Optogenetic inhibitor of the transcription factor CREB

Current approaches for optogenetic control of transcription do not mimic the activity of endogenous transcription factors, which act at numerous sites in the genome in a complex interplay with other factors. Optogenetic control of dominant negative versions of endogenous transcription factors provides a mechanism for mimicking the natural regulation of gene expression. Here we describe opto-DN-CREB, a blue light controlled inhibitor of the transcription factor CREB created by fusing the dominant negative inhibitor A-CREB to photoactive yellow protein (PYP). A light driven conformational change in PYP prevents coiled-coil formation between A-CREB and CREB, thereby activating CREB. Optogenetic control of CREB function was characterized in vitro, in HEK293T cells, and in neurons where blue light enabled control of expression of the CREB targets NR4A2 and c-Fos. Dominant negative inhibitors exist for numerous transcription factors; linking these to optogenetic domains offers a general approach for spatiotemporal control of native transcriptional events

Optogenetic inhibitor of the transcription factor CREB

Current approaches for optogenetic control of transcription do not mimic the activity of endogenous transcription factors, which act at numerous sites in the genome in a complex interplay with other factors. Optogenetic control of dominant negative versions of endogenous transcription factors provides a mechanism for mimicking the natural regulation of gene expression. Here we describe opto-DN-CREB, a blue light controlled inhibitor of the transcription factor CREB created by fusing the dominant negative inhibitor A-CREB to photoactive yellow protein (PYP). A light driven conformational change in PYP prevents coiled-coil formation between A-CREB and CREB, thereby activating CREB. Optogenetic control of CREB function was characterized in vitro, in HEK293T cells, and in neurons where blue light enabled control of expression of the CREB targets NR4A2 and c-Fos. Dominant negative inhibitors exist for numerous transcription factors; linking these to optogenetic domains offers a general approach for spatiotemporal control of native transcriptional events

Xバンド電子スピン共鳴によるGaAs;Er,OのEr濃度およびキャリア依存性の研究

博士（理学）神戸大

Increasing frailty is associated with higher prevalence and reduced recognition of delirium in older hospitalised inpatients: results of a multi-centre study

Purpose: Delirium is a neuropsychiatric disorder delineated by an acute change in cognition, attention, and consciousness. It is common, particularly in older adults, but poorly recognised. Frailty is the accumulation of deficits conferring an increased risk of adverse outcomes. We set out to determine how severity of frailty, as measured using the CFS, affected delirium rates, and recognition in hospitalised older people in the United Kingdom. Methods: Adults over 65 years were included in an observational multi-centre audit across UK hospitals, two prospective rounds, and one retrospective note review. Clinical Frailty Scale (CFS), delirium status, and 30-day outcomes were recorded. Results: The overall prevalence of delirium was 16.3% (483). Patients with delirium were more frail than patients without delirium (median CFS 6 vs 4). The risk of delirium was greater with increasing frailty [OR 2.9 (1.8–4.6) in CFS 4 vs 1–3; OR 12.4 (6.2–24.5) in CFS 8 vs 1–3]. Higher CFS was associated with reduced recognition of delirium (OR of 0.7 (0.3–1.9) in CFS 4 compared to 0.2 (0.1–0.7) in CFS 8). These risks were both independent of age and dementia. Conclusion: We have demonstrated an incremental increase in risk of delirium with increasing frailty. This has important clinical implications, suggesting that frailty may provide a more nuanced measure of vulnerability to delirium and poor outcomes. However, the most frail patients are least likely to have their delirium diagnosed and there is a significant lack of research into the underlying pathophysiology of both of these common geriatric syndromes

Molecular characterization of voltage-gated K+ channels in the weakly electric fish Apteronotusleptorhynchus

A PCR approach using degenerate primers specific for the highly conserved pore and S6 transmembrane domains of K+ channels was used to assess the diversity of K+ channels in the electric fish Apteronotus leptorhynchus. DNA sequence analysis has identified nineteen K+ channel genes, each of which can be classified into one of the four major families in a similar distribution to that in mammals. Also, an Apteronotus brain cDNA library was constructed and screened with mammalian K+ channel probes. The first K+ channel clone obtained displays remarkable amino acid sequence homology to rat Kv1.3 and Northern blot analysis and RNase protection assays have suggested that this channel may play similar roles in both fish and mammals. The results indicate that the duplications which gave rise to multiple genes within each of the K+ channel families predate the divergence of the Actinopterygii and Sarcopterygii lineages during early vertebrate evolution and the function of these K+ channel genes has been conserved between the two lineages

