The Upstream 1350~1250 Nucleotide Sequences of the Human ENDOU-1 Gene Contain Critical Cis-Elements Responsible for Upregulating Its Transcription during ER Stress.

ENDOU-1 encodes an endoribonuclease that overcomes the inhibitory upstream open reading frame (uORF)-trap at 5'-untranslated region (UTR) of the CHOP transcript, allowing the downstream coding sequence of CHOP be translated during endoplasmic reticulum (ER) stress. However, transcriptional control of ENDOU-1 remains enigmatic. To address this, we cloned an upstream 2.1 kb (-2055~+77 bp) of human ENDOU-1 (pE2.1p) fused with reporter luciferase (luc) cDNA. The promoter strength driven by pE2.1p was significantly upregulated in both pE2.1p-transfected cells and pE2.1p-injected zebrafish embryos treated with stress inducers. Comparing the luc activities driven by pE2.1p and -1125~+77 (pE1.2p) segments, we revealed that cis-elements located at the -2055~-1125 segment might play a critical role in ENDOU-1 upregulation during ER stress. Since bioinformatics analysis predicted many cis-elements clustered at the -1850~-1250, we further deconstructed this segment to generate pE2.1p-based derivatives lacking -1850~-1750, -1749~-1650, -1649~-1486, -1485~-1350 or -1350~-1250 segments. Quantification of promoter activities driven by these five internal deletion plasmids suggested a repressor binding element within the -1649~-1486 and an activator binding element within the -1350~-1250. Since luc activities driven by the -1649~-1486 were not significantly different between normal and stress conditions, we herein propose that the stress-inducible activator bound at the -1350~-1250 segment makes a major contribution to the increased expression of human ENDOU-1 upon ER stresses.

The Promising Role of a Zebrafish Model Employed in Neural Regeneration Following a Spinal Cord Injury.

Spinal cord injury (SCI) is a devastating event that results in a wide range of physical impairments and disabilities. Despite the advances in our understanding of the biological response to injured tissue, no effective treatments are available for SCIs at present. Some studies have addressed this issue by exploring the potential of cell transplantation therapy. However, because of the abnormal microenvironment in injured tissue, the survival rate of transplanted cells is often low, thus limiting the efficacy of such treatments. Many studies have attempted to overcome these obstacles using a variety of cell types and animal models. Recent studies have shown the utility of zebrafish as a model of neural regeneration following SCIs, including the proliferation and migration of various cell types and the involvement of various progenitor cells. In this review, we discuss some of the current challenges in SCI research, including the accurate identification of cell types involved in neural regeneration, the adverse microenvironment created by SCIs, attenuated immune responses that inhibit nerve regeneration, and glial scar formation that prevents axonal regeneration. More in-depth studies are needed to fully understand the neural regeneration mechanisms, proteins, and signaling pathways involved in the complex interactions between the SCI microenvironment and transplanted cells in non-mammals, particularly in the zebrafish model, which could, in turn, lead to new therapeutic approaches to treat SCIs in humans and other mammals.

Extracellular Pgk1 interacts neural membrane protein enolase-2 to improve the neurite outgrowth of motor neurons.

Understanding the molecular interaction between ligand and receptor is important for providing the basis for the development of regenerative drugs. Although it has been reported that extracellular phosphoglycerate kinase 1 (Pgk1) can promote the neurite outgrowth of motoneurons, the Pgk1-interacting neural receptor remains unknown. Here we show that neural membranous Enolase-2 exhibits strong affinity with recombinant Pgk1-Flag, which is also evidently demonstrated by immunoelectron microscopy. The 325th-417th domain of Pgk1 interacts with the 405th-431st domain of Enolase-2, but neither Enolase-1 nor Enolase-3, promoting neurite outgrowth. Combining Pgk1 incubation and Enolase-2 overexpression, we demonstrate a highly significant enhancement of neurite outgrowth of motoneurons through a reduced p-P38-T180/p-Limk1-S323/p-Cofilin signaling. Collectively, extracellular Pgk1 interacts neural membrane receptor Enolase-2 to reduce the P38/Limk1/Cofilin signaling which results in promoting neurite outgrowth. The extracellular Pgk1-specific neural receptor found in this study should provide a material for screening potential small molecule drugs that promote motor nerve regeneration.

