Salidroside alleviates dexamethasone-induced inhibition of bone formation via transforming growth factor-beta/Smad2/3 signaling pathway.

Glucocorticoid-induced osteoporosis is the third epidemic osteoporosis following postmenopausal and senileosteoporosis. According to one study, salidroside made ovariectomized rats' bones strong. Salidroside's potential for treating glucocorticoid-induced osteoporosis remains unproven. This study aimed to investigate the protective effect and mechanism of salidroside on dexamethasone-induced osteogenic differentiation and bone formation in MC3T3-E1 cells and zebrafish. The study proved that salindroside had no harmful impact on MC3T3E1 cells. Salidroside significantly relieved dexamethasone-induced inhibition of ALP (alkaline phosphatase) activity and mineralization in MC3T3-E1 cells, and promoted osteogenic differentiation of cells. Salidroside increased the expression of osteopontin (OPN), runt-related transcription factor 2 (Runx2), osterix (Osx), transforming growth factor-beta (TGF-ß) proteins and promoted the phosphorylation of Smad2/3 in MC3T3-E1 cells treated with dexamethasone. In addition, the effect of salidroside in relieving dexamethasone-induced inhibition of osteogenic differentiation in MC3T3-E1 cells can be blocked by TGF-ß receptor type I/II inhibitor (LY2109761). At the same time, we found that salidroside significantly alleviated the inhibition of dexamethasone-induced bone formation in zebrafish and promoted the mineralization of zebrafish skulls. LY2109761 reversed the protective impact of salidroside on dexamethasone-mediated bone impairment in zebrafish. These findings suggested that salidroside alleviated dexamethasone-induced inhibition of osteogenic differentiation and bone formation via TGF-ß/Smad2/3 signaling pathway.

Transplantation of high fat fed mouse microbiota into zebrafish larvae identifies MyD88-dependent acceleration of hyperlipidaemia by Gram-positive cell wall components.

Gut dysbiosis is an important modifier of pathologies including cardiovascular disease but our understanding of the role of individual microbes is limited. Here, we have used transplantation of mouse microbiota into microbiota-deficient zebrafish larvae to study the interaction between members of a mammalian high fat diet-associated gut microbiota with a lipid rich diet challenge in a tractable model species. We find zebrafish larvae are more susceptible to hyperlipidaemia when exposed to the mouse high fat-diet-associated microbiota and that this effect can be driven by two individual bacterial species fractionated from the mouse high fat-diet-associated microbiota. We find Stenotrophomonas maltophilia increases the hyperlipidaemic potential of chicken egg yolk to zebrafish larvae independent of direct interaction between S. maltophilia and the zebrafish host. Colonization by live, or exposure to heat-killed, Enterococcus faecalis accelerates hyperlipidaemia via host MyD88 signaling. The hyperlipidaemic effect is replicated by exposure to the Gram-positive toll-like receptor agonists peptidoglycan and lipoteichoic acid in a MyD88-dependent manner. In this work, we demonstrate the applicability of zebrafish as a tractable host for the identification of gut microbes that can induce conditional host phenotypes via microbiota transplantation and subsequent challenge with a high fat diet.

Theoretical Exploring of a Molecular Mechanism for Melanin Inhibitory Activity of Calycosin in Zebrafish.

Tyrosinase is an oxidase that is the rate-limiting enzyme for controlling the production of melanin in the human body. Overproduction of melanin can lead to a variety of skin disorders. Calycosin is an isoflavone from Astragali Radix, which is a traditional Chinese medicine that exhibits several pharmacological activities including skin whitening. In our study, the inhibitory effect of calycosin on melanin production is confirmed in a zebrafish in vivo model by comparing with hydroquinone, kojic acid, and arbutin, known as tyrosinase inhibitors. Moreover, the inhibitory kinetics of calycosin on tyrosinase and their binding mechanisms are determined using molecular docking techniques, molecular dynamic simulations, and free energy analysis. The results indicate that calycosin has an obvious inhibitory effect on zebrafish pigmentation at the concentration of 7.5 µM, 15 µM, and 30 µM. The IC50 of calycosin is 30.35 µM, which is lower than hydroquinone (37.35 µM), kojic acid (6.51 × 103 µM), and arbutin (3.67 × 104 µM). Furthermore, all the results of molecular docking, molecular dynamics simulations, and free energy analysis suggest that calycosin can directly bind to the active site of tyrosinase with very good binding affinity. The study indicates that the combination of computer molecular modeling and zebrafish in vivo assay would be feasible in confirming the result of the in vitro test and illustrating the target-binding information.

