) diluted 1:1,000, an anti-IRS1 polyclonal antibody (sc-559; Santa Cruz Biotechnology) diluted 1:1,000, an anti–pFOXO1/3 polyclonal antibody (#9464; Cell Signaling Technology), an anti-AKT polyclonal antibody (#4691; Cell Signaling Technology,), an anti–p-AKT polyclonal antibody (#9275; Cell Signaling Technology), an anti-ERK polyclonal antibody (#4695; Cell Signaling Technology), an anti–p-ERK polyclonal antibody (#9101; Cell Signaling Technology), an anti-GSK3b monoclonal antibody (#61021; BD Transduction Laboratories), an anti-GAPDH (800–656–7625; Rockland Immunochemicals) diluted 1:2,000, or an anti-RhoGDi (sc-360; Santa Cruz Biotechnology) diluted 1:1,000.

Techniques: Diagnostic Assay, Control

Journal: Diabetes

Article Title: Seroreactivity Against Tyrosine Phosphatase PTPRN Links Type 2 Diabetes and Colorectal Cancer and Identifies a Potential Diagnostic and Therapeutic Target.

doi: 10.2337/db20-1206

Figure Lengend Snippet: Figure 4—Silencing of PTPRN in KM12C and KM12SM CRC cells and effect on their tumorigenic properties. A: Protein expression levels in CRC cell lines KM12C and KM12SM. RhoGDi was used as loading control. B: Evaluation by PCR and WB of the transient silencing of

Article Snippet: After blocking, membranes were incubated overnight at 4 C with an anti-HaloTag monoclonal antibody (#6921A; Promega) diluted 1:1,000, an anti-PTPRN monoclonal antibody (sc-130570; Santa Cruz Biotechnology) diluted 1:200, an anti–phosphorylated (p-)insulin Rb polyclonal antibody (sc-25103-R; Santa Cruz Biotechnology) diluted 1:1,000, an anti-IRS1 polyclonal antibody (sc-559; Santa Cruz Biotechnology) diluted 1:1,000, an anti–pFOXO1/3 polyclonal antibody (#9464; Cell Signaling Technology), an anti-AKT polyclonal antibody (#4691; Cell Signaling Technology,), an anti–p-AKT polyclonal antibody (#9275; Cell Signaling Technology), an anti-ERK polyclonal antibody (#4695; Cell Signaling Technology), an anti–p-ERK polyclonal antibody (#9101; Cell Signaling Technology), an anti-GSK3b monoclonal antibody (#61021; BD Transduction Laboratories), an anti-GAPDH (800–656–7625; Rockland Immunochemicals) diluted 1:2,000, or an anti-RhoGDi (sc-360; Santa Cruz Biotechnology) diluted 1:1,000.

Techniques: Expressing, Control

Journal: Diabetes

Article Title: Seroreactivity Against Tyrosine Phosphatase PTPRN Links Type 2 Diabetes and Colorectal Cancer and Identifies a Potential Diagnostic and Therapeutic Target.

doi: 10.2337/db20-1206

Figure Lengend Snippet: Figure 5—PTPRN depletion on KM12C and KM12SM CRC cells alters EMT transition and reduces insulin receptor signaling pathway. A and B: Alterations in EMT inducers after PTPRN depletion in KM12C and KM12SM cell lines. cDNA synthesized from total RNA from tran- siently depleted PTPRN and scrambled control cells was subjected to semiquantitative RT-PCR analysis using specific primers for the EMT inducers TGF-b1, SNAI1, Claudin-2, E-cadherin, N-cadherin, and ZO1 using 18S as control and for normalization (A) or qPCR analy- sis using specific primers for TGF-b1, SNAI1, Claudin-2, and E-cadherin using 18S for normalization (B). C–E: Analysis of alterations in the insulin receptor signaling pathway by PCR and WB, respectively, after PTPRN depletion in KM12C and KM12SM cell lines. cDNA synthe- sized from total RNA from transiently depleted PTPRN and scrambled control cells after 48 h posttransfection was subjected to semiquan- titative RT-PCR analysis using specific primers for IRS1, ERK1, ERK2, AKT1, AKT2, mTOR, FOXO1, AS160, and GSK3a using 18S as control and for normalization (C) or qPCR analysis using specific primers for ERK1, ERK2, AKT1, AKT2, mTOR, FOXO1, and GSK3a using 18S for normalization (D). E: Protein expression levels of p-FOXO1/3, GSK3b, p-IRb, AKT, p-AKT, ERK, p-ERK, and IRS1 in CRC cell lines KM12C and KM12SM transiently transfected with indicated siRNAs for 48 h confirmed RT-PCR and/or qPCR results. GAPDH and RhoGDi were used as controls. Red Ponceau staining of each line was used for normalization. A, C, and E: Two replicate experiments (#1 and #2) were analyzed. Representative images are shown. B and D: Data represent the mean ± SD of two experiments. The abundance of each mRNA and protein was quantified by densitometry using ImageJ. a.u., arbitrary units.

