Antibody response in patients undergoing chronic hemodialysis post-severe acute respiratory syndrome coronavirus 2 vaccination: A prospective observational study.

Vaccination is important for patients undergoing hemodialysis (HD) to prevent coronavirus disease 2019 (COVID-19) infection since they are more vulnerable. However, they exhibit a weak response to vaccines, underscoring the importance of understanding whether antibodies are sufficiently produced and their durability post-COVID-19 vaccination. This prospective observational study assessed the antibody response of Korean patients undergoing HD for 1 year. We compared the antibody responses of patients undergoing HD to the COVID-19 vaccine with those of healthy volunteers from 2021 to 2022. The patient and control groups received 2 doses of ChAdOx1 nCoV-19 and mRNA-1273, respectively. Immunoglobulin G (IgG) and neutralizing antibody levels were measured weeks or months apart after 2 doses for 1 year using enzyme-linked immunosorbent and fluorescence-based competitive severe acute respiratory syndrome coronavirus 2 neutralizing assays, respectively. We analyzed the third dose's effect on the patient group by categorizing the group into patients who received the third dose and those who did not since it was initiated midway through the study. In the control group, we enrolled participants who had completed 3 doses of mRNA-1273 since almost all participants received the third dose. Thirty-two patients undergoing HD and 15 healthy participants who received 2 doses of ChAdOx1 nCoV-19 and 3 of mRNA-1273, respectively, were enrolled. Although antibody production was weaker in the patient group than in the control group (P < .001), patients showed an increase in IgG levels (0.408 ± 0.517 optical density (OD) pre-vaccination, 2.175 ± 1.241 OD in patients with 2 doses, and 2.134 ± 1.157 OD in patients with 3 doses 1 year after the second dose) and neutralizing antibodies (23 ± 8% pre-vaccination, 87 ± 23% in patients with 2 doses, and 89 ± 18% in patients with 3 doses 1 year after the second dose) post-vaccination (P < .001). In the patient group, 19 patients received a third dose (BNT162b2 or mRNA-1273); however, it did not increase the antibody levels (P = 1.000). Furthermore, the antibodies produced by the vaccination did not wane until 1 year. Two doses of vaccination resulted in a significant antibody response in patients undergoing HD, and antibody levels did not wane until 1 year.

Antibody response to COVID-19 vaccination in patients on chronic hemodialysis.

Purpose: Since patients on hemodialysis (HD) are known to be vulnerable to coronavirus disease 2019 (COVID-19), many studies were conducted regarding the effectiveness of the COVID-19 vaccine in HD patients in Western countries. Here, we assessed antibody response of HD patients for 6 months post-vaccination to identify the duration and effectiveness of the COVID-19 vaccine in the Asian population. Materials and Methods: We compared antibody response of the COVID-19 vaccine in HD patients with healthy volunteers. Patient and control groups had two doses of ChAdOx1 nCoV-19 and mRNA-1273, respectively. Immunoglobulin G (IgG) was measured before vaccination, 2 weeks after the first dose, 2 and 4 weeks, 3 and 6 months after the second dose. Neutralizing antibody was measured before vaccination and at 2 weeks, 3 and 6 months after second dose. Since the third dose was started in the middle of the study, we analyzed the effect of the third dose as well. Results: Although antibody production was weaker than the control group (n=22), the patient group (n=39) showed an increase in IgG and neutralizing antibody after two doses. And, 21/39 patients and 14/22 participants had a third dose (BNT162b2 or mRNA-1273 in the patient group, mRNA-1273 in the control group), and it did not affect antibody response in both group. Trend analysis showed IgG and neutralizing antibody did not decrease over time. Age, sex, and HD vintage did not affect antibody production in HD patients. Patients with higher body mass index displayed better seroresponse, while those on immunosuppressants showed poor seroresponse. Conclusion: Two doses of vaccination led to significant antibody response in HD patients, and the antibody did not wane until 6 months.

Peptidyl-prolyl cis/trans isomerase Pin1 interacts with hepatitis B virus core particle, but not with HBc protein, to promote HBV replication.

