Evaluation of orange peel-derived activated carbons for treatment of dye-contaminated wastewater tailings.

Dyes are colored compounds which are visible even at trace concentrations. Due to their recalcitrance and esthetic persistence, certain methods are unable to effectively eliminate them. So far, adsorptive treatment using activated carbons (ACs) is one of the most successful methods. In this study, we have employed orange peel (OP) as a cost-effective alternative to the expensive coal- and coir-based precursors to synthesize ACs for cationic methylene blue (MB) and anionic methyl orange (MO) dye adsorption. The pre-carbonized OP was activated via H2SO4, NaOH, KOH, ZnCl2, and H3PO4 to study the effects of activation reagents on dye removal efficiencies and mechanisms. Among several isotherm models employed to fit the adsorption data, the Langmuir and Sips models sufficiently estimated the maximum equilibrium uptakes close to the experimental values of 1012.10 ± 29.13, 339.82 ± 6.98, and 382.15 ± 8.62 mg/g, for ZnCl2-AC (MO), ZnCl2-AC (MB), and KOH-AC (MB), respectively. The adsorption mechanisms were suggested to involve electrostatic binding, pi-pi interactions, hydrogen bonding, and electron donor-acceptor reactions. Consequently, more than 99% removal efficiency was achieved from a laboratory organic wastewater sample bearing ~ 35 mg/L of MB. The results thus suggest that the synthesized ACs from agricultural waste have the tendencies to be applied to real dye wastewater treatment.

Effect of Tea Saponin-Treated Host Plants on Activities of Antioxidant Enzymes in Larvae of the Diamondback Moth Plutella xylostella (Lepidoptera: Plutellidae).

Tea saponin (TS) is extracted from the seeds of the tea plant and is generally regarded as a safe compound that has insecticidal properties and can act synergistically with other compounds. In this study, the activities of antioxidant enzymes superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and the levels of malondialdehyde (MDA) were compared in midgut tissues of third instar larvae of the diamondback moth (DBM), Plutella xylostella L. (Lepidoptera: Plutellidae). The larvae were fed on three different host plants, cabbage (Brassica oleracea L. var. capitata [Capparales: Brassicaceae]), radish (Raphanus sativus L. var. radiculus Persi [Capparales: Brassicaceae]), or rape (Brassica campestris L. [Capparales: Brassicaceae]), that had been treated with TS. Higher SOD, POD, and CAT activities were found in DBM larvae fed on cabbage after LC20 (concentration that induced 20% larval mortality) or LC50 (concentration that induced 50% larval mortality) treatment than on the control. On rape, TS treatments led to lower SOD and CAT activities than in the control and to higher POD activities after 24 h. MDA content increased in larvae fed on rape but decreased in larvae fed on radish after 12 h. Our results indicated that DBM larvae are more susceptible to TS on rape than on cabbage and radish, suggesting that this treatment may be an economic and effective means of controlling DBM on rape.

Effects of tea saponin on growth and development, nutritional indicators, and hormone titers in diamondback moths feeding on different host plant species.

The diamondback moth (DBM) is an important worldwide pest. This insect has been studied for several decades; however, its control remains problematic. Numerous chemical insecticides have become ineffective and chemical residues constitute an important problem. In the present study, we determined the mortality of 3rd instar DBM larvae feeding on three different host plant species and exposed to various concentrations of tea saponin (TS). In addition, we evaluated growth and development parameters, nutritional indicators, and juvenile hormone (JH) and molting hormone (MH) titers in 2nd instar larvae exposed to LC20 and LC50 doses of TS. We found that treatment of DBM larvae with LC20 and LC50 doses of TS led to lower growth rates, decreased feed consumption, reduced frass production, lower pupal weights, reduced percentage pupation, slower adult emergence percentages, and diminished fecundity, but prolonged durations of the larval and pupal periods. The efficiency of conversion of ingested and digested food increased, but the approximate digestibility did not differ significantly between treatments and controls. JH and MH titers were higher after TS treatment; these increases varied according to the host species and TS concentration. Our results indicate that TS represents a potential new alternative insecticide based on its natural origin, low cost, and minimum environmental impact.

Kaempferol Identified by Zebrafish Assay and Fine Fractionations Strategy from Dysosma versipellis Inhibits Angiogenesis through VEGF and FGF Pathways.

