Illuminating the dark depths inside coral.

The ability to observe in situ 3D distribution and dynamics of endosymbionts in corals is crucial for gaining a mechanistic understanding of coral bleaching and reef degradation. Here, we report the development of a tissue clearing (TC) coupled with light sheet fluorescence microscopy (LSFM) method for 3D imaging of the coral holobiont at single-cell resolution. The initial applications have demonstrated the ability of this technique to provide high spatial resolution quantitative information of endosymbiont abundance and distribution within corals. With specific fluorescent probes or assays, TC-LSFM also revealed spatial distribution and dynamics of physiological conditions (such as cell proliferation, apoptosis, and hypoxia response) in both corals and their endosymbionts. This tool is highly promising for in situ and in-depth data acquisition to illuminate coral symbiosis and health conditions in the changing marine environment, providing fundamental information for coral reef conservation and restoration.

Excessive inflammation impairs heart regeneration in zebrafish breakdance mutant after cryoinjury.

Inflammation plays a crucial role in cardiac regeneration. Numerous advantages, including a robust regenerative ability, make the zebrafish a popular model to study cardiovascular diseases. The zebrafish breakdance (bre) mutant shares several key features with human long QT syndrome that predisposes to ventricular arrhythmias and sudden death. However, how inflammatory response and tissue regeneration following cardiac damage occur in bre mutant is unknown. Here, we have found that inflammatory response related genes were markedly expressed in the injured heart and excessive leukocyte accumulation occurred in the injured area of the bre mutant zebrafish. Furthermore, bre mutant zebrafish exhibited aberrant apoptosis and impaired heart regenerative ability after ventricular cryoinjury. Mild dosages of anti-inflammatory or prokinetic drugs protected regenerative cells from undergoing aberrant apoptosis and promoted heart regeneration in bre mutant zebrafish. We propose that immune or prokinetic therapy could be a potential therapeutic regimen for patients with genetic long QT syndrome who suffers from myocardial infarction.

SAGER: a database of Symbiodiniaceae and Algal Genomic Resource.

Symbiodiniaceae dinoflagellates are essential endosymbionts of reef building corals and some other invertebrates. Information of their genome structure and function is critical for understanding coral symbiosis and bleaching. With the rapid development of sequencing technology, genome draft assemblies of several Symbiodiniaceae species and diverse marine algal genomes have become publicly available but spread in multiple separate locations. Here, we present a Symbiodiniaceae and Algal Genomic Resource Database (SAGER), a user-friendly online repository for integrating existing genomic data of Symbiodiniaceae species and diverse marine algal gene sets from MMETSP and PhyloDB databases. Relevant algal data are included to facilitate comparative analyses. The database is freely accessible at http://sampgr.org.cn. It provides comprehensive tools for studying gene function, expression and comparative genomics, including search tools to identify gene information from Symbiodiniaceae species, and BLAST tool to find orthologs from marine algae and protists. Moreover, SAGER integrates transcriptome datasets derived from diverse culture conditions of corresponding Symbiodiniaceae species. SAGER was developed with the capacity to incorporate future Symbiodiniaceae and algal genome and transcriptome data, and will serve as an open-access and sustained platform providing genomic and molecular tools that can be conveniently used to study Symbiodiniaceae and other marine algae. Database URL: http://sampgr.org.cn.

Development of a magnetic microrobot for carrying and delivering targeted cells.

The precise delivery of targeted cells through magnetic field-driven microrobots/carriers is a promising technique for targeted therapy and tissue regeneration. This paper presents a microrobot designed with a burr-like porous spherical structure for carrying and delivering targeted cells in vivo under a magnetic gradient field-driven mechanism. The robot was fabricated by using three-dimensional laser lithography and coated with Ni for magnetic actuation and Ti for biocompatibility. Numerical and experimental studies demonstrated that the proposed microrobot design could enhance magnetic driving capability, promote cell-carrying capacity, and benefit cell viability. Microrobots loaded with cells could be automatically controlled to reach a desired site by using a self-constructed electromagnetic coil system, as verified by in vivo transport of cell-cultured microrobots in zebrafish embryos. The carried cells could be spontaneously released from the microrobot to the surrounding tissues; in vitro experiments showed that cells from the microrobot were directly released onto the desired site or were able to pass through the blood vessel-like microchannel to arrive at the delivery area. Further in vivo cell-releasing tests were performed on nude mice, followed by histological study. This research provides a microrobotic device platform for regenerative medicine and cell-based therapy.

Simultaneous expression of apolipoprotein B mRNA editing enzyme and scavenger receptor BI mediated by a therapeutic gene expression system.

Cardiovascular diseases are often accompanied by elevated LDL particles and endothelial dysfunction. We have examined the possibility of concurrently reducing LDL levels and modulating endothelial function using a single helper-dependent adenovirus vector system to simultaneously express the apolipoprotein B mRNA editing enzyme (Apobec1) and the scavenger receptor, class B, type I (SR-BI) genes under the control of separate promoters (designated HD-C2). Apobec1 edits apoB mRNA at nucleotide C-6666 to produce truncated apoB48 and is normally expressed in small intestine only. SR-BI is a receptor for multiple ligands with distinct tissue-specific functions. Expression of Apobec1 in HepG2 cells resulted in apoB mRNA editing, leading to decreased apoB100 abundance (to 6% of control) and the appearance of apoB48. Editing of apoB mRNA in HepG2 cells resulted in decline in apoB mRNA levels of 50%. This was probably the result of nonsense-mediated decay of edited message, since over-expression of Apobec1 increased neither Apobec1 complementary factor (ACF) mRNA nor protein abundance. Over-expression of SR-BI in human endothelial cells activated endothelial nitric oxide synthase (eNOS) activity by phosphorylation of eNOS at residue Ser-1177 in the presence of HDL, leading to increased production of the anti-atherogenic molecule nitric oxide (NO). Taken together, this study demonstrates that using one vector delivery system to express two genes in two different cell types results in the cell-specific beneficial effects of decreasing apoB100 production and increasing eNOS activities. This combined gene expression approach may provide an improved therapeutic strategy by targeting multiple sites in the mechanism of cardiovascular injury.

Single Cell Transfection through Precise Microinjection with Quantitatively Controlled Injection Volumes.

Cell transfection is a technique wherein foreign genetic molecules are delivered into cells. To elucidate distinct responses during cell genetic modification, methods to achieve transfection at the single-cell level are of great value. Herein, we developed an automated micropipette-based quantitative microinjection technology that can deliver precise amounts of materials into cells. The developed microinjection system achieved precise single-cell microinjection by pre-patterning cells in an array and controlling the amount of substance delivered based on injection pressure and time. The precision of the proposed injection technique was examined by comparing the fluorescence intensities of fluorescent dye droplets with a standard concentration and water droplets with a known injection amount of the dye in oil. Injection of synthetic modified mRNA (modRNA) encoding green fluorescence proteins or a cocktail of plasmids encoding green and red fluorescence proteins into human foreskin fibroblast cells demonstrated that the resulting green fluorescence intensity or green/red fluorescence intensity ratio were well correlated with the amount of genetic material injected into the cells. Single-cell transfection via the developed microinjection technique will be of particular use in cases where cell transfection is challenging and genetically modified of selected cells are desired.