Search

We present here results from the first-ever search for dark photon dark matter that could have coupled to baryons in LISA Pathfinder, the technology demonstrator for a space-based gravitational-wave antenna. After analyzing approximately three months of data taken by LISA Pathfinder in the frequency range $[2\times 10^{-5},5]$ Hz, corresponding to dark photon masses of $[8\times 10^{-20},2\times 10^{-14}]$ eV/$c^2$, we find no evidence of a dark-matter signal, and set upper limits on the strength of the dark photon/baryon coupling. To perform this search, we leveraged methods that search for quasi-monochromatic gravitational-wave signals in ground-based interferometers, and are robust against non-Gaussianities and gaps in the data. Our work therefore represents a proof-of-concept test of search methods in LISA to find persistent, quasi-monochromatic signals, and shows our ability to handle non-Guassian artifacts and gaps while maintaining good sensitivity compared to the optimal matched filter. The results also indicate that these methods can be powerful tools in LISA to not only find dark matter, but also look for other persistent signals from e.g. intermediate-mass black hole inspirals and galactic white dwarf binaries., Comment: 9 pages, 5 figures

A stochastic gravitational-wave background (SGWB) is expected to be produced by the superposition of individually undetectable, unresolved gravitational-wave (GW) signals from cosmological and astrophysical sources. Such a signal can be searched with dedicated techniques using the data acquired by a network of ground-based GW detectors. In this work, we consider the astrophysical SGWB resulting from pulsar glitches, which are sudden increases in the rotational pulsar frequency, within our Galaxy. More specifically, we assume glitches to be associated with quantized, superfluid, vortex-avalanches in the pulsars, and we model the SGWB from the superposition of GW bursts emitted during the glitching phase. We perform a cross-correlation search for this SGWB-like signal employing the data from the first three observation runs of Advanced LIGO and Virgo. Not having found any evidence for a SGWB signal, we set upper limits on the dimensionless energy density parameter $\Omega_{\mathrm{gw}}(f)$ for two different power-law SGWBs, corresponding to two different glitch regimes. We obtain $\Omega_{\mathrm{gw}}(f)\leq 7.5 \times 10^{-10}$ at 25 Hz for a spectral index 5/2, and $\Omega_{\mathrm{gw}}(f)\leq 5.7 \times 10^{-17}$ at 25 Hz for a spectral index 17/2. We then use these results to set constraints on the average glitch duration and the average radial motion of the vortices during the glitches for the population of the glitching Galactic pulsars, as a function of the Galactic glitch rate., Comment: 16 pages, 3 figures, 1 table

Migrating sea trout Salmo trutta L. in the waters of the Shetland Islands (UK) were once found in abundant numbers but they have been in decline since the mid-1980s. Therefore, it is becoming ever more critical to investigate sea trout behaviour with respect to the prevailing environmental conditions in the unique Shetland habitat so that we can determine how this prized and important species might be re-established to more sustainable levels. Here, during a period of four months (July to October) in 2015, environmental and fish catch rate data were collected for a small sample size of sea trout caught at six Shetland locations. We then looked for trends and correlations in the data that might help account for the migratory behaviour of the fish with respect to the environmental conditions at the time of capture. Our data showed that wind speed and pH correlated to the catch rate. However, no significant correlation was found between the water temperature, air temperature, barometric pressure, or humidity, and catch rate. Multivariate regression showed that wind speed and pH explained 26.3% of the variance in the catch rate. The sex ratio of the sea trout caught was skewed towards females at a ratio of 4.7 : 1.0 and the largest trout caught were all female. Recording biological data and understanding the migration patterns and behaviours of the remaining wild populations of sea trout are important for optimizing management plans to support current efforts to increase the numbers of this valuable anadromous species in Shetland waters.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the acute respiratory disease coronavirus disease 2019 (COVID-19), which has resulted in millions of deaths globally. Here, we explored the mechanism of host cell entry of a luciferase-ZsGreen spike (SARS-CoV-2)-pseudotyped lentivirus using zebrafish embryos/larvae as an

COVID-19, derived from SARS-CoV-2, has resulted in millions of deaths and caused unprecedented socioeconomic damage since its outbreak in 2019. Although the vaccines developed against SARS-CoV-2 provide some protection, they have unexpected side effects in some people. Furthermore, new viral mutations reduce the effectiveness of the current vaccines. Thus, there is still an urgent need to develop potent non-vaccine therapeutics against this infectious disease. We recently established a series of detecting platforms to screen a large library of Chinese medicinal herbs and phytochemicals. Here, we reveal that the ethanolic extract of Evodiae Fructus and one of its components, rutaecarpine, showed promising potency in inhibiting the activity of 3C-like (3CL) protease, blocking the entry of the pseudo-typed SARS-CoV-2 (including wild-type and omicron) into cultured cells. In addition, inflammatory responses induced by pseudo-typed SARS-CoV-2 were markedly reduced by Evodiae Fructus extract and rutaecarpine. Together our data indicate that the herbal extract of Evodiae Fructus and rutaecarpine are potent anti-SARS-CoV-2 agents, which might be considered as a treatment against COVID-19 in clinical applications.

