An improved automated zebrafish larva high-throughput imaging system.

As a typical multicellular model organism, the zebrafish has been increasingly used in biological research. Despite the efforts to develop automated zebrafish larva imaging systems, existing ones are still defective in terms of reliability and automation. This paper presents an improved zebrafish larva high-throughput imaging system, which makes improvements to the existing designs in the following aspects. Firstly, a single larva extraction strategy is developed to make larva loading more reliable. The aggregated larvae are identified, classified by their numbers and patterns, and separated by the aspiration pipette or water stream. Secondly, the dynamic model of larva motion in the capillary is established and an adaptive robust controller is designed for decelerating the fast-moving larva to ensure the survival rate. Thirdly, rotating the larva to the desired orientation is automated by developing an algorithm to estimate the larva's initial rotation angle. For validating the improved larva imaging system, a real-time heart rate monitoring experiment is conducted as an application example. Experimental results demonstrate that the goals of the improvements have been achieved. With these improvements, the improved zebrafish larva imaging system remarkably reduces human intervention and increases the efficiency and success/survival rates of larva imaging.

Zebrafish Larva Orientation and Smooth Aspiration Control for Microinjection.

OBJECTIVE: The zebrafish has been proven to be a significant model organism in various research fields. For investigating the in vivo properties of biologics within zebrafish with developed organs, an automated zebrafish larva organ injection system is crucially needed. However, current zebrafish larva manipulation techniques cannot accomplish this operation efficiently and continuously. METHODS: In this paper, we present a novel zebrafish larva manipulation technique with two key steps in the microinjection system: orienting and aspirating zebrafish larvae automatically. The orientation control is realized in a customized microfluidic chip, after which the larva moves tail-first until reaching the channel exit. Then a dynamic model of larva aspiration is established and an adaptive robust controller is designed. RESULTS: Experimental results demonstrate that high success rate can be reached and damage to larva body is reduced. CONCLUSION: The presented strategy is capable of orienting and aspirating zebrafish larvae smoothly and efficiently. SIGNIFICANCE: The proposed methods have the advantage of low cost, easy implementability and good stability.

CNTNAP2 stabilizes interneuron dendritic arbors through CASK.

Contactin associated protein-like 2 (CNTNAP2) has emerged as a prominent susceptibility gene implicated in multiple complex neurodevelopmental disorders, including autism spectrum disorders (ASD), intellectual disability (ID), and schizophrenia (SCZ). The presence of seizure comorbidity in many of these cases, as well as inhibitory neuron dysfunction in Cntnap2 knockout (KO) mice, suggests CNTNAP2 may be crucial for proper inhibitory network function. However, underlying cellular mechanisms are unclear. Here we show that cultured Cntnap2 KO mouse neurons exhibit an inhibitory neuron-specific simplification of the dendritic tree. These alterations can be replicated by acute knockdown of CNTNAP2 in mature wild-type (WT) neurons and are caused by faulty dendrite stabilization rather than outgrowth. Using structured illumination microscopy (SIM) and stimulated-emission depletion microscopy (STED), two super-resolution imaging techniques, we uncovered relationships between nanoscale CNTNAP2 protein localization and dendrite arborization patterns. Employing yeast two-hybrid screening, biochemical analysis, in situ proximity ligation assay (PLA), SIM, and phenotype rescue, we show that these effects are mediated at the membrane by the interaction of CNTNAP2's C-terminus with calcium/calmodulin-dependent serine protein kinase (CASK), another ASD/ID risk gene. Finally, we show that adult Cntnap2 KO mice have reduced interneuron dendritic length and branching in particular cortical regions, as well as decreased CASK levels in the cortical membrane fraction. Taken together, our data reveal an interneuron-specific mechanism for dendrite stabilization that may provide a cellular mechanism for inhibitory circuit dysfunction in CNTNAP2-related disorders.

Visual Servoed Three-Dimensional Rotation Control in Zebrafish Larva Heart Microinjection System.

OBJECTIVE: Zebrafish larva heart microinjection is a widely used technique in cardiac disease study. Compared with intensively researched rotation control of spherical or nearly spherical targets with clear structures, such as cells and embryos, 3-D rotation control of zebrafish larva demands new techniques due to its nontransparent structures and irregular outlines. METHODS: In this paper, we present a vision-servo system to automate the rotation process of zebrafish larva body. A switched control strategy is adopted to rotate zebrafish larva about the optical axis by using two micropipettes. Precisely rolling about larva body is performed, which involves a custom-designed rotational micromanipulator. A vision detection and online tracking algorithm is also developed to meet the requirement of visual servoing. With designed rotation control strategy, zebrafish larva heart can be adjusted to a desired orientation, which is often towards the injection pipette tip. RESULTS: Experimental results show that the designed system is capable of achieving high success rate of 94% about -axis rotation and 100% about -axis with 50 trails. The system also performs an average speed of 44 s/larva with a satisfied rotation accuracy of 0.5 in the horizontal plane and 2.5 about its roll axis. CONCLUSION: The proposed strategy is effective in flexibly manipulating larvae in 3-D. SIGNIFICANCE: The developed 3-D rotation control scheme is able to be applied to injection of various organs in zebrafish larva body for different experimental requirements.

Magnetic lipid nanocapsules (MLNCs): self-assembled lipid-based nanoconstruct for non-invasive theranostic applications.