Genomic Structure, Protein Character, Phylogenic Implication, and Embryonic Expression Pattern of a Zebrafish New Member of Zinc Finger BED-Type Gene Family.

We reported a new member of the C2H2-zinc-finger BED-type (ZBED) protein family found in zebrafish (Danio rerio). It was previously assigned as an uncharacterized protein LOC569044 encoded by the Zgc:161969 gene, the transcripts of which were highly expressed in the CNS after the spinal cord injury of zebrafish. As such, this novel gene deserves a more detailed investigation. The 2.79-kb Zgc:161969 gene contains one intron located on Chromosome 6 at 16,468,776-16,475,879 in the zebrafish genome encoding a 630-aa protein LOC569044. This protein is composed of a DNA-binding BED domain, which is highly conserved among the ZBED protein family, and a catalytic domain consisting of an α-helix structure and an hAT dimerization region. Phylogenetic analysis revealed the LOC569044 protein to be clustered into the monophyletic clade of the ZBED protein family of golden fish. Specifically, the LOC569044 protein was classified as closely related to the monophyletic clades of zebrafish ZBED4-like isoforms and ZBED isoform 2. Furthermore, Zgc:161969 transcripts represented maternal inheritance, expressed in the brain and eyes at early developmental stages and in the telencephalon ventricular zone at late developmental stages. After characterizing the LOC569044 protein encoded by the Zgc:161969 gene, it was identified as a new member of the zebrafish ZBED protein family, named the ZBEDX protein.

Anp32a Promotes Neuronal Regeneration after Spinal Cord Injury of Zebrafish Embryos.

After spinal cord injury (SCI) in mammals, neuronal regeneration is limited; in contrast, such regeneration occurs quickly in zebrafish. Member A of the acidic nuclear phosphoprotein 32 (ANP32a) family is involved in neuronal development, but its function is controversial, and its involvement in zebrafish SCI remains unknown. To determine the role of zebrafish ANP32a in the neuronal regeneration of SCI embryos, we microinjected ANP32a mRNA into embryos from zebrafish transgenic line Tg(mnx1:GFP) prior to SCI. Compared to control SCI embryos, the results showed that the regeneration of spinal cord and resumption of swimming capability were promoted by the overexpression of ANP32a mRNA but reduced by its knockdown. We next combined fluorescence-activated cell sorting with immunochemical staining of anti-GFAP and immunofluorescence staining against anti-PH3 on Tg(gfap:GFP) SCI embryos. The results showed that ANP32a promoted the proliferation and cell number of radial glial cells at the injury epicenter at 24 h post-injury (hpi). Moreover, when we applied BrdU labeling to SCI embryos derived from crossing the Tg(gfap:GFP) and Tg(mnx1:TagRFP) lines, we found that both radial glial cells and motor neurons had proliferated, along with their increased cell numbers in Anp32a-overexpression SCI-embryos. On this basis, we conclude that ANP32a plays a positive role in the regeneration of zebrafish SCI embryos.

Quantification of Idua Enzymatic Activity Combined with Observation of Phenotypic Change in Zebrafish Embryos Provide a Preliminary Assessment of Mutated idua Correlated with Mucopolysaccharidosis Type I.

