Lipocalin-2 activates hepatic stellate cells and promotes nonalcoholic steatohepatitis in high-fat diet-fed Ob/Ob mice.

BACKGROUND AND AIMS: In obesity and type 2 diabetes mellitus, leptin promotes insulin resistance and contributes to the progression of NASH via activation of hepatic stellate cells (HSCs). However, the pathogenic mechanisms that trigger HSC activation in leptin-deficient obesity are still unknown. This study aimed to determine how HSC-targeting lipocalin-2 (LCN2) mediates the transition from simple steatosis to NASH. APPROACH AND RESULTS: Male wild-type (WT) and ob/ob mice were fed a high-fat diet (HFD) for 20 weeks to establish an animal model of NASH with fibrosis. Ob/ob mice were subject to caloric restriction or recombinant leptin treatment. Double knockout (DKO) mice lacking both leptin and lcn2 were also fed an HFD for 20 weeks. In addition, HFD-fed ob/ob mice were treated with gadolinium trichloride to deplete Kupffer cells. The LX-2 human HSCs and primary HSCs from ob/ob mice were used to investigate the effects of LCN2 on HSC activation. Serum and hepatic LCN2 expression levels were prominently increased in HFD-fed ob/ob mice compared with normal diet-fed ob/ob mice or HFD-fed WT mice, and these changes were closely linked to liver fibrosis and increased hepatic α-SMA/matrix metalloproteinase 9 (MMP9)/signal transducer and activator of transcription 3 (STAT3) protein levels. HFD-fed DKO mice showed a marked reduction of α-SMA protein compared with HFD-fed ob/ob mice. In particular, the colocalization of LCN2 and α-SMA was increased in HSCs from HFD-fed ob/ob mice. In primary HSCs from ob/ob mice, exogenous LCN2 treatment induced HSC activation and MMP9 secretion. By contrast, LCN2 receptor 24p3R deficiency or a STAT3 inhibitor reduced the activation and migration of primary HSCs. CONCLUSIONS: LCN2 acts as a key mediator of HSC activation in leptin-deficient obesity via α-SMA/MMP9/STAT3 signaling, thereby exacerbating NASH.

Naegleria fowleri Extracellular Vesicles Induce Proinflammatory Immune Responses in BV-2 Microglial Cells.

Extracellular vesicles (EVs) of protozoan parasites have diverse biological functions that are essential for parasite survival and host-parasite interactions. In this study, we characterized the functional properties of EVs from Naegleria fowleri, a pathogenic amoeba that causes a fatal brain infection called primary amoebic meningoencephalitis (PAM). N. fowleri EVs (NfEVs) have been shown to be internalized by host cells such as C6 glial cells and BV-2 microglial cells without causing direct cell death, indicating their potential roles in modulating host cell functions. NfEVs induced increased expression of proinflammatory cytokines and chemokines such as TNF-α, IL-1α, IL-1ß, IL-6, IL-17, IFN-γ, MIP-1α, and MIP-2 in BV-2 microglial cells; these increases were initiated via MyD88-dependent TLR-2/TLR-4. The production levels of proinflammatory cytokines and chemokines in NfEVs-stimulated BV-2 microglial cells were effectively downregulated by inhibitors of MAPK, NF-κB, or JAK-STAT. Phosphorylation levels of JNK, p38, ERK, p65, JAK-1, and STAT3 were increased in NfEVs-stimulated BV-2 microglial cells but were effectively suppressed by each corresponding inhibitor. These results suggest that NfEVs could induce proinflammatory immune responses in BV-2 microglial cells via the NF-κB-dependent MAPK and JAK-STAT signaling pathways. Taken together, these findings suggest that NfEVs are pathogenic factors involved in the contact-independent pathogenic mechanisms of N. fowleri by inducing proinflammatory immune responses in BV-2 microglial cells, further contributing to deleterious inflammation in infected foci by activating subsequent inflammation cascades in other brain cells.

Current status of Clonorchis sinensis and clonorchiasis in Korea: epidemiological perspectives integrating the data from human and intermediate hosts.

