Role of Gasdermin E in the Biogenesis of Apoptotic Cell-Derived Exosomes.

The gasdermins are a family of pore-forming proteins that has recently been suggested to play a central role in pyroptosis. In this study, we describe the novel roles of gasdermins in the biogenesis of apoptotic cell-derived exosomes. In apoptotic human HeLa and HEK293 cells, GSDMA, GSDMC, GSDMD, and GSDME increased the release of apoptotic exosomes. GSDMB and DFNB59, in contrast, negatively affected the release of apoptotic exosomes. GSDME at its full-length and cleaved forms was localized in the exosomes and exosomal membrane. Full-length and cleaved forms of GSDME are suggested to increase Ca2+ influx to the cytosol through endosomal pores and thus increase the biogenesis of apoptotic exosomes. In addition, the GSDME-mediated biogenesis of apoptotic exosomes depended on the ESCRT-III complex and endosomal recruitment of Ca2+-dependent proteins, that is, annexins A2 and A7, the PEF domain family proteins sorcin and grancalcin, and the Bro1 domain protein HD-PTP. Therefore, we propose that the biogenesis of apoptotic exosomes begins when gasdermin-mediated endosomal pores increase cytosolic Ca2+, continues through the recruitment of annexin-sorcin/grancalcin-HD-PTP, and is completed when the ESCRT-III complex synthesizes intraluminal vesicles in the multivesicular bodies of dying cells. Finally, we found that GSDME-bearing tumors released apoptotic exosomes to induce inflammatory responses in the in vivo mouse 4T1 orthotropic model of BALB/c breast cancer. The data indicate that the switch from apoptosis to pyroptosis could drive the transfer of mass signals to nearby or distant living cells and tissues by way of extracellular vesicles, and that gasdermins play critical roles in that process.

The LRRK2-G2019S mutation attenuates repair of brain injury partially by reducing the release of osteopontin-containing monocytic exosome-like vesicles.

BACKGROUND: Brain injury has been suggested as a risk factor for neurodegenerative diseases. Accordingly, defects in the brain's intrinsic capacity to repair injury may result in the accumulation of damage and a progressive loss of brain function. The G2019S (GS) mutation in LRRK2 (leucine rich repeat kinase 2) is the most prevalent genetic alteration in Parkinson's disease (PD). Here, we sought to investigate how this LRRK2-GS mutation affects repair of the injured brain. METHODS: Brain injury was induced by stereotaxic injection of ATP, a damage-associated molecular pattern (DAMP) component, into the striatum of wild-type (WT) and LRRK2-GS mice. Effects of the LRRK2-GS mutation on brain injury and the recovery from injury were examined by analyzing the molecular and cellular behavior of neurons, astrocytes, and monocytes. RESULTS: Damaged neurons express osteopontin (OPN), a factor associated with brain repair. Following ATP-induced damage, monocytes entered injured brains, phagocytosing damaged neurons and producing exosome-like vesicles (EVs) containing OPN through activation of the inflammasome and subsequent pyroptosis. Following EV production, neurons and astrocytes processes elongated towards injured cores. In LRRK2-GS mice, OPN expression and monocytic pyroptosis were decreased compared with that in WT mice, resulting in diminished release of OPN-containing EVs and attenuated elongation of neuron and astrocyte processes. In addition, exosomes prepared from injured LRRK2-GS brains induced neurite outgrowth less efficiently than those from injured WT brains. CONCLUSIONS: The LRRK2-GS mutation delays repair of injured brains through reduced expression of OPN and diminished release of OPN-containing EVs from monocytes. These findings suggest that the LRRK2-GS mutation may promote the development of PD by delaying the repair of brain injury.

Update on Extracellular Vesicle-Based Vaccines and Therapeutics to Combat COVID-19.

