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Sleep is a fundamental feature of life for virtually all multicellular animals, but many questions remain about how sleep is regulated by circadian rhythms, homeostatic sleep drive that builds up with wakefulness, and modifying factors such as hunger or social interactions, as well as about the biological functions of sleep. Substantial headway has been made in the study of both circadian rhythms and sleep in the fruit flyDrosophila melanogaster, much of it through studies of individual fly activity usingDrosophilaactivity monitors (DAMs). Here, we describe approaches for the activation of specific neurons of interest using optogenetics (involving genetic modifications that allow for light-based neuronal activation) and thermogenetics (involving genetic modifications that allow for temperature-based neuronal activation) so that researchers can evaluate the roles of those neurons in controlling rest and activity behavior. In this protocol, we describe how to set up a rig for simultaneous optogenetic or thermogenetic stimulation and activity monitoring for analysis of sleep and circadian rhythms inDrosophila, how to raise appropriate flies, and how to perform the experiment. This protocol will allow researchers to assess the causative role in the regulation of sleep and activity rhythms of any genetically tractable subset of cells.
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Base Modifications of Nucleosides via the Use of Peptide‐Coupling Agents, and Beyond
Abstract Several naturally occurring purine and pyrimidine nucleosides contain an amide linkage as part of the heterocyclic aglycone. Enolization of the amide and conversion to leaving groups at the amide carbon atom permits base modification by addition‐elimination types of processes. Although a number of methods have been developed over the years for accomplishing such conversions, the present Personal Account describes efforts from the Lakshman laboratories. Facile activation of the amido groups in nucleobases can be achieved with peptide‐coupling agents. Subsequent reaction with nucleophiles then accomplishes the base modifications. In many cases, the activation and displacement steps can be done as two‐step, one‐pot processes, whereas in other cases, discrete storable activated nucleosides can be isolated for subsequent displacement reactions. Using such an approach a wide range of nucleoside base modifications is readily achievable. In many instances, mechanistic investigations have been conducted so as to understand the activation process.
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- Award ID(s):
- 1953574
- PAR ID:
- 10392091
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- The Chemical Record
- Volume:
- 23
- Issue:
- 1
- ISSN:
- 1527-8999
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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