MukB Is a Gene Necessary for Rapid Proliferation of Vibrio vulnificus in the Systemic Circulation but Not at the Local Infection Site in the Mouse Wound Infection Model.

Vibrio vulnificus causes rapid septicemia in susceptible individuals who have ingested contaminated foods or have open wounds exposed to seawater contaminated with the bacteria. Despite antibiotic therapy and aggressive debridement, mortality from septicemia is high. In this study, we showed that MukB mutation (mukB::Tn) affected the proliferation of V. vulnificus in the systemic circulation but not at the inoculation site in the wound infection model. A comparison of mukB::Tn with WT and a mukB complement strain (mukB::Tn/pmukB) on the bacterial burden in the muscle at the infection site showed that spreading and proliferation of the mukB::Tn strain was similar to those of the other strains. However, the bacterial burden of mukB::Tn in the spleen was reduced compared to that of the WT strain in the wound infection model. In a competition experiment, we found a lower bacterial burden of mukB::Tn in the spleen than that of the WT strain infecting the systemic circulation. Here, we report on a gene required for the rapid proliferation of V. vulnificus only in the systemic circulation and potentially required for its survival. Our finding may provide a novel therapeutic target for V. vulnificus septicemia.

Comprehensive Gene Mutation Profiling of Circulating Tumor DNA in Ovarian Cancer: Its Pathological and Prognostic Impact.

Liquid biopsies from circulating tumor DNA (ctDNA) have been employed recently as a non-invasive diagnostic tool for detecting cancer-specific gene mutations. Here, we show the comprehensive gene mutation profiles of ctDNA in 51 patients with different histological subtypes of stage I-IV ovarian cancer, and their association with clinical outcomes. The ctDNA extracted from pre-treatment patients' plasma were analyzed using Cancer Personalized Profiling by Deep Sequencing targeting 197 genes. Of 51 patients, 48 (94%) showed one or more non-synonymous somatic mutations, including TP53 (37.3%), APC (17.6%), KRAS (15.7%), EGFR (13.7%), MET (11.8%), PIK3CA (11.8%), NPAP1 (11.8%), and ALK (9.8%). The most frequently mutated genes were as follows: TP53 in high-grade serous carcinoma (66.7%), APC in clear cell carcinoma (30.8%), PIK3CA in endometrioid carcinoma (40%), and KRAS in mucinous carcinoma (66.7%). Higher cell-free (cf)DNA concentration significantly correlated with worse progression-free survival (PFS) in all patients as well as stage III-IV patients (p = 0.01 and 0.005, respectively). Further, patients with any pathogenic mutations showed significantly worse PFS (p = 0.048). Blood tumor mutational burden detected from ctDNA did not significantly correlate with the histological subtypes or survival. Collectively, clinico-genomic profiles of individual ovarian cancer patients could be identified using ctDNA and may serve as a useful prognostic indicator. These findings suggest that ctDNA-based gene profiling might help in establishing personalized therapeutic strategies.

Vibiro vulnificus hemolysin associates with gangliosides.

BACKGROUND: Vibrio vulnificus hemolysin (VVH) is a pore-forming toxin secreted by Vibrio vulnificus. Cellular cholesterol was believed to be the receptor for VVH, because cholesterol could bind to VVH and preincubation with cholesterol inhibited cytotoxicity. It has been reported that specific glycans such as N-acetyl-D-galactosamine and N-acetyl-D-lactosamine bind to VVH, however, it has not been known whether these glycans could inhibit the cytotoxicity of VVH without oligomer formation. Thus, to date, binding mechanisms of VVH to cellular membrane, including specific receptors have not been elucidated. RESULTS: We show here that VVH associates with ganglioside GM1a, Fucosyl-GM1, GD1a, GT1c, and GD1b by glycan array. Among them, GM1a could pulldown VVH. Moreover, the GD1a inhibited the cytotoxicity of VVH without the formation of oligomers. CONCLUSION: This is the first report of a molecule able to inhibit the binding of VVH to target cells without oligomerization of VVH.

Changes in the gene mutation profiles of circulating tumor DNA detected using CAPP-Seq in neoadjuvant chemotherapy-treated advanced ovarian cancer.

