Biotransformation of flonicamid and sulfoxaflor by multifunctional bacterium Ensifer meliloti CGMCC 7333.

Flonicamid is a novel, selective, systemic pyridinecarboxamide insecticide that effectively controls hemipterous pests. Sulfoxaflor, a sulfoximine insecticide, effectively controls many sap-feeding insect pests. Ensifer meliloti CGMCC 7333 transforms flonicamid into N-(4-trifluoromethylnicotinoyl) glycinamide (TFNG-AM). Resting cells of E. meliloti CGMCC 7333 (optical density at 600 nm [OD600] = 5) transformed 67.20% of the flonicamid in a 200-mg/L solution within 96 h. E. meliloti CGMCC 7333 transforms sulfoxaflor into N-(methyl(oxido){1-[6-(trifluoromethyl) pyridin-3-yl] ethyl}-k4-sulfanylidene) urea (X11719474). E. meliloti CGMCC 7333 resting cells (OD600 = 5) transformed 89.36% of the sulfoxaflor in a 200 mg/L solution within 96 h. On inoculating 2 mL of E. meliloti CGMCC 7333 (OD600 = 10) into soil containing 80 mg/kg flonicamid, 91.1% of the flonicamid was transformed within 9 d (half-life 2.6 d). On inoculating 2 mL of E. meliloti CGMCC 7333 (OD600 = 10) into soil containing 80 mg/kg sulfoxaflor, 83.9% of the sulfoxaflor was transformed within 9 d (half-life 3.4 d). Recombinant Escherichia coli harboring the E. meliloti CGMCC 7333 nitrile hydratase (NHase)-encoding gene and NHase both showed the ability to transform flonicamid or sulfoxaflor into their corresponding amides, TFNG-AM and X11719474, respectively. These findings may help develop a bioremediation agent for the elimination of flonicamid and sulfoxaflor contamination.

Actinomycetes Rhodococcus ruber CGMCC 17550 degrades neonicotinoid insecticide nitenpyram via a novel hydroxylation pathway and remediates nitenpyram in surface water.

Neonicotinoid insecticides are neurotoxicants that cause serious environmental pollution and ecosystem risks. In the present study, a nitenpyram-degrading bacterium, Rhodococcus ruber CGMCC 17550, was isolated from a nitenpyram production sewage treatment tank. Liquid chromatography-mass spectrometry analysis revealed R. ruber degraded nitenpyram via a novel hydroxylation pathway to form three different metabolites, one of which was confirmed to hydroxylate nitenpyram at the C3 site of the 6-chlorpyridine cycle by nuclear magnetic resonance analysis. The nitenpyram degradation rate increased as the biomass of resting R. ruber CGMCC 17550 cells increased, reaching 98.37% at an OD600 of 9 in transformation broth containing 100 mg L-1 nitenpyram after 72 h of incubation. Nitenpyram degradation by R. ruber CGMCC 17550 was insensitive to dissolved oxygen levels. Use of glucose, fructose and pyruvate as co-substrates slightly increased nitenpyram degradation. The cytochrome P450 inhibitor 1-aminobenzotriazole strongly inhibited nitenpyram degradation, indicating that P450 enzymes may mediate nitenpyram hydroxylation. Inoculation of R. ruber CGMCC 17550 enhanced nitenpyram degradation in surface water. Additionally, R. ruber cells immobilized by calcium-alginate remediated 87.11% of 100 mg L-1 NIT in 8 d. Genome sequencing analysis confirmed that R. ruber CGMCC 17550 has metabolic diversity and abundant KEGG genes involved in xenobiotics biodegradation and metabolism. These findings demonstrate that R. ruber CGMCC 17550 is capable of unique biodegradation of nitenpyram via the hydroxylation pathway and is a promising bacterium for bioremediation of contaminants.

Dysfunction of regulatory T cells mediated by AKT-FOXO1 signaling pathway occurs during the development of psoriasis.

Psoriasis is an immune-mediated skin disease with abnormal T cells. Regulatory T cells (Treg) are a kind of cell group with immunosuppressive effects. This study aimed to explore the role of Treg cells in the pathogenesis of psoriasis and its possible mechanism. Imiquimod induced psoriasis mice model was conducted. The skin lesions were evaluated according to the psoriasis area and severity index (PASI). Skin biopsies were taken for HE staining and immunohistochemical staining of IL-23, IL-17, IL-33 and TNF-α. CD4+CD25+ Treg cells were isolated. The proportions of Treg cells, cell proliferation, and immunosuppressive activity were analyzed by flow cytometry. The expression of AKT, Foxo1, pAKT, pFoxo1 protein, and the localization of Foxo1 protein in Treg cells were detected by western blot and immunofluorescence. The results showed that the psoriasis mice model was established successfully. There was no significant difference in the proportion of Treg cells between the two groups (P > 0.05). The cell proliferation abilities were decreased, and the immunosuppressive functions of Treg cells were weakened in the psoriatic group (P < 0.05). Western blot showed that pAKT and pFoxo1 levels of Treg cells were significantly increased in the psoriatic group (P < 0.05). Immunofluorescence showed that Foxo1 was mainly expressed in the nucleus of Treg cells in the control group, whereas expressed in the cytoplasm in the psoriasis group. Therefore, we concluded that the cell proliferation and immunosuppressive dysfunction of Treg cells mediated by AKT-FOXO1 signaling pathway may occurs during the development of psoriasis.