We present a convolutional neural network that is capable of searching for continuous gravitational waves, quasi-monochromatic, persistent signals arising from asymmetrically rotating neutron stars, in $\sim 1$ year of simulated data that is plagued by non-stationary, narrow-band disturbances, i.e., lines. Our network has learned to classify the input strain data into four categories: (1) only Gaussian noise, (2) an astrophysical signal injected into Gaussian noise, (3) a line embedded in Gaussian noise, and (4) an astrophysical signal contaminated by both Gaussian noise and line noise. In our algorithm, different frequencies are treated independently; therefore, our network is robust against sets of evenly-spaced lines, i.e., combs, and we only need to consider perfectly sinusoidal line in this work. We find that our neural network can distinguish between astrophysical signals and lines with high accuracy. In a frequency band without line noise, the sensitivity depth of our network is about $\mathcal{D}^{95\%} \simeq 43.9$ with a false alarm probability of $\sim 0.5\%$, while in the presence of line noise, we can maintain a false alarm probability of $\sim 10\%$ and achieve $\mathcal{D}^\mathrm{95\%} \simeq 3.62$ when the line noise amplitude is $h_0^\mathrm{line}/\sqrt{S_\mathrm{n}(f_k)} = 1.0$. We evaluate the computational cost of our method to be $O(10^{19})$ floating point operations, and compare it to those from standard all-sky searches, putting aside differences between covered parameter spaces. Our results show that our method is more efficient by one or two orders of magnitude than standard searches. Although our neural network takes about $O(10^8)$ sec to employ using our current facilities (a single GPU of GTX1080Ti), we expect that it can be reduced to an acceptable level by utilizing a larger number of improved GPUs., Comment: 17 pages, 11 figures; v2: Virgo's contribution appropriately mentioned, typos fixed

Aims/Introduction β‐Cell dysfunction is a hallmark of type 2 diabetes. In a previous pilot study, we identified an association between genetic variants within the human DACH1 gene and young‐onset type 2 diabetes. Here, we characterized the function of dachb, the only dach homologue to be expressed in the pancreas, in developing zebrafish embryos. Materials and Methods We injected one‐cell stage embryos with a dachb‐morpholino (MO) or with the dachb‐MO and dachb messenger ribonucleic acid, and determined the effect on the development of the pancreatic islet. We also carried out quantitative polymerase chain reaction and ribonucleic acid sequencing on the dachb‐MO group to determine the effect of dachb knockdown on gene expression. Results MO‐mediated dachb knockdown resulted in impaired islet cell development, with a significant decrease in both the β‐cell and islet cell numbers. This islet developmental defect was rescued when embryos were co‐injected with dachb‐MO and dachb messenger ribonucleic acid. Knockdown of dachb was associated with a significant downregulation of the β‐cell specific marker gene, insa, and the somatostatin cell marker, sst2, as well as regulators of pancreas development, ptf1a, neuroD, pax6a and nkx6.1, and the cell cycle gene, insm1a. Furthermore, ribonucleic sequencing analysis showed an upregulation of genes enriched in the forkhead box O and mitogen‐activated protein kinase signaling pathways in the dachb‐MO group, when compared with the control groups. Conclusions Together, our results suggest the possible role of dachb in islet development in zebrafish., Morpholino (MO)‐mediated knock‐down of dachb resulted in impaired islet cell development, with a significant decrease in both the β‐cell and islet cell numbers in zebrafish. Embryos co‐injected with dachb mRNA and dachb‐MO expressed a similar phenotype as the uninjected control groups, indicating rescue of the islet developmental defect. The studies highlight the role of dachb in islet development in zebrafish.

Ground-based gravitational-wave interferometers could directly probe the existence of ultralight dark matter ($\mathcal{O}(10^{-14}-10^{-11})$ eV/$c^2$) that couples to standard-model particles in the detectors. Recently, many techniques have been developed to extract a variety of potential dark-matter signals from noisy gravitational-wave data; however, little effort has gone into ways to distinguish between types of dark matter that could directly interact with the interferometers. In this work, we employ the Wiener filter to follow-up candidate dark-matter interaction signals. The filter captures the stochastic nature of these signals, and, in simulations, successfully identifies which type of dark matter interacts with the interferometers. We apply the Wiener filter to outliers that remained in the LIGO/Virgo/KAGRA search for dark photons in data from the most recent observing (O3), and show that they are consistent with noise disturbances. Our proof-of-concept analysis demonstrates that the Wiener filter can be a powerful technique to confirm or deny the presence of dark-matter interaction signals in gravitational-wave data, and distinguish between scalar and vector dark-matter interactions., Comment: 10 pages, 8 figures, published in PRD

COVID-19, resulting from infection by the SARS-CoV-2 virus, caused a contagious pandemic. Even with the current vaccines, there is still an urgent need to develop effective pharmacological treatments against this deadly disease. Here, we show that the water and ethanol extracts of the root and rhizome of