Natural products are a rich resource for the discovery of therapeutic substances. By directly using 504 fine fractions from isolated traditional Chinese medicine plants, we performed a transgenic zebrafish based screen for anti-angiogenesis substances. One fraction, DYVE-D3, was found to inhibit the growth of intersegmental vessels in the zebrafish vasculature. Bioassay-guided isolation of DYVE-D3 indicates that the flavonoid kaempferol was the active substance. Kaempferol also inhibited the proliferation and migration of HUVECs in vitro. Furthermore, we found that kaempferol suppressed angiogenesis through inhibiting VEGFR2 expression, which can be enhanced by FGF inhibition. In summary, this study shows that the construction of fine fraction libraries allows efficient identification of active substances from natural products.

Saxagliptin is similar in glycaemic variability more effective in metabolic control than acarbose in aged type 2 diabetes inadequately controlled with metformin.

This study compared the effects on glycaemic variability and glucose control between saxagliptin and acarbose as add-on therapies for aged T2DM inadequately controlled with metformin alone. The results showed that compared with acarbose-metformin, saxagliptin-metformin was more effective in glucose control with similar glycaemic variability.

The role of the DNA damage response in zebrafish and cellular models of Diamond Blackfan anemia.

Ribosomal biogenesis involves the processing of pre-ribosomal RNA. A deficiency of some ribosomal proteins (RPs) impairs processing and causes Diamond Blackfan anemia (DBA), which is associated with anemia, congenital malformations and cancer. p53 mediates many features of DBA, but the mechanism of p53 activation remains unclear. Another hallmark of DBA is the upregulation of adenosine deaminase (ADA), indicating changes in nucleotide metabolism. In RP-deficient zebrafish, we found activation of both nucleotide catabolism and biosynthesis, which is consistent with the need to break and replace the faulty ribosomal RNA. We also found upregulation of deoxynucleotide triphosphate (dNTP) synthesis - a typical response to replication stress and DNA damage. Both RP-deficient zebrafish and human hematopoietic cells showed activation of the ATR/ATM-CHK1/CHK2/p53 pathway. Other features of RP deficiency included an imbalanced dNTP pool, ATP depletion and AMPK activation. Replication stress and DNA damage in cultured cells in non-DBA models can be decreased by exogenous nucleosides. Therefore, we treated RP-deficient zebrafish embryos with exogenous nucleosides and observed decreased activation of p53 and AMPK, reduced apoptosis, and rescue of hematopoiesis. Our data suggest that the DNA damage response contributes to p53 activation in cellular and zebrafish models of DBA. Furthermore, the rescue of RP-deficient zebrafish with exogenous nucleosides suggests that nucleoside supplements could be beneficial in the treatment of DBA.

Zebrafish tyrosine hydroxylase 2 gene encodes tryptophan hydroxylase.

The primary pathological hallmark of Parkinson disease (PD) is the profound loss of dopaminergic neurons in the substantia nigra pars compacta. To facilitate the understanding of the underling mechanism of PD, several zebrafish PD models have been generated to recapitulate the characteristics of dopaminergic (DA) neuron loss. In zebrafish studies, tyrosine hydroxylase 1 (th1) has been frequently used as a molecular marker of DA neurons. However, th1 also labels norepinephrine and epinephrine neurons. Recently, a homologue of th1, named tyrosine hydroxylase 2 (th2), was identified based on the sequence homology and subsequently used as a novel marker of DA neurons. In this study, we present evidence that th2 co-localizes with serotonin in the ventral diencephalon and caudal hypothalamus in zebrafish embryos. In addition, knockdown of th2 reduces the level of serotonin in the corresponding th2-positive neurons. This phenotype can be rescued by both zebrafish th2 and mouse tryptophan hydroxylase 1 (Tph1) mRNA as well as by 5-hydroxytryptophan, the product of tryptophan hydroxylase. Moreover, the purified Th2 protein has tryptophan hydroxylase activity comparable with that of the mouse TPH1 protein in vitro. Based on these in vivo and in vitro results, we conclude that th2 is a gene encoding for tryptophan hydroxylase and should be used as a marker gene of serotonergic neurons.

[Study on absorption ingredients of yuanhuzhitong pill by everted intestinal sac method].