Article Snippet: After blocking, membranes were incubated overnight at 4 C with an anti-HaloTag monoclonal antibody (#6921A; Promega) diluted 1:1,000, an anti-PTPRN monoclonal antibody (sc-130570; Santa Cruz Biotechnology) diluted 1:200, an anti–phosphorylated (p-)insulin Rb polyclonal antibody (sc-25103-R; Santa Cruz Biotechnology) diluted 1:1,000, an anti-IRS1 polyclonal antibody (sc-559; Santa Cruz Biotechnology) diluted 1:1,000, an anti–pFOXO1/3 polyclonal antibody (#9464; Cell Signaling Technology), an anti-AKT polyclonal antibody (#4691; Cell Signaling Technology,), an anti–p-AKT polyclonal antibody (#9275; Cell Signaling Technology), an anti-ERK polyclonal antibody (#4695; Cell Signaling Technology), an anti–p-ERK polyclonal antibody (#9101; Cell Signaling Technology), an anti-GSK3b monoclonal antibody (#61021; BD Transduction Laboratories), an anti-GAPDH (800–656–7625; Rockland Immunochemicals) diluted 1:2,000, or an anti-RhoGDi (sc-360; Santa Cruz Biotechnology) diluted 1:1,000.

Techniques: Synthesized, Control, Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection, Staining

Journal: Diabetes

Article Title: Seroreactivity Against Tyrosine Phosphatase PTPRN Links Type 2 Diabetes and Colorectal Cancer and Identifies a Potential Diagnostic and Therapeutic Target.

doi: 10.2337/db20-1206

Figure Lengend Snippet: Figure 6—PTPRN depletion decreases liver homing and liver metastasis in KM12SM CRC cells. A: Nude mice intrasplenically inoculated with KM12SM cells transiently transfected with siScramble (n 5 2) and indicated PTPRN siRNAs (n 5 1/PTPRN siRNA) were sacrificed 24 h after inoculation for analysis of in vivo liver homing. RNA was isolated from the liver and spleen (as control) and directly subjected to RT-PCR to amplify human GAPDH (hGAPDH). Representative experiments out of two are shown. Murine b-actin (mActin) was amplified as control. B: Representative images of luminescence intensity of in vivo luciferase activity from mice injected with KM12SM cells tran- siently transfected with control and PTPRN siRNAs at days 45 and 60 postintrasplenical inoculation. Mice injected with control cells did show detectable bioluminescence by IVIS analyses, in contrast to mice injected with PTPRN-depleted cells (one out of six mice showed

Article Snippet: After blocking, membranes were incubated overnight at 4 C with an anti-HaloTag monoclonal antibody (#6921A; Promega) diluted 1:1,000, an anti-PTPRN monoclonal antibody (sc-130570; Santa Cruz Biotechnology) diluted 1:200, an anti–phosphorylated (p-)insulin Rb polyclonal antibody (sc-25103-R; Santa Cruz Biotechnology) diluted 1:1,000, an anti-IRS1 polyclonal antibody (sc-559; Santa Cruz Biotechnology) diluted 1:1,000, an anti–pFOXO1/3 polyclonal antibody (#9464; Cell Signaling Technology), an anti-AKT polyclonal antibody (#4691; Cell Signaling Technology,), an anti–p-AKT polyclonal antibody (#9275; Cell Signaling Technology), an anti-ERK polyclonal antibody (#4695; Cell Signaling Technology), an anti–p-ERK polyclonal antibody (#9101; Cell Signaling Technology), an anti-GSK3b monoclonal antibody (#61021; BD Transduction Laboratories), an anti-GAPDH (800–656–7625; Rockland Immunochemicals) diluted 1:2,000, or an anti-RhoGDi (sc-360; Santa Cruz Biotechnology) diluted 1:1,000.

Techniques: Transfection, In Vivo, Isolation, Control, Reverse Transcription Polymerase Chain Reaction, Luciferase, Activity Assay, Injection

Journal: Frontiers in Cell and Developmental Biology

Article Title: Overexpression of PTPRN Promotes Metastasis of Lung Adenocarcinoma and Suppresses NK Cell Cytotoxicity

doi: 10.3389/fcell.2021.622018

Figure Lengend Snippet: Antibodies used for WB and IHC.

