Assessing developmental and transcriptional effects of PM2.5 on zebrafish embryos.

Investigating fine particulate matter (PM2.5) toxicity is crucial for health risk assessment and pollution control. This study explores the developmental toxicity of two PM2.5 sources: standard reference material 2786 (NIST, USA) and PM2.5 from Chakri Naruebodindra Medical Institute (CNMI, Thailand) located in the Bangkok Metropolitan area. Zebrafish embryos exposed to these samples exhibited embryonic mortality, with 50% lethal concentration (LC50) values of 1476 µg/mL for standard PM2.5 and 512 µg/mL for CNMI PM2.5. Morphological analysis revealed malformations, including pericardial and yolk sac edema, and blood clotting in both groups. Gene expression analysis highlighted source-specific effects. Standard PM2.5 downregulated sod1 and cat while upregulating gstp2. Inflammatory genes tnf-α and il-1b were upregulated, and nfkbi-αa was downregulated. Apoptosis-related genes bax, bcl-2, and casp3a were downregulated. CNMI PM2.5 consistently downregulated all examined genes. These findings underscore PM2.5 source variability's significance in biological system impact assessment, providing insights into pollutant-gene expression interactions. The study emphasizes the need for source-specific risk assessment and interventions to address PM2.5 exposure's health impacts effectively.

Investigation on the physical properties and biocompatibility of zirconia-alumina-silicate@diopside composite materials and its in vivo toxicity study in embryonic zebrafish.

Bioceramic materials have a wide range of applications in the biomedical field, such as in the repair of bone defects and dental surgery. Silicate-based bioceramics have attracted biomedical researchers' interest due to their bioactivity and biodegradability. In this study, extended the scope of ZAS utilization in bone tissue engineering by introducing calcium-magnesium-silicate (diopside, CMS) as an interface material aim to develop a machinable bioceramic composite (ZASCMS) by the sol-gel method. The physicochemical characterization, in vitro biological properties and in vivo zebrafish cytotoxicity study of ZAS-based composites as a function of CMS contents, 0, 25, 50, 75 and 100 wt%, were performed. Results showed that the as-prepared ZASCMS possessed porous architecture with well-interconnected pore structure. Results also revealed that the mechanical properties of ZASCMS composite materials were gradually improved with increasing CMS contents. The ZASCMS composites with more than 50 wt% CMS had the highest compressive strength and modulus of 6.78 ± 0.62 MPa and 340.10 ± 16.81 MPa, respectively. Regarding in vitro bioactivities, the composite scaffolds were found to stimulate osteoblast-like UMR-106 cell adhesion, growth, and proliferation. The antibacterial activity of the ZASCMS composite scaffolds was tested against Staphylococcus epidermidis (S. epidermidis) and Escherichia coli (E. coli) also exhibited an antibacterial property. Furthermore, the in vivo studies using embryonic zebrafish were exposed to as-prepared particles (0-500 µg mL-1) and showed that the synthesized ZAS, CMS and ZASCMS composite particles were non-toxic based on the evaluation of survivability, hatching rate and embryonic morphology. In conclusions, our results indicated that the synthesized composite exhibited their biological properties and antibacterial activity, which could well be a promising material with high potential to be applied in orthopaedic and dental tissue engineering.

Impact of a real food matrix and in vitro digestion on properties and acute toxicity of polystyrene microparticles.

Although it is proved that humans ingest microplastics via food, and microplastics were found in human tissues, blood and feces, there needs to be more data on the properties and health-related effects of plastic particles that interact with food and undergo digestion. This study aimed to examine the impact of a real food matrix, milk, on the behavior and gastrointestinal fate of polystyrene microparticles (PSMP). In the presence of the food matrix, the net negative ζ-potential values of PSMP (diameter size of 1.823 µm) decreased significantly due to the formation of the corona, mostly consisting of α and ß-casein fragments. Protein corona profiles and morphologies of particles incubated with whole and skim milk were found to be similar, and the protein profiles were completely altered after in vitro digestion simulation. In vitro and in vivo toxicity studies showed that neither bare PSMP nor food-interacted PSMP pose acute toxicity on the Caco-2 cell line and zebrafish embryos under the chosen experimental conditions. In summary, these results may contribute to a better understanding of changes that microplastics undergo in foods. Further studies on repeated exposure or chronic toxicity are needed to fully reveal the effect of food matrix on microplastic toxicity.