We present new constraints on the merging rates of planetary-mass and asteroid-mass primordial black hole binaries using limits on continuous waves(quasi-monochromatic, quasi-infinite duration signals) derived from an all-sky search for isolated compact objects in the first six months of the third observing run (O3a) of LIGO/Virgo. We calculate the merging rates of these binaries in a model-independent way, and convert them to constraints on the primordial black hole abundance with minimal modelling assumptions. Our results show that we are sensitive to sources at most $\mathcal{O}(10$ pc) away for systems with chirp masses of $\mathcal{O}(10^{-5}M_\odot)$ at gravitational-wave frequencies around 30-40 Hz. These results also show that continuous-wave searches could in the future directly probe the existence of planetary-mass and asteroid-mass primordial black holes, especially those in binaries with asymmetric mass ratios. Furthermore, they demonstrate that new methods accounting for the full nonlinear gravitational-wave frequency evolution are needed to improve constraints on primordial black holes., 8 pages, 4 figures

The elasmoid scales of anadromous sea trout Salmo trutta L. represent a significant internal reservoir of Ca2+ . Although more is known about long-term remodelling of scales in response to calciotropic challenges encountered during smoltification and migration, very little is known about the contribution made by scales to the short-term, minute-to-minute regulation of Ca2+ homeostasis in the extracellular fluid (ECF) during these phases of the life cycle. This gap in the knowledge is partly due to the technical challenges involved in measuring small Ca2+ fluxes around the scales of live fish in real time. Here, this study describes exfoliating, mounting and culturing scales and their resident cells from parr, smolt and adult sea trout from a freshwater environment, as well as from adult sea trout caught in sea or brackish water. All the scales were then examined using an extracellular, non-invasive, surface-scanning Ca2+ -sensitive microelectrode. The authors quantified the Ca2+ fluxes, in the absence of any systemic or local regulators, into and out of scales on both the episquamal and hyposquamal sides under different extracellular calcemic challenges set to mimic a variety of ECF-Ca2+ concentrations. Scales from the life-cycle stages as well as from adult fish taken from sea, brackish or fresh water all showed a consistent efflux or influx of Ca2+ under hypo- or hypercalcemic conditions, respectively. What were considered to be isocalcemic conditions resulted in minimal flux of Ca2+ in either direction, or in the case of adult scales, a consistent but small influx. Indeed, adult scales appeared to display the largest flux densities in either direction. These new data extend the current understanding of the role played by fish scales in the short-term, minute-to-minute homeostatic regulation of ECF-Ca2+ concentration, and are similar to those recently reported from zebrafish Danio rerio scales. This suggests that this short-term regulatory response might be a common feature of teleost scales.

Gravitational-wave astronomy allows us to study objects and events invisible to electromagnetic waves. So far, only signals triggered by coalescing binaries have been detected. However, as the interferometers' sensitivities improve over time, we expect to observe weaker signals in the future, e.g. emission of continuous gravitational waves from spinning, isolated neutron stars. Parallax is a well-known method, widely used in electromagnetic astronomical observations, to estimate the distance to a source. In this work, we consider the application of the parallax method to gravitational-wave searches and explore possible distance estimation errors. We show that detection of parallax in the signal from a spinning down source can constrain the neutron star moment of inertia. For instance, we found that the relative error of the moment of inertia estimation is smaller than $10\%$ for all sources closer than 300 pc, for the assumed birth frequency of 700 Hz, ellipticity $\geq 10^{-7}$ and for two years of observations by the Einstein Telescope, assuming spin down due purely to quadrupolar gravitational radiation., Comment: 7 pages, 7 figures, accepted for publication in MNRAS

Rotating neutron stars (NSs) are promising sources of gravitational waves (GWs) in the frequency band of ground-based detectors. They are expected to emit quasi-monochromatic, long-duration GW signals, called continuous waves (CWs), due to their deviations from spherical symmetry. The degree of such deformations, and hence the information about the internal structure of a NS, is encoded in a dimensionless parameter $\varepsilon$ called ellipticity. Searches for CW signals from isolated Galactic NSs have shown to be sensitive to ellipticities as low as $\varepsilon \sim \mathcal{O}(10^{-9})$. These searches are optimal for detecting and characterising GWs from individual NSs, but they are not designed to measure the properties of NSs as population, such as the average ellipticity $\varepsilon_{\mathrm{av}}$. These ensemble properties can be determined by the measurement of the stochastic gravitational-wave background (SGWB) arising from the superposition of GW signals from individually-undetectable NSs. In this work, we perform a cross-correlation search for such a SGWB using the data from the first three observation runs of Advanced LIGO and Virgo. Finding no evidence for a SGWB signal, we set upper limits on the dimensionless energy density parameter $\Omega_{\mathrm{gw}}(f)$. Using these results, we also constrain the average ellipticity of Galactic NSs and five NS ``hotspots'', as a function of the number of NSs emitting GWs within the frequency band of the search $N_{\mathrm{band}}$. We find $\varepsilon_{\mathrm{av}} \lesssim 1.8 \times 10^{-8}$, with $N_{\mathrm{band}}=1.6 \times 10^7$, for Galactic NSs, and $\varepsilon_{\mathrm{av}} \lesssim [3.5-11.8]\times 10^{-7}$, with $N_{\mathrm{band}}=1.6 \times 10^{10}$, for NS hotspots., Comment: 10 pages, 6 figures, 2 tables

