Interior modification of Macrobrachium rosenbergii nodavirus-like particle enhances encapsulation of VP37-dsRNA against shrimp white spot syndrome infection.

BACKGROUND: Application of a virus-like particle (VLP) as a nanocontainer to encapsulate double stranded (ds)RNA to control viral infection in shrimp aquaculture has been extensively reported. In this study, we aimed at improving VLP's encapsulation efficiency which should lead to a superior fighting weapon with disastrous viruses. RESULTS: We constructed 2 variants of chimeric Macrobrachium rosenbergii nodavirus (MrNV)-like particles (V1- and V2-MrN-VLPs) and tested their efficiency to encapsulate VP37 double stranded RNA as well as WSSV protection in P. vannamei. Two types of short peptides, RNA-binding domain (RBD) and deca-arginine (10R) were successfully engineered into the interior surface of VLP, the site where the contact with VP37-dsRNA occurs. TEM and dynamic light scattering (DLS) analyses revealed that the chimeric VLPs remained their assembling property to be an icosahedral symmetric particle with a diameter of about 30 nm, similar to the original MrN-VLP particle. The superior encapsulation efficiency of VP37-dsRNA into V2-MrN-VLP was achieved, which was slightly better than that of V1-MrN-VLP but far better (1.4-fold) than its parental V0-MrN-VLP which the mole ratio of 7.5-10.5 for all VLP variants. The protection effect against challenging WSSV (as gauged from the level of VP37 gene and the remaining viral copy number in shrimp) was significantly improved in both V1- and V2-MrN-VLP compared with an original V0-MrN-VLP template. CONCLUSION: MrN-VLP (V0-) were re-engineered interiorly with RBD (V1-) and 10R (V2-) peptides which had an improved VP37-dsRNA encapsulation capability. The protection effect against WSSV infection through shrimp administration with dsRNA + V1-/V2-MrN VLPs was experimentally evident.

Infectivity and virulence of the infectious Macrobrachium rosenbergii nodavirus produced from Drosophila melanogaster cell using Penaeus merguiensis as an infection model.

It has been established that baculovirus-insect cell line is applicable for shrimp virus replication, propagation and secretion in the in vitro culture system. We thus aimed to produce Macrobrachium rosenbergii nodavirus (MrNV) clone within S2 cell to improve viral production over the previous model using Sf9 cell. Upon the transfection of genomic RNA1 and RNA2 into S2 cells, the recognizable cellular changes including cytoplasmic swelling and clumping of cells were observed within 24 h. The culture media containing secreted MrNV particles were re-transfected into healthy S2 cells and similar cellular changes as with the first transfection were observed. Immunohistochemistry analysis of the re-infecting S2 cell revealed an intense immunoreactivity against MrNV capsid protein confirming that S2 cell was permissive cells for MrNV. In vivo infectivity test using P. merguiensis as a model animal exposed to the secreted MrNV revealed the presence of RNA2 fragment in shrimp tissue accompanied with the sign of whitish abdominal muscle at 24 h post-infection (p.i.). In addition, the number of shrimp hemocytes decreased at 6-24 h p.i. and returned to the normal level at 48 h p.i., whereas a significant up-regulation of immune-related genes including HSP70 and trypsin was noted. These data suggested that rescued MrNV produced in S2 is practically useful for MrNV infection test in which their natural virion inoculae are difficult to obtain. In addition, the molecular basis of viral pathogenesis can further be investigated which should be beneficial for any antiviral therapy developments in the future.

Effect of caffeine on genes expressions of developing retinas in the chick model.

It has been reported that overconsumption of caffeine during pregnancy leads to a deleterious effect within the nervous tissues during embryonic development. In this study, we further extrapolated the effect of caffeine in the developing retinas, which is known to be one of the most sensitive tissues in chick embryos. Morphological changes of retinal thickness and organization of neuroretinal epithelium were monitored using three gene markers, Atoh7, FoxN4, and Lim1. Upon treating with a single dose of caffeine (15 µmol at embryonic day 1 [E1]), relative thicknesses of developing retinas (particularly of E7 and E9) were significantly altered. Among the three genes studied, the expression pattern of Atoh7 was notably altered while those of FoxN4, and Lim1 mRNA showed only a slight change in these developing retinas. Quantitative polymerase chain reaction results supported the most notable changes of Atoh7 but not FoxN4, and Lim1 gene in the developing retinas, particularly at E7. The effect of caffeine towards other organs during development should be extrapolated and the awareness of its intensive consumption should be raised.