Sesamin lacks zebrafish embryotoxicity but exhibits evidence of anti-angiogenesis, anti-oxidant and anti-inflammatory activities.

Sesamin, the major lignan in sesame seeds (Sesamum indicum L.), is known to have several pharmaceutical activities. However, its toxicological profile is still limited, especially regarding embryotoxicity. This study aimed to evaluate the developmental toxicity of sesamin in zebrafish embryos. After 72 h exposure, sesamin did not affect the survival and hatching rates, nor did it cause malformation in zebrafish embryos. Cardiotoxicity was also evaluated by monitoring embryo heartbeats and erythrocyte staining using o-dianisidine. The results showed that sesamin did not affect heart morphology, heart rate, or cardiac output in zebrafish embryos. The present study also evaluated sesamin's anti-angiogenesis, antioxidant and anti-inflammation activities. Sesamin significantly decreased the sub-intestinal vessel plexus as revealed by alkaline phosphatase staining indicating the compound exhibited anti-angiogenesis activity. For the antioxidant and anti-inflammatory assays, oxidative stress and inflammation in zebrafish embryos were induced by hydrogen peroxide and lipopolysaccharide, respectively. The reactive oxygen species (ROS) and nitric oxide (NO) production were detected using a fluorescent dye. Sesamin significantly decreased ROS and NO production in zebrafish embryos. In addition, the transcription examination by qRT-PCR of oxidative- and inflammation-related genes showed that sesamin affected the genes in a manner that correlated with results from the efficacy assays. In conclusion, the present study revealed that sesamin did not cause embryotoxicity and cardiotoxicity in zebrafish embryos. In addition, it exhibited evidence of anti-angiogenesis, antioxidant and anti-inflammatory activities.

Development of Biomaterials Based on Biomimetic Trace Elements Co-Doped Hydroxyapatite: Physical, In Vitro Osteoblast-like Cell Growth and In Vivo Cytotoxicity in Zebrafish Studies.

Synthesized hydroxyapatite (sHA)-calcium phosphate (CaP) based biomaterials play a vital role and have been widely used in the process of bone regeneration for bone defect repair, due to their similarities to the inorganic components of human bones. However, for bone tissue engineering purpose, the composite components, physical and biological properties, efficacy and safety of sHA still need further improvements. In this work, we synthesized inhomogeneous hydroxyapatite based on biomimetic trace elements (Mg, Fe, Zn, Mn, Cu, Ni, Mo, Sr, Co, BO33-, and CO32-) co-doped into HA (THA) (Ca10-δMδ(PO4)5.5(CO3)0.5(OH)2, M = trace elements) via co-precipitation from an ionic solution. The physical properties, their bioactivities using in vitro osteoblast cells, and in vivo cytotoxicity using zebrafish were studied. By introducing biomimetic trace elements, the as-prepared THA samples showed nanorod (needle-like) structures, having a positively charged surface (6.49 meV), and showing paramagnetic behavior. The bioactivity studies demonstrated that the THA substrate can induce apatite particles to cover its surface and be in contact with surrounding simulated body fluid (SBF). In vitro biological assays revealed that the osteoblast-like UMR-106 cells were well-attached with growth and proliferation on the substrate's surface. Upon differentiation, enhanced ALP (alkaline phosphatase) activity was observed for bone cells on the surface of the THA compared with that on the control substrates (sHA). The in vivo performance in embryonic zebrafish studies showed that the synthesized THA particles are nontoxic based on the measurements of essential parameters such as survivability, hatching rate, and the morphology of the embryo. The mechanism of the ions release profile using digital conductivity measurement revealed that sustained controlled release was successfully achieved. These preliminary results indicated that the synthesized THA could be a promising material for potential practical applications in bone tissue engineering.

