Addiction leading to abuse should be treatable by pharmacological approaches, and programs that identify new drugs to treat methamphetamine abuse address an immediate goal of the National Institute on Drug Abuse (NIDA) that new approaches are needed for treating METH addiction. Currently, small molecule drug-like compounds are not available for treating METH abuse. Neurotensin receptor 1 (NTR1) more ..
Related BioAssays
Related BioAssays
Target
BioActive Compounds: 184
Depositor Specified Assays
| AID | Name | Type | Comment |
|---|---|---|---|
| 493055 | Summary Assay for Selective Agonists of NTR1 | summary | Summary AID. |
Description:
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA)
Network: NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R03 MH089653-01
Assay Provider: Dr. Lawrence Barak, Duke University Medical Center
Addiction leading to abuse should be treatable by pharmacological approaches, and programs that identify new drugs to treat methamphetamine abuse address an immediate goal of the National Institute on Drug Abuse (NIDA) that new approaches are needed for treating METH addiction. Currently, small molecule drug-like compounds are not available for treating METH abuse. Neurotensin receptor 1 (NTR1) peptide agonists produce behaviors that are exactly opposite to the psychostimulant effects observed with methamphetamine abuse, such as hyperactivity, neurotoxicity, psychotic episodes, and cognitive deficits, and repeated administrations of NTR1 agonists do not lead to the development of tolerance. These data form the basis of the idea that neurotensin receptors are valid targets for antagonizing drug seeking behaviors and preventing relapses. We propose to identify novel small molecule neurotensin receptor agonists by screening libraries of compounds using a primary assay based upon the ability of a b-arrestin fluorescent reporter to directly recognize the activated state of the NTR1.
The goal of this assay is to confirm hits from Image-Based HTS for Selective Agonists for NTR1 (AID 493036).
References:
Biology of neurotensin: revisited study. Katsanos GS, Anogianaki A, Castellani ML, Ciampoli C, De Amicis D, Orso C, Pollice R, Vecchiet J, Tete S, Salini V, Caraffa A, Patruno A, Shaik YB, Kempuraj D, Doyle R, Antinolfi PL, Cerulli G, Conti CM, Fulcheri M, Neri G, Sabatino G. Int J Immunopathol Pharmacol. 2008 Apr-Jun;21(2):255-9
Biochemical and pharmacological profile of a potent and selective nonpeptide antagonist of the neurotensin receptor.
D Gully, M Canton, R Boigegrain, F Jeanjean, J C Molimard, M Poncelet, C Gueudet, M Heaulme, R Leyris, A Brouard, et al.
Proc Natl Acad Sci U S A. 1993 January 1; 90(1): 65-69.
Neurotensin receptor antagonists and therapeutical perspectives.
Gully D, Maffrand JP, Soubrie P, Rostene W, Kitabgi P, Le Fur G.
Therapie. 1995 Jan-Feb;50(1):5-7.
Neuropharmacological profile of non-peptide neurotensin antagonists.
Gully D, Jeanjean F, Poncelet M, Steinberg R, Soubrie P, Le Fur G, Maffrand JP. Fundam Clin Pharmacol. 1995;9(6):513-21. Review.
Use of nonpeptide antagonists to explore the physiological roles of neurotensin. Focus on brain neurotensin/dopamine interactions.
Rostene W, Azzi M, Boudin H, Lepee I, Souaze F, Mendez-Ubach M, Betancur C, Gully D. Ann N Y Acad Sci. 1997 Apr 24;814:125-41. Review.
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA)
Network: NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R03 MH089653-01
Assay Provider: Dr. Lawrence Barak, Duke University Medical Center
Addiction leading to abuse should be treatable by pharmacological approaches, and programs that identify new drugs to treat methamphetamine abuse address an immediate goal of the National Institute on Drug Abuse (NIDA) that new approaches are needed for treating METH addiction. Currently, small molecule drug-like compounds are not available for treating METH abuse. Neurotensin receptor 1 (NTR1) peptide agonists produce behaviors that are exactly opposite to the psychostimulant effects observed with methamphetamine abuse, such as hyperactivity, neurotoxicity, psychotic episodes, and cognitive deficits, and repeated administrations of NTR1 agonists do not lead to the development of tolerance. These data form the basis of the idea that neurotensin receptors are valid targets for antagonizing drug seeking behaviors and preventing relapses. We propose to identify novel small molecule neurotensin receptor agonists by screening libraries of compounds using a primary assay based upon the ability of a b-arrestin fluorescent reporter to directly recognize the activated state of the NTR1.
