Development and Validation of an Enzyme-Linked Immunosorbent Assay-Based Protocol for Evaluation of Respiratory Syncytial Virus Vaccines.

Recently, respiratory syncytial virus (RSV) vaccines based on the prefusion F (pre-F) antigen were approved in the United States. We aimed to develop an enzyme-linked immunosorbent assay (ELISA)-based protocol for the practical and large-scale evaluation of RSV vaccines. Two modified pre-F proteins (DS-Cav1 and SC-TM) were produced by genetic recombination and replication using an adenoviral vector. The protocol was established by optimizing the concentrations of the coating antigen (pre-F proteins), secondary antibodies, and blocking buffer. To validate the protocol, we examined its accuracy, precision, and specificity using serum samples from 150 participants across various age groups and the standard serum provided by the National Institute of Health. In the linear correlation analysis, coating concentrations of 5 and 2.5 µg/mL of DS-Cav1 and SC-TM showed high coefficients of determination (r > 0.90), respectively. Concentrations of secondary antibodies (alkaline phosphatase-conjugated anti-human immunoglobulin G, diluted 1:2000) and blocking reagents (5% skim milk/PBS-T) were optimized to minimize non-specific reactions. High accuracy was observed for DS-Cav1 (r = 0.90) and SC-TM (r = 0.86). Further, both antigens showed high precision (coefficient of variation < 15%). Inhibition ELISA revealed cross-reactivity of antibodies against DS-Cav1 and SC-TM, but not with the attachment (G) protein.

Age-Stratified Seroprevalence of Respiratory Syncytial Virus: Analysis Using Prefusion F and G Protein Antibodies.

This is a cross-sectional serosurveillance study for RSV. Between June and September of 2021, a total of 150 sera were collected from 30 individuals in each age group (<5, 5-18, 19-49, 50-64, and ≥65 years). Seroprevalence was estimated using enzyme-linked immunosorbent assays targeting two stabilized prefusion F (preF; DS-Cav1 and SC-TM) and G proteins. The overall seroprevalence was low in young children and older adults, despite them having a higher risk of severe RSV infection. There was a remarkable difference in age-stratified seroprevalence rates between anti-preF and anti-G protein antibodies. Given the high disease burden and low seroprevalence in both infants and old adults, RSV vaccination would be crucial for pregnant women and people aged over 60 years.

The combination of calreticulin-targeting L-ASNase and anti-PD-L1 antibody modulates the tumor immune microenvironment to synergistically enhance the antitumor efficacy of radiotherapy.

Radiotherapy (RT) triggers immunogenic cell death (ICD). L-ASNase, which catalyzes the conversion of asparagine (Asn), thereby depleting it, is used in the treatment of blood cancers. In previous work, we showed that CRT3LP and CRT4LP, PASylated L-ASNases conjugated to the calreticulin (CRT)-specific monobodies CRT3 and CRT4, increase the efficacy of ICD-inducing chemotherapy. Here, we assessed their efficacy in tumor-bearing mice treated with RT. Methods: Monobody binding was evaluated by in silico molecular docking analysis. The expression and cellular localization of ecto-CRT were assessed by confocal imaging and flow cytometry. The antitumor effect and the roles of CRT3LP and CRT4LP in irradiation (IR)-induced ICD in tumors were analyzed by ELISA, immunohistochemistry, and immune analysis methods. Results: Molecular docking analysis showed that CRT3 and CRT4 monobodies were stably bound to CRT. Exposure to 10 Gy IR decreased the viability of CT-26 and MC-38 tumor cells in a time-dependent manner until 72 h, and increased the expression of the ICD marker ecto-CRT (CRT exposed on the cell surface) and the immune checkpoint marker PD-L1 until 48 h. IR enhanced the cytotoxicity of CRT3LP and CRT4LP in CT-26 and MC-38 tumor cells, and increased reactive oxygen species (ROS) levels. In mice bearing CT-26 and MC-38 subcutaneous tumors treated with 6 Gy IR, Rluc8-conjugated CRT-specific monobodies (CRT3-Rluc8 and CRT4-Rluc8) specifically targeted tumor tissues, and CRT3LP and CRT4LP increased total ROS levels in tumor tissues, thereby enhancing the antitumor efficacy of RT. Tumor tissues from these mice showed increased mature dendritic, CD4+ T, and CD8+ T cells and pro-inflammatory cytokines (IFNγ and TNFα) and decreased regulatory T cells, and the expression of tumor cell proliferation markers (Ki67 and CD31) was downregulated. These data indicate that the combination of IR and CRT-targeting L-ASNases activated and reprogramed the immune system of the tumor microenvironment. Consistent with these data, an immune checkpoint inhibitor (anti-PD-L1 antibody) markedly increased the therapeutic efficacy of combined IR and CRT-targeting L-ASNases. Conclusion: CRT-specific L-ASNases are useful as additive drug candidates in tumors treated with RT, and combination treatment with anti-PD-L1 antibody increases their therapeutic efficacy.