Effects of ß-eudesmol and atractylodin on target genes and hormone related to cardiotoxicity, hepatotoxicity, and endocrine disruption in developing zebrafish embryos.

Atractylodes lancea, commonly known as Kod-Kamao in Thai, a traditional medicinal herb, is being developed for clinical use in cholangiocarcinoma. ß-eudesmol and atractylodin are the main active components of this herb which possess most of the pharmacological properties. However, the lack of adequate toxicity data would be a significant hindrance to their further development. The present study investigated the toxic effects of selected concentrations of ß-eudesmol and atractylodin in the heart, liver, and endocrine systems of zebrafish embryos. Study endpoints included changes in the expression of genes related to Na/K-ATPase activity in the heart, fatty acid-binding protein 10a and cytochrome P450 family 1 subfamily A member 1 in the liver, and cortisol levels in the endocrine system. Both compounds produced inhibitory effects on the Na/K-ATPase gene expressions in the heart. Both also triggered the biomarkers of liver toxicity. While ß-eudesmol did not alter the expression of the cytochrome P450 family 1 subfamily A member 1 gene, atractylodin at high concentrations upregulated the gene, suggesting its potential enzyme-inducing activity in this gene. ß-eudesmol, but not atractylodin, showed some stress-reducing properties with suppression of cortisol production.

Transparent Anti-SARS COV-2 Film from Copper(I) Oxide Incorporated in Zeolite Nanoparticles.

The high antibacterial and antiviral performance of synthesized copper(I) oxide (Cu2O) incorporated in zeolite nanoparticles (Cu-Z) was determined. Various Cu contents (1-9 wt %) in solutions were loaded in the zeolite matrix under neutral conditions at room temperature. All synthesized Cu-Z nanoparticles showed high selectivity of the cuprous oxide, as confirmed by X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) analysis. An advantage of the prepared Cu-Z over the pristine Cu2O nanoparticles was its high thermal stability. The 7 and 9 wt % Cu contents (07Cu-Z and 09Cu-Z) exhibited the best activities to deactivate Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus bacteria. The film coated with 07Cu-Z nanoparticles also had high antiviral activities against porcine coronavirus (porcine epidemic diarrhea virus, PEDV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Specifically, the 07Cu-Z-coated film could reduce 99.93% of PEDV and 99.94% of SARS-CoV-2 viruses in 5 min of contact time, which were higher efficacies and faster than those of any previously reported works. The anti-SARS-CoV-2 virus film was coated on a low-cost PET or PVC film. A very small amount of cuprous oxide in zeolite was used to fabricate the antivirus film; therefore, the film was more transparent (79.4% transparency) than the cuprous oxide film or other commercial products. The toxicity of 07Cu-Z nanoparticles was determined by a toxicity test on zebrafish embryo and a skin irritation test to reconstruct a human epidermis (RhE) model. It was found that the impact on the aquatic environment and human skin was lower than that of the pristine Cu2O.

Developmental effects of sesamolin on zebrafish (Danio rerio) embryos.

