High-throughput assessment of calcium sensitivity in skinned cardiac myocytes

2001 ◽  
Vol 281 (2) ◽  
pp. H969-H974 ◽  
Author(s):  
Chee Chew Lim ◽  
Michiel H. B. Helmes ◽  
Douglas B. Sawyer ◽  
Mohit Jain ◽  
Ronglih Liao
Keyword(s):  
Cardiac Myocytes ◽  
High Throughput ◽  
High Throughput Screening ◽  
Rapid Determination ◽  
Isometric Force ◽  
Calcium Sensitivity ◽  
Pharmacological Agents ◽  
Length Changes ◽  
Myofilament Calcium Sensitivity ◽  
High Degree

Isolated permeabilized cardiac myocytes have been used in the study of myofilament calcium sensitivity through measurement of the isometric force-pCa curve. Determining this force-pCa relationship in skinned myocytes is relatively expensive and carries a high degree of variability. We therefore attempted to establish an alternative high-throughput method to measure calcium sensitivity in cardiac myocytes. With the use of commercially available software that allows for precise measurement of sarcomere spacing, we measured sarcomere length changes in unloaded skinned cardiac myocytes over a range of calcium concentrations. With the use of this technique, we were able to accurately detect acute increases or decreases in myofilament calcium sensitivity after exposure to 10 mM caffeine or 5 mM 2,3-butanedione monoxime, respectively. This technique allows for the simple and rapid determination of myofilament calcium sensitivity in cardiac myocytes in a reproducible and inexpensive manner and could be used for high-throughput screening of pharmacological agents and/or transgenic mouse models for changes in myofilament calcium sensitivity.

A force transducer for measuring mechanical properties of single cardiac myocytes

1999 ◽  
Vol 277 (6) ◽  
pp. H2400-H2408 ◽  
Author(s):  
C. Tasche ◽  
E. Meyhöfer ◽  
B. Brenner
Keyword(s):  
Mechanical Properties ◽  
Laser Beam ◽  
Cardiac Myocytes ◽  
Cardiac Myocyte ◽  
Isometric Force ◽  
Force Transducer ◽  
Cardiac Cells ◽  
Beam Deflection ◽  
Resonance Frequencies ◽  
Length Changes

We have described a transducer design capable of recording forces generated by single cardiac myocytes with sufficient temporal resolution to detect force responses to rapid length changes. Our force sensors were made from thin steel foils that act as cantilevers whose bending is monitored by reflection off a laser beam. Deflection of the laser beam is measured by a differential photodiode detector. A small, 50-μm-thick tungsten needle attached to the free end of the steel foil allowed us to glue single cardiac cells to the force transducer. The transducers have compliances of ∼0.02 m/N and resonance frequencies between 2 and 3 kHz. The resolution is ∼18 nN rms at a detector bandwidth of 16 kHz, so we were able to resolve 0.2% of the maximum isometric force (∼12 μN) developed by a single cardiac myocyte. We have demonstrated that the transducer is well suited to analysis of mechanical properties of single ventricular myocytes, for example, the recording of isometric forces and rate constants of force redevelopment after rapid release-restretch maneuvers.

Functional mimetics of neurotrophins and their receptors

2006 ◽  
Vol 34 (4) ◽  
pp. 612-617 ◽  
Author(s):  
J. Peleshok ◽  
H.U. Saragovi
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Ligand Binding ◽  
Cell Survival ◽  
High Throughput ◽  
Neurodegenerative Disorders ◽  
High Throughput Screening ◽  
Pharmacological Agents ◽  
Binding Agents ◽  
The Central Nervous System

Neurotrophins regulate cell survival, death, differentiation and growth. Neurotrophins and their receptors have been validated for pathologies including neurodegenerative disorders of the central nervous system and the peripheral nervous system, certain types of cancers, asthma, inflammation and others. Development of neurotrophin-based therapeutics is important due to the limitations of using whole neurotrophins as pharmacological agents. The use of mimicry has proven to be an alternative. Mimetics can be developed through a number of different approaches. To develop receptor-binding agents, we have used anti-receptor antibody mimicry and neurotrophin mimicry. To develop ligand-binding agents, we have used antiligand antibody mimicry and receptor mimicry. High-throughput screening can be incorporated to complement any of these approaches. The end result is small molecule peptidomimetics with properties favourable over proteins. The present review will offer a general overview of these strategies with a few proven examples from our laboratory.

scholarly journals Efficient Management of High-Throughput Screening Libraries with SAVANAH

2016 ◽  
Vol 22 (2) ◽  
pp. 196-202
Author(s):  
Markus List ◽  
Marlene Pedersen Elnegaard ◽  
Steffen Schmidt ◽  
Helle Christiansen ◽  
Qihua Tan ◽  
...  
Keyword(s):  
Pharmaceutical Industry ◽  
High Throughput ◽  
High Throughput Screening ◽  
Molecular Screening ◽  
Microtiter Plates ◽  
Efficient Management ◽  
Indispensable Tool ◽  
High Degree ◽  
Molecular Libraries

