Thyrotroph Embryonic Factor Regulates Light-Induced Transcription of Repair Genes in Zebrafish Embryonic Cells

Numerous responses are triggered by light in the cell. How the light signal is detected and transduced into a cellular response is still an enigma. Each zebrafish cell has the capacity to directly detect light, making this organism particularly suitable for the study of light dependent transcription. To gain insight into the light signalling mechanism we identified genes that are activated by light exposure at an early embryonic stage, when specialised light sensing organs have not yet formed. We screened over 14,900 genes using micro-array GeneChips, and identified 19 light-induced genes that function primarily in light signalling, stress response, and DNA repair. Here we reveal that PAR Response Elements are present in all promoters of the light-induced genes, and demonstrate a pivotal role for the PAR bZip transcription factor Thyrotroph embryonic factor (Tef) in regulating the majority of light-induced genes. We show that tefβ transcription is directly regulated by light while transcription of tefα is under circadian clock control at later stages of development. These data leads us to propose their involvement in light-induced UV tolerance in the zebrafish embryo

A Rapid, Highly Sensitive and Open-Access SARS-CoV-2 Detection Assay for Laboratory and Home Testing

RT-qPCR-based diagnostic tests play important roles in combating virus-caused pandemics such as Covid-19. However, their dependence on sophisticated equipment and the associated costs often limits their widespread use. Loop-mediated isothermal amplification after reverse transcription (RT-LAMP) is an alternative nucleic acid detection method that overcomes these limitations. Here, we present a rapid, robust, and sensitive RT-LAMP-based SARS-CoV-2 detection assay. Our 40-min procedure bypasses the RNA isolation step, is insensitive to carryover contamination, and uses a colorimetric readout that enables robust SARS-CoV-2 detection from various sample types. Based on this assay, we have increased sensitivity and scalability by adding a nucleic acid enrichment step (Bead-LAMP), developed a version for home testing (HomeDip-LAMP), and identified open-source RT-LAMP enzymes that can be produced in any molecular biology laboratory. On a dedicated website, rtlamp.org (DOI: 10.5281/zenodo.6033689), we provide detailed protocols and videos. Our optimized, general-purpose RT-LAMP assay is an important step toward population-scale SARS-CoV-2 testing.MK was supported by the Vienna Science and Technology Fund (WWTF) through project COV20-031 (to JZ) and a Cambridge Trust LMB Cambridge Scholarship. Research in the AP lab is supported by the Austrian Science Fund (START Projekt Y 1031-B28, SFB “RNA-Deco” F 80) and EMBO-YIP; research in the JB lab is supported by the European Research Council (ERC- 2015-CoG - 682181). The IMP receives generous institutional funding from Boehringer Ingelheim and the Austrian Research Promotion Agency (Headquarter grant FFG-852936); IMBA is generously supported by the Austrian Academy of Sciences. Work in the LM-A laboratory is supported by grant PID2019- 104176RB-I00/AEI/10.13039/501100011033 of the Spanish Ministry of Science and Innovation, and an institutional grant of the Fundación Ramón Areces.Peer reviewe

Instrument design and protocol for the study of light controlled processes in aquatic organisms, and its application to examine the effect of infrared light on zebrafish

<div><p>The acquisition of reliable data strongly depends on experimental design. When studying the effects of light on processes such as behaviour and physiology it is crucial to maintain all environmental conditions constant apart from the one under study. Furthermore, the precise values of the environmental factors applied during the experiment should be known. Although seemingly obvious, these conditions are often not met when the effects of light are being studied. Here, we document and discuss the wavelengths and light intensities of natural and artificial light sources. We present standardised experimental protocols together with building plans of a custom made instrument designed to accurately control light and temperature for experiments using fresh water or marine species. Infrared light is commonly used for recording behaviour and in electrophysiological experiments although the properties of fish photoreceptors potentially allow detection into the far red. As an example of our experimental procedure we have applied our protocol and instrument to specifically test the impact of infrared light (840 nm) on the zebrafish circadian clock, which controls many aspects of behaviour, physiology and metabolism. We demonstrate that infrared light does not influence the zebrafish circadian clock. Our results help to provide a solid framework for the future study of light dependent processes in aquatic organisms.</p></div

IR light does not entrain the zebrafish circadian clock.

<p>The transcript levels of three core circadian clock genes and two clock output genes in larvae exposed to six 12–12 hour white light-dark or IR light-dark cycles and larvae raised in constant darkness, all at 28.0 ± 0.2°C, were used as readout. (A) <i>per1b</i> (circadian clock negative feedback loop) transcript levels are compared between larvae kept in constant darkness (DD, black bars) and under an IR light-dark (IRD, red bars) and white light-dark regime (LD, grey bars). The white-black bar below the chart indicates the light-dark interval and the black bar indicates constant darkness. (B) <i>clk1a</i> (circadian clock positive feedback loop), (C) <i>per2</i> (circadian clock directly light regulated), (D) <i>aanat2</i> (circadian clock output) and (E) <i>tefα</i> (circadian clock output) transcript levels under the same conditions as in A. None of the analyzed genes exhibit significant differences in transcript level between the IRD and DD conditions for each ZT (p>0.05, two-sample equal unpaired Student’s t-test). Error bars: standard deviations. (F) Intensities of IR light (154 μW/cm², 10.96 μmol/m²/s photons) in red and white daylight (33 μW/cm², 1.57 μmol/m²/s photons) in grey to which the larvae were exposed in this experiment.</p

Light sources with narrow bandwidth spectra.