Cancer Personalized Profiling by deep Sequencing (CAPP-Seq) is a novel ultrasensitive next-generation sequencing-based approach that is used to detect circulating tumor DNA (ctDNA). The aim of the present study was to compare the gene mutation profiles and blood tumor mutation burden (bTMB) measured between pre- and post-neoadjuvant chemotherapy (NAC), utilizing CAPP-seq for plasma ctDNA in patients with advanced ovarian cancer. The current study included 10 patients (6 NAC-sensitive and 4 NAC-resistant) clinically diagnosed as having stage III or IV ovarian cancer and were administered NAC between May 2017 and February 2019. The plasma ctDNA samples were collected at pre- and post-NAC, and comprehensive gene mutation analysis was performed using CAPP-seq. In 5 out of 6 NAC-sensitive cases, the variant allele frequency (VAF) of non-synonymous somatic mutations decreased following NAC. In 2 out of the 4 NAC-resistant cases, the VAF of non-synonymous somatic mutations increased, and new somatic mutations emerged following NAC. In regard to TP53 mutation, the rate of TP53 mutation in the NAC-resistant cases was significantly higher compared with NAC-sensitive cases. Finally, the bTMB decreased significantly after NAC treatment in the NAC-sensitive cases, even though there were no significant differences in the pretreatment bTMB levels between the NAC-sensitive and NAC-resistant cases. These results indicated that gene mutation can be profiled and monitored using liquid biopsy-based CAPP-Seq in patients with advanced ovarian cancer with NAC treatment, and TP53 mutation in the ctDNA and bTMB may be novel biomarkers that can be used for patient monitoring during NAC treatment.

A combined kinetic and thermodynamic approach for interpreting the complex interactions during chloride volatilization of heavy metals in municipal solid waste fly ash.

This study elucidated complex interactions during the chloride volatilization of heavy metals (Pb, Cu, Zn, Mn, and Cr) from municipal solid waste fly ash by combining thermodynamic and kinetic approaches. Chloride volatilization tests under HCl flow at 900 °C and subsequent rinsing with water achieved almost complete removal of Pb, Zn, and Mn. In contrast, almost 100 % of Cr and ∼40 % of Cu were not removed by either volatilization or rinsing processes. Kinetics indicated that the chlorination of Pb, Zn, and Mn followed a pseudo second order reaction and their apparent activation energies were 96.3, 89.2, and 43.5 kJ/mol, respectively. Further thermodynamic calculation revealed that the components contained in fly ash greatly influenced the chlorination of each heavy metal. Unburned carbon facilitated the chlorination of Pb, Zn, and Mn, while it inhibited Cu chlorination. MgO immobilized Cr and inhibited chlorination. KCl and NaCl promoted Zn and Mn chlorination, respectively. The revealed chloride volatilization behavior and effects of co-existing elements could be useful in the design of high-efficiency recovery process of heavy metals from fly ash and the utilization of residues as raw materials for cement. Furthermore, these findings could guide the realization of a recycling-oriented society in terms of reducing waste disposal.

Identification of in vivo Essential Genes of Vibrio vulnificus for Establishment of Wound Infection by Signature-Tagged Mutagenesis.

Vibrio vulnificus can cause severe necrotic lesions within a short time. Recently, it has been reported that the numbers of wound infection cases in healthy hosts are increasing, for which surgical procedures are essential in many instances to eliminate the pathogen owing to its rapid proliferation. However, the mechanisms by which V. vulnificus can achieve wound infection in healthy hosts have not been elucidated. Here, we advance a systematic understanding of V. vulnificus wound infection through genome-wide identification of the relevant genes. Signature-tagged mutagenesis (STM) has been developed to identify functions required for the establishment of infection including colonization, rapid proliferation, and pathogenicity. Previously, STM had been regarded to be unsuitable for negative selection to detect the virulence genes of V. vulnificus owing to the low colonization and proliferation ability of this pathogen in the intestinal tract and systemic circulation. Alternatively, we successfully identified the virulence genes by applying STM to a murine model of wound infection. We examined a total of 5418 independent transposon insertion mutants by signature-tagged transposon mutagenesis and detected 71 clones as attenuated mutants consequent to disruption of genes by the insertion of a transposon. This is the first report demonstrating that the pathogenicity of V. vulnificus during wound infection is highly dependent on its characteristics: flagellar-based motility, siderophore-mediated iron acquisition system, capsular polysaccharide, lipopolysaccharide, and rapid chromosome partitioning. In particular, these functions during the wound infection process and are indispensable for proliferation in healthy hosts. Our results may thus allow the potential development of new strategies and reagents to control the proliferation of V. vulnificus and prevent human infections.

Accurate prediction of anti-phagocytic activity of Vibrio vulnificus by measurement of bacterial adherence to hydrocarbonsPrediction of Anti-Phagocytic Activity.

Vibrio vulnificus can cause necrotizing soft tissue infection via exposure through an open wound, and the incubation period in cases of wound infection is only about 16 h. These facts strongly suggest that mechanisms to evade innate immune cell phagocytosis are essential for its pathogenicity. Hydrophobic interaction is one of the binding mechanisms between bacteria and phagocytes. Several factors that maintain cell surface hydrophobicity (CSH) can contribute to anti-phagocytic activity. In this study, we tried to identify V. vulnificus genes involved in maintaining the CSH, in order to elucidate mechanisms of anti-phagocytic activity. We obtained 143 mutants that had lost their ability to proliferate in the host, using signature-tagged transposon basis mutagenesis (STM). The CSH of these mutants was measured by the bacterial adherence to hydrocarbons (BATH) assay. The CSH of only four mutants differed significantly from that of wild type (WT). Of these four mutants, degS mutant (degS::Tn) showed lesser anti-phagocytic activity than WT in the opsonophagocytosis assay, even though degS::Tn showed opaque-type colonies. Furthermore, survival times of mice subcutaneously inoculated with degS::Tn were prolonged. These facts indicated that the BATH assay is a more suitable method of analyzing the anti-phagocytic activity of V. vulnificus than the comparison of colony morphology.

Severe pyoderma gangrenosum in association with a flame burn.

We herein report a severe case of pyoderma gangrenosum occurring in a burn patient and discuss the possibility of occult complications of this pathological state in daily treatment.

Algorithm for reconstruction of composite cranial defects using the fascial component of free anterolateral thigh flaps.

BACKGROUND: In case of composite cranial defect including the dura mater, the cranial bone, and the scalp, the fascial component of the anterolateral thigh flap can be used for dural reconstruction. However, the advantages and applications of the fascial component depending on the type of defect have not been thoroughly discussed. We made the algorithm for reconstruction of composite cranial defects using the fascial component of free anterolateral thigh flaps. PATIENTS AND METHODS: Six cases of composite cranial defects were reconstructed using free anterolateral thigh flaps with the fascial component. The type of method used was classified into 3 types. Type 1 involves separating the fascia from the flap completely and using it as a nonvascularized component. In type 2, the fascia is not separated from the flap and is instead used as a vascularized component. Type 3 involves separating the vascularized adipofascial component from the skin paddle and using it as a chimeric pattern flap. The algorithm for determining the type of fascial component is applied depending on the condition of the defect. RESULTS: All flaps were transferred successfully in every case. In 4 cases, the type 1 method was used. The type 2 and 3 methods were used in 1 case each. Cranial bone reconstruction was performed in 3 cases. There were no major complications after the procedures. CONCLUSIONS: The fascial component is useful for dural reconstruction. The type of fascial component used is selected depending on the condition of the defect.

Asynchronous osteoradionecrosis of the mandible treated with sequential fibula osteoseptocutaneous flaps: a report of two cases.

Osteroradionecrosis of the mandible is one of the most serious complications of radiotherapy in head and neck cancer. Once osteoradionecrosis of the mandible has been established, conservative therapy is often useless. Two cases of asynchronous bilateral osteoradionecrosis of the mandible are presented. In both cases, successful reconstruction was performed with 2 free fibula osteoseptocutaneous flap transfers from both legs. We believe that adequate debridement, with removal of dead or hypovascular bone and surrounding soft tissue, followed by vascularized composite bone grafts, is the key for a successful outcome. In our cases, the oral function after 2 reconstructive surgeries with the fibula osteoseptocutaneous flap was acceptable. Furthermore, the patient could walk without difficulty.

CBHA is a family of hybrid polar compounds that inhibit histone deacetylase, and induces growth inhibition, cell cycle arrest and apoptosis in human endometrial and ovarian cancer cells.

OBJECTIVES: We investigated the effect of a novel synthesized histone deacetylase inhibitor (HDACI), CBHA, on three endometrial cancer cell lines, two ovarian cancer cell lines, and normal human endometrial epithelial cells. METHODS: Endometrial and ovarian cancer cells were treated with various concentrations of CBHA, and its effect on cell growth, cell cycle, apoptosis, and related measurements was investigated. RESULTS: The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that all endometrial and ovarian cancer cell lines were sensitive to the growth-inhibitory effect of CBHA, although normal endometrial epithelial cells were viable after treatment with the same doses of CBHA that induced growth inhibition in endometrial and ovarian cancer cells. Cell cycle analysis indicated that their exposure to CBHA decreased the proportion of cells in the S-phase and increased the proportion in the G(0)/G(1) phases of the cell cycle. Induction of apoptosis was confirmed by annexin V staining of externalized phosphatidylserine and loss of the transmembrane potential of mitochondria. This induction occurred in concert with altered expression of genes related to cell growth, malignant phenotype, and apoptosis. Furthermore, CBHA treatment of these cell lines increased acetylation of H3 and H4 histone tails. CONCLUSIONS: These results raise the possibility that CBHA may prove particularly effective in the treatment of endometrial and ovarian cancers.