Alternative Splicing Regulation of Low-Frequency Genetic Variants in Exon 2 of TREM2 in Alzheimer's Disease by Splicing-Based Aggregation.

TREM2 is among the most well-known Alzheimer's disease (AD) risk genes; however, the functional roles of its AD-associated variants remain to be elucidated, and most known risk alleles are low-frequency variants whose investigation is challenging. Here, we utilized a splicing-guided aggregation method in which multiple low-frequency TREM2 variants were bundled together to investigate the functional impact of those variants on alternative splicing in AD. We analyzed whole genome sequencing (WGS) and RNA-seq data generated from cognitively normal elderly controls (CN) and AD patients in two independent cohorts, representing three regions in the frontal lobe of the human brain: the dorsolateral prefrontal cortex (CN = 213 and AD = 376), frontal pole (CN = 72 and AD = 175), and inferior frontal (CN = 63 and AD = 157). We observed an exon skipping event in the second exon of TREM2, with that exon tending to be more frequently skipped (p = 0.0012) in individuals having at least one low-frequency variant that caused loss-of-function for a splicing regulatory element. In addition, genes differentially expressed between AD patients with high vs. low skipping of the second exon (i.e., loss of a TREM2 functional domain) were significantly enriched in immune-related pathways. Our splicing-guided aggregation method thus provides new insight into the regulation of alternative splicing of the second exon of TREM2 by low-frequency variants and could be a useful tool for further exploring the potential molecular mechanisms of multiple, disease-associated, low-frequency variants.

Taurodeoxycholate Increases the Number of Myeloid-Derived Suppressor Cells That Ameliorate Sepsis in Mice.

Bile acids (BAs) control metabolism and inflammation by interacting with several receptors. Here, we report that intravenous infusion of taurodeoxycholate (TDCA) decreases serum pro-inflammatory cytokines, normalizes hypotension, protects against renal injury, and prolongs mouse survival during sepsis. TDCA increases the number of granulocytic myeloid-derived suppressor cells (MDSCLT) distinctive from MDSCs obtained without TDCA treatment (MDSCL) in the spleen of septic mice. FACS-sorted MDSCLT cells suppress T-cell proliferation and confer protection against sepsis when adoptively transferred better than MDSCL. Proteogenomic analysis indicated that TDCA controls chromatin silencing, alternative splicing, and translation of the immune proteome of MDSCLT, which increases the expression of anti-inflammatory molecules such as oncostatin, lactoferrin and CD244. TDCA also decreases the expression of pro-inflammatory molecules such as neutrophil elastase. These findings suggest that TDCA globally edits the proteome to increase the number of MDSCLT cells and affect their immune-regulatory functions to resolve systemic inflammation during sepsis.

Zebrafish Larvae Model of Dilated Cardiomyopathy Induced by Terfenadine.

BACKGROUND AND OBJECTIVES: Dilated cardiomyopathy can be the end-stage of severe cardiac disorders and directly affects the cardiac muscle, inducing cardiomegaly and heart failure (HF). Although a wide variety of animal models are available to study dilated cardiomyopathy, there is no model to assess dilated cardiomyopathy with non-invasive, simple, and large screening methods. METHODS: We developed a dilated cardiomyopathy model in zebrafish larvae using short duration terfenadine, a known cardiotoxic drug that induces ventricular size dilation. Fractional shortening of zebrafish hearts was calculated. RESULTS: We treated zebrafish with 5 to 10 µM terfenadine for 24 hours. In terfenadine-treated zebrafish, blood frequently pooled and clotted in the chamber, and circulation was remarkably reduced. Atria and ventricles were swollen, and fluid was deposited around the heart, mimicking edema. Cardiac contractility was significantly reduced, and ventricular area was significantly enlarged. Heart rate was markedly reduced even after terfenadine withdrawal. Acridine orange staining also showed that terfenadine increased cardiomyocyte apoptosis. A significant increase of natriuretic peptide B (NPPB) mRNA was found in terfenadine-treated zebrafish. A low dose of terfenadine (5-10 µM) did not show mortality in short-term treatment (24 hours). However, moderate dose (35-45 µM) terfenadine treatment reduced zebrafish survival within 1 hour. CONCLUSION: With advantages of rapid sample preparation procedure and transparent observation of the live heart, this model can potentially be applied to large-scale drug screening and toxicity assays for non-ischemic HF.

