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.

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.

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.

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.

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.

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.

Short fish-origin DNA elements served as flanking sequences in a knockdown cloning vector enabling the generation of a functional siRNA molecule in mammalian cells and fish embryos.

Improving the quality of a siRNA-knockdown cloning vector requires simpler, shorter, and more effective flanking sequences. In this study, we designed such flanking sequences based on those found in zebrafish pre-miR3906, namely, internal element (IE) 1 and IE2. We engineered a vegf-shRNA fragment flanked by an 80-bp IE1/IE2 and then inserted into the 3' UTR of GFP reporter cDNA driven by a cytomegalovirus promoter to obtain a plasmid containing gfp-IE-vegf-shRNA-polA. Upon microinjection of this plasmid into zebrafish embryos, we found that IE flanking sequences could effectively induce the production of vegf-shRNA fragment, which was then processed into a functional siRNA to silence the target vegf121 gene. Northern blot showed that the vegf-shRNA fragment was cleaved from gfp-IE-vegf-shRNA-polA, resulting in the loss of polyA tails, subsequently degrading the remaining RNA-containing GFP. Moreover, Western blot revealed that addition of IE-based vegf-shRNA fragment could markedly decrease the expression of VEGF. Finally, to facilitate a more versatile application of the IE-based knockdown vector, we generated an inducible expression vector in which IE-vegf-shRNA was constructed downstream in a Tet-on system to generate a Tet-on-IE-vegf-shRNA construct. After doxycycline induction, the protein level of VEGF in SW620 cells harboring the Tet-on-IE-vegf-shRNA construct was decreased 77%. Interestingly, when SW620 cells harboring Tet-on-IE-vegf-shRNA cells were induced and transplanted into zebrafish embryos, we found that abnormal branch of the sub-intestinal vessels was reduced in the recipient embryos, suggesting that vegf-shRNA cleaved from Tet-on-IE-vegf-shRNA-polA was processed into a functional vegf-siRNA in embryos suppressing endogenous VEGF and reducing tumor angiogenesis. Therefore, we conclude that fish-origin IEs are flanking sequences with short, simple, and effective DNA elements. This IE-based knockdown cloning vector provides a new alternative material to facilitate the generation of functional siRNA with which to perform loss-of-function experiments, both in vitro (mammalian cells) and in vivo (zebrafish embryos).

Zebrafish and Medaka: new model organisms for modern biomedical research.

Although they are primitive vertebrates, zebrafish (Danio rerio) and medaka (Oryzias latipes) have surpassed other animals as the most used model organisms based on their many advantages. Studies on gene expression patterns, regulatory cis-elements identification, and gene functions can be facilitated by using zebrafish embryos via a number of techniques, including transgenesis, in vivo transient assay, overexpression by injection of mRNAs, knockdown by injection of morpholino oligonucleotides, knockout and gene editing by CRISPR/Cas9 system and mutagenesis. In addition, transgenic lines of model fish harboring a tissue-specific reporter have become a powerful tool for the study of biological sciences, since it is possible to visualize the dynamic expression of a specific gene in the transparent embryos. In particular, some transgenic fish lines and mutants display defective phenotypes similar to those of human diseases. Therefore, a wide variety of fish model not only sheds light on the molecular mechanisms underlying disease pathogenesis in vivo but also provides a living platform for high-throughput screening of drug candidates. Interestingly, transgenic model fish lines can also be applied as biosensors to detect environmental pollutants, and even as pet fish to display beautiful fluorescent colors. Therefore, transgenic model fish possess a broad spectrum of applications in modern biomedical research, as exampled in the following review.

Effect of Free-range Rearing on Meat Composition, Physical Properties and Sensory Evaluation in Taiwan Game Hens.

Experiments were conducted to evaluate the effect of an outdoor-grazed raising model on meat composition, physical properties and sensory attributes of Taiwan game hens. Six hundred 1-d old female chicks were raised on a floor for 8 weeks. On day 57, 600 healthy birds, with similar body weight, were selected and randomly assigned to three treatment groups (cage, floor-pen and free-range). The results showed that different feeding models had no effect on drip loss, cooking loss, moisture, crude protein, crude fat, crude ash, zinc and calorie contents in breast meat and moisture content in thigh meat. The free-range group had the lowest fat content in both breast and thigh meat, and the lowest calorie content in thigh meat. The firmness and toughness in both thigh and breast of the free-range group were the highest values (p<0.05). The crude protein, total collagen, zinc and iron contents in thigh meat and total collagen content in breast meat of the free-range group were significantly higher than those of the cage-feeding group (p<0.05). The meat sensory scores of flavor, chewiness and overall acceptability of both thigh and breast meat of the free-range group were significantly (p<0.05) better than those of the other two groups. Moreover, the current findings also indicate that the Taiwan game hens of the free-range feeding model displayed well-received carcass traits and meat quality, with higher scores for flavor, chewiness and overall acceptability for greater sensory satisfaction in both breast and thigh meat. In addition, the thigh meat contained high protein and total collage but low fat, offering a healthier diet choice.