Clonorchis sinensis is a carcinogenic liver fluke that causes clonorchiasis in humans. Clonorchiasis is prevalent in East Asian countries, and approximately 15­20 million individuals are estimated to be infected with this fluke globally. This review highlights the current status of C. sinensis and clonorchiasis in Korea from the epidemiological perspective involving the analysis of humans and intermediate hosts. Despite the recent decline in C. sinensis infection rate in Korea, C. sinensis infections remain endemic in 5 major river basins (Han-gang, Geum-gang, Seomjin-gang, Yeongsan-gang and Nakdong-gang; gang means river) with a high incidence of cholangiocarcinoma. A noticeable pattern involves increasing mild infections among patients diagnosed positive for C. sinensis eggs. The infection rate of C. sinensis metacercariae in the second intermediate host, freshwater fish, is also maintained at a substantial level. Thus, the One Health approach integrating different sectors and disciplines is recommended to accelerate and sustain control of C. sinensis, thereby leading to successful eradication. Health promotion via information dissemination and health education should be extended to prevent the consumption of raw freshwater fish by residents living in high-risk areas.

Mapping of the Complement C9 Binding Region on Clonorchis sinensis Paramyosin.

Heliminthic paramyosin is a multifunctional protein that not only acts as a structural protein in muscle layers but as an immune-modulatory molecule interacting with the host immune system. Previously, we found that paramyosin from Clonorchis sinensis (CsPmy) is bound to human complement C9 protein (C9). To analyze the C9 binding region on CsPmy, overlapping recombinant fragments of CsPmy were produced and their binding activity to human C9 was investigated. The fragmental expression of CsPmy and C9 binding assays revealed that the C9 binding region was located at the C-terminus of CsPmy. Further analysis of the C-terminus of CsPmy to narrow the C9 binding region on CsPmy indicated that the region flanking731Leu-780 Leu was a potent C9 binding region. The CsPmy fragments corresponding to the region effectively inhibited human C9 polymerization. These results provide a precise molecular basis for CsPmy as a potent immunomodulator to evade host immune defenses by inhibiting complement attack.

Genetic variations in histidine-rich protein 2 and histidine-rich protein 3 of Myanmar Plasmodium falciparum isolates.

BACKGROUND: Malaria rapid diagnostic tests (RDTs) are precious tools to diagnose malaria. Most RDTs used currently are based on the detection of Plasmodium falciparum histidine-rich protein 2 (PfHRP2) in a patient's blood. However, concern has been raised in recent years that deletion of pfhrp2 in the parasite could affect the accuracy of PfHRP2-based RDTs. In addition, genetic variation in pfhrp2 might influence the accuracy and sensitivity of RDTs. In this study, the genetic variation in pfhrp2 and pfhrp3 in Myanmar P. falciparum isolates was analysed. METHODS: Blood samples were collected from malaria patients who were infected with P. falciparum in Mandalay, Naung Cho, Tha Beik Kyin, and Pyin Oo Lwin, Upper Myanmar between 2013 and 2015. The pfhrp2 and pfhrp3 were amplified by nested polymerase chain reaction (PCR), cloned and sequenced. Genetic variation in Myanmar pfhrp2 and pfhrp3 was analysed using the DNASTAR program. Comparative analysis of Myanmar and global pfhrp2 and pfhrp3 isolates was also performed. RESULTS: One-hundred and two pfhrp2 and 89 pfhrp3 were amplified from 105 blood samples, of which 84 pfhrp2 and 56 pfhrp3 sequences were obtained successfully. Myanmar pfhrp2 and pfhrp3 showed high levels of genetic variation with different arrangements of distinct repeat types, which further classified Myanmar pfhrp2 and pfhrp3 into 76 and 47 haplotypes, respectively. Novel amino acid changes were also found in Myanmar pfhrp2 and pfhrp3, but their frequencies were very low. Similar structural organization was shared by Myanmar and global pfhrp2 and pfhrp3, and differences in frequencies of repeat types and lengths were also observed between and among global isolates. CONCLUSION: Length polymorphisms and amino acid substitutions generated extensive genetic variation in Myanmar pfhrp2 and pfhrp3. Comparative analysis revealed that global pfhrp2 and pfhrp3 share similar structural features, as well as extensive length polymorphisms and distinct organizations of repeat types. These results provide a better understanding of the genetic structure of pfhrp2 and pfhrp3 in global P. falciparum populations and suggest useful information to develop RDTs with improved quality.