Exploration and comparison of in vitro eye irritation tests with the ISO standard in vivo rabbit test for the evaluation of the ocular irritancy of contact lenses.

In an effort to explore the use of alternative methods to animal testing for the evaluation of the ocular irritancy of medical devices, we evaluated representative contact lenses with the bovine corneal opacity and permeability test (BCOP) and an in vitro eye irritation test using the three-dimensionally-reconstructed human corneal epithelium (RhCE) models, EpiOcular™ and MCTT HCE™. In addition, we compared the obtained results with the ISO standard in vivo rabbit eye irritation test (ISO10993-10). Along with the positive controls (benzalkonium chloride, BAK, 0.02, 0.2, and 1%), the extracts of 4 representative contact lenses (soft, disposable, hard, and colored lenses) and 2 reference lenses (dye-eluting and BAK-coated lenses) were tested. All the lenses, except for the BAK-coated lens, were determined non-irritants in all test methods, while the positive controls yielded relevant results. More importantly, BCOP, EpiOcular™, and MCTT HCE™ yielded a consistent decision for all the tested samples, with the exception of 0.2% BAK in BCOP, for which no prediction could be made. Overall, all the in vitro tests correlated well with the in vivo rabbit eye irritation test, and furthermore, the combination of in vitro tests as a tiered testing strategy was able to produce results similar to those seen in vivo. These observations suggest that such methods can be used as alternative assays to replace the conventional in vivo test method in the evaluation of the ocular irritancy of ophthalmic medical devices, although further study is necessary.

An in vitro model of granuloma-like cell aggregates substantiates early host immune responses against Mycobacterium massiliense infection.

Mycobacterium massiliense (M. mass), belonging to the M. abscessus complex, is a rapidly growing mycobacterium that is known to cause tuberculous-like lesions in humans. To better understand the interaction between host cells and M. mass, we used a recently developed in vitro model of early granuloma-like cell aggregates composed of human peripheral blood mononuclear cells (PBMCs). PBMCs formed granuloma-like, small and rounded cell aggregates when infected by live M. mass Microscopic examination showed monocytes and macrophages surrounded by lymphocytes, which resembled cell aggregation induced by M. tuberculosis (M. tb). M. mass-infected PBMCs exhibited higher expression levels of HLA-DR, CD86 and CD80 on macrophages, and a significant decrease in the populations of CD4+ and CD8+ T cells. Interestingly, low doses of M. mass were sufficient to infect PBMCs, while active host cell death was gradually induced with highly increased bacterial loads, reflecting host destruction and dissemination of virulent rapid-growing mycobacteria (RGM). Collectively, this in vitro model of M. mass infection improves our understanding of the interplay of host immune cells with mycobacteria, and may be useful for developing therapeutics to control bacterial pathogenesis.

Mycobacterium massiliense Induces Macrophage Extracellular Traps with Facilitating Bacterial Growth.

Human neutrophils have been known to release neutrophil extracellular traps (NETs), antimicrobial DNA structures capable of capturing and killing microbes. Recently, a similar phenomenon has been reported in macrophages infected with various pathogens. However, a role for macrophages extracellular traps (METs) in host defense responses against Mycobacterium massiliense (M. mass) has yet to be described. In this study, we show that M. mass, a rapid growing mycobacterium (RGM), also induces the release of METs from PMA-differentiated THP-1 cells. Intriguingly, this process is not dependent on NADPH oxidase activity, which regulates NET formation. Instead, M. mass-induced MET formation partially depends on calcium influx and requires phagocytosis of high bacterial load. The METs consist of a DNA backbone embedded with microbicidal proteins such as histone, MPO and elastase. Released METs entrap M. mass and prevent their dissemination, but do not have bactericidal activity. Instead, they result in enhanced bacterial growth. In this regard, METs were considered to provide interaction of M. mass with cells and an environment for bacterial aggregation, which may facilitate mycobacterial survival and growth. In conclusion, our results demonstrate METs as an innate defense response against M. mass infection, and suggest that extracellular traps play a multifaceted role in the interplay between host and bacteria.