Article Snippet: Anti-human PTPRN (for IHC) , Proteintech , 1:100.

Techniques:

Journal: Frontiers in Cell and Developmental Biology

Article Title: Overexpression of PTPRN Promotes Metastasis of Lung Adenocarcinoma and Suppresses NK Cell Cytotoxicity

doi: 10.3389/fcell.2021.622018

Figure Lengend Snippet: Association of PTPRN expression with OS by IHC. (A) IHC is used to stain for PTPRN in tumor samples from 40 LUAD patients. Representative IHC pictures of the high and low PTPRN expression groups are shown at ×20 and ×40 magnification (scale bars, 100 μm and 50 μm, respectively). (B) Kaplan–Meier survival analysis of 40 LUAD patients shows that the PTPRN high expression is related to unfavorable prognosis.

Article Snippet: Anti-human PTPRN (for IHC) , Proteintech , 1:100.

Techniques: Expressing, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: Overexpression of PTPRN Promotes Metastasis of Lung Adenocarcinoma and Suppresses NK Cell Cytotoxicity

doi: 10.3389/fcell.2021.622018

Figure Lengend Snippet: Effect of overexpression of PTPRN expression on migration of LUAD cells. (A) The expression of PTPRN in A549 and H1299 cells after transfection of the PTPRN plasmid. (B) The effect on migration of A549- and H1299-overexpressed PTPRN is detected by wound healing assay. (C) The effect on migration of A549- and H1299-overexpressed PTPRN is identified by transwell migration assay. Original magnification, ×100. Scale bars, 100 μm. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. oe-NC, negative control; and oe-PTPRN, PTPRN overexpression group.

Article Snippet: Anti-human PTPRN (for IHC) , Proteintech , 1:100.

Techniques: Over Expression, Expressing, Migration, Transfection, Plasmid Preparation, Wound Healing Assay, Transwell Migration Assay, Negative Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Overexpression of PTPRN Promotes Metastasis of Lung Adenocarcinoma and Suppresses NK Cell Cytotoxicity

doi: 10.3389/fcell.2021.622018

Figure Lengend Snippet: PTPRN expression in normal and lung cancer tissues in TCGA and survival time analyzed by Kaplan–Meier Plotter. (A,B) mRNA expression of PTPRN is analyzed in lung cancer in the TCGA-LUNG database. (C,D) mRNA expression of PTPRN is analyzed in LUAD in the TCGA-LUAD database. (E,F) mRNA expression of PTPRN is analyzed in LUSC in the TCGA database. (G) Kaplan–Meier survival analysis of lung cancer patients indicates that the decrease in PTPRN expression is correlated with favorable prognosis ( P = 0.0057). (H) Kaplan–Meier survival analysis of LUAD patients shows that the decrease in PTPRN expression is correlated with favorable prognosis ( P < 0.0001). (I) Kaplan–Meier survival analysis indicates that there is no correlation between PTPRN expression and prognosis of LUSC patients ( P = 0.96). * P < 0.05, ** P < 0.01. # P > 0.05.

Article Snippet: Anti-human PTPRN (for IHC) , Proteintech , 1:100.

Techniques: Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Overexpression of PTPRN Promotes Metastasis of Lung Adenocarcinoma and Suppresses NK Cell Cytotoxicity

doi: 10.3389/fcell.2021.622018

Figure Lengend Snippet: Univariate and multivariate analyses of OS in patients with LUAD in TCGA.

Article Snippet: Anti-human PTPRN (for IHC) , Proteintech , 1:100.

Techniques:

Journal: Frontiers in Cell and Developmental Biology

Article Title: Overexpression of PTPRN Promotes Metastasis of Lung Adenocarcinoma and Suppresses NK Cell Cytotoxicity

doi: 10.3389/fcell.2021.622018

Figure Lengend Snippet: Bioinformatic analysis and western blot indicating that PTPRN expression is related to EMT. (A) The result of GSEA shows that EMT is enriched in the high-PTPRN expression group. PTPRN promotes mRNA expression levels of EMT markers such as (B) N-cadherin, (C) Slug, (D) Snail, (E) Twist, and (F) Vimentin from RNA sequencing data from the TCGA-LUAD database. (G) The PI3K/AKT/mTOR signaling pathway was enriched in the PTPRN high-expression group by GSEA. (H) Left: Expression of EMT markers (Vimentin, N-cadherin, and E-cadherin) is detected in PTPRN-overexpressing A549 and H1299 cells by western blotting. Right: Densitometric analysis of protein expression. (I) Left: Proteins in MAPK/ERK and PI3K/AKT pathways are detected in PTPRN-overexpressing A549 and H1299 cells by western blotting. Right: Densitometric analysis of protein expression. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: Anti-human PTPRN (for IHC) , Proteintech , 1:100.