Here, we demonstrate that the peptidyl-prolyl cis/trans isomerase Pin1 interacts noncovalently with the hepatitis B virus (HBV) core particle through phosphorylated serine/threonine-proline (pS/TP) motifs in the carboxyl-terminal domain (CTD) but not with particle-defective, dimer-positive mutants of HBc. This suggests that neither dimers nor monomers of HBc are Pin1-binding partners. The 162TP, 164SP, and 172SP motifs within the HBc CTD are important for the Pin1/core particle interaction. Although Pin1 dissociated from core particle upon heat treatment, it was detected as an opened-up core particle, demonstrating that Pin1 binds both to the outside and the inside of the core particle. Although the amino-terminal domain S/TP motifs of HBc are not involved in the interaction, 49SP contributes to core particle stability, and 128TP might be involved in core particle assembly, as shown by the decreased core particle level of S49A mutant through repeated freeze and thaw and low-level assembly of the T128A mutant, respectively. Overexpression of Pin1 increased core particle stability through their interactions, HBV DNA synthesis, and virion secretion without concomitant increases in HBV RNA levels, indicating that Pin1 may be involved in core particle assembly and maturation, thereby promoting the later stages of the HBV life cycle. By contrast, parvulin inhibitors and PIN1 knockdown reduced HBV replication. Since more Pin1 proteins bound to immature core particles than to mature core particles, the interaction appears to depend on the stage of virus replication. Taken together, the data suggest that physical association between Pin1 and phosphorylated core particles may induce structural alterations through isomerization by Pin1, induce dephosphorylation by unidentified host phosphatases, and promote completion of virus life cycle.

De Novo Transcriptome Profiling of Naegleria fowleri Trophozoites and Cysts via RNA Sequencing.

Naegleria fowleri is a pathogenic free-living amoeba, commonly found around the world in warm, fresh water and soil. N. fowleri trophozoites can infect humans by entering the brain through the nose and causing usually fatal primary amebic meningoencephalitis (PAM). Trophozoites can encyst to survive under unfavorable conditions such as cold temperature, starvation, and desiccation. Recent technological advances in genomics and bioinformatics have provided unique opportunities for the identification and pre-validation of pathogen-related and environmental resistance through improved understanding of the biology of pathogenic N. fowleri trophozoites and cysts at a molecular level. However, genomic and transcriptomic data on differential expression genes (DEGs) between trophozoites and cysts of N. fowleri are very limited. Here, we report transcriptome Illumina RNA sequencing (RNA-seq) for N. fowleri trophozoites and cysts and de novo transcriptome assembly. RNA-seq libraries were generated from RNA extracted from N. fowleri sampled from cysts, and a reference transcriptome was generated through the assembly of trophozoite data. In the database, the assembly procedure resulted in 42,220 contigs with a mean length of 11,254 nucleotides and a C+G content of 37.21%. RNA sequencing showed that 146 genes in cysts of N. fowleri indicated 2-fold upregulation in comparison with trophozoites of N. fowleri, and 163 genes were downregulated; these genes were found to participate in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. The KEGG pathway included metabolic (131 sequences) and genetic information processing (66 sequences), cellular processing (43 sequences), environmental information processing (22 sequences), and organismal system (20 sequences) pathways. On the other hand, an analysis of 11,254 sequences via the Gene Ontology database showed that their annotations contained 1069 biological processes including the cellular process (228 sequences) and metabolic process (214 sequences); 923 cellular components including cells (240 sequences) and cell parts (225 sequences); and 415 molecular functions including catalytic activities (195 sequences) and binding processes (186 sequences). Differential expression levels increased in cysts of N. fowleri compared to trophozoites of N. fowleri, which were mainly categorized as serine/threonine protease, kinase, and lipid metabolism-related proteins. These results may provide new insights into pathogen-related genes or environment-resistant genes in the pathogenesis of N. fowleri.

PPIases Par14/Par17 Affect HBV Replication in Multiple Ways.

Human parvulin 14 (Par14) and parvulin 17 (Par17) are peptidyl-prolyl cis/trans isomerases that upregulate hepatitis B virus (HBV) replication by binding to the conserved 133Arg-Pro134 (RP) motif of HBc and core particles, and 19RP20-28RP29 motifs of HBx. In the absence of HBx, Par14/Par17 have no effect on HBV replication. Interaction with Par14/Par17 enhances the stability of HBx, core particles, and HBc. Par14/Par17 binds outside and inside core particles and is involved in HBc dimer-dimer interaction to facilitate core particle assembly. Although HBc RP motif is important for HBV replication, R133 residue is solely important for its interaction with Par14/Par17. Interaction of Par14 and Par17 with HBx involves two substrate-binding residues, Glu46/Asp74 (E46/D74) and E71/D99, respectively, and promotes HBx translocation to the nucleus and mitochondria. In the presence of HBx, Par14/Par17 are efficiently recruited to cccDNA and promote transcriptional activation via specific DNA-binding residues Ser19/44 (S19/44). S19 and E46/D74 of Par14, and S44 and E71/D99 of Par17, are also involved in the recruitment of HBc onto cccDNA. Par14/Par17 upregulate HBV replication via various effects that are mediated in part through the HBx-Par14/Par17-cccDNA complex and triple HBc, Par14/Par17, and cccDNA interactions in the nucleus, as well as via core particle-Par14/Par17 interactions in the cytoplasm.