Serotype-Dependent Inhibition of Streptococcus pneumoniae Growth by Short-Chain Fatty Acids.

Streptococcus pneumoniae (pneumococcus) is an opportunistic pathogen that can cause severe infectious diseases such as pneumonia, meningitis, and otitis media. Despite the availability of antibiotics and pneumococcal vaccines against some invasive serotypes, pneumococcal infection remains a tremendous clinical challenge due to the increasing frequency of infection by antimicrobial resistant, nonencapsulated, and/or non-vaccine serotype strains. Short-chain fatty acids (SCFAs), which are produced at various mucosal sites in the body, have potent antimicrobial activity, including inhibition of pathogen growth and/or bacterial biofilm formation. In this study, we investigated the antimicrobial activity of SCFAs (acetate, propionate, and butyrate) against various serotypes pneumococci. Propionate generally inhibited the growth of S. pneumoniae serotypes included in the pneumococcal conjugate vaccine (PCV) 13, except for serotypes 3 and 7F, though butyrate and acetate showed no or low inhibition, depending on the serotypes. Of note, butyrate showed strong inhibition against serotype 3, the most prevalent invasive strain since the introduction of the PCV. No SCFAs showed inhibitory effects against serotype 7F. Remarkably, the nonencapsulated pneumococcal strain had more sensitivity to SCFAs than encapsulated parental strains. Taken together, these results suggest that propionate showing the most potent inhibition of pneumococcal growth may be used as an alternative treatment for pneumococcal infection, and that butyrate could be used against serotype 3, which is becoming a serious threat.

Deinococcus radiodurans-derived membrane vesicles protect HaCaT cells against H2O2-induced oxidative stress via modulation of MAPK and Nrf2/ARE pathways.

BACKGROUND: Deinococcus radiodurans is a robust bacterium that can withstand harsh environments that cause oxidative stress to macromolecules due to its cellular structure and physiological functions. Cells release extracellular vesicles for intercellular communication and the transfer of biological information; their payload reflects the status of the source cells. Yet, the biological role and mechanism of Deinococcus radiodurans-derived extracellular vesicles remain unclear. AIM: This study investigated the protective effects of membrane vesicles derived from D. radiodurans (R1-MVs) against H2O2-induced oxidative stress in HaCaT cells. RESULTS: R1-MVs were identified as 322 nm spherical molecules. Pretreatment with R1-MVs inhibited H2O2-mediated apoptosis in HaCaT cells by suppressing the loss of mitochondrial membrane potential and reactive oxygen species (ROS) production. R1-MVs increased the superoxide dismutase (SOD) and catalase (CAT) activities, restored glutathione (GSH) homeostasis, and reduced malondialdehyde (MDA) production in H2O2-exposed HaCaT cells. Moreover, the protective effect of R1-MVs against H2O2-induced oxidative stress in HaCaT cells was dependent on the downregulation of mitogen-activated protein kinase (MAPK) phosphorylation and the upregulation of the nuclear factor E2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway. Furthermore, the weaker protective capabilities of R1-MVs derived from ΔDR2577 mutant than that of the wild-type R1-MVs confirmed our inferences and indicated that SlpA protein plays a crucial role in R1-MVs against H2O2-induced oxidative stress. CONCLUSION: Taken together, R1-MVs exert significant protective effects against H2O2-induced oxidative stress in keratinocytes and have the potential to be applied in radiation-induced oxidative stress models.