Mucopolysaccharidosis type I (MPS I) is an inherited autosomal recessive disease resulting from mutation of the α-l-Iduronidase (IDUA) gene. New unknown mutated nucleotides of idua have increasingly been discovered in newborn screening, and remain to be elucidated. In this study, we found that the z-Idua enzymatic activity of zebrafish idua-knockdown embryos was reduced, resulting in the accumulation of undegradable metabolite of heparin sulfate, as well as increased mortality and defective phenotypes similar to some symptoms of human MPS I. After microinjecting mutated z-idua-L346R, -T364M, -E398-deleted, and -E540-frameshifted mRNAs, corresponding to mutated human IDUA associated with MPS I, into zebrafish embryos, no increase in z-Idua enzymatic activity, except of z-idua-E540-frameshift-injected embryos, was noted compared with endogenous z-Idua of untreated embryos. Defective phenotypes were observed in the z-idua-L346R-injected embryos, suggesting that failed enzymatic activity of mutated z-Idua-L346R might have a dominant negative effect on endogenous z-Idua function. However, defective phenotypes were not observed in the z-idua-E540-frameshifted-mRNA-injected embryos, which provided partial enzymatic activity. Based on these results, we suggest that the z-Idua enzyme activity assay combined with phenotypic observation of mutated-idua-injected zebrafish embryos could serve as an alternative platform for a preliminary assessment of mutated idua not yet characterized for their role in MPS I.

Cerebroventricular Injection of Pgk1 Attenuates MPTP-Induced Neuronal Toxicity in Dopaminergic Cells in Zebrafish Brain in a Glycolysis-Independent Manner.

Parkinson's disease (PD) is characterized by the degeneration of dopaminergic neurons. While extracellular Pgk1 (ePgk1) is reported to promote neurite outgrowth, it remains unclear if it can affect the survival of dopaminergic cells. To address this, we employed cerebroventricular microinjection (CVMI) to deliver Pgk1 into the brain of larvae and adult zebrafish treated with methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) as a PD-like model. The number of dopamine-producing cells in ventral diencephalon clusters of Pgk1-injected, MPTP-treated embryos increased over that of MPTP-treated embryos. Swimming distances of Pgk1-injected, MPTP-treated larvae and adult zebrafish were much longer compared to MPTP-treated samples. The effect of injected Pgk1 on both dopamine-producing cells and locomotion was time- and dose-dependent. Indeed, injected Pgk1 could be detected, located on dopamine neurons. When the glycolytic mutant Pgk1, Pgk1-T378P, was injected into the brain of MPTP-treated zebrafish groups, the protective ability of dopaminergic neurons did not differ from that of normal Pgk1. Therefore, ePgk1 is functionally independent from intracellular Pgk1 serving as an energy supplier. Furthermore, when Pgk1 was added to the culture medium for culturing dopamine-like SH-SY5Y cells, it could reduce the ROS pathway and apoptosis caused by the neurotoxin MPP+. These results show that ePgk1 benefits the survival of dopamine-producing cells and decreases neurotoxin damage.

High antimicrobial activity of lactoferricin-expressing Bacillus subtilis strains.

The lactoferricin expressed in Bacillus subtilis is relatively low in yield, making it hard to apply in industrial settings. We constructed a six tandem repeat of lactoferricin cDNA driven by promoter PtrnQ. After transformation, two transformants P245 and P263 possessing a stable inheritance of plasmid and high expression of lactoferricin were selected. The bactericidal activities, 1 µl of aliquot of a total 5.5 ml of solution extracted from 5 ml of cultured P245 and P263, were equivalent to the efficacy of 238.25 and 322.7 ng of Ampicillin against Escherichia coli, respectively, and 366.4 and 452.52 ng of Ampicillin against Staphylococcus epidermidis respectively. These extracts were able to kill an Ampicillin-resistant E. coli strain. The bactericidal activities of P245 and P263 equivalent to the efficacy of Tetracycline against Vibrio parahaemolyticus and V. alginolyticus were also determined. Moreover, the bactericidal activities of P245 and P263 were 168.04 and 249.94 ng of Ampicillin against Edwardsiella tarda, respectively, and 219.7 and 252.43 ng of Tetracycline against Streptococcus iniae respectively. Interestingly, the survival rate of E. tarda-infected tilapia fry fed the P263 extract displayed a significantly greater than that of the fry-fed control strain. Collectively, these B. subtilis transgenic strains are highly promising for use in animal husbandry during a disease outbreak.

