Calcineurin subunit B is involved in shell regeneration in Haliotis diversicolor

Funding: This study was supported by the Thailand Research Fund (TRF) through a Royal Golden Jubilee Ph.D. Program (Grant No. PHD/0047/2554).Abalone shells are mainly composed of two major polymorphs of CaCO3 that are distributed in different layers of the shell. The process of shell biomineralization is controlled by genes and proteins expressed within the mantle epithelium. In this present paper, we conducted a shell regeneration experiment to study the role of HcCNA and HcCNB (individual subunits of calcineurin) in shell biomineralization in H. diversicolor. The results of qPCR showed that HcCNB is upregulated to a greater extent than HcCNA in the mantle after shell notching. In vivo study of the effects of rHcCNB injection showed a significantly higher percentage of regenerated shell length, but not area, in the injected group compared to the control group. In addition, SEM observation of the inner surface of the regenerated shells revealed three different zones including prismatic, nacreous, and a distinct transition zone. Changes in the crystal organization and ultrastructure are clearly evident in these three zones, particularly after 3 weeks of rHcCNB administration. We hypothesize that this is due to faster biomineralization rates in the rHcCNB treated group. Taken together, our results demonstrate that HcCNB participates in shell regeneration in H. diversicolor. As calcineurin subunits have also been implicated in shell formation in bivalves, these findings suggest that calcineurin subunits may play important roles in biomineralization in all conchiferans.Publisher PDFPeer reviewe

Chimeric MrNV-GE11-VLPs serve as a nano-container to deliver Doxorubicin into cancer cells

We have reported that virus-like particle from shrimp virus, MrNV-VLP, effectively encapsulates and delivers plasmid DNA and dsRNA into Sf9 insect cells and shrimp tissues. Additionally, modifying VLP with GE-11 peptide extension on the surface (so called, E-MrNV-GE11-VLP) allows them to interact specifically with the EGFR-positive SW480 cancer cells. This work extrapolated the use of E-MrNV-GE11-VLP to encapsulate and deliver doxorubicin (DOX) towards SW480 cells. The results showed that DOX was passively loaded into VLPs in a molar ratio of >200 DOX/VLP equivalent to a loading efficiency of 3%. Specific targeting of E-MrNV-GE11-VLP + DOX and its anti-cancer effect towards SW480 was more pronounced than that of N-MrNV-VLP + DOX, suggesting an interaction and internalization of E-MrNV-GE11-VLP through surface EGFR. This claim was also supported by a lower DOX delivery into MCF7 than SW480 cells. Finally, the cell cytotoxicity assay showed that E-MrNV-GE11-VLP + DOX significantly decreased cell viability in SW480 cells more than that by N-MrNV-VLP + DOX (P<0.05), while its cytotoxicity effect on MFC7 cells was much lower than on SW480 cells. This study provides insights into how to develop target-specific drug delivery for carrying therapeutic agents towards specific tumor cells

Nanocontainer designed from an infectious hypodermal and hematopoietic necrosis virus (IHHNV) has excellent physical stability and ability to deliver shrimp tissues

Background A virus-like particle (VLP) is an excellent tool for a compound delivery system due to its simple composition, symmetrical structure and self-assembly. Its surface modification both chemically and genetically is established, leading to the target-specific delivery and improved encapsulation efficiency. However, its physical stabilities against many harsh conditions that guarantee long term storage and oral administration have been much less studied. Methods IHHNV-VLPs were reconstructed from recombinant IHHNV capsid protein in E. coli. Their physical properties against three strong physical conditions including long term storage (0–30 days) in 4 °C, physical stabilities against broad ranged pH (4–9) and against three types of digestive enzymes were tested. Disassembly and reassembly of VLPs for encapsidating an enhanced green fluorescent protein tagged plasmid DNA (EGFP-VLPs) were controlled by the use of reducing agent (DTT) and calcium specific chelating agent (EGTA). Lastly, delivering ability of EGFP-VLPs was performed in vivo by intramuscular injection and traced the expression of GFP in the shrimp tissues 24 hr post-injection. Results Upon its purification, IHHNV-VLPs were able to be kept at 4 °C up to 30 days with only slight degradation. They were very stable in basic condition (pH 8–9) and to a lesser extent in acidic condition (pH 4–6) while they could stand digestions of trypsin and chymotrypsin better than pepsin. As similar with many other non-enveloped viruses, the assembly of IHHNV-VLPs was dependent on both disulfide bridging and calcium ions which allowed us to control disassembly and reassembly of these VLPs to pack EGFP plasmid DNA. IHHNV-VLPs could deliver EGFP plasmids into shrimp muscles and gills as evident by RT-PCR and confocal microscopy demonstrating the expression of GFP in the targeted tissues. Discussion There are extensive data in which capsid proteins of the non-enveloped viruses in the form of VLPs are constructed and used as nano-containers for therapeutic compound delivery. However, the bottleneck of its application as an excellent delivery container for oral administration would rely solely on physical stability and interacting ability of VLPs to the host cells. These properties are retained for IHHNV-VLPs reported herein. Thus, IHHNV-VLPs would stand as a good applicable nanocontainer to carry therapeutic agents towards the targeting tissues against ionic and digestive conditions via oral administration in aquaculture field