Gamma Irradiation-Inactivated Respiratory Syncytial Virus Vaccine Provides Protection but Exacerbates Pulmonary Inflammation by Switching from Prefusion to Postfusion F Protein.

Respiratory syncytial virus (RSV) is a common respiratory pathogen that causes lower respiratory diseases among infants and elderly people. Moreover, formalin-inactivated RSV (FI-RSV) vaccine induces serious enhanced respiratory disease (ERD). Radiation has been investigated as an alternative approach for producing inactivated or live-attenuated vaccines, which enhance the antigenicity and heterogeneous protective effects of vaccines compared with conventional formalin inactivation. In this study, we developed an RSV vaccine using gamma irradiation and analyzed its efficacy against RSV vaccine-induced ERD in a mouse model. Although gamma irradiation-inactivated RSV (RI-RSV) carbonylation was lower than FI-RSV carbonylation and RI-RSV showed a significant antibody production and viral clearance, RI-RSV caused more obvious body weight loss, pulmonary eosinophil infiltration, and pulmonary mucus secretion. Further, the conversion of prefusion F (pre-F) to postfusion F (post-F) was significant for both RI-RSV and FI-RSV, while that of RI-RSV was significantly higher than that of FI-RSV. We found that the conversion from pre- to post-F during radiation was caused by radiation-induced reactive oxygen species. Although we could not propose an effective RSV vaccine manufacturing method, we found that ERD was induced by RSV vaccine by various biochemical effects that affect antigen modification during RSV vaccine manufacturing, rather than simply by the combination of formalin and alum. Therefore, these biochemical actions should be considered in future developments of RSV vaccine. IMPORTANCE Radiation inactivation for viral vaccine production has been known to elicit a better immune response than other inactivation methods due to less surface protein damage. However, we found in this study that radiation-inactivated RSV (RI-RSV) vaccine induced a level of immune response similar to that induced by formalin-inactivated RSV (FI-RSV). Although RI-RSV vaccine showed less carbonylation than FI-RSV, it induced more conformational changes from pre-F to post-F due to the gamma radiation-induced reactive oxygen species response, which may be a key factor in RI-RSV-induced ERD. Therefore, ERD induced by RSV vaccine may be due to pre-F to post-F denaturation by random protein modifications caused by external stress. Our findings provide new ideas for inactivated vaccines for RSV and other viruses and confirm the importance of pre-F in RSV vaccines.

Low-dose radiation therapy suppresses viral pneumonia by enhancing broad-spectrum anti-inflammatory responses via transforming growth factor-ß production.

Low-dose radiation therapy (LDRT) can suppress intractable inflammation, such as that in rheumatoid arthritis, and is used for treating more than 10,000 rheumatoid arthritis patients annually in Europe. Several recent clinical trials have reported that LDRT can effectively reduce the severity of coronavirus disease (COVID-19) and other cases of viral pneumonia. However, the therapeutic mechanism of LDRT remains unelucidated. Therefore, in the current study, we aimed to investigate the molecular mechanism underlying immunological alterations in influenza pneumonia after LDRT. Mice were irradiated to the whole lung 1 day post-infection. The changes in levels of inflammatory mediators (cytokines and chemokines) and immune cell populations in the bronchoalveolar lavage (BALF), lungs, and serum were examined. LDRT-treated mice displayed markedly increased survival rates and reduced lung edema and airway and vascular inflammation in the lung; however, the viral titers in the lungs were unaffected. Levels of primary inflammatory cytokines were reduced after LDRT, and transforming growth factor-ß (TGF-ß) levels increased significantly on day 1 following LDRT. Levels of chemokines increased from day 3 following LDRT. Additionally, M2 macrophage polarization or recruitment was increased following LDRT. We found that LDRT-induced TGF-ß reduced the levels of cytokines and polarized M2 cells and blocked immune cell infiltration, including neutrophils, in BALF. LDRT-induced early TGF-ß production was shown to be a key regulator involved in broad-spectrum anti-inflammatory activity in virus-infected lungs. Therefore, LDRT or TGF-ß may be an alternative therapy for viral pneumonia.

Clinical implications of gut microbiota and cytokine responses in coronavirus disease prognosis.