Sulfoxaflor Degraded by Aminobacter sp. CGMCC 1.17253 through Hydration Pathway Mediated by Nitrile Hydratase.

Sulfoxaflor, a sulfoximine insecticide, could efficiently control many insect pests of sap-feeding. Microbial degradation of sulfoxaflor and the enzymatic mechanism involved have not been studied to date. A bacterial isolate JW2 that transforms sulfoxaflor to X11719474 was isolated and identified as Aminobacter sp. CGMCC 1.17253. Both the recombinant Escherichia coli strain harboring the Aminobacter sp. CGMCC 1.17253 nitrile hydratase (NHase) gene and the pure NHase acquired sulfoxaflor-degrading ability. Aminobacter sp. CGMCC 1.17253 NHase is a typical cobalt-containing NHase content of subunit α, subunit ß, and an accessory protein, and the three-dimensional homology model of NHase was built. Substrate specificity tests showed that NHase catalyzed the conversion of acetamiprid, thiacloprid, indolyl-3-acetonitrile, 3-cyanopyridine, and benzonitrile into their corresponding amides, indicating its broad substrate specificity. This is the first report of the pure bacteria degradation of the sulfoxaflor residual in the environment and reveals the enzymatic mechanism mediated by Aminobacter sp. CGMCC 1.17253.

A Novel Nutrient Deprivation-Induced Neonicotinoid Insecticide Acetamiprid Degradation by Ensifer adhaerens CGMCC 6315.

Biodegradation of pesticide pollution is often restricted by environmental pressures, such as nutrient deprivation. Ensifer adhaerens CGMCC 6315 could overcome this issue and degrade neonicotinoid acetamiprid (ACE) efficiently under low nutrient stimuli. The ACE degradation rate improved by 33.1-fold when the lysogeny broth content for cell culture was decreased to 1/15-fold. Resting cells of CGMCC 6315 degraded 94.4% of 200 mg/L ACE in 12 h and quickly eliminated 87.8% of 5 mg/kg of residual soil ACE within 2 d. ACE degradation by CGMCC 6315 was via a nitrile hydratase (NHase) pathway. Genome sequencing showed that CGMCC 6315 had two NHase genes ( cnhA and pnhA). PnhA had the highest reported activity of 28.8 U/mg for ACE. QPCR and proteomic analysis showed that the improved ACE degradation ability was attributed to the up-regulated expression of PnhA. This biodegradation system of CGMCC 6315 has great potential for use in pesticide pollution remediation.

Analysis of Immunologic Function Changes in Lichen Planus After Clinical Treatment.

BACKGROUND Lichen planus (LP) is a common chronic superficial skin lesion that causes chronic or sub-acute inflammatory disorders. LP can affect the oral cavity, skin, mucous membrane, and other body parts, and features include repeat attacks and long duration, leading to lower quality of life. This study aimed to analyze the changes of immunologic function before and after treatment of LP. MATERIAL AND METHODS Thirty cutaneous LP patients were selected. Peripheral blood was collected in the morning before and after treatment. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient method. Flow cytometry was used to detect T cell subpopulation CD4⁺ T cells and CD8⁺ T to calculate CD4⁺ T/CD8⁺ T ratio. Enzyme-linked immunosorbent assay (ELISA) was adopted to detect the helper T-cell (Th) factor IL-2, IFN-γ, IL-4, IL-6, IL-17, and IL-22 levels. RESULTS Compared with before treatment, the expressions of CD4⁺ T cells and CD8⁺ T cells were decreased, while the proportion of CD4⁺ T/CD8⁺ T were significantly elevated after treatment. IL-2 and IFN-γ secretion were markedly increased, whereas IL-4, IL-6, IL-17, and IL-22 were significantly reduced after treatment (P<0.05). CONCLUSIONS LP treatment reduces the distribution of CD4⁺ T cells and CD8⁺ T cells, and promotes the changes of Th1, Th2, and Th17 cytokines secretion.

Antitumor Effects of DC Vaccine With ALA-PDT-Induced Immunogenic Apoptotic Cells for Skin Squamous Cell Carcinoma in Mice.