A link between CD8+CD27-CD28- T cells expressing chemerin chemokine-like receptor 1 and rheumatoid arthritis.

OBJECTIVES: Transcription of the chemerin chemokine-like receptor 1 (CMKLR1) has been observed in T cell subsets, but its role in T cells has not been well studied. As previously reported, the levels of its ligand, chemerin, are increased in the plasma and synovial fluid of patients with rheumatoid arthritis (RA); hence, we aimed to explore the expression and role of CMKLR1 in the T cells of these patients. METHODS: Peripheral blood and synovial fluid from patients with RA or osteoarthritis and healthy individuals were collected to analyse the frequency of CD27-CD28- T cells and the expression of CMKLR1 and TNF-α by flow cytometry. Chemotaxis of T cells was assessed using a Transwell migration assay. Chemerin levels were measured using an enzyme-linked immunosorbent assay. RESULTS: CMKLR1 was preferentially expressed in CD27-CD28- T cell subsets. Its surface levels were reduced by stimulation with anti-CD3 antibody or chemerin. We found a correlation between CMKLR1+CD8+CD27-CD28- T cell frequency and disease activity score 28 of RA. Chemerin treatment up-regulated but CMKLR1 inhibitor treatment down-regulated TNF-α expression in CD8+CD27-CD28- T cells, half of which express CMKLR1 on average. Moreover, chemerin induced migration of these cells. Analysis of blood and synovial fluid samples of RA showed a reduction of CMKLR1+CD27-CD28- T cell levels in the synovial fluid, with a few exceptions. CONCLUSIONS: Our results suggest that CMKLR1 expression in T cells may be involved in RA pathogenesis through modulation of TNF-α expression and cell migration.

Ca2+/Calmodulin-Dependent Protein Kinase II Inhibits Hepatitis B Virus Replication from cccDNA via AMPK Activation and AKT/mTOR Suppression.

Ca2+/calmodulin-dependent protein kinase II (CaMKII), which is involved in the calcium signaling pathway, is an important regulator of cancer cell proliferation, motility, growth, and metastasis. The effects of CaMKII on hepatitis B virus (HBV) replication have never been evaluated. Here, we found that phosphorylated, active CaMKII is reduced during HBV replication. Similar to other members of the AMPK/AKT/mTOR signaling pathway associated with HBV replication, CaMKII, which is associated with this pathway, was found to be a novel regulator of HBV replication. Overexpression of CaMKII reduced the expression of covalently closed circular DNA (cccDNA), HBV RNAs, and replicative intermediate (RI) DNAs while activating AMPK and inhibiting the AKT/mTOR signaling pathway. Findings in HBx-deficient mutant-transfected HepG2 cells showed that the CaMKII-mediated AMPK/AKT/mTOR signaling pathway was independent of HBx. Moreover, AMPK overexpression reduced HBV cccDNA, RNAs, and RI DNAs through CaMKII activation. Although AMPK acts downstream of CaMKII, AMPK overexpression altered CaMKII phosphorylation, suggesting that CaMKII and AMPK form a positive feedback loop. These results demonstrate that HBV replication suppresses CaMKII activity, and that CaMKII upregulation suppresses HBV replication from cccDNA via AMPK and the AKT/mTOR signaling pathway. Thus, activation or overexpression of CaMKII may be a new therapeutic target against HBV infection.

The HBV Core Protein and Core Particle Both Bind to the PPiase Par14 and Par17 to Enhance Their Stabilities and HBV Replication.

We recently reported that the PPIase Par14 and Par17 encoded by PIN4 upregulate HBV replication in an HBx-dependent manner by binding to conserved arginine-proline (RP) motifs of HBx. HBV core protein (HBc) has a conserved 133RP134 motif; therefore, we investigated whether Par14/Par17 bind to HBc and/or core particles. Native agarose gel electrophoresis (NAGE) and immunoblotting and co-immunoprecipitation were used. Chromatin immunoprecipitation from HBV-infected HepG2-hNTCP-C9 cells was performed. NAGE and immunoblotting revealed that Par14/Par17 bound to core particles and co-immunoprecipitation revealed that Par14/Par17 interacted with core particle assembly-defective, and dimer-positive HBc-Y132A. Thus, core particles and HBc interact with Par14/Par17. Par14/Par17 interacted with the HBc 133RP134 motif possibly via substrate-binding E46/D74 and E71/D99 motifs. Although Par14/Par17 dissociated from core particles upon heat treatment, they were detected in 0.2 N NaOH-treated opened-up core particles, demonstrating that Par14/Par17 bind outside and inside core particles. Furthermore, these interactions enhanced the stabilities of HBc and core particles. Like HBc-Y132A, HBc-R133D and HBc-R133E were core particle assembly-defective and dimer-positive, demonstrating that a negatively charged residue at position 133 cannot be tolerated for particle assembly. Although positively charged R133 is solely important for Par14/17 interactions, the 133RP134 motif is important for efficient HBV replication. Chromatin immunoprecipitation from HBV-infected cells revealed that the S19 and E46/D74 residues of Par14 and S44 and E71/D99 residues of Par17 were involved in recruitment of 133RP134 motif-containing HBc into cccDNA. Our results demonstrate that interactions of HBc, Par14/Par17, and cccDNA in the nucleus and core particle-Par14/Par17 interactions in the cytoplasm are important for HBV replication.