Objectives: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects gut luminal cells through the angiotensin-converting enzyme-2 receptor and disrupts the gut microbiome. We investigated whether the gut microbiome in the early stage of SARS-CoV-2 infection was associated with the prognosis of coronavirus disease (COVID-19). Methods: Thirty COVID-19 patients and 16 healthy controls were prospectively enrolled. Blood and stool samples and clinical details were collected on days 0 (enrollment), 7, 14, and 28. Participants were categorized into four groups by their clinical course. Results: Gut microbiota composition varied during the clinical course of COVID-19 and was closely associated with cytokine levels (p=0.003). A high abundance of the genus Dialister (linear discriminant analysis [LDA] effect size: 3.97856, p=0.004), species Peptoniphilus lacrimalis (LDA effect size: 4.00551, p=0.020), and Anaerococcus prevotii (LDA effect size: 4.00885, p=0.007) was associated with a good prognosis. Starch, sucrose, and galactose metabolism was highly activated in the gut microbiota of the poor prognosis group. Glucose-lowering diets, including whole grains, were positively correlated with a good prognosis. Conclusion: Gut microbiota may mediate the prognosis of COVID-19 by regulating cytokine responses and controlling glucose metabolism, which is implicated in the host immune response to SARS-CoV-2.

Lipoteichoic acid of Streptococcus gordonii as a negative regulator of human dendritic cell activation.

Streptococcus gordonii, an opportunistic Gram-positive bacterium, causes an infective endocarditis that could be fatal to human health. Dendritic cells (DCs) are known to be involved in disease progression and immune responses in S. gordonii infection. Since lipoteichoic acid (LTA) is a representative virulence factor of S. gordonii, we here investigated its role in the activation of human DCs stimulated with LTA-deficient (ΔltaS) S. gordonii or S. gordonii LTA. DCs were differentiated from human blood-derived monocytes in the presence of GM-CSF and IL-4 for 6 days. DCs treated with heat-killed ΔltaS S. gordonii (ΔltaS HKSG) showed relatively higher binding and phagocytic activities than those treated with heat-killed wild-type S. gordonii (wild-type HKSG). Furthermore, ΔltaS HKSG was superior to wild-type HKSG in inducing phenotypic maturation markers including CD80, CD83, CD86, PD-L1, and PD-L2, antigen-presenting molecule MHC class II, and proinflammatory cytokines such as TNF-α and IL-6. Concomitantly, DCs treated with the ΔltaS HKSG induced better T cell activities, including proliferation and activation marker (CD25) expression, than those treated with the wild-type. LTA, but not lipoproteins, isolated from S. gordonii weakly activated TLR2 and barely affected the expression of phenotypic maturation markers or cytokines in DCs. Collectively, these results demonstrated that LTA is not a major immuno-stimulating agent of S. gordonii but rather it interferes with bacteria-induced DC maturation, suggesting its potential role in immune evasion.

Radioprotection of deinococcal exopolysaccharide BRD125 by regenerating hematopoietic stem cells.

There is a substantial need for the development of biomaterials for protecting hematopoietic stem cells and enhancing hematopoiesis after radiation damage. Bacterial exopolysaccharide (EPS) has been shown to be very attractive to researchers as a radioprotectant owing to its high antioxidant, anti-cancer, and limited adverse effects. In the present study, we isolated EPS from a novel strain, Deinococcus radiodurans BRD125, which produces EPS in high abundance, and investigated its applicability as a radioprotective biomaterial. We found that EPS isolated from EPS-rich D. radiodurans BRD125 (DeinoPol-BRD125) had an excellent free-radical scavenging effect and reduced irradiation-induced apoptosis. In addition, bone-marrow and spleen-cell apoptosis in irradiated mice were significantly reduced by DeinoPol-BRD125 administration. DeinoPol-BRD125 enhanced the expression of hematopoiesis-related cytokines such as GM-CSF, G-GSF, M-CSF, and SCF, thereby enhancing hematopoietic stem cells protection and regeneration. Taken together, our findings are the first to report the immunological mechanism of a novel radioprotectant, DeinoPol-BRD125, which might constitute an ideal radioprotective and radiation mitigating agent as a supplement drug during radiotherapy.

Anti-allergic function of the cell wall (DeinoWall) from Deinococcus radiodurans.

