ALBERT

Description: Magnetic Nanoparticles (MNPs) are widely being investigated as novel promising multifunctional agents, specifically in the fields of development for theranostics, electronics, waste water treatment, cosmetics, and energy storage devices. Unique, superior, and indispensable properties of magnetization, heat transfer, and melting temperature make MNPs emerge in the field of therapeutics in future healthcare industries. However, MNPs ecotoxicity as well as behavioral toxicity is still unexplored. Ecotoxicity analysis may assist investigate MNPs uptake mechanism and its influence on bioavailability under a given set of environmental factors, which can be followed to investigate the biomagnification of MNPs in the environment and health risk possessed by them in an ecological food chain. In this study, we attempted to determine the behavioral changes in zebrafishes at low (1 ppm) or high (10 ppm) concentration levels of Fe3O4 MNPs. The synthesized Fe3O4 MNPs sized at 15 nm were characterized by the transmission electron microscope (TEM), the superconducting quantum interference device (SQUID) magnetometer, and the multiple behavior tests for novel tank, mirror biting, conspecific social interaction, shoaling, circadian rhythm, and short-term memory of zebrafish under MNPs chronic exposure were demonstrated. Low concentration MNP exposure did not trigger alteration for majority behavioral and biochemical tests in adult zebrafish. However, tight shoal groups were observed at a high concentration of MNPs exposure along with a modest reduction in fish exploratory behavior and a significant reduction in conspecific social interaction behavior. By using enzyme-linked immunosorbent assays (ELISA), we found a high dose of MNPs exposure significantly elevated cortisol, acetylcholine, and catalase levels while reducing serotonin, acetylcholine esterase, and dopamine levels in the brain. Our data demonstrates chronic MNPs exposure at an environmentally-relevant dose is relatively safe by supporting evidence from an array of behavioral and biochemical tests. This combinational approach using behavioral and biochemical tests would be helpful for understanding the MNPs association with anticipated colloids and particles effecting bioavailability and uptake into cells and organisms.

Description: Rare earth elements (REEs) or “technology metals” were coined by the U.S. Department of Energy, a group of seventeen elements found in the Earth’s crust. These chemical elements are vital and irreplaceable to the world of technology owing to their unique physical, chemical, and light-emitting properties, all of which are beneficial in modern healthcare, telecommunication, and defense. Rare earth elements are relatively abundant in Earth’s crust, with critical qualities to the device performance. The reuse and recycling of rare earth elements through different technologies can minimize impacts on the environment; however, there is insufficient data about their biological, bioaccumulation, and health effects. The increasing usage of rare earth elements has raised concern about environmental toxicity, which may further cause harmful effects on human health. The study aims to review the toxicity analysis of these rare earth elements concerning aquatic biota, considering it to be the sensitive indicator of the environment. Based on the limited reports of REE effects, the review highlights the need for more detailed studies on the hormetic effects of REEs. Aquatic biota is a cheap, robust, and efficient platform to study REEs’ toxicity, mobility of REEs, and biomagnification in water bodies. REEs’ diverse effects on aquatic life forms have been observed due to the lack of safety limits and extensive use in the various sectors. In accordance with the available data, we have put in efforts to compile all the relevant research results in this paper related to the topic “toxicity effect of REEs on aquatic life”.