Male-specific hepatitis B virus large surface protein variant W4P potentiates tumorigenicity and induces gender disparity.

BACKGROUND: The underlying mechanisms of carcinogenesis and gender disparity in hepatitis B virus (HBV)-induced hepatocellular carcinoma (HCC) remain unclear. Recently, we reported a novel HCC-related W4P/R mutation in the large surface protein (LHB) of HBV genotype C, which was found exclusively in male HCC patients. METHODS: LHB sequences from a carrier (wild type; WT) and W4P variant LHB sequence from an HCC patient were cloned and used to generate NIH3T3 and Huh7 cell lines. Cell proliferation and in vitro tumorigenicity were assessed by cell growth and transformation assays. Male and female nude mice were injected with the cells to determine in vivo tumorigenicity. To confirm the effect of estrogen in W4P-mediated tumorigenicity, male mice were injected with estrogen and challenged with W4P-expressing cells. The serum levels of different cytokines from the mouse model and patients were analyzed by ELISA. A critical role of interleukin (IL)-6 signaling in W4P-mediated tumorigenicity was tested by inhibition of Jak2. RESULTS: Although both WT and W4P variant LHBs enhanced cell proliferation by regulating the cell cycle and facilitated cell colony formation, the W4P variant demonstrated significantly higher activity. NIH3T3 cells expressing variant LHB, but not the WT, induced tumor in a nude mouse model. Tumor masses produced by variant LHB were significantly larger in male than female mice, and significantly reduced by estrogen. IL-6, but not tumor necrosis factor-α, was elevated in male mice harboring W4P-induced tumor, and was reduced by estrogen. IL-6 levels of HCC patients with the W4P variant were significantly higher than those of patients with WT LHB. W4P LHB induced higher production of IL-6 than WT LHB in cell lines, and the level was reduced by estrogen. The ability to reduce cell proliferation and colony formation of W4P LHB was hampered by inhibition of IL-6 signaling. CONCLUSIONS: This study suggests that the W4P mutation during the natural course of chronic hepatitis B infection may contribute to HCC development, particularly in male patients, in an IL-6-dependent manner.

Interleukin-6-induced Twist and N-cadherin enhance melanoma cell metastasis.

Melanoma patients frequently have elevated serum levels of interleukin-6 (IL-6), which is correlated with a poor prognosis. IL-6 activates STAT3 phosphorylation, inducing the transcription of genes that regulate tumor cell proliferation and antiapoptosis. In addition, recent evidence suggests that IL-6 induces the epithelial-to-mesenchymal transition and enhances the invasiveness of tumor cells of epithelial origin. However, it is unknown whether IL-6 affects mesenchymal tumor cells. In this study, we examined the effects of IL-6 on melanoma cells and found that IL-6 can enhance their metastatic potential by regulating the expression of Twist and N-cadherin. First, we confirmed that human melanoma tissues express IL-6 (especially at the lesion site), the IL-6 receptor, N-cadherin, and nuclear Twist. Next, we found that IL-6 induces STAT3 phosphorylation in WM-266-4 human melanoma cells, resulting in transient upregulation of Twist, which is a key regulator of metastasis. Importantly, the expression of N-cadherin, a protein downstream of Twist, was also increased on the cell surface after treatment with IL-6. These cells showed enhanced invasiveness, assessed using an invasion assay, and formed more metastatic nodules in the lungs of NOD-SCID mice after an intravenous injection. Importantly, melanoma cells with knocked-down N-cadherin formed less lung nodules compared with control in the NOD-SCID mouse model. Our data suggest that increased serum IL-6 in cancer patients could increase the invasiveness of melanoma cells and accelerate metastasis. Blocking IL-6 in the melanoma microenvironment may therefore inhibit disease progression.