The goal of this assay is to confirm hits from Image-Based HTS for Selective Agonists for NTR1 (AID 493036).
References:
Biology of neurotensin: revisited study. Katsanos GS, Anogianaki A, Castellani ML, Ciampoli C, De Amicis D, Orso C, Pollice R, Vecchiet J, Tete S, Salini V, Caraffa A, Patruno A, Shaik YB, Kempuraj D, Doyle R, Antinolfi PL, Cerulli G, Conti CM, Fulcheri M, Neri G, Sabatino G. Int J Immunopathol Pharmacol. 2008 Apr-Jun;21(2):255-9
Biochemical and pharmacological profile of a potent and selective nonpeptide antagonist of the neurotensin receptor.
D Gully, M Canton, R Boigegrain, F Jeanjean, J C Molimard, M Poncelet, C Gueudet, M Heaulme, R Leyris, A Brouard, et al.
Proc Natl Acad Sci U S A. 1993 January 1; 90(1): 65-69.
Neurotensin receptor antagonists and therapeutical perspectives.
Gully D, Maffrand JP, Soubrie P, Rostene W, Kitabgi P, Le Fur G.
Therapie. 1995 Jan-Feb;50(1):5-7.
Neuropharmacological profile of non-peptide neurotensin antagonists.
Gully D, Jeanjean F, Poncelet M, Steinberg R, Soubrie P, Le Fur G, Maffrand JP. Fundam Clin Pharmacol. 1995;9(6):513-21. Review.
Use of nonpeptide antagonists to explore the physiological roles of neurotensin. Focus on brain neurotensin/dopamine interactions.
Rostene W, Azzi M, Boudin H, Lepee I, Souaze F, Mendez-Ubach M, Betancur C, Gully D. Ann N Y Acad Sci. 1997 Apr 24;814:125-41. Review.
Protocol
A. Brief Description of the Assay:
This assay is to confirm lead agonists from the primary screening results that activate the neurotensin receptor-1 pathway by detecting of spots of-arrestin-GFP in NTR1-U2OS Osteosarcoma Cells.
B. Materials:
Description Source Cat
NTR1-U2OS osteosarcoma cells Dr. Lawrence Barak, Duke University N/A
MEM medium Cellgro/Mediatech 15-010-CM
Fetal Bovine Serum Hyclone SH30396.03
Penicillin Streptomycin solution Omega Scientific PS-20
L-Glutamine Mediatech 25-005-CL
G418 (100 mg/mL) Invivogen ant-gn-1
Zeocin (100 mg/mL) Invitrogen R250-01
T225 Tissue Culture flasks Corning 431082
DPBS Cellgro 21-031CV
Trypsin-EDTA 0.05% Invitrogen 25300
1536 well black clear bottom plate Aurora Biotechnology 29326
NTR1 peptide Sigma N6383
Paraformaldehyde Acros Organics 30528954
Hoechst 33342 (10 mg/mL) Invitrogen H3570
C. Plate Map:
Positive (High) control (P) in columns 3 and 4, DMSO and 100 nM NTR1
Negative (Low) control (N) in columns 1 and 2, DMSO but No NTR1
Test compound in columns 5 - 48, Test compound but No NTR1
D. Procedures:
Day 1
Step Description
1 Prepare cell suspension as described in section F. Cell Culture.
2 Set up and prime Combi liquid dispenser
3 Dispense 4 uL/well of cells at 3.5X10;5 cells/mL using Combi into a black 1536 well plate with clear bottom.
4 Spin down plates on Eppendorf centrifuge 5810 at 1000 rpm for 1 minute.
5 Put Kalypsys metal lids on plates, and incubate plates at 37 oC with 5% CO2 overnight.
Day 2
Step Description
1 Set up Combi and nL liquid dispensers, Kalypsys plate washer and Opera.
2 Transfer 12.5 nL/well of 10 mM test compounds into assay plates using the Labcyte ECHO 555. The final concentration of compounds is 20 uM with 0.2% DMSO. Compounds are screened in quadruplicates.