It has previously been reported that in ex vivo planar explants prepared from Xenopus laevis embryos, the intracellular pH (pHi) increases in cells of the dorsal ectoderm from stage 10.5 to 11.5 (i.e. 11-12.5 hpf). It was proposed that such increases (potentially due to H+ being extruded, sequestered, or buffered in some manner), play a role in regulating neural induction. Here, we used an extracellular ion-selective electrode to non-invasively measure H+ fluxes at eight locations around the equatorial circumference of intact X. laevis embryos between stages 9-12 (˜7-13.25 hpf). We showed that at stages 9-11, there was a small H+ efflux recorded from all the measuring positions. At stage 12 there was a small, but significant, increase in the efflux of H+ from most locations, but the efflux from the dorsal side of the embryo was significantly greater than from the other positions. Embryos were also treated from stages 9-12 with bafilomycin A1, to block the activity of the ATP-driven H+ pump. By stage 22 (24 hpf), these embryos displayed retarded development, arresting before the end of gastrulation and therefore did not display the usual anterior and neural structures, which were observed in the solvent-control embryos. In addition, expression of the early neural gene, Zic3, was absent in treated embryos compared with the solvent controls. Together, our new in vivo data corroborated and extended the earlier explant-derived report describing changes in pHi that were suggested to play a role during neural induction in X. laevis embryos.

SummaryIt has previously been reported that in ex vivo planar explants prepared from Xenopus laevis embryos, the intracellular pH (pHi) increases in cells of the dorsal ectoderm from stage 10.5 to 11.5 (i.e. 11–12.5 hpf). It was proposed that such increases (potentially due to H+ being extruded, sequestered, or buffered in some manner), play a role in regulating neural induction. Here, we used an extracellular ion-selective electrode to non-invasively measure H+ fluxes at eight locations around the equatorial circumference of intact X. laevis embryos between stages 9–12 (˜7–13.25 hpf). We showed that at stages 9–11, there was a small H+ efflux recorded from all the measuring positions. At stage 12 there was a small, but significant, increase in the efflux of H+ from most locations, but the efflux from the dorsal side of the embryo was significantly greater than from the other positions. Embryos were also treated from stages 9–12 with bafilomycin A1, to block the activity of the ATP-driven H+ pump. By stage 22 (24 hpf), these embryos displayed retarded development, arresting before the end of gastrulation and therefore did not display the usual anterior and neural structures, which were observed in the solvent-control embryos. In addition, expression of the early neural gene, Zic3, was absent in treated embryos compared with the solvent controls. Together, our new in vivo data corroborated and extended the earlier explant-derived report describing changes in pHi that were suggested to play a role during neural induction in X. laevis embryos.

Globally, COVID-19 has caused millions of deaths and led to unprecedented socioeconomic damage. There is therefore, in addition to vaccination, an urgent need to develop complementary effective treatments and/or protective and preventative therapies against this deadly disease.Here, a multi-component testing platform was established to screen a library of herbal extracts from traditional Chinese medicine (TCM), to identify potent herbal extracts/phytochemicals as possible therapeutics for COVID-19. We utilized assays for spike protein (S-protein) binding to angiotensin-converting enzyme II (ACE2); the enzymatic inhibition of 3CL protease; and entry of the SARS-CoV-2 pseudovirus into cultured HEK293T cells and zebrafish larvae.Over a thousand herbal extracts were screened and approximately 20 positive hits were identified. Among these, we found that the water and ethanol extracts of Polygoni Multiflori Radix (PMR) significantly inhibited S-protein binding to ACE2, 3CL protease activity, and viral entry into the cell and fish models. The water extract was more effective than the ethanol extract, with ICOur results indicate that the water and ethanol extracts of PMR have an inhibitory effect on SARS-CoV-2 pseudovirus host-cell entry. Furthermore, EGCG might be an active component of PMR, which blocks SARS-CoV-2 entry to cells. Taken together, our findings suggest that PMR might be considered as a potential treatment for COVID-19.

The stocking of hatchery-origin fish into rivers and lakes has long been used in fisheries management to try to enhance catches, especially for trout and salmon species. Frequently, however, the long-term impacts of stocking programmes have not been evaluated. In this study, the authors investigate the contribution of a stocking programme undertaken to support the rod catch of sea trout in the Shetland Islands, UK. Once a highly productive recreational fishery, Shetland sea trout catches crashed in the mid-1990s. Around the time that stocking began, increases in rod catches were also reported, with advocates of the stocking highlighting the apparent success of the programme. Using a suite of genetic markers (microsatellites), this study explores the contribution of the stocking programme to the Shetland sea trout population. The authors found that the domesticated broodstock and wild spawned brown trout from seven streams were genetically distinct. Despite extensive stocking, wild spawned brown trout dominated, even in those streams with a long history of supplementation. The majority of sea trout caught and analysed were of wild origin - only a single individual was of pure stocked origin, with a small number of fish being of wild × stocked origins. This study suggests that stocking with a domesticated strain of brown trout has made only a very limited contribution to the Shetland Islands rod catch, and that the revival of sea trout numbers appears to be driven almost exclusively by recovery of trout spawned in the wild.

