Hatching gland development and hatching in zebrafish embryos: A role for zinc and its transporters Zip10 and Znt1a.

Zinc transporters of the ZIP (Slc39, importers) and ZnT (Slc30, exporters) protein families have evolutionary conserved roles in biology. The aim of the present study was to explore the role of zinc, and zinc transporters Zip10 and Znt1a in zebrafish hatching gland development and larval hatching. In the study, knockdown of genes for Zip10 and Znt1a in zebrafish embryos was achieved using morpholino-modified oligonucleotides. A partial loss-of-function Znt1a mutant (Znt1asa17) allowed comparison with the Znt1a morphant. Free Zn2+ in embryos and apoptosis were investigated using fluorescent dyes whereas gene expression was investigated by whole-mount in situ hybridization (WISH). The results showed high levels of free Zn2+ in the hatching gland cells (HGC) along with abundant expression of zip10 and znt1a in normal embryo. Knockdown of zip10 reduced free Zn2+ in HGC, ceased their normal developmental apoptosis, and resulted in displacement and later disappearance of hatching glands and hatching enzymes he1a and catL1b, and inability to hatch. Conversely, knockdown of znt1a or the Znt1asa17 mutation accelerated hatching and coincided with high expression of hatching enzymes and free Zn2+ in the HGC. Thus, Zip10 and free Zn2+ in the HGC are required both for their development and function. This study also demonstrated the opposite functions of the two zinc transporters, ZIP10 and ZnT1 as well as shedding light on the role of Zn2+ in regulation of the human hatching enzyme homologue, ovastacin, which is activated by zinc and cleaves the zona pellucida protein, ZP2, to prevent polyspermy.

Anticoccidial effects of Khaya senegalensis aqueous stem bark extract on broiler chickens experimentally infected with Eimeria species.

Graded concentrations (200, 400 and 800 mg/kg) of the aqueous stem bark extract of Khaya senegalensis was evaluated for its therapeutic efficacy against experimentally induced coccidiosis in broiler chicken. The phytochemical analysis shows the presence of tannins, saponins, cardiac glycosides and steroids. There was significant reduction in oocyst count across the groups in a graded dose manner with 800 mg/kg being the most efficacious dose. There was also weight gain across the treatment groups with immuno-modulatory and erythropoetic activities observed. Also, a significant (p < 0.05) graded dose-dependent reduction in the oocyst count in the treatment groups. A significant (p < 0.05) increase in mean weight gain was also recorded across the experimental groups except the negative control. The haematology also showed a dose-dependent increase in red blood cells, haemoglobin and packed cell volume of the treatment groups. The extract had no significant difference (p > 0.05) on the white blood cells, but a slight decrease in the white blood cells and heterophil counts was observed at 400 mg/kg. Furthermore, the aspartate amino transaminase level showed a significant difference (p < 0.05). Fluctuating levels of other serum biochemical parameters such as total protein, albumin and potassium were observed. No significant difference (p > 0.05) in the sodium concentration was observed. In addition, oxidative stress biomarkers such as catalase significantly increased (p < 0.05) in all the experimental groups in addition to the concomitant increase in reduced gluthathione (GSH) and superoxide dismutase (SOD) levels. Conclusively, the aqueous extract of K. senegalensis was effective in the management of coccidiosis thus supporting its folkloric use.

Zinc transporter ZIP10 forms a heteromer with ZIP6 which regulates embryonic development and cell migration.

There is growing evidence that zinc and its transporters are involved in cell migration during development and in cancer. In the present study, we show that zinc transporter ZIP10 (SLC39A10) stimulates cell motility and proliferation, both in mammalian cells and in the zebrafish embryo. This is associated with inactivation of GSK (glycogen synthase kinase)-3α and -3ß and down-regulation of E-cadherin (CDH1). Morpholino-mediated knockdown of zip10 causes delayed epiboly and deformities of the head, eye, heart and tail. Furthermore, zip10 deficiency results in overexpression of cdh1, zip6 and stat3, the latter gene product driving transcription of both zip6 and zip10 The non-redundant requirement of Zip6 and Zip10 for epithelial to mesenchymal transition (EMT) is consistent with our finding that they exist as a heteromer. We postulate that a subset of ZIPs carrying prion protein (PrP)-like ectodomains, including ZIP6 and ZIP10, are integral to cellular pathways and plasticity programmes, such as EMT.

The potential of putative zinc-binding motifs of haemagglutinin (HA) protein for categorization and prediction of pathogenicity of H5 subtypes of avian influenza virus.