Contribution of Kv3 potassium channels to signal processing by electrosensory neurons

Gamma-frequency burst discharge in pyramidal cells of the apteronotid electrosensory lateral line lobe (ELL) is necessary for signal processing. Bursting is dependent on an interaction between somatic and apical dendritic currents, in which spike broadening in the apical dendrite potentiates a somatic afterpotential that follows each rapid somatic spike. Somatic spike repolarization must be consistent in order for the afterpotential to be expressed soon after. The work presented in this thesis describes how the expression and differential subcellular distribution of two Kv3-type K+ channels in ELL pyramidal cells may contribute to this mechanism of burst discharge.I cloned a family of Kv3 channels from an apteronotid brain cDNA library and demonstrated that two of these channels, homologues of the mammalian subtypes Kv3.1 and Kv3.3, are expressed in ELL pyramidal cells. Immunohistochemical analysis demonstrated that the AptKv3.3 K+ channel is distributed throughout the dendrites of pyramidal cells while the AptKv3.1 channel is restricted in its expression to pyramidal cell somata, basilar dendrites and proximal apical dendrites. Heterologous expression of each channel in HEK 293 cells indicated that AptKv3.3 encodes a high-threshold inactivating K + current while AptKv3.1 encodes a high-threshold K+ current which does not display inactivation upon prolonged membrane depolarization. Based on these results as well as pharmacological analysis of native ELL pyramidal cells, I propose that AptKv3.3 mediates spike repolarization in the apical dendrite and inactivation of the channel during repetitive firing allows spike broadening. In contrast, AptKv3.1 likely contributes towards rapid and consistent spike repolarization in the cell soma. Therefore, the expression and differential distribution of these two Kv3 channels in ELL pyramidal cells may underlie the compartmental differences in spike repolarzation that is necessary for burst discharge.The extensive dendritic localization of AptKv3.3 observed in ELL pyramidal cells as well as in other hindbrain neurons has not previously been demonstrated for members of the Kv3 family of K+ channels. The differential localization of AptKv3.1. AptKv3.3 and possibly AptKv3.3 splice variants that I have identified presents an opportunity to examine the molecular mechanisms of Kv3 channel targeting in neurons. Preliminary data is presented which provides the foundation for future studies on channel targeting

Rashid et al Science 2016- Data for Figs 1-4, S1-S9

Excel file with all data presented in manuscript (each sheet corresponds to specific figures as indicated)

1H-imidazole; YM254890

ABSTRACT In this report, we investigated whether the D5 dopamine receptor, given its structural and sequence homology with the D1 receptor, could interact with the D2 receptor to mediate a calcium signal similar to the G q/11 protein-linked phospholipase C-mediated calcium signal resulting from the coactivation of D1 and D2 dopamine receptors within D1-D2 receptor heterooligomers. Fluorescent resonance energy transfer experiments demonstrated close colocalization of cell surface D5 and D2 receptors (Ͻ100 Å), indicating hetero-oligomerization of D5 and D2 receptors in cells coexpressing both receptors. Coactivation of D5 and D2 receptors within the D5-D2 hetero-oligomers activated a calcium signal. However, unlike what is observed for D1 receptors, which activate extensive calcium mobilization only within a complex with the D2 receptors, a robust calcium signal was triggered by D5 receptors expressed alone. Heterooligomerization with the D2 receptor attenuated the ability of the D5 receptor to trigger a calcium signal. The D5 and D5-D2-associated calcium signals were G q/11 protein-linked and phospholipase C-mediated but were also critically dependent on the influx of extracellular calcium through store-operated calcium channels, unlike the calcium release triggered by D1-D2 heterooligomers. Collectively, these results demonstrate that calcium signaling through D5-D2 receptor hetero-oligomers occurred through a distinct mechanism to achieve an increase in intracellular calcium levels. The neurotransmitter dopamine controls a variety of brain functions such as locomotion, cognition, and emotion. Dysfunction of this system has been linked to a number of pathological condition