Raw data for Figure 1

Raw data of Figure 1:  Agarose gel electrophoresis (1%) of dsDNA encapsulated VLP.</p

Kiatmetha_Raw Data

<div><div><b>Figure 1A:</b> Agarose gel electrophoresis showing the expression of IHHNV capsid ORF at 990 bp upon transforming vectors into various transformed <i>E. coli</i>, S1-S4.</div><div><br></div><div><b> Figure 1B:</b> SDS-PAGE protein profiling of a highly purified IHHNV capsid protein at 37.5 kDa (arrowhead) through a Nikel affinity chromatography.</div><div><br></div><div><b>Figure 2A:</b> Physical stability tests of IHHNV-VLPs against long term storage at 4ºC, from 1 day storsge up to 30 days. </div><div><br></div></div><div><b></b><i></i><u></u><b></b><i></i><u></u><b></b><i></i><u></u><br></div><div><b></b><i></i><u></u><b></b><i></i><u></u><br></div><div><b></b><i></i><u></u><b></b><i></i><u></u><br></div><div><br></div

Kiatmetha_Raw Data 3&4

<p><b>Figure 3D</b>: Agarose gel electrophoresis of the encapsidated EGFP-plasmid DNA into the VLP interior showing a major DNA band at 3.1 kbp (lane 4). An additional minor band at 6.2 kbp (lane 3; arrowheads) of unloaded plasmid was also noted.</p> <p><br> <br> </p> <p><b>Figure 4A</b>: Agarose gel electrophoresis of EGFP gene (600 bp product) present in the shrimp muscle, gill and hepatopancreas.</p><p><br></p><p><b>Figure 4B</b>: Confocal microscopy of shrimp tissues injected with EGFP-IHHNV-VLPs (green), and stained with anti-GFP (red). The bottom-most row represents control where the shrimp are injected with unloaded IHHNV-VLPs.</p><p><br></p><p><b>Figure 4C</b>: Western blot showing anti-GFP reactivity in the EGFP-IHHNV-VLPs injected shrimp's tissues.</p

Characterization of calcineurin A and B genes in the abalone, Haliotis diversicolor, and their immune response role during bacterial infection

Calcineurin (CN) is known to be involved in many biological processes, particularly, the immune response mechanism in many invertebrates. In this study, we characterized both HcCNA and HcCNB genes in Haliotis diversicolor, documented their expression in many tissues, and discerned their function as immune responsive genes against Vibrio parahaemolyticus infection. Similar to other mollusk CNs, the HcCNA gene lacked a proline-rich domain and comprised only one isoform of its catalytic unit, in contrast to CNs found in mammals. HcCNB was highly conserved in both sequence and domain architecture. Quantitative PCR and in situ hybridization revealed that the genes were broadly expressed and were not restricted to tissues traditionally associated with immune function. Upon infection of H. diversicolor with V. parahaemolyticus (a bacteria that causes serious disease in crustaceans and mollusks), both HcCNA and HcCNB genes were highly up-regulated at the early phase of bacterial infection. HcCNB was expressed significantly higher than HcCNA in response to bacterial challenge, suggesting its independent or more rapid response to bacterial infection. Together, the two CN genes are unique in their gene structure (particular HcCNA) and distribution in mollusk species and likely function as immune responsive genes along with many other genes that are enhanced in the early phase of V. parahaemolyticus infection in abalone.</p

Morphological and biochemical alterations of abalone testicular germ cells and spawned sperm and their fertilizing ability

In this study, we aimed to detect morphological and biochemical changes in developing germ cells (Gc), testicular sperm (Tsp), and spawned sperm (Ssp) using capacitation-associated characteristics. Gradual changes in the profiles of two membrane proteins, namely NaCl- and detergent-extractable proteins, were observed as compared Gc with Tsp and Tsp with Ssp. These membrane modifications were accomplished mostly through the introduction of new protein sets, both peripheral and integral, into Tsp and Ssp membranes. Activation of serine proteases, particularly in Ssp detergent-extracted proteins with the molecular masses of 38&ndash;130 kDa was evident and marked a major difference between Ssp and Tsp. An increase in the level of tyrosine phosphorylation of the proteins ranging from 15 to 20 kDa was noted in Tsp and remained constant in Ssp. Specifically, these three capacitation-associated characteristics could be detected in Ssp, possessing full fertilizing capacity. The lack of an activated proteolytic activity in Tsp resulted in a delayed fertilization, but not affected fertilizing ability. We believe that these characteristics should be advantageous in predicting abalone sperm fertilizing capability, particularly in cases when isolated germ cells or purified Tsp are used in place of spawned sperm in abalone aquaculture.<br /