<p>For comparison all spectra were measured from LEDs (Winger Electronics GmbH) of the same manufacturer near their maximum power (1W, 340 mA) and at a distance of 60 cm in air. (A) Spectrum of a blue LED (39 μW/cm²,1.7 μmol/m²/s photons), green LED (11 μW/cm², 0.5 μmol/m²/s photons), red LED (23 μW/cm², 1.2 μmol/m²/s photons), and IR LED (20 μW/cm², 1.4 μmol/m²/s photons). (B) The same spectra as in A, with instead of absolute irradiance the photon flux plotted on the y-axis. Note when comparing two LEDs that emit at different wavelengths but produce similar absolute irradiance (compare blue with IR), that the photon flux of the photons with a larger wavelength will be higher. (C) Reducing the intensity narrows the bandwidth of the spectrum. Compare blue LED driven at a current of 340 mA (39 μW/cm², 1.69 μmol/m²/s photons) in dark blue with the same LED at 170 mA (21 μW/cm²,0.94 μmol/m²/s photons) and at 50 mA (6 μW/cm², 0.29 μmol/m²/s photons) in light blue. This chart shows that the light intensity affects the bandwidth of the spectrum disproportionally. Also note the near linear correlation between the current and the light intensity. (D) A long-pass filter (Lee filter #87) is inserted between the camera and the lens to block visible light below 730 nm. Note that the spectra of the IR LED (red) and the white LED (grey) are on opposite sides of the filter. As the white LED does not emit IR, a LD cycle will not produce intensity fluctuations in the recording and thereby affect the tracking.</p

Construction plan for an instrument to control environmental conditions, and an infrared background lighting design to track small aquatic animals.

<p>(A) Technical drawing of the light-sealed temperature regulated instrument: 1) Front cover. 2) Side panels. 3) Rear panel. 4) Top panel. 5) Base. 6) Air vent cover. 7) Air vent flange. 8) Cap on air outlet. 9) Power supply cover. 10) Reflector plate (optional). For detailed technical drawings see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172038#pone.0172038.s001" target="_blank">S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172038#pone.0172038.s002" target="_blank">S2</a> Figs. (B) Section through infrared light box (left) and outside (right), which is designed to emit a particularly even distribution of light. 1) IR light strip around the inside of the box. 2) White rigid projector screen on bottom and sides. 3) Two layers of opaque plexiglass separated by 3 mm. 4) Top aluminium frame. 5) Bottom aluminium frame. 6) Base. 7) Aluminium profiles on corners that connect the top and bottom frames. 8) Screws with nuts.</p

Spectra of sunlight and artificial light sources.

<p>For light dependent processes the photon flux has a higher relevance than the irradiance since photoreceptors are activated by photons, thus we compared spectra plotted in μmol/m²/s photons versus the wavelength. (A) Spectrum of sunlight on a cloudless summer day in Vienna at 12:00 hours in grey (22437 μmol/m²/s photons) and at 19:00 hours during sunset in orange (1268 μmol/m²/s photons). (B) Spectrum of a Philips compact fluorescent lamp (14W, 5.1 μmol/m²/s photons). Red arrowheads indicate gaps in the spectrum. (C) Spectrum of an Osram incandescent lamp (15W, 5.8 μmol/m²/s photons). (D) Spectra of a Winger white daylight LED (1W, 6500K, 1.4 μmol/m²/s photons) in grey, and warm white LED (1W, 3200K, 1.1 μmol/m²/s photons) in orange. The main difference between a daylight and a warm white LED is the light intensity around 450 nm. Note the similarity of these light sources to respectively daylight and twilight in A. All measurements except for sunlight were performed at a distance of 60 cm from the light source, which corresponds to the distance between light source and sample in our experimental instrument.</p

A rapid, highly sensitive and open-access SARS-CoV-2 detection assay for laboratory and home testing

Global efforts to combat the Covid-19 pandemic caused by the beta coronavirus SARS-CoV-2 are currently based on RT-qPCR-based diagnostic tests. However, their high cost, moderate throughput and reliance on sophisticated equipment limit widespread implementation. Loop-mediated isothermal amplification after reverse transcription (RT-LAMP) is an alternative detection method that has the potential to overcome these limitations. Here we present a rapid, robust, highly sensitive and versatile RT-LAMP based SARS-CoV-2 detection assay. Our forty-minute procedure bypasses a dedicated RNA isolation step, is insensitive to carry-over contamination, and uses a hydroxynaphthol blue (HNB)-based colorimetric readout, which allows robust SARS-CoV-2 detection from various sample types. Based on this assay, we have substantially increased sensitivity and scalability by a simple nucleic acid enrichment step (bead-LAMP), established a pipette-free version for home testing (HomeDip-LAMP), and developed a version with open source enzymes that can be produced in any molecular biology setting. Our advanced, universally applicable RT-LAMP assay is a major step towards population-scale SARS-CoV-2 testing.N