Bcl-xL deamidation is regulated by multiple ion transporters and is intramolecularly catalyzed.

In susceptible tumor cells, DNA-damaging antineoplastic agents induce an increase in intracellular pH during the premitochondrial stage of apoptosis. The rate of nonenzymatic deamidation of two asparagines in the anti-apoptotic protein Bcl-xL is accelerated by this increase in pH. Deamidation of these asparagines is a signal for the degradation of Bcl-xL, which is a component of the apoptotic response to DNA damage. It has previously been shown that the increase in pH is mediated by the ion transporter Na+/H+ exchanger 1 in some cells. Here we demonstrate that one or more additional ion transporters also have a role in the regulation of Bcl-xL deamidation in at least some tumor cell lines and fibroblasts. As a second, independent finding, we report that there are histidines in close proximity to the Bcl-xL deamidation sites that are highly conserved in land-dwelling species and we present evidence that deamidation of human Bcl-xL is intramolecularly catalyzed in a manner that is dependent upon these histidines. Further, we present evidence that these histidines act as a pH-sensitive switch that enhances the effect of the increase in pH on the rate of Bcl-xL deamidation. The conservation of such histidines implies that human Bcl-xL is in essence "designed" to be deamidated, which provides further evidence that deamidation serves as a bona fide regulatory post-translational modification of Bcl-xL.

Ultrasound-enhanced scintillation proximity assay for rapid diagnostics.

Scintillation proximity assay (SPA) is a type of radioimmunoassay (RIA). We apply ultrasound enhancement to the general SPA. All assay procedures, including the antibody coating and radiolabeled antigen binding are achieved by simply mixing then standing for 5 min in an ultrasound chamber. No additional incubation time is required. To further demonstrate the capability of the UE-SPA, a quantitative measurement of CD55 in various grades of colon tumors was assessed on human tissue slides. The results showed a significant correlation between CD55 expression and tumorigenesis. In conclusion, we confirmed that UE-SPA is a reliable, rapid and alternative to RIA.

Functional analysis and gene expression profiling of extracellular cathepsin Z in red sea bream, Pagrus major.

Cathepsin Z (CTSZ) is a lysosomal cysteine protease that is known to be involved in the maintenance of homeostasis and the biological mechanisms of immune cells. In this study, we have confirmed the tissue specific expression of the cathepsin Z (PmCTSZ) gene in Pagrus major, and confirmed its biological function after producing recombinant protein using Escherichia coli (E. coli). Multiple sequence alignment analysis revealed that the active site of the cysteine proteases and three N-glycosylation sites of the deduced protein sequence were highly conserved among all of the organisms. Phylogenetic analysis revealed that PmCTSZ was included in the clusters of CTSZ and the cysteine proteases of other bony fish and is most closely related to Japanese flounder CTSZ. PmCTSZ was distributed in all of the tissues from healthy red sea bream that were used in the experiment and was most abundantly found in the spleen and gill. Analysis of mRNA expression after bacterial (Edwardsiella piscicida: E. piscicida and Streptococcus iniae: S. iniae) or viral (red seabream iridovirus: RSIV) challenge showed significant gene expression regulation in immune-related tissues, but they maintained relatively normal levels of expression. We produced recombinant PmCTSZ (rPmCTSZ) using an E. coli expression system and confirmed the biological function of extracellular rPmCTSZ in vitro. We found that bacterial proliferation was significantly inhibited by rPmCTSZ, and the leukocytes of red sea bream also induced apoptosis and viability reduction.

Molecular characterization, expression and functional analysis of peptidoglycan recognition protein-SC2 from rock bream, Oplegnathus fasciatus.