Role of BMP signaling during early development of the annelid Capitella teleta.

The mechanisms regulating nervous system development are still unknown for a wide variety of taxa. In insects and vertebrates, bone morphogenetic protein (BMP) signaling plays a key role in establishing the dorsal-ventral (D-V) axis and limiting the neuroectoderm to one side of that axis, leading to speculation about the conserved evolution of centralized nervous systems. Studies outside of insects and vertebrates show a more diverse picture of what, if any role, BMP signaling plays in neural development across Bilateria. This is especially true in the morphologically diverse Spiralia (≈Lophotrochozoa). Despite several studies of D-V axis formation and neural induction in spiralians, there is no consensus for how these two processes are related, or whether BMP signaling may have played an ancestral role in either process. To determine the function of BMP signaling during early development of the spiralian annelid Capitella teleta, we incubated embryos and larvae in BMP4 protein for different amounts of time. Adding exogenous BMP protein to early-cleaving C. teleta embryos had a striking effect on formation of the brain, eyes, foregut, and ventral midline in a time-dependent manner. However, adding BMP did not block brain or VNC formation or majorly disrupt the D-V axis. We identified three key time windows of BMP activity. 1) BMP treatment around birth of the 3rd-quartet micromeres caused the loss of the eyes, radialization of the brain, and a reduction of the foregut, which we interpret as a loss of A- and C-quadrant identities with a possible trans-fate switch to a D-quadrant identity. 2) Treatment after the birth of micromere 4d induced formation of a third ectopic brain lobe, eye, and foregut lobe, which we interpret as a trans-fate switch of B-quadrant micromeres to a C-quadrant identity. 3) Continuous BMP treatment from late cleavage (4d â€‹+ â€‹12 â€‹h) through mid-larval stages resulted in a modest expansion of Ct-chrdl expression in the dorsal ectoderm and a concomitant loss of the ventral midline (neurotroch ciliary band). Loss of the ventral midline was accompanied by a collapse of the bilaterally-symmetric ventral nerve cord, although the total amount of neural tissue was not greatly affected. Our results compared with those from other annelids and molluscs suggest that BMP signaling was not ancestrally involved in delimiting neural tissue to one region of the D-V axis. However, the effects of ectopic BMP on quadrant-identity during cleavage stages may represent a non-axial organizing signal that was present in the last common ancestor of annelids and mollusks. Furthermore, in the last common ancestor of annelids, BMP signaling may have functioned in patterning ectodermal fates along the D-V axis in the trunk. Ultimately, studies on a wider range of spiralian taxa are needed to determine the role of BMP signaling during neural induction and neural patterning in the last common ancestor of this group. Ultimately, these comparisons will give us insight into the evolutionary origins of centralized nervous systems and body plans.

Habenula kisspeptin retrieves morphine impaired fear memory in zebrafish.

The habenula is an evolutionarily conserved brain structure, which has recently been implicated in fear memory. In the zebrafish, kisspeptin (Kiss1) is predominantly expressed in the habenula, which has been implicated as a modulator of fear response. Hence, in the present study, we questioned whether Kiss1 has a role in fear memory and morphine-induced fear memory impairment using an odorant cue (alarm substances, AS)-induced fear avoidance paradigm in adult zebrafish, whereby the fear-conditioned memory can be assessed by a change of basal place preference (= avoidance) of fish due to AS-induced fear experience. Subsequently, to examine the possible role of Kiss1 neurons-serotonergic pathway, kiss1 mRNA and serotonin levels were measured. AS exposure triggered fear episodes and fear-conditioned place avoidance. Morphine treatment followed by AS exposure, significantly impaired fear memory with increased time-spent in AS-paired compartment. However, fish administered with Kiss1 (10-21 mol/fish) after morphine treatment had significantly lower kiss1 mRNA levels but retained fear memory. In addition, the total brain serotonin levels were significantly increased in AS- and Kiss1-treated groups as compared to control and morphine treated group. These results suggest that habenular Kiss1 might be involved in consolidation or retrieval of fear memory through the serotonin system.

Folding of Truncated Granulin Peptides.