Establishment of an Acanthamoeba keratitis mouse model confirmed by amoebic DNA amplification.

Acanthamoeba castellanii, the causative agent of Acanthamoeba keratitis (AK), occurs mainly in contact lens users with poor eye hygiene. The findings of many in vitro studies of AK, as well as the testing of therapeutic drugs, need validation in in vivo experiments. BALB/c mice were used in this study to establish in vivo AK model. A. castellanii cell suspensions (equal mixtures of trophozoites and cysts) were loaded onto 2-mm contact lens pieces and inserted into mouse eyes that were scratched using an ophthalmic surgical blade under anesthesia and the eyelids of the mice were sutured. The AK signs were grossly observed and PCR was performed using P-FLA primers to amplify the Acanthamoeba 18S-rRNA gene from mouse ocular tissue. The experimental AK mouse model was characterized by typical hazy blurring and melting of the mouse cornea established on day 1 post-inoculation. AK was induced with at least 0.3 × 105 A. castellanii cells (optimal number, 5 × 104), and the infection persisted for two months. The PCR products amplified from the extracted mouse eye DNA confirmed the development of Acanthamoeba-induced keratitis during the infection periods. In conclusion, the present AK mouse model may serve as an important in vivo model for the development of various therapeutic drugs against AK.

Modulation of the Gut Microbiota Alters the Tumour-Suppressive Efficacy of Tim-3 Pathway Blockade in a Bacterial Species- and Host Factor-Dependent Manner.

T cell immunoglobulin and mucin domain-containing protein-3 (Tim-3) is an immune checkpoint molecule and a target for anti-cancer therapy. In this study, we examined whether gut microbiota manipulation altered the anti-tumour efficacy of Tim-3 blockade. The gut microbiota of mice was manipulated through the administration of antibiotics and oral gavage of bacteria. Alterations in the gut microbiome were analysed by 16S rRNA gene sequencing. Gut dysbiosis triggered by antibiotics attenuated the anti-tumour efficacy of Tim-3 blockade in both C57BL/6 and BALB/c mice. Anti-tumour efficacy was restored following oral gavage of faecal bacteria even as antibiotic administration continued. In the case of oral gavage of Enterococcus hirae or Lactobacillus johnsonii, transferred bacterial species and host mouse strain were critical determinants of the anti-tumour efficacy of Tim-3 blockade. Bacterial gavage did not increase the alpha diversity of gut microbiota in antibiotic-treated mice but did alter the microbiome composition, which was associated with the restoration of the anti-tumour efficacy of Tim-3 blockade. Conclusively, our results indicate that gut microbiota modulation may improve the therapeutic efficacy of Tim-3 blockade during concomitant antibiotic treatment. The administered bacterial species and host factors should be considered in order to achieve therapeutically beneficial modulation of the microbiota.

Association of TIM-3 expression with glucose metabolism in Jurkat T cells.

BACKGROUND: T cell activation is associated with increase in glycolysis and glutaminolysis. T cell immunoglobulin and mucin domain containing protein-3 (TIM-3), a T cell surface molecule, downregulates T cell activation and leads to insufficient immunity in cancer and chronic infection. TIM-3 regulates T cell activation possibly through alterations in metabolism; however, the relationship between TIM-3 expression and T cell metabolic changes has not been well studied. RESULTS: We investigated the association between TIM-3 expression and metabolic changes by analyzing glucose metabolism, glutamine metabolism, and mitochondrial function in TIM-3 overexpressing or knockout Jurkat T cell lines relative to their control cell lines. Glucose uptake and consumption, and lactate release were downregulated by TIM-3 expression but upregulated by TIM-3 knockout. Concomitantly, the expression of the glucose transporter, Glut1, but not Glut2, 3, or 4 was altered by TIM-3 expression. However, TIM-3 expression alone could not account for the change in glutamine consumption, glutamate release, and mitochondrial mass, ROS production or membrane potential in these cell lines. CONCLUSION: Our results show the association of TIM-3 expression with T cell glucose metabolism. These results are significant in chronic infections and cancers where it is necessary to control TIM-3 expressing T cells.