Description: Abstract 2393 Somatic mutations of ASXL1 gene have been described in patients with myeloid malignancies and were associated with inferior outcomes. ASXL1 mutations have also been detected in myeloid blast crisis of chronic myeloid leukemia (CML) patients. The mechanisms of acute myeloid leukemia (AML) transformation and functional role of ASXL1 mutations in the leukemogenesis remain to be determined. Recently, we identified PHD domain deletion mutations (R693X and L885X) in patients with CML in myeloid blast crisis and/or AML with minimal differentiation (M0). In the present study, we aimed to investigate the role of PHD domain deletion mutations in the pathogenesis of AML transformation. The K562 cells carrying Philadelphia chromosome, serves as a model to study the molecular mechanisms associated with leukemogenesis. Our result showed that R693X/L885X mutations inhibited PMA-treated megakaryocytic differentiation with the change of physiological characteristic features and suppressed the induction of CD61, a specific cell surface marker of megakaryocytes. We also found that FOSB, a member of Fos family of AP-1 transcription factors was down-regulated in K562 cells expressing R693X and L885X compared to wild-type ASXL1 during PMA-mediated megakaryocytic differentiation. Examination of intracellular signaling pathways showed that the mutant ASXL1 protein prevented PMA-induced megakaryocytic differentiation through the inactivation of ERK, AKT and STAT5 which are required for differentiation. Further, ASXL1 depletion by shRNA in K562 cells led to enhanced cell proliferation, increased colony formation and impaired PMA-mediated differentiation. Previous studies in Drosophila had revealed that Asxl forms the protein complexes of both Trithorax and Polycomb groups that are required for maintaining chromatin in both activated and repressed transcriptional states. By using Western blot analysis, we demonstrated that PHD domain deletion mutations of ASXL1 significantly suppressed the transcriptionally repressive mark H3K27 trimethylation, however no effect on methylated H3K4 (H3K4me2 and H3K4me3), an active histone mark in K562 cells. Co-immunoprecipitation analysis revealed that wild-type, but not PHD domain deletion mutations of ASXL1 interact with EZH2, a member of the polycomb repressive complex 2 (PRC2). Importantly, PHD deletion mutations or downregulation of ASXL1 resulted in the suppression of EZH2 in K562 cells. Our study demonstrated that PHD deletion mutations of ASXL1 resulted in a loss-of-function which exhibited direct effects on the proliferation and differentiation and also proposed a specific role for ASXL1 in epigenetic regulation of gene expression in K562 cells. Disclosures: No relevant conflicts of interest to declare.

Description: Background and purpose Abnormalities of genes regulating DNA methylation have been described in acute myeloid leukemia (AML). MLL protein is a transcriptional regulator and governs proper hematopoiesis through its histone methyltransferase activity. AML with partial tandem duplication of MLL (MLL-PTD) was associated with an unfavorable prognosis. The cooperating roles of MLL-PTD with other mutated genes regulating DNA methylation have not been comprehensively studied in AML. We aimed to determine the prevalence and clinical impact of mutations of DNA methylation regulators in AML with MLL-PTD. Materials and methods Bone marrow samples from 98 AML patients with MLL-PTD were analyzed for gene mutations of TET2, DNMT3A, IDH1 and IDH2. MLL-PTD was screened by RT-PCR and confirmed by real-time quantitative PCR assays. The mutational analysis was performed with PCR assays followed by direct sequencing for TET2 (whole coding exons 3–11) and IDH1/2 (hotspots exon 4). For the detection of DNMT3A mutations, the PCR products amplified for entire coding exons 2 to 23 were first screened with denaturing high-performance liquid chromatography followed by direct sequencing for the abnormal profiles. Results The frequency of TET2, IDH1, IDH2 and DNMT3A mutations in AML patients with MLL-PTD was 17.0% (16/94), 10.2% (10/98), 18.4% (18/98), and 31.6% (31/98), respectively. Taken together, 61.1% of patients with MLL-PTD had at least