Granulins are a family of unique protein growth factors which are found in a range of species and have several bioactivities that include cell proliferation and wound healing. They typically contain six disulfide bonds, but the sequences, structures and bioactivities vary significantly. We have previously shown that an N-terminally truncated version of a granulin from the human liver fluke, Opisthorchis viverrini, can fold independently into a "mini-granulin" structure and has potent wound healing properties in vivo. The incorporation of a non-native third disulfide bond, with respect to the full-length granulin module, was critical for the formation of regular secondary structure in the liver fluke derived peptide. By contrast, this third disulfide bond is not required for a carp granulin-1 truncated peptide to fold independently. This distinction led us to explore granulins from the zebrafish model organism. Here we show that the mini-granulin fold occurs in a naturally occurring paragranulin (half-domain) from zebrafish, and is also present in a truncated form of a full-length zebrafish granulin, suggesting this structure might be a common property in either naturally occurring or engineered N-terminally truncated granulins and the carp granulin-1 folding is an anomaly. The in vitro folding yield is significantly higher in the naturally occurring paragranulin, but only the truncated zebrafish granulin peptide promoted the proliferation of fibroblasts consistent with a growth factor function, and therefore the function of the paragranulin remains unknown. These findings provide insight into the folding and evolution of granulin domains and might be useful in the elucidation of the structural features important for bioactivity to aid the design of more potent and stable analogues for the development of novel wound healing agents.

The Use of Stem Cell Differentiation Stage Factors (SCDSFs) Taken from Zebrafish Embryos during Organogenesis and Their Role in Regulating the Gene Expression of Normal and Pathological (Stem) Cells.

Studies conducted on Zebrafish embryos in our laboratory have allowed for the identification of precise moments of organogenesis in which a lot of genes are switched on and off, a sign that the genome is undergoing substantial changes in gene expression. Stem cell growth and differentiation stage-factors present in different moments of organogenesis have proven to have different specific functions in gene regulation. The substances present in the first stages of cell differentiation in Zebrafish embryos have demonstrated an ability to counteract the senescence of stem cells, reducing the expression of the beta-galactosidase marker, enhancing the genes Oct-4, Sox-2, c-Myc, TERT, and the transcription of Bmi-1, which act as key telomerase-independent repressors of cell aging. The molecules present in the intermediate to late stages of cell differentiation have proven to be able to reprogram pathological human cells, such as cancer cells and those of the basal layer of the epidermis in psoriasis, which present a higher multiplication rate than normal cells. The factors present in all the stages of cell differentiation are able to counteract neurodegeneration, and to regenerate tissues: It has been possible to regenerate hair follicles in many patients with androgenetic alopecia through transdermal administration of stem cell differentiation stage factors (SCDSFs) by means of cryopass-laser.

Nesfatin-1-like peptide suppresses hypothalamo-pituitary-gonadal mRNAs, gonadal steroidogenesis, and oocyte maturation in fish†.

Nucleobindin (Nucb)-1 and Nucb2 are DNA and Ca2+ binding proteins with multiple functions in vertebrates. Prohormone convertase-mediated processing of Nucb2 results in the production of biologically active nesfatin-1. Nesfatin-1 is involved in the regulation of reproduction in many vertebrates, including fish. Our lab originally reported a nesfatin-1-like peptide (Nlp) encoded in Nucb1 that exhibits nesfatin-1-like metabolic effects. We hypothesized that Nlp has a suppressive role in the reproductive physiology of fish. In this research, whether Nlp regulates reproductive hormones and oocyte maturation in fish were determined. Single intraperitoneal (IP) injection of goldfish Nlp (50 ng/g body weight) suppressed salmon and chicken gonadotropin-releasing hormone (sgnrh and cgnrh2), gonadotropin-inhibiting hormone (gnih) and its receptor (gnihr), and kisspeptin and brain aromatase mRNA expression in the hypothalamus of both male and female goldfish. In the pituitary, Nlp decreased mRNAs encoding lhb, fshb and kisspeptin and its receptor, while a significant increase in gnih and gnihr was observed. In the gonads, lh (only in male fish) and fsh receptor mRNAs were also significantly downregulated in Nlp-injected fish. Sex-specific modulation of gnih, gnihr, and kisspeptin system in the gonads was also observed. Nlp decreased sex steroidogenic enzyme encoding mRNAs and circulating levels of testosterone and estradiol. In addition, incubation of zebrafish ovarian follicles with Nlp resulted in a reduction in oocyte maturation. These results provide evidence for a robust role for Nlp in regulating reproductive hormones in goldfish and oocyte maturation in zebrafish, and these effects resemble that of nesfatin-1.