Shrimp thrombospondin (TSP): presence of O-ß1,4 N-acetylglucosamine polymers and its function in TSP chain association in egg extracellular matrix.

We characterized the existence of O-ß(1,4)-GlcNAc polymers (ß1,4GNP) that were anchored on the O-linked glycosylation sites of shrimp thrombospondin (pmTSP-II). There were five putative ß1,4GNP linkages on the epithelial growth factor-like domain of pmTSP-II. Antibody against O-ß-GlcNAc (CTD110.6) was used to prove the existence of linear and complex ß1,4GNP. The antibody well reacted with linear chito-triose, -tetraose and -pentaose conjugated with phosphatidylethanolamine lipid. The immunoreactivity could also be detected with a complex ß1,4GNP within pmTSP-II (at MW > 250 kDa). Upon denaturing the protein with SDS-PAGE buffer, the size of pmTSP-II was shifted to be 250 kDa, approximately 2.5 folds larger than the deduced molecular mass of pmTSP-II (110 kDa), suggesting additional association of pmTSP-II apart from its known disulfide bridging. This was confirmed by chitinase digestion on pmTSP-II protein leading to the subsequent smaller protein bands at 110-170 kDa in time- and concentration-dependent manners. These bands well reacted with CTD110.6 antibody and disappeared after extensive chitinase hydrolysis. Together, we believe that ß1,4GNP on pmTSP-II serve the function in an inter-chain association to provide structural architecture of egg extracellular matrix, a novel function of pmTSP-II in reproductive biology.

Anatomical variants identified on chest computed tomography of 1000+ COVID-19 patients from an open-access dataset.

Chest computed tomography (CT) has been the preferred imaging modality during the pandemic owing to its sensitivity in detecting COVID-19 infections. Recently, a large number of COVID-19 imaging datasets have been deposited in public databases, leading to rapid advances in COVID-19 research. However, the application of these datasets beyond COVID-19-related research has been little explored. The authors believe that they could be used in anatomical research to elucidate the link between anatomy and disease and to study disease-related alterations to normal anatomy. Therefore, the present study was designed to investigate the prevalence of six well-known anatomical variants in the thorax using open-access CT images obtained from over 1000 Iranian COVID-19 patients aged between 6 and 89 years (60.9% male and 39.1% female). In brief, we found that the azygos lobe, tracheal bronchus, and cardiac bronchus were present in 0.8%, 0.2%, and 0% of the patients, respectively. Variations of the sternum, including sternal foramen, episternal ossicles, and sternalis muscle, were observed in 9.6%, 2.9%, and 1.5%, respectively. We believe anatomists could benefit from using open-access datasets as raw materials for research because these datasets are freely accessible and are abundant, though further research is needed to evaluate the uses of other datasets from different body regions and imaging modalities. Radiologists should also be aware of these common anatomical variants when examining lung CTs, especially since the use of this imaging modality has increased during the pandemic.

Infectious hypodermal and hematopoietic necrosis virus-like particle (IHHNV-VLP) induces peroxiredoxin expression and activity in Fenneropenaeus merguiensis.

Virus like particles (VLPs) are non-infectious nanoparticles containing repetitive, high density viral epitopes on the surface and can prevent viral infections in aquatic animals. Here, we evaluated the immuno-stimulation effect of infectious hypodermal and hematopoietic necrosis virus like particle (IHHNV-VLP) using a next generation sequencing in Fenneropenaeus merguiensis to identify the important immune-related genes that may prevent viral infection. The in situ target of IHHNV was predominantly found in gill tissue following IHHNV-VLP administration in juvenile shrimp. Comparative transcriptome analysis in the injected gills showed that there were 326 unigenes expressed differently than the mock-injected samples. One of the most differential genes between the two animal groups was the antioxidative gene, peroxiredoxin (FmPrx), that was up-regulated after 6 h post-VLP injection. Phylogenetic tree analysis showed that this gene could be found among many shrimp species and was closely clustered among Prx families. The expression of FmPrx was also detected in all tissues examined, thus suggesting the multi-functional roles of this gene in many tissues. Administration of IHHNV-VLP in vivo led to a significant increase in peroxidase activity in gill tissue-approximately two-fold versus control animals; the WSSV copy number was significantly reduced. These data suggest that IHHNV-VLP exerts an immune-stimulating effect by enhancing the level of immune-related genes including FmPrx and its corresponding peroxidase activity, which are a well-known part of the shrimp innate immune system.