Genetic diversity of Plasmodium vivax and Plasmodium falciparum lactate dehydrogenases in Myanmar isolates.

BACKGROUND: Plasmodium lactate dehydrogenase (pLDH) is a major target in diagnosing the erythrocytic stage of malaria parasites because it is highly expressed during blood-stage parasites and is distinguished from human LDH. Rapid diagnostic tests (RDTs) for malaria use pLDH as a target antigen; however, genetic variations in pLDH within the natural population threaten the efficacy of pLDH-based RDTs. METHODS: Genetic polymorphisms of Plasmodium vivax LDH (PvLDH) and Plasmodium falciparum LDH (PfLDH) in Myanmar isolates were analysed by nucleotide sequencing analysis. Genetic polymorphisms and the natural selection of PvLDH and PfLDH were analysed using DNASTAR, MEGA6, and DnaSP ver. 5.10.00 programs. The genetic diversity and natural selection of global PvLDH and PfLDH were also analysed. The haplotype network of global PvLDH and PfLDH was constructed using NETWORK ver. 5.0.0.3. Three-dimensional structures of PvLDH and PfLDH were built with YASARA Structure ver. 18.4.24 and the impact of mutations on structural change and stability was evaluated with SDM ver. 2, CUPSAT and MAESTROweb. RESULTS: Forty-nine PvLDH and 52 PfLDH sequences were obtained from Myanmar P. vivax and P. falciparum isolates. Non-synonymous nucleotide substitutions resulting in amino acid changes were identified in both Myanmar PvLDH and PfLDH. Amino acid changes were also identified in the global PvLDH and PfLDH populations, but they did not produce structural alterations in either protein. Low genetic diversity was observed in global PvLDH and PfLDH, which may be maintained by a strong purifying selection. CONCLUSION: This study extends knowledge for genetic diversity and natural selection of global PvLDH and PfLDH. Although amino acid changes were observed in global PvLDH and PfLDH, they did not alter the conformational structures of the proteins. These suggest that PvLDH and PfLDH are genetically well-conserved in global populations, which indicates that they are suitable antigens for diagnostic purpose and attractive targets for drug development.

Bile Ductal Transcriptome Identifies Key Pathways and Hub Genes in Clonorchis sinensis-Infected Sprague-Dawley Rats.

Clonorchis sinensis is a food-borne trematode that infects more than 15 million people. The liver fluke causes clonorchiasis and chronical cholangitis, and promotes cholangiocarcinoma. The underlying molecular pathogenesis occurring in the bile duct by the infection is little known. In this study, transcriptome profile in the bile ducts infected with C. sinensis were analyzed using microarray methods. Differentially expressed genes (DEGs) were 1,563 and 1,457 at 2 and 4 weeks after infection. Majority of the DEGs were temporally dysregulated at 2 weeks, but 519 DEGs showed monotonically changing expression patterns that formed seven distinct expression profiles. Protein-protein interaction (PPI) analysis of the DEG products revealed 5 sub-networks and 10 key hub proteins while weighted co-expression network analysis (WGCNA)-derived gene-gene interaction exhibited 16 co-expression modules and 13 key hub genes. The DEGs were significantly enriched in 16 Kyoto Encyclopedia of Genes and Genomes pathways, which were related to original systems, cellular process, environmental information processing, and human diseases. This study uncovered a global picture of gene expression profiles in the bile ducts infected with C. sinensis, and provided a set of potent predictive biomarkers for early diagnosis of clonorchiasis.

Cytokine Production in Cholangiocarcinoma Cells in Response to Clonorchis sinensis Excretory-Secretory Products and Their Putative Protein Components.