Targeted immunotherapy using dendritic cell vaccine has been employed for the treatment of solid tumors. Topical 5-aminolevulinic acid-mediated photodynamic therapy, an established approach for topical cancers, can induce an effective antitumor immune response. We have previously shown that 5-aminolevulinic acid-mediated photodynamic therapy-induced tumor lysates could considerably enhance antigen-presenting capacity of ex vivo-generated dendritic cells. The current study further demonstrates that 5-aminolevulinic acid-mediated photodynamic therapy dendritic cell vaccine can induce immune responses against cancers. Dendritic cells pulsed by photodynamic therapy-treated skin squamous cell carcinoma cells inhibited squamous cell carcinoma to a greater extent than tumor lysates treated by photodynamic therapy alone or dendritic cells pulsed by freeze-thawed treated tumor cells. Immunohistochemistry showed that photodynamic therapy dendritic cell vaccine could increase the activity of CD4+ and CD8+ T cells in the tumor implantation sites. Flow cytometry assays showed that CD4+ and CD8+ T cells in the spleens of photodynamic therapy dendritic cell vaccine immunized mice increased significantly. Furthermore, we observed increased amounts of interleukin 12 and Interferon gamma (IFN-γ) and decreased amounts of interleukin 10 in the splenocytes and peripheral blood of photodynamic therapy dendritic cell vaccine immunized mice by enzyme linked immunosorbent assay (ELISA). Taken together, our findings suggest that photodynamic therapy dendritic cell vaccination is an effective prophylactic therapy for squamous cell carcinoma.

Stimulation of dendritic cells by DAMPs in ALA-PDT treated SCC tumor cells.

Photodynamic therapy (PDT) not only kills tumor cells directly but also rapidly recruits and activates immune cells favoring the development of antitumor adaptive immunity. It is believed that Topical 5-aminolevulinic acid mediated photodynamic therapy (ALA-PDT) can induce anti-tumor immune responses through dangerous signals damage-associated molecular patterns (DAMPs). In this study, we investigated the effect of ALA-PDT induced DAMPs on immune cells. We focused on the stimulation of dendritic cells by major DAMPs, enhanced the expression of calreticulin (CRT), heat shock proteins 70 (HSP70), and high mobility group box 1 (HMGB1), either individually or in combination. We evaluated in vitro and in vivo expressions of DAMPs induced by ALA-PDT using immunohistochemistry, western blot, and ELISA in a squamous cell carcinoma (SCC) mouse model. The role of DAMPs in the maturation of DCs potentiated by ALA-PDT-treated tumor cells was detected by FACS and ELISA. Our results showed that ALA-PDT enhanced the expression of CRT, HSP70, and HMGB1. These induced DAMPs played an important part in activating DCs by PDT-treated tumor cells, including phenotypic maturation (increase of surface expression of MHC-II, CD80, and CD86) and functional maturation (enhanced capability to secrete IFN-γ and IL-12). Furthermore, injecting ALA-PDT-treated tumor cells into naïve mice resulted in complete protection against cancer cells of the same origin. Our findings indicate that ALA-PDT can increase DAMPs and enhance tumor immunogenicity, providing a promising strategy for inducing a systemic anticancer immune response.

Improvement of DC vaccine with ALA-PDT induced immunogenic apoptotic cells for skin squamous cell carcinoma.

Dendritic cell (DC) based vaccines have emerged as a promising immunotherapy for cancers. However, most DC vaccines so far have achieved only limited success in cancer treatment. Photodynamic therapy (PDT), an established cancer treatment strategy, can cause immunogenic apoptosis to induce an effective antitumor immune response. In this study, we developed a DC-based cancer vaccine using immunogenic apoptotic tumor cells induced by 5-aminolevulinic acid (ALA) mediated PDT. The maturation of DCs induced by PDT-treated apoptotic cells was evaluated using electron microscopy, FACS, and ELISA. The anti-tumor immunity of ALA-PDT-DC vaccine was tested with a mouse model. We observed the maturations of DCs potentiated by ALA-PDT treated tumor cells, including morphology maturation (enlargement of dendrites and increase of lysosomes), phenotypic maturation (upregulation of surface expression of MHC-II, DC80, and CD86), and functional maturation (enhanced capability to secrete IFN-γ and IL-12, and to induce T cell proliferation). Most interestingly, PDT-induced apoptotic tumor cells are more capable of potentiating maturation of DCs than PDT-treated or freeze/thaw treated necrotic tumor cells. ALA-PDT-DC vaccine mediated by apoptotic cells provided protection against tumors in mice, far stronger than that of DC vaccine obtained from freeze/thaw treated tumor cells. Our results indicate that immunogenic apoptotic tumor cells can be more effective in enhancing a DC-based cancer vaccine, which could improve the clinical application of PDT-DC vaccines.