Phoenixin-20 suppresses food intake, modulates glucoregulatory enzymes, and enhances glycolysis in zebrafish.

Phoenixin is a 20-amino acid peptide (PNX-20) cleaved from the small integral membrane protein 20 (SMIM20), with multiple biological roles in mammals. However, its role in nonmammalian vertebrates is poorly understood. This research aimed to determine whether PNX-20 influences feeding and metabolism in zebrafish. The mRNAs encoding SMIM20 and its putative receptor, super conserved receptor expressed in brain 3 (SREB3), are present in both central and peripheral tissues of zebrafish. Immunohistochemical analysis confirmed the presence of PNX-like immunoreactivity in the gut and in zebrafish liver (ZFL) cell line. We also found that short-term fasting (7 days) significantly decreased smim20 mRNA expression in the brain, gut, liver, gonads, and muscle, which suggests a role for PNX-20 in food intake regulation. Indeed, single intraperitoneal injection of 1,000 ng/g body wt PNX-20 reduced feeding in both male and female zebrafish, likely in part by enhancing hypothalamic cart and reducing hypothalamic/gut preproghrelin mRNAs. Furthermore, the present results demonstrated that PNX-20 modulates the expression of genes involved in glucose transport and metabolism in ZFL cells. In general terms, such PNX-induced modulation of gene expression was characterized by the upregulation of glycolytic genes and the downregulation of gluconeogenic genes. A kinetic study of the ATP production rate from both glycolytic and mitochondrial pathways demonstrated that PNX-20-treated ZFL cells exhibited significantly higher ATP production rate associated with glycolysis than control cells. This confirms a positive role for PNX-20 on glycolysis. Together, these results indicate that PNX-20 is an anorexigen with important metabolic roles in zebrafish.

Zebrafish C-reactive protein isoforms inhibit SVCV replication by blocking autophagy through interactions with cell membrane cholesterol.

In the present work, the mechanisms involved in the recently reported antiviral activity of zebrafish C-reactive protein-like protein (CRP1-7) against the spring viraemia of carp rhabdovirus (SVCV) in fish are explored. The results neither indicate blocking of the attachment or the binding step of the viral replication cycle nor suggest the direct inhibition of G protein fusion activity or the stimulation of the host's interferon system. However, an antiviral state in the host is induced. Further results showed that the antiviral protection conferred by CRP1-7 was mainly due to the inhibition of autophagic processes. Thus, given the high affinity of CRPs for cholesterol and the recently described influence of the cholesterol balance in lipid rafts on autophagy, both methyl-ß-cyclodextrin (a cholesterol-complexing agent) and 25-hydroxycholesterol (a cholesterol molecule with antiviral properties) were used to further describe CRP activity. All the tested compounds exerted antiviral activity by affecting autophagy in a similar manner. Further assays indicate that CRP reduces autophagy activity by initially disturbing the cholesterol ratios in the host cellular membranes, which in turn negatively affects the intracellular regulation of reactive oxygen species (ROS) and increases lysosomal pH as a consequence. Ultimately, here we propose that such pH changes exert an inhibitory direct effect on SVCV replication by disrupting the pH-dependent membrane-fusogenic ability of the viral glycoprotein G, which allows the release of the virus from endosomes into cytoplasm during its entry phase.

Yap1 promotes sprouting and proliferation of lymphatic progenitors downstream of Vegfc in the zebrafish trunk.

Lymphatic vascular development involves specification of lymphatic endothelial progenitors that subsequently undergo sprouting, proliferation and tissue growth to form a complex second vasculature. The Hippo pathway and effectors Yap and Taz control organ growth and regulate morphogenesis and cellular proliferation. Yap and Taz control angiogenesis but a role in lymphangiogenesis remains to be fully elucidated. Here we show that YAP displays dynamic changes in lymphatic progenitors and Yap1 is essential for lymphatic vascular development in zebrafish. Maternal and Zygotic (MZ) yap1 mutants show normal specification of lymphatic progenitors, abnormal cellular sprouting and reduced numbers of lymphatic progenitors emerging from the cardinal vein during lymphangiogenesis. Furthermore, Yap1 is indispensable for Vegfc-induced proliferation in a transgenic model of Vegfc overexpression. Paracrine Vegfc-signalling ultimately increases nuclear YAP in lymphatic progenitors to control lymphatic development. We thus identify a role for Yap in lymphangiogenesis, acting downstream of Vegfc to promote expansion of this vascular lineage.