The role of Korean Medicine in the post-COVID-19 era: an online panel discussion part 2 - basic research and education.

BACKGROUND: Experiencing difficulties and challenges though COVID-19 pandemic, there are voices that it needs to be discussed to seek direction of basic research and college education of Korean Medicine (KM) so that KM community can play a significant role in the future infectious disease outbreaks. METHODS: This paper summarizes the edited highlights of an online video meeting by Google meet on May 19, 2020, organized by the Korean Medicine Convergence Research Information Center. Five researchers specialized in immunology, microbiology, virology, preventive medicine, and herbology, respectively, presented what KM community should prepare for the future acute infectious disease outbreaks by learning from the previous research on antiviral effect of herbs for coronavirus and the experiences of the present COVID-19 pandemic. RESULTS: There are a lot of herbs or natural products with potential anti-coronavirus effects reported from in vitro experiments and despite criticism, many clinical trials on traditional herbal medicine for COVID-19 are being conducted. In addition to establishing research evidence, KM community should train and produce public health professionals among Korean Medicine Doctors (KMDs) and official participation in public healthcare system should be ensured in terms of regulation and policy. Newly developed KM treatments can be interpreted by the KM theories and also should be allowed by regulations for KMDs to utilize them. CONCLUSION: The present online discussion suggested directions of basic research for acute viral infections diseases utilizing KM and how to enforce relevant education and regulations in the post-COVID-19 era.

An Alternatively Spliced Sirtuin 2 Isoform 5 Inhibits Hepatitis B Virus Replication from cccDNA by Repressing Epigenetic Modifications Made by Histone Lysine Methyltransferases.

Sirtuin 2 (Sirt2), an NAD+-dependent protein deacetylase, deacetylates tubulin, AKT, and other proteins. Previously, we showed that Sirt2 isoform 1 (Sirt2.1) increased replication of hepatitis B virus (HBV). Here, we show that HBV replication upregulates the expression of Sirt2 primary and alternatively spliced transcripts and their respective isoforms, 1, 2, and 5. Since Sirt2 isoform 5 (Sirt2.5) is a catalytically inactive nuclear protein with a spliced-out nuclear export signal (NES), we speculated that its different localization affects its activity. The overexpression of Sirt2.5 reduced expression of HBV mRNAs, replicative intermediate DNAs, and covalently closed circular DNA (cccDNA), an activity opposite that of Sirt2.1 and Sirt2.2. Unlike the Sirt2.1-AKT interaction, the Sirt2.5-AKT interaction was weakened by HBV replication. Unlike Sirt2.1, Sirt2.5 activated the AKT/GSK-3ß/ß-catenin signaling pathway very weakly and independently of HBV replication. When the NES and an N-terminal truncated catalytic domain were added to the Sirt2.5 construct, it localized in the cytoplasm and increased HBV replication (like Sirt2.1 and Sirt2.2). Chromatin immunoprecipitation assays revealed that more Sirt2.5 was recruited to cccDNA than Sirt2.1. The recruitment of histone lysine methyltransferases (HKMTs), such as SETDB1, SUV39H1, EZH2, and PR-Set7, and their respective transcriptional repressive markers, H3K9me3, H3K27me3, and H4K20me1, to cccDNA also increased in Sirt2.5-overexpressing cells. Among these, the Sirt2.5-PR-Set7 and -SETDB1 interactions increased upon HBV replication. These results demonstrate that Sirt2.5 reduces cccDNA levels and viral transcription through epigenetic modification of cccDNA via direct and/or indirect association with HKMTs, thereby exhibiting anti-HBV activity.IMPORTANCE Sirt2, a predominant cytoplasmic α-tubulin deacetylase, promotes the growth of hepatocellular carcinoma; indeed, HBV replication increases Sirt2 expression, and overexpression of Sirt2 is associated with hepatic fibrosis and epithelial-to-mesenchymal transition. Increased amounts of Sirt2 isoforms 1, 2, and 5 upon HBV replication might further upregulate HBV replication, leading to a vicious cycle of virus replication/disease progression. However, we show here that catalytically inactive nuclear Sirt2.5 antagonizes the effects of Sirt2.1 and Sirt2.2 on HBV replication, thereby inhibiting cccDNA level, transcription of cccDNA, and subsequent synthesis of replicative intermediate DNA. More Sirt2.5 was recruited to cccDNA than Sirt2.1, thereby increasing epigenetic modification by depositing transcriptional repressive markers, possibly through direct and/or indirect association with histone lysine methyltransferases, such as SETDB1, SUV39H1, EZH2, and/or PR-Set7, which represses HBV transcription. Thus, Sirt2.5 might provide a functional cure for HBV by silencing the transcription of HBV.

