This study developed novel luminescence-based assays called MeNArC and EeNArC to measure β-arrestin recruitment to G protein-coupled receptors (GPCRs) without requiring fusion tags. MeNArC detects plasma membrane recruitment for class A receptors like dopamine D2 receptor. EeNArC detects both plasma membrane and early endosomal recruitment for class B receptors like angiotensin II receptor. They validated the assays in cell lines and showed they can detect partial agonism and different receptor types. The assays may serve as a generic screening method for measuring β-arrestin recruitment to unmodified receptors.
An overview of the history of structural studies of G protein-coupled receptors, written by Prof. Tony Harmar prior to his retirement from the University of Edinburgh and as chair of the Guide to PHARMACOLOGY.
An overview of the history of structural studies of G protein-coupled receptors, written by Prof. Tony Harmar prior to his retirement from the University of Edinburgh and as chair of the Guide to PHARMACOLOGY.
Purification of total RNA from peripheral blood mononuclear cells - Download ...QIAGEN
Peripheral blood is often used for in vitro studies of the human immune system or immune responses, such as inflammation. An important part of the human immune system is represented by the peripheral blood mononuclear cells (PBMC). PBMC are blood cells characterized by a round nucleus and consist mainly of lymphocytes (T cells, B cells, and NK cells), macrophages and dendritic cells. Here, we describe the analysis of lipopolysaccharide-induced transcriptional response of isolated PBMC from whole blood using the RNeasy® Mini Kit or RNeasy Micro Kit, RT2 First Strand Kit, RT2 SYBR® Green ROX™ qPCR Mastermix, and RT2 Profiler PCR Arrays.
Two dimensional Nuclear Magnetic Resonance (2D NMR) refers to a set of multi pulse techniques which were introduced to overcome the complex spectra obtained with NMR.
It is a set of NMR methods which give data plotted in a space defined by two frequency axes rather than one.
A novel label-free cocaine assay based on aptamer-wrapped single-walled carbo...Nanomedicine Journal (NMJ)
Objective(s):
This paper describes a selective and sensitive biosensor based on the dissolution and aggregation of aptamer wrapped single-walled carbon nanotubes. We report on the direct detection of aptamer–cocaine interactions, namely between a DNA aptamer and cocaine molecules based on near-infrared absorption at λ807.
Materials and Methods:
First a DNA aptamer recognizing cocaine was non-covalently immobilized on the surface of single walled carbon nanotubes and consequently dissolution of SWNTs was occurred. Vis-NIR absorption (A807nm) of dispersed, soluble aptamer-SWNTs hybrid, before and after incubation with cocaine was measured using a CECIL9000 spectrophotometer.
Results:
This carbon nanotube setup enabled the reliable monitoring of the interaction of cocaine with its cognate aptamer by aggregation of SWNTs in the presence of cocaine.
Disscusion:
This assay system provides a mean for the label-free, concentration-dependent, and selective detection of cocaine with an observed detection limit of 49.5 nM.
Francesca Gottschalk - How can education support child empowerment.pptxEduSkills OECD
Francesca Gottschalk from the OECD’s Centre for Educational Research and Innovation presents at the Ask an Expert Webinar: How can education support child empowerment?
Purification of total RNA from peripheral blood mononuclear cells - Download ...QIAGEN
Peripheral blood is often used for in vitro studies of the human immune system or immune responses, such as inflammation. An important part of the human immune system is represented by the peripheral blood mononuclear cells (PBMC). PBMC are blood cells characterized by a round nucleus and consist mainly of lymphocytes (T cells, B cells, and NK cells), macrophages and dendritic cells. Here, we describe the analysis of lipopolysaccharide-induced transcriptional response of isolated PBMC from whole blood using the RNeasy® Mini Kit or RNeasy Micro Kit, RT2 First Strand Kit, RT2 SYBR® Green ROX™ qPCR Mastermix, and RT2 Profiler PCR Arrays.