Peptidoglycan recognition proteins are members of the family of pattern recognition receptors (PRRs), that play important roles in the recognition of peptidoglycan and various biological processes. In this study, we have characterized peptidoglycan recognition protein-SC2 (PGRP-SC2) in rock bream (Oplegnathus fasciatus) (RbPGRP-SC2) and analysed its expression in various tissues after pathogen challenge. A sequence alignment revealed that the residues essential to zinc binding of the deduced protein were highly conserved among all the organisms. Phylogenetic analysis revealed that RbPGRP-SC2 is most closely related to the large yellow croaker PGRP-SC2. RbPGRP-SC2 was ubiquitously expressed in all tissues analysed, predominantly distributed in muscle and skin. After challenge with microbial pathogens (Edwardsiella piscicida), Streptococcus iniae or red seabream iridovirus [RSIV]), RbPGRP-SC2 was up-regulated in all the tissues examined, especially in liver. We produced recombinant RbPGRP-SC2 (rRbPGRP-SC2) using an Escherichia coli expression system. The rRbPGRP-SC2 had agglutination activity towards both Gram-negative (E. piscicida) and Gram-positive bacteria (S. iniae). In addition, rRbPGRP-SC2 induced leukocyte apoptosis and promoted leukocyte phagocytosis. These results suggest that the RbPGRP-SC2 plays an important role in the immune system and in maintaining cellular homeostasis of rock bream.

Nocardia seriolae infection in the cultured eel Anguilla japonica in Korea.

Mass mortality occurred at an Anguilla japonica eel farm equipped with a recirculating aquaculture system in Gimcheon, Korea, from late spring to early summer 2015. The cumulative 3-month mortality was 16% (approximately 24,300-150,000 fish). The majority of affected fish displayed ulcerative lesions that progressed to petechial haemorrhages and small white granulomas in the major organs. A Gram-positive, acid-fast, nonmotile bacterium was isolated from internal organ lesions. Phylogenetic analysis of 16S rRNA identified the species as Nocardia seriolae and the strain was designated EM150506. Afterwards, naïve eels were injected with 1.8 × 107 colony-forming units per fish to confirm the strain's pathogenicity, which resulted in a 20% mortality rate within 4 weeks. However, surviving fish still exhibited white N. seriolae colonies in internal organs. To our knowledge, this is the first report of a N. seriolae infection in cultured eel.

Montanide IMS 1312 VG adjuvant enhances the efficacy of immersion vaccine of inactivated viral hemorrhagic septicemia virus (VHSV) in olive flounder, Paralichthys olivaceus.

Vaccination by immersion is suitable for mass vaccination of small size fish. However, no viral vaccine has been developed for immersion applications, because of low efficacy. In this study, we evaluated the efficacy and safety of immersion vaccine against viral hemorrhagic septicemia (VHS) containing Montanide IMS 1312 VG adjuvant in olive flounder (Paralichthys olivaceus). Healthy fish were vaccinated by an immersion method with a heat-inactivated FP-VHS2010-1 strain of VHS virus (VHSV) in combination with Montanide IMS 1312 VG for 5 min at 20 ± 2 °C. The control group was vaccinated with sterile PBS. No toxicity of immersion vaccine with Montanide IMS 1312 VG adjuvant was observed by hematological and histopathological analysis. Immersion vaccine with adjuvant enhanced gene expression of immune-associated genes, i.e., genes encoding interleukin (IL)-1ß, IL-6, IL-8, and Toll-like receptor (TLR) 3. Relative percent survival (RPS) of fish was measured on weeks 4 and 8 post vaccination. In fish vaccinated with adjuvant, RPS was significantly higher than that of fish vaccinated without adjuvant. The results of the present study provide evidence that the VHSV immersion vaccine with Montanide IMS 1312 VG induces protective immunity in olive flounder against VHS.

RNA-Seq transcriptome analysis of the olive flounder (Paralichthys olivaceus) kidney response to vaccination with heat-inactivated viral hemorrhagic septicemia virus.

