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. In this new screen, rutaecarpine (an alkaloid from Evodia rutaecarpa) was identified as exhibiting anti-SARS-CoV-2 activity. Therefore, we conducted multiple rounds of structure-activity-relationship (SAR) studies around this phytochemical and generated several rutaecarpine analogs that were subjected to in vitro evaluations. Among these derivatives, RU-75 and RU-184 displayed remarkable inhibitory activity when tested in the 3CL protease assay, S-protein-based ELISA, and pseudovirus entry assay (for both wild-type and omicron variants), and they attenuated the inflammatory response induced by SARS-CoV-2. Interestingly, RU-75 and RU-184 both appeared to be more potent than rutaecarpine itself, and this suggests that they might be considered as lead candidates for future pharmacological elaboration.

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). DM was confirmed using a body-mass index, assessment of adipose deposit areas, two glucose assays, and one insulin assay. An analytical technique, whereby GI motility was quantified using pixel differences to track displacement along the centreline of the anterior, middle, and posterior intestine (AI, MI, and PI, respectively), was developed. Our results indicated that clear DM-like traits were observed in the HFD and SGD models, but not the ED model. In the SD controls, the AI showed similar anterograde and retrograde contractions indicating normal GI mixing; the MI exhibited more prominent forward contractions, and the PI showed distinct rectal waves. Compared to the SD, the HFD and SDG models exhibited significantly increased and decreased contraction velocities and could be used as models of diarrhoea and constipation in DM, respectively, while the ED model showed comparatively little change in motility. Together, these data indicate that complex changes in GI motility are associated with diet and therapeutics used to alleviate GI complications in DM should take these into account. Ultimately, the HFD and SDG models can be used to investigate different aspects of GI motility in association to DM. Hence, zebrafish are a useful model for studying GI dysfunctions due to DM and/or DM medication side-effects.

We have previously shown that in the developing trunk of zebrafish embryos, two-pore channel type 2 (TPC2)-mediated Ca 2+ release from endolysosomes plays a role in the formation of the skeletal slow muscle. In addition, TPC2-mediated Ca 2+ 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. For example, when tpcn2 was knocked down or out, increased numbers of small vacuoles were formed in the inner notochord cells, compared with the single large vacuole in the notochord of control embryos. This abnormal vacuolation was associated with embryos displaying attenuated body axis straightening. We also showed that TPC2 has a distinct pattern of localization in the notochord in embryos at ∼24 hpf. Finally, we conducted RNAseq to identify differentially expressed genes in tpcn2 mutants compared to wild-type controls, and found that those involved in actin filament severing, cellular component morphogenesis, Ca 2+ binding, and structural constituent of cytoskeleton were downregulated in the mutants. Together, our data suggest that TPC2 activity plays a key role in notochord biogenesis in zebrafish embryos.

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.

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 region of membrane surface protrusions was relatively wide at 30% epiboly but narrowed as epiboly progressed. This narrowing was coincident with the formation of the punctate actin band. We also demonstrated using immunogold transmission EM that actin clusters were localized at the membrane surface mainly within the protrusions as well as in deeper locations of the E-YSL. Furthermore, during the latter part of epiboly, the punctate actin band was coincident with a region of highly dynamic endocytosis. Treatment with cytochalasin B led to the disruption of the punctate actin band and the membrane surface protrusions, as well as the attenuation of endocytosis. Together, our data suggest that, in the E-YSL, the region encompassing the membrane surface protrusions and its associated punctate actin band are likely to be an integral part of the localized endocytosis, which is important for the progression of epiboly in zebrafish embryos.

Despite the development of effective vaccines to combat the COVID-19 pandemic, which help prevent the most life-threatening symptoms, full protection cannot be guaranteed, especially with the emergence of new viral variants. Moreover, some resistance to vaccination remains in certain age groups and cultures.

In the trunk of developing zebrafish embryos, adjacent myotome blocks transmit contractile force via myoseptal junctions (MJs), 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), 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 (SMCs) at MJs. Use of antisense morpholinos or CRISPR/Cas9 gene editing to disrupt tpcn1 gene expression resulted in abnormal MJ phenotypes including SMCs detaching from or crossing the myosepta. We also report that TPC1-decorated endolysosomes are dynamically associated with MJs in a microtubule-dependent manner, and that attenuating tpcn1 expression or function disrupted endolysosomal trafficking and resulted in an abnormal distribution of β-dystroglycan (a key transmembrane component of the dystrophin-associated protein complex). Together, our data suggest that localized TPC1-generated Ca2+ signals facilitate essential endolysosomal trafficking and membrane contact events, which help form and maintain MJs following the onset of SMC contractile activity.

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. 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. Results: 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 IC50 values of 25 to 500 mu g/ml. In addition, the polysaccharide-depleted fraction of the former, and epigallocatechin gallate (EGCG) which was found in both extracts, displayed significant antiviral activity. Conclusions: Our 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.