Clonorchis sinensis is a carcinogenic human liver fluke that promotes hepatic inflammatory environments via direct contact or through their excretory-secretory products (ESPs), subsequently leading to cholangitis, periductal fibrosis, liver cirrhosis, and even cholangiocarcinoma (CCA). This study was conducted to examine the host inflammatory responses to C. sinensis ESPs and their putative protein components selected from C. sinensis expressed sequenced tag (EST) pool databases, including TGF-ß receptor interacting protein 1(CsTRIP1), legumain (CsLeg), and growth factor binding protein 2 (CsGrb2). Treatment of CCA cells (HuCCT1) with the ESPs or bacterial recombinant C. sinensis proteins differentially promoted the secretion of proinflammatory cytokines (IL-1ß, IL-6, and TNF-α) as well as anti-inflammatory cytokines (IL-10, TGF-ß1, and TGF-ß2) in a time-dependent manner. In particular, recombinant C. sinensis protein treatment resulted in increase (at maximum) of ~7-fold in TGF-ß1, ~30-fold in TGF-ß2, and ~3-fold in TNF-α compared with the increase produced by ESPs, indicating that CsTrip1, CsLeg, and CsGrb2 function as strong inducers for secretion of these cytokines in host cells. These results suggest that C. sinensis ESPs contribute to the immunopathological response in host cells, leading to clonorchiasis-associated hepatobiliary abnormalities of greater severity.

Partial Characterization of Two Cathepsin D Family Aspartic Peptidases of Clonorchis sinensis.

Cathepsin D (CatD, EC 3.4.23.5) is a member belonging to the subfamily of aspartic endopeptidases, which are classified into the MEROPS clan AA, family A1. Helminth parasites express a large set of different peptidases that play pivotal roles in parasite biology and pathophysiology. However, CatD is less well known than the other classes of peptidases in terms of biochemical properties and biological functions. In this study, we identified 2 novel CatDs (CsCatD1 and CsCatD2) of Clonorchis sinensis and partially characterized their properties. Both CsCatDs represent typical enzymes sharing amino acid residues and motifs that are tightly conserved in the CatD superfamily of proteins. Both CsCatDs showed similar patterns of expression in different developmental stages of C. sinensis, but CsCatD2 was also expressed in metacercariae. CsCatD2 was mainly expressed in the intestines and eggs of C. sinensis. Sera obtained from rats experimentally infected with C. sinensis reacted with recombinant CsCatD2 beginning 2 weeks after infection and the antibody titers were gradually increased by maturation of the parasite. Structural analysis of CsCatD2 revealed a bilobed enzyme structure consisting of 2 antiparallel ß-sheet domains packed against each other forming a homodimeric structure. These results suggested a plausible biological role of CsCatD2 in the nutrition and reproduction of parasite and its potential utility as a serodiagnostic antigen in clonorchiasis.

Plasmodium vivax Merozoite Surface Protein 1 Paralog as a Mediator of Parasite Adherence to Reticulocytes.

Plasmodium vivax parasites preferentially invade reticulocytes in human beings. P. vivax merozoite surface protein 1 (PvMSP1) and PvMSP1 paralog (PvMSP1P) may have important functions in reticulocyte adherence during invasion. These proteins share similar structures, including the presence of two epidermal growth factor (EGF)-like and glycosylphosphatidylinositol (GPI)-anchored domains at the C terminus. However, there have been no reports concerning the functional activity of PvMSP1P in reticulocyte adherence during P. vivax invasion. In this study, the ability of PvMSP1P-19 to bind to reticulocytes and normocytes was analyzed. The reticulocyte binding activity of PvMSP1P-19 was 4.0-fold higher than its normocyte binding activity. The binding of PvMSP1P-19 to reticulocytes and normocytes was inhibited in a dose-dependent manner by antibodies from immunized rabbits and by antibodies from vivax parasite-infected patients. Consistently, antibodies against PvMSP1P inhibited parasite invasion during short-term in vitro cultivation. Similar to the case for PvDBPII binding activity, PvMSP1P-19 binding activity was reduced in chymotrypsin-treated reticulocytes. However, no significant difference between the binding of PvMSP1P-19 to Duffy-positive and Duffy-negative erythrocytes was found. The minimal binding motif of PvMSP1P-19 was characterized using synthetic peptides. The results showed that the residues at amino acid positions 1791 to 1808 may have an important function in mediating merozoite adherence to reticulocytes. The positively charged residues within the EGF-like domain were shown to constitute a key binding motif. This work presents strong evidence supporting the role of PvMSP1P in host target cell selection and invasion of Duffy-independent pathway in P. vivax Moreover, PvMSP1P-19-specific antibodies may confer protection against P. vivax reinvasion.

