TRIM22 facilitates autophagosome-lysosome fusion by mediating the association of GABARAPs and PLEKHM1.

Tripartite motif (TRIM) proteins are a large family of E3 ubiquitin ligases implicated in antiviral defense systems, tumorigenesis, and protein quality control. TRIM proteins contribute to protein quality control by regulating the ubiquitin-proteasome system, endoplasmic reticulum-associated degradation, and macroautophagy/autophagy. However, the detailed mechanisms through which various TRIM proteins regulate downstream events have not yet been fully elucidated. Herein, we identified a novel function of TRIM22 in the regulation of autophagy. TRIM22 promotes autophagosome-lysosome fusion by mediating the association of GABARAP family proteins with PLEKHM1, thereby inducing the autophagic clearance of protein aggregates, independent of its E3 ubiquitin ligase activity. Furthermore, a TRIM22 variant associated with early-onset familial Alzheimer disease interferes with autophagosome-lysosome fusion and autophagic clearance. These findings suggest TRIM22 as a critical autophagic regulator that orchestrates autophagosome-lysosome fusion by scaffolding autophagy-related proteins, thus representing a potential therapeutic target in neurodegenerative diseases.Abbreviations: AD: Alzheimer disease; ADAOO: AD age of onset; AICD: APP intracellular domain; APP: amyloid beta precursor protein; BSA: bovine serum albumin; cDNAs: complementary DNAs; CQ: chloroquine; CTF: carboxyl-terminal fragment; EBSS: Earle's balanced salt solution; GABARAP: GABA type A receptor-associated protein; GST: glutathione S-transferase; HA: hemagglutinin; HOPS: homotypic fusion and protein sorting; IFN: interferon; IL1A/IL-1α: interleukin 1 alpha; KO: knockout; MTORC1: mechanistic target of rapamycin kinase complex 1; NFKBIA/IκBα: NFKB inhibitor alpha; NFE2L2/NRF2: NFE2 like bZIP transcription factor; PBS: phosphate-buffered saline; PI3K: class I phosphoinositide 3-kinase; PLA: proximity ligation assay; PLEKHM1: pleckstrin homology and RUN domain containing M1; PSEN1: presenilin 1; SEM: standard errors of the means; SNAREs: soluble N-ethylmaleimide-sensitive factor attachment protein receptors; SNCA: synuclein alpha; SNP: single nucleotide polymorphism; TBS: tris-buffered saline; TNF/TNF-α: tumor necrosis factor; TRIM: tripartite motif; ULK1: unc-51 like autophagy activating kinase 1; WT: wild-type.

Effects of klotho protein or klotho knockdown in porcine oocytes at different stages.

Klotho is a protein that plays different functions in female fertility. We have previously reported that klotho protein supplementation during in vitro maturation improves porcine embryo development, while klotho knockout for somatic cell cloning completely blocks full-term pregnancy in vivo. However, the effects of the microinjection of klotho protein or klotho knockdown dual vector in porcine embryos at different time points and the specific molecular mechanisms remain largely unknown. In this study, we injected the preassembled cas9 + sgRNA dual vector, for klotho knockdown, into the cytoplasm of the germinal vesicle stage of oocytes and into porcine embryos after 6-h parthenogenetic activation. Similarly, the klotho protein was inserted into the cytoplasm of germinal vesicle stage oocytes and porcine embryos after 6-h parthenogenetic activation. Compared with the controls, the microinjection of klotho dual vector markedly decreased the blastocyst formation rates in germinal vesicle stage oocytes and activated embryos. However, the efficiency of blastocyst formation when klotho protein was inserted before in vitro maturation was significantly higher than that after klotho protein insertion into parthenogenetically activated embryos. These results indicated that klotho knockdown may impair embryo development into blastocyst irrespective of injection timing. In addition, klotho protein injection timing in pig embryos may be an important factor for regulating embryo development.

Pre-analytical handling conditions and protein marker recovery from urine extracellular vesicles for bladder cancer diagnosis.

