First report of Perkinsus olseni infections in blood cockles Tegillarca granosa on the south coast of Korea.

The protozoan parasite Perkinsus olseni has become a focus of attention since it has been responsible for mass mortalities and economic losses in a wide range of bivalve hosts globally. The P. olseni host range along the south coast of Korea may extend beyond what was previously understood, and blood cockles in the Family Arcidae are also suggested to be potential hosts of P. olseni. In the present study, we applied histology and molecular techniques to identify Perkinsus sp. infections in the blood cockles Tegillarca granosa, which have been commercially exploited on the south coast of Korea for several decades. Histology and molecular techniques, including genus-specific immunofluorescence assay, species-specific fluorescence in situ hybridization, and phylogeny based on the ribosomal DNA internal transcribed spacer region revealed that T. granosa is infected by P. olseni, although the prevalence was low (0.5%). Histology revealed massive hemocyte infiltrations in the mantle, gill, and digestive gland connective tissues, indicating that the infection exerts negative impacts on the host cockles.

First Report of Enterocytozoon hepatopenaei Infection in Giant Freshwater Prawn (Macrobrachium rosenbergii de Man) Cultured in the Republic of Korea.

In the Republic of Korea, Enterocytozoon hepatopenaei (EHP) was first isolated from Pacific whiteleg shrimp in April 2020; however, there are no existing reports of EHP infection in other shrimp or prawns. Here, we aimed to investigate EHP infection and its prevalence in giant freshwater prawn farms in the Republic of Korea. We tested prawns from 22 farms for EHP infection, and samples from eight farms showed positive EHP infection results in 2021. In EHP-infected prawn farms, the prevalence ranged from 4.9% to 18.2%. The prevalence of EHP infection in the Republic of Korea, derived from the prevalence in prawn farms, was estimated to be 0.8% in 2021. The proliferation of EHP was observed within the hepatopancreatic epithelial cells of prawns using H&E and Giemsa staining. Mature EHP was observed in the sinus between epithelial cells of the digestive tubules. Phylogenetic analysis revealed a clade distinct from the previously reported EHP in Pacific whiteleg shrimps. This is the first report of EHP infection in a giant freshwater prawn in the Republic of Korea, where the prevalence of EHP infection is not high, but it is recognized as an emerging disease that requires periodic monitoring and quarantine management in giant freshwater prawns.

A multipathogen DNA vaccine elicits protective immune responses against two class A bioterrorism agents, anthrax and botulism.

The potential use of biological agents has become a major public health concern worldwide. According to the CDC classification, Bacillus anthracis and Clostridium botulinum, the bacterial pathogens that cause anthrax and botulism, respectively, are considered to be the most dangerous potential biological agents. Currently, there is no licensed vaccine that is well suited for mass immunization in the event of an anthrax or botulism epidemic. In the present study, we developed a dual-expression system-based multipathogen DNA vaccine that encodes the PA-D4 gene of B. anthracis and the HCt gene of C. botulinum. When the multipathogen DNA vaccine was administered to mice and guinea pigs, high level antibody responses were elicited against both PA-D4 and HCt. Analysis of the serum IgG subtype implied a combined Th1/Th2 response to both antigens, but one that was Th2 skewed. In addition, immunization with the multipathogen DNA vaccine induced effective neutralizing antibody activity against both PA-D4 and HCt. Finally, the protection efficiency of the multipathogen DNA vaccine was determined by sequential challenge with 10 LD50 of B. anthracis spores and 10 LD50 of botulinum toxin, or vice versa, and the multipathogen DNA vaccine provided higher than 50% protection against lethal challenge with both high-risk biothreat agents. Our studies suggest the strategy used for this anthrax-botulinum multipathogen DNA vaccine as a prospective approach for developing emergency vaccines that can be immediately distributed on a massive scale in response to a biothreat emergency or infectious disease outbreak. Key points • A novel multipathogen DNA vaccine was constructed against anthrax and botulism. • Robust immune responses were induced following vaccination. • Suggests a potential vaccine development strategy against biothreat agents.

Immunogenicity and Biodistribution of Anthrax DNA Vaccine Delivered by Intradermal Electroporation.

PURPOSE: Anthrax is a lethal bacterial disease caused by gram-positive bacterium Bacillus anthracis and vaccination is a desirable method to prevent anthrax infections. In the present study, DNA vaccine encoding a protective antigen of Bacillus anthracis was prepared and we investigated the influence of DNA electrotransfer in the skin on the induced immune response and biodistribution. METHODS AND RESULTS: The tdTomato reporter gene for the whole animal in vivo imaging was used to assess gene transfer efficiency into the skin as a function of electrical parameters. Compared to that with 25 V, the transgene expression of red fluorescent protein increased significantly when a voltage of 90 V was used. Delivery of DNA vaccines expressing Bacillus anthracis protective antigen domain 4 (PAD4) with an applied voltage of 90 V induced robust PA-D4-specific antibody responses. In addition, the in vivo fate of anthrax DNA vaccine was studied after intradermal administration into the mouse. DNA plasmids remained at the skin injection site for an appropriate period of time after immunization. Intradermal administration of DNA vaccine resulted in detection in various organs (viz., lung, heart, kidney, spleen, brain, and liver), although the levels were significantly reduced. CONCLUSION: Our results offer important insights into how anthrax DNA vaccine delivery by intradermal electroporation affects the immune response and biodistribution of DNA vaccine. Therefore, it may provide valuable information for the development of effective DNA vaccines against anthrax infection.