Venom allergen-like protein 28 in Clonorchis sinensis: four epitopes on its surface and the potential role of Cys124 for its conformational stability.

Venom allergen-like (VAL) proteins are important to host-parasite interactions. We previously demonstrated that a Clonorchis sinensis VAL (CsVAL) protein-derived synthetic peptide suppresses allergic and inflammatory responses. However, little is known regarding the physicochemical and antigenic properties of CsVAL proteins. Here, we identified a novel 194 amino acid VAL protein, named C. sinensis VAL 28 (CsVAL28), and characterized its functional motifs and structural details as a new member of the CAP superfamily. Unlike members of the Schistosoma mansoni VAL (SmVAL) family, CsVAL28 has a single CAP1 motif and six highly conserved disulfide bond-forming cysteines. Tertiary models of wild-type CsVAL28 and mutants were built using SmVAL4 as template via homology modeling. Normal mode analysis predicted that disulfide bond breaking by mutation of cysteine 124 to serine would greatly affect protein mobility. Four major immunoreactive linear epitopes were identified in the surface-exposed region or its vicinity via epitope mapping, using sera from clonorchiasis patients and healthy controls. Our findings provide in-depth knowledge on the structure-function properties of VAL proteins and may help determine highly antigenic regions for developing new diagnostic approaches.

Tissue Fluid Enzyme-Linked Immunosorbant Assay for Piglets Experimentally Infected with Toxoplasma gondii and Survey on Local and Imported Pork in Korean Retail Meat Markets.

To investigate the prevalence of Toxoplasma gondii in pork on the market in Korea, an in-house enzyme-linked immunosorbent assay for tissue fluid (CAU-tf-ELISA) was developed using a soluble extract of T. gondii RH strain tachyzoites. As the standard positive controls, the piglets were experimentally infected with T. gondii: Group A (1,000 cysts-containing bradyzoites), Group B (500 cysts-containing bradyzoites) and Group C (1.0×103 or 1.0×104 tachyzoites). The CAU-tf-ELISA demonstrated infection intensity-dependent positivity toward tissue fluids with average cut-off value 0.15: 100% for Group A, 93.8% for Group B and 40.6% for Group C. When tissue-specific cut-off values 0.066-0.199 were applied, CAU-tf-ELISA showed 96.7% sensitivity, 100% specificity, 100% positive and 90.0% negative predictive values. When compared with the same tissue fluids, performance of CAU-tf-ELISA was better than that of a commercial ELISA kit. Of the 583 Korea domestic pork samples tested, anti-T. gondii antibodies were detected from 9.1% of whole samples and 37.9% from skirt meat highest among pork parts. In the 386 imported frozen pork samples, 1.8% (skirt meat and shoulder blade) were positive for anti-T. gondii antibodies. In Korea, prevalence of anti-T. gondii antibodies in the pork on retail markets appeared high, suggesting that regulations on pig farming and facilities are necessary to supply safe pork on the tables.

Molecular and structural characteristics of multidrug resistance-associated protein 7 in Chinese liver fluke Clonorchis sinensis.