Viral Capsid Change upon Encapsulation of Double-Stranded DNA into an Infectious Hypodermal and Hematopoietic Necrosis Virus-like Particle.

In this study, we aimed to encapsulate the sizable double-stranded DNA (dsDNA, 3.9 kbp) into a small-sized infectious hypodermal and hematopoietic necrosis virus-like particle (IHHNV-VLP; T = 1) and compared the changes in capsid structure between dsDNA-filled VLP and empty VLP. Based on our encapsulation protocol, IHHNV-VLP was able to load dsDNA at an efficiency of 30-40% (w/w) into its cavity. Structural analysis revealed two subclasses of IHHNV-VLP, so-called empty and dsDNA-filled VLPs. The three-dimensional (3D) structure of the empty VLP produced in E. coli was similar to that of the empty IHHNV-VLP produced in Sf9 insect cells. The size of the dsDNA-filled VLP was slightly bigger (50 Å) than its empty VLP counterpart; however, the capsid structure was drastically altered. The capsid was about 1.5-fold thicker due to the thickening of the capsid interior, presumably from DNA-capsid interaction evident from capsid protrusions or nodules on the interior surface. In addition, the morphological changes of the capsid exterior were particularly observed in the vicinity of the five-fold axes, where the counter-clockwise twisting of the "tripod" structure at the vertex of the five-fold channel was evident, resulting in a widening of the channel's opening. Whether these capsid changes are similar to virion capsid maturation in the host cells remains to be investigated. Nevertheless, the ability of IHHNV-VLP to encapsulate the sizable dsDNA has opened up the opportunity to package a dsDNA vector that can insert exogenous genes and target susceptible shrimp cells in order to halt viral infection.

Bone Morphogenetic Protein 2/4 in Mollusk, Haliotis diversicolor: Its Expression and Osteoinductive Function In Vitro.

Bone morphogenetic proteins (BMPs), which are members of the superfamily of transforming growth factor-ß (TGF-ß), are known both in vitro and in vivo for their osteoinduction properties on the osteoblastic cells. Its role in the mollusk shell formation has also been gradually established. Using Haliotis diversicolor as a model, we characterized the HdBMP2/4 gene in the mantle tissue and showed its expression in the outer fold epithelium (particularly at the periostracal groove) the epithelial site which is involved in shell formation, both prismatic and nacreous layers. Shell notching experiments following gene analysis by qPCR revealed the upregulation of the HdBMP2/4 gene up to 3.2-fold than that of the control animals. In vitro treatments of the preosteoblastic cells, MC3T3-E1 with HdBMP2/4 synthetic peptide demonstrated the enhanced effect of many osteogenic genes that are known to regulate bone and shell biomineralization including ALP, Runx2, and OCN with 2-4 fold-change throughout 14 days of culture. In addition, the increased deposition of calcium-based mineral (as assessed by Alizarin red staining) of the treated cells was comparable to the ascorbic acid (Vit C) + glycerophosphate positive control which revealed the enhanced effect of HdBMP2/4 peptide on matrix biomineralization of the preosteoblastic cells. In conclusion, these results indicated the presence of the HdBMP2/4 gene in the mantle tissue at the site involved in shell formation and the effect of the HdBMP2/4 knuckle epitope peptide in osteoinduction in vitro.

Characterization of Thrombospondin Type 1 Repeat in Haliotis diversicolor and Its Possible Role in Osteoinduction.

