scholarly journals Evaluation of a high-throughput deorphanization strategy to identify cytochrome p450s important for odor degradation in Drosophila

2021 ◽  
Author(s):  
Shane R Baldwin ◽  
Pratyajit Mohapatra ◽  
Monica Nagalla ◽  
Rhea Sindvani ◽  
Desiree Amaya ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
High Throughput ◽  
Transcriptional Profiling ◽  
Expression Patterns ◽  
Neuronal Cells ◽  
Cytochrome P450s ◽  
Olfactory Neuron ◽  
Plant Volatile ◽  
Electrophysiological Recordings

Members of the cytochrome p450 (CYP) enzyme family are abundantly expressed in insect olfactory tissues, where they are thought to act as Odorant Degrading Enzymes (ODEs). However, their contribution to olfactory signaling in vivo is poorly understood. This is due in part to the challenge of identifying which of the dozens of antennal-expressed CYPs might inactivate a given odorant. Here, we tested a high-throughput deorphanization strategy in Drosophila to identify CYPs that are transcriptionally induced by exposure to a plant volatile. We discovered three CYPs selectively upregulated by the odorant using transcriptional profiling. Although these CYPs are shown to be broadly expressed in the antenna in non-neuronal cells, electrophysiological recordings from CYP mutants did not reveal any changes in olfactory neuron responses to the odorant. Neurons were desensitized by pre-exposing flies to the odorant, but this effect was similar in CYP mutants. Together, our data suggest that this transcriptomic approach may not be useful for identifying CYPs that contribute to olfactory signaling. We go on to show that some CYPs have highly restricted expression patterns in the antenna, and suggest that such CYPs may be useful candidates for further studies on olfactory CYP function.

Development of the “Inje Cocktail” for High-throughput Evaluation of Five Human Cytochrome P450 Isoforms in vivo

2007 ◽  
Vol 82 (5) ◽  
pp. 531-540 ◽  
Author(s):  
J Y Ryu ◽  
I S Song ◽  
Y E Sunwoo ◽  
J H Shon ◽  
K H Liu ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
High Throughput ◽  
Human Cytochrome ◽  
Cytochrome P450 Isoforms

scholarly journals A calibrated optogenetic toolbox of stable zebrafish opsin lines

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Paride Antinucci ◽  
Adna Dumitrescu ◽  
Charlotte Deleuze ◽  
Holly J Morley ◽  
Kristie Leung ◽  
...  
Keyword(s):  
High Throughput ◽  
Neural Activity ◽  
Ion Selectivity ◽  
Transgenic Zebrafish ◽  
Substantial Variation ◽  
Opsin Expression ◽  
Electrophysiological Recordings ◽  
Subcellular Resolution ◽  
Kinetics Of

Optogenetic actuators with diverse spectral tuning, ion selectivity and kinetics are constantly being engineered providing powerful tools for controlling neural activity with subcellular resolution and millisecond precision. Achieving reliable and interpretable in vivo optogenetic manipulations requires reproducible actuator expression and calibration of photocurrents in target neurons. Here, we developed nine transgenic zebrafish lines for stable opsin expression and calibrated their efficacy in vivo. We first used high-throughput behavioural assays to compare opsin ability to elicit or silence neural activity. Next, we performed in vivo whole-cell electrophysiological recordings to quantify the amplitude and kinetics of photocurrents and test opsin ability to precisely control spiking. We observed substantial variation in efficacy, associated with differences in both opsin expression level and photocurrent characteristics, and identified conditions for optimal use of the most efficient opsins. Overall, our calibrated optogenetic toolkit will facilitate the design of controlled optogenetic circuit manipulations.

scholarly journals Transcriptional Profiling of Bacillus anthracis during Infection of Host Macrophages