Multidrug resistance-associated protein 7 (MRP7, ABCC10) is a C subfamily member of the ATP-binding cassette (ABC) superfamily. MRP7 is a lipophilic anion transporter that pumps endogenous and xenobiotic substrates from the cytoplasm to the extracellular milieu. Here, we cloned and characterized CsMRP7 as a novel ABC transporter from the Chinese liver fluke, Clonorchis sinensis. Full-length cDNA of CsMRP7 was 5174 nt, encoded 1636 amino acids (aa), and harbored a 147-bp 5'-untranslated region (5'-UTR) and 116-bp 3'-UTR. Phylogenetic analysis confirmed that CsMRP7 was closer to the ABCC subfamily than the ABCB subfamily. Tertiary structures of the N-terminal region (1-322 aa) and core region (323-1621 aa) of CsMRP7 were generated by homology modeling using glucagon receptor (PDB ID: 5ee7_A) and P-glycoprotein (PDB ID: 4f4c_A) as templates, respectively. CsMRP7 nucleotide-binding domain 2 (NBD2) was conserved more than NBD1, which was the sites of ATP binding and hydrolysis. Like typical long MRPs, CsMRP7 has an additional membrane-spanning domain 0 (MSD0) and cytoplasmic loop, along with a common structural fold consisting of MSD1-NBD1-MSD2-NBD2 as a single polypeptide assembly. MSD0, MSD1, and MSD2 consisted of TM1-7, TM8-13, and TM14-19, respectively. The CsMRP7 transcript was more abundant in the metacercariae than in the adult worms. Truncated NBD1 (39 kDa) and NBD2 (44 kDa) were produced in bacteria and mouse immune sera were raised. CsMRP7 was localized in the apical side of the intestinal epithelium, sperm in the testes and seminal receptacle, receptacle membrane, and mesenchymal tissue around intestine in the adult worm. These results provide molecular information and insights into structural and functional characteristics of CsMRP7 and homologs of flukes.

Molecular and Structural Characterization of the Tegumental 20.6-kDa Protein in Clonorchis sinensis as a Potential Druggable Target.

The tegument, representing the membrane-bound outer surface of platyhelminth parasites, plays an important role for the regulation of the host immune response and parasite survival. A comprehensive understanding of tegumental proteins can provide drug candidates for use against helminth-associated diseases, such as clonorchiasis caused by the liver fluke Clonorchis sinensis. However, little is known regarding the physicochemical properties of C. sinensis teguments. In this study, a novel 20.6-kDa tegumental protein of the C. sinensis adult worm (CsTegu20.6) was identified and characterized by molecular and in silico methods. The complete coding sequence of 525 bp was derived from cDNA clones and encodes a protein of 175 amino acids. Homology search using BLASTX showed CsTegu20.6 identity ranging from 29% to 39% with previously-known tegumental proteins in C. sinensis. Domain analysis indicated the presence of a calcium-binding EF-hand domain containing a basic helix-loop-helix structure and a dynein light chain domain exhibiting a ferredoxin fold. We used a modified method to obtain the accurate tertiary structure of the CsTegu20.6 protein because of the unavailability of appropriate templates. The CsTegu20.6 protein sequence was split into two domains based on the disordered region, and then, the structure of each domain was modeled using I-TASSER. A final full-length structure was obtained by combining two structures and refining the whole structure. A refined CsTegu20.6 structure was used to identify a potential CsTegu20.6 inhibitor based on protein structure-compound interaction analysis. The recombinant proteins were expressed in Escherichia coli and purified by nickel-nitrilotriacetic acid affinity chromatography. In C. sinensis, CsTegu20.6 mRNAs were abundant in adult and metacercariae, but not in the egg. Immunohistochemistry revealed that CsTegu20.6 localized to the surface of the tegument in the adult fluke. Collectively, our results contribute to a better understanding of the structural and functional characteristics of CsTegu20.6 and homologs of flukes. One compound is proposed as a putative inhibitor of CsTegu20.6 to facilitate further studies for anthelmintics.

Antimicrobial peptides in the centipede Scolopendra subspinipes mutilans.