Viral hemorrhagic septicemia (VHS) is a highly contagious disease of cultured flounder caused by VHS virus (VHSV). To develop effective VHSV vaccines, it is essential to understand the molecular mechanisms underlying the host's protective response against VHSV. The purpose of this study is to clarify which genes are involved in the protective response of olive flounder after VHSV vaccination. We first injected olive flounder intraperitoneally with 107 TCID50 heat-inactivated VHSV vaccine and evaluated the vaccine efficacy at 20 °C. Fish vaccinated with heat-inactivated VHSV were significantly protected compared to non-vaccinated fish, with a relative percentage survival of 83%. To analyze the vaccination-induced changes in the expression profiles of genes, kidneys were collected from control and vaccinated fish at days 1, 3, and 7 after vaccination and global gene expression profiling was carried out by RNA sequencing. The analysis revealed that 15,001 genes were differentially expressed by at least 2-fold between vaccinated fish and non-vaccinated controls. Of these, 58 genes clustered into the acute phase response, Toll-like receptor, interferon-inducible/regulatory proteins, and apoptosis pathways. These data provided insights into the molecular mechanisms underlying the protective immune response of olive flounder against heat-inactivated VHSV vaccine and might aid future studies to develop a highly immunogenic vaccine against VHSV in flounder.

The first report of CD2 associated protein gene, in a teleost (Rock bream, Oplegnathus fasciatus): An investigation of the immune response upon infection with several pathogens.

CD2 is expressed on the surfaces of virtually all T cells and natural killer (NK) cells. In mammals, the CD2 molecule is 50 kDa. The cytoplasmic tail of CD2 interacts with CD2-associated protein (CD2AP), which plays an important role in mediating the trigger signal in outer magnetic pole cells. In this study, we identified CD2AP from rock bream and investigated its gene expression. The ORF of CD2AP (1950 bp) encodes 650 amino acids (aa). CD2AP has a Src homology 3 (SH3) domain. Quantitative real-time PCR analysis revealed that CD2AP shows higher expression in the gills and skin. Under experimental challenge, CD2AP gene expression was increased as relative to the control after 7 days. This result will improve our understanding of blood vessels in teleost fish, and will provide a basis for the study of CD2-related genes.

First Report of Clavinema mariae (Nematoda: Philometridae) in Cultured Rockfish, Sebastes schlegeli, in Cheonsuman (Bay), the Republic of Korea.

In July 2012, philometrid nematodes were discovered in cultured rockfish (Sebastes schlegeli) in Cheonsuman (Bay), the Republic of Korea. The nematodes were detected in the epithelial tissues of the rockfish and were identified as Clavinema mariae based on morphological studies using light and scanning electron microscopy. They revealed the characteristics same as previously identified C. mariae, notably having a long body with narrow posterior half, no caudal projection, a cylindrical-shaped esophagus, a well-developed anterior bulbous part of the esophagus, cephalic papillae, and a dorsal esophageal gland. This is the first confirmation of C. mariae infection in rockfish in Korea.

Coagulation factor II from rock bream (Oplegnathus fasciatus): First report on the molecular biological function and expression analysis in the teleost.

The rapid haemostasis of fish prevents bleeding or infection that could be caused by physical properties of the aquatic environment. Additionally, the innate immune system is the first line of defence against infection and is responsible for the recognition of pathogen-associated molecular patterns, which are important for the activation of acquired immune responses. Coagulation factor II (CFII) is an important factor in the coagulation system and is involved in recognition and interaction with various bacterial and extracellular proteins. In this study, we identified and characterised the gene encoding CFII in rock bream (Oplegnathus fasciatus) (RbCFII) and analysed its expression in various tissues after a pathogen challenge. The full-length RbCFII cDNA (2079 bp) contained an open reading frame of 1854 bp encoding 617 amino acids. Alignment analysis revealed that a gamma-carboxyglutamic acid-rich domain, two kringle domains, and a trypsin-like serine protease domain of the deduced protein were well conserved. RbCFII was ubiquitously expressed in all tissues examined but, predominantly detected in the liver and skin. RbCFII expression was dramatically up-regulated in the kidney, spleen and liver after infection with Edwardsiella tarda, Streptococcus iniae, or red seabream iridovirus. The recombinant protein RbCFII (rRbCFII) produced using an Escherichia coli expression system was able to bind all examined bacteria. Interestingly, rRbCFII has agglutination activities towards E. coli and E. tarda, while no agglutination was shown toward Vibrio ordalii and S. iniae. These findings indicate that rRbCFII performs an immunological function in the immune response, and might be involved in innate immunity as well as blood coagulation.

An assessment tumor targeting ability of (177)Lu labeled cyclic CCK analogue peptide by binding with cholecystokinin receptor.

