Imaging analytical technique to assess gastrointestinal motility in vivo using zebrafish larvae with diabetes mellitus-like traits.

In diabetes mellitus (DM), the prevalence of gastrointestinal (GI) complications, including constipation, diarrhoea, gastroparesis, and/or enteropathy, can be up to ~75%. In this study, we compared three zebrafish larvae models of DM and established an analytical protocol for GI motility. Larvae were fed with either a standard diet (SD; control), or one of three diets to induce a DM-like phenotype: excessive feeding of SD food (ED), a high-fat diet (HFD), or exposing SD-fed larvae to 30 mmol/L glucose (SDG).

Computational Docking as a Tool in Guiding the Drug Design of Rutaecarpine Derivatives as Potential SARS-CoV-2 Inhibitors.

COVID-19 continues to spread around the world. This is mainly because new variants of the SARS-CoV-2 virus emerge due to genomic mutations, evade the immune system and result in the effectiveness of current therapeutics being reduced. We previously established a series of detection platforms, comprising computational docking analysis, S-protein-based ELISA, pseudovirus entry, and 3CL protease activity assays, which allow us to screen a large library of phytochemicals from natural products and to determine their potential in blocking the entry of SARS-CoV-2.

A Role for Two-Pore Channel Type 2 (TPC2)-Mediated Regulation of Membrane Contact Sites During Zebrafish Notochord Biogenesis?

We have previously shown that in the developing trunk of zebrafish embryos, two-pore channel type 2 (TPC2)-mediated Ca release from endolysosomes plays a role in the formation of the skeletal slow muscle. In addition, TPC2-mediated Ca signaling is required for axon extension and the establishment of synchronized activity in the primary motor neurons. Here, we report that TPC2 might also play a role in the development of the notochord of zebrafish embryos.

Actin-mediated endocytosis in the E-YSL helps drive epiboly in zebrafish.

In zebrafish, a punctate band of F-actin is reported to develop in the external yolk syncytial layer (E-YSL) during the latter part of epiboly in zebrafish embryos. Here, electron microscopy (EM) and fluorescence confocal microscopy were conducted to investigate dynamic changes in the E-YSL membrane during epiboly. Using scanning EM, we report that the surface of the E-YSL is highly convoluted, consisting of a complex interwoven network of branching membrane surface microvilli-like protrusions.

The Ethanol Extract of Evodiae Fructus and Its Ingredient, Rutaecarpine, Inhibit Infection of SARS-CoV-2 and Inflammatory Responses.

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.

Neuromasts and Olfactory Organs of Zebrafish Larvae Represent Possible Sites of SARS-CoV-2 Pseudovirus Host Cell Entry.

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 model. Successful pseudovirus entry was demonstrated via the expression of the luciferase () gene, which was validated by reverse transcription-PCR (RT-PCR).

The Extracts of Root and Rhizome Block the Entry of SARS-CoV-2 Wild-Type and Omicron Pseudotyped Viruses via Inhibition of the S-Protein and 3CL Protease.

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 (Polygoni Cuspidati Rhizoma et Radix), a common Chinese herbal medicine, blocked the entry of wild-type and the omicron variant of the SARS-CoV-2 pseudotyped virus into fibroblasts or zebrafish larvae, with IC values ranging from 0.

Polygoni multiflori radix extracts inhibit SARS-CoV-2 pseudovirus entry in HEK293T cells and zebrafish larvae.

Background: 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.

Methods: 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.

Localized TPC1-mediated Ca2+ release from endolysosomes contributes to myoseptal junction development in zebrafish.

In the trunk of developing zebrafish embryos, adjacent myotome blocks transmit contractile force via myoseptal junctions (MJs), which are dynamic structures that connect the actin cytoskeleton of skeletal muscle cells to extracellular matrix components via transmembrane protein complexes in the sarcolemma. Here, we report that the endolysosomal ion channel, two-pore channel type 1 (TPC1, encoded by tpcn1), generates highly localized non-propagating Ca2+ transients that play a distinct and required role in the capture and attachment of superficial slow skeletal muscle cells at MJs. Use of antisense morpholinos or CRISPR/Cas9 gene editing to disrupt tpcn1 gene expression resulted in abnormal MJ phenotypes, including slow skeletal muscle cells detaching from or crossing the myosepta.

Transmembrane H fluxes and the regulation of neural induction in .

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.

Daily rhythms in heartbeat rate are intrinsic to the zebrafish heart.

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. 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. 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.

Investigating the role of dachshund b in the development of the pancreatic islet in zebrafish.

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.

Short-term homeostatic regulation of blood/interstitial fluid Ca concentration by the scales of anadromous sea trout Salmo trutta L. during smoltification and migration.

The elasmoid scales of anadromous sea trout Salmo trutta L. represent a significant internal reservoir of Ca . 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 Ca homeostasis in the extracellular fluid (ECF) during these phases of the life cycle.

TPC2-mediated Ca signaling is required for axon extension in caudal primary motor neurons in zebrafish embryos.

