317 research outputs found
Organic anions exhibit distinct inhibition patterns on the low-Km and high-Km transport of S-(2,4-dinitrophenyl)glutathione through the human erythrocyte membrane
Simultaneous whole-animal 3D-imaging of neuronal activity using light field microscopy
3D functional imaging of neuronal activity in entire organisms at single cell
level and physiologically relevant time scales faces major obstacles due to
trade-offs between the size of the imaged volumes, and spatial and temporal
resolution. Here, using light-field microscopy in combination with 3D
deconvolution, we demonstrate intrinsically simultaneous volumetric functional
imaging of neuronal population activity at single neuron resolution for an
entire organism, the nematode Caenorhabditis elegans. The simplicity of our
technique and possibility of the integration into epi-fluoresence microscopes
makes it an attractive tool for high-speed volumetric calcium imaging.Comment: 25 pages, 7 figures, incl. supplementary informatio
Design and mechanistic insight into ultrafast calcium indicators for monitoring intracellular calcium dynamics.
Calmodulin-based genetically encoded fluorescent calcium indicators (GCaMP-s) are powerful tools of imaging calcium dynamics from cells to freely moving animals. High affinity indicators with slow kinetics however distort the temporal profile of calcium transients. Here we report the development of reduced affinity ultrafast variants of GCaMP6s and GCaMP6f. We hypothesized that GCaMP-s have a common kinetic mechanism with a rate-limiting process in the interaction of the RS20 peptide and calcium-calmodulin. Therefore we targeted specific residues in the binding interface by rational design generating improved indicators with GCaMP6fu displaying fluorescence rise and decay times (t1/2) of 1 and 3 ms (37â°C) in vitro, 9 and 22-fold faster than GCaMP6f respectively. In HEK293T cells, GCaMP6fu revealed a 4-fold faster decay of ATP-evoked intracellular calcium transients than GCaMP6f. Stimulation of hippocampal CA1 pyramidal neurons with five action potentials fired at 100âHz resulted in a single dendritic calcium transient with a 2-fold faster rise and 7-fold faster decay time (t1/2 of 40âms) than GCaMP6f, indicating that tracking high frequency action potentials may be limited by calcium dynamics. We propose that the design strategy used for generating GCaMP6fu is applicable for the acceleration of the response kinetics of GCaMP-type calcium indicators
Mitochondrial and cytosolic ATP/ADP ratios in isolated hepatocytes. A comparison of the digitonin method and the non-aqueous fractionation procedure
Fast-Response Calmodulin-Based Fluorescent Indicators Reveal Rapid Intracellular Calcium Dynamics
Faithful reporting of temporal patterns of intracellular Ca
2
+
dynamics requires the working range
of indicators to match the signals. Current genetically encoded calmodulin-based fluorescent
indicators are likely to distort fast Ca
2
+
signals by apparent saturation and integration due to their
limiting fluorescence rise and decay kinetics. A series of probes was engineered with a range of
Ca
2
+
affinities and accelerated kinetics by weakening the Ca
2
+
-calmodulin-peptide interactions. At
37
°C, the GCaMP3-derived probe termed GCaMP3
fast
is 40-fold faster than GCaMP3 with Ca
2
+
decay
and rise times,
t
1/2
, of 3.3
ms and 0.9
ms, respectively, making it the fastest to-date. GCaMP3
fast
revealed discreet transients with significantly faster Ca
2
+
dynamics in neonatal cardiac myocytes
than GCaMP6f. With 5-fold increased two-photon fluorescence cross-section for Ca
2
+
at 940
nm,
GCaMP3
fast
is suitable for deep tissue studies. The green fluorescent protein serves as a reporter
providing important novel insights into the kinetic mechanism of target recognition by calmodulin.