It is a well-established fact that different tissues within the body contain their own circadian clocks or pacemakers, where it is proposed that the clock controls the local, daily cell biology of that organ.1,2 In mammals, these peripheral clocks work in concert with and are entrained by rhythmic signals arising from the suprachiasmatic nucleus (SCN) in the hypothalamus of the animal, among other systemic cues.2 In the case of zebrafish, the circadian system appears to be highly decentralized with each tissue not only having an internal circadian clock, but also being directly light entrained.1 Several years ago, we showed that the zebrafish heart contains its own circadian pacemaker at the gene expression level.1 This is also the case in mammals, where the circadian clock controls approximately 10% of the genes expressed in the heart.3 However, heart rate itself is generally thought to be regulated by several well-described autonomic cues, neurotransmitters, and hormones. In this study, we report that, for larval zebrafish hearts, the daily change in heartbeat rate is not only clock-controlled in vivo, but that this rhythm also persists in vitro, indicating that the cardiac circadian clock itself can directly drive this major physiological oscillation.

The elasmoid scales of anadromous sea trout Salmo trutta L. represent a significant internal reservoir of Ca

The role of two-pore channel type 2 (TPC2, encoded by

The role of TPC2-mediated Ca2+ release was recently characterized in zebrafish during the establishment of the early spinal circuitry, one of the key events in the coordination of neuromuscular activity. Here, we extend our study to investigate the in vivo role of TPC2 in the regulation of caudal primary motor neuron (CaP) axon extension. We used a combination of: TPC2 knock-down with a translational-blocking morpholino antisense oligonucleotide (MO); TPC2 knock-out via the generation of a tpcn2dhkz1a mutant line of zebrafish using CRISPR/Cas9 gene-editing; and pharmacological inhibition of TPC2 via incubation with bafilomycin A1 (an H+-ATPase inhibitor) or trans-ned-19 (an NAADP receptor antagonist), and showed that these treatments attenuated CaP Ca2+ signaling and inhibited axon extension. We also characterized the expression of an arc1-like transcript in CaPs grown in primary culture. MO-mediated knock-down of ARC1-like in vivo led to an attenuation of the Ca2+ transients in the CaP growth cones, and an inhibition of axon extension. Together our new data suggest a link between ARC1-like, TPC2 and Ca2+ signaling during axon extension in zebrafish.

Calcium ion concentration ([Ca(2+)]) in the systemic extracellular fluid, ECF-[Ca(2+)], is maintained around a genetically predetermined set-point, which combines the operational level of the kidney/ and bone/ECF interfaces. The ECF-[Ca(2+)] is maintained within a narrow oscillation range by the regulatory action of Parathyroid Hormone (PTH), Calcitonin, FGF-23, and 1,25(OH)(2)D(3). This model implies two correction mechanisms, i.e. tubular Ca(2+) reabsorption and osteoclast Ca(2+) resorption. Although their alterations have an effect on the ECF-[Ca(2+)] maintenance, they cannot fully account for rapid correction of the continuing perturbations of plasma [Ca(2+)], which occur daily in life. The existence of Ca(2+) fluxes at quiescent bone surfaces fulfills the role of a short-term error correction mechanism in Ca(2+) homeostasis. To explore the hypothesis that PTH regulates the cell system responsible for the fast Ca(2+) fluxes at the bone/ECF interface, we have performed direct real-time measurements of Ca(2+) fluxes at the surface of ex-vivo metatarsal bones maintained in physiological conditions mimicking ECF, and exposed to PTH. To further characterize whether the PTH receptor on osteocytes is a critical component of the minute-to-minute ECF-[Ca(2+)] regulation, metatarsal bones from mice lacking the PTH receptor in these cells were tested ex vivo for rapid Ca(2+) exchange. We performed direct real-time measurements of Ca(2+) fluxes and concentration gradients by a scanning ion-selective electrode technique (SIET). To validate ex vivo measurements, we also evaluated acute calcemic response to PTH in vivo in mice lacking PTH receptors in osteocytes vs littermate controls. Our data demonstrated that Ca(2+) fluxes at the bone-ECF interface in excised bones as well as acute calcemic response in the short-term were unaffected by PTH exposure and its signaling through its receptor in osteocytes. Rapid minute-to-minute regulation of the ECF-[Ca(2+)] was found to be independent of PTH actions on osteocytes. Similarly, mice lacking PTH receptor in osteocytes, responded to PTH challenge with similar calcemic increases.

Aims MicroRNAs (miRNAs) are crucial for the post-transcriptional control of protein-encoding genes and together with transcription factors (TFs) regulate gene expression; however, the regulatory activities of miRNAs during cardiac development are only partially understood. In this study, we tested the hypothesis that integrative computational approaches could identify miRNAs that experimentally could be shown to regulate cardiomyogenesis. Methods and results We integrated expression profiles with bioinformatics analyses of miRNA and TF regulatory programs to identify candidate miRNAs involved with cardiac development. Expression profiling showed that miR-200c, which is not normally detected in adult heart, is progressively down-regulated both during cardiac development and in vitro differentiation of human embryonic stem cells (hESCs) to cardiomyocytes (CMs). We employed computational methodologies to predict target genes of both miR-200c and five key cardiac TFs to identify co-regulated gene networks. The inferred cardiac networks revealed that the cooperative action of miR-200c with these five key TFs, including three (GATA4, SRF and TBX5) targeted by miR-200c, should modulate key processes and pathways necessary for CM development and function. Experimentally, over-expression (OE) of miR-200c in hESC-CMs reduced the mRNA levels of GATA4, SRF and TBX5. Cardiac expression of Ca2+, K+ and Na+ ion channel genes (CACNA1C, KCNJ2 and SCN5A) were also significantly altered by knockdown or OE of miR-200c. Luciferase reporter assays validated miR-200c binding sites on the 3' untranslated region of CACNA1C. In hESC-CMs, elevated miR-200c increased beating frequency, and repressed both Ca2+ influx, mediated by the L-type Ca2+ channel and Ca2+ transients. Conclusions Our analyses demonstrate that miR-200c represses hESC-CM differentiation and maturation. The integrative computation and experimental approaches described here, when applied more broadly, will enhance our understanding of the interplays between miRNAs and TFs in controlling cardiac development and disease processes.