The centipede Scolopendra subspinipes mutilans is an environmentally beneficial and medically important arthropod species. Although this species is increasingly applied as a reliable source of new antimicrobial peptides, the transcriptome of this species is a prerequisite for more rational selection of antimicrobial peptides. In this report, we isolated total RNA from the whole body of adult centipedes, S. subspinipes mutilans, that were nonimmunized and immunized against Escherichia coli, and we generated a total of 77,063 pooled contigs and singletons using high-throughput sequencing. To screen putative antimicrobial peptides, in silico analyses of the S. subspinipes mutilans transcriptome were performed based on the physicochemical evidence of length, charge, isoelectric point, and in vitro and in vivo aggregation scores together with the existence of continuous antimicrobial peptide stretches. Moreover, we excluded some transcripts that showed similarity with both previously known antimicrobial peptides and the human proteome, had a proteolytic cleavage site, and had downregulated expression compared with the nonimmunized sample. As a result, we selected 17 transcripts and tested their antimicrobial activity with a radial diffusion assay. Among them, ten synthetic peptides experimentally showed antimicrobial activity against microbes and no toxicity to mouse erythrocytes. Our results provide not only a useful set of antimicrobial peptide candidates and an efficient strategy for novel antimicrobial peptide development but also the transcriptome data of a big centipede as a valuable resource.

Identification of CDH23 mutations in Korean families with hearing loss by whole-exome sequencing.

BACKGROUND: Patient genetic heterogeneity renders it difficult to discover disease-cause genes. Whole-exome sequencing is a powerful new strategy that can be used to this end. The purpose of the present study was to identify a hitherto unknown mutation causing autosomal recessive nonsyndromic hearing loss (ARNSHL) in Korean families. METHODS: We performed whole-exome sequencing in 16 individuals from 13 unrelated small families with ARNSHL. After filtering out population-specific polymorphisms, we focused on known deafness genes. Pathogenic effects of the detected mutations on protein structure or function were predicted via in silico analysis. RESULTS: We identified compound heterozygous CDH23 mutations in hearing-loss genes of two families. These include two previously reported pathological mutations, p.Pro240Leu and p.Glu1595Lys, as well as one novel mutation, p.Asn342Ser. The p.Pro240Leu mutation was found in both families. We also identified 26 non-synonymous variants in CDH23 coding exons from 16 hearing-loss patients and 30 Korean exomes. CONCLUSION: The present study is the first to show that CDH23 mutations cause hearing loss in Koreans. Although the precise contribution made by such mutations needs to be determined using a larger patient cohort, our data indicate that mutations in the CDH23 gene are one of the most important causes of non-syndromic hearing loss in East Asians. Further exome sequencing will identify common mutations or polymorphisms and contribute to the molecular diagnosis of, and development of new therapies for, hereditary hearing loss.

Molecular characterization of voltage-gated calcium channel ß-subunits of Clonorchis sinensis.

The voltage-gated Ca(2+) channel ß-subunit is a member of the membrane-associated guanylate kinase family and modulates kinetic properties of the Ca(2+) channels, such as their voltage-dependent activation and inactivation rates. Two cDNA clones were identified to encode each ß-subunit isotype of the voltage-gated Ca(2+) channel of Clonorchis sinensis, CsCavß1 and CsCavß2, which consist of 606 and 887 amino acids, respectively. CsCavß1 was found to be similar to the ß-subunit containing two conserved serine residues that constitute the consensus protein kinase C phosphorylation site in the ß-interaction domain (BID). CsCavß2 had cysteine and alanine residues instead of the two serine residues conserved in BID and was homologous to variant ß-subunit of Schistosoma mansoni and Schistosoma japonicum. CsCavß1 and CsCavß2 were almost equally expressed in the adults and metacercariae, but were more expressed in adult C. sinensis than in metacercariae. Collectively, our findings suggest that substitution of the two serine residues in BID of CsCavß2 may render C. sinensis sensitive to praziquantel.

(‒)-Epicatechin reveals amoebicidal activity against Acanthamoeba castellanii by activating the programmed cell death pathway.