2007 ◽  
Vol 75 (7) ◽  
pp. 3434-3444 ◽  
Author(s):  
Nicholas H. Bergman ◽  
Erica C. Anderson ◽  
Ellen E. Swenson ◽  
Brian K. Janes ◽  
Nathan Fisher ◽  
...  
Keyword(s):  
Bacillus Anthracis ◽  
Host Cell ◽  
Transcriptional Profiling ◽  
Expression Patterns ◽  
Survival Strategies ◽  
Reverse Transcription Pcr ◽  
Bacterial Rna ◽  
Definition Of

ABSTRACT The interaction between Bacillus anthracis and the mammalian phagocyte is one of the central stages in the progression of inhalational anthrax, and it is commonly believed that the host cell plays a key role in facilitating germination and dissemination of inhaled B. anthracis spores. Given this, a detailed definition of the survival strategies used by B. anthracis within the phagocyte is critical for our understanding of anthrax. In this study, we report the first genome-wide analysis of B. anthracis gene expression during infection of host phagocytes. We developed a technique for specific isolation of bacterial RNA from within infected murine macrophages, and we used custom B. anthracis microarrays to characterize the expression patterns occurring within intracellular bacteria throughout infection of the host phagocyte. We found that B. anthracis adapts very quickly to the intracellular environment, and our analyses identified metabolic pathways that appear to be important to the bacterium during intracellular growth, as well as individual genes that show significant induction in vivo. We used quantitative reverse transcription-PCR to verify that the expression trends that we observed by microarray analysis were valid, and we chose one gene (GBAA1941, encoding a putative transcriptional regulator) for further characterization. A deletion strain missing this gene showed no phenotype in vitro but was significantly attenuated in a mouse model of inhalational anthrax, suggesting that the microarray data described here provide not only the first comprehensive view of how B. anthracis survives within the host cell but also a number of promising leads for further research in anthrax.

The Participation of Cytochrome P450 3A4 in Clozapine Biotransformation Is Detected in People With Schizophrenia by High-Throughput In Vivo Phenotyping

2010 ◽  
Vol 30 (5) ◽  
pp. 629-631 ◽  
Author(s):  
Sussan Ghassabian ◽  
Manoranjenni Chetty ◽  
Bruce N. Tattam ◽  
John Glen ◽  
Jeannie Rahme ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
High Throughput ◽  
Cytochrome P450 3A4

scholarly journals CCA1 and ATAF2 differentially suppress cytochrome P450-mediated brassinosteroid inactivation in Arabidopsis

2018 ◽  
Author(s):  
Hao Peng ◽  
Michael M. Neff
Keyword(s):  
Cytochrome P450 ◽  
Binding Sites ◽  
Expression Patterns ◽  
Regulatory Protein ◽  
Cytochrome P450s ◽  
Null Mutant ◽  
Yeast Two Hybrid ◽  
Protein Levels ◽  
The Core ◽  
Two Hybrid

AbstractBrassinosteroids (BRs) are a group of steroid hormones regulating plant growth and development. Since BRs do not undergo transport among plant tissues, their metabolism is tightly regulated by transcription factors (TFs) and feedback loops. BAS1 (CYP734A1, formerly CYP72B1) and SOB7 (CYP72C1) are two BR-inactivating cytochrome P450s identified in Arabidopsis thaliana. We previously found that a TF ATAF2 (ANAC081) suppresses BAS1 and SOB7 expression by binding to the Evening Element (EE) and CCA1-binding sites (CBS) on their promoters. Both EE and CBS are known binding targets of the core circadian clock regulatory protein CCA1. Here, we confirm that CCA1 binds the EE and CBS motifs on BAS1 and SOB7 promoters, respectively. Elevated accumulations of BAS1 and SOB7 transcripts in the CCA1 null mutant cca1-1 indicate that CCA1 is a repressor of their expression. When compared to either cca1-1 or the ATAF2 null mutant ataf2-2, the cca1-1 ataf2-2 double mutant shows higher SOB7 transcript accumulations and stronger BR-insensitive phenotype of hypocotyl elongation in white light. CCA1 interacts with ATAF2 at both DNA-protein and protein-protein levels. ATAF2, BAS1 and SOB7 are all circadian-regulated with distinct expression patterns. These results demonstrate that CCA1 and ATAF2 differentially suppress BAS1- and SOB7-mediated BR inactivation.HighlightThe core circadian regulator CCA1 is a direct repressor of brassinosteroid inactivating genes BAS1 and SOB7, and interact with another repressor, ATAF2. Their differential suppressing effects are regulated by light.Abbreviations3-aminotriazole (3-AT), brassinolide (BL), brassinosteroid (BR), CCA1-binding site (CBS), cytochrome P450 (P450), Evening Element (EE), transcription factor (TF), yeast one-hybrid (Y1H), yeast two-hybrid (Y2H)

