Clostridium perfringens α-toxin is one of the major virulence factors during C. perfringens infection, causing hemolysis of erythrocytes in various species. Here, genetically engineered Lactobacillus casei (pPG-α/L. casei 393) constitutively expressing the toxoid of C. perfringens α-toxin was generated and its immunogenicity in mice for induction of protective immunity against the α-toxin was evaluated via oral immunization. The α-toxoid was constitutively expressed by pPG-α/L. casei 393 without a specific inducer, as confirmed by western blotting, laser confocal microscopy, and flow cytometry. In an experiment on BALB/c mice to evaluate the oral immunogenicity of pPG-α/L. casei 393, significant levels of a specific secretory IgA (sIgA) antibody in the intestinal mucus and feces and an IgG antibody in the serum of the probiotic vaccine group were detected after booster immunization (p < 0.05) as compared with the pPG/L. casei 393 and PBS control groups. These antibodies effectively neutralized C. perfringens natural α-toxin. Moreover, significantly higher levels of cytokines IL-2, IL-4, IL-10, IL-12, IL-17, and interferon (IFN) γ in the serum and increased proliferation of spleen lymphocytes obtained from mice orally immunized with pPG-α/L. casei 393 were detected. With a commercial C. perfringens type A inactivated vaccine as a control, immune protection provided by the probiotic vaccine against C. perfringens α-toxin was evaluated, and 90% and 80% protection rates were observed, respectively. Therefore, strain pPG-α/L. casei 393 effectively elicited mucosal, humoral, and cellular immunity, suggesting that pPG-α/L. casei 393 is a promising candidate for development of a vaccine against C. perfringens α-toxin. Clostridium perfringens, a gram-positive, spore-forming anaerobic bacterium, is ubiquitous in the environment, e.g. in soil, feces, sewage, and human and animal intestinal tracts [C. perfringens toxinotyping scheme has been helpful for diagnosing C. perfringens infections in humans and animals. On the basis of the traditional scheme of a combination of four typing toxins , C. perfringens strains are classified into five toxinotypes: A to E [C. perfringens strains be classified into seven toxinotypes: A to G [C. perfringens in sheep, cattle, goats, and other animal species are called enterotoxemias.l tracts ,2. It isl tracts ,4. The C: A to E . Recentl: A to G . GeneralC. perfringens may proliferate to large numbers when the intestinal environment is altered by sudden changes in diet or other factors. As a result, potent toxins are produced and absorbed into the systemic circulation or act locally, having devastating effects on the host. Among these toxins, C. perfringens α-toxin is one of the major virulence factors, has both enzymatic and toxin properties [C. perfringens α-toxin are critical for its biological activities. When these histidines are replaced by other amino acid residues, such as glycine, the hemolytic activity and lethality of the α-toxin are significantly reduced or even eliminated. Nonetheless, its antigenicity can be retained [C. perfringens α-toxin [As a typical inhabitant of the intestinal tract of many animal species, operties , and plaoperties ,9. Histooperties ,11. Someretained –14, poin α-toxin ,16.C. perfringens infections in livestock and poultry. Nevertheless, antibiotics can have many negative effects on the environment and human health. According to the characteristics of C. perfringens intestinal infections and intestinal absorption of C. perfringens enterotoxin, an effective oral vaccine that can induce specific secretory IgA (sIgA)-based mucosal and IgG-based humoral immunity against a C. perfringens α-toxin challenge is important for clinical practice. Lactic acid bacteria (LAB), a type of facultative anaerobic gram-positive bacteria, are widely distributed in the digestive tract, respiratory tract, and genitourinary system of humans and animals [Currently, in-feed antibiotics, such as virginiamycin and tylosin, are commonly used to control  animals  and play animals ,19. Furt animals ,21. LAB  animals ,23. FurtLactobacillus casei, a potential antigen delivery vehicle for the development of oral vaccines [C. perfringens α-toxin. Immunogenicity of this vaccine in mice for induction of protective immunity against C. perfringens α-toxin was evaluated via oral immunization.In this study, vaccines , was empC. perfringens toxinotype A (C57-1) was obtained from the China Institute of Veterinary Drug Control  and was grown anaerobically at 37°C in Schaedler Anaerobe Broth . Escherichia coli strains JM109 and TG1 and engineered strain pMD19-T-α/JM109 that carries the gene encoding the α-toxoid (in which histidine-68 in the α-toxin was mutated to a glycine residue) and strain pPG-T7g10/TG1 carrying constitutive expression plasmid pPG-T7g10 constructed by our laboratory were cultured overnight in Luria–Bertani (LB) broth at 37°C. Lactobacillus casei ATCC 393 kept in our laboratory was grown anaerobically in de Man, Rogosa, and Sharpe (MRS) broth  at 37°C without shaking. The details of all plasmids used in this study are listed in Sac I and Apa I , the gene fragment encoding the α-toxoid of C. perfringens was subcloned into constitutive expression vector pPG-T7g10 constructed in our laboratory, giving rise to recombinant plasmid pPG-α  of approximately 0.5 and was centrifuged at 3000 × g for 10 min at 4°C. After that, the cell pellets were washed thrice with precooled EPWB  and twice with precooled EPB (EPWB supplemented with 0.3 M sucrose). The cell pellets were resuspended in EPB and stored at – 80°C until use. For construction of engineered L. casei bearing the pPG-α plasmid, electroporation was carried out as previously described [For the construction of recombinant expression plasmid pPG-α, the ampicillin (Amp)-resistant pMD19-T-α/JM109 strain and chloramphenicol (Cm)-resistant pPG-T7g10/TG1 strain were cultured overnight in LB broth at 37°C. Following the extraction of plasmids pMD19-T-α and pPG-T7g10 with the Plasmid DNA Extraction Kit  and double-enzyme digestion with id pPG-α . For preescribed , and engL. casei strain carrying plasmid pPG-α (pPG-α/L. casei 393) was cultured in MRS broth without a specific inducer at 37°C for 12 h. After that, the bacterial cultures were centrifuged at 12,000 × g for 5 min, and the cells were lysed with 2× SDS buffer followed by boiling for 10 min and centrifugation at 12,000 × g for 10 min. The supernatant was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in a 10% gel followed by western blot analysis using a mouse anti–α-toxoid monoclonal antibody diluted at 1:500 as the primary antibody  and a horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody  diluted 1:2000 as the secondary antibody. Next, the immunoblot was visualized with the chemiluminescent substrate reagent . In addition, to identify the α-toxoid protein expressed on the cell surface of pPG-α/L. casei 393, laser confocal microscopy and flow cytometry were performed. In brief, 1 mL of pPG-α/L. casei 393 (OD600 ≈ 1) was centrifuged at 5000 × g for 5 min, and the cells were washed thrice with sterile PBS. Then, 500 μL of a mouse anti–α-toxoid monoclonal antibody (diluted at 1:100) was added to the cells, gently mixed, and incubated at 37°C for 30 min. After centrifugation and washing with sterile PBS, 500 μL of a fluorescein isothiocyanate (FITC)-labeled goat anti-mouse IgG antibody  was added, mixed, and incubated at 37°C for 60 min in the dark. After a wash and centrifugation, the cell pellets were resuspended in sterile PBS and used for laser confocal microscopy examination and flow-cytometric analysis.The engineered L. casei 393 serving as an oral vaccine, 5-week-old specific pathogen-free (SPF) BALB/c mice obtained from Liaoning Changsheng Biotechnology Co., Ltd.  were chosen as an animal model and were kept under SPF conditions with free access to a standard diet and water. Before oral immunization, engineered strain pPG-α/L. casei 393 was cultured in MRS broth for 16 h without shaking followed by washing with PBS and was resuspended in PBS at a concentration of 1010 CFU/mL. The probiotic vaccine group (41 mice) was immunized with 200 μL of pPG-α/L. casei 393 per mouse [L. casei 393, and mice (n = 41) in the mock control group received 200 μL of PBS. In parallel, mice (n = 41) were injected intramuscularly with a commercial inactivated C. perfringens type A vaccine obtained from China Animal Husbandry Industry Co., Ltd.  and served as the vaccine control group. All mice were immunized via oral administration, and the immunization protocol was carried out on three consecutive days: days 1, 2, and 3. Booster immunization was administered on days 14, 15, and 16, and a second booster was given on days 28, 29, and 30  and national guidelines (CNAS-CL06:2018) for the care and use of laboratory animals were followed. Approval was obtained from the Institutional Committee of Northeast Agricultural University for the animal experiments. In this study, to evaluate the immunogenicity of engineered strain pPG-α/er mouse . The con490 was measured. Moreover, the cytokine levels in serum samples collected on day 35 post-immunization were determined by means of an ELISA Kit , including interleukin 2 (IL-2), IL-4, IL-10, IL-12, IL-17, and interferon γ (IFN-γ).On days 0, 7, 14, 21, 28, 35, and 42 after immunization, serum, feces, and intestinal-mucus samples were collected from three mice selected randomly in each group, and the immune samples were prepared as described elsewhere ,27. In bC. perfringens natural α-toxin obtained from mice orally immunized with pPG-α/L. casei 393 was determined by means of the IgG and sIgA antibodies obtained from pPG/L. casei 393, with PBS groups as a control. Briefly, serum IgG and intestinal mucosal sIgA antibodies collected from the mice immunized with pPG-α/L. casei 393, pPG/L. casei 393, and PBS on day 35 after immunization were mixed with an equal volume  of a solution of the natural α-toxin produced by C. perfringens toxinotype A (C57-1) and were incubated at 37°C for 1 h. Each group of 20 five-week-old SPF BALB/c mice was challenged with 300 μL of the α-toxin treated with the IgG antibody (by injection)/sIgA antibody  to examine survival of the mice.The neutralizing ability of the serum IgG antibody and of intestinal mucosal sIgA antibody toward 6 cells/mL) was incubated in a 96-well plate containing the RPMI 1640 medium supplemented with 10% of fetal calf serum at 37°C in a 5% CO2 incubator. After that, splenocytes were stimulated with the purified α-toxoid protein expressed by pET-32a-α/BL21 at a final concentration of 1, 25, and 50 μg/ml for 72 h. Finally, lymphocyte proliferation was assessed by a CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (MTT) , with absorbance measured at 570 nm.On day 42 post-immunization, splenocytes were obtained from the mice in each group and were subjected to a lymphocyte proliferation assay. In brief, 100 μL of the splenocyte suspension  in each group were challenged with 1 × LD100 of the natural α-toxin combined with 200 μL of C. perfringens type A (~109 CFU/mL) by oral administration via an oral gavage needle. Animal health was monitored daily for