Hypoxia-Responsive Subtype Cells Differentiate Into Neurons in the Brain of Zebrafish Embryos Exposed to Hypoxic Stress.

Severe hypoxia results in complete loss of central nervous system (CNS) function in mammals, while several other vertebrates, such as zebrafish, can regenerate after hypoxia-induced injury of CNS. Since the cellular mechanism involved in this remarkable feature of other vertebrates is still unclear, we studied the cellular regeneration of zebrafish brain, employing zebrafish embryos from transgenic line huORFZ exposed to hypoxia and then oxygen recovery. GFP-expressing cells, identified in some cells of the CNS, including some brain cells, were termed as hypoxia-responsive recovering cells (HrRCs). After hypoxia, HrRCs did not undergo apoptosis, while most non-GFP-expressing cells, including neurons, did. Major cell types of HrRCs found in the brain of zebrafish embryos induced by hypoxic stress were neural stem/progenitor cells (NSPCs) and radial glia cells (RGs), that is, subtypes of NSPCs (NSPCs-HrRCs) and RGs (RGs-HrRCs) that were induced by and sensitively responded to hypoxic stress. Interestingly, among HrRCs, subtypes of NSPCs- or RGs-HrRCs could proliferate and differentiate into early neurons during oxygen recovery, suggesting that these subtype cells might play a critical role in brain regeneration of zebrafish embryos after hypoxic stress.

KEPI plays a negative role in the repression that accompanies translational inhibition guided by the uORF element of human CHOP transcript during stress response.

Translation of the downstream coding sequence of some mRNAs may be repressed by the upstream open reading frame (uORF) at their 5'-end. The mechanism underlying this uORF-mediated translational inhibition (uORF-MTI) is not fully understood in vivo. Recently, it was found that zebrafish Endouc or its human orthologue ENDOU (Endouc/ENDOU) plays a positive role in repressing the uORF-MTI of human CHOP (uORFchop-MTI) during stress by blocking its activity However, the repression of uORFchop-MTI assisted by an as-yet unidentified negative effector remains to be elucidated. Compared to the upregulated CHOP transcript, we herein report that the kepi (kinase-enhanced PP1 inhibitor) transcript was downregulated in the zebrafish embryos treated with both heat shock and hypoxia. Quantitative RT-PCR also revealed that the level of kepi mRNA was noticeably decreased in both heat-shock-treated and hypoxia-exposed embryos. When kepi mRNA was microinjected into the one-celled embryos from transgenic line huORFZ, the translation of downstream GFP reporter controlled by the uORFchop-MTI was reduced in the hypoxia-exposed embryos. In contrast, when kepi was knocked down by injection of antisense Morpholino oligonucleotide, the translation of downstream GFP reporter was induced and expressed in the brain and spinal cord of injected embryos in the absence of stress. During normal condition, overexpression of KEPI increased eIF2α phosphorylation, resulting in inducing the translation of uORF-tag mRNA, such as ATF4 and CHOP mRNAs. However, during stress condition, overexpression of KEPI decreased eIF2α phosphorylation, resulting in reducing the GFP reporter and CHOP proteins. This is the first report to demonstrate that KEPI plays a negative role in uORFchop - mediated translation during ER stress.

Zebrafish, an In Vivo Platform to Screen Drugs and Proteins for Biomedical Use.

The nearly simultaneous convergence of human genetics and advanced molecular technologies has led to an improved understanding of human diseases. At the same time, the demand for drug screening and gene function identification has also increased, albeit time- and labor-intensive. However, bridging the gap between in vitro evidence from cell lines and in vivo evidence, the lower vertebrate zebrafish possesses many advantages over higher vertebrates, such as low maintenance, high fecundity, light-induced spawning, transparent embryos, short generation interval, rapid embryonic development, fully sequenced genome, and some phenotypes similar to human diseases. Such merits have popularized the zebrafish as a model system for biomedical and pharmaceutical studies, including drug screening. Here, we reviewed the various ways in which zebrafish serve as an in vivo platform to perform drug and protein screening in the fields of rare human diseases, social behavior and cancer studies. Since zebrafish mutations faithfully phenocopy many human disorders, many compounds identified from zebrafish screening systems have advanced to early clinical trials, such as those for Adenoid cystic carcinoma, Dravet syndrome and Diamond-Blackfan anemia. We also reviewed and described how zebrafish are used to carry out environmental pollutant detection and assessment of nanoparticle biosafety and QT prolongation.