High cholesterol diet induces IL-1ß expression in adult but not larval zebrafish.

Recently, it has been demonstrated that high cholesterol diet induced hypercholesterolemia and vascular lipid oxidation and accumulation in zebrafish larvae, suggesting that zebrafish is a new promising atherosclerosis model in addition to mouse models. However, up to date, there was no report regarding inflammatory cytokine expression during the lipid accumulation in zebrafish larva and adult fish. In this study, we first demonstrated the expression levels of IL-1ß and TNF-α in high cholesterol diet (HCD)-fed zebrafish larvae, and found that although HCD induced vascular lipid accumulation, the cytokine expressions in the larvae were not changed by HCD. Furthermore, there was no significant difference in leukocyte accumulation in vessels between control and HCD fed group. But prolonged HCD induced IL-1ß expression in spleen and liver compared to those of control zebrafish, and produced very early stage of fatty streak lesion in dorsal aorta of 19 week HCD-fed zebrafish. These results indicate that HCD induced hypercholesterolemia and atherosclerotic changes in zebrafish are very early stage, and suggest the necessity of the generation of mutant zebrafish having a disruption in a lipid metabolism-related gene leading to severe hypercholesterolemia and advanced atherosclerosis.

Orthotopic transplantation of retinoblastoma cells into vitreous cavity of zebrafish for screening of anticancer drugs.

BACKGROUND: With high throughput screening, novel therapeutic agents can be efficiently identified. Unfortunately, researchers only resort to in vitro cell viability assays for screening of anticancer drugs for retinoblastoma, the most common intraocular cancer in the childhood. Current available animal models of retinoblastoma require more than 2 weeks for tumour formation and the investigation of the efficacy of therapeutic agents. In this study, we established a novel orthotopic transplantation model of retinoblastoma in zebrafish as an in vivo animal model for screening of anticancer drugs. METHODS: We injected retinoblastoma cells into the vitreous cavity of zebrafish at 48 hours after fertilization. Eyeballs of zebrafish were scanned daily under the confocal laser microscope, and the tumor population was quantitatively analyzed by measuring the mean intensity of green fluorescent protein (GFP). Transplanted retinoblastoma cells were isolated to perform further analyses including Western blotting and reverse transcriptase-polymerase chain reaction to confirm that retinoblastoma cells maintained their characteristics as tumor cells even after transplantation and further isolation. To figure out the potential of this model for screening of anticancer drugs, zebrafish were cultured in Ringer's solution containing carboplatin and melphalan after the injection of retinoblastoma cells. RESULTS: The degree of the tumor population was dependent on the number of retinoblastoma cells injected and maintained stably for at least 4 days. Transplanted retinoblastoma cells maintain their proliferative potential and characteristics as retinoblastoma cells after isolation. Interestingly, systemic application of carboplatin and melphalan demonstrated significant reduction in the tumor population, which could be quantitatively analyzed by the estimation of the mean intensity of GFP. CONCLUSIONS: This orthotopic retinoblastoma model in zebrafish is expected to be utilized for the screening of anticancer drugs for the treatment of retinoblastoma.

Angiopoietin-1 elicits pro-inflammatory responses in monocytes and differentiating macrophages.

The angiopoietin/Tie2 system is an important regulator of angiogenesis and inflammation. In addition to its functions in endothelial cells, Tie2 expression on non-endothelial cells allows for angiopoietin ligands to stimulate the cells. Although Ang1 is a strong Tie2 receptor agonist, little is known regarding the effect of Ang1 on non-endothelial cells, such as monocytes and macrophages. In this study, we found that Ang1 functionally binds to and stimulates monocytes via p38 and Erk1/2 phosphorylation. Ang1-mediated monocyte stimulation is associated with proinflammatory cytokine TNF-α expression. We also determined that Ang1 switched macrophage differentiation toward a pro-inflammatory phenotype, even in the presence of an anti-inflammatory mediator. These findings suggest that Ang1 plays a role in stimulating pro-inflammatory responses and could provide a new strategy by which to manage inflammatory responses.

Cyclooxygenase-2 inhibition blocks M2 macrophage differentiation and suppresses metastasis in murine breast cancer model.

