Identification and Characterization of an Affimer Affinity Reagent for the Detection of the cAMP Sensor, EPAC1

An exchange protein directly activated by cAMP 1 (EPAC1) is an intracellular sensor for cAMP that is involved in a wide variety of cellular and physiological processes in health and disease. However, reagents are lacking to study its association with intracellular cAMP nanodomains. Here, we use non-antibody Affimer protein scaffolds to develop isoform-selective protein binders of EPAC1. Phage-display screens were carried out against purified, biotinylated human recombinant EPAC1ΔDEP protein (amino acids 149–811), which identified five potential EPAC1-selective Affimer binders. Dot blots and indirect ELISA assays were next used to identify Affimer 780A as the top EPAC1 binder. Mutagenesis studies further revealed a potential interaction site for 780A within the EPAC1 cyclic nucleotide binding domain (CNBD). In addition, 780A was shown to co-precipitate EPAC1 from transfected cells and co-localize with both wild-type EPAC1 and a mis-targeting mutant of EPAC1(K212R), predominantly in perinuclear and cytosolic regions of cells, respectively. As a novel EPAC1-selective binder, 780A therefore has the potential to be used in future studies to further understand compartmentalization of the cAMP-EPAC1 signaling system.

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Chromatin accessibility is dynamically regulated across C. elegans development and ageing

10.1101/279158 ◽

2018 ◽

Cited By ~ 1

Author(s):

Jürgen Jänes ◽

Yan Dong ◽

Michael Schoof ◽

Jacques Serizay ◽

Alex Appert ◽

...

Keyword(s):

Regulatory Mechanism ◽

Regulatory Elements ◽

Chromatin Accessibility ◽

Protein Coding ◽

C Elegans ◽

Transcription Profiles ◽

Physiological Processes ◽

Global Identification ◽

Identification And Characterization

AbstractAn essential step for understanding the transcriptional circuits that control development and physiology is the global identification and characterization of regulatory elements. Here we present the first map of regulatory elements across the development and ageing of an animal, identifying 42,245 elements accessible in at least one C. elegans stage. Based on nuclear transcription profiles, we define 15,714 protein-coding promoters and 19,231 putative enhancers, and find that both types of element can drive orientation-independent transcription. Additionally, hundreds of promoters produce transcripts antisense to protein coding genes, suggesting involvement in a widespread regulatory mechanism. We find that the accessibility of most elements is regulated during development and/or ageing and that patterns of accessibility change are linked to specific developmental or physiological processes. The map and characterization of regulatory elements across C. elegans life provides a platform for understanding how transcription controls development and ageing.

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Identification of Patulin from Penicillium coprobium as a Toxin for Enteric Neurons

Molecules ◽

10.3390/molecules24152776 ◽

2019 ◽

Vol 24 (15) ◽

pp. 2776 ◽

Cited By ~ 3

Author(s):

Brand ◽

Stoye ◽

Guilherme ◽

Nguyen ◽

Baumgaertner ◽

...

Keyword(s):

Nervous System ◽

Neuronal Cell ◽

Enteric Neurons ◽

The Central Nervous System ◽

Health And Disease ◽

Gut Microorganisms ◽

Identification And Characterization ◽

Effective Compound ◽

Specific Impact

The identification and characterization of fungal commensals of the human gut (the mycobiota) is ongoing, and the effects of their various secondary metabolites on the health and disease of the host is a matter of current research. While the neurons of the central nervous system might be affected indirectly by compounds from gut microorganisms, the largest peripheral neuronal network (the enteric nervous system) is located within the gut and is exposed directly to such metabolites. We analyzed 320 fungal extracts and their effect on the viability of a human neuronal cell line (SH-SY5Y), as well as their effects on the viability and functionality of the most effective compound on primary enteric neurons of murine origin. An extract from P. coprobium was identified to decrease viability with an EC50 of 0.23 ng/µL in SH-SY5Y cells and an EC50 of 1 ng/µL in enteric neurons. Further spectral analysis revealed that the effective compound was patulin, and that this polyketide lactone is not only capable of evoking ROS production in SH-SY5Y cells, but also diverse functional disabilities in primary enteric neurons such as altered calcium signaling. As patulin can be found as a common contaminant on fruit and vegetables and causes intestinal injury, deciphering its specific impact on enteric neurons might help in the elaboration of preventive strategies.

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Ripening related processes in strawberry, a nonclimacteric fruit: a short overview

International Journal of Horticultural Science ◽

10.31421/ijhs/15/1-2/821 ◽

2009 ◽

Vol 15 (1-2) ◽

Author(s):

V. Tisza ◽

L. Kovács ◽

L. Heszky ◽

E. Kiss

Keyword(s):

Fruit Development ◽

Ethylene Biosynthesis ◽

Strawberry Fruit ◽

Human Diet ◽

Short Overview ◽

Physiological Processes ◽

Fruit Development And Ripening ◽

Nonclimacteric Fruit ◽

Identification And Characterization

Fruits are essential part of the human diet: they provide vitamins, minerals, antioxidants to the mankind. Physiologically they can be divided into two groups-climacteric and nonclimacteric - depending if they display any respiratory peak and dramatic increase in ethylene biosynthesis or do not. Ethylene is a gaseous hormone playing a very important role in several physiological processes in plants. While climacteric fruits, like apples, bananas, tomatoes, peaches, apricots show increased ethylene biosynthesis and dramatic respiratory peak during their ripening, nonclimacteric fruits, like strawberries, grapes, citrus do not. The most widely used fruits for studying nonclimacteric ripening are strawberries: several papers are focusing on the identification and characterization of ripening related genes from this plant. Therefore here we attempt to summarize the most important advances in strawberry fruit development, and ripening.