Using Bacillus subtilis as a Host Cell to Express an Antimicrobial Peptide from the Marine Chordate Ciona intestinalis.

Ciona molecule against microbes-A24 (CiMAM) isolated from the marine chordate Ciona intestinalis is an antimicrobial peptide. To generate CiMAM-expressing transgenic Bacillus subtilis, we constructed a plasmid expressing recombinant CiMAM (rCiMAM) and introduced it into B. subtilis. Transgenic strains C117 and C166 were selected since they were able to highly and stably express rCiMAM. We studied the bactericidal activity of pepsin-digested extracts from rCiMAM-expressing strains against freshwater and euryhaline pathogens that commonly occur in aquaculture ponds and found no difference from that of lactoferricin-expressing strains. The bactericidal activity of 1-µL aliquot from a total 5.5 mL extracted from 5 mL of cultured C117 (1.45 × 108 CFU·mL-1) and C166 (2.17 × 108 CFU·mL-1) against halophilic bacteria was equivalent to the efficacy of 57.06 and 32.35 ng of Tetracycline against Vibrio natriegens, 47.07 and 25.2 ng against V. parahaemolyticus, and 58.17 and 36.55 ng against V. alginolyticus, respectively, indicating higher bactericidal activity of pepsin-extracts from rCiMAM-containing strains against halophilic bacteria compared to that from lactoferricin-containing strains. Since the antibacterial activity of rCiMAM-expressing B. subtilis strains shows higher competence against halophilic pathogens compared to that against freshwater and euryhaline pathogens, these strains are promising candidates to protect marine fish and shellfish from halophilic bacterial infection.

Injury-induced Cavl-expressing cells at lesion rostral side play major roles in spinal cord regeneration.

The extent of cellular heterogeneity involved in neuronal regeneration after spinal cord injury (SCI) remains unclear. Therefore, we established stress-responsive transgenic zebrafish embryos with SCI. As a result, we found an SCI-induced cell population, termed SCI stress-responsive regenerating cells (SrRCs), essential for neuronal regeneration post-SCI. SrRCs were mostly composed of subtypes of radial glia (RGs-SrRCs) and neuron stem/progenitor cells (NSPCs-SrRCs) that are able to differentiate into neurons, and they formed a bridge across the lesion and connected with neighbouring undamaged motor neurons post-SCI. Compared to SrRCs at the caudal side of the SCI site (caudal-SrRCs), rostral-SrRCs participated more actively in neuronal regeneration. After RNA-seq analysis, we discovered that caveolin 1 (cav1) was significantly upregulated in rostral-SrRCs and that cav1 was responsible for the axonal regrowth and regenerative capability of rostral-SrRCs. Collectively, we define a specific SCI-induced cell population, SrRCs, involved in neuronal regeneration, demonstrate that rostral-SrRCs exhibit higher neuronal differentiation capability and prove that cav1 is predominantly expressed in rostral-SrRCs, playing a major role in neuronal regeneration after SCI.

Poly(U)-specific endoribonuclease ENDOU promotes translation of human CHOP mRNA by releasing uORF element-mediated inhibition.