OBJECTIVE: To establish an everted intestinal sac method for determining absorption ingredients of yuanhuzhitong pill and study absorption characteristics of major chemical constituents of yuanhuzhitong pill. METHOD: The everted intestinal sac model was adopted. Intestinal sac fluid samples at different time points after administration of three concentrations of Yuanhuzhitong pill were collected and determined by HPLC. The accumulative absorbed doses of active constituents were calculated, while the proportion between samples of yuanhuzhitong pill and absorption ingredients was compared. RESULT: Eight ingredients of Yuanhuzhitong pill can be detected in intestinal sac, they are protopine, palmatine, coptisine, imperatorin, berberine, byakangelicin, alpha-allocryptopine and tetrahydropalmatine. The absorption rate constants (Ka) of eight constituents increased in jejunum and ileum with the increase in concentration of Yuanhuzhitong pill extracts (P < 0. 05), indicating a passive absorption. CONCLUSION: As ingredients are selectively absorbed in intestinal sac, the everted intestinal sac method is selected to assess the intestinal absorption characteristics of ingredients of Yuanhuzhitong prescription.

[Application of zebrafish models in drug screening].

Due to its small size, fast external development, transparent embryos, and amenability to genetic analysis, zebrafish has become an ideal vertebrate animal model. In addition to studies in genetics and developmental biology, zebrafish has also been widely used in human disease modeling and drug screening. As a small whole-organism model, zebrafish can be used to comprehensively test and evaluate the activity and side effect of a compound at the same time, fulfilling high content screening. Recently, new zebrafish disease models and screening technologies have been developed. A number of active compounds were identified and most of them have similar functions in mammal models. One compound prostaglandin E2 has been subjected to clinical trial to test if it can promote the growth of umbilical cord blood units after transplantation. Another compound leflunomide has also been approved in clinical trial to cure melanoma in combination with vemurafenib. These findings demonstrate that zebrafish model is appropriate for drug screening. This review summarizes the unique features of zebrafish model and the recent progresses of zebrafish based drug screening.

High-throughput screening for bioactive molecules using primary cell culture of transgenic zebrafish embryos.

Transgenic zebrafish embryos expressing tissue-specific green fluorescent protein (GFP) can provide an unlimited supply of primary embryonic cells. Agents that promote the differentiation of these cells may be beneficial for therapeutics. We report a high-throughput approach for screening small molecules that regulate cell differentiation using lineage-specific GFP transgenic zebrafish embryonic cells. After validating several known regulators of the differentiation of endothelial and other cell types, we performed a screen for proangiogenic molecules using undifferentiated primary cells from flk1-GFP transgenic zebrafish embryos. Cells were grown in 384-well plates with 12,128 individual small molecules, and GFP expression was analyzed by means of an automated imaging system, which allowed us to screen thousands of compounds weekly. As a result, 23 molecules were confirmed to enhance angiogenesis, and 11 of them were validated to promote the proliferation of mammalian human umbilical vascular endothelial cells and induce Flk1+ cells from murine embryonic stem cells. We demonstrated the general applicability of this strategy by analyzing additional cell lineages using zebrafish expressing GFP in pancreatic, cardiac, and dopaminergic cells.

Chemical screening with zebrafish embryos.

Functional chemicals are very useful tools for molecular biology studies. Due to its small size, large progeny clutch, and embryonic transparency, zebrafish serves as a superb in vivo animal model for chemical compound screens and characterization. During zebrafish embryogenesis, multiple developmental phenotypes can be easily examined under the microscope, therefore allowing a more comprehensive evaluation for identifying novel functional chemicals than cell-based assays. Ever since the first zebrafish-based chemical screen was conducted in the year 2000, many functional chemicals have been discovered using this strategy. In this chapter, we describe how to perform a typical zebrafish-based chemical screen and discuss the details of the protocol by using the example of the identification and characterization of two new Smo inhibitors with a Gli:GFP transgenic line.

Use of a high-throughput screening approach coupled with in vivo zebrafish embryo screening to develop hazard ranking for engineered nanomaterials.