In the present study, we used the potential of bioinformatics and computational analysis to predict the existence and biological relevance of zinc finger (ZF) motifs in heamagglutinin (HA) protein of Avian Influenza (AI) virus. Sequence data of Avian Influenza (AI) viruses were retrieved from accessible databases (GenBank, GISAID, IRD) and analyzed for the existence, as well as functional prediction of the putative zinc finger or ''zinc-binding'' motif(s) of HA protein. It is hypothesized that the ZF motif(s) in HA of AI virus can be used as a ''novel'' biomarker for categorization of the virus and/or its virulence. As a model for analysis, we used the H5 subtypes of highly pathogenic, non-pathogenic and low pathogenic avian influenza (HPAI, NPAI and LPAI) viruses of H5N1 and H5N2 of avian and human origins. Interestingly, our method of characterization using the zinc-finger agrees with the existing classification in distinguishing between highly pathogenic and non-pathogenic or low pathogenic subtypes. The new method also clearly distinguished between low and non-pathogenic strains of H5N2 and H5N1 which are indistinguishable by the existing method that utilizes the sequence of the polybasic amino acids of the proteolytic cleavage site for pathogenicity. It is hypothesized that zinc through the activities of zinc-binding proteins modulates the virulence property of the viral subtypes. Our observation further revealed that only the HA protein among the eight encoded proteins of influenza viruses contain high numbers of Cys-His residues. It is expected that the information gathered from the analysis of the data will be useful to generate more research hypotheses/designs that will give further insight towards the identification and control of avian influenza virus through the molecular manipulation of zinc finger motifs present in viral HA protein.

The zebrafish Znt1asa17 mutant reveals roles of zinc transporter-1a in embryonic development.

BACKGROUND: Zinc is one of the vital micronutrients required through various developmental stages in animals. Zinc transporter-1 (ZnT1; Slc30a1) is essential in vertebrates for nutritional zinc uptake and cellular zinc extrusion. Knockout of ZnT1 is lethal in vertebrates and there are therefore few functional studies of this protein in vivo. METHODS: In the present study we characterised the embryonic development in a zebrafish Znt1a mutant (Znt1asa17) which is lacking the last 40 amino acids of Znt1a as generated by TILLING. In parallel experiments, we compared the development of a zebrafish embryo Znt1a morphant (Znt1aMO) which was generated by knockdown of Znt1a using morpholino-modified oligonucliotides. RESULTS: The homozygous Znt1asa17 embryo is viable, but displays a subtle phenotype informing on the biological roles of Znt1a. The Znt1asa17 fish have delayed development, including attenuated epiboly. They further show a decrease in phosphorylated extracellular signal-regulated kinases 1 and 2 (pERK1/2), retarded yolk resorption, and impaired clearance of free Zn2+ from the vitelline fluid and its storage in hatching gland cells. All these aberrations are milder versions of those observed upon knockdown of Znt1a by morpholinos. Interestingly, the phenotype could be rescued by addition of the cell-permeable zinc chelator, N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN) to the incubation medium and was aggravated by addition of zinc(II). Thus, the Znt1asa17 mutant has a reduced ability to handle zinc and can be characterised as a hypomorph. CONCLUSION: This study is the first to show that the last 40 amino acids of Znt1a are of importance for its role in zinc homeostasis and ability to activate the MAPK/ERK pathway contrary to what was previously thought.

Phosphine poisoning in free-range local chickens: a case report.

Phosphine poisoning is rarely reported in poultry, and its diagnosis is a great challenge for veterinary toxicologists and pathologists. A case of phosphine toxicosis in local, free range chickens is reported. Fourteen dead chickens (age ≥6 months old) were presented to the veterinary clinic for necropsy. The history revealed that the chickens were normally fed with guinea corn grains, but were suspected to have been fed boiled rice laced with poison. The clinical signs observed were ruffled feathers, somnolence, anorexia and high mortality. The disease ran a 2-day course with mortality pattern of four chickens the first day, six overnight and 14 the following day. Necropsy findings showed generalized vascular congestion and haemorrhage in the lungs and visceral organs, with the crop and gizzard filled with guinea corn and rice grains and greenish-yellow faecal material in the intestinal lumen. The presence of widespread congestion and petechial haemorrhages on visceral organs with the microscopic pulmonary congestion, and diffuse intraparabronchial presence of air sac macrophages, strongly suggested an acute toxic cause of death. Chemical tests on the crop contents of the dead chickens were positive for phosphine gas. This report will contribute to a better understanding of the clinical signs and lesions presented in cases of acute phosphide rodenticide exposure in domestic chickens, with a brief review of the forensic literature.

Time-series analysis of ruminant foetal wastage at a slaughterhouse in North Central Nigeria between 2001 and 2012.

In developing countries, foetal wastage from slaughtered ruminants and the associated economic losses appear to be substantial. However, only a limited number of studies have comprehensively evaluated these trends. In the current study, secondary (retrospective) and primary data were collected and evaluated to estimate the prevalence of foetal wastage from cattle, sheep and goats slaughtered at an abattoir in Minna, Nigeria, over a 12-year period (January 2001-December 2012). Time-series modelling revealed substantial differences in the rate of foetal wastage amongst the slaughtered species, with more lambs having been wasted than calves or kids. Seasonal effects seem to influence rates of foetal wastage and certain months in the year appear to be associated with higher odds of foetal wastage. Improved management systems are suggested to reduce the risk of foetal losses.