We have visualized many of the Ca

We recently demonstrated the requirement of two-pore channel type 2 (TPC2)-mediated Ca2+ release during slow muscle cell differentiation and motor circuit maturation in intact zebrafish embryos. However, the upstream trigger(s) of TPC2/Ca2+ signaling during these developmental processes remains unclear. Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca2+ mobilizing messenger, which is suggested to target TPC2 in mediating the release of Ca2+ from acidic vesicles. Here, we report the molecular cloning of the zebrafish ADP ribosyl cyclase (ARC) homolog (i.e., ARC1-like), which is a putative enzyme for generating NAADP. We characterized the expression of the arc1-like transcript and the NAADP levels between ~ 16 h post-fertilization (hpf) and ~ 48 hpf in whole zebrafish embryos. We showed that if ARC1-like (when fused with either EGFP or tdTomato) was overexpressed it localized in the plasma membrane, and associated with intracellular organelles, such as the acidic vesicles, Golgi complex and sarcoplasmic reticulum, in primary muscle cell cultures. Morpholino (MO)-mediated knockdown of arc1-like or pharmacological inhibition of ARC1-like (via treatment with nicotinamide), led to an attenuation of Ca2+ signaling and disruption of slow muscle cell development. In addition, the injection of arc1-like mRNA into ARC1-like morphants partially rescued the Ca2+ signals and slow muscle cell development. Together, our data might suggest a link between ARC1-like, NAADP, TPC2 and Ca2+ signaling during zebrafish myogenesis.

The elasmoid scales of fish represent a significant internal reservoir of calcium ions (Ca2+), but little is known about the contribution of these scales to the short-term regulation of Ca2+ homeostasis in the extracellular fluid (ECF). This gap in our knowledge is partly due to the technical challenges involved in measuring small Ca2+ fluxes around the scales of live fish in real time. Here, we describe a technique for exfoliating, mounting, and culturing intact living zebrafish Danio rerio scales, then subjecting them to examination using an extracellular, non-invasive, surface-scanning ion-selective electrode technique (SIET). In a Ca2+-sensitive configuration, the SIET can resolve Ca2+ flux values in the low-to-sub picomole/square centimeter/second range, with a spatial resolution of approximately 5 μm. We quantified the Ca2+ fluxes into and out of scales under different extracellular calcemic challenges set to mimic a variety of Ca2+ concentrations in the ECF and showed that the results were similar to those previously reported from isolated mouse metatarsal bone. Our new data extend our current understanding of the role played by fish scales in the short-term homeostatic regulation of Ca2+ concentration in the ECF. They also support the suggestion that scales might provide an inexpensive and complementary model for studying the fundamentals of bone-mediated homeostatic Ca2+ regulation and the diseases that result from its dysregulation.

We have visualized many of the Ca2+ signaling events that occur during the early stages of zebrafish development using complementary luminescent and fluorescent imaging techniques. We initially microinject embryos with the luminescent Ca2+ reporter, f-holo-aequorin, and using a custom-designed luminescent imaging system, we can obtain pan-embryonic visual information continually for up to the first ~24 h postfertilization (hpf). Once we know approximately when and where to look for these Ca2+ signaling events within a complex developing embryo, we then repeat the experiment using a fluorescent Ca2+ reporter such as calcium green-1 dextran and use confocal laser scanning microscopy to provide time-lapse series of higher-resolution images. These protocols allow us to identify the specific cell types and even the particular subcellular domain (e.g., nucleus or cytoplasm) generating the Ca2+ signal. Here, we outline the techniques we use to precisely microinject f-holo-aequorin or calcium green-1 dextran into embryos without affecting their viability or development. We also describe how to inject specific regions of early embryos in order to load localized embryonic domains with a particular Ca2+ reporter. These same techniques can also be used to introduce other membrane-impermeable reagents into embryos, including Ca2+ channel antagonists, Ca2+ chelators, fluorescent dyes, RNA, and DNA.

SummaryIn zebrafish embryos, distinct Ca2+transients are localized to the early cleavage furrows during the first few cell division cycles. These transients are generated mainly by release via IP3Rs in the endoplasmic reticulum, and they are necessary for furrow positioning, propagation, deepening and apposition. We previously showed, via the use of inhibitors, that store-operated Ca2+entry (SOCE) also appears to be essential for maintaining the IP3R-mediated elevated levels of [Ca2+]ifor the extended periods required for the completion of successful furrow deepening and daughter cell apposition in these large embryonic cells. Here, newly fertilized, dechorionated embryos were fixed at various times during the first and second cell division cycles and immunolabelled with antibodies to STIM1 and/or Orai1 (key components of SOCE). We show that both of these proteins have a dynamic pattern of localization during cytokinesis of the first two cell division cycles. These new data help to support our previous inhibitor results, and provide additional evidence that SOCE contributes to the maintenance of the sustained elevated Ca2+that is required for the successful completion of cytokinesis in the large cells of embryonic zebrafish.