Extracellular vesicles (EVs) contain a variety of biomolecules and provide information about the cells that produce them. EVs from cancer cells found in urine can be used as biomarkers to detect cancer, enabling early diagnosis and treatment. The potential of alpha-2-macroglobulin (A2M) and clusterin (CLU) as novel diagnostic urinary EV (uEV) biomarkers for bladder cancer (BC) was demonstrated previously. To validate the diagnostic value of these proteins in uEVs in a large BC cohort, urine handling conditions before uEV isolation should be optimized during sample transportation from medical centers. In this study, we analyzed the uEV protein quantity, EV particle number, and uEV-A2M/CLU after urine storage at 20°C and 4°C for 0-6 days, each. A2M and CLU levels in uEVs were relatively stable when stored at 4°C for a maximum of three days and at 20°C for up to 24 h, with minimal impact on analysis results. Interestingly, pre-processing to remove debris and cells by centrifugation and filtration of urine did not show any beneficial effects on the preservation of protein biomarkers of uEVs during storage. Here, the importance of optimizing shipping conditions to minimize the impact of pre-analytical handling on the uEVs protein biomarkers was emphasized. These findings provide insights for the development of clinical protocols that use uEVs for diagnostic purposes.

A novel chimeric vaccine candidate for porcine reproductive and respiratory syndrome virus (PRRSV) I and II elicits neutralizing antibodies against both types.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically important virus within the swine industry. The virus causes respiratory disease and reproductive failure. Two species of PRRSV-I and II are co-dominant, yet no effective vaccination strategy has been developed to protect against these two types. With an aim to develop a chimeric vaccine strain to protect against both types, in this study, a chimeric porcine reproductive and respiratory syndrome virus (PRRSV) type I and II was rescued using reverse genetics for the first time. Four chimeric infectious clones were designed based on the genomic arrangement of the structural proteins. However, only the clone carrying the transcriptional regulatory sequence (TRS) and ORF6 of a PRRSV-I and ORF6 of a PRRSV-II generated a viable recombinant virus, suggesting that concurrent expression of ORF6 from both parental viruses is essential for the recovery of type I and II chimeric PRRSV. The chimeric virus showed significantly lower replication ability than its parental strains in vitro, which was improved by serial passaging. In vivo, groups of pigs were inoculated with either the chimeric virus, one of the parental strains, or PBS. The chimeric virus replicated in pig tissue and was detected in serum 7 days post-inoculation. Serum neutralization tests indicated that pigs inoculated with the chimeric virus elicited neutralizing antibodies that inhibited infection with strains of both species and with greater coverage than the parental viruses. In conclusion, the application of this technique to construct a chimeric PRRSV holds promise for the development of a highly effective modified live vaccine candidate. This is particularly significant since there are currently no approved commercial divalent vaccines available to combat PRRSV-I and II co-infections.

Efficient reverse genetics approach involving infectious subgenomic amplicon for engineering dengue virus.

Dengue virus, which belongs to the Flaviviridae family, can induce a range of symptoms from mild to severe, including dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. While infectious cloning technology is a useful tool for understanding viral pathogenesis and symptoms, it exhibits limitations when constructing the entire Flavivirus genome. The instability and toxicity of the genome to bacteria make its full-length construction in bacterial vectors a time-consuming and laborious process. To address these challenges, we employed the modified infectious subgenomic amplicon (ISA) method in this study, which can potentially be a superior tool for reverse genetic studies on the dengue virus. Using ISA, we generated recombinant dengue viruses de novo and validated their robust replication in both human and insect cell lines, which was comparable to that of the original strains. Moreover, the efficiency of ISA in genetically modifying the dengue virus was elucidated by successfully inserting the gene for green fluorescence protein into the genome of dengue virus serotype 4. Overall, this study highlighted the effectiveness of ISA for genetically engineering the dengue virus and provided a technical basis for a convenient reverse genetics system that could expedite investigations into the dengue virus.

HMGB1, a potential regulator of tumor microenvironment in KSHV-infected endothelial cells.