Thrombospondin repeats (TSR) are important peptide domains present in the sequences of many extracellular and transmembrane proteins with which a variety of ligands interact. In this study, we characterized HdTSR domains in the ADAMTS3 protein of Thai abalone, Haliotis diversicolor, based on the transcriptomic analysis of its mantle tissues. PCR amplification and localization studies demonstrated the existence of HdTSR transcript and protein in H. diversicolor tissues, particularly in both the inner and outer mantle epithelial folds. We, therefore, generated a short recombinant protein, termed HdTSR1/2, based on the existence of the WxxWxxW or WxxxxW motif (which binds to TGF-ß, a known signaling in bone formation/repair) in HdTSR1 and HdTSR2 sequences and used it to test the osteoinduction function in the pre-osteoblastic cell line, MC3T3-E1. This recombinant protein demonstrated the ability to induce the differentiation of MC3T3-E1 cells by the concentration- and time-dependent upregulation of many known osteogenic markers, including RUNX2, COL1A1, OCN, and OPN. We also demonstrated the upregulation of the SMAD2 gene after cell treatment with HdTSR1/2 proteinindicating its possible interaction through TGF-ß, which thus activates its downstream signaling cascade and triggers the biomineralization process in the differentiated osteoblastic cells. Together, HdTSR domains existed in an extracellular ADAMTS3 protein in the mantle epithelium of H. diversicolor and played a role in osteoinduction as similar to the other nacreous proteins, opening up its possibility to be developed as an inducing agent of bone repair.

Chimeric virus-like particles (VLPs) designed from shrimp nodavirus (MrNV) capsid protein specifically target EGFR-positive human colorectal cancer cells.

Recombinant MrNV capsid protein has been shown to effectively deliver plasmid DNA and dsRNA into Sf9 insect cells and shrimp tissues. To extend its application to cancer cell-targeting drug delivery, we created three different types of chimeric MrNV virus-like particles (VLPs) (R-MrNV, I-MrNV, and E-MrNV) that have specificity toward the epidermal growth factor receptor (EGFR), a cancer cell biomarker, by incorporating the EGFR-specific GE11 peptide at 3 different locations within the host cell recognition site of the capsid. All three chimeric MrNV-VLPs preserved the ability to form a mulberry-like VLP structure and to encapsulate EGFP DNA plasmid with an efficiency comparable to that previously reported for normal MrNV (N-MrNV). Compared to N-MrNV, the chimeric R-MrNV and E-MrNV carrying the exposed GE-11 peptide showed a significantly enhanced binding and internalization abilities that were specific towards EGFR expression in colorectal cancer cells (SW480). Specific targeting of chimeric MrNV to EGFR was proven by both EGFR silencing with siRNA vector and a competition with excess GE-11 peptide as well as the use of EGFR-negative colorectal cells (SW620) and breast cancer cells (MCF7). We demonstrated here that both chimeric R-MrNV and E-MrNV could be used to encapsulate cargo such as exogenous DNA and deliver it specifically to EGFR-positive cells. Our study presents the potential use of surface-modified VLPs of shrimp virus origin as nanocontainers for targeted cancer drug delivery.

Dual VP28 and VP37 dsRNA encapsulation in IHHNV virus-like particles enhances shrimp protection against white spot syndrome virus.

Accumulative evidence of using double stranded (ds) RNA encapsulated into virus like particle (VLP) nanocarrier has open feasibility to fight against shrimp viral infection in aquaculture field. In this study, we co-encapsulated VP37 and VP28 dsRNA into hypodermal and hematopoietic necrosis virus (IHHNV) like particle and investigated its protection against white spot syndrome virus (WSSV). Five micrograms of each dsRNA were used as starting materials to load into VLP, while the loading efficiency was slightly different, i.e, VP37 dsRNA had somewhat a better load into VLP's cavity. It was apparent that co-encapsulation of dual dsRNA showed a superior WSSV silencing ability than the single dsRNA counterpart as evidence by the lower WSSV gene expression and its copy number in the gill tissues. Besides, we also demonstrated that co-encapsulated dual dsRNA into IHHNV-VLP stimulated the increased number of hemocytes and the corresponding PO activity as well as up-regulated proPO gene expression in hemocytes to resist viral invasion after an acute stage of WSSV infection. This synergistic action of dual dsRNA encapsulated into IHHNV-VLPs could thus act to delay time of shrimp death and reduced shrimp cumulative mortality greater than the single, naked dsRNA treatment and positive control groups. The obtaining results would encourage the feasibility to use it as a new weapon to fight WSSV infection in shrimp aquaculture.

Calcineurin subunit B is involved in shell regeneration in Haliotis diversicolor.

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.

Mannosylated glycoconjugates on the surface of activated sperm in the giant freshwater prawn are crucial for sperm binding with the egg vitelline envelop.