The role of two-pore channel type 2 (TPC2, encoded by )-mediated Ca release was recently characterized in zebrafish during 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 role of TPC2 in the regulation of caudal primary motor neuron (CaP) axon extension. We used a combination of TPC2 knockdown with a translation-blocking morpholino antisense oligonucleotide (MO), TPC2 knockout via the generation of a mutant line of zebrafish using CRISPR/Cas9 gene-editing and pharmacological inhibition of TPC2 via incubation with bafilomycin A1 (an H-ATPase inhibitor) or -ned-19 (an NAADP receptor antagonist), and showed that these treatments attenuated CaP Ca signaling and inhibited axon extension.

Trpc1 as the Missing Link Between the Bmp and Ca Signalling Pathways During Neural Specification in Amphibians.

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 Ca through voltage-dependent L-type Ca channels (LTCCs), mainly via Ca1.2, and we showed that this influx constitutes a necessary and sufficient signal for triggering the expression of neural genes.

Role of Two-Pore Channels in Embryonic Development and Cellular Differentiation.

Since the identification of nicotinic acid adenine dinucleotide phosphate (NAADP) and its putative target, the two-pore channel (TPC), the NAADP/TPC/Ca signaling pathway has been reported to play a role in a diverse range of functions in a variety of different cell types. TPCs have also been associated with a number of diseases, which arise when their activity is perturbed. In addition, TPCs have been shown to play key roles in various embryological processes and during the differentiation of a variety of cell types.

The Use of Complementary Luminescent and Fluorescent Techniques for Imaging Ca Signaling Events During the Early Development of Zebrafish (Danio rerio).

We have visualized many of the Ca 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 Ca 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 Ca signaling events within a complex developing embryo, we then repeat the experiment using a fluorescent Ca reporter such as calcium green-1 dextran and use confocal laser scanning microscopy to provide time-lapse series of higher-resolution images.

Characterization of ADP-ribosyl cyclase 1-like (ARC1-like) activity and NAADP signaling during slow muscle cell development in zebrafish embryos.

We recently demonstrated the requirement of two-pore channel type 2 (TPC2)-mediated Ca release during slow muscle cell differentiation and motor circuit maturation in intact zebrafish embryos. However, the upstream trigger(s) of TPC2/Ca signaling during these developmental processes remains unclear. Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca mobilizing messenger, which is suggested to target TPC2 in mediating the release of Ca from acidic vesicles.

Identification of Ca signaling components in neural stem/progenitor cells during differentiation into neurons and glia in intact and dissociated zebrafish neurospheres.

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.

Matrix metalloproteinases (MMPs) mediate leukocyte recruitment during the inflammatory phase of zebrafish heart regeneration.

In zebrafish, the role of matrix metalloproteinases (MMPs) in the inflammatory phase of heart regeneration following cryoinjury remains poorly understood. Here, we demonstrated an increase in MMP enzymatic activity and elevated expression of mmp9 and mmp13 in the injured area (IA) of hearts from as early as 1 day post-cryoinjury (dpc). Treatment with the broad-spectrum MMP inhibitor, GM6001, during the first week after cryoinjury resulted in impaired heart regeneration, as indicated by the larger scar and reduced numbers of proliferating cardiomyocytes.

TPC2-mediated Ca signaling is required for the establishment of synchronized activity in developing zebrafish primary motor neurons.

During the development of the early spinal circuitry in zebrafish, spontaneous Ca 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, Ca 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, Ca transients were visualized in the caudal PMNs (CaPs).

TRPC3 is required for the survival, pluripotency and neural differentiation of mouse embryonic stem cells (mESCs).

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.

Integrated transcriptomic and regulatory network analyses identify microRNA-200c as a novel repressor of human pluripotent stem cell-derived cardiomyocyte differentiation and maturation.

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.

Ca release via two-pore channel type 2 (TPC2) is required for slow muscle cell myofibrillogenesis and myotomal patterning in intact zebrafish embryos.

We recently demonstrated a critical role for two-pore channel type 2 (TPC2)-mediated Ca release during the differentiation of slow (skeletal) muscle cells (SMC) in intact zebrafish embryos, via the introduction of a translational-blocking morpholino antisense oligonucleotide (MO). Here, we extend our study and demonstrate that knockdown of TPC2 with a non-overlapping splice-blocking MO, knockout of TPC2 (via the generation of a tpcn2 mutant line of zebrafish using CRISPR/Cas9 gene-editing), or the pharmacological inhibition of TPC2 action with bafilomycin A1 or trans-ned-19, also lead to a significant attenuation of SMC differentiation, characterized by a disruption of SMC myofibrillogenesis and gross morphological changes in the trunk musculature. When the morphants were injected with tpcn2-mRNA or were treated with IP/BM or caffeine (agonists of the inositol 1,4,5-trisphosphate receptor (IPR) and ryanodine receptor (RyR), respectively), many aspects of myofibrillogenesis and myotomal patterning (and in the case of the pharmacological treatments, the Ca signals generated in the SMCs), were rescued.