Sesamolin is one of the major active compounds found in sesame seeds (Sesamum indicum L.) that are commonly and increasingly used as an ingredient in cuisines and various food products. The compound has been reported to have several pharmaceutical activities such as antioxidant, antimicrobial, neuroprotective, and anticancer. However, the toxicological profile of sesamolin does not currently include developmental toxicity. In this study, we assessed sesamolin toxicity to embryonic development of zebrafish by exposure for 72 h at concentrations ranging from 10 to 50 µM. The evaluation revealed that sesamolin did not affect survival and hatching rates. However, it did induce embryo malformations and reduced embryonic heart rates in a dose-dependent manner. By qRT-PCR analysis, it downregulated the expression of oxidative stress-related genes, including superoxide dismutase 1 (sod1), catalase (cat), and glutathione S-transferase pi 2 (gstp2). Alkaline phosphatase staining of embryos revealed that sesamolin inhibited the development of subintestinal vessels, and hemoglobin staining revealed a negative impact on embryonic erythropoiesis. These findings showed that sesamolin affected genes related to angiogenesis and erythropoiesis. The risks of sesamolin to embryonic development found in this study may imply similar effects in humans and other mammals.

Protective effects of Thai silk sericins and their related mechanisms on UVA-induced phototoxicity and melanogenesis: Investigation in primary melanocyte cells using a proteomic approach.

UV radiation causes excess production of melanin as a result of hyperpigmentation and skin disorders. Silk sericin exhibited bioactivities to skin and inhibited UV-induced phototoxicity and melanogenesis in skin cells; however, the mechanism related to sericin against UV-induced melanogenesis has not been investigated. This study aimed to investigate the protective effects of Thai silk sericins against UVA-induced phototoxicity and melanogenesis and their related mechanisms. Thai silk sericins exhibited cytoprotective effects against UV-induced toxicity in human primary melanocytes by attenuation of cytotoxicity, intracellular ROS generation, and mitochondrial potential impairment. Pre- and post-treatment with sericin significantly inhibited melanin synthesis and tyrosinase activity against UVA exposure. In addition, sericin S2 could reduce the basal melanin content in zebrafish embryos. The proteomic analysis demonstrated that Thai silk sericins altered the protein expression in melanocytes especially proteins related to stress, inflammatory, cytokine stimulation, cell proliferation, and cell survival processes that contribute to cytoprotective effect and inhibitory effect on melanogenesis of sericin. Moreover, we demonstrated the novel mechanism of Thai silk sericins in inhibiting UVA-induced melanogenesis via increasing BMP4 expression in MAPK/ERK signaling pathway. These evidences support the potential use of Thai silk sericins in prevention of hyperpigmentation in skin disorders especially after UVA exposure.

Anti-angiogenic effects of beta-eudesmol and atractylodin in developing zebrafish embryos.

Angiogenesis is the process of formation of new blood vessels which plays an essential role in the normal physiological development of the organs and systems. Several factors contribute to and regulate this process. Unregulated angiogenesis, however, is harmful and is usually found in tumors and cancerous cells. ß-Eudesmol and atractylodin are sesquiterpenoid contents extracted from the rhizome of Atractylodes lancea (AL). Reports suggest potential anti-angiogenic activities of both compounds. In this study, the anti-angiogenic activities of both compounds were investigated using the well-established zebrafish in vivo model. Zebrafish embryos were treated with a series of concentrations (6.3, 12.5, 25, and 50 µM) of ß-eudesmol and (6.3, 12.5, and 25 µM) of atractylodin up to 72 h post-fertilization. Assessment of the effects on phenotypic blood vessel development (sub-intestinal vessel intersection count) revealed that both the compounds inhibited vessel development, particularly at higher concentrations. At the genetic levels, only ß-eudesmol significantly downregulated the expression of the Vegfaa gene and also its receptor Vegfr2. ß-Eudesmol also affected the expression of Vegfaa protein in a concentration-dependent manner. Results indicate that ß-eudesmol exerts anti-angiogenic property through inhibition of Vegfaa at both the gene and protein levels. However, atractylodin does not possess this property.

Fabrication of biocompatible magneto-fluorescence nanoparticles as a platform for fluorescent sensor and magnetic hyperthermia applications.