Two dimensional Nuclear Magnetic Resonance (2D NMR) refers to a set of multi pulse techniques which were introduced to overcome the complex spectra obtained with NMR.
It is a set of NMR methods which give data plotted in a space defined by two frequency axes rather than one.
A novel label-free cocaine assay based on aptamer-wrapped single-walled carbo...Nanomedicine Journal (NMJ)
Objective(s):
This paper describes a selective and sensitive biosensor based on the dissolution and aggregation of aptamer wrapped single-walled carbon nanotubes. We report on the direct detection of aptamer–cocaine interactions, namely between a DNA aptamer and cocaine molecules based on near-infrared absorption at λ807.
Materials and Methods:
First a DNA aptamer recognizing cocaine was non-covalently immobilized on the surface of single walled carbon nanotubes and consequently dissolution of SWNTs was occurred. Vis-NIR absorption (A807nm) of dispersed, soluble aptamer-SWNTs hybrid, before and after incubation with cocaine was measured using a CECIL9000 spectrophotometer.
Results:
This carbon nanotube setup enabled the reliable monitoring of the interaction of cocaine with its cognate aptamer by aggregation of SWNTs in the presence of cocaine.
Disscusion:
This assay system provides a mean for the label-free, concentration-dependent, and selective detection of cocaine with an observed detection limit of 49.5 nM.
Francesca Gottschalk - How can education support child empowerment.pptxEduSkills OECD
Francesca Gottschalk from the OECD’s Centre for Educational Research and Innovation presents at the Ask an Expert Webinar: How can education support child empowerment?
The French Revolution, which began in 1789, was a period of radical social and political upheaval in France. It marked the decline of absolute monarchies, the rise of secular and democratic republics, and the eventual rise of Napoleon Bonaparte. This revolutionary period is crucial in understanding the transition from feudalism to modernity in Europe.
For more information, visit-www.vavaclasses.com
June 3, 2024 Anti-Semitism Letter Sent to MIT President Kornbluth and MIT Cor...Levi Shapiro
Letter from the Congress of the United States regarding Anti-Semitism sent June 3rd to MIT President Sally Kornbluth, MIT Corp Chair, Mark Gorenberg
Dear Dr. Kornbluth and Mr. Gorenberg,
The US House of Representatives is deeply concerned by ongoing and pervasive acts of antisemitic
harassment and intimidation at the Massachusetts Institute of Technology (MIT). Failing to act decisively to ensure a safe learning environment for all students would be a grave dereliction of your responsibilities as President of MIT and Chair of the MIT Corporation.
This Congress will not stand idly by and allow an environment hostile to Jewish students to persist. The House believes that your institution is in violation of Title VI of the Civil Rights Act, and the inability or
unwillingness to rectify this violation through action requires accountability.
Postsecondary education is a unique opportunity for students to learn and have their ideas and beliefs challenged. However, universities receiving hundreds of millions of federal funds annually have denied
students that opportunity and have been hijacked to become venues for the promotion of terrorism, antisemitic harassment and intimidation, unlawful encampments, and in some cases, assaults and riots.
The House of Representatives will not countenance the use of federal funds to indoctrinate students into hateful, antisemitic, anti-American supporters of terrorism. Investigations into campus antisemitism by the Committee on Education and the Workforce and the Committee on Ways and Means have been expanded into a Congress-wide probe across all relevant jurisdictions to address this national crisis. The undersigned Committees will conduct oversight into the use of federal funds at MIT and its learning environment under authorities granted to each Committee.
• The Committee on Education and the Workforce has been investigating your institution since December 7, 2023. The Committee has broad jurisdiction over postsecondary education, including its compliance with Title VI of the Civil Rights Act, campus safety concerns over disruptions to the learning environment, and the awarding of federal student aid under the Higher Education Act.
• The Committee on Oversight and Accountability is investigating the sources of funding and other support flowing to groups espousing pro-Hamas propaganda and engaged in antisemitic harassment and intimidation of students. The Committee on Oversight and Accountability is the principal oversight committee of the US House of Representatives and has broad authority to investigate “any matter” at “any time” under House Rule X.