The cholecystokinin (CCK) receptor is known as a receptor that is overexpressed in many human tumors. The present study was designed to investigate the targeting ability of cyclic CCK analogue in AR42J pancreatic cells. The CCK analogues, DOTA-K(glucose)-Gly-Trp-Nle-Asp-Phe (DOTA-glucose-CCK) and DOTA-Nle-cyclo(Glu-Trp-Nle-Asp-Phe-Lys-NH2) (DOTA-[Nle]-cCCK), were synthesized and radiolabeled with (177)Lu, and competitive binding was evaluated. The binding appearance of synthesized peptide with AR42J cells was evaluated by confocal microscopy. And bio-distribution was performed in AR42J xenografted mice. Synthesized peptides were prepared by a solid phase synthesis method, and their purity was over 98%. DOTA is the chelating agent for (177)Lu-labeling, in which the peptides were radiolabeled with (177)Lu by a high radiolabeling yield. A competitive displacement of (125)I-CCK8 on the AR42J cells revealed that the 50% inhibitory concentration value (IC50) was 12.3 nM of DOTA-glucose-CCK and 1.7 nM of DOTA-[Nle]-cCCK. Radio-labeled peptides were accumulated in AR42J tumor in vivo, and %ID/g of the tumor was 0.4 and 0.9 at 2 h p.i. It was concluded that (177)Lu-DOTA-[Nle]-cCCK has higher binding affinity than (177)Lu-DOTA-glucose-CCK and can be a potential candidate as a targeting modality for a CCK receptor over-expressing tumors.

Molecular cloning and expression analysis of peptidase genes in the fish-pathogenic scuticociliate Miamiensis avidus.

BACKGROUND: Parasite peptidases have been actively studied as vaccine candidates or drug targets for prevention or treatment of parasitic diseases because of their important roles for survival and/or invasion in the host. Like other parasites, the facultative histophagous ciliate Miamiensis avidus would possess peptidases that are closely associated with the invasion into the host tissue and survival in the host. RESULTS: The 17 genes encoding peptidases, including seven cathepsin-like cysteine peptidases, four serine carboxypeptidases, a eukaryotic aspartyl protease family protein, an ATP-dependent metalloprotease FtsH family protein, three leishmanolysin family proteins and a peptidase family M49 protein were identified from a Miamiensis avidus cDNA library by BLAST X search. Expression of genes encoding two cysteine peptidases, three leishmanolysin-like peptidases and a peptidase family M49 protein was up-regulated in the cell-fed ciliates compared to the starved ciliates. Especially, one cysteine peptidase (MaPro 4) and one leishmanolysin-like peptidase (MaPro 14) were transcribed more than 100-folds in the cell-fed ciliates. CONCLUSIONS: The genetic information and transcriptional characteristics of the peptidases in the present results would be helpful to elucidate the role of peptidases in the invasion of scuticociliates into their hosts.

Molecular identification and real-time quantitative PCR (qPCR) for rapid detection of Thelohanellus kitauei, a Myxozoan parasite causing intestinal giant cystic disease in the Israel carp.

Intestinal giant-cystic disease (IGCD) of the Israel carp (Cyprinus carpio nudus) has been recognized as one of the most serious diseases afflicting inland farmed fish in the Republic of Korea, and Thelohanellus kitauei has been identified as the causative agent of the disease. Until now, studies concerning IGCD caused by T. kitauei in the Israel carp have been limited to morphological and histopathological examinations. However, these types of diagnostic examinations are relatively time-consuming, and the infection frequently cannot be detected in its early stages. In this study, we cloned the full-length 18S rRNA gene of T. kitauei isolated from diseased Israel carps, and carried out molecular identification by comparing the sequence with those of other myxosporeans. Moreover, conventional PCR and real-time quantitative PCR (qPCR) using oligonucleotide primers for the amplification of 18S rRNA gene fragment were established for further use as methods for rapid diagnosis of IGCD. Our results demonstrated that both the conventional PCR and real-time quantitative PCR systems applied herein are effective for rapid detection of T. kitauei spores in fish tissues and environmental water.