Deinococcus radiodurans is an extremophile, well known to be extremely resistant to external stresses due to its unique physiological system and structure of cellular components. Although the proportion of D. radiodurans has been reported to be negatively correlated with atopic dermatitis, the exact function of D. radiodurans in allergic diseases and its precise mechanisms have not been studied. In the present study, we hypothesize that D. radiodurans or its cellular constituents play a critical role in the skin to prevent allergic inflammatory responses by modulating immunity. Heat-killed D. radiodurans inhibited the production of Th2 cytokines, such as IL-4 and IL-5, induced by ovalbumin (OVA) stimulation in splenocytes from OVA-sensitized mice. Among the cellular constituents of D. radiodurans, such as cell wall (DeinoWall), cell membrane (DeinoMem), and exopolysaccharide (DeinoPol), only DeinoWall inhibited the production of Th2 cytokines and 2,4-dinitrochlorobenzene (DNCB)-induced atopic dermatitis (AD), a Th2-predominant allergic disease in mice. Moreover, serum IgE levels and infiltration of mast cells into skin lesions, the markers of Th2 response induced by DNCB application, were significantly inhibited by treatment with DeinoWall. Remarkably, DeinoWall induced the maturation of bone marrow-derived dendritic cells (BMDCs) that promote Th1-biased immunity, which might balance Th1/Th2 and regulate allergic inflammatory responses. Collectively, these results suggest that DeinoWall acts as a major cellular constituent in the negative regulation of allergic inflammatory responses by D. radiodurans and might be a viable candidate for the treatment of allergic diseases.

The effects of air pollutants exposure on the transmission and severity of invasive infection caused by an opportunistic pathogen Streptococcus pyogenes.

Currently, urbanization is associated with an increase in air pollutants that contribute to invasive pathogen infections by altering the host's innate immunity and antimicrobial resistance capability. Streptococcus pyogenes, also known as Group A Streptococcus (GAS), is a gram-positive opportunistic pathogen that causes a wide range of diseases, especially in children and immunosuppressed individuals. Diesel exhaust particle (DEP), a significant constituent of particulate matter (PM), are considered a prominent risk factor for respiratory illness and circulatory diseases worldwide. Several clinical and epidemiological studies have identified a close association between PM and the prevalence of viral and bacterial infections. This study investigated the role of DEP exposure in increasing pulmonary and blood bacterial counts and mortality during GAS M1 strain infection in mice. Thus, we characterized the upregulation of reactive oxygen species production and disruption of tight junctions in the A549 lung epithelial cell line due to DEP exposure, leading to the upregulation of GAS adhesion and invasion. Furthermore, DEP exposure altered the leukocyte components of infiltrated cells in bronchoalveolar lavage fluid, as determined by Diff-Quik staining. The results highlighted the DEP-related macrophage dysfunction, neutrophil impairment, and imbalance in pro-inflammatory cytokine production via the toll-like receptor 4/mitogen-activated protein kinase signaling axis. Notably, the tolerance of the GAS biofilms toward potent antibiotics and bacterial resistance against environmental stresses was also significantly enhanced by DEP. This study aimed to provide a better understanding of the physiological and molecular interactions between exposure to invasive air pollutants and susceptibility to invasive GAS infections.

Development of Live Attenuated Salmonella Typhimurium Vaccine Strain Using Radiation Mutation Enhancement Technology (R-MET).