• The Committee on Ways and Means has been investigating several universities since November 15, 2023, when the Committee held a hearing entitled From Ivory Towers to Dark Corners: Investigating the Nexus Between Antisemitism, Tax-Exempt Universities, and Terror Financing. The Committee followed the hearing with letters to those institutions on January 10, 202
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Read| The latest issue of The Challenger is here! We are thrilled to announce that our school paper has qualified for the NATIONAL SCHOOLS PRESS CONFERENCE (NSPC) 2024. Thank you for your unwavering support and trust. Dive into the stories that made us stand out!
1. Pedersen, et. al. J. Biol. Chem. 2021
Departments of Psychiatry & Molecular Pharmacology and Therapeutics,
Colombia University
22/04/19 SB8
1
A novel luminescence-based β-arrestin
recruitment assay for unmodified receptors
2. Introduction
• GPCR: G protein-coupled receptors (GPCRs) and interaction with β-arrestin-
mediated signaling have involved signaling in more targeted therapeutics and
biological phenomena, e. g: heart failure (β-adrenergic receptor),
neuropathology (μ-opioid receptor), and behavior (dopamine D2 receptor).
2
heart failure (β-adrenergic receptor)
neuropathology
(μ-opioid
receptor),
4. The most well-established techniques to
investigate β-arrestin recruitment
4
PathHunter
The Tango-GPCR assay
5. Introduction
• GPCR: G protein-coupled receptors (GPCRs) and interaction with β-arrestin-
mediated signaling have involved signaling in more targeted therapeutics and
biological phenomena, e. g: heart failure (β-adrenergic receptor),
neuropathology (μ-opioid receptor), and behavior (dopamine D2 receptor).
5
Why the assay need to bind to membrane not in GPCR receptor? Notably,
all of these β-arrestin assays require that fusion tags be directly attached to the
C terminus of receptors of interest, which could alter GRK, arrestin, or other
protein interactions with the receptors and therefore impact signaling
Purpose: developed the assay which avoids potential artifacts related to C-
terminal receptor modification for measuring β-arrestin recruitment to diverse
GPCR types in heterologous or native cells.
6. Flow this research
• Models: HEK293 cells (ATTC CRL-1573) and CHO-K1 cell lines
• Plasmid construction: using standard techniques in molecular biology,
including overlapping-PCR and Gibson Assembly
• β-Arrestin complementation assay:
• For initial testing: transfected cells were measured in 96-well black-white
using PHERAstar FS (BMG labtech) plate reader
• For stable cell line testing: in 384 well plate and luminescence was read on a
Flexstation III.
6
7. Validation of a unique NanoLuc split for
complementation assays (1)
Namkung, et. al. Nat. Comm. 2016 7
Preliminary data, They used NanoBiT to
detect membrane recruitment of β-arrestin.
No luminescence detected upon agonist
stimulation
8. Validation of a unique NanoLuc split for
complementation assays (2)
8
They selected a site in a loop region that
divides NanoLuc into two almost equally
sized fragments (N: 1–102; C: 103–172),
without disrupting any secondary structural
element
Cotransfection of both fragments increased
~30-fold luminescence signal by the
addition of rapamycin
9. 9
Validation of a unique NanoLuc split for
complementation assays (3)
the N-terminal Nanoluc fragment was
attached to the C-terminal tail of D2R
and the C-terminal fragment was fused
to the N terminus of β- arrestin2
In this configuration, the endogenous
agonist dopamine produced a time- and
dose-dependent increase in
luminescence
10. Detection of β-arrestin plasma membrane
recruitment by class A receptors (1)
10
The N-terminal fragment of NanoLuc is
anchored to the plasma membrane
through a double palmitoylated domain
of GAP43 (MeN)
the C-terminal fragment is fused to the N
terminus of β-arrestin1 or β-arrestin2
(ArC)
Increase in luminescence upon agonist
treatment (QP/quinpirole) and decay in
luminescence after antagonist stimulation
(SP/sulpiride)
11. Detection of β-arrestin plasma membrane
recruitment by class A receptors (2)
11
To assess the MeNArC assay’s dynamic
range, different level of efficacy agonist
were tested.