High-throughput screening (HTS) has become an indispensable tool for the pharmaceutical industry and for biomedical research. A high degree of automation allows for experiments in the range of a few hundred up to several hundred thousand to be performed in close succession. The basis for such screens are molecular libraries, that is, microtiter plates with solubilized reagents such as siRNAs, shRNAs, miRNA inhibitors or mimics, and sgRNAs, or small compounds, that is, drugs. These reagents are typically condensed to provide enough material for covering several screens. Library plates thus need to be serially diluted before they can be used as assay plates. This process, however, leads to an explosion in the number of plates and samples to be tracked. Here, we present SAVANAH, the first tool to effectively manage molecular screening libraries across dilution series. It conveniently links (connects) sample information from the library to experimental results from the assay plates. All results can be exported to the R statistical environment or piped into HiTSeekR ( http://hitseekr.compbio.sdu.dk ) for comprehensive follow-up analyses. In summary, SAVANAH supports the HTS community in managing and analyzing HTS experiments with an emphasis on serially diluted molecular libraries.

scholarly journals Development of an HTRF Assay for the Detection and Characterization of Inhibitors of Catechol-O-Methyltransferase

2015 ◽  
Vol 21 (5) ◽  
pp. 490-495 ◽  
Author(s):  
Martha Kimos ◽  
Maggi Burton ◽  
David Urbain ◽  
Didier Caudron ◽  
Murielle Martini ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Common Property ◽  
Kinetic Studies ◽  
Fluorescence Ratio ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Membrane Bound ◽  
High Degree

Catechol-O-methyltransferase (COMT) plays an important role in the deactivation of catecholamine neurotransmitters and hormones. Inhibitors of COMT, such as tolcapone and entacapone, are used clinically in the treatment of Parkinson’s disease. Discovery of novel inhibitors has been hampered by a lack of suitable assays for high-throughput screening (HTS). Although assays using esculetin have been developed, these are affected by fluorescence, a common property of catechol-type compounds. We have therefore evaluated a new homogenous time-resolved fluorescence (HTRF)–based assay from CisBio (Codolet, France), which measures the production of S-adenosyl-L-homocysteine (SAH). The assay has been run in both HTS and medium-throughput screening (MTS) modes. The assay was established using membranes expressing human membrane-bound COMT and was optimized for protein and time to give an acceptable signal window, good potency for tolcapone, and a high degree of translation between data in fluorescence ratio and data in terms of [SAH] produced. pIC50 values for the hits from the HTS mode were determined in the MTS mode. The assay also proved suitable for kinetic studies such as Km,app determination.

scholarly journals High-Throughput Screens to Discover Small-Molecule Modulators of Ryanodine Receptor Calcium Release Channels

2016 ◽  
Vol 22 (2) ◽  
pp. 176-186 ◽  
Author(s):  
Robyn T. Rebbeck ◽  
Maram M. Essawy ◽  
Florentin R. Nitu ◽  
Benjamin D. Grant ◽  
Gregory D. Gillispie ◽  
...  
Keyword(s):  
Ryanodine Receptor ◽  
High Throughput ◽  
High Throughput Screening ◽  
Calcium Release ◽  
Striated Muscle ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Pathological State ◽  
Pharmacological Agents ◽  
Intracellular Ca

Using time-resolved fluorescence resonance energy transfer (FRET), we have developed and validated the first high-throughput screening (HTS) method to discover compounds that modulate an intracellular Ca2+ channel, the ryanodine receptor (RyR), for therapeutic applications. Intracellular Ca2+ regulation is critical for striated muscle function, and RyR is a central player. At resting [Ca2+], an increased propensity of channel opening due to RyR dysregulation is associated with severe cardiac and skeletal myopathies, diabetes, and neurological disorders. This leaky state of the RyR is an attractive target for pharmacological agents to treat such pathologies. Our FRET-based HTS detects RyR binding of accessory proteins calmodulin (CaM) or FKBP12.6. Under conditions that mimic a pathological state, we carried out a screen of the 727-compound NIH Clinical Collection, which yielded six compounds that reproducibly changed FRET by >3 SD. Dose–response of FRET and [3H]ryanodine binding readouts reveal that five hits reproducibly alter RyR1 structure and activity. One compound increased FRET and inhibited RyR1, which was only significant at nM [Ca2+], and accentuated without CaM present. These properties characterize a compound that could mitigate RyR1 leak. An excellent Z′ factor and the tight correlation between structural and functional readouts validate this first HTS method to identify RyR modulators.

High-throughput screening program for botanical extracts

Planta Medica ◽  
2012 ◽  
Vol 78 (11) ◽  
Author(s):  
L Hingorani ◽  
NP Seeram ◽  
B Ebersole
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Screening Program ◽  
Botanical Extracts

High throughput screening of microbial biodiversity for the discovery of novel cosmeceutical agents

Planta Medica ◽  
2015 ◽  
Vol 81 (16) ◽  
Author(s):  
K Georgousaki ◽  
N DePedro ◽  
AM Chinchilla ◽  
N Aliagiannis ◽  
F Vicente ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Microbial Biodiversity
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