Regulating TKT activity inhibits proliferation of human acute lymphoblastic leukemia cells.

Among pediatric blood cancers, acute lymphoblastic leukemia (ALL) is the most common hematologic malignancy. Within ALL, T-cell acute lymphoblastic leukemia (T-ALL) accounts for 10 to 15% of all pediatric cases, and ~25% of adult cases. For T-ALL, its recurrence and relapse after treatment remain problematic. Therefore, it is necessary to develop new therapies for T-ALL. Recent studies suggested regulating energy metabolism is a novel approach to inhibit tumor growth, likely a promising treatment. Transketolase (TKT) is an important enzyme for modulating glucose metabolize in the pentose phosphate pathway (PPP). In this study, we treated T-ALL cells with different doses of niclosamide and primary T-ALL PBMCs were analyzed by RNA sequencing. T-ALL cells treated with niclosamide were analyzed with the Western blotting and TKT activity assay. Metabolism of T-ALL cells was evaluated by ATP assay and seahorse analyses. Lastly, we used a T-ALL xenograft murine model to determine effects of TKT knockdown on T-ALL tumor growth. Tumor samples were analyzed by H&E and IHC stainings. We found that niclosamide reduced T-ALL cell viability, and reduced expressions of TKT, Transketolase-Like Protein 1/2 (TKTL1/2) and transaldolase. In addition, niclosamide inhibited TKT enzyme activity, aerobic metabolism and glycolysis, finally leading to lower production of ATP. TKT knockdown inhibited tumor growth of xenograft T-ALL mice. Findings showed that niclosamide inhibits T-ALL cell growth by inhibiting TKT and energy metabolism.

Blu-ray disk lens as the objective of a miniaturized two-photon fluorescence microscope.

In this paper, we examine the performance of a Blu-ray disk (BD) aspheric lens as the objective of a miniaturized scanning nonlinear optical microscope. By combining a single 2D micro-electro mechanical system (MEMS) mirror as the scanner and with different tube lens pairs, the field of view (FOV) of the studied microscope varies from 59 µm × 93 µm up to 178 µm × 280 µm, while the corresponding lateral resolution varies from 0.6 µm to 2 µm for two-photon fluorescence (2PF) signals. With a 34/s video frame rate, in vivo dynamic observation of zebrafish heartbeat through 2PF of the excited green fluorescence protein (GFP) is demonstrated.

Transgenic zebrafish model to study translational control mediated by upstream open reading frame of human chop gene.

Upstream open reading frame (uORF)-mediated translational inhibition is important in controlling key regulatory genes expression. However, understanding the underlying molecular mechanism of such uORF-mediated control system in vivo is challenging in the absence of an animal model. Therefore, we generated a zebrafish transgenic line, termed huORFZ, harboring a construct in which the uORF sequence from human CCAAT/enhancer-binding protein homologous protein gene (huORF(chop)) is added to the leader of GFP and is driven by a cytomegalovirus promoter. The translation of transgenic huORF(chop)-gfp mRNA was absolutely inhibited by the huORF(chop) cassette in huORFZ embryos during normal conditions, but the downstream GFP was only apparent when the huORFZ embryos were treated with endoplasmic reticulum (ER) stresses. Interestingly, the number and location of GFP-responsive embryonic cells were dependent on the developmental stage and type of ER stresses encountered. These results indicate that the translation of the huORF(chop)-tag downstream reporter gene is controlled in the huORFZ line. Moreover, using cell sorting and microarray analysis of huORFZ embryos, we identified such putative factors as Nrg/ErbB, PI3K and hsp90, which are involved in huORF(chop)-mediated translational control under heat-shock stress. Therefore, using the huORFZ embryos allows us to study the regulatory network involved in human uORF(chop)-mediated translational inhibition.

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.

Genetic Diversity and Population Structure in Captive Populations of Formosan Sambar Deer (Rusa unicolor swinhoei).

Formosan sambar deer (Rusa unicolor swinhoei) are of great economic significance in Taiwan, resulting in a substantial increase in deer farming to meet the high demand for velvet antlers. Inbreeding depression and reduced genetic variability can lead to the deterioration of captive populations. In this study, 239 Formosan sambar deer were genotyped using 13 microsatellites to analyze their genetic diversity and population genetic structure. Our results indicate a high-resolution power of these microsatellites in individual discrimination and parentage analysis. However, captive populations exhibit a low level of genetic diversity, likely because of inbreeding and bottleneck effects. Both principal coordinate analysis (PCoA) and STRUCTURE analyses revealed two distinct and segregated genetic groups within the captive populations and indicated no clear population genetic structure among the captive populations. Introducing new genetic material from the wild through translocation offers a potential solution for mitigating the impact of inbreeding and enhancing genetic diversity. The comprehensive information obtained from these genetic analyses is crucial for the development of effective breeding strategies aimed at preserving and enhancing Formosan sambar deer populations.