a 10-day observation period, and the cumulative mortality of mice in each group was recorded. Next, histopathological changes in the heart, liver, spleen, kidneys, brain, and gut were examined with special attention to color and structure, the presence of congestion and hemorrhage, necrosis, and inflammatory-cell infiltration.To evaluate the immune protection of mice orally vaccinated with pPG-α/P < 0.05 was assumed to indicate statistical significance, and P < 0.01 high significance.Data are shown as mean ± standard error of three replicates per test in a single experiment repeated three times. Kaplan–Meier survival analysis was conducted too. Tukey’s multiple-comparison tests were performed to analyze the differences between groups. L. casei 393 constructed in this study, cell lysates of pPG-α/L. casei 393 were analyzed by a western blot assay. As shown in L. casei 393 but not from pPG/L. casei 393 or L. casei 393, indicating that the α-toxoid protein was expressed by the genetically engineered pPG-α/L. casei 393 and was recognized by the anti–α-toxoid monoclonal antibody. Moreover, expression vector pPG-T7g10 is a cell surface expression system. Therefore, to analyze cell surface expression of the α-toxoid protein, laser confocal microscopy and flow cytometry assays were performed, and the results suggested that there were clear-cut green fluorescent signals on the cell surface of pPG-α/L. casei 393 observed by laser confocal microscopy, but not on pPG/L. casei 393  in sIgA and IgG antibody levels among the groups before immunization. After a second booster immunization, anti–α-toxoid specific mucosal sIgA antibody levels in the intestinal mucus (L. casei 393 group were significantly higher (p ˂ 0.05) as compared to groups “pPG/L. casei 393” and “PBS.” Nonetheless, no significant changes in anti–α-toxoid sIgA or IgG levels were observed in mice that received pPG/L. casei 393 or PBS  were observed in mice orally immunized with pPG-α/L. casei 393 as compared to group pPG/L. casei 393 or PBS  than that among mice of the pPG/L. casei 393 group or PBS group at each time point. In contrast, mice challenged with C. perfringens natural α-toxin – treated with antibodies obtained from mice orally immunized with pPG/L. casei 393 or PBS – all died within 24 h.In this experiment, mice were challenged with IgG 85%;  and α-toIgG 85%; . At the L. casei 393 but not in the group of mice that received pPG/L. casei 393 or PBS , the survival rates of mice in the probiotic vaccine (pPG-α/L. casei 393) group and in the commercially available inactivated C. perfringens toxinotype A vaccine group were 90% and 80%, respectively, whereas the mice in the pPG/L. casei 393 group and PBS group all died  of sIgA and IgG antibody levels in the mice that received pPG/L. casei 393 or PBS. Moreover, mucosal sIgA and serum IgG antibodies induced by the probiotic vaccine turned out to have good abilities to neutralize C. perfringens natural α-toxin. Our results indicate that genetically engineered Lactobacillus strain pPG-α/L. casei 393 as an oral vaccine can effectively induce antigen-specific mucosal and systemic immune responses against C. perfringens α-toxin and has good immunogenicity.Oral vaccination can induce mucosal immune responses, especially an intestinal one, and an effective intestinal mucosal immune response requires production of both mucosal sIgA and serum IgG antibodies against the infectious agent, which have been proven as a valid approach against the colonization by pathogens and against their further spread to the systemic circulation ,33. C. pfections ,35. In tL. casei 393 was confirmed, suggesting that a cellular immune response can be induced by the probiotic vaccine. The balance or ratio of T helper 1 (Th1) and Th2 cells is important for a healthy immune response. One study suggests that L. casei can promote the process of innate immune responses and activate Th1-mediated immune responses [+ T cells and certain CD4+ T cells  can enhance the activity of Th1 cells and promote cellular immune function [+ T helper cells, and the frequency of Th1 and Th2 cell responses after oral immunization may determine the magnitude and isotype of mucosal antibody responses. In this study, we examined Th1-type  and Th2-type (IL-4 and IL-10) cytokine levels in the serum of mice immunized with pPG-α/L. casei 393 by ELISAs. Significantly higher amounts (p < 0.01) of these cytokines were found to be secreted into the serum of mice in the pPG-α/L. casei 393 group compared to group pPG/L. casei 393 or PBS. In addition, cytokine IL-17 was effectively induced by the oral probiotic vaccine; this mechanism may fight against pathogen invasion at different phases and locations of infection. Moreover, our results confirmed that a mixed Th1- and Th2-type immune response can be induced by the genetically engineered Lactobacillus.Lymphocyte proliferation positively correlates with cellular immunity. In this study, proliferation of spleen lymphocytes from mice orally immunized with pPG-α/esponses . IFN-γ pfunction . The Th2function . AntigenC. perfringens should be capable of eliciting potent immune responses against exotoxins: the main virulence factors of C. perfringens. Therefore, to confirm the protection efficacy of pPG-α/L. casei 393 as an oral vaccine, a challenge test was performed on mice by means of the crude α-toxin of C. perfringens coupled with C. perfringens type A after the second booster immunization. A commercial inactivated C. perfringens type A vaccine served as a vaccine control. Our results revealed that the survival rates of mice in the probiotic vaccine group and in the commercial vaccine group reached 90% and 80%, respectively, whereas none of the mice in the pPG/L. casei 393 and PBS groups survived. This finding indicates that the mucosal and systemic immune responses induced by pPG-α/L. casei 393 via oral immunization can provide effective protection in mice to neutralize an attack by C. perfringens type A together with its crude α-toxin. Moreover, histopathological changes were detected in the liver, spleen, kidneys, and colon of mice in the pPG/L. casei 393 and PBS groups exposed to the α-toxin challenge, whereas no significant changes were observed in the tissues of mice from the probiotic vaccine group before and after the challenge. All these results confirm that the toxoid of C. perfringens α-toxin as an immunogen expressed by strain pPG-α/L. casei 393 induced effective protective immunity against the crude α-toxin challenge.An effective vaccine against L. casei 393 constructed in this study expressing the toxoid of C. perfringens α-toxin was able to effectively induce potent antigen-specific mucosal and humoral immune responses and to protect murine hosts from a C. perfringens natural α-toxin challenge, suggesting that the engineered strain is a promising candidate vaccine against the α-toxin.In summary, the genetically engineered strain pPG-α/


The decreased levels of pro-inflammatory markers, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), provided evidence for the anti-inflammatory effects of GC/L/C treatment. Chronic wounds treated with GC/L/C achieved better performance with a 58 ± 7% increase in recovery rate on the seventh day. Based on its anti-inflammatory and wound-healing effects, the GC/L/C bilayer nanofibrous scaffolds can be potential materials for chronic wound treatment.Impaired growth factor production, angiogenic response, macrophage function, and collagen accumulation have been shown to delay wound healing. Delayed wound healing is a debilitating complication of diabetes that leads to significant morbidity. In this study, curcumin and Lithospermi radix (LR) extract, which are used in traditional Chinese herbal medicine, were added within nanofibrous membranes to improve wound healing in a streptozotocin (STZ)-induced diabetic rat model. Gelatin-based nanofibers, which were constructed with curcumin and LR extract at a flow rate of 0.1 mL/hour and an applied voltage of 20 kV, were electrospun onto chitosan scaffolds to produce bilayer nanofibrous scaffolds (GC/L/C). The wounds treated with GC/L/C exhibited a higher recovery rate and transforming growth factor-beta (TGF- Wound healing is a complex event, requiring the interactions of many cell types, including inflammatory cells, fibroblasts, keratinocytes, and endothelial cells, as well as the involvement of growth factors and enzymes ,2. The p− of gelatin [The ideal biomaterial should mimic the characteristics of the desired in vivo environments for tissue engineering. In recent years, natural materials have drawn attention in their application as membranes for wound healing. Gelatin is a partially degraded product of collagen, and the most abundant protein in the extracellular matrix (ECM). Gelatin nanofiber, manufactured by an electrospinning technique, is a biodegradable membrane-forming material with considerable properties, including small pores, a high surface area, physical stability, and promotes cell adhesion, differentiation, and proliferation . Its dis gelatin . Therefoβ-hydroxy-isovaleryl-shikonin, shikonin, quinones, isobutyl-shikonin, and α-methyl-n-butyl-shikonin [β expression in the LR-treated fibroblasts [Curcuma longa (turmeric). It has been shown to have a wide range of pharmacological activities, including anti-inflammatory, anti-oxidant, anti-bacterial effects, and wound healing effects [Gelatin nanofibers have also been studied as potential drug carriers due to their large surface area and high porosity. Pharmaceutical ingredients which are commonly delivered using electrospun membranes include antibiotics, anti-inflammatory drugs, proteins, nucleic acids, and herbal extracts ,11. Lithshikonin . The benshikonin . Evidencroblasts ,16,17. A effects ,19. LR aα) are involved in the up-regulation of inflammatory responses [α indicates the suppression of the inflammatory response. TGF-β, a family of growth factors, exerts pleiotropic effects on wound healing by regulating cell proliferation, differentiation, extracellular matrix production, and modulating the immune response. An effective treatment could help cells precisely react to inflammatory mediators, growth factors, and cytokines, which are essential for effective wound recovery [In the wound healing process, several cytokines have been reported to play an essential role in the inflammatory reaction. Evidence has shown that pro-inflammatory cytokines  alcohol (PVA) (MW = 1400) was purchased from SHOWA . Chitosan (>75% deacetylated), glutaraldehyde, DAPI, and curcumin were acquired by Sigma . Mouse fibroblasts (L929) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). The reagents for cell culture were supplied from Invitrogen .L929 cells were cultured at 1 × 104 cells/well in a 96 well-culture dish. The cell viability was performed by MTT -2,5-diphenyltetrazolium bromide) assay. Add MTT made up in the medium to a final concentration of 0.5 mg/mL. The microplate was incubated at 37 °C for 4 h. After removing the medium from the microplate, the solubilizing solution was added to each well. After shaking for 3 min, the optical density was read at a wavelength of 570 nm using an ELISA reader .4 cell/well in the 12 well-culture