The COVID-19 pandemic has had a deep impact on people worldwide since late 2019 when SARS-CoV-2 was first identified in Wuhan, China. In addition to its effect on public health, it has affected humans in various aspects of life, including social, economic, cultural, and political. It is also true that researchers have made vigorous efforts to overcome COVID-19 throughout the world, but they still have a long way to go. Accordingly, innumerable therapeutics and vaccine candidates have been studied for their efficacies and have been tried clinically in a very short span of time. For example, the versatility of extracellular vesicles, which are membrane-bound particles released from all types of cells, have recently been highlighted in terms of their effectiveness, biocompatibility, and safety in the fight against COVID-19. Thus, here, we tried to explain the use of extracellular vesicles as therapeutics and for the development of vaccines against COVID-19. Along with the mechanisms and a comprehensive background of their application in trapping the coronavirus or controlling the cytokine storm, we also discuss the obstacles to the clinical use of extracellular vesicles and how these could be resolved in the future.

Dying neurons conduct repair processes in the injured brain through osteopontin expression in cooperation with infiltrated blood monocytes.

The brain has an intrinsic capacity to repair injury, but the specific mechanisms are largely unknown. In this study, we found that, despite their incipient death, damaged neurons play a key repair role with the help of monocytes infiltrated from blood. Monocytes phagocytosed damaged and/or dying neurons that expressed osteopontin (OPN), with possible subsequent activation of their inflammasome pathway, resulting in pyroptosis. During this process, monocytes released CD63-positive exosome-like vesicles containing OPN. Importantly, following the exosome-like vesicles, neuron and astrocyte processes elongated toward the injury core. In addition, exosomes prepared from the injured brain contained OPN, and enhanced neurite outgrowth of cultured neurons in an OPN-dependent manner. Thus, our results introduce the concept that neurons in the injured brain that are destined to die perceive the stressful condition and begin the regeneration processes through induction of OPN, ultimately executing the repair process with the help of monocytes recruited from the circulation.

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.

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.

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.

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.

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.

NLRP3 Inflammasome Activation in THP-1 Target Cells Triggered by Pathogenic Naegleria fowleri.

Naegleria fowleri, known as the brain-eating amoeba, causes acute primary amoebic meningoencephalitis. During swimming and other recreational water activities, N. fowleri trophozoites penetrate the nasal mucosa and invade the olfactory bulbs, resulting in intense inflammatory reactions in the forebrain tissue. To investigate what kinds of inflammasome molecules are expressed in target cells due to N. fowleri infection, human macrophage cells (THP-1 cells) were cocultured with N. fowleri trophozoites in a noncontact system, and consequently, interleukin-1ß (IL-1ß) production was estimated. Caspase-1 activation and IL-1ß production from THP-1 cells by Western blotting and the culture supernatant by enzyme-linked immunosorbent assay analysis were observed at 3 h after cocultivation. In addition, the increased expression of ASC and NLRP3, which make up an inflammasome complex, was also observed at 3 h after cocultivation. To confirm the caspase-1 activation and IL-1ß production via the NLRP3 inflammasome in THP-1 cells triggered by N. fowleri trophozoites, THP-1 cells were pretreated with several inhibitors. The inhibition assay showed that CA-074 (a cathepsin B inhibitor), glybenclamide (an NLRP3 molecule inhibitor), and N-benzyloxycarbony-Val-Ala-Asp(O-methyl)-fluoromethylketone (Z-VAD-FMK; a caspase-1 inhibitor) reduced the levels of caspase-1 activation and IL-1ß production from THP-1 cells. This study suggests that N. fowleri infection induces the NLRP3 inflammasome, which activates caspase-1 and subsequently produces IL-1ß, thus resulting in inflammation.

Hepatitis C virus attenuates interferon-induced major histocompatibility complex class I expression and decreases CD8+ T cell effector functions.