scholarly journals A calibrated optogenetic toolbox of stable zebrafish opsin lines

2020 ◽  
Author(s):  
P Antinucci ◽  
AS Dumitrescu ◽  
C Deleuze ◽  
HJ Morley ◽  
K Leung ◽  
...  
Keyword(s):  
High Throughput ◽  
Neural Activity ◽  
Ion Selectivity ◽  
Transgenic Zebrafish ◽  
Substantial Variation ◽  
Opsin Expression ◽  
Electrophysiological Recordings ◽  
Subcellular Resolution ◽  
Kinetics Of

AbstractOptogenetic actuators with diverse spectral tuning, ion selectivity and kinetics are constantly being engineered providing powerful tools for controlling neural activity with subcellular resolution and millisecond precision. Achieving reliable and interpretable in vivo optogenetic manipulations requires reproducible actuator expression and calibration of photocurrents in target neurons. Here, we developed nine transgenic zebrafish lines for stable opsin expression and calibrated their efficacy in vivo. We first used high-throughput behavioural assays to compare opsin ability to elicit or silence neural activity. Next, we performed in vivo whole-cell electrophysiological recordings to quantify the amplitude and kinetics of photocurrents and test opsin ability to precisely control spiking. We observed substantial variation in efficacy, associated with differences in both opsin expression level and photocurrent characteristics, and identified conditions for optimal use of the most efficient opsins. Overall, our calibrated optogenetic toolkit will facilitate the design of controlled optogenetic circuit manipulations.

scholarly journals Single Cell Transcriptomics Reveals Global Markers of Transcriptional Diversity Across Different Forms of Cellular Senescence

2021 ◽  
Author(s):  
Shane A Evans ◽  
Yee Voan Teo ◽  
Kelly Klark ◽  
Takahiro Ito ◽  
John M Sedivy ◽  
...  
Keyword(s):  
Single Cell ◽  
Cellular Senescence ◽  
Replicative Senescence ◽  
Expression Profiles ◽  
Transcriptional Profiling ◽  
Expression Patterns ◽  
Cell Contact ◽  
Matrix Remodeling

Cellular Senescence is a state of irreversible cell cycle arrest, and the accumulation of senescent cells contributes to agerelated organismal decline. The detrimental effects of cellular senescence are due to the senescence associated secretory phenotype (SASP), an array of signaling molecules and growth factors secreted by senescent cells that contribute to the terile inflammation associated with aging tissues. Recent studies, both in vivo and in vitro, have highlighted the heterogeneous nature of the senescence phenotype. In particular, single cell transcriptomics has revealed that Oncogene Induced Senescence (OIS) is characterized by the presence of subpopulations of cells expressing different SASP profiles. We have generated a comprehensive dataset via single-cell transcriptional profiling of genetically homogenous clonal cell lines from different forms of senescence, including OIS, Replicative Senescence (RS), and DNA Damage Induced Senescence (DDIS). We identified subpopulations of cells that are common to all three major forms of senescence and show that the expression profiles of these subpopulations are driven by markers formerly identified in individual forms of senescence. These common signatures are characterized by chromatin modifiers, inflammation, extracellular matrix remodeling, and Ribosomal protein expression. The expression patterns of these subpopulations recapitulate primary and secondary senescence, a phenomenon where a preexisting (primary) senescent cell induces senescence in a neighboring (secondary) cell through cell-to-cell contact. Since it is still unclear what type of senescence occurs in-vivo with age, it is important to know that the formation of primary and secondary populations is common to multiple types of senescence since this mechanism could help explain how senescent cells accumulate in aged organisms. Finally, we show that these subpopulations show differential susceptibility to the senolytic agent Navitoclax, suggesting that senolytic agents targeting the apoptotic pathways may be clearing only a subset of senescent cells based on their inflammatory profiles in-vivo.