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Chromatin accessibility dynamics across C. elegans development and ageing

eLife ◽

10.7554/elife.37344 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 22

Author(s):

Jürgen Jänes ◽

Yan Dong ◽

Michael Schoof ◽

Jacques Serizay ◽

Alex Appert ◽

...

Keyword(s):

Regulatory Mechanism ◽

Regulatory Elements ◽

Chromatin Accessibility ◽

Protein Coding ◽

C Elegans ◽

Transcription Profiles ◽

Physiological Processes ◽

Global Identification ◽

Identification And Characterization

An essential step for understanding the transcriptional circuits that control development and physiology is the global identification and characterization of regulatory elements. Here, we present the first map of regulatory elements across the development and ageing of an animal, identifying 42,245 elements accessible in at least one Caenorhabditis elegans stage. Based on nuclear transcription profiles, we define 15,714 protein-coding promoters and 19,231 putative enhancers, and find that both types of element can drive orientation-independent transcription. Additionally, more than 1000 promoters produce transcripts antisense to protein coding genes, suggesting involvement in a widespread regulatory mechanism. We find that the accessibility of most elements changes during development and/or ageing and that patterns of accessibility change are linked to specific developmental or physiological processes. The map and characterization of regulatory elements across C. elegans life provides a platform for understanding how transcription controls development and ageing.

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d-Amino Acids in Plants: Sources, Metabolism, and Functions

International Journal of Molecular Sciences ◽

10.3390/ijms21155421 ◽

2020 ◽

Vol 21 (15) ◽

pp. 5421

Author(s):

Üner Kolukisaoglu

Keyword(s):

Amino Acids ◽

Side Reactions ◽

Inhibitory Effects ◽

Physiological Processes ◽

Long Time ◽

Critical Revision ◽

Metabolizing Enzyme ◽

First Time ◽

Identification And Characterization

Although plants are permanently exposed to d-amino acids (d-AAs) in the rhizosphere, these compounds were for a long time regarded as generally detrimental, due to their inhibitory effects on plant growth. Recent studies showed that this statement needs a critical revision. There were several reports of active uptake by and transport of d-AAs in plants, leading to the question whether these processes happened just as side reactions or even on purpose. The identification and characterization of various transporter proteins and enzymes in plants with considerable affinities or specificities for d-AAs also pointed in the direction of their targeted uptake and utilization. This attracted more interest, as d-AAs were shown to be involved in different physiological processes in plants. Especially, the recent characterization of d-AA stimulated ethylene production in Arabidopsis thaliana revealed for the first time a physiological function for a specific d-AA and its metabolizing enzyme in plants. This finding opened the question regarding the physiological or developmental contexts in which d-AA stimulated ethylene synthesis are involved in. This question and the ones about the transport characteristics of d-AAs, their metabolism, and their different physiological effects, are the focus of this review.

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A TALE of shrimps: Genome-wide survey of homeobox genes in 120 species from diverse crustacean taxa

F1000Research ◽

10.12688/f1000research.13636.1 ◽

2018 ◽

Vol 7 ◽

pp. 71

Author(s):

Wai Hoong Chang ◽

Alvina G. Lai

Keyword(s):

Homeobox Genes ◽

Whole Body ◽

Model Organisms ◽

Data Sets ◽

Crop Species ◽

Physiological Processes ◽

Genome Wide ◽

Wide Range ◽

Identification And Characterization

The homeodomain-containing proteins are an important group of transcription factors found in most eukaryotes including animals, plants and fungi. Homeobox genes are responsible for a wide range of critical developmental and physiological processes, ranging from embryonic development, innate immune homeostasis to whole-body regeneration. With continued fascination on this key class of proteins by developmental and evolutionary biologists, multiple efforts have thus far focused on the identification and characterization of homeobox orthologs from key model organisms in attempts to infer their evolutionary origin and how this underpins the evolution of complex body plans. Despite their importance, the genetic complement of homeobox genes has yet been described in one of the most valuable groups of animals representing economically important food crops. With crustacean aquaculture being a growing industry worldwide, it is clear that systematic and cross-species identification of crustacean homeobox orthologs is necessary in order to harness this genetic circuitry for the improvement of aquaculture sustainability. Using publicly available transcriptome data sets, we identified a total of 4183 putative homeobox genes from 120 crustacean species that include food crop species, such as lobsters, shrimps, crayfish and crabs. Additionally, we identified 717 homeobox orthologs from 6 other non-crustacean arthropods, which include the scorpion, deer tick, mosquitoes and centipede. This high confidence set of homeobox genes will now serve as a key resource to the broader community for future functional and comparative genomics studies.

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