Silver nanoparticles induce reactive oxygen species-mediated cell cycle delay and synergistic cytotoxicity with 3-bromopyruvate in Candida albicans, but not in Saccharomyces cerevisiae.

Background: Silver nanoparticles (AgNPs) inhibit the proliferation of various fungi; however, their mechanisms of action remain poorly understood. To better understand the inhibitory mechanisms, we focused on the early events elicited by 5 nm AgNPs in pathogenic Candida albicans and non-pathogenic Saccharomyces cerevisiae. Methods: The effect of 5 nm and 100 nm AgNPs on fungus cell proliferation was analyzed by growth kinetics monitoring and spot assay. We examined cell cycle progression, reactive oxygen species (ROS) production, and cell death using flow cytometry. Glucose uptake was assessed using tritium-labeled 2-deoxyglucose. Results: The growth of both C. albicans and S. cerevisiae was suppressed by treatment with 5 nm AgNPs but not with 100 nm AgNPs. In addition, 5 nm AgNPs induced cell cycle arrest and a reduction in glucose uptake in both fungi after 30 minutes of culture in a dose-dependent manner (P<0.05). However, in C. albicans only, an increase in ROS production was detected after exposure to 5 nm AgNPs. Concordantly, an ROS scavenger blocked the effect of 5 nm AgNPs on the cell cycle and glucose uptake in C. albicans only. Furthermore, the growth-inhibition effect of 5 nm AgNPs was not greater in S. cerevisiae mutant strains deficient in oxidative stress response genes than it was in wild type. Finally, 5 nm AgNPs together with a glycolysis inhibitor, 3-bromopyruvate, synergistically enhanced cell death in C. albicans (P<0.05) but not in S. cerevisiae. Conclusion: AgNPs exhibit antifungal activity in a manner that may or may not be ROS dependent, according to the fungal species. The combination of AgNPs with 3-bromopyruvate may be more useful against infection with C. albicans.

Cellular characterization of actin gene concerned with contact-dependent mechanisms in Naegleria fowleri.

Free-living amoeba, Naegleria fowleri, destroys target cells through contact-dependent mechanisms, such as phagocytosis and/or trogocytosis. A previous experiment showed that the nf-actin gene consisted of 1.2 kbp, produced a 50.1 kDa recombinant protein (Nf-actin), and was localized on the cytoskeleton, pseudopodia and amoebastome. In this study, cellular characterization of the nf-actin gene concerned with contact-dependent mechanisms in N fowleri was performed. The nf-actin gene was amplified from a gene-cloned vector, pEXQP5-T7/NT TOPO. The nf-actin gene was introduced into the Ubi-pEGFP-C2 vector, and Ubi-pEGFP-C2/nf-actin was transfected into N fowleri trophozoites. Strong GFP fluorescence was detected in N fowleri trophozoites transfected with Ubi-pEGFP-C2/nf-actin. Expression of EGFP-Nf-actin protein was detected by Western blot analysis. The nf-actin-overexpressing N fowleri showed significantly increased adhesion activity against extracellular matrix components, fibronectin, collagen I and fibrinogen, compared with wild-type N fowleri. Moreover, nf-actin-overexpressing N fowleri showed increased phagocytic activity and cytotoxicity in comparison with wild-type N fowleri. In summary, the overexpressed nf-actin gene has an important function in ability to increase cell adhesion, cytotoxicity and phagocytosis by N fowleri.

Regulation of TIM-3 expression in a human T cell line by tumor-conditioned media and cyclic AMP-dependent signaling.

T cell immunoglobulin and mucin domain-3 (TIM-3) expression increases in exhausted T cells, which inhibits T cell function. TIM-3 expression is supposedly up-regulated in tumor-bearing individuals via chronic antigenic stimulation of T cells. Considering the immunosuppressive nature of the tumor microenvironment, we investigated whether tumor-secreted molecules might enhance TIM-3 expression in Jurkat T cells. We observed that TIM-3 expression was increased by the activation of prostaglandin (PG) E2 and cyclic AMP (cAMP) signaling pathways. Adenylate cyclase activation led to protein kinase A (PKA)-dependent upregulation of the TIM-3 minimal promoter region and of upstream conserved non-coding sequences. TIM-3 expression in Jurkat T cells was increased by the exposure to breast tumor cell-conditioned media partially through the interaction between PGE2 and its receptor, EP4. Our results propose that tumor-secreted molecules such as PGE2, which activates PKA and EPAC, may regulate TIM-3 expression in T cells.