Clinical Features and Long-term Prognosis of Crohn's Disease in Korea: Results from the Prospective CONNECT Study.

Background/Aims: The prospective Crohn's Disease Clinical Network and Cohort Study is a nationwide multicenter cohort study of patients with Crohn's disease (CD) in Korea, aiming to prospectively investigate the clinical features and long-term prognosis associated with CD. Methods: Patients diagnosed with CD between January 2009 and September 2019 were prospectively enrolled. They were divided into two cohorts according to the year of diagnosis: cohort 1 (diagnosed between 2009 and 2011) versus cohort 2 (between 2012 and 2019). Results: A total of 1,175 patients were included, and the median follow-up duration was 68 months (interquartile range, 39.0 to 91.0 months). The treatment-free durations for thiopurines (p<0.001) and anti-tumor necrosis factor agents (p=0.018) of cohort 2 were shorter than those of cohort 1. Among 887 patients with B1 behavior at diagnosis, 149 patients (16.8%) progressed to either B2 or B3 behavior during follow-up. Early use of thiopurine was associated with a reduced risk of behavioral progression (adjusted hazard ratio [aHR], 0.69; 95% confidence interval [CI], 0.50 to 0.90), and family history of inflammatory bowel disease was associated with an increased risk of behavioral progression (aHR, 2.29; 95% CI, 1.16 to 4.50). One hundred forty-one patients (12.0%) underwent intestinal resection, and the intestinal resection-free survival time was significantly longer in cohort 2 than in cohort 1 (p=0.003). The early use of thiopurines (aHR, 0.35; 95% CI, 0.23 to 0.51) was independently associated with a reduced risk of intestinal resection. Conclusions: The prognosis of CD in Korea appears to have improved over time, as evidenced by the decreasing intestinal resection rate. Early use of thiopurines was associated with an improved prognosis represented by a reduced risk of intestinal resection.

C4 Bacterial Volatiles Improve Plant Health.

Plant growth-promoting rhizobacteria (PGPR) associated with plant roots can trigger plant growth promotion and induced systemic resistance. Several bacterial determinants including cell-wall components and secreted compounds have been identified to date. Here, we review a group of low-molecular-weight volatile compounds released by PGPR, which improve plant health, mostly by protecting plants against pathogen attack under greenhouse and field conditions. We particularly focus on C4 bacterial volatile compounds (BVCs), such as 2,3-butanediol and acetoin, which have been shown to activate the plant immune response and to promote plant growth at the molecular level as well as in large-scale field applications. We also disc/ uss the potential applications, metabolic engineering, and large-scale fermentation of C4 BVCs. The C4 bacterial volatiles act as airborne signals and therefore represent a new type of biocontrol agent. Further advances in the encapsulation procedure, together with the development of standards and guidelines, will promote the application of C4 volatiles in the field.

Simultaneous profiling of Arabidopsis thaliana and Vibrio vulnificus MO6-24/O transcriptomes by dual RNA-seq analysis.

We previously demonstrated that a marine bacterial pathogen Vibrio vulnificus isolated from sea foods modulated gene expression levels and defense responses of a land plant Arabidopsis thaliana. Although the interaction between V. vulnificus and A. thaliana was verified under artificial and greenhouse conditions, the simultaneous changes in host and pathogen transcriptomes remained obscure. In this study, we simultaneously analyzed the transcriptome of V. vulnificus MO6-24/O and A. thaliana by dual RNA-sequencing analysis. Disease symptoms appeared at 5 and 7 days post-inoculation in vitro and post-infiltration in planta, respectively. A total of 31, 128, 303, 219, and 130 differentially expressed genes (DEGs) were identified in V. vulnificus MO6-24/O at 3, 6, 12, 24, and 48 h post-infiltration. Out of these, 14 genes involved in the virulence and pathogenicity of V. vulnificus MO6 were characterized. These genes were clustered into six categories, including adherence, antiphagocytosis, chemotaxis and motility, iron uptake, toxin and secretion system. In plant side, the bacterium DEGs potentially played a pivotal role in activating pattern recognition receptors (PRRs)-mediated defense responses. A. thaliana genes related to PRRs, reactive oxygen species burst, mitogen-activated protein kinase cascade induction, salicylic acid, jasmonic acid, ethylene, abscisic acid, auxin, gibberellin, and cytokinin were highly induced by V. vulnificus MO6-24/O challenge. Taken together, our results indicate that the sophisticated communication between a marine bacterial pathogen V. vulnificus and A. thaliana occurs. It is the first report demonstration that V. vulnificus actively modulates its virulence factors and potential host immune regulator in a land plant species.