dishes and using a tip to draw a line in the middle of culture dish after 24 h incubation. Each well was washed with PBS buffer for 3 times and added 9:1 culture medium with different concentrations of curcumin. After incubation for 48 h, L929 cells were stained with Liu’s stain and observed using an inverted microscope .L929 cell was planted at 8 × 10Tyrosinase inhibitory assay was performed with l-3,4-dihydroxyphenylalanine (L-DOPA) as a substrate. The inhibition activity was measured by a spectrophotometric method that used a modified dopachrome method. Forty microliters of curcumin solutions at different concentrations were added into each well of a 96-well plate with 140 μL of 2 mg/mL L-DOPA and 20 μL of 10 U/μL tyrosinase. After incubation at room temperature for 30 min, the absorbance was read using an ELISA reader at a wavelength of 475 nm. The percentage of tyrosinase inhibition was calculated as follows:v/v% acetic acid under magnetic stirring at 200 rpm for 4 h at room temperature. Planar membranes were performed by pouring five milliliters of the chitosan solution into a 6-cm-diameter dish and frozen at −80 °C for 24 h before lyophilization. The dried chitosan scaffolds were neutralized with 1 N NaOH for 1 h and then copiously rinsed with deionized water. After freeze-dried, the chitosan scaffolds were cross-linked with 50% glutaraldehyde for 45 min.Chitosan solution with the concentration of 2 wt% was prepared by dissolving the chitosan powder in 2 v/v. The solution was agitated at room temperature for 1 h. Four hundred μg/mL curcumin and 625 μg/mL LR extract were added to the gelatin/PVA solution to produce gelatin/PVA/L/C solution. Then the gelatin/PVA solution containing curcumin and LR extract was electrospun onto the chitosan scaffolds to produce the GC/L/C bilayer scaffold. The collection distance was 10 cm. The applied voltage was controlled at 20 kV. The solution flow rate was 0.1 mL/h. After electrospinning, the nanofibers were cross-linked in 50 wt% glutaraldehyde vapor for 45 min. The prepared GC/L/C bilayer nanofibrous scaffolds were sterilized with ultraviolet (UV) radiation for 1h.Gelatin solution with a concentration of 17 wt% was prepared by dissolving 1.53 g gelatin powders in 9 mL formic acid (85%). A clear aqueous solution of polyvinyl alcohol (10 wt% PVA) solution prepared by dissolving 1.0 g PVA in 9 mL deionized water at 70 °C was added to the gelatin solution with a gelatin/PVA ratio of 9/1 The structure of dry GEL nanofibers was observed by using a Field emission scanning electron microscopy . The fiber diameters were measured as mean values of 20 measurements in the SEM images and analyzed using Image J.4 cells/well in a 24-well, flat-bottomed tissue culture plate in triplicate. After 48 h-incubation, the MTT assay was performed to evaluate the cell viability.The biocompatibility of the membranes was performed on mouse fibroblasts (L929). The electrospun membrane was cut into a circle with a diameter of 1.5 cm in a 24-well plate and sterilized with UV radiation for 1 h. The L929 cells were grown in triplicate with the membrane-conditioned well at a density of 1 × 104/well and grown on GEL nanofibers at 37 °C for 48 h. After removing the supernatant and washing with PBS for 3 times, the L929 cells were fixed using methanol: acetone 1:1 for 10 min and soaking with 3% milk for 30 min. For the immunofluorescence staining, the nanofibrous membranes were labeled with anti-β-actin antibodies as 1st antibody. After removing the 1st antibody, washed by PBS solution for 3 times and added FITC as the 2nd antibody for 1 h. Then 4′,6-diamidino-2-phenylindole (DAPI) staining for 10 min was performed for quantitative analysis.Gelatin/PVA solution containing curcumin and LR extract were electrospun on the coverslip. L929 cells were seeded on nanofibers in a density of 1 × 10Male Sprague–Dawley (SD) rats weighing 350–400 g were used and treated according to the Ethical Guidelines of the Animal Center at China Medical University (CMU) . The animal use protocol (protocol ID: CMUIACUC-2017-253) approved by the Institutional Animal Care and Use Committee (IACUC) of CMU on 15 February 2017.SD rats weighing 350–400 g were used. On experimental day 1, all rats were fasted for 24 h prior to STZ treatment. The STZ was dissolved in 50 mM sodium citrate and injected at 55 mg/kg. On experiment day 4, the blood glucose of STZ-injected rats was measured to confirm diabetes condition.2) on the back of each rat under anesthesia with ether was removed using a hair remover. After removing the skin of four areas (15 × 15 mm2), the wound surfaces were disinfected with I2. A GC membrane, GC/L membrane, GC/C membrane, GC/L/C scaffold, a commercial wound dressing (Comfeel®), and gauze (control) were placed on the wound surfaces randomly on each rat for 4, 7 and 14 days. The recovery ratio was calculated as (Ai − Au)/Ai × 100%, where Ai is the initial area of the wound, and Au is the area of the unrecovered wound surface.The hair  staining; the skin specimens were immersed in Bouin’s solution for Masson’s trichrome staining. The fixed skin specimens were processed routinely and viewed under a light microscope to evaluate collagen formation and wound-healing processes.Skin samples from the wound area were dried and ground into powder. Ten percent pepsin was mixed with the skin sample powder and incubated at 4 °C overnight for protein extraction. A QuickZyme collagen assay kit was applied for collagen quantitation.w/v) blocking buffer to shake at room temperature for one hour. The PVDF membrane was placed reacted with primary antibody (TGF-β) overnight at 4 °C. After washing three times, added the secondary antibody  and put it at 4 °C for 2 h. After removing the PVDF membrane by washing buffer for 3 times, add Chemiluminescence Reagent Plus was added to capture images with a cold light and fluorescent image processing system.Eighty μg of each sample was mixed with 5X loading dye and heated at 95 °C for 5 min. The upper layer of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is 3.75% Stacking gel, and the lower layer is 12% separating gel. After loading the protein samples, the electrophoresis runs 120 mV electrophoresis and run for 2–3 h. After electrophoresis, the protein was transferred to the methanol activated polyvinylidene difluoride (PVDF) membrane at 100 volts, 4 °C for 2 h. Remove the PVDF membrane and immerse it in a 5%  was incubated for at least 2 h. Then, 100 μL of the Late Color Development Enzyme was added and incubated for 30 min. After adding 100 μL of TMP solution for 5 min, 100 μL stop solution was added to stop the reaction. The absorbance was measured by ELISA at a wavelength of 450 nm .The inflammatory index was detected using TNF-p < 0.05.The results are presented as the mean ± standard deviation. Statistical analysis was conducted using Student’s t-test or one-way analysis of variance followed by a post hoc Fisher’s least significant difference test for multiple comparisons. Levels of statistical significance were set at p < 0.05%) increase in cell viability was observed. L929 cultures treated with curcumin at a concentration of 20 μg/mL showed 84.7% inhibition in cell growth.L929 cells incubated with curcumin for 24 h at concentrations of 0.002, 0.02, 0.2, 2 and 20 μg/mL. The viability of the cultures was shown in L929 cells were planted in 12-well culture dishes and using a tip to draw a line in the middle of the culture dish. Cultures treated with various concentrations of curcumin were observed after 48 h incubation. Tyrosinase inhibitory activity was determined by a spectrophotometric method using 2 mg/mL L-DOPA as the substrate. As shown in In our previous study, the LR extract at a concentration of 3.12–6.25 μg/mL, accelerated the cell viability of L929 fibroblasts and demonstrated antimicrobial activity . CurcumiThe various gelatin nanofibers electrospun at 0.1 mL/h at 20 kV had filamentous and highly porous structure. The SEM images of nanofibers were observed by SEM A–D and aβ-actin antibodies and labeled with goat anti-mouse FITC antibodies  or gauze (control) at seventh day (a), 14th day (b) and 28th day (c) in STZ-induced diabetic rats. On the seventh day, the recovery rates of diabetic rats treated with GC, GC/L, GC/C and GC/L/C scaffold, Comfeel® or gauze (control) were 43 ± 10%, 53 ± 3%, 54 ± 12%, and 58 ± 7% (p < 0.05), 41 ± 11% and 40 ± 16%, respectively (®) or gauze (control) were 75 ± 3%, 76 ± 7% and 79 ± 1%, 76 ± 5%, 66 ± 5%, and 68 ± 6%, respectively. Treatments with GC, GC/L, GC/C, and GC/L/C bilayer scaffold showed a higher recovery rate than the control group. On the 28th day, the recovery rates of diabetic rats treated with GC, GC/L, GC/C, and GC/L/C scaffold, Comfeel® or gauze (control) were 83 ± 7%, 87 ± 3%, 86 ± 4%, and 87 ± 4%, 81 ± 3% and 85 ± 4% respectively. The recovery rates of all treatments are higher than 80%, with no visible difference between them.ectively B. Treatmp < 0.05), 41.59% (p < 0.05), and 47.58% (p < 0.05) in the wound on the seventh, 14th and 28th days.In β protein in wounds with various treatments. The ratio of TGF-β protein band areas are shown in β protein increased by 70.3% (p < 0.05%) and 40.15% (p < 0.05%) with GC/C and GC/L/C treatment, respectively, on seventh day. On the 14th day, the TGF-β protein increased by 189.7% (p < 0.05%), 59.3% (p < 0.05%) and 107.5% with GC/C, GC/L and GC/L/C treatment, respectively. The TGF-β protein contents show no obvious difference in wounds treated with commercial dressing and GC nanofibers at seventh and 14th days.p < 0.05) on the 14th day in α level in the specimen treated with GC/L/C was reduced by 6.45% (p < 0.05) on the 14th day, in the specimen treated with GC/L, it was reduced by 13.6% (p < 0.05) on the 28th day , it is appropriate to produce fibers with diameters thinner than 100 nm . The varp < 0.05%) on the seventh day , the wound area treated with GC/L/C nanofibers showed the highest recovery rate  promote TGF-β release in wounds [β release on the 14th day in the Western blotting analysis  (α level on the 14th day (p < 0.05%)  A, GC/L/C< 0.05%) B. These thy rats . It mighβ protein secretion (Curcumin and LR extracts performed different effects in wound treatments. According to the results of the Western blotting assay and histopathological studies, GC/L treatment obtained better performance in increasing collagen synthesis , and GC/ecretion . The treβ production, anti-inflammatory effect, and promoted the wound healing process. Based on their wound healing activity, the GC/L/C bilayer nanofibrous scaffold could be an excellent candidate for wound dressing.Gelatin nanofibers containing curcumin and LR extract were electrospun onto chitosan scaffolds to manufacture bilayer nanofibrous scaffolds with a healing effect. The benefits of the highly porous GC/L/C bilayer scaffold could reduce the frequency of dressing changes and minimize discomfort. In an STZ diabetic rat model, the GC/L/C bilayer scaffold out-performed the control groups in terms of collagen synthesis, TGF-