Embryonic stem cells (ESCs) are promising resources for both scientific research and clinical regenerative medicine. With regards to the latter, ESCs are especially useful for treating several neurodegenerative disorders. Two significant characteristics of ESCs, which make them so valuable, are their capacity for self-renewal and their pluripotency, both of which are regulated by the integration of various signaling pathways. Intracellular Ca(2+) signaling is involved in several of these pathways. It is known to be precisely controlled by different Ca(2+) channels and pumps, which play an important role in a variety of cellular activities, including proliferation, differentiation and apoptosis. Here, we provide a review of the recent work conducted to investigate the function of Ca(2+) signaling in the self-renewal and the neural differentiation of ESCs. Specifically, we describe the role of intracellular Ca(2+) mobilization mediated by RyRs (ryanodine receptors); by cADPR (cyclic adenosine 5'-diphosphate ribose) and CD38 (cluster of differentiation 38/cADPR hydrolase); and by NAADP (nicotinic acid adenine dinucleotide phosphate) and TPC2 (two pore channel 2). We also discuss the Ca(2+) influx mediated by SOCs (store-operated Ca(2+) channels), TRPCs (transient receptor potential cation channels) and LTCC (L-type Ca(2+) channels) in the pluripotent ESCs as well as in neural differentiation of ESCs. Moreover, we describe the integration of Ca(2+) signaling in the other signaling pathways that are known to regulate the fate of ESCs.

The development of the CNS in vertebrate embryos involves the generation of different sub-types of neurons and glia in a complex but highly-ordered spatio-temporal manner. Zebrafish are commonly used for exploring the development, plasticity and regeneration of the CNS, and the recent development of reliable protocols for isolating and culturing neural stem/progenitor cells (NSCs/NPCs) from the brain of adult fish now enables the exploration of mechanisms underlying the induction/specification/differentiation of these cells. Here, we refined a protocol to generate proliferating and differentiating neurospheres from the entire brain of adult zebrafish. We demonstrated via RT-qPCR that some isoforms of ip3r, ryr and stim are upregulated/downregulated significantly in differentiating neurospheres, and via immunolabelling that 1,4,5-inositol trisphosphate receptor (IP3R) type-1 and ryanodine receptor (RyR) type-2 are differentially expressed in cells with neuron- or radial glial-like properties. Furthermore, ATP but not caffeine (IP3R and RyR agonists, respectively), induced the generation of Ca2+ transients in cells exhibiting neuron- or glial-like morphology. These results indicate the differential expression of components of the Ca2+-signaling toolkit in proliferating and differentiating cells. Thus, given the complexity of the intact vertebrate brain, neurospheres might be a useful system for exploring neurodegenerative disease diagnosis protocols and drug development using Ca2+ signaling as a read-out.

In amphibians, the inhibition of bone morphogenetic protein (BMP) in the dorsal ectoderm has been proposed to be responsible for the first step of neural specification, called neural induction. We previously demonstrated that in Xenopus laevis embryos, the BMP signalling antagonist, noggin, triggers an influx of Ca2+ through voltage-dependent L-type Ca2+ channels (LTCCs), mainly via CaV1.2, and we showed that this influx constitutes a necessary and sufficient signal for triggering the expression of neural genes. However, the mechanism linking the inhibition of BMP signalling with the activation of LTCCs remained unknown. Here, we demonstrate that the transient receptor potential canonical subfamily member 1, (Trpc1), is an intermediate between BMP receptor type II (BMPRII) and the CaV1.2 channel. We show that noggin induces a physical interaction between BMPRII and Trpc1 channels. This interaction leads to the activation of Trpc1 channels and to an influx of cations, which depolarizes the plasma membrane up to a threshold sufficient to activate Cav1.2. Together, our results demonstrate for the first time that during neural induction, Ca2+ entry through the CaV1.2 channel results from the noggin-induced interaction between Trpc1 and BMPRII.

Interest in the role of Ca

During the development of the early spinal circuitry in zebrafish, spontaneous Ca2+ transients in the primary motor neurons (PMNs) are reported to transform from being slow and uncorrelated, to being rapid, synchronized and patterned. In this study, we demonstrated that in intact zebrafish, Ca2+ release via two-pore channel type 2 (TPC2) from acidic stores/endolysosomes is required for the establishment of synchronized activity in the PMNs. Using the SAIGFF213A;UAS:GCaMP7a double-transgenic zebrafish line, Ca2+ transients were visualized in the caudal PMNs (CaPs). TPC2 inhibition via molecular, genetic or pharmacological means attenuated the CaP Ca2+ transients, and decreased the normal ipsilateral correlation and contralateral anti-correlation, indicating a disruption in normal spinal circuitry maturation. Furthermore, treatment with MS-222 resulted in a complete (but reversible) inhibition of the CaP Ca2+ transients, as well as a significant decrease in the concentration of the Ca2+ mobilizing messenger, nicotinic acid adenine diphosphate (NAADP) in whole embryo extract. Together, our new data suggest a novel function for NAADP/TPC2-mediated Ca2+ signaling in the development, coordination, and maturation of the spinal network in zebrafish embryos.