Tumor cells are often associated with abundant macrophages that resemble the alternatively activated M2 subset. Tumor-associated macrophages (TAMs) inhibit anti-tumor immune responses and promote metastasis. Cyclooxygenase-2 (COX-2) inhibition is known to prevent breast cancer metastasis. This study hypothesized that COX-2 inhibition affects TAM characteristics potentially relevant to tumor cell metastasis. We found that the specific COX-2 inhibitor, etodolac, inhibited human M2 macrophage differentiation, as determined by decreased CD14 and CD163 expressions and increased TNFα production. Several key metastasis-related mediators, such as vascular endothelial growth factor-A, vascular endothelial growth factor-C, and matrix metalloproteinase-9, were inhibited in the presence of etodolac as compared to untreated M2 macrophages. Murine bone marrow derived M2 macrophages also showed enhanced surface MHCII IA/IE and CD80, CD86 expressions together with enhanced TNFα expressions with etodolac treatment during differentiation. Using a BALB/c breast cancer model, we found that etodolac significantly reduced lung metastasis, possibly due to macrophages expressing increased IA/IE and TNFα, but decreased M2 macrophage-related genes expressions (Ym1, TGFß). In conclusion, COX-2 inhibition caused loss of the M2 macrophage characteristics of TAMs and may assist prevention of breast cancer metastasis.

Effects of dietary high fat on prostate intraepithelial neoplasia in TRAMP mice.

Increased fat intake is known to be a major cause of prostate cancer. In this study, we investigated the effect of dietary high fat on prostate intraepithelial neoplasia using transgenic adenocarcinoma mouse prostate (TRAMP) mice. Six-week-old male TRAMP mice were fed AIN93G (control group, 4.0 kcal/kg, n=6) and AIN93G-HFD (experimental group, 4.8 kcal/kg, n=7) for 10 weeks. Prostate histopathology, urogenital tract (UGT) weight, epididymal white adipose tissue weight, argyrophilic nucleolar organizer regions (AgNORs) counts, and serum leptin levels were examined. AIN93G-HFD fed group showed progressed neoplastic lesions in the prostate (P<0.05) compared to AIN93G fed group. AIN93G-HFD intake resulted in a increase in the weight of UGT (P<0.05) and epididymal white adipose tissue. The number of Ag-NOR positive dots significantly increased in each prostate lobe and final serum leptin levels in AIN93G-HFD fed group were about twice those of AIN93G fed group (P<0.05). Dietary high fat was related to the prostate cancer progression in the early stage of TRAMP mice and increased serum leptin levels, suggesting that the regulation of dietary components could delay the progression of prostate cancer.

Rat pancreatitis produced by 13-week administration of zinc oxide nanoparticles: biopersistence of nanoparticles and possible solutions.

Zinc oxide (ZnO) nanoparticles (NPs) are used in diverse applications ranging from paints and cosmetics to biomedicine and food. Although micron-sized ZnO is a traditional food supplement, ZnO NPs are an unknown public health risk because of their unique physicochemical properties. Herein, we studied the 13-week subchronic toxicity of ZnO NPs administered via the oral route according to Organization for Economic Cooperation and Development (OECD) test guideline 408. Well-dispersed ZnO NPs were administered to Sprague-Dawley (SD) rats (11/sex/group) at doses of 67.1, 134.2, 268.4 or 536.8 mg kg(-1) per body weight over a 13-week period. The mean body weight gain in males given 536.8 mg kg(-1) ZnO NPs was significantly lower than that of control male rats, whereas no significant differences were observed between the other treatment groups and the controls. Male and female rats dosed at 536.8 mg kg(-1) ZnO NPs had significant changes in anemia-related hematologic parameters. Mild to moderate pancreatitis also developed in both sexes dosed at 536.8 mg kg(-1) , whereas no histological changes were observed in the other treatment groups. To evaluate the mechanism of toxicity, we performed a bio-persistence study and evaluated the effects of the ZnO NPs on cell proliferation. The treatment of a human gastric adenocarcinoma cell line with ZnO NPs resulted in a significant inhibition of cellular proliferation. The anti-proliferative effect of ZnO NPs or Zn(2+) was effectively blocked by treatment with chelators. These results indicate that the bio-persistence of ZnO NPs after ingestion is key to their toxicity; the no-observed-adverse effect level (NOAEL) of ZnO NPs was found to be 268.4 mg kg(-1) per day for both sexes.