High-mobility group box 1 (HMGB1) is a protein that binds to DNA and participates in various cellular processes, including DNA repair, transcription, and inflammation. It is also associated with cancer progression and therapeutic resistance. Despite its known role in promoting tumor growth and immune evasion in the tumor microenvironment, the contribution of HMGB1 to the development of Kaposi's sarcoma (KS) is not well understood. We investigated the effect of HMGB1 on KS pathogenesis using immortalized human endothelial cells infected with Kaposi's sarcoma-associated human herpes virus (KSHV). Our results showed that a higher amount of HMGB1 was detected in the supernatant of KSHV-infected cells compared to that of mock-infected cells, indicating that KSHV infection induced the secretion of HMGB1 in human endothelial cells. By generating HMGB1 knockout clones from immortalized human endothelial cells using CRISPR/Cas9, we elucidated the role of HMGB1 in KSHV-infected endothelial cells. Our findings indicate that the absence of HMGB1 did not induce lytic replication in KSHV-infected cells, but the cell viability of KSHV-infected cells was decreased in both 2D and 3D cultures. Through the antibody array for cytokines and growth factors, CXCL5, PDGF-AA, G-CSF, Emmprin, IL-17A, and VEGF were found to be suppressed in HMGB1 KO KSHV-infected cells compared to the KSHV-infected wild-type control. Mechanistically, phosphorylation of p38 would be associated with transcriptional regulation of CXCL5, PDGF-A and VEGF. These observations suggest that HMGB1 may play a critical role in KS pathogenesis by regulating cytokine and growth factor secretion and emphasize its potential as a therapeutic target for KS by modulating the tumor microenvironment.

Porcine Follicular Fluid-Derived Exosome: The Pivotal Material for Porcine Oocyte Maturation in Lipid Antioxidant Activity.

Several studies have examined exosomes derived from porcine follicular fluid (FF), but few have reported their application in controlled experiments. The main concern in the field of embryology may be that controlled conditions, such as using a defined medium intermittently, cause poor results in mammalian oocyte maturation and embryo development. The first reason is the absence of the FF, which copes with the majority of the processes emerging in oocytes and embryos. Therefore, we added exosomes derived from porcine FF to the maturation medium of porcine oocytes. For morphological assessment, cumulus cell expansion and subsequent embryonic development were evaluated. Moreover, several stainings, such as glutathione (GSH) and reactive oxygen species (ROS), fatty acid, ATP, and mitochondrial activity, as well as evaluations of gene expression and protein analysis, were used for the functional verification of exosomes. When the oocytes were treated with exosomes, the lipid metabolism and cell survival of the oocytes were fully recovered, as well as morphological evaluations compared to the porcine FF-excluded defined medium. Therefore, controlled experiments may produce reliable data if the exosomes are treated with the desired amounts, and we suggest applying FF-derived exosomes to promote experimental data when performing controlled experiments in embryology.

Application of codon pair deoptimization for ORF7-induced attenuation of type I porcine reproductive and respiratory syndrome virus without reduced immune responses.

Codon pair deoptimization (CPD) attenuated type I porcine reproductive and respiratory syndrome virus (PRRSV). Infectious clones covering the full genome of a Korean type I PRRSV (E38) were synthesized, and CPD induced nine synonymous mutants of NSP1 (n = 1) and ORF7 (n = 8). In a trial to rescue live viruses from infectious clones, only four clones with mutations at nt 177 downstream of ORF7 were rescued, which showed a substantial decrease in cellular replication ability. The rescue-failed clones had two common mutation sites with a high minimum free energy and significantly modified RNA secondary structure relative to the original virus. In infected pigs, CPD viruses demonstrated significantly lower replication ability and pathogenicity than the original virus. However, immune response level induced by the attenuated viruses and the original virus was similar. This is the first study to demonstrate that type I PRRSV virulence can be attenuated through CPD application to ORF7.

CXCL1 confers a survival advantage in Kaposi's sarcoma-associated herpesvirus-infected human endothelial cells through STAT3 phosphorylation.

Many cytokines produced by Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells have been shown to participate in the pathogenesis of KSHV. Determination of the exact role of cytokines in Kaposi's sarcoma (KS) pathogenesis is limited, however, by the difficulty to manipulate the target genes in human endothelial cells. In this study, we sought to elucidate the role of cytokines in KSHV-infected human immortalized endothelial cell line (HuARLT cells) by knockout (KO) of the corresponding target genes using the CRISPR/Cas9 system. The cytokine production profile of KSHV-infected HuARLT cells was analyzed using a protein array, and several cytokines were found to be highly upregulated following KSHV infection. This study focused on CXCL1, which was investigated by knocked out in HuARLT cells. KSHV-infected CXCL1 KO cells underwent increased cell death compared to KSHV-infected wild-type (WT) cells and mock-infected CXCL1 KO cells. Lytic replication was not observed in KSHV-infected WT nor CXCL1 KO cells. Phosphorylation of STAT3 was significantly suppressed in KSHV-infected CXCL1 KO cells. Additionally, inhibitors of STAT3 and CXCL1 induced cell death in KSHV-infected endothelial cells. Our results show that CXCL1 production is required for the survival of KSHV-infected endothelial cells, and the CXCL1 to STAT3 phosphorylation signaling pathway may be a therapeutic target for KS.