BACKGROUND: Acanthamoeba is an opportunistic pathogen that can cause human infections such as granulomatous amebic encephalitis and acanthamoeba keratitis. However, no specific drug to treat the diseases has been developed. Therefore, the discovery or development of novel drugs for treating Acanthamoeba infections is urgently needed. The anti-protozoan activity of (‒)-epicatechin (EC) has been reported, suggesting it is an attractive anti-protozoal drug candidate. In this study, the amoebicidal activity of EC against A. castellanii was assessed and its mechanism of action was unveiled. METHODS: The amoebicidal activity of EC against A. castellanii trophozoites and the cytotoxicity of EC in HCE-2 and C6 cells were determined with cell viability assay. The underlying amoebicidal mechanism of EC against A. castellanii was analyzed by the apoptosis/necrosis assay, TUNEL assay, mitochondrial dysfunction assay, caspase-3 assay, and quantitative reverse transcription polymerase chain reaction. The cysticidal activity of EC was also investigated. RESULTS: EC revealed amoebicidal activity against A. castellanii trophozoites with an IC50 of 37.01 ± 3.96 µM, but was not cytotoxic to HCE-2 or C6 cells. EC induced apoptotic events such as increases in DNA fragmentation and intracellular reactive oxygen species production in A. castellanii. EC also caused mitochondrial dysfunction in the amoebae, as evidenced by the loss of mitochondrial membrane potential and reductions in ATP production. Caspase-3 activity, autophagosome formation, and the expression levels of autophagy-related genes were also increased in EC-treated amoebae. EC led to the partial death of cysts and the inhibition of excystation. CONCLUSION: EC revealed promising amoebicidal activity against A. castellanii trophozoites via programmed cell death events. EC could be a candidate drug or supplemental compound for treating Acanthamoeba infections.

The anti-amoebic activity of Pinus densiflora leaf extract against the brain-eating amoeba Naegleria fowleri.

Naegleria fowleri invades the brain and causes a fatal primary amoebic meningoencephalitis (PAM). Despite its high mortality rate of approximately 97%, an effective therapeutic drug for PAM has not been developed. Approaches with miltefosine, amphotericin B, and other antimicrobials have been clinically attempted to treat PAM, but their therapeutic efficacy remains unclear. The development of an effective and safe therapeutic drug for PAM is urgently needed. In this study, we investigated the anti-amoebic activity of Pinus densiflora leaf extract (PLE) against N. fowleri. PLE induced significant morphological changes in N. fowleri trophozoites, resulting in the death of the amoeba. The IC50 of PLE on N. fowleri was 62.3±0.95 µg/ml. Alternatively, PLE did not significantly affect the viability of the rat glial cell line C6. Transcriptome analysis revealed differentially expressed genes (DEGs) between PLE-treated and non-treated amoebae. A total of 5,846 DEGs were identified, of which 2,189 were upregulated, and 3,657 were downregulated in the PLE-treated amoebae. The DEGs were categorized into biological process (1,742 genes), cellular component (1,237 genes), and molecular function (846 genes) based on the gene ontology analysis, indicating that PLE may have dramatically altered the biological and cellular functions of the amoeba and contributed to their death. These results suggest that PLE has anti-N. fowleri activity and may be considered as a potential candidate for the development of therapeutic drugs for PAM. It may also be used as a supplement compound to enhance the therapeutic efficacy of drugs currently used to treat PAM.

Genetic Characterization and Phylogeographic Analysis of the First H13N6 Avian Influenza Virus Isolated from Vega Gull in South Korea.

Avian influenza virus (AIV) is a pathogen with zoonotic and pandemic potential. Migratory birds are natural reservoirs of all known subtypes of AIVs, except for H17N10 and H18N11, and they have been implicated in previous highly pathogenic avian influenza outbreaks worldwide. This study identified and characterized the first isolate of the H13N6 subtype from a Vega gull (Larus vegae mongolicus) in South Korea. The amino acid sequence of hemagglutinin gene showed a low pathogenic AIV subtype and various amino acid substitutions were found in the sequence compared to the reference sequence and known H13 isolates. High sequence homology with other H13N6 isolates was found in HA, NA, PB1, and PA genes, but not for PB2, NP, M, and NS genes. Interestingly, various point amino acid mutations were found on all gene segments, and some are linked to an increased binding to human-type receptors, resistance to antivirals, and virulence. Evolutionary and phylogenetic analyses showed that all gene segments are gull-adapted, with a phylogeographic origin of mostly Eurasian, except for PB2, PA, and M. Findings from this study support the evidence that reassortment of AIVs continuously occurs in nature, and migratory birds are vital in the intercontinental spread of avian influenza viruses.