Differentiation of skin sensitizers from irritant chemicals by interleukin-1α and macrophage inflammatory protein-2 in murine keratinocytes.

The development of novel alternative testing methods is required to identify the sensitizing capacity of chemicals as a replacement for animal experimentation. We aimed to evaluate in vitro assays as screening tools for detecting skin sensitizers. The murine epidermal keratinocyte cell line HEL-30 was exposed to 16 relevant skin sensitizers and 6 skin irritants. The dose causing 75% cell viability (CV(75)) measured by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was chosen as a highest dose and three more doses (0.5×, 0.1×, and 0.01× of CV(75)) were tested. As parameters, levels of interleukin 1α (IL-1α), macrophage inflammatory protein 2 (MIP-2), IL-6, and IL-18 production were measured using 4 different doses. The accuracy of detecting sensitizers or irritants by IL-1α or MIP-2 alone was exactly same: 75% (12 out of 16) for sensitizers, 83% (5 out of 6) for irritants, and overall 77% (17 out of 22). However, combination of IL-1α and MIP-2 showed better accuracy: 94% (15 out of 16), 67% (4 out of 6), and overall 86% (19 out of 22). IL-6 and IL-18 could not differentiate sensitizers from irritants. This study suggests that the combination of pro-inflammatory cytokines IL-1α and MIP-2 in murine HEL-30 cells can be a reliable in vitro method for identifying chemicals that may act as skin sensitizers.

Prevalence of murine norovirus infection in Korean laboratory animal facilities.

Currently, murine noroviruses (MNV) are the most prevalent viral pathogens identified in laboratory animal facilities. While several reports exist concerning the prevalence of MNV in North American research facilities, very few reports are available for other parts of the world, including Korea. This study evaluated the prevalence of MNV infection in 745 murine sera collected from 15 animal facilities in Korea by enzyme linked immunosorbent assay (ELISA). Positive cases were subcategorized by murine strain/genetics, housing environments and animal sources. In summary, 6.6% of inbred/outbred mice purchased from commercial vendors were seropositive, 9.6% of in-house colonies were seropositive and 27.0% of genetically modified mice (GMM) were seropositive. Partial gene amplification of fecal isolates from infected animals showed that they were homologous (100%) with MNV-4.

Microbiological quality assessment of laboratory mice in Korea and recommendations for quality improvement.

Regular monitoring of commercial laboratory rodents and institutional research animal residents is essential for microbiological quality control programs. The objective of our study was to investigate the recent prevalence of infectious pathogens in laboratory mice from eight experimental animal vendors and 56 institutional animal facilities in Korea. Our investigation was conducted in 2006-2007. Specific Pathogen Free (SPF) mice from four commercial breeders were clean according to serological, bacteriological, parasitological, and histopathological examination results. However, mice from one intermediate vendor that distributed SPF animals from main commercial vendors to local districts had Syphacia obvelata and Mycoptes musculinus infections. Additionally, mice from conventional animal breeders were highly contaminated. Among the 56 institutional animal facilities, mouse hepatitis virus (MHV), Sendai virus and Mycoplasma pulmonis positive results were obtained in 23.2, 8.9, and 1.8% of animals tested, respectively. These results indicate that quarantine and eradication efforts of infectious pathogens in these facilities are sub-optimal and need to be improved. The use of commercial conventional mice for research should be eliminated and appropriate vendor selection as well as thorough quarantine before releasing animals into a facility are needed. Finally we suggest qualified veterinary experts are needed at each animal facility to ensure an adequate health surveillance program.

Cre/loxP-regulated transgenic zebrafish model for neural progenitor-specific oncogenic Kras expression.