The unusual morphology and poorly defined acrosome-like structure in the mature sperm of the giant freshwater prawn Macrobrachium rosenbergii has led to difficulties in identifying the state of sperm activation. Mature distal vas deferens sperm (dVSp) can be activated by the calcium ionophore A23187 to show acrosome reaction-like enzymatic activities that increase their binding and penetration capabilities. However, these short-lived enzymatic activities limit their usefulness as a marker of sperm activation for further qualitative and quantitative analyses, leading to our examining the alterations in the exposure of sperm surface glycoconjugates both as markers of sperm activation and for their role in gamete interaction. Our results showed that after A23187 treatment, there was an increased exposure of mannosylated glycoconjugates on the sperm surface revealed by significant Concanavalin A (Con A) staining. Furthermore, sodium metaperiodate pre-treatment, Con A pre-incubation, or co-incubation with α-mannose monosaccharides all significantly reduced A23187-induced dVSp binding to the egg vitelline envelop, demonstrating the importance of sperm surface mannosylated glycoconjugates in the binding process. These same pre-treatments of sperm also resulted in the inhibition of the binding of soluble vitelline envelop proteins (MrVE) to both the sperm surface and to mannosylated dVSp protein bands. Therefore, the present study demonstrated the importance of the exposure of mannosylated glycoconjugates on the surface of activated dVSp, both as a reliable marker of sperm activation and as a binding factor in the gamete interaction process. Furthermore, these findings allow for a better understanding of the surface glycoconjugate-mediated interaction process between gametes in this species of prawn.

Cathepsin D in prawn reproductive system: its localization and function in actin degradation.

Cathepsin D (CAT-D) is a well-known aspartic protease that serves a function as house-keeping lysosomal enzyme in all somatic cells. Its existence in reproductive tissues is highly variable, even in the somatic derived epithelial cells of reproductive tract. In Macrobrachium rosenbergii, existence of MrCAT-D and its translational product was detected in both somatic cells (Sertoli-like supporting cells) and developing spermatogenic cells as well as along accessory spermatic ducts. Specifically, MrCAT-D was localized onto the sperm surface rather than within the acrosomal matrix, as evident by similar staining pattern of anti-CAT-D on live and aldehyde fixed sperm. MrCAT-D in testicular extracts and sperm isolates showed active enzyme activities towards its specific fluorogenic substrate (MCA-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys (Dnp)-D-Arg-NH2). MrCAT-D also exerted its function towards hydrolyzing filamentous actin, the meshwork of which is shown to be localized at the junction between germ cells and supporting cells and spermatogonia in M. rosenbergii testicular epithelium. Together, we have localized MrCAT-D transcript and its translational product in both supporting and germ cells of testis and claimed its enzymatic function towards actin degradation, which may be related to sperm release from the epithelial cell interaction.

Macrobrachium rosenbergii nodavirus virus-like particles attach to fucosylated glycans in the gills of the giant freshwater prawn.

The Macrobrachium rosenbergii nodavirus (MrNV), the causative agent of white-tail disease (WTD) in many species of shrimp and prawn, has been shown to infect hemocytes and tissues such as the gills and muscles. However, little is known about the host surface molecules to which MrNV attach to initiate infection. Therefore, the present study investigated the role of glycans as binding molecules for virus attachment in susceptible tissues such as the gills. We established that MrNV in their virus-like particle (MrNV-VLP) form exhibited strong binding to gill tissues and lysates, which was highly reduced by the glycan-reducing periodate and PNGase F. The broad, fucose-binding Aleuria Aurantia lectin (AAL) highly reduced MrNV-VLPs binding to gill tissue sections and lysates, and efficiently disrupted the specific interactions between the VLPs and gill glycoproteins. Furthermore, mass spectroscopy revealed the existence of unique fucosylated LacdiNAc-extended N-linked and O-linked glycans in the gill tissues, whereas beta-elimination experiments showed that MrNV-VLPs demonstrated a binding preference for N-glycans. Therefore, the results from this study highly suggested that MrNV-VLPs preferentially attach to fucosylated N-glycans in the susceptible gill tissues, and these findings could lead to the development of strategies that target virus-host surface glycan interactions to reduce MrNV infections.

Enhancement of shrimp health and immunity with diets supplemented with combined probiotics: application to Vibrio parahaemolyticus infections.