Granulin 1 Promotes Retinal Regeneration in Zebrafish.

Purpose: Retinal degenerative diseases can progress to severe reductions of vision. In general, the changes are permanent in higher vertebrates, including humans; however, retinal regeneration can occur in lower vertebrates, such as amphibians and teleost fish. Progranulin is a secreted growth factor that is involved in normal development and wound-healing processes. We have shown that progranulin promotes the proliferation of retinal precursor cells in mouse retinas. The purpose of this study was to investigate the role played by granulin 1 (grn1) in the retinal regeneration in zebrafish. Methods: We injured the retina of zebrafish with needle puncturing, and the retinas were examined at different times after the injury. We also checked the proliferation and the expression of retinal regeneration-related genes after knockdown of grn1 by electroporation with morpholino oligonucleotides (MO) and intravitreal injection of recombinant grn1. Results: Our results showed that the level of grn1 was highly increased after retinal injury, and it was expressed in various types of retinal cells. A knockdown of grn1 reduced the proliferation of Müller glial cells in zebrafish eyes undergoing retinal regeneration. The knockdown of grn1 also reduced the expression of achaete-scute homolog 1a (ascl1a), an important factor in retinal regeneration. An intravitreal injection of recombinant grn1 led to a proliferation of Müller glial cells and an increase in the expression of retinal regeneration-related genes, such as ascl1a and lin28. Conclusions: These findings suggested that grn1 should be considered as a target for stimulating the dedifferentiation of Müller glial cells and retinal regeneration.

Igf3 activates ß-catenin signaling to stimulate spermatogonial differentiation in zebrafish.

Follicle-stimulating hormone (Fsh) is a major regulator of spermatogenesis, targeting somatic cell functions in the testes. We reported previously that zebrafish Fsh promoted the differentiation of type A undifferentiated spermatogonia (Aund) by stimulating the production of factors that advance germ cell differentiation, such as androgens, insulin-like peptide 3 (Insl3) and insulin-like growth factor 3 (Igf3). In addition, Fsh also modulated the transcript levels of several other genes, including some belonging to the Wnt signaling pathway. Here, we evaluated if and how Fsh utilizes part of the canonical Wnt pathway to regulate the development of spermatogonia. We quantified the proliferation activity and relative section areas occupied by Aund and type A differentiating (Adiff) spermatogonia and we analyzed the expression of selected genes in response to recombinant proteins and pharmacological inhibitors. We found that from the three downstream mediators of Fsh activity we examined, Igf3, but not 11-ketotestosterone or Insl3, modulated the transcript levels of two ß-catenin sensitive genes (cyclinD1 and axin2). Using a zebrafish ß-catenin signaling reporter line, we showed that Igf3 activated ß-catenin signaling in type A spermatogonia and that this activation did not depend on the release of Wnt ligands. Pharmacological inhibition of the ß-catenin or of the phosphoinositide 3-kinase (PI3K) pathways revealed that Igf3 activated ß-catenin signaling in a manner involving PI3K to promote the differentiation of Aund to Adiff spermatogonia. This mechanism represents an intriguing example for a pituitary hormone like Fsh using Igf signaling to recruit the evolutionary conserved, local ß-catenin signaling pathway to regulate spermatogenesis.

Secretogranin-II plays a critical role in zebrafish neurovascular modeling.

Secretoneurin (SN) is a neuropeptide derived from specific proteolytic processing of the precursor secretogranin II (SgII). In zebrafish and other teleosts, there are two paralogs named sgIIa and sgIIb. Our results showed that neurons expressing sgIIb were aligned with central arteries in the hindbrain, demonstrating a close neurovascular association. Both sgIIb-/- and sgIIa-/-/sgIIb-/- mutant embryos were defective in hindbrain central artery development due to impairment of migration and proliferation of central artery cells. Further study revealed that sgIIb is non-cell autonomous and required for central artery development. Hindbrain arterial and venous network identities were not affected in sgIIb-/- mutant embryos, and the mRNA levels of Notch and VEGF pathway-related genes were not altered. However, the activation of MAPK and PI3K/AKT pathways was inhibited in sgIIb-/- mutant embryos. Reactivation of MAPK or PI3K/AKT in endothelial cells could partially rescue the central artery developmental defects in the sgIIb mutants. This study provides the first in vivo evidence that sgIIb plays a critical role in neurovascular modeling of the hindbrain. Targeting the SgII system may, therefore, represent a new avenue for the treatment of vascular defects in the central nervous system.

