Narrow Results

Revealing biological responses of organisms in responding to environmental stressors is the critical issue in contemporary ecological sciences. Markov processes in behavioral data were unraveled by utilizing the hidden Markov model (HMM). Individual organisms of daphnia (Daphnia magna) and zebrafish (Danio rerio) were exposed to diazinon at low concentrations. The transition probability matrix (TPM) and the emission probability matrix (EPM) were accordingly estimated by training with the HMM and were verified before and after the treatments with 10^-6 tolerance in 10³ iterations. Structured property in behavioral changes was accordingly revealed to characterize dynamic processes in movement patterns. Parameters and sequences produced through the HMM training could be a suitable means of monitoring toxic chemicals in environment.

Based on computer vision techniques, the movement tracks of an indicator species (zebrafish) were continuously observed in two dimensions before and after the treatments with a toxic chemical (formaldehyde, 2.5 ppm). Behavioral patterns based on the shape of movement segments were regarded as states, while linear and angular speeds measured from the movement segments were used as observed events for training with a hidden Markov model (HMM). The state sequences were estimated by HMM based on transition and emission probability matrices, and observed events. The movement tracks were further reconstructed based on behavior state sequences generated by HMM. Subsequently, permutation entropy and fractal dimension were calculated to monitor behavioral changes before and after the treatments. Both parameters based on the real and reconstructed data significantly decreased after the treatments, and individual variability was minimized with the parameters obtained from the reconstructed tracks. The parameter extraction based on optimal state sequence by HMM was suitable for resolving the problem of variability in behavioral data, and would be an effective means of monitoring chemical stress in the environment.

Zebrafish is emerging as a species of choice in alcohol-related pharmacological studies. In these studies, zebrafish are often exposed to acute ethanol treatments and their activity scored during behavioral assays. Computational modeling of zebrafish behavior is expected to positively impact these efforts by offering a predictive toolbox to plan hypothesis-driven studies, reduce the number of subjects, perform pilot trials, and refine behavioral screening. In this work, we demonstrate the use of the recently proposed jump persistent turning walker to model the turning rate dynamics of zebrafish exposed to acute ethanol administration. This modeling framework is based on a stochastic mean reverting jump process to capture the sudden and large changes in orientation of swimming zebrafish. The model is calibrated on an available experimental dataset of 40 subjects, tested at different ethanol concentrations. We demonstrate that model parameters are modulated by ethanol administration, whereby both the relaxation rate and jump frequency of the turning rate dynamics are influenced by ethanol concentration. This effort offers a first evidence for the possibility of complementing zebrafish pharmacological research with computational modeling of animal behavior.

The metabolic process of steroidogenesis exhibits a complex biochemical network topology as the activity of various steroidogenic enzymes control cholesterol metabolism to steroid hormone derivatives. In this paper, a stoichiometric reconstruction of a sub-set of 65 reactions from the zebrafish (Danio rerio) steroidogenic network is presented and simulated using uniform reaction constraints. The reconstruction defined a set of 65 enzyme catalyzed reactions and 37 exchange or transport reactions for steroid metabolites. The reconstructed reactions were inclusive of cholesterol and androgen/estrogen metabolism. Biased (statement of network objective function) and un-biased (no statement of objective function) analyses were applied to identify network properties dependent on reaction stoichiometry. Random sampling of flux distributions through the network identified highly-correlated reaction sets that corresponded to the catalysis of steroid metabolites of physiological relevance. Subsequently, optimal flux distributions through network pathways were determined for the production of the three steroidogenic metabolites of: 11-deoxycorticosterone, testosterone and 17β-estradiol. Furthermore, flux variability analyses revealed and confirmed optimal network fluxes through physiologically feasible pathways. The stoichiometric dependence of reactions was also confirmed by conducting deletions of reactions utilized for the optimal production of 17β-estradiol. This paper demonstrates the potential application of constraint-based reconstruction and simulation techniques in enabling the construction of deterministic and predictive physiological models. This acknowledgement is poignant considering the susceptibility of the steroidogenic network to environmental and anthropogenic stressors.

Major Breakthrough in Cancer Treatment.