In vivo Preclinical Tumor-Specific Imaging of Superparamagnetic Iron Oxide Nanoparticles Using Magnetic Particle Imaging for Cancer Diagnosis.

Purpose: Magnetic particle imaging (MPI) is an emerging radiation-free, non-invasive three-dimensional tomographic technology that can visualize the concentrations of superparamagnetic iron oxide nanoparticles (SPIONs). To verify the applicability of the previously proposed point-of-care testing MPI (PoCT-MPI) in medical diagnosis and therapeutics, we imaged SPIONs in animal tumor models. Methods: CT26 or MC38 mouse colon carcinoma cells (2 × 106 cells) were subcutaneously injected into the right flank of BALB/c mice. SPIONs were either injected directly into the tumor lesions in the intratumoral group or through tail veins in the intravenous group. CT26 and MC38 tumor models were examined both intratumorally and intravenously to confirm the biological availability of SPIONs using PoCT-MPI. Results: Signals were observed in the tumor lesions from day 1 to day 7. This is the first study to successfully image the pathological region and show the biodistribution of SPIONs in CT26 tumor models using the recently developed PoCT-MPI technology. Furthermore, MC38 tumor models were examined, resulting in similar images to those of the CT26 tumor model in both intratumoral and intravenous groups. Conclusion: The present study demonstrates the biological applicability of PoCT-MPI, which promises to be a powerful diagnostic and therapeutic technique in biomedical imaging.

Alpha-2-macroglobulin as a novel diagnostic biomarker for human bladder cancer in urinary extracellular vesicles.

Extracellular vesicles (EVs) derived from urine are promising tools for the diagnosis of urogenital cancers. Urinary EVs (uEVs) are considered potential biomarkers for bladder cancer (BC) because urine is in direct contact with the BC tumor microenvironment and thus reflects the current state of the disease. However, challenges associated with the effective isolation and analysis of uEVs complicate the clinical detection of uEV-associated protein biomarkers. Herein, we identified uEV-derived alpha-2-macroglobulin (a2M) as a novel diagnostic biomarker for BC through comparative analysis of uEVs obtained from patients with BC pre- and post-operation using an antibody array. Furthermore, enzyme-linked immunosorbent assay of uEVs isolated from patients with BC (n=60) and non-cancer control subjects (n=23) validated the significant upregulation of a2M expression in patient uEVs (p<0.0001). There was no significant difference in whole urine a2M levels between patients with BC and controls (p=0.317). We observed that compared to classical differential centrifugation, ExoDisc, a centrifugal microfluidic tangential flow filtration device, was a significantly more effective separation method for uEV protein analysis. We expect that our approach for EV analysis will provide an efficient route for the identification of clinically meaningful uEV-based biomarkers for cancer diagnosis.

Minimal Dosage of Porcine Circovirus Type 2d Based Virus-like Particles to Induce Stable Protective Immunity against Infection.

In recent years, porcine circovirus type 2d (PCV2d) has achieved a dominant position worldwide. Various PCV2d capsid-based vaccines have been used to alleviate concerns regarding the emergence of the variant. This study aimed to determine the dosage of recombinant PCV2d capsid protein to induce protective efficacy against experimental challenge with a virulent PCV2d strain. Conventional 3-week-old pigs were intramuscularly inoculated with different doses of the protein (60, 20, 10 and 2 µg). Four weeks after vaccination, all pigs were challenged with pathogenic PCV2d (SNU140003), which was isolated from a farm severely experiencing PCV2-associated disease in Korea. Vaccination with greater than 10 µg of the capsid protein caused a significant (p < 0.05) reduction in PCV2d viremia, lymphoid lesions and lymphoid PCV2 antigen levels in vaccinated challenged pigs compared to unvaccinated challenged pigs. The vaccination also resulted in significantly higher (p < 0.05) titers of neutralizing antibodies against PCV2d. However, the pigs vaccinated with 2 µg had significantly lower neutralizing antibody titers than the other vaccinated groups. They showed a similar level of challenged PCV2d in serum and lymphoid lesion score compared to unvaccinated challenged pigs. The difference in efficacy among the vaccinated groups indicates that there may be a baseline dosage to induce sufficient neutralizing antibodies to prevent viral replication in pigs. In conclusion, at least 10 µg dosage of capsid protein is essential for stable protective efficacy against PCV2d in a pig model.