Ras proteins regulate signaling pathways that control many cellular responses, such as proliferation, survival, and differentiation. However, there are intriguing questions about the relationship between the developmental timing of specific mutations and the resultant phenotypes in individual cells. In this study, we used the Cre/loxP system for maintaining transgenic zebrafish lines harboring oncogenic Kras(V12) under the nestin promoter, and investigated the developmental effects of Ras activation in neural progenitor cells. Activated human Kras(V12) was induced within pDSNesLCherryLEGFPKRas(V12) transgenic fish by Cre mRNA injection. Cre-mediated gene excision was confirmed by polymerase chain reaction, and the injected embryos were investigated for Kras(V12) effects using the hemotoxylin-eosin staining, terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling assay, and in situ hybridization. pDSNesLCherryLEGFPKRas(V12) transgenic embryos normally expressed mCherry in their central nervous system throughout the developmental stage. However, Cre mRNA injection efficiently excised the flanking stop sequence, and the injected embryos expressed enhanced green fluorescent protein in their brain with severe edema. Brain histology showed that neuronal cell differentiation could occur in spite of oncogenic Kras(V12) overexpression, but massive apoptosis and brain edema caused early embryonal death. In summary, the overexpression of Kras(V12) induces extensive apoptosis of neural progenitor cells followed by severe edema of the brain. However, some neural progenitor cells are resistant to Kras(V12) and can retain their ability to differentiate into neurons. Finally, our transgenic model demonstrates the inability of Kras(V12) alone to induce brain tumors at the early stage of development.

Estrogen-responsive transient expression assay using a brain aromatase-based reporter gene in zebrafish (Danio rerio).

Whereas endogenous estrogens play an important role in the development, maintenance, and function of female and male reproductive organs, xenoestrogens present in the environment disrupt normal endocrine function in humans and wildlife. Various in vivo and in vitro assays have been developed to screen these xenoestrogens. However, traditional in vivo assays are laborious and unsuitable for large-scale screening, and in vitro assays do not necessarily replicate in vivo functioning. To overcome these limitations, we developed a transient expression assay in zebrafish, into which a brain aromatase (cyp19a1b)-based estrogen-responsive reporter gene was introduced. In response to 17beta-estradiol (10(-6) M) and heptachlor (10(-6) M), zebrafish embryos carrying the reporter construct expressed enhanced green fluorescent protein in the olfactory bulb, telencephalon, preoptic area, and mediobasal hypothalamus. This system will serve to model the in vivo conversion and breakdown of estrogenic compounds and thus provide a rapid preliminary screening method to estimate their estrogenicity.

Bone morphogenetic protein 7 induces mesenchymal-to-epithelial transition in melanoma cells, leading to inhibition of metastasis.

Bone morphogenetic protein (BMP) 7 counteracts physiological epithelial-to-mesenchymal transition, a process that is indicative of epithelial plasticity in developmental stages. Because epithelial-to-mesenchymal transition and its reversed process mesenchymal-to-epithelial transition (MET) are also involved in cancer progression, we investigated whether BMP7 plays a role in WM-266-4 melanoma cell growth and metastasis. An MTT assay was conducted in WM-266-4 and HEK293T cell lines to show the cell growth inhibition ability of BMP7 and cisplatin. Semiquantitative RT-PCR was used to determine MET in morphologically changed BMP7-treated melanoma cells. MET-induced cells expressed less a basic helix-loop-helix transcription factor (TWIST) in western blot analysis, and we confirm that BMP receptor (Alk2) siRNA transduction could restore TWIST protein expression via blocking of Smad 1, 5 and 8 signaling. Matrigel invasion and cell migration assays were done to investigate the BMP7-induced metastasis inhibition ability. BMP7 treatment only slightly reduced cell growth rate, but induced apparent MET. BMP7 also reduced the invasion and migration ability. Furthermore, BMP7 reduced the resistance of WM-266-4 cells to cisplatin. Collectively, our findings indicate that the metastatis inhibition ability of BMP7 is involved in MET, and that BMP7 could be used as a potential metastasis inhibitor in human melanoma cells.