MeNArC assay is also capable of
measuring different degrees of partial
agonism of unmodified receptors
Quinpirole (High-efficacy)
Bromocriptine and Terguride (Moderate)
PPP (Low)
12. Detection of β-arrestin plasma membrane and early
endosomal recruitment by a class B receptor (1)
12
Unlike class A receptors, AT1R and
other class B receptors bind β-arrestin
with higher affinity and form longer-lived
complexes that persist after endocytosis
The ArC component described is
coexpressed with the N-terminal
NanoLuc fragment attached to the
FYVE domain of endofin (EeN)
(EeNArC), which was shown previously
to selectively bind endosomes and used
in a direct receptor recruitment assay
13. Detection of β-arrestin plasma membrane and early
endosomal recruitment by a class B receptor (2)
13
As expected, the agonist effect was
dramatically blunted by sucrose for the
EeNArC assay but not for the MeNArC
assay.
14. Development of a polycistronic MeNArC expression
in stably transfected CHO-K1 cells
14
Schematic of the polycistronic MeNArC
Dose–response curves of the MeNArC assay for the
MOR and KOR cells showing increase in luminescence
(au) with increasing concentration of agonist
β-arrestin2 recruitment tested with the MOR
partial agonists morphine and buprenorphine
normalized to the full agonist DAMGO
15. In Conclusion
• MeNArC and EeNArC is β-arrestin membrane and endosomal recruitment
assays using complementation of novel NanoLuc fragments with the potential of
serving as a generic and readily adapted screening method for detecting β-
arrestin recruitment to unmodified receptors.
15
• Note for reader:
• This assays cannot rule out action of a tested agonist on endogenous
receptors or possible off-target receptors when tested in native tissue unless
knockout models are available.
• Need to test the utility of MeNArC and EeNArC in primary cell cultures for
measuring β-arrestin recruitment to endogenous receptors.
• This assay reports on β-arrestin recruitment, like all β-arrestin recruitment
assays, it does not measure β-arrestin “activity” and therefore does not
directly report on downstream signaling, which can involve several
different pathways such as ERK and Src
16. Note
• Imaging data
• Part fragment not lebih berat dari b arrestin
• Check endosomal activity
16
Editor's Notes
“Today, I‘d like to talk about my theme:
Nowadays, increased attention of GPCR for signaling purpose.
(GRK)-mediated phosphorylation of serine and threonine residues, most notably in the C-terminal tail of receptors but also in the intracellular loops of some receptors, which are the
primary receptor recognition regions for β-arrestin binding.
In addition to its role in signaling, β-arrestin also regulates receptor internalization and for some receptors stays bound to endosomes where receptor-mediated signaling may continue to occur
The PathHunter assay is an enzyme complementation assay in which split enzyme fragments are fused to the receptor and to β-arrestin and complementation of the functional enzyme creates a chemiluminescence readout
The Tango-GPCR assay is a reporter gene assay where a transcription factor fused to the receptor C terminus is cleaved off by a proteasetagged β-arrestin, leading to the expression of a reporter that
creates a luminescence readout
LinkLight uses a modified luciferase attached to β-arrestin that is cleaved off by a protease fused to the C terminus of the GPCR of interest, thereby activating the luciferase and producing light
Nowadays, increased attention of GPCR for signaling purpose.
They assumed that the nature of the NanoBiT split might prevent proper complementation when used in our bystander membrane recruitment assay
The addition of sucrose, a known inhibitor of receptor endocytosis
Stimulation of the receptors with Ang II resulted in a robust increase in luminescence over time that reached a
plateau after 60 min, consistent with β-arrestin recruitment to early endosomes.
The μ, δ, κ, and nociception opioid receptor (MOR, DOR, KOR, and NOP, respectively