A Novel Xenograft Model in Zebrafish for High-Resolution Investigating Dynamics of Neovascularization in Tumors

Tumor neovascularization is a highly complex process including multiple steps. Understanding this process, especially the initial stage, has been limited by the difficulties of real-time visualizing the neovascularization embedded in tumor tissues in living animal models. In the present study, we have established a xenograft model in zebrafish by implanting mammalian tumor cells into the perivitelline space of 48 hours old Tg(Flk1:EGFP) transgenic zebrafish embryos. With this model, we dynamically visualized the process of tumor neovascularization, with unprecedented high-resolution, including new sprouts from the host vessels and the origination from VEGFR2+ individual endothelial cells. Moreover, we quantified their contributions during the formation of vascular network in tumor. Real-time observations revealed that angiogenic sprouts in tumors preferred to connect each other to form endothelial loops, and more and more endothelial loops accumulated into the irregular and chaotic vascular network. The over-expression of VEGF165 in tumor cells significantly affected the vascularization in xenografts, not only the number and size of neo-vessels but the abnormalities of tumor vascular architecture. The specific inhibitor of VEGFR2, SU5416, significantly inhibited the vascularization and the growth of melanoma xenografts, but had little affects to normal vessels in zebrafish. Thus, this zebrafish/tumor xenograft model not only provides a unique window to investigate the earliest events of tumoral neoangiogenesis, but is sensitive to be used as an experimental platform to rapidly and visually evaluate functions of angiogenic-related genes. Finally, it also offers an efficient and cost-effective means for the rapid evaluation of anti-angiogenic chemicals

Cardiac Hypertrophy Involves Both Myocyte Hypertrophy and Hyperplasia in Anemic Zebrafish

Background: An adult zebrafish heart possesses a high capacity of regeneration. However, it has been unclear whether and how myocyte hyperplasia contributes to cardiac remodeling in response to biomechanical stress and whether myocyte hypertrophy exists in the zebrafish. To address these questions, we characterized the zebrafish mutant tr265/tr265, whose Band 3 mutation disrupts erythrocyte formation and results in anemia. Although Band 3 does not express and function in the heart, the chronic anemia imposes a sequential biomechanical stress towards the heart. Methodology/principal findings: Hearts of the tr265/tr265 Danio rerio mutant become larger than those of the sibling by week 4 post fertilization and gradually exhibit characteristics of human cardiomyopathy, such as muscular disarray, re-activated fetal gene expression, and severe arrhythmia. At the cellular level, we found both increased individual cardiomyocyte size and increased myocyte proliferation can be detected in week 4 to week 12 tr265/tr265 fish. Interestingly, all tr265/tr265 fish that survive after week-12 have many more cardiomyocytes of smaller size than those in the sibling, suggesting that myocyte hyperplasia allows the long-term survival of these fish. We also show the cardiac hypertrophy process can be recapitulated in wild-type fish using the anemia-inducing drug phenylhydrazine (PHZ). Conclusions/significance: The anemia-induced cardiac hypertrophy models reported here are the first adult zebrafish cardiac hypertrophy models characterized. Unlike mammalian models, both cardiomyocyte hypertrophy and hyperplasia contribute to the cardiac remodeling process in these models, thus allowing the effects of cardiomyocyte hyperplasia on cardiac remodeling to be studied. However, since anemia can induce effects on the heart other than biomechanical, non-anemic zebrafish cardiac hypertrophy models shall be generated and characterized

Lobe Specific Ca2+-Calmodulin Nano-Domain in Neuronal Spines: A Single Molecule Level Analysis

Calmodulin (CaM) is a ubiquitous Ca2+ buffer and second messenger that affects cellular function as diverse as cardiac excitability, synaptic plasticity, and gene transcription. In CA1 pyramidal neurons, CaM regulates two opposing Ca2+-dependent processes that underlie memory formation: long-term potentiation (LTP) and long-term depression (LTD). Induction of LTP and LTD require activation of Ca2+-CaM-dependent enzymes: Ca2+/CaM-dependent kinase II (CaMKII) and calcineurin, respectively. Yet, it remains unclear as to how Ca2+ and CaM produce these two opposing effects, LTP and LTD. CaM binds 4 Ca2+ ions: two in its N-terminal lobe and two in its C-terminal lobe. Experimental studies have shown that the N- and C-terminal lobes of CaM have different binding kinetics toward Ca2+ and its downstream targets. This may suggest that each lobe of CaM differentially responds to Ca2+ signal patterns. Here, we use a novel event-driven particle-based Monte Carlo simulation and statistical point pattern analysis to explore the spatial and temporal dynamics of lobe-specific Ca2+-CaM interaction at the single molecule level. We show that the N-lobe of CaM, but not the C-lobe, exhibits a nano-scale domain of activation that is highly sensitive to the location of Ca2+ channels, and to the microscopic injection rate of Ca2+ ions. We also demonstrate that Ca2+ saturation takes place via two different pathways depending on the Ca2+ injection rate, one dominated by the N-terminal lobe, and the other one by the C-terminal lobe. Taken together, these results suggest that the two lobes of CaM function as distinct Ca2+ sensors that can differentially transduce Ca2+ influx to downstream targets. We discuss a possible role of the N-terminal lobe-specific Ca2+-CaM nano-domain in CaMKII activation required for the induction of synaptic plasticity