3 Add 2 uL/well of 300 nM NTR1 peptide in DPBS to columns 3 and 4 of the assay plates and then add 2 uL/well of DPBS (no peptide) to columns 1, 2, 5-48 using Combi. The final concentration of NTR1 peptide is 100nM.
4 Spin down plates on Eppendorf centrifuge 5810 at 1000 rpm for 1 minute.
5 Incubate plates at 37 oC with 5% CO2 for 1 hour.
6 Fix cells by adding 4 uL/well of 6% Paraformaldehyde into all wells using Combi. The final concentration of Paraformaldehyde is 2.4%.
7 Spin down plates on Eppendorf centrifuge 5810 at 1000 rpm for 1 minute, and Incubate for one hour at room temperature.
8 Wash plates with 1X PBS on the Kalypsys waher by aspirating leaving ~2.5 uL/well and dispensing 11 uL/well DPBS twice, and then aspirating leaving ~2.5 uL/well.
9 Add 5 uL/well of 5 ug/mL Hoechst 33342 diluted in DPBS to assay plates by using Combi nL dispenser. The final concentration of Hoechst 33342 is 3.3 ug/mL.
10 Spin down plates on Eppendorf centrifuge 5810 at 1000 rpm for 1 minute. Then seal plates.
11 Incubate plates for one hour prior to loading to PerkinElmer Opera, or store plates at 4 oC until imaging.
E. Recipes:
(1) Cell Growth Media and Cell Culture
NTR1-U2OS osteosarcoma cells are maintained in MEM growth medium containing 10% Fetal Bovine Serum, 1% Penicillin Streptomycin solution, 1% L-Glutamine with 400 u g/mL G418 and 200ug/mL Zeocin at 37 degrees C with 5% CO2. Discard cells after 20 passages.
(2) Assay Media
MEM medium containing 2.5 % Fetal Bovine Serum, 1 % Penicillin Streptomycin solution, 1 % L-Glutamine with 400 ug/mL G418 and 200 ug/mL Zeocin.
(3) NTR1 peptide solution
Step Description
1 Dissolve the powder in 50% Glycerol-H2O at 1 mM.
2 Store it as stock solution at 4 degrees until until just before use.
3 Dilute stock solution (1 mM) to the working conc. of 300 nM in DPBS.
*Prepare working solution (300 nM) just before use due to stability issue of the peptide.