Transient receptor potential canonical subfamily member 3 (TRPC3) is known to be important for neural development and the formation of neuronal networks. Here, we investigated the role of TRPC3 in undifferentiated mouse embryonic stem cells (mESCs) and during the differentiation of mESCs into neurons. CRISPR/Cas9-mediated knockout (KO) of TRPC3 induced apoptosis and the disruption of mitochondrial membrane potential both in undifferentiated mESCs and in those undergoing neural differentiation. In addition, TRPC3 KO impaired the pluripotency of mESCs. TRPC3 KO also dramatically repressed the neural differentiation of mESCs by inhibiting the expression of markers for neural progenitors, neurons, astrocytes and oligodendrocytes. Taken together, our new data demonstrate an important function of TRPC3 with regards to the survival, pluripotency and neural differentiation of mESCs.

The development of the CNS in vertebrate embryos involves the generation of different sub-types of neurons and glia in a complex but highly-ordered spatio-temporal manner. Zebrafish are commonly used for exploring the development, plasticity and regeneration of the CNS, and the recent development of reliable protocols for isolating and culturing neural stem/progenitor cells (NSCs/NPCs) from the brain of adult fish now enables the exploration of mechanisms underlying the induction/specification/differentiation of these cells. Here, we refined a protocol to generate proliferating and differentiating neurospheres from the entire brain of adult zebrafish. We demonstrated via RT-qPCR that some isoforms of ip3r, ryr and stim are upregulated/downregulated significantly in differentiating neurospheres, and via immunolabelling that 1,4,5-inositol trisphosphate receptor (IP

We examined 15 embryos and saw regular, periodic pulses of light in 9 of them. These pulses arose in the pacemaker region, spread across the blastoderm, and ended on the opposite side of the blastoderm, near the embryonic axis. We saw no pulses in two eggs that had not quite developed to the stage at which they begin to contract rhythmically. The interval between calcium pulses at the pacemaker (155 f 11 s, x + SEM, n = 9 embryos) was estimated by counting photons for 9-32 min in a 15-pixel-square box drawn over this region (Fig. 1); the interval between mechanical pulses at the pacemaker (146 t 7 s, X f SEM, n = 6 embryos) was estimated from an analysis of timelapse video tape recordings. The velocity of the calcium wave (11.9 f 1.1 p/s, X + SEM, n = 7 embryos) was estimated by comparing the timing of the pulses at the pacemaker with those near the embryonic axis. This velocity is in the range of the fast calcium waves that are propagated by calcium-induced calcium release or by some related reaction-diffusion process (5). The calcium pulses and waves described herein are similar to the contraction waves in the following ways: 1) they both originate in the pacemaker region of the blastoderm, propagate across the blastodisc, and extinguish near the embryonic axis; 2) they have similar periods; and 3) they propagate at similar velocities.

Vincent et al. (1) reported that injections of EGTA giving final cytosolic concentrations of about 8 mM had no detectable effects on cytokinesis; but final concentrations of about 30 n-f the true figure seems close to a quarter (2, 3)]. Inhibition at such extremely high buffer concentrations might well suggest that calcium is not involved in the natural control of cytokinesis in Xenopus eggs. However, the results of our study of buffer inhibition in fucoid eggs (4) suggested a reinvestigation with another buffer more closely matched to the calcium level in the cytosolic region responsible for initiating and driving the division process (5). Table I shows the results of injecting 5,5’-dibromo-BAPTA, a buffer with a cytosolic Kn of about 5 pM. The lower figure in each range corrects for total accessible volume, on the assumption that about 20% of the egg volume is accessible to the injected buffer; buffer accessibility was calculated, in turn by taking 70% of the egg volume as inaccessible yolk (2) and about 70% of the cytosol as water (3). The higher figure in each range takes into account the likelihood that, at the time of action, the buffer had diffused out through a sphere only about 800 pm in diameter instead of filling the whole 1200 pm diameter egg. This buffer is about one hundred times more effective than EGTA in delaying or blocking cytokinesis. Detectable delays occur in response to final cytosolic buffer concentrations as low as 0.1 mM. This finding supports both the hypothesis that calcium controls cytokinesis, and the validity of the following equation (4) in Xenopus eggs

Factors that promote (low pH and fusicoccin) or reduce (high pH and IAA) the elongation rate of intact primary roots of Zea mays cv. Golden Bantam increased or decreased, respectively, the density of positive current flowing into the meristematic and elongating tissues of the developing root apex. Mature non-growing root regions generated the outward limb of the current loop. Ion-substitution and pH-profile experiments suggested that the bulk of the ion current was carried by H+. Calcium ions did not carry a significant portion of the current, but calcium appeared to regulate the proton circulation since the current density was larger in calcium-depleted media. Increased root elongation at low pH was associated with increased current density and a basipetally extended zone of inward current. Conversely, decreased elongation at high pH was associated with a reduced current density and a more restricted zone of inward current. The fungal toxin fusicoccin increased the current density of the inward limb of the ion current and increased root extension. Concentrations of IAA that reduced growth also reduced the density of the inward current and shortened the inward current zone. The effects of both fusicoccin and IAA were dependent on the pH of the bathing medium.