Thus, the composite shows excellent potential applications towards the areas such as aeronautics, mobile phones, radars, and military.MXenes, carbon nanotubes, and nanoparticles are attractive candidates for electromagnetic interference (EMI) shielding. The composites were prepared through a filtration technique and spray coating process. The functionalization of non-woven carbon fabric is an attractive strategy. The prepared composite was characterized using X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), scanning electron microscope (SEM), energy-dispersive X-ray spectroscopy (EDX), and Raman spectroscopy. The MXene-oxidized carbon nanotube-sodium dodecyl sulfate composite (MXCS) exhibited 50.5 dB (99.999%), and the whole nanoparticle-based composite blocked 99.99% of the electromagnetic radiation. The functionalization increased the shielding by 15.4%. The composite possessed good thermal stability, and the maximum electric conductivity achieved was 12.5 S·cm Electromagnetic interference (EMI) leads to inevitable interactions in electronic devices. Smaller and high-speed electronic systems are susceptible to issues related to EMI which can affect either adjacent electronic items or humans, thus potentially affecting the security of the nation ,2. In waVarious substances have been used for EMI shielding, such as MXenes, graphene (GN), graphene oxide, carbon nanotubes (CNTs), nanoparticles, polymers, fabrics, textiles, composites, and metals in various frequency ranges ,4,5,6. T(n+1)XnTx) are two dimensional (2D) material derived from a corresponding three dimensional (3D) MAX phase (M(n+1)AXn), where M is early transition elements , A includes group 13/14 elements, X represents carbon or nitrogen, and Tx is surface functional groups  [4HF2, and FeF3/HCl have also been practiced. During the etching, the weaker M–A bonds are eradicated while the strong M–C bond remains with newly formed functionalities [2CTx, Ti3CNTx, and Ti2CTx. In addition, MXenes are used to make hybrid composites such as TiO2–Ti3C2TX/graphene, Ti3C2TX–sodium alginate, Ti3C2Tx/PVA, cellulose nanofibers–Ti3C2Tx and Ti3C2TX/paraffin. MXenes exhibit maximum EMI shielding of 92 dB with 45 μm thickness. Thus, the loading amount of MXenes in different polymers matrices, the morphology of composite, and the thickness influence the EMI shielding of the MXene [MXenes  and sodium dodecyl sulfate (SDS), respectively. MC and FC indicated uncoated carbon fabric and functionalized carbon fabric, respectively. The parameters of EMI shielding, elementals analysis, morphology, structural analysis, electric conductivity, surface property, magnetic property, and thermal stability were investigated in detail.In this study, we developed a layered pliable composite with different surface morphology and magnetic composite. Each layer of the composite consists of a layered and magnetic domain for which we employed spray coating and filtration technique under gravity. The functionalization of carbon fabric intercalation of the MXene, CNTs, and magnetic nanoparticle dramatically changed the EMI shielding. The spray coating samples were denoted as MXCNTCx, where x  was the number of coating cycles, and Ni coated fabric was expressed as MXCNTNi25. The composite fabricated using the filtration method was denoted as MXCBCM, where M is the type of metal nanoparticle such as Ni, Co, Fe, Cu, and Fe4), polyacrylamide (PAM), anhydrous FeCl2, FeCl3, NiCl2, CuCl2, CoCl2, and cetyltrimethylammonium bromide (CTAB) were obtained from Sigma Aldrich . Wet laid nickel coated non-woven carbon fabric  was acquired from Clean & Science Co. Ltd., . Nitric acid (HNO3-70%) and hydrochloric acid (HCl-35%) were obtained from Samchun Chemical Co., Ltd. . Chitooligosaccharide (Mw 5000) was issued by biomedical polymer lab, Sunchon National University .Multiwall carbon nanotube (MWCNTs)  were purchased from Applied Carbon Technology Co. Ltd. . Carbon fiber  and polyethylene terephthalate (PET) binder  were collected from TORAY product, . Sodium dodecyl sulfate (SDS) (98%), lithium fluoride (LiF) , sodium borohydride  in ethanol medium at 200 rpm for 1 h in a nitrogen environment. The resultant mixture was dried at 80 °C for 12 h. Then, 3 g of 12 mm diameter disc was prepared by applying 27.6 MPa pressure for 5 min in a laboratory press. The resultant disc was treated at 1350 °C with a heating rate of 20 °C/minute in argon gas for 2 h, then cooled down to room temperature. The treated disc was again ball milled in ethanol medium at 300 rpm for 3 h in a nitrogen environment. The powder yield was then dried at 80 °C for 12 h, and the obtained product was directly used for MXene synthesis .3 were sonicated by using a mini ultrasonic cleaner  for 5 h at room temperature. The volume of the reaction mixture was doubled by adding deionized (DI) water and filtered. The acid solution and reacted CNT were separated by filtration. The resultant black color product known as CNTO was washed until reaching a neutral pH, then dried at 80 °C for 24 h. This CNTO was used for the coating process and synthesis decorated CNTO.1 g of MWCNT (CNT) and 90 mL of HNO3+, 100 ml of 0.1 M of Fe2+, and 0.5 g·L−1 of CNTO were stirred together at 30 °C for 30 min. A total of 10% of NH4OH was added dropwise until the pH of the solution reached 11–12, along with 2 mL of 0.2 M SDS. Then, the temperature of the reaction mixture was raised up to 80 °C and stirred for 2 h. The volume of the solution was reduced by half via evaporation and then cooled down to room temperature. The resultant product was washed until reaching a neutral pH in a vacuum filter, then dried at 105 °C for 12 h.100 mL of 0.1 M of Fe2+ of metal ion solution  and 0.5 g·L−1 of CNTO were stirred for 30 min. 100 mL of 0.2 M cold NaBH4 was added drop wisely in to the mixture, along with 0.2 g·L−1 of SDS. Fe, Ni, Co, and Cu decorated CNTO were successfully synthesized in a nitrogen environment. The product was filtered and washed by an ample amount of DI water. Then, the product was dried at 80 °C and 0.8 atm in a vacuum oven for 12 h. The obtained product was denoted as CM, where C is CNTO and M is nanoparticles. The corresponding decorated CNTOs were denoted as CFe, CNi, CCo, and CCu.100 mL of 0.1 M0.1 g of CNTO and 0.1 g of SDS were mixed in 100 mL of deionized water and sonicated for 3 h. Then, the dispersed mixture was refluxed at 120 °C for 12 h. The obtained well dispersed solution was then used for the spray coating process. In addition, 0.4 g of CNTO and 0.4 g of CTAB were added together in 100 mL of deionized water and sonicated for 5 h (CTAB–CNTO). This procedure was repeated using SDS and CNTO (SDS–CNTO). The decorated nanoparticle dispersed solution was prepared accordingly; equal amounts of CTAB and CM (0.1 g) were sonicated in 25 mL of deionized water for 30 min. The obtained dispersed solutions were directly used for the filtration process.3AlC2 and LiF were mixed together in 20 mL of 9 M HCl solution. This mixture was stirred at 35 °C for 24 h. The etched product was washed with DI water up to approximately pH 6 by centrifuging at 3500 rpm for 5 min, then Ti3C2Tx was dried in a vacuum oven for 12 h. Then, 0.1 g of MXene was sonicated in 10 mL of DI water in a nitrogen environment at about 15–17 °C for 2 h. The sonicated MXene solution was centrifuged at 3500 rpm for 30 min, and the supernatant was collected in a Teflon container stored at 5 °C for the coating process. The concentration of the MXene colloidal solution was 0.175 g·L−1. 100 mL of colloidal solution was filtered by using 0.45 μm of 47 mm of Nylon supported filter paper and dried at 80 °C (0.8 atm) until obtained constant weight. The concentration was calculated based on the weight differences.Equal amounts (1 g) of Ti− speed. The areal density of obtained fabric was 30 g·m−2 [We reported that 0.6 kg of carbon fiber, 0.15 kg of PET binder and 0.3 weight percent of dispersant (PAM) were dispersed in a sufficient amount of deionized (DI) water at 500 rpm for 10 min. The general wet laid method was used to produce the web. During the process, the drum dryer was used with a 140 °C surface temperature and 7 m·min30 g·m−2 .2 and a basic density of 30 g·m−2 was dipped in the chitooligosaccharide solution for 2 min and dried at 100 °C for 12 h. This fabric was directly used for the preparation of the filtration-based composite.0.2 g of chitooligosaccharide was dissolved in 100 mL of deionized water and stirred for 15 min. Then, carbon fabric with a dimension of 29 × 21 cm−2 of MC with a dimension of 29 × 21 cm2 was used, where one CNTO layer was sandwiched between two MXene layers. The thickness of the fabric was adjusted by a spraying and drying process. The drying was done using an air-drying gun while spraying was done by using an air compressor (Keyang compressors (KAC-25), Sichuan, China). This process was repeated for Ni coated fabric in order to compare the EMI shielding of carbon fabric. The coating on carbon fabric was denoted as MXCNTCx, while the Ni coated carbon fabric was denoted as MXCNTNiCx, where x was the number of coating cycles. MXCNTC30, MXCNTC25, MXCNTC10 and MXCNTNiC25 were successfully manufactured.A series of MXene–CNTO composites were prepared by the spray coating process, for which 30 g·m100 mL of MXene colloidal solution, 100 mL of CM dispersed solution and 70 mL of dispersed CNTO were alternatively filtered through FC under gravity and dried using an air gun. The resultant composite was denoted as MXCBCM. Further, MXCB was prepared by using 100 mL of MXene colloidal solution and 70 mL of CTAB–CNTO dispersed while 100 mL of MXene colloidal solution and 70 mL of SDS–CNTO dispersed mixture were used to prepare MXCS. Finally, MXCBCFeO, MXCBCFe, MXCBCNi, MXCBCCo, and MXCBCCu were successfully prepared.The structural features of the composites were investigated using a high-resolution Raman spectrophotometer . A Laser Flash Apparatus LFA457  was used to measure the density of the composites. A field emission scanning electron microscope  was used to examine the surface morphology of the composites. XPS with a 30–400 µm spot size at 100 W of Emax (Al anode)  was used to analyze the chemical environment and elemental percentage of the composites. A High-power X-ray Diffractometer D/max-2500V/PC,  with Cu(Kα) was used to record the X-ray diffraction patterns of the composites. The EMI shielding effectiveness (SE) of the composite in S-band (1–3 GHz) was recorded using an EMI shielding tent ASTM-D4935-10, ASTM International  at room temperature while X-band (8.2–12.4 GHz) EMI shielding was measured using a vector network analyzer  with a sample size of 22.16 mm × 10.16 mm. The four-probe method FPP-RS8, DASOL ENG  was used to measure the electric conductivity of the composites. The thermal stability of the composites was tested using a Thermal Analyzer DSC TMA Q400 . A Mitutoyo thickness 2046S dial gage  was used to measure the thickness of the composites. The surface property was measured using a Phoenix-300A contact angle meter . The magnetic property was measured using SQUID—VSM . The graphs were plotted using a Savitzky–Golay function .3O4, Fe, and Cu nanoparticles behaved in a similar manner, whereas the Ni nanoparticle encircled all of the carbon nanotube and was densely packed like a cauliflower. The self-assembling of the Co nanoparticles was completely different from that