Salmonella enterica is a leading cause of food-borne diseases in humans worldwide, resulting in severe morbidity and mortality. They are carried asymptomatically in the intestine or gallbladder of livestock, and are transmitted predominantly from animals to humans via the fecal-oral route. Thus, the best preventive strategy is to preemptively prevent transmission to humans by vaccinating livestock. Live attenuated vaccines have been mostly favored because they elicit both cellular and humoral immunity and provide long-term protective immunity. However, developing these vaccines is a laborious and time-consuming process. Therefore, most live attenuated vaccines have been mainly used for phenotypic screening using the auxotrophic replica plate method, and new types of vaccines have not been sufficiently explored. In this study, we used Radiation-Mutation Enhancement Technology (R-MET) to introduce a wide variety of mutations and attenuate the virulence of Salmonella spp. to develop live vaccine strains. The Salmonella Typhimurium, ST454 strain (ST WT) was irradiated with Cobalt60 gamma-irradiator at 1.5 kGy for 1 h to maximize the mutation rate, and attenuated daughter colonies were screened using in vitro macrophage replication capacity and in vivo mouse infection assays. Among 30 candidates, ATOMSal-L6, with 9,961-fold lower virulence than the parent strain (ST454) in the mouse LD50 model, was chosen. This vaccine candidate was mutated at 71 sites, and in particular, lost one bacteriophage. As a vaccine, ATOMSal-L6 induced a Salmonella-specific IgG response to provide effective protective immunity upon intramuscular vaccination of mice. Furthermore, when mice and sows were orally immunized with ATOMSal-L6, we found a strong protective immune response, including multifunctional cellular immunity. These results indicate that ATOMSal-L6 is the first live vaccine candidate to be developed using R-MET, to the best of our knowledge. R-MET can be used as a fast and effective live vaccine development technology that can be used to develop vaccine strains against emerging or serotype-shifting pathogens.

Induction of Apoptotic Cell Death by Oral Streptococci in Human Periodontal Ligament Cells.

Initiation and progression of oral infectious diseases are associated with streptococcal species. Bacterial infection induces inflammatory responses together with reactive oxygen species (ROS), often causing cell death and tissue damage in the host. In the present study, we investigated the effects of oral streptococci on cytotoxicity and ROS production in human periodontal ligament (PDL) cells. Streptococcus gordonii showed cell cytotoxicity in a dose- and time-dependent manner. The cytotoxicity might be due to apoptosis since S. gordonii increased annexin V-positive cells, and the cytotoxicity was reduced by an apoptosis inhibitor, Z-VAD-FMK. Other oral streptococci such as Streptococcus mitis, Streptococcus sanguinis, and Streptococcus sobrinus also induced apoptosis, whereas Streptococcus mutans did not. All streptococci tested except S. mutans triggered ROS production in human PDL cells. Interestingly, however, streptococci-induced apoptosis appears to be ROS-independent, as the cell death induced by S. gordonii was not recovered by the ROS inhibitor, resveratrol or n-acetylcysteine. Instead, hydrogen peroxide (H2O2) appears to be important for the cytotoxic effects of streptococci since most oral streptococci except S. mutans generated H2O2, and the cytotoxicity was dramatically reduced by catalase. Furthermore, streptococcal lipoproteins are involved in cytotoxicity, as we observed that cytotoxicity induced by the lipoprotein-deficient S. gordonii mutant was less potent than that by the wild-type and was attenuated by anti-TLR2-neutralizing antibody. Indeed, lipoproteins purified from S. gordonii alone were sufficient to induce cytotoxicity. Notably, S. gordonii lipoproteins did not induce H2O2 or ROS but cooperatively induced cell death when co-treated with H2O2. Taken together, these results suggest that most oral streptococci except S. mutans efficiently induce damage to human PDL cells by inducing apoptotic cell death with bacterial H2O2 and lipoproteins, which might contribute to the progression of oral infectious diseases such as apical periodontitis.

Structural and Biochemical Characterization of Thioredoxin-2 from Deinococcus radiodurans.

Thioredoxin (Trx), a ubiquitous protein showing disulfide reductase activity, plays critical roles in cellular redox control and oxidative stress response. Trx is a member of the Trx system, comprising Trx, Trx reductase (TrxR), and a cognate reductant (generally reduced nicotinamide adenine dinucleotide phosphate, NADPH). Bacterial Trx1 contains only the Trx-fold domain, in which the active site CXXC motif that is critical for the disulfide reduction activity is located. Bacterial Trx2 contains an N-terminal extension, which forms a zinc-finger domain, including two additional CXXC motifs. The multi-stress resistant bacterium Deinococcus radiodurans encodes both Trx1 (DrTrx1) and Trx2 (DrTrx2), which act as members of the enzymatic antioxidant systems. In this study, we constructed Δdrtrx1 and Δdrtrx2 mutants and examined their survival rates under H2O2 treated conditions. Both drtrx1 and drtrx2 genes were induced following H2O2 treatment, and the Δdrtrx1 and Δdrtrx2 mutants showed a decrease in resistance toward H2O2, compared to the wild-type. Native DrTrx1 and DrTrx2 clearly displayed insulin and DTNB reduction activity, whereas mutant DrTrx1 and DrTrx2, which harbors the substitution of conserved cysteine to serine in its active site CXXC motif, showed almost no reduction activity. Mutations in the zinc binding cysteines did not fully eliminate the reduction activities of DrTrx2. Furthermore, we solved the crystal structure of full-length DrTrx2 at 1.96 Å resolution. The N-terminal zinc-finger domain of Trx2 is thought to be involved in Trx-target interaction and, from our DrTrx2 structure, the orientation of the zinc-finger domain of DrTrx2 and its interdomain interaction, between the Trx-fold domain and the zinc-finger domain, is clearly distinguished from those of the other Trx2 structures.