Parvulin 14 and Parvulin 17 Bind to HBx and cccDNA and Upregulate Hepatitis B Virus Replication from cccDNA to Virion in an HBx-Dependent Manner.

The parvulin 14 (Par14) and parvulin 17 (Par17) proteins, which are both encoded by the PIN4 gene, play roles in protein folding, chromatin remodeling, DNA binding, ribosome biogenesis, and cell cycle progression. However, the effects of Par14 and Par17 on viral replication have never been explored. In this study, we found that, in the presence of HBx, either Par14 or Par17 could upregulate hepatitis B virus (HBV) replication, whereas in the absence of HBx, neither Par14 nor Par17 had any effect on replication. Overexpression of Par14/Par17 markedly increased the formation of covalently closed circular DNA (cccDNA), synthesis of HBV RNA and DNA, and virion secretion. Conversely, PIN4 knockdown significantly decreased HBV replication in HBV-transfected and -infected cells. Coimmunoprecipitation revealed that Par14/Par17 engaged in direct physical interactions with HBx in the cytoplasm, nucleus, and mitochondria, possibly mediated through substrate-binding residues on Par14/Par17 (E46/D74 and E71/D99, respectively) and conserved 19R20P-28R29P motifs on HBx. Furthermore, these interactions enhanced HBx stability, promoted HBx translocation to the nuclear and mitochondrial fractions, and increased HBV replication. Chromatin immunoprecipitation assays revealed that, in the presence of HBx, Par14/Par17 were efficiently recruited to cccDNA and promoted transcriptional activation via specific DNA-binding residues (S19/44). In contrast, in the absence of HBx, Par14/Par17 bound cccDNA only at the basal level and did not promote transcriptional activation. Taken together, our results demonstrate that Par14 and Par17 upregulate HBV RNA transcription and DNA synthesis, thereby increasing the HBV cccDNA level, through formation of the cccDNA-Par14/17-HBx complex.IMPORTANCE The HBx protein plays an essential regulatory role in HBV replication. We found that substrate-binding residues on the human parvulin peptidylprolyl cis/trans isomerase proteins Par14 and Par17 bound to conserved arginine-proline (RP) motifs on HBx in the cytoplasm, nucleus, and mitochondria. The HBx-Par14/Par17 interaction stabilized HBx; promoted its translocation to the nucleus and mitochondria; and stimulated multiple steps of HBV replication, including cccDNA formation, HBV RNA and DNA synthesis, and virion secretion. In addition, in the presence of HBx, the Par14 and Par17 proteins bound to cccDNA and promoted its transcriptional activation. Our results suggest that inhibition or knockdown of Par14 and Par17 may represent a novel therapeutic option against HBV infection.

Molecular mechanisms of biogenesis of apoptotic exosome-like vesicles and their roles as damage-associated molecular patterns.

Recent research has led to contradictory notions regarding the conventional theory that apoptotic cell death can evoke inflammatory or immunogenic responses orchestrated by released damage-associated patterns (DAMPs). By inducing IL-1ß from bone marrow-derived macrophages in an effort to determine the inflammatory mediators released from apoptotic cells, we found that exosomal fractions called "apoptotic exosome-like vesicles" (AEVs) prepared from apoptotic-conditioned medium were the main inflammatory factors. These AEVs showed characteristics of exosomes in their size, density, morphology, and protein expression but had unique marker proteins, sphingosine-1-phosphate receptors 1 and 3 (S1PR1 and 3). Their biogenesis was completely dependent on cellular sphingosine-1-phosphate (S1P)/S1PRs signaling from multiple fine spindles of plasma membrane accompanied by F-actin, S1PR1, S1PR3, and CD63 at the early apoptotic phase and progressing to the maturation of F-actin-guided multivesicular endosomes mediated by Gßγ subunits of S1PRs downstream. S1P-loaded S1PRs on AEVs were critical factors for inducing IL-1ß via NF-κB transcriptional factor and p38 MAPK, possibly through the RHOA/NOD2 axis, in differentiating macrophages. The AEVs induced genes of proinflammatory cytokines, chemokines, and mediators in both in vitro and in vivo models. In conclusion, AEVs could be key inflammatory mediators, acting as DAMPs that could explain the pathogeneses of various chronic inflammations, autoimmune diseases, or cancers in the future.

Sirtuin 2 Isoform 1 Enhances Hepatitis B Virus RNA Transcription and DNA Synthesis through the AKT/GSK-3ß/ß-Catenin Signaling Pathway.