of another nanoparticle used. This is because it consumed CNTO as a template and formed a structure-like bacterial chain , indicating the presence of the graphite-like structure and the generation of a feeble G’ band at 2908.2 cm−1. The ID/IG values of the MC, MXCS, MXCBCCu, MXCBCCo, MXCBCNi, MXCBCFe, MXCBCFeO, and MXCB were 0.91, 1.03, 0.81, 0.87, 1.01, 0.86, 0.81, and 1.01, respectively, and the corresponding ID/IG’ values were 4.13, 2.33, 1.48, 2.12, 1.88, 1.96, 2.09, and 2.04, respectively , Ti2+(2p3/2), TiO2 (2p3/2), Ti2+ (2p1/2), and TiO2(2p1/2), respectively . In the fitting curve of MWCNT, the intense peak at 284.13 eV was raised due to the C–C bond of graphite, while the presence of the oxygen generated weaker peaks between 287–291 eV, and its corresponding oxygenic species such as C=O, C–O, and carbonate were triggered. The fitting peak at 285.86 eV confirmed the presence of the defects, which was further backed by the ID/IG ratio and amount of oxygen [The functionalities, surface elemental composition, structure, and bonding nature of the composites can be explained using XPS. The fitting curve of MXene was plotted using overlapping curves of the Gaussian–Lorentzian function and the overlapping curve of the composites was plotted using origin pro. The fitting curves of Ti2p, O1s, and C1s, the F1s of TInd bonds  [56,57,5ectively a. The bictively, b–d. The ctively, c. The C1ctively, d [33,35,f oxygen ,64. The s) of the fabric significantly changed due to the introduction of the MXene, CNTOs, and nanoparticles in the non-woven fabric matrix  EMI-SE was performed for all composite, while S band (1–3 GHz) EMI-SE was only measured for spray coated composites  and absorption (SEA) prevented 90% and 99.9%, respectively. MXCNTC30 and MXCNTC25 displayed 99.99% of maximum, minimum, and average shielding whereas the others showed 99.9% of shielding in the X-band region. In addition, spray coated composites SEA were in the range of 99–99.9% and 90% of reflection. The spray coated composite showed maximum shielding of 99.9%  showed a maximum of 33 dB, which is substantially lower than that of the MXene–CNTO–nanoparticle composite [−1 electrical conductivity [omposite . The polomposite ,13,74,75omposite . Most ofuctivity . The MXeuctivity . Hence, 2O3 crucible in a nitrogen environment with a heating rate of 10 °C·min−1. The composites exhibited an outstanding thermal stability over 100 °C. Most of the composites had a starting point of degradation above 140 °C, and the degradation beginning lay between 120–255 °C, where MXCNTC30 showed 120 °C. In addition, MXCB, MXCBCFeO, MXCBCFe, MXCBCNi, MXCBCCo, and MXCNTNiC25 prohibited degradation above 150 °C among mushroom gill like MXCBCCu decomposed at lower temperature of 127.5 °C, whereas the degradation temperatures of the other composites were below 149 °C and above 127.5 °C (except MXCNTC30 at 120 °C). The rapid mass changing percentage of composites occurred between 120–897.5 °C where spray coated composite hit the higher value (above 700 °C), while filtered gave rise below 653 °C  and differential thermal analysis (DTA) were used to investigate the thermal stabilities of the composites. TGA graphs were plotted in the temperature range of 30–1000 °C while DTA graphs were plotted in the range of 30–900 °C a–d. The w 653 °C . The temw 653 °C .The MC aw 653 °C . The funw 653 °C . The DTAw 653 °C b,d. Strow 653 °C . It was −3 while the thickness of the composites ranged between 0.266–0.408 mm. The ranges of Ms and Mr were 0.0099–0.86 emu·g−1 and 0.00022–0.44 emu·g−1, respectively. The MXCBCNi displayed a higher Ms of 0.86 emu·g−1 and a 0.44 emu·g−1 of Mr was shown by MXCBCFeO. The MXCCFeO exhibited a maximum Kp of 1.128 while FC was 0.022. The functionalized carbon fabric displayed a hydrophobic nature with a 134.18° contact angle, whereas MXCNTC25 displayed a contact angle of 131.3°. The electric conductivities of the composites varied between 2.65–12 S/cm, whereas the surface resistances were in the range of 2.08–13.98 Ω/sq. The composites showed good thermal stability and resisted complete thermal degradation above 120°. In addition, functionalization increased the thermal stability and prevented degradation up to 255.5°. The maximum EMI-SE shown by MXCS was 50.5 dB, and the EMI shielding range of the composite was 50.5–28.5 dB. The maximum SER and SEA were shown by MXCB and MXCS, respectively. The ranges of SSE and SSE/t were 149.37–401.93 dB cm3·g−1 and 4330.82–12,639.35 dB cm2·g−1, respectively, where FC showed maximums of both. Hence, the manufactured fabric composite presented high EMI shielding, magnetic behavior, low density, smaller thickness, and flexibility.The filtration-based nanocomposite and spray coated nanocomposite were successfully prepared. The effect of the functionalization of non-woven carbon fabric was analyzed as well. The composites were lightweight and thinner. The density of the composites ranged from 0.108 to 0.288 g·cm


A new ability to monitor volcanic HCl using miniature electrochemical sensors is here demonstrated. A “direct-exposure” Multi-Gas instrument contained SO2, H2S and HCl sensors, whose sensitivities, cross-sensitivities and response times were characterized by laboratory calibration. SRM analysis of the field data yields H2S/SO2 and HCl/SO2 molar ratios, finding H2S/SO2 = 0.02 (0.01–0.03), with distinct HCl/SO2 for the VOR, NEC and BN crater emissions of 0.41 (0.38–0.43), 0.58 (0.54–0.60) and 0.20 (0.17–0.33). A second Multi-Gas instrument provided CO2/SO2 and H2O/SO2 and enabled cross-comparison of SO2. The Multi-Gas-measured SO2-HCl-H2S-CO2-H2O compositions provide insights into volcanic outgassing. H2S/SO2 ratios indicate gas equilibration at slightly below magmatic temperatures, assuming that the magmatic redox state is preserved. Low SO2/HCl alongside low CO2/SO2 indicates a partially outgassed magma source. We highlight the potential for low-cost HCl sensing of H2S-poor HCl-rich volcanic emissions elsewhere. Further tests are needed for H2S-rich plumes and for long-term monitoring. Our study brings two new advances to volcano hazard monitoring: real-time in situ measurement of HCl and improved Multi-Gas SRM measurements of gas ratios.Volcanic gas emission measurements inform predictions of hazard and atmospheric impacts. For these measurements, Multi-Gas sensors provide low-cost in situ monitoring of gas composition but to date have lacked the ability to detect halogens. Here, two Multi-Gas instruments characterized passive outgassing emissions from Mt. Etna’s  three summit craters, Voragine (VOR), North-east Crater (NEC) and Bocca Nuova (BN) on 2 October 2013. Signal processing  approaches are used to analyse HThe online version of this article (doi:10.1007/s00445-017-1114-z) contains supplementary material, which is available to authorized users. Fischer  even when well-calibrated. This is due to the non-instantaneous response times of the sensors. This study demonstrates a new capacity for in situ HCl monitoring by low-cost electrochemical sensors, demonstrated at quiescently outgassing Mt. Etna volcano, Italy, with improved accuracy of H2S/SO2 and HCl/SO2 gas ratios achieved by signal processing methods.Monitoring of volcanic gas emissions provides insight into subsurface degassing and outgassing processes with the aim of improved prediction of volcanic activity and hazards  can be further characterized by in situ methods (e.g. gas/aerosol sampling onto filters) or remote sensing (e.g. Fourier transform infra-red spectroscopy (FTIR)), with data analysed to yield gas ratios relative to SO2. Combining these gas ratios with the SO2 gas flux thereby provides a comprehensive emission flux.To quantify volcanic emissions requires both the gas flux and emission composition to be characterized. Volcanic SO2 ratios in the summit emissions and in sustained grounding downwind plume. However, deployment requires hazardous visits to the volcano summit followed by further costs in time-intensive laboratory analysis. Also, infrequent campaign-based monitoring might miss some composition changes. Remote sensing of HCl (e.g. by active FTIR with IR source) at individual summit craters is similarly typically limited to occasional field campaigns , including long-term installations with data telemetry, e.g. at Italian volcanoes Mt. Etna and Stromboli, Aiuppa et al.  in the volcanic emission is of strong interest. HCl outgasses at shallower depths than SO2 and CO2, thus HCl/SO2 can be an informative indicator of magma state and potentially might be used to improve monitoring and prediction of volcanic eruption hazards: Studies report both increasing and decreasing trends in HCl/SO2 related to volcanic activity, for example S/Cl mass ratios rising from 5 to 25 prior to an eruption event at Asama volcano , Aiuppa  implicitly assumes instantaneous or identical sensor response times. Through forward modelling of sensor response, Roberts et al. (2 on the H2S measurement) in data post-processing. At Mijake-jima Volcano, this led to 30–50% errors in standard analysis of Multi-Gas H2S/SO2 measured at the crater-rim, but not in the sustained (and more slowly fluctuating) downwind plume. This systematic error is independent of any calibration errors and is particularly large at low H2S/SO2. As consequence, Multi-Gas H2S/SO2 ratios are rarely reported from H2S-poor volcanoes.A source of error in gas ratios from Multi-Gas arises from differing sensor response times, which depend on both sensor and gas properties. “Standard” analysis of Multi-Gas data  gas pulse events, Roberts et al. . Here, s2 gas abundance, [SO2(t)] in ppmv, is determined by Eq. SO2-AE(t) is the signal of the SO2-AE sensor (with any baseline removed), whose sensitivity, sensSO2, is determined by calibration. Typically, sensSO2 is in nA/ppmv. The sensor SignalSO2-AE(t) is in nA, which is converted to a voltage and recorded by the Multi-Gas.Standard analysis of Multi-Gas data implicitly assumes an instantaneous sensor response. The SO2S sensors such as H2S-AE exhibit cross-sensitivity to SO2 as well as sensitivity to H2S. This interference is subtracted from the sensor signal in data post-processing, following Eq. H2S is the sensitivity to H2S and xsensSO2 is the cross-sensitivity to SO2, [SO2(t)], which is provided by 1.Multi-Gas H2S(t)] versus [SO2(t)] determines the gas ratio. However, this ratio is prone to biases if sensors have non-identical response times, see “Introduction”. A similar analysis of HCl/SO2 by equivalent equations to Eqs. 2S interference from the HCl-A1 signal) could also incur similar errors. This study attempts to reduce such biases in Multi-Gas gas ratios by modelling the sensor response.Linear regression on a scatter plot of  to [Xfinal]. The time constant parameter, τ, is the time to reach 1/e of the signal change. The response time to reach 90% of the signal change, T90, is related to τ by T90 = Loge[10] τ. Typically, T90 and τ are independent of the gas abundance change, but see “Sensor characterization: sensitivity, cross-sensitivities, T90 and SRM” for further discussion.The rise/decay response curve of an electrochemical sensor responding to a step-change in gas abundance is broadly exponential, i.e. follows Eq. t-Δt as Eq. F = Exp[−Δt/τ] and both adding and subtracting [Xfinal]∙sens from the right-hand-side yields Eq. Equation t), as a function of [Xfinal] and sens and the previously recorded signal, Signal(t-Δt).Finally, substituting Eq. F (0 ≤ F ≤ 1) describes the amount of decay between successive samples and is related to the time-constant T90 or τ, in seconds, by Eq. t is the sampling period in seconds.Parameter t-2Δt) terms. Mathematically, this is equivalent to the weighted addition of two first-order SRMs (labelled A and B), Eqs. W is a weighting factor (between 0 and 1).