Immunostimulatory Potential of Extracellular Vesicles Isolated from an Edible Plant, Petasites japonicus, via the Induction of Murine Dendritic Cell Maturation.

Extracellular vesicles (EVs) have recently been isolated from different plants. Plant-derived EVs have been proposed as potent therapeutics and drug-delivery nanoplatforms for delivering biomolecules, including proteins, RNAs, DNAs, and lipids. Herein, Petasites japonicus-derived EVs (PJ-EVs) were isolated through a series of centrifugation steps and characterized using dynamic light scattering and transmission electron microscopy. Immunomodulatory effects of PJ-EVs were assessed using dendritic cells (DCs). PJ-EVs exhibited a spherical morphology with an average size of 122.6 nm. They induced the maturation of DCs via an increase in the expression of surface molecules (CD80, CD86, MHC-I, and MHC-II), production of Th1-polarizing cytokines (TNF-α and IL-12p70), and antigen-presenting ability; however, they reduced the antigen-uptake ability. Furthermore, maturation of DCs induced by PJ-EVs was dependent on the activation and phosphorylation of MAPK and NF-κB signal pathways. Notably, PJ-EV-treated DCs strongly induced the proliferation and differentiation of naïve T cells toward Th1-type T cells and cytotoxic CD8+ T cells along with robust secretion of IFN-γ and IL-2. In conclusion, our study indicates that PJ-EVs can be potent immunostimulatory candidates with an ability of strongly inducing the maturation of DCs.

Radiation-Inactivated S. gallinarum Vaccine Provides a High Protective Immune Response by Activating Both Humoral and Cellular Immunity.

Salmonella enterica subsp. enterica serovar Gallinarum (SG) is a common pathogen in chickens, and causes an acute systemic disease that leads to high mortality. The live attenuated vaccine 9R is able to successfully protect chickens older than six weeks by activating a robust cell-mediated immune response, but its safety and efficacy in young chickens remains controversial. An inactivated SG vaccine is being used as an alternative, but because of its low cellular immune response, it cannot be used as a replacement for live attenuated 9R vaccine. In this study, we employed gamma irradiation instead of formalin as an inactivation method to increase the efficacy of the inactivated SG vaccine. Humoral, cellular, and protective immune responses were compared in both mouse and chicken models. The radiation-inactivated SG vaccine (r-SG) induced production of significantly higher levels of IgG2b and IgG3 antibodies than the formalin-inactivated vaccine (f-SG), and provided a homogeneous functional antibody response against group D, but not group B Salmonella. Moreover, we found that r-SG vaccination could provide a higher protective immune response than f-SG by inducing higher Th17 activation. These results indicate that r-SG can provide a protective immune response similar to the live attenuated 9R vaccine by activating a higher humoral immunity and a lower, but still protective, cellular immune response. Therefore, we expect that the radiation inactivation method might substitute for the 9R vaccine with little or no side effects in chickens younger than six weeks.

Lack of the Bacterial Phytochrome Protein Decreases Deinococcus radiodurans Resistance to Mitomycin C.