Augmentation of the antibacterial activities of Pt5-derived antimicrobial peptides (AMPs) by amino acid substitutions: Design of novel AMPs against MDR bacteria.

The ever-growing concerns on multi-drug resistant (MDR) bacteria lead to urgent demands for novel antibiotics including antimicrobial peptides (AMPs). Pt5, a peptide consisting of the C-terminal 55 residues of zebrafish phosvitin, has been shown to function as an antibacterial agent. Here we used Pt5 as a template to design new AMPs by shortening the sequence and substituting with tryptophan (W) and lysine (K) at selected positions. Among the resultant Pt5-derived peptides, Pt5-1c showed the strongest antimicrobial activity against both Gram-negative and Gram-positive bacteria, including MDR bacteia, with the minimum inhibitory concentrations (MICs) ranging from 1.2 µM to 4.8 µM. Electron microscopic examination showed that Pt5-1c was able to kill the bacteria directly. ELISA revealed that Pt5-1c possessed high affinity to lipopolysaccharide (LPS), lipoteichoic acid (LTA) and peptidoglycan (PGN). Importantly, Pt5-1c was able to disrupt the bacterial membrane by a combined action of membrane depolarization and permeabilization, with little cytotoxicity to mammalian cells. Taken together, these findings suggest that Pt5-1c has considerable potential for future development as novel peptide antibiotics against MDR bacteria.

Zebrafish phosvitin-derived peptide Pt5 inhibits melanogenesis via cAMP pathway.

Zebrafish phosvitin-derived peptide Pt5, consisting of the C-terminal 55 residues of phosvitin, has been shown to have an antimicrobial-immunomodulatory activity comparable to phosvitin. Here, we showed clearly that Pt5 had the capacity to inhibit tyrosinase (TYR) activity and melanin biosynthesis, and this inhibition was independent of cell proliferation and cytotoxic effects. Incubation of fluorescein isothiocyanate (FITC)-labeled Pt5 with B16F10 melanoma cells revealed that Pt5 was localized in the cytoplasm of the cells. In addition, Pt5 inhibited the expression of TYR, tyrosinase-related protein-1 (TRP-1), tyrosinase-related protein-2 (TRP-2), and microphthalmia-associated transcription factor (MITF) in B16F10 melanoma cells and reduced the intracellular cyclic adenosine monophosphate (cAMP) concentration in the cells, but it did not affect the cellular contents of pERK1/2 and ß-catenin, suggesting that Pt5 regulates melanin biosynthesis via cAMP signaling pathway rather than Wnt and MAPK pathways. Collectively, these data indicate that Pt5 has the potential to be used as a melanogenesis inhibitor in medical and cosmetic industry, a novel role ever reported.

Osteocrin, a peptide secreted from the heart and other tissues, contributes to cranial osteogenesis and chondrogenesis in zebrafish.

The heart is an endocrine organ, as cardiomyocytes (CMs) secrete natriuretic peptide (NP) hormones. Since the discovery of NPs, no other peptide hormones that affect remote organs have been identified from the heart. We identified osteocrin (Ostn) as an osteogenesis/chondrogenesis regulatory hormone secreted from CMs in zebrafish. ostn mutant larvae exhibit impaired membranous and chondral bone formation. The impaired bones were recovered by CM-specific overexpression of OSTN. We analyzed the parasphenoid (ps) as a representative of membranous bones. In the shortened ps of ostn morphants, nuclear Yap1/Wwtr1-dependent transcription was increased, suggesting that Ostn might induce the nuclear export of Yap1/Wwtr1 in osteoblasts. Although OSTN is proposed to bind to NPR3 (clearance receptor for NPs) to enhance the binding of NPs to NPR1 or NPR2, OSTN enhanced C-type NP (CNP)-dependent nuclear export of YAP1/WWTR1 of cultured mouse osteoblasts stimulated with saturable CNP. OSTN might therefore activate unidentified receptors that augment protein kinase G signaling mediated by a CNP-NPR2 signaling axis. These data demonstrate that Ostn secreted from the heart contributes to bone formation as an endocrine hormone.