Zebrafish Model for Leukemia Research.

Singapore Scientists Embark on Human Gene Study with the Zebrafish.

Singapore May Soon Have the Answer to the Cure for SARS.

Novel Liver Cancer Gene Identified.

Orchid’s Unique Nutrient Transformation Mechanism Revealed.

Vitamin Fortification Could Play a Role in Genetic Selection.

Using the Zebrafish to Study Human Diseases.

New Bone Implants That Resemble Natural Bone.

Exploring the Role of Glutathione in the Regulation of Immune Cell Function.

Does Oxidative Damage Cause Poor Healing?

Pathogenesis of Atopic Dermatitis in Singapore.

Proteomics and Colorectal Cancer Metastasis: Bird's-Eye View on Current Scenario and Our Contribution.

Zebrafish: A Small Fish Model for a Big Human Disease.

The Reign of a New Dictator: Circulating MicroRNA in Diabetes.

Engineering Artificial Vascularized Bone Grafts for the Repair of Large Bone Defects.

A 'Nano' Era for Blood Glucose Sensing.

Ancient Medicine with Newer Roles: Potential Role of Celastrol in the Treatment of Multiple Myeloma.

Proteins, Proteome and Proteomics.

A Novel Promising Biomarker and Therapy Target of Liver Cancer.

Training experimental animals to discriminate between different visual stimuli has been an important tool in cognitive neuroscience as well as in vision research for many decades. Current methods used for visual choice discrimination training of zebrafish require human observers for response tracking, stimulus presentation and reward delivery and, consequently, are very labor intensive and possibly experimenter biased. By combining video tracking of fish positions, stimulus presentation on computer monitors and food delivery by computer-controlled electromagnetic valves, we developed a method that allows for a fully automated training of multiple adult zebrafish to arbitrary visual stimuli in parallel. The standardized training procedure facilitates the comparison of results across different experiments and laboratories and contributes to the usability of zebrafish as vertebrate model organisms in behavioral brain research and vision research.

In recent times, zebrafish has garnered lot of popularity as model organism to study human cancers. Despite high evolutionary divergence from humans, zebrafish develops almost all types of human tumors when induced. However, mechanistic details of tumor formation have remained largely unknown. Present study is aimed at analysis of repertoire of kinases in zebrafish proteome to provide insights into various cellular components. Annotation using highly sensitive remote homology detection methods revealed "substantial expansion" of Ser/Thr/Tyr kinase family in zebrafish compared to humans, constituting over 3% of proteome. Subsequent classification of kinases into subfamilies revealed presence of large number of CAMK group of kinases, with massive representation of PIM kinases, important for cell cycle regulation and growth. Extensive sequence comparison between human and zebrafish PIM kinases revealed high conservation of functionally important residues with a few organism specific variations. There are about 300 PIM kinases in zebrafish kinome, while human genome codes for only about 500 kinases altogether. PIM kinases have been implicated in various human cancers and are currently being targeted to explore their therapeutic potentials. Hence, in depth analysis of PIM kinases in zebrafish has opened up new avenues of research to verify the model organism status of zebrafish.

Zebrafish is an important animal model, which is used to study development, pathology, and genetic research. The zebrafish skin model is widely used in cutaneous research, and angiogenesis is critical for cutaneous wound healing. However, limited by the penetration depth, the available optical methods are difficult to describe the internal skin structure and the connection of blood vessels between the skin and subcutaneous tissue. By a homemade high-resolution polarization-sensitive optical coherence tomography (PS-OCT) system, we imaged the polarization contrast of zebrafish skin and the zebrafish skin vasculature with optical coherence tomography angiography (OCTA). Based on these OCT images, the spatial distribution of the zebrafish skin vasculature was described. Furthermore, we monitored the healing process of zebrafish cutaneous wounds. We think the high-resolution PS-OCT system will be a promising tool in studying cutaneous models of zebrafish.