Deinococcus radiodurans known for its extraordinary resistance to ionizing radiation contains bacterial phytochrome (BphP), a member of the family of red/far-red light-sensing proteins. In this study, we constructed a bphP mutant strain (ΔbphP) to investigate the role of D. radiodurans BphP (DrBphP) in the DNA damage response. When cells were incubated under light and dark conditions following exposure to DNA damaging agents, such as γ- and UV-radiation and mitomycin C (MMC), no significant difference in cell survival was observed between the wild-type D. radiodurans strain (WT) and ΔbphP. However, when continuously exposed to MMC under light conditions, the WT strain notably exhibited increased survival compared to cells grown in the dark. The increased survival was not observed in the ΔbphP strain. These results are indicative of the protective role of light-activated DrBphP in the presence of MMC. Site-directed mutagenesis revealed that the conserved amino acids Cys-24 and His-532 involved in chromophore binding and signal transduction, respectively, were essential for the protective function of DrBphP. Inactivation of the cognate response regulator (RR; DrBphR) of DrBphP increased MMC resistance in the dark. In trans complementation of the bphP bphR double mutant strain (ΔbphPR) with DrBphR decreased MMC resistance. Considering that DrBphP acts as a light-activated phosphatase that dephosphorylates DrBphR, it appears that phosphorylated DrBphR exerts a negative effect on cell survival in the presence of MMC. DrBphP overexpression resulted in an increase in MMC resistance of ΔbphPR, implying that other RRs might be involved in the DrBphP-mediated signaling pathway. A mutant lacking the dr_0781 gene (Δdr_0781) demonstrated the same MMC phenotype as ΔbphR. Survival was further increased in the bphR dr_0781 double mutant strain compared to each single mutant ΔbphR or Δdr_0781, suggesting that DR_0781 is also involved in the DrBphP-dependent MMC sensitivity. This study uncovered a previously unknown phenomenon of red/far-red light-dependent DNA damage survival mediated by BphP by identifying the conditions under which DrBphP exhibits a fitness advantage.

Protective Effect of Polysaccharides Extracted from Cudrania tricuspidata Fruit against Cisplatin-Induced Cytotoxicity in Macrophages and a Mouse Model.

Although cisplatin is one of most effective chemotherapeutic drugs that is widely used to treat various types of cancer, it can cause undesirable damage in immune cells and normal tissue because of its strong cytotoxicity and non-selectivity. This study was conducted to investigate the cytoprotective effects of Cudrania tricuspidata fruit-derived polysaccharides (CTPS) against cisplatin-induced cytotoxicity in macrophages, lung cancer cell lines, and a mouse model, and to explore the possibility of application of CTPS as a supplement for anticancer therapy. Both cisplatin alone and cisplatin with CTPS induced a significant cytotoxicity in A549 and H460 lung cancer cells, whereas cytotoxicity was suppressed by CTPS in cisplatin-treated RAW264.7 cells. CTPS significantly attenuated the apoptotic and necrotic population, as well as cell penetration in cisplatin-treated RAW264.7 cells, which ultimately inhibited the upregulation of Bcl-2-associated X protein (Bax), cytosolic cytochrome c, poly (adenosine diphosphateribose) polymerase (PARP) cleavage, and caspases-3, -8, and -9, and the downregulation of B cell lymphoma-2 (Bcl-2). The CTPS-induced cytoprotective action was mediated with a reduction in reactive oxygen species production and mitochondrial transmembrane potential loss in cisplatin-treated RAW264.7 cells. In agreement with the results obtained above, CTPS induced the attenuation of cell damage in cisplatin-treated bone marrow-derived macrophages (primary cells). In in vivo studies, CTPS significantly inhibited metastatic colonies and bodyweight loss as well as immunotoxicity in splenic T cells compared to the cisplatin-treated group in lung metastasis-induced mice. Furthermore, CTPS decreased the level of CRE and BUN in serum. In summation, these results suggest that CTPS-induced cytoprotective action may play a role in alleviating the side effects induced by chemotherapeutic drugs.

Complete Genome Sequence of Streptococcus oralis SF100, Isolated from Blood Cultures from a Patient with Infective Endocarditis.

Streptococcus oralis is a commensal viridans group streptococcus of the human oral cavity and a frequent cause of endovascular infection. Here, we report the complete whole-genome sequence of S. oralis strain SF100, which was originally isolated from the blood of a patient with infective endocarditis. This strain contains the lysogenic bacteriophage SM1, which enhances the virulence of SF100 in animal models of endocardial infection.