“Sensor characterization: sensitivity, cross-sensitivities, T90 and SRM” section finds the signal is slightly over-damped compared to Eq. X(t)] is an interference rather than target gas of the sensor, the sensitivity, sens, is replaced by the cross-sensitivity, xsens, in Eqs. If , Eq. Firstly, for H2inv(t)] is used with (forward modelled) SRM2 to simulate the interference from SO2 on H2S-AE, Eqs. (2) [SO2S abundance [H2S(t)est] is estimated by the product of [SO2(t)inv] with a range of possible RH2S/SO2, Eq. (3) The H2Sest(t)] is used with SRM3 to simulate the sensitivity signal of H2S on H2S-AE, Eqs. (4)  with RH2S/SO2 provided above. (2) The interference of H2S on the HCl-A1 signal is simulated by SRM4, Eqs. Secondly for HCl/SOHCl-A1 to yield the sensitivity signal of HCl-A1, Eq. slow(t)], Eq. (3) This interference signal is subtracted from the observed Signal2 time series, [SO2slow(t)], is simulated by sensor response modelling by applying the time response properties of SRM5 to [SO2inv(t)], Eqs. 2] from standard analysis as input given T90 for HCl-A1 > > T90 for SO2-AE).(4) A comparable slow SOslow(t)] vs [SO2slow(t)] with linear regression is used to determine the gas ratio RHCl/SO2. Further illustration is given in Fig. (5) Finally, a scatter plot of  and [SO2slow] outputs that can be directly compared in a scatter plot, Fig. 2S/SO2 ratio of RH2S/SO2 = 0.02 (range 0.01–0.03) and H2S-A1 cross-sensitivity to H2S of 210% (range 170–250%), following “Sensor characterization: sensitivity, cross-sensitivities, T90 and SRM” and “Analysis of H2S/SO2 in Mt. Etna plume”.The SRM analysis approach outlined in “Development of SRM data analysis approach for H2 for VOR, NEC and BN. [HClslow] and [SO2slow] are well-correlated  and 0.58 (0.54–0.60), respectively (range reflects possible H2S/SO2 of 0.01–0.03 and cross-sensitivity of 170–250%). These can be correspondingly written as SO2/HCl molar ratios of 2.45 (2.33–2.63) and 1.72 (1.66–1.85) for VOR and NEC, respectively. The (more dilute) BN plume exhibited much poorer correlation in [HClslow] and [SO2slow]. BN HCl/SO2 is thus more uncertain, but estimated as 0.20 (0.17–0.33), i.e. SO2/HCl of 5.0 (3.0–5.8). Time series of [SO2] and [HCl] , [SO2slow], [HClslow] (and with interference), 2slow than SO2 . Response over longer timescales than simulated here  might additionally contribute to the observed rising HCl signal , can be used to estimate the temperature at which the H2S-SO2 magmatic gas equilibrium, R1, is quenched at Mt. Etna. Our calculation follows Aiuppa et al. . Assuming a H2O content of 80% , i.e. fH2O of 0.8 (1 bar) pressure, rearrangement of Eq. 2 which is at Ni-NiO buffer +0.35, . Regardless of associated uncertainties, such a broad agreement in temperatures supports the notion that, to a first order, measured H2S/SO2 ratios by Multi-Gas correctly capture magmatic conditions, hence likely to give insight to deep seated processes. Whilst Aiuppa et al. .Our Mt. Etna SOa et al.  used a s2 at the three craters in good agreement to recent observations  gas depletion of magma has previously been suggested at Mt. Etna, e.g. Burton et al.  with Mt. Etna SO2 gas flux (1800–2100 t day−1 during the campaign), monitored by the INGV Ultraviolet scanning spectrometer FLAME network , Ambrym (Vanuatu) and Villarrica (Chile). Further tests are being undertaken for HCl sensing in more H2S-rich plumes that should use sensor-specific (rather than batch) cross-sensitivities to remove the H2S interference. Use of filters to remove H2S but not HCl will also be considered but is challenging. Integration of HCl sensors into permanent Multi-Gas installations for continuous emissions monitoring would be of interest to trace in real-time changes in Cl/S that can be associated with changing volcanic activity . However, the feasibility of continuous and long-term HCl monitoring by Multi-Gas requires further in-field tests of HCl-A1 sensor performance, stability and response. There is furthermore a general need to test the (wide-spread) use of laboratory sensor characterisations in the analysis of Multi-Gas field measurements at volcanoes.The low cost of the HCl sensors (~100 euros) can facilitate wider application of the technology beyond Mt. Etna to other H2S/SO2 and HCl/SO2 gas ratios by modelling the sensor response. The techniques are demonstrated in a field campaign at Mt. Etna on 2 October 2013 when two Multi-Gas instruments operating at 0.1–0.5 Hz were co-deployed to consecutively sample emissions from the three summit craters, Voragine (VOR), North-East Crater (NEC) and Bocca Nuova (BN), respectively.Measurements of the composition of volcanic emissions help researchers monitor and predict hazardous volcanic eruptions and assess downwind plume impacts. This study introduces real-time in situ HCl detection by Multi-Gas electrochemical sensors, with improvements made to analytical accuracy of Multi-Gas-measured HDirect, contains electrochemical sensors for HCl, SO2 and H2S, which were directly exposed to the atmosphere. This removes the need for a pump, enabling a lighter and lower power instrument (hence easier in-field deployment with longer battery lifetime). The Multi-Gas SO2 sensor has negligible interferences, but laboratory calibrations show that the H2S sensor has a 14 ± 0.5% cross-sensitivity to SO2 and the HCl sensor has a 170–250% cross-sensitivity to H2S. The HCl sensor also exhibits a ~50% cross-sensitivity to HBr, but this can be neglected given HCl > > HBr in the volcanic emission. Laboratory characterization of the sensor response times found T90 = 12 s for SO2-AE, T90 = 20–50 s for H2S-AE and T90 = 100–250 s for HCl-A1. The combined effects of sensor response times, sensitivities and cross-sensitivities in the Multi-Gas field-data were deconvolved by signal processing algorithms to yield HCl/SO2 and H2S/SO2 molar gas ratios, finding H2S/SO2 = 0.02 (0.01–0.03) and HCl/SO2 = 0.44 (0.43–0.45), 0.61 (0.60–0.61) and 0.29 (0.23–0.34) for VOR, NEC and BN, respectively. These gas ratios agree with recent time-averaged sampling . Future work will evaluate sensor performance in H2S-rich plumes and over longer timescales. We emphasize that accurate determination of Multi-Gas gas ratios requires inclusion of the effects of differing sensor response times when data is analysed (achieved here using Sensor Response Modelling). We encourage further application of signal processing/systems engineering in this area and also emphasize the need for field validation of laboratory-derived Multi-Gas sensor properties.This study demonstrates Multi-Gas sensing of HCl emissions at Mt. Etna craters. The low-cost (~100 euros) of the HCl sensors can facilitate application to HESM 1(DOCX 1653 kb)


Obesity has been identified as a major risk factor for a large number of chronic diseases. Understanding factors related to adolescent obesity is critical for prevention of chronic diseases. The associations between sleep duration and obesity among adolescents in the existing literature are controversial. Our study was designed to determine the prevalence of short sleep duration, and assess the association of sleep duration and obesity, among middle and high school students in Zhejiang, China.18,403 Students in 442 schools were recruited and surveyed using an anonymous, self-administered questionnaires. Weighted multivariable logistic regression models were used for data analyses.P < 0.0001). The figures for middle school, academic high school, and vocational high school were 59.0, 82.4 and 59.7%, respectively (P < 0.0001). As compared with girls who sleep 8 h per day (reference), the odds ratios (95% CI) of obesity for girls who sleep < 7 h, 7 h, 9 h and ≥ 10 h were 1.97 (1.15–3.38), 1.90 (1.18–3.04), 1.38 (0.86–2.20) and 2.12 (1.22–3.67) respectively, after adjustment for socio-demographic status, lifestyle factors, and mental health. The corresponding figures among boys were 1.45 (0.97–2.16), 1.13 (0.81–1.57), 1.25 (0.89–1.74), and 1.12 (0.81–1.54), respectively.The mean (SD) age of the students was 15.9 (1.8) years. 49.7% of students were girls. The mean (SD) height and weight were 166.2 (8.5) cm and 54.6 (11.1) kg, respectively. The overall prevalence of obesity and overweight were 3.4% (95% CI: 3.0–3.8) and 7.8% (95% CI: 7.4–8.3), respectively. The overall prevalence of short sleep duration among students was 66.0% (95% CI: 63.8–68.1), higher among girls than boys (69.8% vs. 62.1%)  contains supplementary material, which is available to authorized users. Worldwide, the prevalence of overweight and obesity have steadily increased over the last several decades, with the number of obese individuals estimated to reach 711.4 million in 2015, of which 107.7 million were children [In China, the prevalence of overweight and obesity among 7–18-year students increased from 1.1 to 9.6% and from 0.1 to 4.9%, respectively, between 1985 and 2010, and in 2010 the prevalence of obesity varied from 0.4 to 21.7% across different provinces in China . ZhejianOverweight and obesity are well-documented risk factors for a large number of chronic diseases, including diabetes, cancers, strokes, and coronary heart disease –7. HenceDetails of study design, sample, and participants have been reported previously . BrieflyHeight was assessed by the question “How tall are you without your shoe on? □□□.