BACKGROUND & AIMS: Major histocompatibility complex (MHC) class I-restricted CD8(+) T cells are required for clearance of hepatitis C virus (HCV) infection. MHC class I expression is up-regulated by type I and II interferons (IFNs). However, little is known about the effects of HCV infection on IFN-induced expression of MHC class I. METHODS: We used the HCV cell culture system (HCVcc) with the genotype 2a Japanese fulminant hepatitis-1 strain to investigate IFN-induced expression of MHC class I and its regulatory mechanisms. HCVcc-infected Huh-7.5 cells were analyzed by flow cytometry, metabolic labeling, immunoprecipitation, and immunoblotting analyses. Protein kinase R (PKR) was knocked down with lentiviruses that express small hairpin RNAs. The functional effects of MHC class I regulation by HCV were demonstrated in co-culture studies, using HCV-specific CD8(+) T cells. RESULTS: Although the baseline level of MHC class I was not affected by HCV infection, IFN-induced expression of MHC class I was notably attenuated in HCV-infected cells. This was associated with replicating HCV RNA, not with viral protein. HCV infection reduced IFN-induced synthesis of MHC class I protein and induced phosphorylation of PKR and eIF2α. IFN-induced MHC class I expression was restored by small hairpin RNA-mediated knockdown of PKR in HCV-infected cells. Co-culture of HCV-specific CD8(+) T cells and HCV-infected cells that expressed HLA-A2 demonstrated that HCV infection reduced the effector functions of HCV-specific CD8(+) T cells; these functions were restored by small hairpin RNA-mediated knockdown of PKR. CONCLUSIONS: IFN-induced expression of MHC class I is attenuated in HCV-infected cells by activation of PKR, which reduces the effector functions of HCV-specific CD8(+) T cells. This appears to be an important mechanism by which HCV circumvents antiviral adaptive immune responses.

Phosphoacceptors threonine 162 and serines 170 and 178 within the carboxyl-terminal RRRS/T motif of the hepatitis B virus core protein make multiple contributions to hepatitis B virus replication.

UNLABELLED: Phosphorylation of serines 157, 164, and 172 within the carboxyl-terminal SPRRR motif of the hepatitis B virus (HBV) core (C) protein modulates HBV replication at multiple stages. Threonine 162 and serines 170 and 178, located within the carboxyl-terminal conserved RRRS/T motif of HBV C protein, have been proposed to be protein kinase A phosphorylation sites. However, in vivo phosphorylation of these residues has never been observed, and their contribution to HBV replication remains unknown. In this study, [(32)P]orthophosphate labeling of cells expressing C proteins followed by immunoprecipitation with anti-HBc antibody revealed that threonine 162 and serines 170 and 178 are phosphoacceptor residues. A triple-alanine-substituted mutant, mimicking dephosphorylation of all three residues, drastically decreased pregenomic RNA (pgRNA) encapsidation, thereby decreasing HBV DNA synthesis. In contrast, a triple-glutamate-substituted mutant, mimicking phosphorylation of these residues, decreased DNA synthesis without significantly decreasing encapsidation. Neither triple mutant affected C protein expression or core particle assembly. Individual alanine substitution of threonine 162 significantly decreased minus-strand, plus-strand, and relaxed-circular DNA synthesis, demonstrating that this residue plays multiple roles in HBV DNA synthesis. Double-alanine substitution of serines 170 and 178 reduced HBV replication at multiple stages, indicating that these residues also contribute to HBV replication. Thus, in addition to serines 157, 164, and 172, threonine 162 and serines 170 and 178 of HBV C protein are also phosphorylated in cells, and phosphorylation and dephosphorylation of these residues play multiple roles in modulation of HBV replication. IMPORTANCE: Threonine 162, within the carboxyl-terminal end of the hepatitis B virus (HBV adw) core (C) protein, has long been ignored as a phosphoacceptor, even though it is highly conserved among mammalian hepadnaviruses and in the overlapping consensus RxxS/T, RRxS/T, and TP motifs. Here we show, for the first time, that in addition to the well-known phosphoacceptor serines 157, 164, and 172 in SPRRR motifs, threonine 162 and serines 170 and 178 in the RRRS/T motif are phosphorylated in cells. We also show that, like serines 157, 164, and 172, phosphorylated and dephosphorylated threonine 162 and serines 170 and 178 contribute to multiple steps of HBV replication, including pgRNA encapsidation, minus-strand and plus-strand DNA synthesis, and relaxed-circular DNA synthesis. Of these residues, threonine 162 is the most important. Furthermore, we show that phosphorylation of C protein is required for efficient completion of HBV replication.

Novel cathepsin B and cathepsin B-like cysteine protease of Naegleria fowleri excretory-secretory proteins and their biochemical properties.