(4) 6% Paraformaldehyde Solution
Step Description
1 Place 2.4 L of MilliQ H2O on a container and pH to 11 using NaOH while stirring.
2 Add 180 g of Paraformaldehyde powder gently into solution.
3 Keep stirring at room temp until it is completely dissolved (this can take several hours).
4 Adjust pH to 7.4 using H2SO4 (not HCL).
5 Add MilliQ H2O to have the solution final volume 3L.
6 Store the solution at 4 degrees. Protect from light. It can be stored up to a week.
(5) Hoechst solution
Step Description
1 Dilute stock solution (10 mg/mL) to the working conc. of 5 ug/mL in DPBS.
J. Image Acquisition and Analysis:
1) Image acquisition was performed on an Opera QEHS (Perkin Elmer) with 45 plate capacity loader/stacker and the following settings:
- 40x 0.6 NA air objective
- Acquisition camera set to 2-by-2 binning for an image size of 688 by 512 pixels
- 2 channels acquired sequentially: 1) Beta-arrestin-GFP using 488 nm laser excitation and 540/75 nm emisssion filters, 2) DAPI (nuclei) using 365 nm Xenon lamp excitation and 450/50 nm emission filters
- 3 fields per well for Primary screen
2) Image analysis was performed using the Acapella Spot Detection Algorithm with the following analysis settings:
NUCLEI DETECTION
- Threshold Adjustment: 1.5
- Minimum Nuclei distance: 7
- Nuclear Splitting Adjustment: 7
-Individual Threshold Adjustment: 0.4
- Minimum Nuclear Area: 70
- Minimum Nuclear Contrast: 0.1
CYTOPLASM DETECTION
- Cytoplasm Individual Threshold Adjustment: 0.1
SPOT DETECTION
- Spot Minimum Distance: 3
- Spot Peak Radius: 0
- Spot Reference Radius: 3
- Spot Minimum Contrast: 0.4
- Spot Minimum to Cell Intensity: 1
3) Metrics calculated from...
NUCLEI IMAGES:
Cell Count ("CellCount"),
Nuclei Area ("NuclearArea"),
Integrated Intensity of the Nuclei ("TotNucIntensity"),
Average Intensity of the Nuclei ("AvgNucIntensity");
GFP IMAGES:
Average Intensity of the Cytoplasm ("AvgCytoIntensity"),
Average Intensity of the Cell ("AvgCellIntensity"),
Integrated Intensity of the Cell ("TotCellIntensity"),
Integrated Intensity of the Cytoplasm ("TotCytoIntensity"),
Integrated Intensity of the Detected Spots ("TotSpotIntensity"),
Ratio of the Integrated Spot to Integrated Cytoplasm Intensities ("RatioTotSpotIntensity2CellIntensity"),
Number of Spots per Cell ("AvgSpotPerCell"),
Average Area of Spots ("AvgSpotAreaPerCell").
This assay is to confirm lead agonists from the primary screening results that activate the neurotensin receptor-1 pathway by detecting of spots of-arrestin-GFP in NTR1-U2OS Osteosarcoma Cells.
B. Materials:
Description Source Cat
NTR1-U2OS osteosarcoma cells Dr. Lawrence Barak, Duke University N/A
MEM medium Cellgro/Mediatech 15-010-CM
Fetal Bovine Serum Hyclone SH30396.03
Penicillin Streptomycin solution Omega Scientific PS-20
L-Glutamine Mediatech 25-005-CL
G418 (100 mg/mL) Invivogen ant-gn-1
Zeocin (100 mg/mL) Invitrogen R250-01
T225 Tissue Culture flasks Corning 431082
DPBS Cellgro 21-031CV
Trypsin-EDTA 0.05% Invitrogen 25300
1536 well black clear bottom plate Aurora Biotechnology 29326
NTR1 peptide Sigma N6383
Paraformaldehyde Acros Organics 30528954
Hoechst 33342 (10 mg/mL) Invitrogen H3570
C. Plate Map:
Positive (High) control (P) in columns 3 and 4, DMSO and 100 nM NTR1
Negative (Low) control (N) in columns 1 and 2, DMSO but No NTR1
Test compound in columns 5 - 48, Test compound but No NTR1
D. Procedures:
Day 1
Step Description
1 Prepare cell suspension as described in section F. Cell Culture.
2 Set up and prime Combi liquid dispenser
3 Dispense 4 uL/well of cells at 3.5X10;5 cells/mL using Combi into a black 1536 well plate with clear bottom.
4 Spin down plates on Eppendorf centrifuge 5810 at 1000 rpm for 1 minute.
5 Put Kalypsys metal lids on plates, and incubate plates at 37 oC with 5% CO2 overnight.
Day 2
Step Description
1 Set up Combi and nL liquid dispensers, Kalypsys plate washer and Opera.
2 Transfer 12.5 nL/well of 10 mM test compounds into assay plates using the Labcyte ECHO 555. The final concentration of compounds is 20 uM with 0.2% DMSO. Compounds are screened in quadruplicates.
3 Add 2 uL/well of 300 nM NTR1 peptide in DPBS to columns 3 and 4 of the assay plates and then add 2 uL/well of DPBS (no peptide) to columns 1, 2, 5-48 using Combi. The final concentration of NTR1 peptide is 100nM.
