Here we design and optimize a genetically encoded fluorescent indicator, iAChSnFR, for the ubiquitous neurotransmitter acetylcholine, based on a bacterial periplasmic binding protein. iAChSnFR shows large fluorescence changes, rapid rise and decay kinetics, and insensitivity to most cholinergic drugs. iAChSnFR revealed large transients in a variety of slice and in vivo preparations in mouse, fish, fly and worm. iAChSnFR will be useful for the study of acetylcholine in all animals. IntroductionAcetylcholine (ACh) is a critical neurotransmitter in all animals. Among invertebrates, it is the most prevalent excitatory transmitter in the brain, sensory ganglia, and frequently the neuromuscular junction (NMJ). Among vertebrates, only a minority of neurons release ACh, but these signals play varying key roles. For instance, ACh signals at the NMJ, in the autonomic nervous system, and in subsets of the central nervous system, particularly projections arising from the brainstem and basal forebrain. Other cholinergic neuron populations in the brain include striatal interneurons, the stria vascularis-medial habenula-interpeduncular nucleus pathway, and sparse, incompletely characterized cell types such as intrinsic cholinergic interneurons in cortex 1 and hippocampus 2 . ACh helps to regulate attention 3 and wakefulness 4 , and participates in memory formation and consolidation 5 . ACh is also an important transmitter in glia, and between the nervous and immune systems 6 .Acetylcholine is synthesized pre-synaptically from choline and acetyl-CoA by choline acetyltransferase (ChAT), then packaged into synaptic vesicles by the vesicular acetylcholine transporter (VAChT). A key, partially understood aspect of cholinergic signaling is co-release with other neurotransmitters, including GABA, ATP, and glutamate 7,8 . To understand the role of co-release, one must measure ACh release alongside emerging measurements of other neurotransmitters.Acetylcholine receptors are among the most diverse neurotransmitter receptor families. Humans possess five muscarinic G protein-coupled receptors (GPCRs) for ACh (mAChRs) with diverse expression in the brain and smooth, cardiac, and skeletal muscle. Vertebrate nicotinic ACh receptors (nAChRs) are pentameric ligand-gated cation channels. Humans have a total of 17 nAChR subunit genes, in five classes: 10 a, 4 b, and one each of g, d, and e. nAChRs occur with many subunit combinations 9 , and others may be undiscovered. Invertebrates also have AChgated chloride channels. On neurons, receptors can be localized pre-, post-, and extrasynaptically, often with different isoforms in each place 10

NCT02234635, post-results.

PurposeTo evaluate the repeatability, reproducibility, and agreement of thickness profile measurements of eight intra-retinal layers determined by an automated algorithm applied to optical coherence tomography (OCT) images from two different instruments.MethodsTwenty normal subjects (12 males, 8 females; 24 to 32 years old) were enrolled. Imaging was performed with a custom built ultra-high resolution OCT instrument (UHR-OCT, ∼3 µm resolution) and a commercial RTVue100 OCT (∼5 µm resolution) instrument. An automated algorithm was developed to segment the macular retina into eight layers and quantitate the thickness of each layer. The right eye of each subject was imaged two times by the first examiner using each instrument to assess intra-observer repeatability and once by the second examiner to assess inter-observer reproducibility. The intraclass correlation coefficient (ICC) and coefficients of repeatability and reproducibility (COR) were analyzed to evaluate the reliability.ResultsThe ICCs for the intra-observer repeatability and inter-observer reproducibility of both SD-OCT instruments were greater than 0.945 for the total retina and all intra-retinal layers, except the photoreceptor inner segments, which ranged from 0.051 to 0.643, and the outer segments, which ranged from 0.709 to 0.959. The CORs were less than 6.73% for the total retina and all intra-retinal layers. The total retinal thickness measured by the UHR-OCT was significantly thinner than that measured by the RTVue100. However, the ICC for agreement of the thickness profiles between UHR-OCT and RTVue OCT were greater than 0.80 except for the inner segment and outer segment layers.ConclusionsThickness measurements of the intra-retinal layers determined by the automated algorithm are reliable when applied to images acquired by the UHR-OCT and RTVue100 instruments.

Increasing evidence indicates that long noncoding RNA SPRY4 intronic transcript 1 (lncRNA SPRY4-IT1 ) has been reported to be associated with the progression of several cancers, but its expression level and the function of SPRY4-IT1 in the progression of gastric cancer (GC) have been rarely reported. Here we found that SPRY4-IT1 was upregulated in GC. In vitro experiments revealed that SPRY4-IT1 knockdown significantly inhibited GC cell proliferation by causing G1 arrest and promoting apoptosis, whereas SPRY4-IT1 overexpression promoted cell growth. Further functional assays indicated that SPRY4-IT1 overexpression significantly promoted cell migration and invasion. Bioinformatics analysis predicted that there is a SPRY4-IT1/miR-101-3p/AMPK axis in GC progression. A dual-luciferase reporter system validated the direct interaction of SPRY4-IT1, miR-101-3p, and AMPK. Western blot verified that the inhibition of SPRY4-IT1 decreased AMPK expression. Furthermore, silencing SPRY4-IT1 suppressed GC growth in vivo . Importantly, we demonstrated that SPRY4-IT1 was upregulated in serum exosomes from GC patients and correlated with cancer metastasis. Altogether, silencing SPRY4-IT1 suppresses the progression of GC by interacting with miR-101-3p and decreasing inhibiting AMPK expression. Taken together, our study demonstrates that SPRY4-IT1 could act as a potential therapeutic target for GC patients.