Multifunctional nanoparticles with special magnetic and optical properties have been attracting a great deal of attention due to their important applications in the bioanalytical and biomedical fields. In this study, we report the fabrication of biocompatible magneto-fluorescence nanoparticles consisting of carbon dots (CDots) and silica-coated cobalt-manganese nanoferrites (Co0.5Mn0.5Fe2O4) (CoMnF@Si@CDots) (MagSiCDots) by a facile hydrothermal method. The as-prepared MagSiCDots have a particle size of 100-120 nm and show a negative zeta potential of -35.50 mV at a neutral pH. The fluorescence spectrum of the MagSiCDots nanoparticles consists of sharp excitation at 365 nm and broad blue light emission with a maximum wavelength of 442.5 nm and the MagSiCDots exhibit superparamagnetic behaviour with a saturation magnetization of 11.6 emu g-1. The potential of MagSiCDots as a fluorescent sensor and be used for magnetic hyperthermia applications. It is seen that the fluorescent intensity of a colloidal solution (a hydrogen sulfide (H2S) solution containing MagSiCDots nanoparticles) has a linear relationship with the H2S concentration range of 0.2-2 µM. The limit of detection (LOD) of H2S by our MagSiCDots particles is 0.26 µM and they remain stable for at least 90 min. To test the suitability of the MagSiCDots nanoparticles for use in hyperthermia application, induction heating using an AMF was done. It was observed that these nanoparticles had a specific absorption rate (SAR) of 28.25 W g-1. The in vitro and in vivo cytotoxicity of MagSiCDots were tested on HeLa cells lines. The results show a cell viability of about 85% when exposed to 100 µg mL-1 concentration of the particles. The in vivo cytotoxicity using zebrafish assay also confirmed the non-toxicity and biocompatibility of the nanoparticles to living cells. The reported data demonstrate that by combining CoMnF@Si and fluorescent CDots into a single system, not only nontoxic multifunctional nanomaterials but also multimodal nanoparticles for several applications, such as hazard gas detection and acting as a biocompatible heat source for therapeutic treatment of cancer, are provided.

Embryotoxicity evaluation of atractylodin and ß-eudesmol using the zebrafish model.

Atractylodin and ß-eudesmol are the major active ingredients of Atractylodes lancea (Thunb) DC. (AL). Both compounds exhibit various pharmacological activities, including anticancer activity against cholangiocarcinoma. Despite the widespread use of this plant in traditional medicine in China, Japan, Korea, and Thailand, studies of their toxicological profiles are limited. The present study aimed to evaluate the embryotoxicity of atractylodin and ß-eudesmol using the zebrafish model. Zebrafish embryos were exposed to a series of concentrations (6.3, 12.5, 25, 50, and 100 µM) of each compound up to 72 h post-fertilization (hpf). The results showed that atractylodin and ß-eudesmol induced mortality of zebrafish embryos with the 50% lethal concentration (LC50) of 36.8 and 53.0 µM, respectively. Both compounds also caused embryonic deformities, including pericardial edema, malformed head, yolk sac edema, and truncated body. Only ß-eudesmol decreased the hatching rates, while atractylodin reduced the heart rates of the zebrafish embryos. Additionally, both compounds increased reactive oxygen species (ROS) production and altered the transcriptional expression levels of superoxide dismutase 1 (sod1), catalase (cat), and glutathione S-transferase pi 2 (gstp2) genes. In conclusion, atractylodin and ß-eudesmol induce mortality, developmental toxicity, and oxidative stress in zebrafish embryos. These findings may imply similar toxicity of both compounds in humans.

A Phenotypic and Genotypic Evaluation of Developmental Toxicity of Polyhexamethylene Guanidine Phosphate Using Zebrafish Embryo/Larvae.