Upstream open reading frames (uORFs) are known to negatively affect translation of the downstream ORF. The regulatory proteins involved in relieving this inhibition are however poorly characterized. In response to cellular stress, eIF2α phosphorylation leads to an inhibition of global protein synthesis, while translation of specific factors such as CHOP is induced. We analyzed a 105-nt inhibitory uORF in the transcript of human CHOP (huORFchop ) and found that overexpression of the zebrafish or human ENDOU poly(U)-endoribonuclease (Endouc or ENDOU-1, respectively) increases CHOP mRNA translation also in the absence of stress. We also found that Endouc/ENDOU-1 binds and cleaves the huORFchop transcript at position 80G-81U, which induces CHOP translation independently of phosphorylated eIF2α. However, both ENDOU and phospho-eIF2α are nonetheless required for maximal translation of CHOP mRNA. Increased levels of ENDOU shift a huORFchop reporter as well as endogenous CHOP transcripts from the monosome to polysome fraction, indicating an increase in translation. Furthermore, we found that the uncapped truncated huORFchop -69-105-nt transcript contains an internal ribosome entry site (IRES), facilitating translation of the cleaved transcript. Therefore, we propose a model where ENDOU-mediated transcript cleavage positively regulates CHOP translation resulting in increased CHOP protein levels upon stress. Specifically, CHOP transcript cleavage changes the configuration of huORFchop thereby releasing its inhibition and allowing the stalled ribosomes to resume translation of the downstream ORF.

Effect of Mutated ids Overexpression on IDS Enzyme Activity and Developmental Phenotypes in Zebrafish Embryos: A Valuable Index for Assessing Critical Point-Mutations Associated with Mucopolysaccharidosis Type II Occurrence in Humans.

Mucopolysaccharidosis type II (MPS II) is an X-linked disorder resulting from a deficiency in iduronate 2-sulfatase (IDS), which is reported to be caused by gene mutations in the iduronate 2-sulfatase (IDS) gene. Many IDS mutation sites have not yet had their causal relationship with MPS II characterized. We employed a gain-of-function strategy whereby we microinjected different mutated zebrafish ids (z-ids) mRNAs corresponded to human IDS gene into zebrafish embryos, and then measured their total IDS enzymatic activity and observed the occurrence of defective phenotypes during embryonic development. We examined three known mutation sites for human IDS genes (h-IDS) associated with MPS II symptoms, including h-IDS-P86L, -S333L and -R468W, which corresponded to z-ids-P80L, -S327L and -R454W. When these three mutated z-ids mRNAs were overexpressed in zebrafish embryos, the IDS enzymatic activity of the total proteins extracted from the injected embryos was not increased compared with the endogenous IDS of the untreated embryos, which suggests that the IDS enzymatic activity of these three mutated z-ids was totally lost, as expected. Additionally, we observed defective phenotypes in these injected embryos, resulting from the failed IDS enzyme breakdown, which, in turn, has a dominant negative effect on the endogenous wild-type IDS function. These phenotypes were similar to the clinical symptoms observed in MPS II pathogenesis. We further studied six uncharacterized IDS mutation sites as identified by the Taiwanese MPS newborn screening programs. We propose a novel IDS enzyme activity assay combined with phenotypic observation in zebrafish embryos, as an alternative platform for quickly providing a valuable index for preliminarily assessment of any identified IDS point mutation gene that has not yet been characterized, in the context of its role in MPS II development.

The Neuronal Regeneration of Adult Zebrafish After Spinal Cord Injury Is Enhanced by Transplanting Optimized Number of Neural Progenitor Cells.

