Establishment of a transgenic zebrafish line for superficial skin ablation and functional validation of apoptosis modulators in vivo

Background

Zebrafish skin is composed of enveloping and basal layers which form a first-line defense system against pathogens. Zebrafish epidermis contains ionocytes and mucous cells that aid secretion of acid/ions or mucous through skin. Previous studies demonstrated that fish skin is extremely sensitive to external stimuli. However, little is known about the molecular mechanisms that modulate skin cell apoptosis in zebrafish.

Methodology/Principal Findings

This study aimed to create a platform to conduct conditional skin ablation and determine if it is possible to attenuate apoptotic stimuli by overexpressing potential apoptosis modulating genes in the skin of live animals. A transgenic zebrafish line of Tg(krt4:NTR-hKikGR)^cy17 (killer line), which can conditionally trigger apoptosis in superficial skin cells, was first established. When the killer line was incubated with the prodrug metrodinazole, the superficial skin displayed extensive apoptosis as judged by detection of massive TUNEL- and active caspase 3-positive signals. Great reductions in NTR-hKikGR⁺ fluorescent signals accompanied epidermal cell apoptosis. This indicated that NTR-hKikGR⁺ signal fluorescence can be utilized to evaluate apoptotic events in vivo. After removal of metrodinazole, the skin integrity progressively recovered and NTR-hKikGR⁺ fluorescent signals gradually restored. In contrast, either crossing the killer line with testing lines or transiently injecting the killer line with testing vectors that expressed human constitutive active Akt1, mouse constitutive active Stat3, or HPV16 E6 element displayed apoptosis-resistant phenotypes to cytotoxic metrodinazole as judged by the loss of reduction in NTR-hKikGR⁺ fluorescent signaling.

Conclusion/Significance

The killer/testing line binary system established in the current study demonstrates a nitroreductase/metrodinazole system that can be utilized to conditionally perform skin ablation in a real-time manner, and provides a valuable tool to visualize and quantify the anti-apoptotic potential of interesting target genes in vivo. The current work identifies a potential use for transgenic zebrafish as a high-throughput platform to validate potential apoptosis modulators in vivo.     

The Susceptive Alendronate-Treatment Timing and Dosage for Osteogenesis Enhancement in Human Bone Marrow-Derived Stem Cells

Recent studies indicated that alendronate enhanced osteogenesis in osteoblasts and human bone marrow-derived stem cells. However, the time- and dose-dependent effects of Aln on ostegenic differentiation and cytotoxicity of hBMSCs remain undefined. In present study, we investigated the effective dose range and timing of hBMSCs. hBMSCs were treated with various Aln doses (1, 5 and 10 µM) according to the following groups: group A was treated with Aln during the first five days of bone medium, groups B, C and D were treated during the first, second, and final five days of osteo-induction medium and group E was treated throughout the entire experiment. The mineralization level and cytotoxicity were measured by quantified Alizarin Red S staining and MTT assay. In addition, the reversal effects of farnesyl pyrophosphate and geranylgeranyl pyrophosphate replenishment in group B were also investigated. The results showed that Aln treatment in groups A, B and E enhanced hBMSC mineralization in a dose-dependent manner, and the most pronounced effects were observed in groups B and E. The higher dose of Aln simultaneously enhanced mineralization and caused cytotoxicity in groups B, C and E. Replenishment of FPP or GGPP resulted in partial or complete reverse of the Aln-induced mineralization respectively. Furthermore, the addition of FPP or GGPP also eliminated the Aln-induced cytotoxicity. We demonstrated that hBMSCs are susceptible to 5 µM Aln during the initiation stage of osteogenic differentiation and that a 10 µM dose is cytotoxic.     

Analysis of landscape spatial pattern changes in urban fringe area: a case study of Hunhe Niaodao Area in Shenyang City

The urban fringe is areas where land use changes and develops rapidly, analyzing land use changes and driving forces in urban fringe area is of great significance for landscape pattern and ecological environment construction. This study takes the Hunhe Bird Island area of Shenyang City as the research object, uses Landsat remote sensing images from 2005 to 2019 as the basic data, and uses the land transfer matrix and landscape index method to analyze the overall landscape environment and six land types. Using government policies and statistical data to analyze the driving forces of landscape changes. The research results show that the landscape matrix had changed, the overall landscape structure had changed drastically, the ecological environment was unstable, and human interference factors had increased, and the fragmentation phenomenon had become more and more serious. Each land type has different environmental changes. Except for the relatively stable water environment, other land types have different degrees of fragmentation. The reason is that the driving force of landscape pattern changes mainly comes from the influence of human factors, which is closely related to population, economic development and government policies. Therefore, future development and protection strategies are proposed for each land type in the urban fringe area. For the overall development of urban fringe area, it is advocated to make full use of land resources, strengthen the correct judgment and forecast of urban population and economy, formulate the correct urban boundary strategy for sustainable development, avoid the ecological environment problems faced by excessive land development, and realize the “Rational expansion” of cities.