Sound Vibration-Triggered Epigenetic Modulation Induces Plant Root Immunity Against Ralstonia solanacearum.

Sound vibration (SV) is one of the several environmental stimuli that induce physiological changes in plants including changes in plant immunity. Immune activation is a complicated process involving epigenetic modifications, however, SV-induced epigenetic modifications remain unexplored. Here, we performed an integrative analysis comprising chromatin immunoprecipitation (ChIP) and microRNA sequencing (miRNA-seq) to understand the role of SV-mediated epigenetic modifications in immune activation in Arabidopsis thaliana against the root pathogen Ralstonia solanacearum. Plants exposed to SV (10 kHz) showed abundant H3K27me3 modification in the promoter regions of aliphatic glucosinolate biosynthesis and cytokinin signaling genes, leading to transcriptional changes that promote immunity. Additionally, 10 kHz SV down-regulated miR397b expression, thus activating three target LACCASE transcripts that mediate cell wall reinforcement via lignin accumulation. Taken together, SV triggers epigenetic modification of genes involved in secondary metabolite biosynthesis, defense hormone signaling, and pre-formed defense in A. thaliana, leading to the activation of plant immunity against R. solanacearum.

Transcriptome analysis based on RNA-seq of common innate immune responses of flounder cells to IHNV, VHSV, and HIRRV.

Viral hemorrhagic septicemia virus (VHSV) and hirame rhabdovirus (HIRRV) belong to the genus Novirhabdovirus and are the causative agents of a serious disease in cultured flounder. However, infectious hematopoietic necrosis virus (IHNV), a prototype of the genus Novirhabdovirus, does not cause disease in flounder. To determine whether IHNV growth is restricted in flounder cells, we compared the growth of IHNV with that of VHSV and HIRRV in hirame natural embryo (HINAE) cells infected with novirhabdoviruses at 1 multiplicity of infection. Unexpectedly, we found that IHNV grew as well as VHSV and HIRRV. For successful growth in host cells, viruses modulate innate immune responses exerted by virus-infected cells. Our results suggest that IHNV, like VHSV and HIRRV, has evolved the ability to overcome the innate immune response of flounder cells. To determine the innate immune response genes of virus-infected HINAE cells which are commonly modulated by the three novirhabdoviruses, we infected HINAE cells with novirhabdoviruses at multiplicity of infection (MOI) 1 and performed an RNA sequencing-based transcriptome analysis at 24 h post-infection. We discovered ~12,500 unigenes altered by novirhabdovirus infection and found that many of these were involved in multiple cellular pathways. After novirhabdovirus infection, 170 genes involved in the innate immune response were differentially expressed compared to uninfected cells. Among them, 9 genes changed expression by more than 2-fold and were commonly modulated by all three novirhabdoviruses. Interferon regulatory factor 8 (IRF8), C-X-C motif chemokine receptor 1 (CXCR1), Toll/interleukin-1 receptor domain-containing adapter protein (TIRAP), cholesterol 25-hydroxylase (CH25H), C-X-C motif chemokine ligand 11, duplicate 5 (CXCL11.5), and Toll-like receptor 2 (TLR2) were up-regulated, whereas C-C motif chemokine receptor 6a (CCR6a), interleukin-12a (IL12a), and Toll-like receptor 1 (TLR1) were down-regulated. These genes have been reported to be involved in antiviral responses and, thus, their modulation may be critical for the growth of novirhabdovirus in flounder cells. This is the first report to identify innate immune response genes in flounder that are commonly modulated by IHNV, VHSV, and HIRRV. These data will provide new insights into how novirhabdoviruses survive the innate immune response of flounder cells.