Our strategy to match the probe to the signal by tuning the affinity and hence the Ca
2
+
kinetics of
the indicator is applicable to the emerging new generations of calmodulin-based probe
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Optimization of a GCaMP Calcium Indicator for Neural Activity Imaging
Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Recent efforts in protein engineering have significantly increased the performance of GECIs. The state-of-the art single-wavelength GECI, GCaMP3, has been deployed in a number of model organisms and can reliably detect three or more action potentials in short bursts in several systems in vivo. Through protein structure determination, targeted mutagenesis, high-throughput screening, and a battery of in vitro assays, we have increased the dynamic range of GCaMP3 by severalfold, creating a family of âGCaMP5â sensors. We tested GCaMP5s in several systems: cultured neurons and astrocytes, mouse retina, and in vivo in Caenorhabditis chemosensory neurons, Drosophila larval neuromuscular junction and adult antennal lobe, zebrafish retina and tectum, and mouse visual cortex. Signal-to-noise ratio was improved by at least 2- to 3-fold. In the visual cortex, two GCaMP5 variants detected twice as many visual stimulus-responsive cells as GCaMP3. By combining in vivo imaging with electrophysiology we show that GCaMP5 fluorescence provides a more reliable measure of neuronal activity than its predecessor GCaMP3. GCaMP5 allows more sensitive detection of neural activity in vivo and may find widespread applications for cellular imaging in general.Molecular and Cellular Biolog
Social Sciences and Humanities priority 100 research questions for renewable energy in Horizon Europe
Generation of a Homozygous Transgenic Rat Strain Stably Expressing a Calcium Sensor Protein for Direct Examination of Calcium Signaling
In drug discovery, prediction of selectivity and toxicity
require the evaluation of cellular calcium homeostasis. The rat
is a preferred laboratory animal for pharmacology and
toxicology studies, while currently no calcium indicator
protein expressing rat model is available. We established a
transgenic rat strain stably expressing the GCaMP2
fluorescent calcium sensor by a transposon-based methodology.
Zygotes were co-injected with mRNA of transposase and a CAG-
GCaMP2 expressing construct, and animals with one
transgene copy were pre-selected by measuring fluorescence in
blood cells. A homozygous rat strain was generated with high
sensor protein expression in the heart, kidney, liver, and
blood cells. No pathological alterations were found in these
animals, and fluorescence measurements in cardiac tissue slices
and primary cultures demonstrated the applicability of this
system for studying calcium signaling. We show here that the
GCaMP2 expressing rat cardiomyocytes allow the
prediction of cardiotoxic drug side-effects, and provide
evidence for the role of Na+/Ca2+ exchanger and its beneficial
pharmacological modulation in cardiac reperfusion. Our data
indicate that drug-induced alterations and pathological
processes can be followed by using this rat model, suggesting
that transgenic rats expressing a calcium-sensitive protein
provide a valuable system for pharmacological and toxicological
studies
Work characteristics and determinants of job satisfaction in four age groups: university employeesâ point of view
Contains fulltext :
79843.pdf (publisher's version ) (Closed access)PURPOSE: To investigate (a) differences in work characteristics and (b) determinants of job satisfaction among employees in different age groups. METHODS: A cross-sectional questionnaire was filled in by 1,112 university employees, classified into four age groups. (a) Work characteristics were analysed with ANOVA while adjusting for sex and job classification. (b) Job satisfaction was regressed against job demands and job resources adapted from the Job Demands-Resources model. Results : Statistically significant differences concerning work characteristics between age groups are present, but rather small. Regression analyses revealed that negative association of the job demands workload and conflicts at work with job satisfaction faded by adding job resources. Job resources were most correlated with more job satisfaction, especially more skill discretion and more relations with colleagues. CONCLUSIONS: Skill discretion and relations with colleagues are major determinants of job satisfaction. However, attention should also be given to conflicts at work, support from supervisor and opportunities for further education, because the mean scores of these work characteristics were disappointing in almost all age groups. The latter two characteristics were found to be associated significantly to job satisfaction in older workers
A microfluidics-based method for measuring neuronal activity in Drosophila chemosensory neurons
Monitoring neuronal responses to defined sensory stimuli is a powerful and widely used approach for understanding sensory coding in the nervous system. However, providing precise, stereotypic and reproducible cues while concomitantly recording neuronal activity remains technically challenging. Here we describe the fabrication and use of a microfluidics system that allows precise temporally restricted stimulation of Drosophila chemosensory neurons with an array of different chemical cues. The system can easily be combined with genetically encoded calcium sensors, and it can measure neuronal activity at single-cell resolution in larval sense organs and in the proboscis or leg of the adult fly. We describe the design of the master mold, the production of the microfluidic chip and live imaging using the calcium sensor GCaMP, expressed in distinct types of Drosophila chemosensory neurons. Fabrication of the master mold and microfluidic chips requires basic skills in photolithography and takes ~2 weeks; the same devices can be used repeatedly over several months. Flies can be prepared for measurements in minutes and imaged for up to 1 h
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