Sirtuin 2 (Sirt2), a NAD+-dependent protein deacetylase, is overexpressed in many hepatocellular carcinomas (HCCs) and can deacetylate many proteins, including tubulins and AKT, prior to AKT activation. Here, we found that endogenous Sirt2 was upregulated in wild-type hepatitis B virus (HBV WT)-replicating cells, leading to tubulin deacetylation; however, this was not the case in HBV replication-deficient-mutant-transfected cells and 1.3-mer HBV WT-transfected and reverse transcriptase inhibitor (entecavir or lamivudine)-treated cells, but all HBV proteins were expressed. In HBV WT-replicating cells, upregulation of Sirt2 induced AKT activation, which consequently downregulated glycogen synthase kinase 3ß (GSK-3ß) and increased ß-catenin levels; however, downregulation of Sirt2 in HBV-nonreplicating cells impaired AKT/GSK-3ß/ß-catenin signaling. Overexpression of Sirt2 isoform 1 stimulated HBV transcription and consequently HBV DNA synthesis, which in turn activated AKT and consequently increased ß-catenin levels, possibly through physical interactions with Sirt2 and AKT. Knockdown of Sirt2 by short hairpin RNAs (shRNAs), inhibition by 2-cyano-3-[5-(2,5-dichlorophenyl)-2-furanyl]-N-5-quinolinyl-2-propenamide (AGK2), or dominant negative mutant expression inhibited HBV replication, reduced AKT activation, and decreased ß-catenin levels. Through HBV infection, we demonstrated that Sirt2 knockdown inhibited HBV replication from transcription. Although HBx itself activates AKT and upregulates ß-catenin, Sirt2-mediated signaling and upregulated HBV replication were HBx independent. Since constitutively active AKT inhibits HBV replication, the results suggest that upregulated Sirt2 and activated AKT may balance HBV replication to prolong viral replication, eventually leading to the development of HCC. Also, the results indicate that Sirt2 inhibition may be a new therapeutic option for controlling HBV infection and preventing HCC.IMPORTANCE Even though Sirt2, a NAD+-dependent protein deacetylase, is overexpressed in many HCCs, and overexpressed Sirt2 promotes hepatic fibrosis and associates positively with vascular invasion by primary HCCs through AKT/GSK-3ß/ß-catenin signaling, the relationship between Sirt2, HBV, HBx, and/or HBV-associated hepatocarcinogenesis is unclear. Here, we show that HBV DNA replication, not HBV expression, correlates positively with Sirt2 upregulation and AKT activation. We demonstrate that overexpression of Sirt2 further increases HBV replication, increases AKT activation, downregulates GSK-3ß, and increases ß-catenin levels. Conversely, inhibiting Sirt2 decreases HBV replication, reduces AKT activation, and decreases ß-catenin levels. Although HBx activates AKT to upregulate ß-catenin, Sirt2-mediated effects were not dependent on HBx. The results also indicate that a Sirt2 inhibitor may control HBV infection and prevent the development of hepatic fibrosis and HCC.

The Suppressive Effect of Butyrate and Bromopyruvate on Inflammatory Cytokine Production and Short Chain Fatty Acid Receptor Expression by Blood Mononuclear Cells in Patients with Behçet's Disease.

BACKGROUND: Controlling inflammation is a therapeutic goal of various autoimmune/autoinflammatory diseases including Behçet's disease (BD). The immunomodulatory effect of metabolites or metabolic analogs such as butyrate and 3-bromopyruvate has been observed in animal disease models. OBJECTIVE: We attempted to evaluate the effect of butyrate and 3-bromopyruvate on the inflammatory cytokine production by peripheral blood mononuclear cells (PBMCs) isolated from patients with mucocutaneous involvement of BD. METHODS: PBMCs isolated from 11 patients with BD and 10 healthy controls were stimulated with lipopolysaccharide in the presence of butyrate or 3-bromopyruvate. Butyrate receptor and cytokine messenger ribonucleic acid (mRNA) expression was analyzed by real-time reverse transcription polymerase chain reaction. Cytokine secretion was assessed by enzyme-linked immunosorbent assay. PBMCs survival was analyzed by flow cytometry. RESULTS: Bromopyruvate or butyrate treatment suppressed inflammatory cytokine production in PBMCs from all our subjects. Bromopyruvate also reduced PBMCs survival while butyrate did not. As the effect of butyrate was slightly greater in BD patients than in healthy controls, we analyzed butyrate receptor expression and found that lipopolysaccharide-induced free fatty acid receptor 2 mRNA level in PBMCs was higher in BD patients than in controls. CONCLUSION: We propose bromopyruvate and butyrate as supplementary therapeutic candidates to control inflammation in patients with BD.