Cell transplantation is commonly used to study the regeneration and repair of the nervous system in animals. However, a technical platform used to evaluate the optimum number of transplanted cells in the recipient's spinal cord is little reported. Therefore, to develop such platform, we used a zebrafish model, which has transparent embryos, and transgenic line huORFZ, which generates green fluorescent protein (GFP)-expressing cells in the central nervous system under hypoxic stress. After GFP-expressing cells, also termed as hypoxia-responsive recovering cells, were obtained from hypoxia-exposed huORFZ embryos, we transplanted these GFP-(+) cells into the site of spinal cord injury (SCI) in adult wild-type zebrafish, followed by assessing the relationship between number of transplanted cells and the survival rate of recipients. When 100, 300, 500, and 1,000 GFP-(+) donor cells were transplanted into the lesion site of SCI-treated recipients, we found that recipient adult zebrafish transplanted with 300 donor cells had the highest survival rate. Those GFP-(+) donor cells could undergo proliferation and differentiation into neuron in recipients. Furthermore, transplantation of GFP-(+) cells into adult zebrafish treated with SCI was able to enhance the neuronal regeneration of recipients. In contrast, those fish transplanted with over 500 cells showed signs of inflammation around the SCI site, resulting in higher mortality. In this study, we developed a technological platform for transplanting cells into the lesion site of SCI-treated adult zebrafish and defined the optimum number of successfully transplanted cells into recipients, as 300, and those GFP-(+) donor cells could enhance recipient's spinal cord regeneration. Thus, we provided a practical methodology for studying cell transplantation therapy in neuronal regeneration of zebrafish after SCI.

A new member of the forkhead box protein family in zebrafish: Domain composition, phylogenetic implication and embryonic expression pattern.

In this study, we reported a novel member of Forkhead box (Fox) proteins found in model zebrafish (Danio rerio). This new gene we cloned was primarily assigned as zgc:162612, which locates on Chromosome 3 at 26,108,033-26,109,322 in the zebrafish genome, but encodes an uncharacterized protein, LOC100037333. After we determined the nucleotide and deduced amino acid sequences of zgc:162612, we found that zgc:162612 is an intronless gene and contains 1290 base pairs encoding 308 amino acid residues. Zgc:162612 protein is composed of a highly conserved DNA-binding domain similar to that of the Fox protein family, but with variable terminal domains. Based on phylogenetic analysis of all known members within the zebrafish Fox protein family, zgc:162612 was clustered into the zebrafish FoxD isoform subfamily. Thus, we confirmed zgc:162612 as the zebrafish FoxD7 gene. The deduced amino acid sequence of zebrafish FoxD7 shared 49, 49, 74, 63 and 74% identities with that of zebrafish FoxD1, FoxD2, FoxD3, FoxD4 and FoxD6, respectively. Compared with all known FoxD proteins in invertebrate and vertebrate species, the zebrafish FoxD7 is categorized in the same monophyletic group along with FoxD of cephalochordate and sea urchin. Whole-mount in situ hybridization demonstrated that zebrafish FoxD7 transcripts represented maternal inheritance and were ubiquitously expressed throughout the whole embryo at 12hpf. Moreover, while FoxD7 transcripts were expressed in the brain, spinal cord, fins and eyes at early developmental stages, they were mainly presented in the telencephalon ventricular zone at late developmental stages, suggesting that FoxD7 may play roles in neurogenesis and organogenesis during development of zebrafish. Taken together, we have defined a previously uncharacterized gene in the zebrafish genome, zgc:162612, and revealed that Zgc:162612 encodes a novel putative transcription factor, thus becoming a new member of the zebrafish FoxD isoform subfamily.

Oral administration of transgenic biosafe microorganism containing antimicrobial peptide enhances the survival of tilapia fry infected bacterial pathogen.