Embryos develop robust spatiotemporal patterns by encoding and interpreting biological signals in real time. Developmental patterns often scale with body or tissue size even when total cell number, cell size or growth rate are changed. A striking example of patterning is the segmentation of somites — the precursors of vertebral column. Despite decade-long efforts, how positional information for segmentation is encoded by cell signaling remained elusive. To address this fundamental question, we studied a novel zebrafish tail explant model that recapitulated the scaling of somite sizes with the length of unsegmented tissue in growing intact embryos. This paper provides an algorithm written in MATLAB as well as Python and finally finding a way to write an efficient algorithm to be able to answer the question described above. Information encoding by spatial fold-change of cell signaling is a remarkable strategy that could be utilized for engineering precisely patterned tissues or organs. We also discuss the limitations of simulations performed using MATLAB with performance decreasing with the large data sets. So, we tried to analyze the factors that impacted the performance of the algorithm. Finally, we tried to answer questions regarding the language selection in which a simulation method can be written efficiently.

Optical imaging uses nonionizing radiation to obtain images of tissues and cells inside the body. The approach reduces exposure to harmful radiation and is suitable for lengthy and repetitive imaging procedures. Development of strongly fluorescent imaging agents will help in the detection of signal through thick tissues. Presence of such biocompatible imaging agent has potential clinical applications as it gives real-time information on disease progression and therapeutic response. We report here a nanoformulation-based strategy to synthesize a strongly fluorescent imaging agent. The fabrication procedure uses silica nanocapsules (SNC) to trap and concentrate highly fluorescent Coumarin 545T fluorophore. Biocompatibility of synthesized SNC–Coumarin was tested in cell lines and zebrafish. In vivo detection of fluorescent signal was validated in optically translucent zebrafish larvae and adult casper mutant. Nonbiased labeling of all cell types was detected in both young and adult zebrafish. The ability to differentiate fluid filled cavities from cells was also highlighted during in vivo imaging. Concomitant assessment of internalized SNC–Coumarin through acquired fluorescent intensity and associated biocompatibility in zebrafish supports its use as an in vivo imaging agent.

The zebrafish has been an increasingly popular animal model for human diseases as it offers the combined advantages compared to various animal models and cell based assays; in particular, the feasibility of high throughput studies as an economical vertebrate model. In this past decade, we and several other laboratories have developed various hepatocellular carcinoma (HCC) models using the zebrafish and demonstrated the conservation of HCC between zebrafish and human at both histopathological and molecular levels. In this review, we focus on the conservation of signal transductions during hepatocarcinogenesis between zebrafish and human. Based on existing zebrafish HCC models, indeed many alterations of signal pathways that cause human liver cancers can also result in HCC in zebrafish, such as Ras pathway, EGFR pathway, Wng/β-catenin pathway, TGF-β pathway, PI3K/AKT pathway, JAK/STAT pathway, Hippo pathway, src tyrosine kinase pathway, etc. In future, zebrafish may be used for better quantification of signaling molecules and thus to aid development of more effective therapeutic methods.

Cell migration plays an important role in a wide variety of biological processes. In the developing central nervous system (CNS), many neuronal precursor cells migrate from their site of origin to the final position in which they differentiate. In recent years, studies have often focused on stationary or in vitro systems to analyze neuronal migration. However, to understand the cellular and molecular mechanisms of neuronal migration, as well as the malfunction of this process in the diseased brain, it is of great importance to study it in real time in the live intact organism. Advances in microscopy techniques and the development of new dyes and genetically encoded markers have enormously improved in vivo time-lapse studies in the last few years. Here, we will describe the zebrafish as a model system to study the migration of a group of motor neurons in the hindbrain. These neurons move from the place where they are born and specified to more caudal regions in the brain. In order to study the cellular dynamics and molecular mechanisms of this process, we are using a stable transgenic line that expresses a green fluorescent protein (GFP) specifically in a subset of the hindbrain motor neurons. Using multiphoton excitation microscopy we can analyze their migration deep within the live tissue over extended periods of time.