4 Spin down plates on Eppendorf centrifuge 5810 at 1000 rpm for 1 minute.
5 Incubate plates at 37 oC with 5% CO2 for 1 hour.
6 Fix cells by adding 4 uL/well of 6% Paraformaldehyde into all wells using Combi. The final concentration of Paraformaldehyde is 2.4%.
7 Spin down plates on Eppendorf centrifuge 5810 at 1000 rpm for 1 minute, and Incubate for one hour at room temperature.
8 Wash plates with 1X PBS on the Kalypsys waher by aspirating leaving ~2.5 uL/well and dispensing 11 uL/well DPBS twice, and then aspirating leaving ~2.5 uL/well.
9 Add 5 uL/well of 5 ug/mL Hoechst 33342 diluted in DPBS to assay plates by using Combi nL dispenser. The final concentration of Hoechst 33342 is 3.3 ug/mL.
10 Spin down plates on Eppendorf centrifuge 5810 at 1000 rpm for 1 minute. Then seal plates.
11 Incubate plates for one hour prior to loading to PerkinElmer Opera, or store plates at 4 oC until imaging.
E. Recipes:
(1) Cell Growth Media and Cell Culture
NTR1-U2OS osteosarcoma cells are maintained in MEM growth medium containing 10% Fetal Bovine Serum, 1% Penicillin Streptomycin solution, 1% L-Glutamine with 400 u g/mL G418 and 200ug/mL Zeocin at 37 degrees C with 5% CO2. Discard cells after 20 passages.
(2) Assay Media
MEM medium containing 2.5 % Fetal Bovine Serum, 1 % Penicillin Streptomycin solution, 1 % L-Glutamine with 400 ug/mL G418 and 200 ug/mL Zeocin.
(3) NTR1 peptide solution
Step Description
1 Dissolve the powder in 50% Glycerol-H2O at 1 mM.
2 Store it as stock solution at 4 degrees until until just before use.
3 Dilute stock solution (1 mM) to the working conc. of 300 nM in DPBS.
*Prepare working solution (300 nM) just before use due to stability issue of the peptide.
(4) 6% Paraformaldehyde Solution
Step Description
1 Place 2.4 L of MilliQ H2O on a container and pH to 11 using NaOH while stirring.
2 Add 180 g of Paraformaldehyde powder gently into solution.
3 Keep stirring at room temp until it is completely dissolved (this can take several hours).
4 Adjust pH to 7.4 using H2SO4 (not HCL).
5 Add MilliQ H2O to have the solution final volume 3L.
6 Store the solution at 4 degrees. Protect from light. It can be stored up to a week.
(5) Hoechst solution
Step Description
1 Dilute stock solution (10 mg/mL) to the working conc. of 5 ug/mL in DPBS.
J. Image Acquisition and Analysis:
1) Image acquisition was performed on an Opera QEHS (Perkin Elmer) with 45 plate capacity loader/stacker and the following settings:
- 40x 0.6 NA air objective
- Acquisition camera set to 2-by-2 binning for an image size of 688 by 512 pixels
- 2 channels acquired sequentially: 1) Beta-arrestin-GFP using 488 nm laser excitation and 540/75 nm emisssion filters, 2) DAPI (nuclei) using 365 nm Xenon lamp excitation and 450/50 nm emission filters
- 3 fields per well for Primary screen
2) Image analysis was performed using the Acapella Spot Detection Algorithm with the following analysis settings:
NUCLEI DETECTION
- Threshold Adjustment: 1.5
- Minimum Nuclei distance: 7
- Nuclear Splitting Adjustment: 7
-Individual Threshold Adjustment: 0.4
- Minimum Nuclear Area: 70
- Minimum Nuclear Contrast: 0.1
CYTOPLASM DETECTION
- Cytoplasm Individual Threshold Adjustment: 0.1
SPOT DETECTION
- Spot Minimum Distance: 3
- Spot Peak Radius: 0
- Spot Reference Radius: 3
- Spot Minimum Contrast: 0.4
- Spot Minimum to Cell Intensity: 1
3) Metrics calculated from...
NUCLEI IMAGES:
Cell Count ("CellCount"),
Nuclei Area ("NuclearArea"),
Integrated Intensity of the Nuclei ("TotNucIntensity"),
Average Intensity of the Nuclei ("AvgNucIntensity");
GFP IMAGES:
Average Intensity of the Cytoplasm ("AvgCytoIntensity"),
Average Intensity of the Cell ("AvgCellIntensity"),
Integrated Intensity of the Cell ("TotCellIntensity"),
Integrated Intensity of the Cytoplasm ("TotCytoIntensity"),
Integrated Intensity of the Detected Spots ("TotSpotIntensity"),
Ratio of the Integrated Spot to Integrated Cytoplasm Intensities ("RatioTotSpotIntensity2CellIntensity"),
Number of Spots per Cell ("AvgSpotPerCell"),
Average Area of Spots ("AvgSpotAreaPerCell").
Comment
Compounds with %Activity at 20 uM_Mean >= 40% are considered to be active in this assay.
Activity Scoring
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data.
The score is correlated with % activity in the assay:
a. If outcome of the primary screen is inactive, then the assigned score is 0
b. If outcome of the primary screen is inconclusive, then the assigned score is 10
c. If outcome of the primary screen is active, then the assigned score is 20
Scoring for Single concentration confirmation screening
d. If outcome of the single-concentration confirmation screen is inactive, then the assigned score is 21
e. If outcome of the single-concentration confirmation screen is inconclusive, then the assigned score is 25
f. If outcome of the single-concentration confirmation screen is active, then the assigned score is 30
This scoring system helps track the stage of the testing of a particular SID. For the primary hits which are available for confirmation, their scores will be greater than 20. For those which are not further confirmed, their score will stay under 21.
2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable in this assay
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable in this assay
Activity Scoring
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data.
The score is correlated with % activity in the assay:
a. If outcome of the primary screen is inactive, then the assigned score is 0
b. If outcome of the primary screen is inconclusive, then the assigned score is 10
c. If outcome of the primary screen is active, then the assigned score is 20
Scoring for Single concentration confirmation screening
d. If outcome of the single-concentration confirmation screen is inactive, then the assigned score is 21
e. If outcome of the single-concentration confirmation screen is inconclusive, then the assigned score is 25
f. If outcome of the single-concentration confirmation screen is active, then the assigned score is 30
This scoring system helps track the stage of the testing of a particular SID. For the primary hits which are available for confirmation, their scores will be greater than 20. For those which are not further confirmed, their score will stay under 21.
2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable in this assay
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable in this assay
Result Definitions
Show more |
| TID | Name | Description | Histogram | Type | Unit | |
|---|---|---|---|---|---|---|
| Outcome | The BioAssay activity outcome | Outcome | ||||
| Score | The BioAssay activity ranking score |  | Integer | |||
| 1 | %Activity at 20 uM_Mean (20μM**) | Mean %Activity at 20 uM for the four replicates | | Float | % | |
| 2 | %Activity at 20 uM_1 (20μM**) | Mean %Activity at 20 uM for the first replicate | | Float | % | |
| 3 | %Activity at 20 uM_2 (20μM**) | Mean %Activity at 20 uM for the second replicates | | Float | % | |
| 4 | %Activity at 20 uM_3 (20μM**) | Mean %Activity at 20 uM for the third replicates | | Float | % | |
| 5 | %Activity at 20 uM_4 (20μM**) | Mean %Activity at 20 uM for the fourth replicate | | Float | % | |
| 6 | Mean_PC | Mean fluorescence spot detection signal of positive controls in the corresponding plate | | Float | RU | |
| 7 | StdDev_PC | Standard deviation (n=64) of positive controls in the corresponding plate | | Float | RU | |
| 8 | Mean_NC | Mean fluorescence spot detection signal of negative controls in the corresponding plate | | Float | RU | |
| 9 | StdDev_NC | Standard deviation (n=64) of negative controls in the corresponding plate | | Float | RU |
** Test Concentration.
Additional Information
Grant Number: 1 R03 MH089653-01
Data Table (Concise)
Classification
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