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 Polygonum cuspidatum (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 IC50 values ranging from 0.015 to 0.04 mg/mL. The extracts were shown to inhibit various aspects of the pseudovirus entry, including the interaction between the spike protein (S-protein) and the angiotensin-converting enzyme II (ACE2) receptor, and the 3CL protease activity. Out of the chemical compounds tested in this report, gallic acid, a phytochemical in P. cuspidatum, was shown to have a significant anti-viral effect. Therefore, this might be responsible, at least in part, for the anti-viral efficacy of the herbal extract. Together, our data suggest that the extracts of P. cuspidatum inhibit the entry of wild-type and the omicron variant of SARS-CoV-2, and so they could be considered as potent treatments against COVID-19.

Neurospheres derived from the adult zebrafish brain are a convenient model for investigating the initial stages of neurogenesis. Neurospheres are composed of stem cells, which have the ability to either self-renew or differentiate into specialized cells (i.e., neurons and glial cells) if cultured in an appropriate medium. It has previously been suggested that Ca 2+ signaling in the cytosol and mitochondria might play some role in neurogenesis. Ca 2+ signals can be recorded using the non-toxic bioluminescent Ca 2+ reporter complex, holoaequorin. We have developed a novel electroporation protocol to load neurospheres with cDNA encoding either mitochondrial-targeted EGFP-apoaequorin (MitGA) or the cytosolic-targeted EGFP-apoaequorin (CytGA). We show that within ~24 h after electroporation, each of these apoaequorin proteins were expressed in ~20% of the neurosphere cells. In addition, after reconstitution of holoaequorin by incubation of the cells with its luminophore, coelenterazine, dynamic changes in [Ca 2+ ] in the cytosol and mitochondria were imaged using a custom-built electron multiplying charge coupled device (EMCCD)-based photon imaging microscope (PIM) system or measured using a photomultiplier tube (PMT)-based luminescence detection system, after activation of store-operated Ca 2+ entry (SOCE).

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.

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.

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.

Abstract 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.

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.

Dear Editor, Human pluripotent stem cell (hPSC)-derived cardiomyocytes (CMs) are of significant translational value to in vitro studies of human cardiac development, drug and cardiotoxicity testing and cardiac disease modelling.Differentiation of hPSCs to CMs,however, yields mixed cultures of atrial-, ventricular-, and pacemaker-like cells as well as non-CMs in variable proportions.1

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.

Since the identification of nicotinic acid adenine dinucleotide phosphate (NAADP) and its putative target, the two-pore channel (TPC), the NAADP/TPC/Ca2+ 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. Here, we review the role of NAADP/TPC/Ca2+ signaling during early embryonic development and cellular differentiation. We pay particular attention to the role of TPC2 in the development and maturation of early neuromuscular activity in zebrafish, and during the differentiation of isolated osteoclasts, endothelial cells, and keratinocytes. Our aim is to emphasize the conserved features of TPC-mediated Ca2+ signaling in a number of selected examples.

In this chapter, the authors discuss the techniques used when investigating the role of Two-Pore Channel 2 (TPC2) in zebrafish neuromuscular development. The role of TPC2 in the differentiation of slow muscle cells and PMNs in intact zebrafish embryos has been investigated using various approaches. The remaining injected embryos were raised to adulthood, and the founder fish were identified using the restriction endonuclease digestion assay, by excising the tip of the caudal fin as a source of DNA. The founders were then outcrossed with wild-type adults to test for the germline transmission of the tpcn2 mutation. Zebrafish embryos are reported to demonstrate sequential motile behaviours such as spontaneous coiling, and touch-evoked behaviour, during development. The effect of TPC2 knock down on the spontaneous coiling behaviour was determined by video microscopy, whereas the touch-evoked response was determined by gently touching the embryos on the head twice and recording their response.

天然河川における Flavobacterium psychrophilum の分布と 分離株の gyrA 遺伝子型 永田恵里奈,進藤有章,山本充孝, 岡村貴司,竹上健太郎,江口 充 冷水病菌は,gyrA 遺伝子多型から,G-C 型(アユ型),A-C 型(多魚種型),A-T 型(サケマス型)に分けることができ る。琵琶湖水系の河川の下流域において,20102013 年に採 取した様々な試料から冷水病菌を分離し gyrA 遺伝子型を調べ た。アユが遡上する 6 月には全ての型の本菌が分離された。 アユの産卵期である 9 月になると分離株のほとんどを G-C 型 が占め,A-T 型は分離されなかった。12 月には A-T 型のみが 分離された。この時ビワマスが産卵のため遡上してきていた。 河川に存在する宿主魚種の季節的変化に従って,異なる遺伝子 型の本菌が分離されたと考える。 85(6), 913923 (2019)