Techniques: Western Blot, Expressing, RNA Sequencing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Overexpression of PTPRN Promotes Metastasis of Lung Adenocarcinoma and Suppresses NK Cell Cytotoxicity

doi: 10.3389/fcell.2021.622018

Figure Lengend Snippet: The association between PTPRN expression and immune response by GSEA in the TCGA-LUAD dataset. GSEA shows that (A) inflammatory response, (B) TNFα signaling via NFκB, (C) TGFβ signaling, (D) negative regulation of immune response, (E) regulation of immune effector process, and (F) regulation production involved in immune response are enriched in high PTPRN expression. (G) PTPRN expression is negatively related to ImmuneScore. (H) There is no correlation between PTPRN expression and StromalScore. (I) PTPRN expression is not related to ESTIMATEScore.

Article Snippet: Anti-human PTPRN (for IHC) , Proteintech , 1:100.

Techniques: Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Overexpression of PTPRN Promotes Metastasis of Lung Adenocarcinoma and Suppresses NK Cell Cytotoxicity

doi: 10.3389/fcell.2021.622018

Figure Lengend Snippet: Gene set enriched in LUAD samples with high-expression PTPRN.

Article Snippet: Anti-human PTPRN (for IHC) , Proteintech , 1:100.

Techniques:

Journal: Frontiers in Cell and Developmental Biology

Article Title: Overexpression of PTPRN Promotes Metastasis of Lung Adenocarcinoma and Suppresses NK Cell Cytotoxicity

doi: 10.3389/fcell.2021.622018

Figure Lengend Snippet: PTPRN-related immune infiltration alteration. (A) The fractions of activated memory CD4 T cells, resting NK cells, M0 macrophages, activated mast cells, and neutrophils are higher in the high PTPRN expression group. The proportions of naive B cells, CD8 T cells, resting memory T cells, monocytes, and resting mast cells are lower in high-expression PTPRN. (B) The ratios of different types of immune cells are correlated in the tumor microenvironment.

Article Snippet: Anti-human PTPRN (for IHC) , Proteintech , 1:100.

Techniques: Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Overexpression of PTPRN Promotes Metastasis of Lung Adenocarcinoma and Suppresses NK Cell Cytotoxicity

doi: 10.3389/fcell.2021.622018

Figure Lengend Snippet: The potential affection of PTPRN on NK cells. PTPRN affects mRNA expression levels of NK cell inhibitory receptors such as (A) LILRB1 and (B) KLRC1 from RNA-sequencing data from the TCGA-LUAD database. (C) Percentages of CD3 – CD56 + cells of NK cells are determined by flow cytometry. (D) NK cytotoxicity assay is conducted with A549 target cells that overexpressed PTPRN on Day 14. The effector-to-target (E: T) ratio is 5:1. A similar assay is conducted with H1299 target cells on Day 14. The E:T ratio is 5:1. NK cells are treated with LUAD cells that overexpressed PTPRN. IFN-γ (E) and TNF-α (F) secretion is analyzed using ELISA. Data are presented as the mean ± SD of three independent experiments performed in triplicate. * P < 0.05, ** P < 0.01.

Article Snippet: Anti-human PTPRN (for IHC) , Proteintech , 1:100.

Techniques: Expressing, RNA Sequencing, Flow Cytometry, Cytotoxicity Assay, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Overexpression of PTPRN Promotes Metastasis of Lung Adenocarcinoma and Suppresses NK Cell Cytotoxicity

doi: 10.3389/fcell.2021.622018

Figure Lengend Snippet: The effect of PTPRN on EMT and NK cell inactivation in vivo. (A) Images of oe-PTPRN LLC tumor tissues. (B) Average tumor volumes are measured in xenograft mice every 2 days. (C) IHC analysis of vimentin and KLRA1 protein levels in tumor tissues formed from PTPRN-overexpressing cells or control cells. Original magnification, ×400. Scale bars, 50 μm. Data are presented as the mean ± SD of three independent experiments performed in triplicate. * P < 0.05, *** P < 0.001. oe-NC, NC-cDNA group; oe-PTPRN, PTPRN-cDNA group; TGF-β, NC-cDNA with the TGF-β group; and oe-PTPRN+ TGF-β, PTPRN-cDNA with the TGF-β group.

Article Snippet: Anti-human PTPRN (for IHC) , Proteintech , 1:100.

Techniques: In Vivo, Control