Because of concerns about the safety of a growing number of engineered nanomaterials (ENM), it is necessary to develop high-throughput screening and in silico data transformation tools that can speed up in vitro hazard ranking. Here, we report the use of a multiparametric, automated screening assay that incorporates sublethal and lethal cellular injury responses to perform high-throughput analysis of a batch of commercial metal/metal oxide nanoparticles (NP) with the inclusion of a quantum dot (QD1). The responses chosen for tracking cellular injury through automated epifluorescence microscopy included ROS production, intracellular calcium flux, mitochondrial depolarization, and plasma membrane permeability. The z-score transformed high volume data set was used to construct heat maps for in vitro hazard ranking as well as showing the similarity patterns of NPs and response parameters through the use of self-organizing maps (SOM). Among the materials analyzed, QD1 and nano-ZnO showed the most prominent lethality, while Pt, Ag, SiO2, Al2O3, and Au triggered sublethal effects but without cytotoxicity. In order to compare the in vitro with the in vivo response outcomes in zebrafish embryos, NPs were used to assess their impact on mortality rate, hatching rate, cardiac rate, and morphological defects. While QDs, ZnO, and Ag induced morphological abnormalities or interfered in embryo hatching, Pt and Ag exerted inhibitory effects on cardiac rate. Ag toxicity in zebrafish differed from the in vitro results, which is congruent with this material's designation as extremely dangerous in the environment. Interestingly, while toxicity in the initially selected QD formulation was due to a solvent (toluene), supplementary testing of additional QDs selections yielded in vitro hazard profiling that reflect the release of chalcogenides. In conclusion, the use of a high-throughput screening, in silico data handling and zebrafish testing may constitute a paradigm for rapid and integrated ENM toxicological screening.

Genetic approach to evaluate specificity of small molecule drug candidates inhibiting PLK1 using zebrafish.

During the preclinical drug discovery process it remains a challenge to enable early elimination of candidate molecules that may have non-specific, off-target activities. Here, we use whole zebrafish embryo assays coupled with genetic analysis to address this issue. PLK1 (Polo-like kinase 1) is one of the key regulators that control mitotic entry, spindle assembly, chromosome segregation, and cytokinesis in the cell cycle. Since plk1 expression is abnormally up-regulated in several tumors, it is regarded as a good target for cancer therapy. A number of small-molecule inhibitors targeting PLK1 have been developed as reagents and anticancer drug candidates. It will be interesting to determine if these inhibitors indeed specifically target PLK1 in vivo. Bioinformatics analysis revealed that the zebrafish and human genomes share high homology across all PLK family members. In particular, PLK1 has a nearly identical 3-D structure between zebrafish and human. We selected three published PLK1 inhibitors, LFM-A13, ON01910, and thiazole-carboxamide 10A in our assay. When added at 2-cell stage, all of these inhibitors prevented embryos from dividing and caused cells to fuse into one large cell. When added at the later stage during zygotic mRNA transcription program initiation, embryos survived for 3 days but showed different phenotypes for each compound. Embryos treated with LFM-A13 appeared relatively normal. Embryos treated with ON01910 failed to properly develop trunk and tail regions while the head structure was unaffected. Embryos treated with thiazole-carboxamide 10A had a shorter body axis and deformed head structure. To determine which inhibitor is more selectively targeting PLK1, we inhibited PLK1 activity using anti-sense morpholino. Comparative analysis indicated that thiazole-carboxamide 10A could faithfully phenocopy zebrafish embryos genetically deficient of plk1. These findings demonstrate that these three PLK1 inhibitors, although well established by in vitro studies, have different off-target activities in vivo, and that thiazole-carboxamide 10A appears most specific to PLK1. Our studies suggest that zebrafish should be generally useful as an efficient in vivo model to evaluate specificity of small molecules designed to regulate any conserved target proteins through comparative analysis of genetic phenotypes.

Microarray analysis of zebrafish cloche mutant using amplified cDNA and identification of potential downstream target genes.

Zebrafish is an excellent model organism for studying vertebrate development and human disease. With the availability of increased numbers of zebrafish mutants and microarray chips, gene expression profiling has become a powerful tool for identification of downstream target genes perturbed by a specific mutation. One of the obstacles often encountered, however, is to isolate large numbers of zebrafish mutant embryos that are indistinguishable in morphology from the wild-type siblings for microarray analysis. Here, we report a method using amplified cDNA derived from five embryos for gene expression profiling of the 18-somite zebrafish cloche (clo) mutant, in which development of hematopoietic and endothelial lineages is severely impaired. In total, 31 differentially expressed target genes are identified, of which 13 have not been reported previously. We further determine that of these 13 new targets, 8 genes, including coproporphyrinogen oxidase (cpo), carbonic anhydrase (cahz), claudin g (cldn g), zinc-finger-like gene 2 (znfl2), neutrophil cytosol factor 1 (ncf1), matrix metalloproteinase 13 (mmp13), dual specificity phosphatase 5 (dusp5), and a novel gene referred as zebrafish vessel-specific gene 1 (zvsg1) are predominantly expressed in hematopoietic and endothelial cells. Comparative analysis demonstrates that this method is comparable and complementary to that of the conventional approach using unamplified sample. Our study provides valuable information for studying hematopoiesis and vessel formation. The method described here offers a powerful tool for gene expression profiling of zebrafish mutants in general.