Purification and cDNA cloning of the antimicrobial peptide apMolluscidin from the pen shell, Atrina pectinata.

A 5.6 kDa antimicrobial peptide (AMP) was purified from acidified gill extract of the pen shell, Atrina pectinata, by cation exchange and C18 reversed-phase high performance liquid chromatography. Comparison of the amino acid sequences and molecular weight of this peptide with those of other known AMPs revealed that it had high sequence homology with that of cgMolluscidin or hdMolluscidin; it was designated apMolluscidin. apMolluscidin comprises 59 amino acid residues containing several dibasic residue repeats and sequence repeats such as Lys-Lys and Lys-Gly. apMolluscidin exhibited potent antimicrobial activity against both Gram-positive bacteria including Bacillus subtilis (minimal effective concentration [MEC], 2.1 µg/mL), and Gram-negative bacteria including E. coli D31 (MEC, 0.5 µg/mL), without hemolytic activity. However, it did not show any activity against fungi such as Candida albicans. Secondary structure prediction suggested that it might form two helical regions and have an amphipathic structure. Full-length apMolluscidin cDNA contained 812 base pairs (bp), including a 5'-untranslated region (UTR) of 82 bp, a 3'-UTR of 547 bp, and a coding sequence of 183 bp encoding 60 amino acids (containing Met). Furthermore, qPCR analyses revealed that the mature peptide translated from apMolluscidin mRNA is expressed in a tissue-specific manner in locations such as the gill and siphon. These results indicate that apMolluscidin might be related to the innate immune defense system of abalone and may not act directly on the bacterial membrane. This is the first report of an AMP from the pen shell with a fully identified amino acid sequence.

Immunoglobulin can be functionally regulated by protein carboxylmethylation in Fc region.

Protein carboxylmethylation methylates the free carboxyl groups in various substrate proteins by protein carboxyl O-methyltransferase (PCMT) and is one of the post-translational modifications. There have been many studies on protein carboxylmethylation. However, the precise functional role in mammalian systems is unclear. In this study, immunoglobulin, a specific form of gamma-globulin, which is a well-known substrate for PCMT, was chosen to investigate the regulatory roles of protein carboxylmethylation in the immune system. It was found that the anti-BSA antibody could be carboxylmethylated via spleen PCMT to a level similar to gamma-globulin. This carboxylmethylation increased the hydrophobicity of the anti-BSA antibody up to 11.4%, and enhanced the antigen-binding activity of this antibody up to 24.6%. In particular, the Fc region showed a higher methyl accepting capacity with 80% of the whole structure level. According to the amino acid sequence alignment, indeed, 7 aspartic acids and 5 glutamic acids, as potential carboxylmethylation sites, were found to be conserved in the Fc portion in the human, mouse and rabbit. The carboxylmethylation of the anti-BSA antibody was reversibly demethylated under a higher pH and long incubation time. Therefore, these results suggest that protein carboxylmethylation may reversibly regulate the antibody-mediated immunological events via the Fc region.

cAMP induces neuronal differentiation of mesenchymal stem cells via activation of extracellular signal-regulated kinase/MAPK.

Mesenchymal stem cells are able to trans-differentiate into nonmesodermal lineage cells. Here, we identified downstream signaling molecules required for acquisition of neuron-like traits by mesenchymal stem cells following the elevation of intracellular cAMP levels. We found that forskolin induced neuron-like morphology and expression of neuron-specific enolase and neurofilament-200 in mesenchymal stem cells. Forskolin sequentially activated protein kinase A and B-regulation of alpha-fetoprotein (Raf), which led to phosphorylation of extracellular signal-regulated kinase. Importantly, blockade of extracellular signal-regulated kinase phosphorylation with a mitogen-activated protein kinase (MAPK) kinase inhibitor abrogated the forskolin-induced morphological changes and induction of neuronal proteins. These results indicate that extracellular signal-regulated kinase/MAPK mediates both cAMP-induced early cytoskeletal rearrangement and the later induction of neuronal markers in mesenchymal stem cells.

Modulation of the N-type calcium channel gene expression by the alpha subunit of Go.

Go, a heterotrimeric G-protein, is enriched in brain and neuronal growth cones. Although several reports suggest that Go may be involved in modulation of neuronal differentiation, the precise role of Go is not clear. To investigate the function of Go in neuronal differentiation, we determined the effect of Goalpha, the alpha subunit of Go, on the expression of Ca(v)2.2, the pore-forming unit of N-type calcium channels, at the transcription level. Treatment with cyclic AMP (cAMP), which triggers neurite outgrowth in neuroblastoma F11 cells, increased the mRNA level and the promoter activity of the Ca(v)2.2 gene. Overexpression of Goalpha inhibited neurite extension in F11 cells and simultaneously repressed the stimulatory effect of cAMP on the Ca(v)2.2 gene expression to the basal level. Targeted mutation of the Goalpha gene also increased the level of Ca(v)2.2 in the brain. These results suggest that Go may regulate neuronal differentiation through modulation of gene expression of target genes such as N-type calcium channels.