Biphasic Regulation of Mitogen-Activated Protein Kinase Phosphatase 3 in Hypoxic Colon Cancer Cells.

Hypoxia, or low oxygen tension, is a hallmark of the tumor microenvironment. The hypoxia-inducible factor-1α (HIF-1α) subunit plays a critical role in the adaptive cellular response of hypoxic tumor cells to low oxygen tension by activating gene-expression programs that control cancer cell metabolism, angiogenesis, and therapy resistance. Phosphorylation is involved in the stabilization and regulation of HIF-1α transcriptional activity. HIF-1α is activated by several factors, including the mitogen-activated protein kinase (MAPK) superfamily. MAPK phosphatase 3 (MKP-3) is a cytoplasmic dual-specificity phosphatase specific for extracellular signal-regulated kinase 1/2 (Erk1/2). Recent evidence indicates that hypoxia increases the endogenous levels of both MKP-3 mRNA and protein. However, its role in the response of cells to hypoxia is poorly understood. Herein, we demonstrated that small-interfering RNA (siRNA)-mediated knockdown of MKP-3 enhanced HIF-1α (not HIF-2α) levels. Conversely, MKP-3 overexpression suppressed HIF-1α (not HIF-2α) levels, as well as the expression levels of hypoxia-responsive genes (LDHA, CA9, GLUT-1, and VEGF), in hypoxic colon cancer cells. These findings indicated that MKP-3, induced by HIF-1α in hypoxia, negatively regulates HIF-1α protein levels and hypoxia-responsive genes. However, we also found that long-term hypoxia (>12 h) induced proteasomal degradation of MKP-3 in a lactic acid-dependent manner. Taken together, MKP-3 expression is modulated by the hypoxic conditions prevailing in colon cancer, and plays a role in cellular adaptation to tumor hypoxia and tumor progression. Thus, MKP-3 may serve as a potential therapeutic target for colon cancer treatment.

Composition of Vaginal Microbiota in Pregnant Women With Aerobic Vaginitis.

Vaginal dysbiosis, such as bacterial vaginosis (BV) and aerobic vaginitis (AV), is an important cause of premature birth in pregnant women. However, there is very little research on vaginal microbial distribution in AV compared to that in BV. This study aimed to analyze the composition of the vaginal microbiota of pregnant women with AV using microbial community analysis and identify the causative organism using each criterion of the AV scoring system. Also, we compared the quantification of aerobic bacteria using quantitative polymerase chain reaction (qPCR) and their relative abundances (RA) using metagenomics. This prospective case-control study included 228 pregnant Korean women from our previous study. A wet mount test was conducted on 159 women to diagnose AV using the AV scoring system. Vaginal samples were analyzed using metagenomics, Gram staining for Nugent score determination, conventional culture, and qPCR for Staphylococcus spp., Streptococcus spp., and Enterobacteriaceae. The relative abundances (RAs) of eleven species showed significant differences among the three groups (Normal flora (NF), mild AV, and moderate AV). Three species including Lactobacillus crispatus were significantly lower in the AV groups than in the NF group, while eight species were higher in the AV groups, particularly moderate AV. The decrease in the RA of L. crispatus was common in three criteria of the AV scoring system (Lactobacillary, WBC, and background flora grades), while it did not show a significant difference among the three grade groups of the toxic leukocyte criterion. Also, the RAs of anaerobes, such as Gardnerella and Megasphaera, were higher in the AV groups, particularly moderate AV, while the RAs of aerobes were very low (RA < 0.01). Therefore, qPCR was performed for aerobes (Staphylococcus spp., Streptococcus spp., and Enterobacteriaceae); however, their quantification did not show a higher level in the AV groups when compared to that in the NF group. Therefore, AV might be affected by the RA of Lactobacillus spp. and the main anaerobes, such as Gardnerella spp. Activation of leukocytes under specific conditions might convert them to toxic leukocytes, despite high levels of L. crispatus. Thus, the pathogenesis of AV can be evaluated under such conditions.

Enhanced eosinophil-mediated inflammation associated with antibody and complement-dependent pneumonic insults in critical COVID-19.