     

Expression of a cloned sweet potato catalase SPCAT1 alleviates ethephon-mediated leaf senescence and H₂O₂ elevation

In this report a full-length cDNA, SPCAT1, was isolated from ethephon-treated mature L3 leaves of sweet potato. SPCAT1 contained 1479 nucleotides (492 amino acids) in its open reading frame, and exhibited high amino acid sequence identities (ca. 71.2-80.9%) with several plant catalases, including Arabidopsis, eggplant, grey mangrove, pea, potato, tobacco and tomato. Gene structural analysis showed that SPCAT1 encoded a catalase and contained a putative conserved internal peroxisomal targeting signal PTS1 motif and calmodulin binding domain around its C-terminus. RT-PCR showed that SPCAT1 gene expression was enhanced significantly in mature L3 and early senescent L4 leaves and was much reduced in immature L1, L2 and completely yellowing senescent L5 leaves. In dark- and ethephon-treated L3 leaves, SPCAT1 expression was significantly enhanced temporarily from 0 to 24 h, then decreased gradually until 72 h after treatment. SPCAT1 gene expression levels also exhibited approximately inverse correlation with the qualitative and quantitative H₂O₂ amounts. Effector treatment showed that ethephon-enhanced SPCAT1 expression was repressed by antioxidant reduced glutathione, NADPH oxidase inhibitor diphenylene iodonium (DPI), calcium ion chelator EGTA and de novo protein synthesis inhibitor cycloheximide. These data suggest that elevated reactive oxygen species H₂O₂, NADPH oxidase, external calcium influx and de novo synthesized proteins are required and associated with ethephon-mediated enhancement of sweet potato catalase SPCAT1 expression. Exogenous application of expressed catalase SPCAT1 fusion protein delayed or alleviated ethephon-mediated leaf senescence and H₂O₂ elevation. Based on these data we conclude that sweet potato SPCAT1 is an ethephon-inducible peroxisomal catalase, and its expression is regulated by reduced glutathione, DPI, EGTA and cycloheximide. Sweet potato catalase SPCAT1 may play a physiological role or function in cope with H₂O₂ homeostasis in leaves caused by developmental cues and environmental stimuli.     

Fishery policy when considering the future opportunity of harvesting

Free access to a common pool of resource in a country may lead to over-exploitation and sacrifice future opportunities of harvesting. As such, the protection of a common fishery resource is worth investigating. In this paper we develop a two-period model and a multi-period model to analyze the optimal inter-temporal utilization of a finite resource of stock and propose to impose a tax on the harvest rate as an efficient mechanism with an aim at economic sustainability by incorporating the future opportunity of harvesting into the models as a major component of social objectives. The sensitivity analysis of the two-period model shows that (1) labor inputs for harvesting in Period 1 should be reduced, the biomass of fishery stock will increase, but the harvesting in Period 2 should be amplified and the biomass of fishery stock in Period 2 will not be affected if the current generation owns a higher valuation on the future opportunity of harvesting; (2) a higher internal regeneration rate leads to higher harvesting in each period and a higher level of fishery stock in Period 1, but an uncertain level of fishery stock in Period 2; (3) with a higher discount rate the harvesting in Period 1 should increase, but the harvesting in Period 2 should fall and the level of fishery stock in each period will be reduced; (4) a higher fish price in Period 1 leads to higher harvesting in Period 1, but reduced harvesting in Period 2. As a consequence, the level of fishery stock in each period will be reduced; (5) the effect of a change in fish prices in Period 2 on the harvesting and the level of fishery stock in Period 1 is uncertain, but the change in fish prices in Period 2 gives a positive effect on harvesting in Period 2 and a negative effect on the level of fishery stock in Period 2; (6) higher labor wages in Period 1 lead to lower harvesting, but a higher level of fishery stock in Period 1. This encourages an increase in harvesting in Period 2 and leads to a higher level of fishery stock in Period 2; and (7) a change of the labor wage in Period 2 affects the harvesting and the level of fishery stock in Period 1 indecisively, but it gives negative effects on the harvesting in Period 2 and positive effects on the level of fishery stock in Period 2.     