Naegleria fowleri causes a lethal primary amoebic meningoencephalitis (PAM) in humans and experimental animals, which leads to death within 7-14 days. Cysteine proteases of parasites play key roles in nutrient uptake, excystment/encystment, host tissue invasion, and immune evasion. In this study, we cloned N. fowleri cathepsin B (nfcpb) and cathepsin B-like (nfcpb-L) genes from our cDNA library of N. fowleri. The full-length sequences of genes were 1,038 and 939 bp (encoded 345 and 313 amino acids), and molecular weights were 38.4 and 34 kDa, respectively. Also, nfcpb and nfcpb-L showed a 56 and 46 % identity to Naegleria gruberi cathepsin B and cathepsin B-like enzyme, respectively. Recombinant NfCPB (rNfCPB) and NfCPB-L (rNfCPB-L) proteins were expressed by the pEX5-NT/TOPO vector that was transformed into Escherichia coli BL21, and they showed 38.4 and 34 kDa bands on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis using their respective antibodies. Proteolytic activity of refolded rNfCPB and rNfCPB-L was maximum at a pH of 4.5, and the most effective substrate was Z-LR-MCA. rNfCPB and rNfCPB-L showed proteolytic activity for several proteins such as IgA, IgG, IgM, collagen, fibronectin, hemoglobin, and albumin. These results suggested that NfCPB and NfCPB-L cysteine protease are important components of the N. fowleri ESP, and they may play important roles in host tissue invasion and immune evasion as pathogens that cause N. fowleri PAM.

Genomic copy number alterations with transcriptional deregulation at 6p identify an aggressive HCC phenotype.

Genomic analyses have revealed the enormous heterogeneity in essentially all cancer types. However, the identification of precise subtypes, which are biologically informative and clinically useful, remains a challenge. The application of integrative analysis of multilayered genomic profiles to define the chromosomal regions of genomic copy number alterations with concomitant transcriptional deregulation is posited to provide a promising strategy to identify driver targets. In this study, we performed an integrative analysis of the DNA copy numbers and gene expression profiles of hepatocellular carcinoma (HCC). By comparing DNA copy numbers between HCC subtypes based on gene expression pattern, we revealed the DNA copy number alteration with concordant gene expression changes at 6p21-p24 particularly in the HCC subtype of aggressive phenotype without expressing stemness genes. Among the genes at 6p21-p24, we identified IER3 as a potential driver. The clinical utility of IER3 copy numbers was demonstrated by validating its clinical correlation with independent cohorts. In addition, short hairpin RNA-mediated knock-down experiment revealed the functional relevance of IER3 in liver cancer progression. In conclusion, our results suggest that genomic copy number alterations with transcriptional deregulation at 6p21-p24 identify an aggressive HCC phenotype and a novel functional biomarker.

Exosomal delivery of TRAIL and miR­335 for the treatment of hepatocellular carcinoma (Review).

Liver cancer is the sixth most prevalent type of cancer worldwide and accounts for the third most frequent cause of cancer­associated mortality. Conventional anticancer drugs display limited efficacy owing to their short half­life, poor solubility and inefficient drug delivery. Despite advancements being made in drug discovery and development for the treatment of hepatocellular carcinoma (HCC), drug inefficacy and drug continue to pose significant obstacles to effective treatment. Therefore, it is imperative that novel treatment strategies be developed with the aim of developing anticancer treatments without any side­effects and with long­term durability. Extracellular vesicles, such as exosomes, intercellular communication agents which have the ability to carry heterogenous molecules with high penetrability, low immunogenicity and longer durability, may provide a versatile natural delivery system. The present review article illustrates the innovative treatment strategy using exosomes as a delivery agent for two distinct anticancer candidates, i.e., tumor necrosis factor­related apoptosis­inducing ligand and microRNA­335. The aim of the present review was to present a unique strategy for the development of an exceptional anticancer treatment therapy exploiting exosomes as a delivery vehicle which may be used for HCC.

The immune response induced by DNA vaccine expressing nfa1 gene against Naegleria fowleri.