Assessing the impact of driver advisory systems on train driver workload, attention allocation and safety performance

Netherlands Railways has developed driver advisory systems (DAS) to provide the train driver with route context information and coasting advice in order to benefit punctuality and energy efficiency. However, the impact of these DAS on human factors aspects and safety performance is unclear. The current study assesses the impact of two DAS levels (route context information and coasting advice) on mental workload, attention allocation and safety performance, using eye tracking, a subjective mental workload rating scale (RSME) and simulator data. The overall findings suggest that the application of DAS levels has no negative impact on safety performance and attention allocation towards the trackside compared to a control condition with static timetable information. Furthermore, safety performance benefits significantly from DAS with route context information. DAS were originally developed to benefit punctuality and energy efficiency goals. This study implicates that DAS can also benefit safety performance. The current study found that DAS could decrease workload when the functionalities meet the requirements of the situation. The possible presence of mental underload and its effect on driving performance should be taken into consideration when implementing DAS. It is essential in the development of DAS that it meaningfully enriches the train driving task in stead of simply increasing mental workload

Modulation by Cocaine of Dopamine Receptors through miRNA-133b in Zebrafish Embryos

The use of cocaine during pregnancy can affect the mother and indirectly might alter the development of the embryo/foetus. Accordingly, in the present work our aim was to study in vivo (in zebrafish embryos) the effects of cocaine on the expression of dopamine receptors and on miR-133b. These embryos were exposed to cocaine hydrochloride (HCl) at 5 hours post-fertilization (hpf) and were then collected at 8, 16, 24, 48 and 72 hpf to study the expression of dopamine receptors, drd1, drd2a, drd2b and drd3, by quantitative real time PCR (qPCR) and in situ hybridization (ISH, only at 24 hpf). Our results indicate that cocaine alters the expression of the genes studied, depending on the stage of the developing embryo and the type of dopamine receptor. We found that cocaine reduced the expression of miR-133b at 24 and 48 hpf in the central nervous system (CNS) and at the periphery by qPCR and also that the spatial distribution of miR-133b was mainly seen in somites, a finding that suggests the involvement of miR-133b in the development of the skeletal muscle. In contrast, at the level of the CNS miR-133b had a weak and moderate expression at 24 and 48 hpf. We also analysed the interaction of miR-133b with the Pitx3 and Pitx3 target genes drd2a and drd2b, tyrosine hydroxylase (th) and dopamine transporter (dat) by microinjection of the Pitx3-3'UTR sequence. Microinjection of Pitx3-3'UTR affected the expression of pitx3, drd2a, drd2b, th and dat. In conclusion, in the present work we describe a possible mechanism to account for cocaine activity by controlling miR-133b transcription in zebrafish. Via miR-133b cocaine would modulate the expression of pitx3 and subsequently of dopamine receptors, dat and th. These results indicate that miRNAs can play an important role during embryogenesis and in drug addiction

Cloning and expression of the UGA4 gene coding for the inducible GABA-specific transport protein of Saccharomyces cerevisiae.

Transport of 4-aminobutyric acid (GABA) in Saccharomyces cerevisiae is mediated by three transport systems: the general amino acid permease (GAP1 gene), the proline permease (PUT4 gene), and a specific GABA permease (UGA4 gene) which is induced in the presence of GABA. The UGA4 gene encoding the inducible GABA-specific transporter was cloned and sequenced and its expression analyzed. The predicted amino acid sequence shows that UGA4 encodes a 62 kDa protein having 9-12 putative membrane-spanning regions. The predicted UGA4 protein shares significant sequence similarity with the yeast choline transporter (CTR gene), exhibiting but limited similarity to the previously reported GABA transporters, i.e. the yeast GAP1 and PUT4 permeases and the rat brain GAT-1 transporter. Induction of UGA4 in the presence of GABA is exerted at the level of UGA4 mRNA accumulation, most probably at the level of transcription itself. This induction is conferred by the 5' flanking region and requires the integrity of two positive regulatory proteins, the inducer-specific factor UGA3 and the pleiotropic factor UGA35/DURL/DAL81. In the absence of the pleiotropic UGA43/DAL80 repressor, UGA4 is constitutively expressed at high level.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe