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ABSTRACT Scrub typhus is a mite-borne disease caused by the obligately intracellular bacterium Orientia tsutsugamushi. We previously demonstrated that ScaA, an autotransporter membrane protein of O. tsutsugamushi, is commonly shared in various genotypes and involved in adherence to host cells. Here, we identified a mixed-lineage leukemia 5 (MLL5) mammalian trithorax group protein as a host receptor that interacts with ScaA. MLL5, identified by yeast two-hybrid screening, is an alternative splicing variant of MLL5 (vMLL5) which contains 13 exons with additional intron sequences encoding a tentative transmembrane domain. Indeed, vMLL5 is expressed on the plasma membrane as well as in intracellular compartments in eukaryotic cells and colocalized with adherent O. tsutsugamushi. In addition, ScaA-expressing Escherichia coli showed significantly increased adherence to vMLL5-overexpressing cells compared with vector control cells. We mapped the C-terminal region of the passenger domain of ScaA as a ligand for vMLL5 and determined that the Su(var)3-9, Enhancer of zeste, Trithorax (SET) domain of MLL5 is an essential and sufficient motif for ScaA binding. We observed significant and specific inhibition of bacterial adhesion to host cells in competitive inhibition assays using the C-terminal fragment of ScaA or the SET domain of vMLL5. Moreover, immunization with the C-terminal fragment of ScaA provided neutralizing activity and protective immunity against lethal challenge with O. tsutsugamushi as efficiently as vaccination with the whole passenger domain of ScaA. These results indicate that vMLL5 is a novel cellular receptor for ScaA-mediated adhesion of O. tsutsugamushi and facilitates bacterial adhesion to host cells, thereby enhancing bacterial infection. IMPORTANCE O. tsutsugamushi is a mite-borne pathogen that causes scrub typhus. As an obligately intracellular pathogen, its adhesion to and invasion of host cells are critical steps for bacterial growth. However, the molecular basis of the bacterial ligand and host receptor interaction is poorly defined. Here, we identified a splicing variant of MLL5 (vMLL5) as a cellular adhesion receptor of ScaA, an outer membrane autotransporter protein of O. tsutsugamushi. We mapped the interacting domains in the bacterial ligand and host receptor and confirmed their functional interaction. In addition, immunization with the C-terminal region of ScaA, which involves an interaction with the SET domain of vMLL5, not only induces enhanced neutralizing antibodies but also provides protective immunity against lethal challenge with O. tsutsugamushi.

The aim of this study was to investigate the relationship between the collagen type I/III ratio and scarring in patients who underwent immediate reconstruction with the round block technique (RBT) after breast conservation surgery. Seventy-eight patients were included, and demographic and clinical characteristics were recorded. The collagen type I/III ratio was measured using immunofluorescence staining and digital imaging, and scarring was assessed using the Vancouver Scar Scale (VSS). The mean VSS scores were 1.92 ± 2.01 and 1.79 ± 1.89, as assessed by two independent plastic surgeons, with good reliability of the scores. A significant positive correlation was found between VSS and the collagen type I/III ratio (r = 0.552, p < 0.01), and a significant negative correlation was found between VSS and the collagen type III content (r = −0.326, p < 0.05). Multiple linear regression analysis showed that the collagen type I/III ratio had a significant positive effect on VSS (β = 0.415, p = 0.028), whereas the collagen type I and collagen type III content had no significant effect on VSS. These findings suggest that the collagen type I/III ratio is associated with scar development in patients undergoing RBT after breast conservation surgery. Further research is needed to develop a patient-specific scar prediction model based on genetic factors affecting the collagen type I/III ratio.

Background and Objectives: Infections and capsular contractures remain unresolved issues in implant-based breast reconstruction. Capsular contractures are thought to be caused by the endogenous flora of the nipple duct. However, little is known about the antibiotic susceptibility of the microorganisms involved. This study aimed to evaluate the composition of endogenous breast flora and its antimicrobial susceptibility in patients with breast cancer. This study will aid in selecting a prophylactic antibiotic regimen for breast reconstruction surgery. Materials and Methods: We obtained bacteriologic swabs from the nipple intraoperatively in patients who underwent implant-based breast reconstruction following nipple-sparing mastectomy between January 2019 and August 2021. Antibiotic susceptibility tests were performed according to the isolated bacteriology. Statistical analysis was performed based on several patient variables to identify which factors influence the antibiotic resistance rate of endogenous flora. Results: A total of 125 of 220 patients had positive results, of which 106 had positive culture results for coagulase-negative Staphylococcus species (CoNS). Among these 106 patients, 50 (47%) were found to have methicillin-resistant staphylococci, and 56 (53%) were found to have methicillin-susceptible staphylococci. The methicillin resistance rate in the neoadjuvant chemotherapy group (56.3%) was significantly higher (OR, 2.3; p = 0.039) than that in the non-neoadjuvant chemotherapy group (35.5%). Conclusions: Based on the results, demonstrating high and rising incidence of methicillin-resistant staphylococci of nipple endogenous flora in patients with breast cancer compared to the past, it is necessary to consider the selection of prophylactic antibiotics to reduce infections and capsular contracture after implant-based breast reconstruction.

Autotransporter proteins are widely present in Gram-negative bacteria. They play a pivotal role in processes related to bacterial pathogenesis, including adhesion, invasion, colonization, biofilm formation, and cellular toxicity. Bioinformatics analysis revealed that Orientia tsutsugamushi, the causative agent of scrub typhus, encodes six different autotransporter genes (scaA–scaF). Although four of these genes (scaA, scaC, scaD, and scaE) are present in diverse strains, scaB and scaF have been detected in only a limited number of strains. Previous studies have demonstrated that ScaA and ScaC are involved in the adherence of host cells. However, the putative function of other O. tsutsugamushi Sca proteins has not been studied yet. In this study, we show that scaB is transcribed and expressed on the surface of O. tsutsugamushi Boryong strain. Using a heterologous Escherichia coli expression system, we demonstrated that ScaB-expressing E. coli can successfully mediate adherence to and invasion into non-phagocytic cells, including epithelial and endothelial cells. In addition, pretreatment with a recombinant ScaB polypeptide inhibits the entry of O. tsutsugamushi into cultured mammalian cells. Finally, we also identified the scaB gene in the Kuroki and TA686 strains and observed high levels of sequence variation in the passenger domains. Here, we propose that the ScaB protein of O. tsutsugamushi can mediate both adhesion to and invasion into host cells in the absence of other O. tsutsugamushi genes and may play important roles in bacterial pathogenesis.

ABSTRACTScrub typhus is an acute febrile disease caused by Orientia tsutsugamushi infection. Despite the wide range of approaches explored during the last seventy years, an effective prophylactic vaccine is not yet available. Here, we developed a novel recombinant antigen derived from conserved regions of 56 kDa type-specific antigen (TSA56), a major outer membrane protein responsible for genetic heterogeneity and antigenicity, and evaluated it as a protective vaccine antigen. Our findings demonstrate that immunization with conserved blocks of TSA56 (cTSA56) not only provides protective immunity against lethal challenges with the homologous genotype, but also confers significantly better protection against heterologous genotypes than TSA56. Adoptive transfer of CD4+ or CD8+ T cells from immunized mice provided significantly enhanced protection against lethal challenge, whereas immune B cells failed to do so, indicating that cellular immunity against the conserved epitopes plays a protective role. Moreover, immunization with a 10-mer peptide mixture, screened from CD8+ T cell epitopes within the conserved region of TSA56, provided enhanced protection against lethal challenge with O. tsutsugamushi. Therefore, this novel recombinant antigen is a promising candidate for scrub typhus vaccine against a wide range of O. tsutsugamushi genotypes.

Orientia tsutsugamushi infection can cause acute lung injury and high mortality in humans; however, the underlying mechanisms are unclear. Here, we tested a hypothesis that dysregulated pulmonary inflammation and Tie2-mediated endothelial malfunction contribute to lung damage. Using a murine model of lethal O. tsutsugamushi infection, we demonstrated pathological characteristics of vascular activation and tissue damage: 1) a significant increase of ICAM-1 and angiopoietin-2 (Ang2) proteins in inflamed tissues and lung-derived endothelial cells (EC), 2) a progressive loss of endothelial quiescent and junction proteins (Ang1, VE-cadherin/CD144, occuludin), and 3) a profound impairment of Tie2 receptor at the transcriptional and functional levels. In vitro infection of primary human EC cultures and serum Ang2 proteins in scrub typhus patients support our animal studies, implying endothelial dysfunction in severe scrub typhus. Flow cytometric analyses of lung-recovered cells further revealed that pulmonary macrophages (MΦ) were polarized toward an M1-like phenotype (CD80+CD64+CD11b+Ly6G-) during the onset of disease and prior to host death, which correlated with the significant loss of CD31+CD45- ECs and M2-like (CD206+CD64+CD11b+Ly6G-) cells. In vitro studies indicated extensive bacterial replication in M2-type, but not M1-type, MΦs, implying the protective and pathogenic roles of M1-skewed responses. This is the first detailed investigation of lung cellular immune responses during acute O. tsutsugamushi infection. It uncovers specific biomarkers for vascular dysfunction and M1-skewed inflammatory responses, highlighting future therapeutic research for the control of this neglected tropical disease.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne disease caused by SFTS virus (SFTSV) infection. Despite a gradual increase of SFTS cases and high mortality in endemic regions, no specific viral therapy nor vaccine is available. Here, we developed a single recombinant plasmid DNA encoding SFTSV genes, Gn and Gc together with NP-NS fusion antigen, as a vaccine candidate. The viral antigens were fused with Fms-like tyrosine kinase-3 ligand (Flt3L) and IL-12 gene was incorporated into the plasmid to enhance cell-mediated immunity. Vaccination with the DNA provides complete protection of IFNAR KO mice upon lethal SFTSV challenge, whereas immunization with a plasmid without IL-12 gene resulted in partial protection. Since we failed to detect antibodies against surface glycoproteins, Gn and Gc, in the immunized mice, antigen-specific cellular immunity, as confirmed by enhanced antigen-specific T cell responses, might play major role in protection. Finally, we evaluated the degree of protective immunity provided by protein immunization of the individual glycoprotein, Gn or Gc. Although both protein antigens induced a significant level of neutralizing activity against SFTSV, Gn vaccination resulted in relatively higher neutralizing activity and better protection than Gc vaccination. However, both antigens failed to provide complete protection. Given that DNA vaccines have failed to induce sufficient immunogenicity in human trials when compared to protein vaccines, optimal combinations of DNA and protein elements, proper selection of target antigens, and incorporation of efficient adjuvant, need to be further investigated for SFTSV vaccine development.

Wet snow may cause significant damage to humans and property, and thus, it is necessary to estimate the corresponding liquid fraction (FL). Consequently, the FL of wet snow was estimated using a novel technique; specifically, the particle shape irregularity (Ir) was estimated through the particle coordinate information obtained using 2-D video disdrometer (2DVD) measurements. Moreover, the possibility of quantitively estimating FL via Ir, based on the temperature (T), was examined. Eight snowfall cases from 2014 to 2016 were observed through a 2DVD installed in Jincheon, South Korea, to analyze the dominant properties of physical variables of snowflakes (i.e., the terminal velocity (VT), particle density (ρs), Ir, and FL) and the corresponding relationships according to the T ranges (−4.5 < T (°C) < 2.5) in which wet snow can occur. It was clarified that the volume-equivalent particle diameter (D)–FL and D–Ir relationships depended on T, and a relationship existed between Ir and FL. The analysis results were verified using the Yong-In Testbed (YIT) S-band weather radar and T-matrix scattering simulation. The D–FL relationship was implemented in the scattering simulation, and the results indicated that the simulated reflectivity (ZS) was highly correlated with the observed reflectivity (ZO) under all T classes. These features can provide a basis for radar analysis and quantitative snowfall estimation for wet snow with various FL values.

Scrub typhus, caused by Orientia tsutsugamushi infection, has been a serious public health issue in the Asia-Pacific region, with rising incidence and sporadic outbreaks. However, human protective immunity against specific antigens has been poorly characterized for this bacterium. In addition, immunity produced in early vaccine trials or even after natural infections, did not last long and had poor cross-reactivity among various genotypes. Here, we systematically investigated the kinetics and magnitude of specific adaptive immunity against two membrane antigens, 56 kDa type-specific antigen (TSA56) and surface cell antigen A (ScaA), that are involved in bacterial adhesion and invasion of the host in 64 recovered scrub typhus patients. Antibody responses to the bacterial antigens in patients were generally short-lived and waned to baseline levels 2 years after recovery. The anti-TSA56 IgG responses were predominantly composed of the IgG1 and IgG3 subclasses and persisted for up to 1 year after recovery, whereas IgG specific to ScaA primarily consisted of more transient IgG1, with limited responses by other subclasses. Cellular immunity, including CD4 and CD8 T-cells specific to membrane antigens, also rapidly declined from 1 year after infection, as measured by enzyme-linked immunospot (ELISPOT) assays and flow cytometry. The short longevity of antigen-specific adaptive immunity might be attributable to limited memory responses, as observed in earlier vaccine studies using whole bacterial antigens. Finally, we identified HLA-A*0201-restricted and highly conserved CD8 T-cell epitopes in the TSA56 antigen, which may be valuable tools for assessing cellular immunity against O. tsutsugamushi and developing an effective scrub typhus vaccine.Emerging Microbes & Infections (2017) 6, e116 doi:10.1038/emi.2017.106; published online 20 December 2017

Abstract Background Zinc oxide nanoparticle (ZNP) has been applied in various biomedical fields. Here, we investigated the usage of ZNP as an antigen carrier for vaccine development by combining a high affinity peptide to ZNP. Results A novel zinc oxide-binding peptide (ZBP), FPYPGGDA, with high affinity to ZNP (K a = 2.26 × 106 M−1) was isolated from a random peptide library and fused with a bacterial antigen, ScaA of Orientia tsutsugamushi, the causative agent of scrub typhus. The ZNP/ZBP-ScaA complex was efficiently phagocytosed by a dendritic cell line, DC2.4, in vitro and significantly enhanced anti-ScaA antibody responses in vivo compared to control groups. In addition, immunization with the ZNP/ZBP-ScaA complex promoted the generation of IFN-γ-secreting T cells in an antigen-dependent manner. Finally, we observed that ZNP/ZBP-ScaA immunization provided protective immunity against lethal challenge of O. tsutsugamushi, indicating that ZNP can be used as a potent adjuvant when complexed with ZBP-conjugated antigen. Conclusions ZNPs possess good adjuvant potential as a vaccine carrier when combined with an antigen having a high affinity to ZNP. When complexed with ZBP-ScaA antigen, ZNPs could induce strong antibody responses as well as protective immunity against lethal challenges of O. tsutsugamushi. Therefore, application of ZNPs combined with a specific soluble antigen could be a promising strategy as a novel vaccine carrier system.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial leaf blight (BLB) in rice (Oryza sativa L.). In this study, we investigated the effect of a mutation in opsX (XOO1056), which encodes a saccharide biosynthesis regulatory protein, on the virulence and bacterial chemotaxis of Xoo. We performed DNA microarray analysis, which showed that 63 of 2,678 genes, including genes related to bacterial motility (flagellar and chemotaxis proteins) were significantly downregulated (

Background Adipose tissue damage of cryopreserved fat after autologous fat transfer is inevitable in several processes of re-transplantation. This study aims to compare and analyze the survivability of adipocytes after thawing fat cryopreserved at -20℃ by using thawing methods used in clinics. Methods The survival rates of adipocytes in the following thawing groups were measured: natural thawing at 25℃ for 15 minutes; natural thawing at 25℃ for 5 minutes, followed by rapid thawing at 37℃ in a water bath for 5 minutes; and rapid thawing at 37℃ for 10 minutes in a water bath. The survival rates of adipocytes were assessed by measuring the volume of the fat layer in the top layers separated after centrifugation, counting the number of live adipocytes after staining with trypan blue, and measuring the activity of mitochondria in the adipocytes. Results In the group with rapid thawing for 10 minutes in a water bath, it was observed that the cell count of live adipocytes and the activity of the adipocyte mitochondria were significantly higher than in the other two groups (P

Blepharoplasty is one of the most common anti-aging operations. Although rare, complications such as ectropion may occur. Thus, we introduced an operative technique to prevent ectropion of the lower lid after blepharoplasty. From January 2012 to August 2013, we performed a nonincisional suspension suture (NISS) technique for 30 patients who visited our clinic for lower blepharoplasty. These patients had a distance of greater than 7 mm on the distraction test and were suspected of having horizontal lid laxity. We performed a slit incision 3 mm superior to the junction between the lateral epicanthus and the orbital bone during lower blepharoplasty. We passed a 7-0 nylon suture through the subcutaneous layer and the orbicularis oculi muscle. Then, we punctured the tarsal plate at the lateral limbus and fixed it to the lateral orbital rim by puncturing the periosteum. We tied a suspension knot through the slit incision. Thirty patients had satisfactory results without major complications, such as scleral exposure or ectropion. The NISS technique could be an effective method by which to prevent postoperative ectropion in cases with a mild to moderate degree lower lid laxity. The use of a NISS procedure is also a simple surgical technique, which saves time and is minimally invasive.

Background Liposuction is a procedure to reduce the volume of subcutaneous fat by physical force. Intracellular storage fat is composed of triglyceride, whereas circulating fat particles exist as cholesterol or triglycerol bound to carrier proteins. It is unavoidable that the storage form of fat particles enters the circulation system after these particles are physiologically destroyed. To date, however, no studies have clarified the fatal characteristics of fat embolism that occurs after the subclinical phase of free fat particles. Methods A mixture of human lipoaspirate and normal saline (1:100, 0.2 mL) was injected into the external jugular vein of rats, weighing 200 g on average. Biopsy specimens of the lung and kidney were examined at 12-hour intervals until postoperative 72 hours. The deposit location and transport of the injected free fat particles were confirmed histologically by an Oil Red O stain. Results Inconsistent with previous reports, free fat particles were transported from the intravascular space to the parenchyma. At 24 hours after infusion, free fat particles deposited in the vascular lumen were confirmed on the Oil Red O stain. At 72 hours after infusion, free fat particles were accumulated compactly within the parenchymal space near the perivascular area. Conclusions Many surgeons are aware of the fatal results and undiscovered pathophysiologic mechanisms of free fat particles. Our results indicate that free fat particles, the storage form of fat that has been degraded through a physiological process, might be removed through a direct transport mechanism and phagocytotic uptake.

Background Depressed scars are usually corrected using subcutaneous fillers such a dermal fat grafting or an autologous fat grafting. But, fillers are absorbed over time and cannot be appropriately used for the correction of large or deep depressed scars. Therefore, we tried new methods to correct challenging cases of deep depressed scars. Methods From 2005 to 2013, deep depressed scar were corrected in 10 patients. The location of the scars were as follows: 6 neck, 4 lower extremities. Preoperative marking was done involving the scar on the skin in an oval shape including the long axis of relaxed skin tension line, and the epithelium of the scar was removed. Both ends of the dermal layer of the residual scar were dissected and then isolated from the subcutaneous layer of the adjacent skin. Then, we folded both ends of the flap like hinge flaps and thereby appropriately restored the depressed part of the dermal layer. And we directly closed skin for scar revision. Results All ten patients had a good result and satisfaction without complication, such as wound infection, dehiscence, at a mean follow-up of seven months. Correction of depression area made the depressed scar less noticeable. Moreover, the problems like donor morbidity, color and texture matching in case of using our techniques, are not issued. Conclusions We folded residual scar tissue like a hinge flap in the correction of depressed scar. We obtained good outcomes and report our new methods and their outcomes with a review of literatures.

Background Burn injuries of the extremity can result in cosmetically offensive appearance. Postburn scar can improve by the combination of tangential excision and thin split-thickness skin graft. Methods From January 2005 and December 2012, 17 patients (10 males and 7 females) with postburn scar of the extremity underwent the combined techniques. The median time from initial injury to surgery was 66.2 months (range: 11-288 months). In the operation, burn scar was excised until viable dermis could be observed using electrical dermatome, after which thin split thickness skin graft was performed. Results Tangential excision and thin split thickness skin graft gave excellent grafting results without the need of reoperation. Depigmentation in the majority of patients were recovered within a year by the operation of thin split thickness skin graft. Recovery of range of motion and cosmetic results were good in all patients. Conclusions Tangential excision and thin split thickness skin graft are a good way of the reconstruction of deformed and depigmented skin from burns on extremity.

Keloid is a scar tissue that undergoes recovery and excessive growth over the original wounds when the collagen is excessively accumulated in the dermis during the wound healing process. The common sites of keloid occurrence include the anterior thorax, shoulder, upper extremities and ear. To our knowledge, however, there are few cases of keloid that occurs in the axilla. In addition, there are fewer cases of keloid that bilaterally occurs at the operated sites postoperatively in individuals with no past or family history. 21-year-old male had undergone subdermal shaving for the management of osmidrosis but had not received appropriate scar management during the military service. He visited us with a keloid-like scar formed in the bilateral axillae. We excised the scar tissue and treated it with local steroid injection and postoperative axilla compression. In histopathological report, no flattening of the overlying epidermis, and presence of keloid collagen which confirms diagnosis of keloid. We report our clinical experience with a rare case of keloid occurring in the axilla and treatment process.

BACKGROUND:Scrub typhus is a mite-borne febrile disease caused by O. tsutsugamushi infection. Recently, emergence of scrub typhus has attracted considerable attention in several endemic countries in Asia and the western Pacific. In addition, the antigenic diversity of the intracellular pathogen has been a serious obstacle for developing effective diagnostics and vaccine. METHODOLOGY/PRINCIPAL FINDINGS:To understand the evolutionary pathway of genotypic diversification of O. tsutsugamushi and the environmental factors associated with the epidemiological features of scrub typhus, we analyzed sequence data, including spatiotemporal information, of the tsa56 gene encoding a major outer membrane protein responsible for antigenic variation. A total of 324 tsa56 sequences covering more than 85% of its open reading frame were analyzed and classified into 17 genotypes based on phylogenetic relationship. Extensive sequence analysis of tsa56 genes using diverse informatics tools revealed multiple intragenic recombination events, as well as a substantially higher mutation rate than other house-keeping genes. This suggests that genetic diversification occurred via frequent point mutations and subsequent genetic recombination. Interestingly, more diverse bacterial genotypes and dominant vector species prevail in Taiwan compared to other endemic regions. Furthermore, the co-presence of identical and sub-identical clones of tsa56 gene in geographically distant areas implies potential spread of O. tsutsugamushi genotypes. CONCLUSIONS/SIGNIFICANCE:Fluctuation and diversification of vector species harboring O. tsutsugamushi in local endemic areas may facilitate genetic recombination among diverse genotypes. Therefore, careful monitoring of dominant vector species, as well as the prevalence of O. tsutsugamushi genotypes may be advisable to enable proper anticipation of epidemiological changes of scrub typhus.

Flammulina velutipes is a fungus with health and medicinal benefits that has been used for consumption and cultivation in East Asia. F. velutipes is also known to degrade lignocellulose and produce ethanol. The overlapping interests of mushroom production and wood bioconversion make F. velutipes an attractive new model for fungal wood related studies. Here, we present the complete sequence of the F. velutipes genome. This is the first sequenced genome for a commercially produced edible mushroom that also degrades wood. The 35.6-Mb genome contained 12,218 predicted protein-encoding genes and 287 tRNA genes assembled into 11 scaffolds corresponding with the 11 chromosomes of strain KACC42780. The 88.4-kb mitochondrial genome contained 35 genes. Well-developed wood degrading machinery with strong potential for lignin degradation (69 auxiliary activities, formerly FOLymes) and carbohydrate degradation (392 CAZymes), along with 58 alcohol dehydrogenase genes were highly expressed in the mycelium, demonstrating the potential application of this organism to bioethanol production. Thus, the newly uncovered wood degrading capacity and sequential nature of this process in F. velutipes, offer interesting possibilities for more detailed studies on either lignin or (hemi-) cellulose degradation in complex wood substrates. The mutual interest in wood degradation by the mushroom industry and (ligno-)cellulose biomass related industries further increase the significance of F. velutipes as a new model.