Novel intronic microRNA represses zebrafish myf5 promoter activity through silencing dickkopf-3 gene.

A strong, negative cis-element located at the first intron +502/+835 (I300) of zebrafish myf5 has been reported. To elucidate the molecular mechanism underlying this repression network, we microinjected zebrafish single-cell embryos with I300 RNA, resulting in the dramatic reduction of luciferase activity driven by the myf5 promoter. Within this I300 segment, we identified an intronic microRNA (miR-In300) located at +609/+632 and found that it was more highly expressed in the older mature somites than those newly formed, which negatively correlated with the distribution of zebrafish myf5 transcripts. We proved that miR-In300 suppressed the transcription of myf5 through abolishing myf5 promoter activity, and we subsequently identified the long isoform of the Dickkopf-3 gene (dkk3) as the target gene of miR-In300. We further found that injection of the dkk3-morpholinos (MOs) resulted in downregulation of myf5 transcripts in somites, whereas co-injection of myf5 mRNA with dkk3-MO1 enabled rescue of the defects induced by dkk3-MO1 alone. Finally, injection of miR-In300-MO enhanced both myf5 transcripts in somites and the level of Dkk3 protein in zebrafish embryos. Based on these findings, we concluded that miR-In300 binds to its target gene dkk3, which inhibits the translation of dkk3 mRNA and, in turn, suppresses zebrafish myf5 promoter activity.

Effect of age and caponization on blood parameters and bone development of male native chickens in taiwan.

An experiment was carried out to determine the effect of age and caponization on the development blood and bone characteristics development in male country chickens in Taiwan. A total of two hundred 8-wk-old LRI native chicken cockerels, Taishi meat No.13 from LRI-COA, were used as experimental animals. Cockerels were surgically caponized at 8 wks of age. Twelve birds in each group were bled and dressed from 8 wks to 35 wks of age at 1 to 5 wk intervals. The results indicated that the plasma testosterone concentration was significantly (p<0.05) lower in capons after 12 wks of age (caponized treatment after 4 wks) than that of the intact males. The relative tibia weight, bone breaking strength, cortical thickness, bone ash, bone calcium, bone phosphorus and bone magnesium contents were significantly (p<0.05) higher in intact males, while capons had higher (p<0.05) plasma ionized calcium, inorganic phosphorus and alkaline phosphatase concentration. The plasma testosterone concentration, relative tibia weight, tibia length, breaking strength, cortical thickness, bone ash, calcium, and phosphorus contents of intact males chickens increased significantly (p<0.05) with the advance of age. In addition, the relative tibia weight of capons peaked at 18 wks of age, and declined at 35 wks of age. The bone ash, calcium and phosphorus content increased most after 14 wks of age in male native chickens in Taiwan. Also, tibia length and cortical thickness peaked at 22 wks of age. However, the peak of bone strength was found at 26 wks of age. These findings support the assertion that androgens can directly influence bone composition fluxes in male chickens. Caponization caused a significant increase in bone loss at 4 wks post treatment, which reflected bone cell damage, and demonstrated reductions in the relative tibia weight, breaking strength, cortical thickness, bone ash, calcium, phosphorus and magnesium contents, and increases in plasma ionized calcium, inorganic phosphorus and alkaline phosphatase concentration.

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.

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.

Direct-Maternal Genetic Parameters for Litter Size and Body Weight of Piglets of a New Black Breed for the Taiwan Black Hog Market.

The objective of this study was to estimate the genetic parameters of litter size and piglet weight from farrowing to weaning in KHAPS Black sows. The genetic parameters investigated were the direct (h2d), maternal (h2m), realized (h2r), and total (h2T) heritability, as well as correlations (rd, rm, and rdm) within and between traits. The analyses were performed using single- and three-trait animal models with and without maternal genetic effects. In the three-trait model with maternal genetic effect, all estimates of h2d and h2m were significantly different from zero except the h2d of mean birth weight. Positive values of rd and rm between traits were observed as expected in the range of 0.322-1.000. Negative values of rdm were found within and between traits and were less associated with mean piglet weight traits than litter size traits. Estimates of h2T were consistently larger than those of h2r in both the single- and three-trait model analyses. In addition, the three-trait model can take into account the association between the traits, so the estimates are more accurate with smaller SEs. In conclusion, maternal genetic effects were not negligible in this study, and thus, a multiple-trait animal model with maternal genetic effects and full pedigree is recommended to assist future pig breeding decisions in this new breed.

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.