□ cm  was calculated as the ratio of self-reported weight (kg) to the square of self-reported height (m). Standard cut-offs for adolescent overweight and obesity Table , establiSleep duration was assessed through the question: “During the past 30 days, on average, how long did you sleep every day (including daytime rest)? □□ Hours □□ Minutes” Sleep duration (hours) was calculated as: hours + (minutes/60). Short sleep duration was defined as sleep duration < 9 h per day for children aged 6–12 years or < 8 h per day for teens aged ≥13 years, as recommended by American Academy of Sleep Medicine (AASM) .Some covariate question and response options  have been reported previously . Breakfa2, and comparison of weighted mean was performed using design based linear regression.All analyses were performed using SAS software V.9.3. A weighting factor was applied to each student record to adjust for non-response and for the varying probabilities of selection. The weight used for estimation in this survey is given by: W = W1 × W2 × f1 × f2. W1 = the inverse of the probability of selecting the county. W2 = the inverse of the probability of selecting the classroom within the county. f1 = a student-level nonresponse adjustment factor calculated by class. f2 = a post-stratification adjustment factor calculated by grade . ContinuP-values < 0.05 were considered statistically significant.To examine the associations of sleep duration and obesity, weighted multivariable logistic regression analyses were performed using the PROC SURVEYLOGISTC procedure. In multivariable logistic regression, sleep duration (continuous) was transformed into a categorical variable comprising five groups: “< 7 h”, “7 h”, “8 h”, “9 h”, and “≥10 h”. Students who slept 8 h per day were selected as reference group. Three logistic regression models were fitted to assess adjusted odds ratios (ORs) and their 95% CI. In model 1, ORs were adjusted for age group , sex (boys and girls), area , types of school , paternal and maternal education level . In model 2, ORs were additionally adjusted for cigarette use (current smoker and non-smoker), alcohol use (current drinker and non-drinker), breakfast consumption (daily and nondaily), fruit consumption (daily and nondaily), vegetable consumption (≥2 times/day and < 2 times/day), milk consumption (≥3 days/week and < 3 days/week), carbonated drinks consumption (≥4 times/week and < 4 times/week), physical activity (daily and nondaily), and screen-time (≥2 h/day and < 2 h/day). In model 3, ORs were additionally adjusted for loneliness . All statistical tests were two tailed, and P < 0.0001), shorter screen-time (24.2% vs.16.6%) (P < 0.0001), less frequent consumption of breakfast (74.0% vs.69.4%) (P < 0.0001) and fruits (74.7% vs. 69.8%) (P < 0.0001), and more frequent feelings of loneliness (7.4% vs. 13.2%) (P < 0.0001). Mean student height and weight were 166.2 ± 8.5 cm and 54.6 ± 11.1 kg, respectively. Mean sleep duration was 8.3 ± 1.6 h per day. Comparison of included and excluded participants showed no statistically significant differences for the majority of variables examined . The corresponding figures for overweight were 10.4 and 5.3%, respectively (P < 0.0001). The prevalence of overweight in urban areas was higher than in rural areas (9.3% vs. 7.1%). The prevalence of obesity among middle school, academic high school, and vocational high school students were 4.1, 2.2, and 3.6%, respectively, with corresponding figures for overweight of 7.4, 8.2, and 8.3%, respectively.The overall prevalence of obesity and overweight were 3.4% (95% CI: 3.0–3.8) and 7.8% (95% CI: 7.4–8.3), respectively (Table P < 0.0001), and marginally higher in urban, than rural, areas (69.5% vs. 64.2%) (P = 0.05) (Table P < .0001). The prevalence of short sleep duration increased with the increasing age (P < .0001).The overall prevalence of short sleep duration was 66.0% (95% CI: 63.8–68.1), significantly higher among girls than boys (69.8% vs. 62.1%)  for the development of obesity among adolescents , consistHowever, contrary to another cross-sectional study showing short sleep duration was associated with obesity only in adolescent boys , the posMeanwhile, our study found that students with a sleep duration ≥10 h per day had 39% greater odds of obesity compared with those with sleep duration of 8 h per day. The possible mechanisms underlying the association of prolonged sleep duration with obesity among adolescents remain unclear. One possible explanation is that prolonged sleep duration reflects compensation for poor-quality and/or interrupted sleep, which may be more common among overweight and obese person. A study showed that, compared with normal-weight children, overweight and obese children had less slow-wave sleep (SWS), which plays a major role in body restorative processes and energy metabolism .In the present study, a U-shaped association was found between sleep duration and risk of obesity only among girls, and not among boys. This sex-difference may reflect sex-differences in the physiology of puberty, particularly in body composition. For example, during puberty, boys rapidly increase muscle mass and reduce body fat mass due to increase of testosterone and growth hormone, while girls increase fat mass due to increase of estradiol .Our study had several strengths. First, it included a large and provincially representative sample. Second, a wide range of covariates including socio-demographic status, lifestyle factors  and mental health (loneliness) were adjusted for in analyses examining the associations of sleep duration and obesity. However, our study also had several limitations. First, the cross-sectional design limits causal inference. Second, all data was self-reported by participants and not objectively measured, which might increase the risk of information bias, including through underestimation of BMI. Third, although analyses were adjusted for most established potential confounding factors, residual confounding might still exist.In summary, our study found a U-shaped relationship of sleep duration and obesity among girls, but not among boys, and 8 h sleep was associated with the lowest risk of obesity among middle and high school students.Additional file 1:Table S1. Comparison of characteristics between included and excluded participants. (DOCX 32 kb)


Premna odorata Blanco (Lamiaceae) organs, bark, wood, young stems, flowers, and fruits dereplicated 20, 20, 10, 20, and 20 compounds, respectively, using LC–HRESIMS. The identified metabolites (1–34) belonged to different chemical classes, including iridoids, flavones, phenyl ethanoids, and lignans. A phytochemical investigation of P. odorata bark afforded one new tetrahydrofurofuran lignan, 4β-hydroxyasarinin 35, along with fourteen known compounds. The structure of the new compound was confirmed using extensive 1D and 2D NMR, and HRESIMS analyses. A cytotoxic investigation of compounds 35–38 against the HL-60, HT-29, and MCF-7 cancer cell lines, using the MTT assay showed that compound 35 had cytotoxic effects against HL-60 and MCF-7 with IC50 values of 2.7 and 4.2 µg/mL, respectively. A pharmacophore map of compounds 35 showed two hydrogen bond acceptor (HBA) aligning the phenoxy oxygen atoms of benzodioxole moieties, two aromatic ring features vectored on the two phenyl rings, one hydrogen bond donor (HBD) feature aligning the central hydroxyl group and thirteen exclusion spheres which limit the boundaries of sterically inaccessible regions of the target’s active site.Metabolomic profiling of different  Cancer is one of the most threatening diseases worldwide that affect human health and quality of life ,2,3. CheP. odorata, popularly known as ‘Alagaw’ is a small tree native to the tropical regions + .v/v) CO2 in an RPMI 1640 medium at 37 °C, supplemented with 10% (v/v) 1%(w/v) l-glutamine, and 0.4% (w/v) antibiotics (50 U/mL penicillin and 50 mg/mL streptomycin). The HT-29 cells (human colon adenocarcinoma cell) were cultured at 37 °C, 5% (v/v) CO2 in Dulbecco’s Modified Eagle Medium (DMEM) with high glucose (4.5 g/L), supplemented with 10% (v/v) fetal bovine serum (FBS), 1% (w/v) l-glutamine, and 0.4% (w/v) antibiotics . MCF-7 cells (Human breast adenocarcinoma cell) were cultured at 37 °C, 5% (v/v) CO2 in an RPMI1640 medium, supplemented with 10% (v/v) fetal bovine serum (FBS), 1% (w/v) L-glutamine, 1% sodium pyruvate, and 0.4% (w/v) antibiotics .HL-60 cells (human promyelocytic cell) were grown in 5%  at 37 °C with 5% CO2. After treatment, the cells were immediately incubated for 4 h at 37 °C. The cells were lysed in 100 μL of lysis buffer  for 10 min at room temperature and 300 rpm/min. The enzymatic reduction of MTT to formazan crystals that dissolved in DMSO was quantified by a Varioskan LUX multimode reader  at 570 nm. Dose-response curves were generated and the IC50 values were defined as the concentration of compound required to inhibit cell proliferation by 50%. Doxirubicin was used as a positive control.The cytotoxic properties of compounds ll lines . Cell prn = 3). One-way analysis of variance (ANOVA) followed by Dunnett’s test was applied. Graph Pad Prism 5 was used for statistical calculations . Results were regarded as significant at p ˂ 0.05.Data were expressed as mean ± S.E.M , hydrogen bond acceptor (HBA), hydrophobic (Hbic), and ring aromatic (Ring Arom). The number of features was allowed to vary from 1–2 for HBD, 1–3 for HBA, 0–3 for Hbic, and 0–3 for Ring Arom. HipHop uses inactive compounds to create excluded volumes that resemble the sterically inaccessible regions of the binding pocket. HipHop was configured to allow a maximum of 20 exclusion spheres to be added to the generated pharmacophoric hypotheses. The user defines the degree of molecules mapping completely or partially to the hypothesis, via the Principal and MaxOmitFeat parameters of the software. The conformational space of each compound was explored applying the ‘‘best conformer generation’’ option. The default values were applied for other parameters.ChemBioDraw Ultra 12.0  was used to sketch the 2D chemical structures of compounds ructures ,30. The P. odorata different organs  which derplicated different chemical metabolomic compounds belonging to different classes, including iridoids, flavones, phenyl ethanoids, and lignans. A phytochemical investigation of the P. odorata bark afforded one new tetrahydrofurofuran lignan, 4β-hydroxyasarinin 35, along with fourteen known compounds. Investigating the cytotoxic activities of compounds 35–38 against the HL-60, HT-29, and MCF-7 cancer cell lines, using the MTT assay showed that only compound 35 had cytotoxic effects against HL-60 and the MCF-7 cell lines, with IC50 values of 2.7 and 4.2 mg/mL, respectively, using a concentration 10 µL MTT (5.0 mg/mL). The HipHop module of 4β-hydroxyasarinin showed two HBAs aligning with the phenoxy oxygen atoms of benzodioxole moieties, two Ring Arom features vectored on the two phenyl rings, one HBD feature aligning the central hydroxyl group, and thirteen exclusion spheres limiting the boundaries of the sterically inaccessible regions of the target’s active site.This study dealt with LC–HRESIMS metabolomic profiling of