The pathogenic free-living amoeba, Naegleria fowleri, causes fatal primary amoebic meningoencephalitis in experimental animals and in humans. The nfa1 gene that was cloned from N. fowleri is located on pseudopodia, especially amoebic food cups and plays an important role in the pathogenesis of N. fowleri. In this study, we constructed and characterized retroviral vector and lentiviral vector systems for nfa1 DNA vaccination in mice. We constructed the retroviral vector (pQCXIN) and the lentiviral vector (pCDH) cloned with the egfp-nfa1 gene. The expression of nfa1 gene in Chinese hamster ovary cell and human primary nasal epithelial cell transfected with the pQCXIN/egfp-nfa1 vector or pCDH/egfp-nfa1 vector was observed by fluorescent microscopy and Western blotting analysis. Our viral vector systems effectively delivered the nfa1 gene to the target cells and expressed the Nfa1 protein within the target cells. To evaluate immune responses of nfa1-vaccinated mice, BALB/c mice were intranasally vaccinated with viral particles of each retro- or lentiviral vector expressing nfa1 gene. DNA vaccination using viral vectors expressing nfa1 significantly stimulated the production of Nfa1-specific IgG subclass, as well as IgG levels. In particular, both levels of IgG2a (Th1) and IgG1 (Th2) were significantly increased in mice vaccinated with viral vectors. These results show the nfa1-vaccination induce efficiently Th1 type, as well as Th2 type immune responses. This is the first report to construct viral vector systems and to evaluate immune responses as DNA vaccination in N. fowleri infection. Furthermore, these results suggest that nfal vaccination may be an effective method for treatment of N. fowleri infection.

Transforming growth factor­ß family and stem cell­derived exosome therapeutic treatment in osteoarthritis (Review).

Osteoarthritis (OA), although extensively researched, still lacks an effective and safe treatment. The only current treatment option available for advanced OA is joint replacement surgery. This surgery may pose the risks of persistent pain, surgical complications and limited implant lifespan. Transforming growth factor (TGF)­ß has a crucial role in multiple cellular processes such as cell proliferation. Any deterioration in TGF­ß signaling pathways can have an immense impact on OA. Owing to the crucial role of TGF­ß in cartilage homeostasis, targeting it could be an alternative therapeutic approach. Additionally, stem cell­based therapy has recently emerged as an effective treatment strategy that could replace surgery. A number of recent findings suggest that the tissue regeneration effect of stem cells is attributed to the paracrine secretion of anti­inflammatory and chondroprotective mediators or trophic factors, particularly nanosized extracellular vesicles (i.e., exosomes). Literature searches were performed in the MEDLINE, EMBASE, Cochrane Library and PubMed electronic database for relevant articles published before September 2021. Multiple investigators have confirmed TGF­ß3 as a promising candidate which has the chondrogenic potential to repair articular cartilage degeneration. Combining TGF­ß3 with bone morphogenetic proteins­6, which has synergistic effect on chondrogenesis, with an efficient platform such as exosomes, which themselves possess a chondroprotective function, offers an innovative and more efficient approach to treat injured cartilage. In addition, multiple findings stating the role of exosomes in chondroprotection has also verified a similar fact showing exosomes may be a more favorable choice than the source itself. In the present review, the importance of TGF­ß family in OA and the possibility of therapeutic treatment using stem cell­derived exosomes are described.

Possibility of exosome­based coronavirus disease 2019 vaccine (Review).

Coronavirus disease 2019 (COVID­19) is a global pandemic that can have a long­lasting impact on public health if not properly managed. Ongoing vaccine development trials involve classical molecular strategies based on inactivated or attenuated viruses, single peptides or viral vectors. However, there are multiple issues, such as the risk of reversion to virulence, inability to provide long­lasting protection and limited protective immunity. To overcome the aforementioned drawbacks of currently available COVID­19 vaccines, an alternative strategy is required to produce safe and efficacious vaccines that impart long­term immunity. Exosomes (key intercellular communicators characterized by low immunogenicity, high biocompatibility and innate cargo­loading capacity) offer a novel approach for effective COVID­19 vaccine development. An engineered exosome­based vaccine displaying the four primary structural proteins of SARS­CoV­2 (spike, membrane, nucleocapside and envelope proteins) induces humoral and cell mediated immunity and triggers long­lasting immunity. The present review investigated the prospective use of exosomes in the development of COVID­19 vaccines; moreover, exosome­based vaccines may be key to control the COVID­19 pandemic by providing enhanced protection compared with existing vaccines.

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.