one mutated gene of DNA methylation regulators. TET2, IDH1 and IDH2 mutations were mutually exclusive with each other whereas DNMT3A mutations frequently co-existed with other DNA methylation modifiers:TET2 (n=8), IDH1 (n=5) and IDH2 (n=4). No differences were observed between the mutation status of the DNA methylation modifiers and clinico-hematologic features of patients with MLL-PTD except that TET2 (P=0.012) and DNMT3A (P=0.024) mutations were associated with older age. Of the 55 MLL-PTD patients who received standard chemotherapy, IDH2 mutation was associated with a lower complete remission rate (25.0% vs 67.8%, P=0.018), while DNMT3A mutations conferred an inferior event-free survival (0.0 vs 6.8 months, P=0.027) and overall survival (6.0 vs 11.5 months, P=0.032). In multivariate analysis, older age (P=0.008) and DNMT3A mutations (P=0.049) were independent adverse factors for overall survival. The crosstalk between MLL-PTD and genes involving DNA methylation in the leukemogenesis of AML warrants further investigation. Conclusions Gene mutations involving DNA methylation frequently co-existed in AML patients with MLL-PTD, especially DNMT3A mutations which conferred a poor outcome. Our study demonstrated the importance of genetic alterations involving DNA methylation in the pathogenesis of MLL-PTD AML and provided potential epigenetic-targeted therapy. Grant support The work was supported by NHRI-EX93-9011SL, NSC95-2314-B-195-001, NSC96-2314-B-195-006-MY3, NSC97-2314-B-182-011-MY3 and MMH-E-101-09. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3468 Poster Board III-356 Background Transcription factor RUNX1 is essential for normal hematopoiesis. RUNX1 mutations, mainly at Runt homology domain (RHD), have been described in patients with AML-M0 and were rarely found in non-M0 AML. Aim We aimed to analyze the RUNX1 mutations in AML patients with partial tandem duplication of MLL (MLL-PTD) and to investigate the biological functions of the mutants detected. Patients and methods Bone marrow samples from 93 patients with MLL-PTD were analyzed for RUNX1 mutations. MLL-PTD was screened by Southern-blot analysis followed by RT-PCR or detected by real-time quantitative PCR. Mutational analysis of RUNX1 gene was performed by sequencing of all RT-PCR products amplified from exon 3 through exon 8. Each mutation was reconfirmed with alternative primers. The wild-type, all mutants of RUNX1 (except those truncated at RHD or silent) and pcDNA3.1 were transiently transfected into Cos-7 cells. Immunoblot analysis after immunoprecipitation with anti-FLAG RUNX1 antibody and electrophoretic mobility shift assay were used to determine the interaction with CBFβ and DNA-binding ability of the RUNX1 mutants. Dual luciferase assay system was used to analyze the transactivation potential of RUNX1 mutants in K562 cells. Results RUNX1 mutations were detected in 23 patients (24.7%) at diagnosis, with 3 patients carrying double mutations; 14 mutations were located in RHD (exons 3-5) and 12 at C-terminal region (exons 6-8). In addition, one patient acquired a C-terminal mutation at relapse. The patterns of 27 mutations consisted of 6 missense mutations, 3 nonsense mutations, 17 frame-shift mutations, and 1 silent mutation; all were heterozygous. Of the 3 patients with double mutations, clonal analysis showed that one patient had combined missense and frame-shift mutations on the same allele, the other patient had two missense mutations on different alleles, and another patient had a missense mutation and a silent mutation on the same allele. Functional analyses revealed significant difference among mutants. Two missense RUNX1 mutants at RHD (G108D and R174L) and all of the frame-shift mutants in the transactivation domain (TAD) ( S287fsX571, S295fsX571, L300fsX570, V333fsX574, I339fsX569 and P355fsX572) exhibited lack of DNA-binding ability and inhibited transactivation activity of wild-type RUNX1 in a dominant-negative effect. All frame-shift mutants distal to the TAD generated termination codons within the 3'-untranslated region (H377fsX565, Q388fsX572, L414fsX569, L414fsX567 and V425fsX576), they all retained the normal transactivation activity as the wild-type. R177X and R205W retained the ability of heterodimerization with CBFβ but they had markedly reduced DNA-binding and no transactivation potential without inhibitory effect on wild-type RUNX1. L183fsX185 could bind DNA but lacked transactivation activity. S114P and Q370R had normal transactivation activity. Conclusions Our results showed that patients with de novo AML with MLL-PTD had a high frequency of frame-shift mutations at C-terminal region of RUNX1; those within TAD had dominant-negative effects whereas those distal to TAD retained the normal transactivation potential. Supported by grants NHRI-EX96-9434SI, NSC97-2314-B-182 -011-MY3 and MMH-E-96009. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 3064 Poster Board III-1 Both ETV6-RUNX1 (TEL-AML1)fusion and hyperdiploidy (〉50 chromosomes) of lymphoblasts are favorable outcome predictors in childhood acute lymphoblastic leukemia (ALL). In 433 children with ALL diagnosed at our hospitals between 1997 and 2007 in Taiwan, the frequency of ETV6-RUNX1 fusion was 15.8%, and the frequency of hyperdiploidy (〉50 chromosomes) was 14.1%, both were lower than those of the West. While ETV6-RUNX1 fusion had borderline favorable impact on outcome (p=0.053-0.061), hyperdiploidy showed significant favorable impact on event-free survival (91.1% vs 76.6 %, p= 0.016) in our patients. A meta-analysis from literature enrolled reports in which the case numbers and frequency of ETV6-RUNX1 fusion or hyperdiploidy in childhood ALL were described. It revealed that the frequency of ETV6-RUNX1 fusion in childhood ALL in Far East (Japan, Korea, Hong Kong, Chinese in Singapore, and Taiwan) was 14.2% (127/893, range 10-17%), significantly lower than 21.8% (152/697, range 19-27%) in the West (USA, Germany, Italy, France and Chile) (p 〈 0.0001). The frequency of hyperdiploidy in Japan and Taiwan was 15.2% (140/921, range 13-20%), significantly lower than 31.6% in the West (977/3,158, range 19-34%) (USA, UK and Germany) (p 〈 0.0001). So far as we know, there were several articles, including ours, addressing that the frequency of ETV6-RUNX1 fusion in childhood ALL was lower in a Far East country. This is the first meta-analysis to demonstrate that the frequency of ETV6-RUNX1 fusion in childhood ALL in Far East was lower than that in the West. There was no report on that the frequency of hyperdiploidy in Far East was lower than that in the West. This is also the first meta-analysis to demonstrate that the frequency of hyperdiploidy in childhood ALL in Far East is significantly lower than that in the West. The nature of these differences, probably due to racial, needs further study. In Far East, with both a lower frequency of ETV6-RUNX1 fusion, and a lower frequency of hyperdiploidy, it warrants renewed effort to cure a higher proportion of children with ALL. Disclosures No relevant conflicts of interest to declare.

Description: A T-maze test is an experimental approach that is used in congenital research. However, the food reward-based protocol for the T-maze test in fish has low efficiency and a long training period. The aim of this study is to facilitate the T-maze conditions by using a combination of the principles of passive avoidance and a spatial memory test. In our modified T-maze settings, electric shock punishment (1–2 V, 0.3–0.5 mA) is given at the left arm, with a green cue at the right arm. Also, the depth of both arms of the T-maze was increased. The parameters measured in our T-maze design were latency, freezing time, and time spent in different areas of the T-maze. We validated the utility of our modified T-maze protocol by showing the consistent finding of memory impairment in ZnCl2−treated fish, which has been previously detected with the passive avoidance test. In addition, we also tested the spatial memory performance of leptin a (lepa) mutants which displayed an obesity phenotype. The results showed that although the learning and memory performance for lepa KO fish were similar to control fish, they displayed a higher freezing behavior during the training phase. In conclusion, we have established a modified T-maze protocol that can be used to evaluate the anxiety, learning, and memory capacity of adult zebrafish within three days, for the first time.