To develop an alternative to conventional antibiotics used in the aquaculture and livestock industries, we employed Bacillus subtilis, considered a biosafe microorganism, to express the degradable antimicrobial peptide lactoferricin. An expression plasmid pP43-6LFBII-GFP, in which reporter GFP cDNA was fused downstream of lactoferricin cDNA driven by an endogenous constitutive P43 promoter was electroporated into B. subtilis, followed by regeneration and cultivation. The putative colonies harboring plasmids were primarily screened by PCR-amplification of lactoferricin cDNA. Four transformants which were stable inheritance of plasmid containing lactoferricin cDNA included strains T1, T4, T7 and T13. Based on Western blot and Southern blot analyses, we found that transgenic strains T1 and T13 not only highly expressed exogenous recombinant lactoferricin, but also exhibited more stable inheritance of plasmids with 931 and 647 copies per cell, respectively. In the antibacterial in vitro experiment, the bactericidal activity of each microliter of cell lysate from transgenic strains T1 and T13 (5 × 108 CFU) for Escherichia coli was equivalent to 56 and 53 ng of Ampicillin dosage, respectively, while for Staphylococcus epidermidis, the equivalency T1 and T13 was 154 and 130 ng of Ampicillin dosage, respectively. Equivalencies of bacterial activity for Vibrio parahaemolyticus and Edwardsiella tarda followed suit. In the antibacterial in vivo experiment, we oral-in-tube fed tilapia fry (Oreochromis mossambicus X O. niloticus) with cell lysate from transgenic strain T1 and T13 individually. After 1-h of incubation, we immersed these treated fish fry in a water tank containing E. tarda (5 × 1011 CFU) for a 5-hr bacterial challenge. After one month cultivation, an average survival rate of 63 and 67% was observed after having fed the fish fry with transgenic strains T1 and T13, respectively. However, the average survival rate of fish fry fed with B. subtilis WT strain and transgenic strain T19 without expressing recombinant lactoferricin reached only 5 and 9%, respectively. These data indicate that the survival of fish fry infected by the intestinal pathogen tested could be significantly enhanced by feeding transgenic B. subtilis containing antibacterial peptide. Therefore, we suggest that this strategy could be applied to both aquaculture and livestock industries to (i) reduce the dependency on conventional antibiotics during seasonal outbreaks and (ii) eliminate the problem of antibiotic resistance.

Garcimultiflorone K inhibits angiogenesis through Akt/eNOS- and mTOR-dependent pathways in human endothelial progenitor cells.

Background Garcimultiflorone K is a novel polyprenylated polycyclic acylphloroglucinol isolated from the stems of Garcinia multiflora that exhibits promising anti-angiogenic activity in human endothelial progenitor cells (EPCs). Purpose This study sought to determine the underlying anti-angiogenic mechanisms and pharmacological properties of garcimultiflorone K. Methods We examined the anti-angiogenic effects of garcimultiflorone K and its mechanisms of action using in vitro EPC models and in vivo zebrafish embryos. Results EPCs proliferation, migration, differentiation and capillary-like tube formation were effectively and concentration-dependently inhibited by garcimultiflorone K without any signs of cytotoxicity. Our investigations revealed that garcimultiflorone K suppressed EPCs angiogenesis through Akt, mTOR, p70S6K, and eNOS signaling cascades. Notably, garcimultiflorone K dose-dependently impeded angiogenesis in zebrafish embryos. Conclusion Our data demonstrate the anti-angiogneic effects of garcimultiflorone K in both in vitro and in vivo models. Garcimultiflorone K appears to have potential in the treatment of angiogenesis-related diseases.

Extracellular Pgk1 enhances neurite outgrowth of motoneurons through Nogo66/NgR-independent targeting of NogoA.

NogoA inhibits neurite outgrowth of motoneurons (NOM) through interaction with its receptors, Nogo66/NgR. Inhibition of Nogo receptors rescues NOM, but not to the extent exhibited by NogoA-knockout mice, suggesting the presence of other pathways. We found that NogoA-overexpressing muscle cells reduced phosphoglycerate kinase 1 (Pgk1) secretion, resulting in inhibiting NOM. Apart from its glycolytic role and independent of the Nogo66 pathway, extracellular Pgk1 stimulated NOM by triggering a reduction of p-Cofilin-S3, a growth cone collapse marker, through decreasing a novel Rac1-GTP/p-Pak1-T423/p-P38-T180/p-MK2-T334/p-Limk1-S323/p-Cofilin-S3 molecular pathway. Not only did supplementary Pgk1 enhance NOM in defective cells, but injection of Pgk1 rescued denervation in muscle-specific NogoA-overexpression of zebrafish and an Amyotrophic Lateral Sclerosis mouse model, SOD1 G93A. Thus, Pgk1 secreted from muscle is detrimental to motoneuron neurite outgrowth and maintenance.