Formation of a functional nervous system requires the coordinated interaction of neurons and glial cells. These cells often migrate considerable distances from their origins, recognize each other and form intimate and very specific physical connections. The highly dynamic behaviors of neural cells during development are not adequately captured by standard experimental approaches of fixed tissue analysis and in vitro methods. To facilitate direct observation of neural cells within intact embryos, we created transgenic zebrafish that express fluorescent proteins under control of cell type-specific promoters and developed methods for long term, in vivo, time-lapse imaging. In this chapter, we present a brief overview of central and peripheral nervous system development and describe a few of our studies that have revealed new cell origins and behaviors and our investigations of gene functions that direct glial cell specification and differentiation.

The migration of immature neurons from proliferation zones to their place of final differentiation occurs in many areas throughout the developing vertebrate central nervous system. As migration represents a key step in neuronal differentiation it has been modeled extensively in cell culture, matrix gels or explanted tissues. Due to the many complex interactions that are involved in this dynamic process, it would be ideal though to observe neuronal migration non-invasively directly in the developing organism. Furthermore, not only migratory pathways have to be characterized in detail, but also the underlying cell biology of neuronal migration needs to be revealed. For example, addressing how signal transduction is mediated into directed cellular movement at the level of different cellular organelles is not only crucial for understanding the cellular and molecular interplay of dynamic brain differentiation but also for revealing the etiology of neuronal migration diseases. Recent advances in zebrafish conditional genetics and simultaneous multi-cistron expression combined with the progress in high-resolution high-speed bio-optics promise that neuronal migration can be resolved at unprecedented detail in this organism. Thus the stage is set in zebrafish for true in vivo cell biology, which will allow for the re-addressing of the many models of neuronal migration derived from in vitro data of cultured cells or tissue explants. Hence zebrafish can serve to fuse the large fields of developmental genetics and cell biology in vertebrates.

Recent advances in light microscopy and spectroscopy make it possible to study single molecule behavior in an intact 3D multicellular organism. Taking advantage of the transparent tissues of zebrafish embryos as well as the established molecular and genetic approaches in this animal model, we present in this chapter some examples of studying molecular dynamics and interactions in living zebrafish embryos by fluorescence correlation spectroscopy (FCS) and single wavelength fluorescence cross-correlation spectroscopy (SWFCCS). FCS/SW-FCCS are ultra-sensitive experimental techniques that analyze fluorescence fluctuations from a static observation volume and provide information about the concentration, diffusion constant and biophysical properties of the fluorescent particle. A short introduction of the theory and instrumentations of FCS/FCCS is provided. We then give an overview of new FCS modalities that are designed to cope with emerging challenges in intracellular applications, e.g. correction of cell membrane movement during measurement and correction of spectral cross-talk of fluorescent proteins. A short description of the preparation of zebrafish embryos for FCS measurements is included. In the last section we demonstrate measurements of blood flow velocities with high spatial resolution, measurements of diffusion coefficients of fluorescently labeled cytosolic and membrane-bound proteins within living zebrafish embryos, and the determination of dissociation constants of interacting proteins by SW-FCCS. The extension of this biophysical tool into living organism allows answering developmental biology questions directly on a molecular basis, and provides a platform to address the potential artifacts arising from Petri dish-based in vitro studies.

Cells of the innate immune system perform a number for functions that range from clearing apoptotic cell corpses during normal organ development to removing invading pathogens and helping regulate the immune response to infection. Traditional approaches to assessment of these immune cell behaviors have relied upon histological analysis of fixed tissue samples complemented by in vitro functional data. Despite providing significant insights, translating the results from such studies into a multicellular whole animal context is difficult. Recent advances in cell labeling techniques and imaging technologies has given researchers unprecedented ability to directly observe immune cell activities within a live whole animal context. Such live imaging approaches are essential to completely appreciate these activities within their intact normal physiological setting. The zebrafish embryo, with its optical transparency and fully functional innate and adaptive immune systems, represents an ideal system in which to live image innate immune cells. When this is coupled with the relative ease with which specific immune cells can be fluorescently labeled within transgenic reporter lines and the ability to both genetically and chemically interfere with genetic pathways, the zebrafish is emerging as a strong vertebrate platform in which to make novel contributions to understanding immune cell biology. This chapter will discuss how our group and others use live imaging to assess the behavior of myeloid leukocytes during normal development and in response to different inflammatory stimuli, including wounding and pathogenic challenge.