Polyhexamethylene guanidine-phosphate (PHMG-P), a guanidine-based cationic antimicrobial polymer, is an effective antimicrobial biocide, potent even at low concentrations. Due to its resilient bactericidal properties, it has been used extensively in consumer products. It was safely used until its use in humidifiers led to a catastrophic event in South Korea. Epidemiological studies have linked the use of PHMG-P as a humidifier disinfectant to pulmonary fibrosis. However, little is known about its harmful impacts other than pulmonary fibrosis. Thus, we applied a zebrafish embryo/larvae model to evaluate developmental and cardiotoxic effects and transcriptome changes using RNA-sequencing. Zebrafish embryos were exposed to 0.1, 0.2, 0.3, 0.4, 0.5, 1, and 2 mg/L of PHMG-P from 3 h to 96 h post fertilization. 2 mg/L of PHMG-P resulted in total mortality and an LC50 value at 96 h was determined at 1.18 mg/L. Significant developmental changes were not observed but the heart rate of zebrafish larvae was significantly altered. In transcriptome analysis, immune and inflammatory responses were significantly affected similarly to those in epidemiological studies. Our qPCR analysis (Itgb1b, TNC, Arg1, Arg2, IL-1ß, Serpine-1, and Ptgs2b) also confirmed this following a 96 h exposure to 0.4 mg/L of PHMG-P. Based on our results, PHMG-P might induce lethal and cardiotoxic effects in zebrafish, and crucial transcriptome changes were linked to immune and inflammatory response.

Crinamine Induces Apoptosis and Inhibits Proliferation, Migration, and Angiogenesis in Cervical Cancer SiHa Cells.

Crinumasiaticum is a perennial herb widely distributed in many warmer regions, including Thailand, and is well-known for its medicinal and ornamental values. Crinum alkaloids contain numerous compounds, such as crinamine. Even though its mechanism of action is still unknown, crinamine was previously shown to possess anticancer activity. In this study, we demonstrate that crinamine was more cytotoxic to cervical cancer cells than normal cells. It also inhibited anchorage-independent tumor spheroid growth more effectively than existing chemotherapeutic drugs carboplatin and 5-fluorouracil or the CDK9 inhibitor FIT-039. Additionally, unlike cisplatin, crinamine induced apoptosis without promoting DNA double-strand breaks. It suppressed cervical cancer cell migration by inhibiting the expression of positive regulators of epithelial-mesenchymal transition SNAI1 and VIM. Importantly, crinamine also exerted anti-angiogenic activities by inhibiting secretion of VEGF-A protein in cervical cancer cells and blood vessel development in zebrafish embryos. Gene expression analysis revealed that its mechanism of action might be attributed, in part, to downregulation of cancer-related genes, such as AKT1, BCL2L1, CCND1, CDK4, PLK1, and RHOA. Our findings provide a first insight into crinamine's anticancer activity, highlighting its potential use as an alternative bioactive compound for cervical cancer chemoprevention and therapy.

Acute toxicity and teratogenicity of α-mangostin in zebrafish embryos.

IMPACT STATEMENT: α-Mangostin has been reported to have anticancer properties both in vitro and in vivo models. Although there are several studies that evaluated the toxicity of the compound in rodent models, we are the first to evaluate the teratogenicity of α-mangostin. In the present work, we found that α-mangostin induced mortality and malformations in zebrafish embryos. In addition, we exhibited that the compound also disrupted the reactive oxygen species and hemoglobin levels. These findings suggest that α-mangostin may possibly cause the same adverse effects on human health. The mechanisms of these toxicological effects of the compound will be further elucidated and the effects found in zebrafish embryos need to be verified in other animal models.

Surface modification of nanostructure lipid carrier (NLC) by oleoyl-quaternized-chitosan as a mucoadhesive nanocarrier.

A nanostructure lipid carrier (NLC) composed of solid, and liquid lipid as a core has been developed as a delivery system for hydrophobic drug molecules. The aim of this research was to fabricate an oleoyl-quaternized-chitosan (CS)-coated NLC, where the mucoadhesive property of nanoparticles is enhanced for more efficient drug delivery. NLC loaded with alpha-mangostin (AP), a model hydrophobic drug, were fabricated using a high pressure homogenization process and subsequently coated with CS. The fabricated nanoparticles showed particle sizes in the range of 200-400nm, with low polydispersity, high physical stability and excellent encapsulation efficiency (EE>90%). Additionally, in vitro viability, cytotoxicity and ability of NLC and CS-NLC to affect apoptosis in carcinoma Caco-2 cells were determined using the Triplex assay. Gene expressiom analysis were performed using quantitative reverse transcription Polymerase Chain Reaction (RT-qPCR). Moreover, in vivo toxicological testing of NLCs was conducted in zebrafish embryos. Results indicated that CS-NLC provieded high cytotoxicity than NLC itself. In the case of AP loaded nanoparticles, NLC loaded with AP (AP-NLC), and CS-NLC loaded with AP (CS-AP-NLC) exhibited higher cytotoxicity to Caco-2 over Hela cells. These results indicate that CS-NLC shows enhanced cellular uptake but increased cytotoxicity characteristics over NLC and therefore careful optimization of dosage and loading levels in CS-NLC is needed to allow cancer cell targeting, and for exploiting the potential of these systems in cancer therapy.

In vitro and in vivo characterization of the anticancer activity of Thai stingless bee (Tetragonula laeviceps) cerumen.

Tetragonula laeviceps cerumen was sequentially extracted with 80% (v/v) methanol, dichloromethane, and hexane and also in the reverse order. By the MTT assay and the respective 50% inhibition concentration value, the most active fraction was further purified to apparent homogeneity by bioassay-guided silica gel column chromatography. α-Mangostin was identified by high-resolution electrospray ionization mass spectrometry and nuclear magnetic resonance analyses. It had a potent cytotoxicity against the BT474, Chago, Hep-G2, KATO-III, and SW620 cell lines (IC50 values of 1.22 ± 0.03, 2.25 ± 0.20, 0.94 ± 0.01, 0.88 ± 0.16, and 1.50 ± 0.39 µmol/L, respectively). The in vitro cytotoxicity of α-mangostin against the five human cancer cell lines and primary fibroblasts was further characterized by real-time impedance-based analysis. Interestingly, α-mangostin was more cytotoxic against the cancer-derived cell lines than against the primary fibroblasts. Later, the migration assay was performed by continuously measuring the attachment of cells to the plate electrodes at the bottom of the transwell membrane. The combined caspase-3 and -7 activities were assayed by the Caspase-Glo® 3/7 kit. It showed that the cytotoxic mechanism involved caspase-independent apoptosis, while at low (non-toxic) concentrations α-mangostin did not significantly alter cell migration. Furthermore, the in vivo cytotoxicity and angiogenesis were determined by alkaline phosphatase staining in zebrafish embryos along with monitoring changes in the transcript expression level of two genes involved in angiogenesis (vegfaa and vegfr2) by quantitative real-time reverse transcriptase- polymerase chain reaction. It was found that the in vivo cytotoxicity of α-mangostin against zebrafish embryos had a 50% lethal concentration of 9.4 µM, but no anti-angiogenic properties were observed in zebrafish embryos at 9 and 12 µM even though it downregulated the expression of vegfaa and vegfr2 transcripts. Thus, α-mangostin is a major active compound with a potential anticancer activity in T. laeviceps cerumen in Thailand.

Drl.3 governs primitive hematopoiesis in zebrafish.

The molecular program controlling hematopoietic differentiation is not fully understood. Here, we describe a family of zebrafish genes that includes a novel hematopoietic regulator, draculin-like 3 (drl.3). We found that drl.3 is expressed in mesoderm-derived hematopoietic cells and is retained during erythroid maturation. Moreover, drl.3 expression correlated with erythroid development in gata1a- and spi1b-depleted embryos. Loss-of-function analysis indicated that drl.3 plays an essential role in primitive erythropoiesis and, to a lesser extent, myelopoiesis that is independent of effects on vasculature, emergence of primitive and definitive progenitor cells and cell viability. While drl.3 depletion reduced gata1a expression and inhibited erythroid development, enforced expression of gata1a was not sufficient to rescue erythropoiesis, indicating that the regulation of hematopoiesis by drl.3 extends beyond control of gata1a expression. Knockdown of drl.3 increased the proportion of less differentiated, primitive hematopoietic cells without affecting proliferation, establishing drl.3 as an important regulator of primitive hematopoietic cell differentiation.

mTORC2 is required for proliferation and survival of TSC2-null cells.