Identification of Ly2 members as antimicrobial peptides from zebrafish Danio rerio.

The emergence of multidrug-resistant (MDR) microbes caused by overuse of antibiotics leads to urgent demands for novel antibiotics exploration. Our recent data showed that Ly2.1-3 (a novel lymphocyte antigen 6 (Ly6) gene cluster) were proteins with cationic nature and rich in cysteine content, that are characteristic of antimicrobial peptides (AMPs) and their expression were all significantly up-regulated after challenge with lipopolysaccharide (LPS). These strongly suggested that Ly2.1-3 are potential AMPs, but firm evidence are lacking. Here, we clearly showed that the recombinant proteins of Ly2.1-3 were capable of killing Gram-negative bacteria Aeromonas hydrophila and Escherichia coli, while they had little bactericidal activity against the Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis We also showed that recombinant proteins Ly2.1-3 (rLy2.1-3) were able to bind to the Gram-negative bacteria A. hydrophila, E. coli and the microbial signature molecule LPS, but not to the Gram-positive bacteria S. aureus and B. subtilis as well as the microbial signature molecule LTA. Moreover, the Scatchard analysis revealed that rLy2.1-3 could specifically bind to LPS. Finally, we found that Ly2.1-3 were not cytotoxic to mammalian cells. All these together indicate that Ly2.1-3 can function as AMPs.

Antibacterial activity and modes of action of phosvitin-derived peptide Pt5e against clinical multi-drug resistance bacteria.

Pt5e, a mutant peptide derived from the C-terminal 55 residues of zebrafish phosvitin, has been suggested to be a novel antibacterial peptide. However, if it is applicable to clinical MDR bacteria remains to be tested. In this study, high-purity Pt5e was first expressed and purified by fusion with cationic elastin-like polypeptide. Pt5e was then shown to be capable of effectively killing all the five clinical MDR bacteria tested. Pt5e kill the MDR bacteria at several levels, including inserting into the bacterial membranes, causing the membrane depolarization and permeabilization, and inducing the intracellular apoptosis/necrosis. All these data suggest that Pt5e is a promising therapeutic potential as an antibiotics against clinical MDR bacteria.

Releasing prophase arrest in zebrafish oocyte: synergism between maturational steroid and Igf1.

Binding of 17ß-estradiol (E2) to novel G-protein coupled receptor, Gper1, promotes intra-oocyte adenylyl cyclase activity and transactivates epidermal growth factor receptor to ensure prophase-I arrest. Although involvement of either membrane progestin receptor (mPR) or Igf system has been implicated in regulation of meiosis resumption, possibility of concurrent activation and potential synergism between 17α,20ß-dihydroxy-4-pregnen-3-one (DHP)- and Igf-mediated signalling cascades in alleviating E2 inhibition of oocyte maturation (OM) has not been investigated. Here using zebrafish (Danio rerio) defolliculated oocytes, we examined the effect of DHP and Igf1, either alone or in combination, in presence or absence of E2, on OM in vitro. While priming of denuded oocytes with E2 blocked spontaneous maturation, co-treatment with DHP (3 nM) and Igf1 (10 nM), but not alone, reversed E2 inhibition and promoted a robust increase in germinal vesicle breakdown (GVBD). Although stimulation with either Igf1 or DHP promoted Akt phosphorylation, pharmacological inhibition of PI3K/Akt signalling prevented Igf1-induced GVBD but delayed DHP action till 4-5 h of incubation. Moreover, high intra-oocyte cAMP attenuates both DHP and Igf1-mediated OM and co-stimulation with DHP and Igf1 could effectively reverse E2 action on PKA phosphorylation. Interestingly, data from in vivo studies reveal that heightened expression of igf1, igf3 transcripts in intact follicles corresponded well with elevated phosphorylation of Igf1r and Akt, mPRa immunoreactivity, PKA inhibition and accelerated GVBD response just prior to ovulation. This indicates potential synergism between maturational steroid and Igf1 which might have physiological relevance in overcoming E2 inhibition of meiosis resumption in zebrafish oocytes.