Application of fractals: create an artificial habitat with several small (SS) strategy in marine environment

In 2006, Lan and Hsui applied perspectives on landscape ecology to propose a spatially explicit model, and suggested that the cluster type deployment of artificial habitat in a marine environment might be better than other configurations in maximizing habitat complexity with a fractals approach. The limitation is that, however, it could only design a single large island pattern subject to its solving algorithm. Hence, by applying more sophisticated algorithms, Evolution computational (EC) algorithm, we mimic more spatially explicit structural patterns to conduct several small (SS) clusters pattern in an artificial habitat such as those found in nature landscape; additionally, the deployment in a community follows the concept of DARCs model, which applied the cellular automata (CA) concept with Moore neighborhood rule. The results show that with all the new knowledge that has been gained through the appropriate application of fractal geometry to natural sciences, it is clear that understanding how landscape ecology influences population ecology has allowed population ecologists to gain new insights into their field.     

Molecular cloning of a new immunomodulatory protein from Anoectochilus formosanus which induces B cell IgM secretion through a T-independent mechanism

An immunomodulatory protein (IPAF) was purified and cloned from Anoectochilus formosanus, an Orchidaceae herbal plant in Asia. The major targeting immune cells of IPAF and its modulating effects toward B lymphocytes were investigated. Rapid amplification of cDNA ends (RACE) was conducted to clone the IPAF gene, and the obtained sequence was BLAST compared on the NCBI database. MACS-purified mouse T and B lymphocytes were stimulated with IPAF and the cell proliferation, activation, and Igs production were examined. IPAF comprised a 25 amino acids signal peptide and a 138 amino acids protein which was homologous to the lectins from Orchidaceae plant. IPAF selectively induced the cell proliferation in mouse splenic B lymphocytes but not T lymphocytes. The IPAF-induced B cells exhibited increased CD69 and MHC class II expression, and a dose- and time-dependent enhancement in IgM production. These results suggested potential benefits of IPAF to strengthen the humoral immunity.     

Synthesis and structures of three,four, and six-coordinate monomeric tin(II) and tin(IV) compounds containing η2-ketiminate ligands

A series of Sn(II) and Sn(IV) compounds containing ketiminate ligands were synthesized. Reactions of SnCl₂ with 1 or 2 equiv. Li[OCMeCHCMeNAr] (where Ar = 2,6-diisopropylphenyl) generate [OCMeCHCMeNAr]SnCl (1) and [OCMeCHCMeNAr]₂Sn (2) in moderate yield, respectively. Similarly, reacting SnCl₄ with 2 equiv. Li[OCMeCHCMeNAr] yields a six-coordinated [OCMeCHCMeNAr]₂SnCl₂ (3). Divalent tin compound 2 can be oxidized with I₂ in diethyl ether to generate tetravalent tin compound [OCMeCHCMeNAr]₂SnI₂ (4) in moderate yield. Compounds 1–4 have been characterized by ¹H and ¹³C NMR spectroscopy and have been analyzed by X-ray crystallography. Theoretical calculation found that the bonding of ketiminate ligands and tin atom in compound 2 has a strong ionic character.     

Ectopic Expression of Sweet Potato Cysteine Protease SPCP3 Alters Phenotypic Traits and Enhances Drought Stress Sensitivity in Transgenic Arabidopsis Plants

In this report sweet potato cysteine protease SPCP3 cDNAs, with or without the corresponding granulin-like domain, were overexpressed in transgenic Arabidopsis plants. Transgenic Arabidopsis plants with ectopic expression of full-length SPCP3 exhibited slight promotion of earlier floral transition from vegetative to reproductive growth and a higher percentage of yellowing siliques per plant. Transgenic progeny seeds showed similar patterns of germination rates and germination curves but lower germination percentages compared to those of wild-type control seeds. During drought treatment, photochemical F _v/F _m values and relative water content of transgenic plants were significantly reduced compared to those of wild-type controls. Transgenic Arabidopsis plants with ectopic expression of sweet potato SPCP3 with or without the corresponding C-terminal granulin-like domain exhibited similar drought-stress sensitivity patterns. Drought stress also enhanced SPCP3 gene expression, photochemical F _v/F _m reduction, and wilting in sweet potato detached leaves. Based on these data, we conclude that sweet potato granulin-containing cysteine protease SPCP3 is a functional gene, and its ectopic expression alters phenotypic traits and enhances drought-stress sensitivity in transgenic Arabidopsis plants. The presence of the C-terminal granulin-like domain has no significant influence on SPCP3-mediated drought-stress sensitivity in transgenic Arabidopsis plants.