The application of probiotics for disease control in aquaculture is now a convincing approach towards replacement of antibiotics, which can cause adverse effects in aquatic animals and humans. In this study, we combined 2 probiotics, Lactobacillus acidophilus and Saccharomyces cerevisiae, with shrimp feed to create 2 formulas (WU8 and WU9), which were fed for 10 d to juvenile shrimp Penaeus vannamei. The shrimps were then subjected to a challenge infection with Vibrio parahaemolyticus, the causative agent of acute hepatopancreas necrosis disease (AHPND). The protective effects of probiotics against bacterial infection were investigated through histopathology of the hepatopancrease and immunological evaluation of shrimp. Both WU8 and WU9 probiotic mixtures (1:1, at 108 and 109 CFU kg diet-1) increased blasenzellen hepatopancreatic epithelial cells and reduced pathology caused by AHPND. After 10 d of feeding, hemocyte parameters, including the total hemocyte count, percent of granular hemocytes, and phenoloxidase activity, increased significantly and were still increasing at 24 h post infection. Crustin and penaeidin 3 genes were also highly upregulated in hemocytes before and after 24 h of bacterial challenge and significantly upregulated in the hepatopancreas 1 to 5 d post-infection. A significantly higher survival rate was observed in shrimp fed with the probiotic supplemented diet (>90%) in comparison to the control group (60%). In conclusion, probiotic mixtures of L. acidophilus and S. cerevisiae reduced hepatopancreas pathology and protected shrimp from a challenge with AHPND.

Existence and distribution of Niemann-Pick type 2C (NPC2) in prawn reproductive tract and its putative role as a cholesterol modulator during sperm transit in the vas deferens.

Sequestering of cholesterol (CHO) is a hallmark molecular event that is known to be associated with sperm gaining their fertilizing ability in a broad array of animals. We have shown previously that the level of CHO declines in the Macrobrachium rosenbergii sperm membrane when they are migrating into the vas deferens, prompting us to search for CHO transporters, one of which is Niemann-Pick type 2C (NPC2), within the prawn male reproductive tract. Sequence comparison of MrNPC2 with other NPC2, from crustaceans to mammals, revealed its conserved features in the hydrophobic cavity with 3 amino acids forming a CHO lid that is identical in all species analyzed. Expressions of MrNPC2 transcript and protein were detected in testicular supporting and interstitial cells and along the epithelial cells of the vas deferens. As confirmed by live cell staining, the testicular sperm (Tsp) surface was devoid of MrNPC2 but it first existed on the vas deferens sperm, suggesting its acquisition from the luminal fluid, possibly through trafficking of multi-lamellar vesicles during sperm transit in the vas deferens. We further showed that recombinant MrNPC2 had a high affinity towards CHO in the lipid extracts, either from Tsp or from lipid vesicles in the vas deferens. Together, our results indicated the presence of MrNPC2 in the male reproductive tract, which may play an important role as a CHO modulator between the sperm membrane and vas deferens epithelial communication.

Late administration of caffeine affects cardiac maturation in chick embryos: a combined two and threedimensional morphogenetic and gene analyses

Cardiac malformations are very prevalent and can be caused both by defective genes and envi-ronmental teratogens. Among the latter, caffeine causes malformations when exposed during early cardiac development, whereas its later effects are still unclear. We exposed three-day incubated (D3) chick embryos to 2 mg caffeine and analyzed them at D5, D7 and D9. The embryos were serially sec-tioned and analyzed two-dimensionally. Alternatively, the sections of D9 embryos were reconstructed three-dimensionally using Amira® software and analyzed volumetrically. The expres-sion of genes involved in endothelial-mesenchymal transformation (EMT) was studied by real-time PCR. Interestingly, caffeine treatment at D3 em-bryos did not induce cardiac malformations, but did delay growth, in particular that of the ventricles and ventricular trabeculae. Furthermore, it affected EMT in the endocardial cushion and atrioventricu-lar valves. Gene-expression analysis revealed that caffeine had a progressively deleterious effect on the expressions of GATA4, MMP2, SNAIL1, TWIST1, and VIMENTIN. The effect of late caf-feine administration on the chicken embryos would provide suggestive evident towards a possible heart developmental defect in humans, particularly heavy caffeine consumers during pregnancy

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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.