Despite the worldwide effect of the coronavirus disease 2019 (COVID-19) pandemic, the underlying mechanisms of fatal viral pneumonia remain elusive. Here, we show that critical COVID-19 is associated with enhanced eosinophil-mediated inflammation when compared to non-critical cases. In addition, we confirm increased T helper (Th)2-biased adaptive immune responses, accompanying overt complement activation, in the critical group. Moreover, enhanced antibody responses and complement activation are associated with disease pathogenesis as evidenced by formation of immune complexes and membrane attack complexes in airways and vasculature of lung biopsies from six fatal cases, as well as by enhanced hallmark gene set signatures of Fcγ receptor (FcγR) signaling and complement activation in myeloid cells of respiratory specimens from critical COVID-19 patients. These results suggest that SARS-CoV-2 infection may drive specific innate immune responses, including eosinophil-mediated inflammation, and subsequent pulmonary pathogenesis via enhanced Th2-biased immune responses, which might be crucial drivers of critical disease in COVID-19 patients.

HMGB1 Knockout Decreases Kaposi's Sarcoma-Associated Herpesvirus Virion Production in iSLK BAC16 Cells by Attenuating Viral Gene Expression.

Multiple host proteins affect the gene expression of Kaposi's sarcoma-associated herpesvirus (KSHV) during latent and lytic replication. High-mobility group box 1 (HMGB1) serves as a highly conserved chromosomal protein inside the cell and a prototypical damage-associated molecular pattern molecule outside the cell. HMGB1 has been shown to play a pathogenic role in viral infectious diseases and to regulate the lytic replication of KSHV. However, its functional effects on the KSHV life cycle in KSHV-infected cells have not been fully elucidated. Here, we explored the role of intracellular and extracellular HMGB1 in KSHV virion production by employing CRISPR/Cas9-mediated HMGB1 knockout in the KSHV-producing iSLK BAC16 cell line. Intracellular HMGB1 formed complexes with various proteins, and the abundance of HMGB1-interacting proteins changed during latent and lytic replication. Moreover, extracellular HMGB1 was found to enhance lytic replication by phosphorylating JNK. Of note, the expression of viral genes was attenuated during lytic replication in HMGB1 knockout iSLK BAC16 cells, with significantly decreased production of infectious virions compared to that of wild-type cells. Collectively, our results demonstrate that HMGB1 is an important cellular cofactor that affects the generation of infectious KSHV progeny during lytic replication. IMPORTANCE The high-mobility group box 1 (HMGB1) protein has many intra- and extracellular biological functions with an intricate role in various diseases. In certain viral infections, HMGB1 affects the viral life cycle and pathogenesis. In this study, we explored the effects of HMGB1 knockout on the production of Kaposi's sarcoma-associated herpesvirus (KSHV). HMGB1 knockout decreased virion production in KSHV-producing cells by decreasing the expression of viral genes. The processes by which HMGB1 affects KSHV production may occur inside or outside infected cells. For instance, several cellular and viral proteins interacted with intracellular HMGB1 in a nucleosomal complex, whereas extracellular HMGB1 induced JNK phosphorylation, thereby enhancing lytic replication. Our results suggest that both intracellular and extracellular HMGB1 are necessary for efficient KSHV replication. Thus, HMGB1 may represent an effective therapeutic target for the regulation of KSHV production.

Activation of the Complement System on Human Endothelial Cells by Urban Particulate Matter Triggers Inflammation-Related Protein Production.

Exposure to particulate matter (PM) is becoming a major global health issue. The amount and time of exposure to PM are known to be closely associated with cardiovascular diseases. However, the mechanism through which PM affects the vascular system is still not clear. Endothelial cells line the interior surface of blood vessels and actively interact with plasma proteins, including the complement system. Unregulated complement activation caused by invaders, such as pollutants, may promote endothelial inflammation. In the present study, we sought to investigate whether urban PM (UPM) acts on the endothelial environment via the complement system. UPM-treated human endothelial cells with normal human serum showed the deposition of membrane attack complexes (MACs) on the cell surface via the alternative pathway of the complement system. Despite the formation of MACs, cell death was not observed, and cell proliferation was increased in UPM-mediated complement activation. Furthermore, complement activation on endothelial cells stimulated the production of inflammation-related proteins. Our results revealed that UPM could activate the complement system in human endothelial cells and that complement activation regulated inflammatory reaction in microenvironment. These findings provide clues with regard to the role of the complement system in pathophysiologic events of vascular disease elicited by air pollution.