Mutational inactivation of the tumor suppressor tuberous sclerosis complex 2 (TSC2) constitutively activates mTORC1, increases cell proliferation, and induces the pathological manifestations observed in tuberous sclerosis (TS) and in pulmonary lymphangioleiomyomatosis (LAM). While the role of mTORC1 in TSC2-dependent growth has been extensively characterized, little is known about the role of mTORC2. Our data demonstrate that mTORC2 modulates TSC2-null cell proliferation and survival through RhoA GTPase and Bcl2 proteins. TSC2-null cell proliferation was inhibited not only by reexpression of TSC2 or small interfering RNA (siRNA)-induced downregulation of Rheb, mTOR, or raptor, but also by siRNA for rictor. Increased RhoA GTPase activity and P-Ser473 Akt were inhibited by siRNA for rictor. Importantly, constitutively active V14RhoA reversed growth inhibition induced by siRNA for rictor, siRNA TSC1, reexpression of TSC2, or simvastatin. While siRNA for RhoA had a modest effect on growth inhibition, downregulation of RhoA markedly increased TSC2-null cell apoptosis. Inhibition of RhoA activity downregulated antiapoptotic Bcl2 and upregulated proapoptotic Bim, Bok, and Puma. In vitro and in vivo, simvastatin alone or in combination with rapamycin inhibited cell growth and induced TSC2-null cell apoptosis, abrogated TSC2-null tumor growth, improved animal survival, and prevented tumor recurrence by inhibiting cell growth and promoting apoptosis. Our data demonstrate that mTORC2-dependent activation of RhoA is required for TSC2-null cell growth and survival and suggest that targeting both mTORC2 and mTORC1 by a combination of proapoptotic simvastatin and cytostatic rapamycin shows promise for combinational therapeutic intervention in diseases with TSC2 dysfunction.

A key role for the integrin alpha2beta1 in experimental and developmental angiogenesis.

The alpha2beta1 integrin receptor plays a key role in angiogenesis. Here we investigated the effects of small molecule inhibitors (SMIs) designed to disrupt integrin alpha2 I or beta1 I-like domain function on angiogenesis. In unchallenged endothelial cells, fibrillar collagen induced robust capillary morphogenesis. In contrast, tube formation was significantly reduced by SMI496, a beta1 I-like domain inhibitor and by function-blocking anti-alpha2beta1 but not -alpha1beta1 antibodies. Endothelial cells bound fluorescein-labeled collagen I fibrils, an interaction specifically inhibited by SMI496. Moreover, SMI496 caused cell retraction and cytoskeletal collapse of endothelial cells as well as delayed endothelial cell wound healing. SMI activities were examined in vivo by supplementing the growth medium of zebrafish embryos expressing green fluorescent protein under the control of the vascular endothelial growth factor receptor-2 promoter. SMI496, but not a control compound, interfered with angiogenesis in vivo by reversibly inhibiting sprouting from the axial vessels. We further characterized zebrafish alpha2 integrin and discovered that this integrin is highly conserved, especially the I domain. Notably, a similar vascular phenotype was induced by morpholino-mediated knockdown of the integrin alpha2 subunit. By live videomicroscopy, we confirmed that the vessels were largely nonfunctional in the absence of alpha2beta1 integrin. Collectively, our results provide strong biochemical and genetic evidence of a central role for alpha2beta1 integrin in experimental and developmental angiogenesis.