We report magnetic nanostructure-stabilized lipid nanocapsules (MLNCs) that show superior structural stability and theranostic properties compared to conventional lipid-based nanocarriers. As therapeutic nanocarriers, the MLNCs exhibit a therapeutic efficacy that is 16 times greater than that of free drugs due to their high payload capacity and actuated drug release ability. In addition, the magnetic resonance contrast enhancement of the MLNCs is nine times higher than that of a clinically approved T2 MRI contrast agent (ferumoxytol), demonstrating the diagnostic imaging capability of the MLNCs in MRI. The self-assembly method to synthesize the lipid nanocapsules is extended to other types of nanoparticles (gold nanoparticles and quantum dots) to produce lipid nanohybrids with distinct physical properties.

Construction and evaluation of DNA vaccine encoding Hantavirus glycoprotein N-terminal fused with lysosome-associated membrane protein.

BACKGROUND: Hantaviral diseases can have a high case fatality rate within the absence of broadly effective antiviral treatments or vaccines. We developed a DNA vaccine targeting the Hantavirus glycoprotein N-terminal (Gn) to major histocompatibility complex class II compartment by fusing the antigen with lysosome-associated membrane protein 1 (LAMP1), which altered antigen presenting pathway and activated the CD4+ T cells. METHODS: The segments of Gn and LAMP1 were cloned into vector pVAX1, and recombinant plasmid was constructed by inserting Gn sequence into LAMP1, between luminal and the transmembrane/cytoplasmic domains. Subsequently, the protein expression was identified through immunoprecipitation, western blot and Immunofluorescent assay. Adaptive immune responses were assessed by the presence of specific and neutralizing antibodies, interferon (ELISpot results, and cytotoxic T-lymphocyte (CTL) cytotoxicity. Epitope mapping was performed to study the T-cell epitopes. Protective immunity in vivo was evaluated using a novel HTNV-challenging model, and safety evaluation was based on histological and behavioral observations. RESULTS: Native or LAMP1 targeting HTNV Gn was successfully identified. Humoral immune responses were enhanced, featuring with satisfying titers of specific and neutralizing antibody production. The boosted activities of IFN-γ and CTL cytotoxicity witnessed enhanced cellular immune responses. Effective protection against HTNV in vivo was conferred in all three vaccine groups by the challenge model. Safety was confirmed and one dominant T-cell epitope screened from immunized mice overlapped the specific T-cell hot spot in HFRS patients. CONCLUSION: LAMP1 targeting strategy successfully enhanced the efficacy of HTNV Gn-based vaccine, which is highly immunogenic and safe, showing promise for immunoprophylaxis against HFRS. Further investigations are warranted in the future.

Intraspecific competition and light effect on reproduction of Ligularia virgaurea, an invasive native alpine grassland clonal herb.

The relationship between sexual reproduction and clonal growth in clonal plants often shows up at the ramet level. However, only a few studies focus on the relationship at the genet level, which could finally account for evolution. The sexual reproduction and clonal growth of Ligularia virgaurea, a perennial herb widely distributed in the alpine grasslands of the Qinghai-Tibetan Plateau of China, were studied under different competition intensities and light conditions at the genet level through a potted experiment. The results showed that: (1) sexual reproduction did not depend on density or light, and increasing clonal growth with decreasing density and increasing light intensity indicated that intraspecific competition and light intensity may affect the clonal life history of L. virgaurea; (2) both sexual reproduction and clonal growth show a positive linear relationship with genet size under different densities and light conditions; (3) a threshold size is required for sexual reproduction and no evidence of a threshold size for clonal growth under different densities and light conditions; (4) light level affected the allocation of total biomass to clonal and sexual structures, with less allocation to clonal structures and more allocation to sexual structures in full sunlight than in shade; (5) light determined the onset of sexual reproduction, and the genets in the shade required a smaller threshold size for sexual reproduction to occur than the plants in full sunlight; and (6) no evidence was found of trade-offs between clonal growth and sexual reproduction under different densities and light conditions at the genet level, and the positive correlation between two reproductive modes indicated that these are two integrated processes. Clonal growth in this species may be viewed as a growth strategy that tends to maximize genet fitness.

Variation in floral sex allocation, female success, and seed predation within racemiform synflorescence in the gynomonoecious Ligularia virgaurea (Asteraceae).

Studies of diclinous species have showed that floral sex allocation and female reproductive success were quite variable within inflorescences. However, little attention has been paid to gynomonoecious species, in which individuals produce both female and bisexual flowers. The purpose of this study was to investigate the variations in reproductive patterns at different capitulum positions within racemiform synflorescence in Ligularia virgaurea, and to determine selective mechanisms of variations in reproductive patterns. We conducted observational and experimental studies in natural populations of the gynomonoecious composite L. virgaurea. Floral sex allocation, seed production and pre-dispersal seed predation were quantified in the field. The results showed several patterns of variation from top to bottom capitula, including an increase in bisexual flowers and flower number per capitulum, but a decrease in seed set and size. Removing earlier capitula during bud stage did not change floral sex allocation in later capitula. And no effect was found on seed set under supplemental pollination. Thus, although it has been reported many times in previous studies, the variation of floral sex allocation in L. virgaurea may not result from architectural effect or mating environment, and the variation of seed production could not be fully explained by pollination success. Additionally, our results showed that L. virgaurea was susceptible to high levels of bisexual biased predation, which was greater for top capitula. We therefore suggest that these variations may help to enhance reproductive success of L. virgaurea in the face of bisexual-biased seed predation.