Description: Notch signaling is one of the evolutionarily conserved signaling pathways in multicellular organisms. It plays an important role in embryonic development. During skeletal development of vertebrates, it regulates bone homeostasis by manipulating both osteoblastogenesis and osteoclastogenesis through different mechanisms. However, due to the different nature of Notch signaling in mesenchymal stem cell and osteoblast, regulation of Notch signaling in bone-related diseases remains unsettled. Previous studies by cell culture and mouse models showed contradictory results regarding the role of Notch signaling in bone homeostasis. To clarify the role of Notch signaling in osteogenesis, we established a zebrafish model, in which Notch1a intracellular domain (N1aICD) was specifically expressed in the osteoblasts. We found that overexpression of N1aICD in osteoblasts caused hyperosteogeny in the column region of zebrafish with the morphology of narrowed neural/hemal canals. Moreover, increased metabolic activity of osteoblasts instead of augmenting osteoblast number led to hyperosteogeny in N1aICD-overexpressed zebrafish. In summary, we successfully established a transgenic zebrafish line overexpressing N1aICD to clarify the in-vivo function of Notch signaling during osteoblastogenesis. In the future, this fish line can serve as a valuable tool to test the therapeutic drugs for hyperosteogeny.

Description: Lead and lead-derived compounds have been extensively utilized in industry, and their chronic toxicity towards aquatic animals has not been thoroughly addressed at a behavioral level. In this study, we assessed the risk of exposure to lead at a waterborne environmental concentration in adult zebrafish by behavioral and biochemical analyses. Nine tests, including three-dimension (3D) locomotion, novel tank exploration, mirror biting, predator avoidance, social interaction, shoaling, circadian rhythm locomotor activity, color preference, and a short-term memory test, were performed to assess the behavior of adult zebrafish after the exposure to 50 ppb PbCl2 for one month. The brain tissues were dissected and subjected to biochemical assays to measure the relative expression of stress biomarkers and neurotransmitters to elucidate the underlying mechanisms for behavioral alterations. The results of the behavioral tests showed that chronic exposure to lead could elevate the stress and anxiety levels characterized by elevated freezing and reduced exploratory behaviors. The chronic exposure to PbCl2 at a low concentration also induced a sharp reduction of aggressiveness and short-term memory. However, no significant change was found in predator avoidance, social interaction, shoaling, or color preference. The biochemical assays showed elevated cortisol and reduced serotonin and melatonin levels in the brain, thus, altering the behavior of the PbCl2-exposed zebrafish. In general, this study determined the potential ecotoxicity of long-term lead exposure in adult zebrafish through multiple behavioral assessments. The significant findings were that even at a low concentration, long-term exposure to lead could impair the memory and cause a decrease in the aggressiveness and exploratory activities of zebrafish, which may reduce their survival fitness.

Description: In this study, we evaluated the acute (24, 48, 72, and 96 h) and chronic (21 days) adverse effects induced by low doses (0.1, 0.5, 1, and 1.5 mg/L) of zinc chloride (ZnCl2) exposure in adult zebrafish by using behavioral endpoints like three-dimensional (3D) locomotion, passive avoidance, aggression, circadian rhythm, and predator avoidance tests. Also, brain tissues were dissected and subjected to analysis of multiple parameters related to oxidative stress, antioxidant responses, superoxide dismutase (SOD), neurotoxicity, and neurotransmitters. The results showed that ZnCl2-exposed fishes displayed decreased locomotor behavior and impaired short-term memory, which caused an Alzheimer’s Disease (AD)-like syndrome. In addition, low concentrations of ZnCl2 induced amyloid beta (amyloid β) and phosphorylated Tau (p-Tau) protein levels in brains. In addition, significant induction in oxidative stress indices (reactive oxygen species (ROS) and malondialdehyde (MDA)), reduction in antioxidant defense system (glutathione (GSH), GSH peroxidase (GSH-Px) and SOD) and changes in neurotransmitters were observed at low concentrations of ZnCl2. Neurotoxic effects of ZnCl2 were observed with significant inhibition of acetylcholine (ACh) activity when the exposure dose was higher than 1 ppm. Furthermore, we found that zinc, metallothionein (MT), and cortisol levels in brain were elevated compared to the control group. A significantly negative correlation was observed between memory and acetylcholinesterase (AChE) activity. In summary, these findings revealed that exposure to ZnCl2 affected the behavior profile of zebrafish, and induced neurotoxicity which may be associated with damaged brain areas related to memory. Moreover, our ZnCl2-induced zebrafish model may have potential for AD-associated research in the future.