Induction of reversible hemolytic anemia in living zebrafish using a novel small molecule.

We used zebrafish to screen and identify small molecules that affect the process of vertebrate hematopoietic development. Zebrafish embryos were exposed to a library of 5000 synthetic compounds and screened for defects in primitive erythropoiesis. Here, we present the characterization of hemolytic anemia induced in zebrafish by the small molecule 5115318 (3-[5-methyl-furan 2-yl]-propionic acid N'-phenyl-hydrazide). This compound is capable of generating hemoglobin aggregates and Heinz bodies in red cells in vivo only. The induced anemia is reversible and treated fish recover in about 4 days. This study shows the feasibility of using zebrafish to screen for small molecules that can modulate the specific process of erythropoiesis.

IGF-I signaling prevents dehydroepiandrosterone (DHEA)-induced apoptosis in hypothalamic neurons.

Dehydroepiandrosterone (DHEA) is synthesized in the brain, but whether DHEA is involved in modulating neuronal cell survival is not yet fully understood. Herein we show that when deprived of trophic support, GT1-7 hypothalamic neurons undergo apoptosis following exposure to DHEA, as demonstrated both by morphological and biochemical criteria. This proapoptotic effect appeared to be specific to DHEA itself, and not through conversion of DHEA to other steroids such as androgen or estrogen. Importantly, we determined that IGF-I protects GT1-7 neurons from DHEA-induced cell death. DHEA-induced apoptosis was associated with increased activation of caspase 3 and decreased PARP, which were both attenuated with addition of IGF-I. Addition of DHEA prevented phosphorylation of both Akt and glycogen synthase kinase-3 beta (GSK-3beta), downstream effector molecules of the phosphatidylinositol 3-kinase (PI3K) pathway. Further IGF-I was able to sustain Akt activity and thus preventing GSK-3beta activation in the presence of DHEA. On the other hand, the MAP kinases, ERK, p38, and JNK, were not affected by DHEA. These findings suggest that in GT1-7 hypothalamic neurons, DHEA acts detrimentally to induce cell death and IGF-I is able to rescue the neurons by preserving the activity of Akt, and therefore maintaining the proapoptotic kinase GSK-3beta, in a phosphorylated catalytically inactive state.

Evidence that dehydroepiandrosterone, DHEA, directly inhibits GnRH gene expression in GT1-7 hypothalamic neurons.

Dehydroepiandrosterone (DHEA) has been reported to have diverse effects on overall physiology, although its mechanism of action and specific receptor are not yet known. We have used the immortalized, clonal GT1-7 hypothalamic neurons to study DHEA effects on gonadotropin-releasing hormone (GnRH) gene expression. DHEA (10(-4) M) downregulates GnRH transcription by 39, 70 and 83% at 24, 36, and 48 h, respectively, while DHEA-sulphate had no effect. Hydroxyflutamide a specific androgen receptor (AR) antagonist, and cyproterone acetate or trilostane, both inhibitors of 3 beta-hydroxysteroid dehydrogenase/delta 4,5 isomerase, the rate-limiting enzyme for the conversion of DHEA to sex steroids, did not affect the ability of DHEA to downregulate GnRH gene expression. We found that GT1-7 cells did not express aromatase, thereby precluding conversion to estrogen. Analysis of [(14)C] DHEA metabolism by thin layer chromatography indicates that the main metabolites produced are 7 alpha- and 7 beta-hydroxy DHEA, and 7-oxo DHEA, although these steroids were not able to repress GnRH gene expression alone. Cell viability studies indicated that the transcriptional repression observed is not due to GT1-7 cell death. Interestingly, SV40 T-antigen mRNA levels, under the control of 2.3 kb of the rat GnRH gene 5' regulatory region, are also repressed by DHEA. Our studies indicate that DHEA has direct effects on GnRH transcription that appear to be unique from those observed after conversion to other steroidogenic compounds.