Rab27b regulates extracellular vesicle production in cells infected with Kaposi's sarcoma-associated herpesvirus to promote cell survival and persistent infection.

Extracellular vesicles (EVs) play a crucial role in cell-to-cell communication. EVs and viruses share several properties related to their structure and the biogenesis machinery in cells. EVs from virus-infected cells play a key role in virus spread and suppression using various loading molecules, such as viral proteins, host proteins, and microRNAs. However, it remains unclear how and why viruses regulate EV production inside host cells. The purpose of this study is to investigate the molecular mechanisms underlying EV production and their roles in Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells. Here, we found that KSHV induced EV production in human endothelial cells via Rab-27b upregulation. The suppression of Rab27b expression in KSHV-infected cells enhanced cell death by increasing autophagic flux and autolysosome formation. Our results indicate that Rab27b regulates EV biogenesis to promote cell survival and persistent viral infection during KSHV infection, thereby providing novel insights into the crucial role of Rab-27b in the KSHV life cycle.

Intradermal co-inoculation of codon pair deoptimization (CPD)-attenuated chimeric porcine reproductive and respiratory syndrome virus (PRRSV) with Toll like receptor (TLR) agonists enhanced the protective effects in pigs against heterologous challenge.

The objective of this study was to assess protective efficacy of vaccination using CPD-attenuated chimeric PRRSV and Toll like receptor (TLR) agonists (HSP70 c-terminal domain and HSPX) as adjuvants through different inoculation routes. In this study, a chimeric PRRSV composed of two field isolates was synthesized and attenuated by CPD in NSP1 as described in the previous study. The infection of the CPD-attenuated chimeric PRRSV to pigs of 3 weeks-old showed no clinical signs without pathological lesions in necropsy, while it induced improved cross immunity between its parent strains. The TLR agonists were expressed in E. coli and purified to be used. In challenge experiment, pigs of 3 weeks-old were vaccinated using the CPD-attenuated chimeric virus with the prepared TLR agonists through intramuscular or intradermal route, following heterologous challenge after 4 weeks of vaccination. In results, intramuscular or intradermal inoculation of the CPD-attenuated chimeric virus demonstrated excellent protective efficacy against heterologous challenges. Importantly, intradermal inoculation with the TLR agonists enhanced protective effects as shown in the significantly increased level of PRRSV-specific IFN-γ-SCs and cytokines in sera, and the significant reduction of pathological lesion and viral load in lung. This study suggested that the intradermal inoculation of CPD-attenuated chimeric PRRSV plus TLR agonists should be more effective for protection of pigs against diverse PRRS field viruses.

Dimethyl Sulfoxide Enhances Kaposi's Sarcoma-Associated Herpesvirus Production During Lytic Replication.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an etiologic agent of Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman disease. In studies of KSHV, efficient virus production and isolation are essential. Reactivation of KSHV can be initiated by treating latently infected cells with chemicals, such as 12-O-tetradecanoyl-phorbol-13-acetate and sodium butyrate. These chemicals have been used as tools to induce lytic replication and viral production in KSHV-producing cell lines. Dimethyl sulfoxide (DMSO) is an organosulfur compound that is frequently used as an aprotic solvent similar to water. In experiments exploring signaling pathways in KSHV-infected cells, DMSO treatment alone as a vehicle affected the lytic gene expression of KSHV. However, to the best of our knowledge, the effects of DMSO on KSHV-producing cells have not yet been reported. Therefore, in this study, we investigated whether DMSO could be used as a reagent to enhance viral production during lytic replication in KSHV-producing cells and assessed the underlying mechanisms. The effects of DMSO on KSHV production were analyzed in iSLK BAC16 cells, which have been widely used for recombinant KSHV production. We found that the production of KSHV virions was significantly increased by treatment with DMSO during the induction of lytic replication. Mechanistically, lytic genes of KSHV were enhanced by DMSO treatment, which was correlated with virion production. Additionally, DMSO induced the phosphorylation of JNK during lytic replication, and inhibition of JNK abolished the effects of DMSO on lytic replication and virion production. Our findings showed that additional treatment with DMSO during the induction of lytic replication significantly improved the yield of KSHV production.