Pharmacogenetics of Clozapine: In Vivo Role of Cytochrome P450 Isoforms and P-Glycoprotein

2008 ◽  
Vol 41 (05) ◽  
Author(s):  
E Jaquenoud-Sirot ◽  
B Knezevic ◽  
G Perla Morena ◽  
P Baumann ◽  
CB Eap
Keyword(s):  
Cytochrome P450 ◽  
P Glycoprotein ◽  
Cytochrome P450 Isoforms

The metalloproteinase ADAM10 is required for retinal ganglion cell axon guidance in the developing visual systems

2007 ◽  
Vol 30 (4) ◽  
pp. 77
Author(s):  
Y. Y. Chen ◽  
C. L. Hehr ◽  
K. Atkinson-Leadbeater ◽  
J. C. Hocking ◽  
S. McFarlane
Keyword(s):  
Ganglion Cell ◽  
Axon Guidance ◽  
Retinal Ganglion Cell ◽  
Growth Cone ◽  
Expression Patterns ◽  
Optic Tract ◽  
Axon Growth ◽  
Proteolytic Processing ◽  
Retinal Ganglion

Background: The growth cone interprets cues in its environment in order to reach its target. We want to identify molecules that regulate growth cone behaviour in the developing embryo. We investigated the role of A disintegrin and metalloproteinase 10 (ADAM10) in axon guidance in the developing visual system of African frog, Xenopus laevis. Methods: We first examined the expression patterns of adam10 mRNA by in situ hybridization. We then exposed the developing optic tract to an ADAM10 inhibitor, GI254023X, in vivo. Lastly, we inhibited ADAM10 function in diencephalic neuroepithelial cells (through which retinal ganglion cell (RGC) axons extend) or RGCs by electroporating or transfecting an ADAM10 dominant negative (dn-adam10). Results: We show that adam10 mRNA is expressed in the dorsal neuroepithelium over the time RGC axons extend towards their target, the optic tectum. Second, pharmacological inhibition of ADAM10 in an in vivo exposed brain preparation causes the failure of RGC axons to recognize their target at low concentrations (0.5, 1 μM), and the failure of the axons to make a caudal turn in the mid-diencephalon at higher concentration (5 μM). Thus, ADAM10 function is required for RGC axon guidance at two key guidance decisions. Finally, molecular inhibition of ADAM10 function by electroporating dn-adam10 in the brain neuroepithelium causes defects in RGC axon target recognition (57%) and/or defects in caudal turn (12%), as seen with the pharmacological inhibitor. In contrast, molecular inhibition of ADAM10 within the RGC axons has no effect. Conclusions: These data argue strongly that ADAM10 acts cell non-autonomously within the neuroepithelium to regulate the guidance of RGC axons. This study shows for the first time that a metalloproteinase acts in a cell non-autonomous fashion to direct vertebrate axon growth. It will provide important insights into candidate molecules that could be used to reform nerve connections if destroyed because of injury or disease. References Hattori M, Osterfield M, Flanagan JG. Regulated cleavage of a contact-mediated axon repellent. Science 2000; 289(5483):1360-5. Janes PW, Saha N, Barton WA, Kolev MV, Wimmer-Kleikamp SH, Nievergall E, Blobel CP, Himanen JP, Lackmann M, Nikolov DB. Adam meets Eph: an ADAM substrate recognition module acts as a molecular switch for ephrin cleavage in trans. Cell 2005; 123(2):291-304. Pan D, Rubin GM. Kuzbanian controls proteolytic processing of Notch and mediates lateral inhibition during Drosophila and vertebrate neurogenesis. Cell 1997; 90(2):271-80.

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