To assess the efficacy and safety of stereotactic body radiation therapy (SBRT) in the management of adrenal gland metastases (AGMs) from lung cancer. Moreover, it is the first two-institutional experience and the largest-to-date study to report the safety and efficacy of SBRT for inoperable AGM from lung cancer.In this retrospective study, 30 patients  with 32 AGMs were treated by SBRT from October 2006 to June 2016. Of these, 11 patients were treated with the intent of controlling all known metastatic sites and 19 for palliation of bulky AGMs. Follow-up was performed every 3 months for evaluations of efficacy and safety. Factors predictive of overall survival (OS) and local control (LC) were identified with univariate and then multivariate analysis.10, α/β = 10) ≥85.5Gy (P = 0.007) and gross tumor volume < 30 ml (P = 0.003) correlated with LC only after univariate analysis.Median follow-up time was 10.7 months (2.9–96.4 months). The complete response (CR), partial response (PR), stable disease (SD) and progressive disease (PD) rates were 23.3%, 33.3%, 33.3% and 16.7% respectively. The 6-month, 1, and 2-year LC rates were 96.9%, 96.9%, and 72.7% respectively. Additionally, the 6-month, 1, and 2-year OS rates were 85.6%, 58.1%, and 54.0% respectively while 6-month, 1, and 2-year progression free survival (PFS) rates were 39.5%, 24.6%, and 8.2%, respectively. All the patients with cancer-induced pain  had significant alleviations after SBRT. The treatment was well tolerated with only 1 patient reporting grade-3 diarrhoea. No predictors of OS and LC were found after multivariate analysis, while it was demonstrated that biologic equivalent dose (BEDSBRT is a safe and effective treatment modality in the management of AGMs from lung cancer with high LC rates and acceptable toxicity. The adrenal gland is one of the most frequently involved sites of metastases in lung cancer. Adrenal gland metastases (AGMs) from lung cancer are usually characterized by insidious onset and patients only occasionally have back or abdominal pain due to a large or rapidly growing tumor. Rarely, in the case of bilateral adrenal involvement, patients may develop adrenal insufficiency, which may result in attenuation of quality of life (QOL) and often worse survival . A new rAn obvious increase in tumor burden may prompt systemic aggressive treatment. However, for those with AGMs who may have already received first-line chemotherapy and have disease progressions thereafter, alternative regimens might not provide survival benefits, as well as probably contribute to high incidences of toxicity. Furthermore, though the adverse effects of targeted therapy for patients with EGFR mutation are mild, drug resistance remains the biggest problem. Surgery is considered as a curative option for some isolated metastases, but the adrenalectomy sometimes may result in adrenal insufficiency and perioperative complications . PercutaRecently, due to the advantages of stereotactic body radiation therapy (SBRT), including precise delivery and abrupt dose fall-off outside targets and high local dose conformation, it has been commonly used in selected patients with and without metastatic lesions, with high local control (LC) rates and acceptable toxicity, especially when surgery is declined or contraindicated –7. High SBRT was delivered for 30 patients with AGMs from lung cancer  from October 2006 to June 2016 at Tianjin Medical University Cancer Institute and Hospital, and Changhai Hospital of the Second Military Medical University. All patients were examined by an oncologist before the study. Patients with AGMs diagnosed by biopsy or at least imaging examinations. Besides, those with a Karnofsky performance score ≥ 70, a life expectancy of > 3 months and who declined or were not amenable to surgical resections due to comorbidities were included in the study. Informed consent was obtained from all patients prior to the enrolment and the study was conducted according to the Declaration of Helsinki. The review and use of medical records of patients for analysis were approved by the ethical committee.23 < 2/3 volume; renal cortex (right and left), V17.5 < 200 mL; duodenum, D5mL < 18 Gy, D10mL < 12.5 Gy and Dmax < 32 Gy; liver V21 < 700 mL; spinal cord, D0.35mL < 23 Gy, D1.2mL < 14.5 Gy and Dmax < 30 Gy; medulla spinal cord (5-6 mm above and below level treated per Ryu), V23 < 10% of subvolume and Dmax < 30 Gy; stomach, D10mL < 18 Gy and Dmax < 32 Gy; duodenum, D5mL < 18 Gy, D10mL < 12.5 Gy and Dmax < 32 Gy.Of the 30 patients, 11 were treated with the intent of controlling all known metastatic lesions, and 19 underwent SBRT for palliation of bulky adrenal metastases. SBRT was delivered by CyberKnife . All patients were immobilized in supine position with arms by their sides using thermoplastic body mask. Computed tomography (CT) scan was performed with a slice thickness of 1.5 mm, with the scan range of at least 10 cm below and above the tumor. According to the adrenal gland motions during radiotherapy reported by Wang et al. , for patPatients were re-evaluated every 3 months after radiotherapy with contrast-enhanced CT scans, PET-CT scans or contrast-enhanced MRI. Adverse events, amelioration of symptoms and sequential treatment were recorded. Acute and late toxicity was scored according to the Common Terminology Criteria for Adverse Events (CTCAE) version 4.0 . Tumor rP values < 0.05 were considered statistically significant.LC, OS and PFS curves were calculated by the Kaplan-Meier method. Potential factors associated with LC, OS and PFS were identified with univariate log-rank comparisons and then multivariate proportional hazards regression model. Statistical analyses were performed using SPSS 18.0 . Two-sided n = 7, 23.3%), adenocarcinoma , small cell lung cancer , adenosquamous carcinoma , and unknown type . Among all the patients, 15 (50.0%) had other site metastases in addition to AGMs. Eighteen patients (60.0%) had received chemotherapy after SBRT, and 9 (30.0%) had more than one lesions treated. Furthermore, 16 patients (53.3%) were symptom-free, while 14 (46.7%) suffered from abdominal pain or lumbar back pain.Patient characteristics were shown in Table 10) of 85.5 Gy (range: 44.8–112.5 Gy). The median prescription dose for control and palliation intent was 45Gy (range: 42-50Gy) and 42Gy (range: 32-50Gy), respectively. The median GTV was 23.9 ml (range: 2.5–115.3 ml). Treatment planning parameters were demonstrated in Table The median dose was 44.4 Gy range 32–50 Gy), which was delivered in 5 fractions (3–8 fractions), with a corresponding biologic effective dose , 31.2% (n = 10), and 31.2% (n = 10) respectively, while 5 (15.7%) foci showed disease progression (PD).All patients were followed up until death or June 2018. By the last follow-up, 19 patients (63.3%) died while 11 were alive. One patient died of heart disease, whereas 18 patients died of distant metastasis. Hence, local failure and radiation-induced toxicity did not contribute to the death. The median follow-up was 10.7 months (2.9–96.4 months), and median OS and PFS was 24.4 months (95% CI: 4.4–44.4 months) and 3.5 months (95% CI: 1.3–5.7), respectively. The 6-month, 1-year, and 2-year LC rates were 96.9%, 96.9%, and 72.7%, respectively Fig. . FurtherNineteen of thirty patients (21/32 targets) were treated for palliation purpose. The 6-month, 1, and 2-year LC rates of these targets were 95.2%, 95.2%, and 47.6% respectively, whereas the 6-month, 1, and 2-year OS rates of these patients were 76.0%, 54.9%, and 54.9% respectively. Elven of thirty patients (11/32 targets) were treated for radical purpose. The 6-month, 1, and 2-year LC rates of these targets were all 100%, whereas the 6-month, 1, and 2-year OS rates of these patients were 100%, 63.6%, and 54.5% respectively.P = 0.003, Table 10 ≥ 85.5Gy was predictive of better LC , all of them (100.0%) had alleviations of symptoms after SBRT.The treatment was well-tolerated. No grade 4 or higher acute toxicity was observed and only 1 patient had grade 3 diarrhea. Fatigue, poor appetite, nausea, thrombocytopenia, leucopenia, vomiting and pain were the most common side effects during treatment. There were no grade 3 or higher late gastrointestinal toxicities. Furthermore, even grade 1 or 2 late gastrointestinal toxicity was not recorded during follow-up.. All the adverse effects were temporary, reversible and ameliorated by medication, which did not prevent patients from completing the treatment Table . AdditioThe study investigated the efficacy of SBRT for AGMs from lung cancer. Overall, SBRT may provide high LC rates, survival benefits with the median OS of 24.4 months and symptom relief without serious toxicities. Additionally, no grade 4 or above acute toxicities were reported. Therefore, it may be implied that SBRT was a promising modality in the management of AGMs from lung cancer, especially for a palliative purpose to relieve various symptoms caused by the metastases.Surgery was considered as a standard curative-intent strategy for isolated adrenal metastasis from non-small cell lung cancer (NSCLC). In the analysis of 13 retrospective studies, the median OS of patients with NSCLC and isolated adrenal metastasis treated with segmentectomy, lobectomy or pneumonectomy, and adrenalectomy was 18 months, and the 1-, 2- and 5-year survival rates were 66.5, 40.5 and 28.2%, respectively . Another10 compared with those of SBRT. The findings in current study were consistent with previous studies. The 0.5, 1, and 2-year LC rates were 96.9%, 96.9% and 72.7% respectively. The 0.5, 1, and 2-year OS rates were 85.6%, 58.1%, and 54.0% respectively, and similarly the corresponding PFS rates were 39.5%, 24.6%, and 8.2% respectively. Moreover, it was the first multicenter study to report the safety and efficacy of SBRT for inoperable AGMs from lung cancer.Recently, SBRT has been an alternative for patients with AGMs from different types of tumor –24. In a10 < 60Gy was predictive of lower 1-year LC rates [10 > 85Gy correlated with better LC [10 ≥ 85.5Gy had higher 1- and 2-year LC rates compared with those with BED10 < 85.5Gy (P = 0.007).A dose-response relationship has been observed in several studies about SBRT for AGMs. Previous studies demonstrated that BEDLC rates , 29, 30 etter LC , 31, 32.In our study, no late gastrointestinal or renal toxicities were found, which was consistent with the previous studies –28. Only3 (P = 0.05) and PTV less than 12 cm3 (P = 0.005) were associated with improved LC [P = 0.003) than those who had bigger ones (GTV ≥ 30 ml). Notably, it must be noted that high LC rates cannot be achieved with the compromise of protections of organs at risk. Therefore, high radiation doses may not be prescribed in the case of large tumors due to dose constraints of surrounding normal tissues, while smaller lesions may receive higher doses. Hence, a prospective trial is needed to confirm these preliminary observations.In Zishan’ s study, they evaluated and examined the influence of tumor size on outcomes for medically inoperable early-stage non-small cell lung cancer after SBRT. As a result, tumor size was not associated with local failure but regional failure and distant failure . Howeverroved LC . In our Typically, abdominal pain and lumbar back pain were the two most common symptoms in patients with AGMs. We found that all of the patients had symptomatic relief after SBRT. A previous study reported that all 3 patients with AGMs rated their score as 0 on a 10-point scale, which were 4–5 before SBRT . The resThere were some limitations of this study. It was retrospective, and with a limited number of patients or targets. In addition, the treatment schedules were heterogeneous: there was a wide range of doses prescribed with various fractionations. Furthermore, a longer follow-up is warranted to examine the impact of LC on regional or distant disease control and survival.10 ≥ 85.5 Gy and GTV < 30 ml had a better LC rate. Because most patients died of or were at high risk of distant metastasis, local treatment combined with systemic treatment may contribute to a better outcome. Moreover, the efficacy and safety of SBRT for patients with AGMs from lung cancer needed to be validated in the prospective studies.SBRT is an effective modality with fast symptom relief, and acceptable toxicity for lung cancer patients with AGMs. Patients with BED


Agelas sp.) metabolite agelasine D, as well as other agelasine analogs and related structures were screened for inhibitory activity against Plasmodium falciparum, Leishmania infantum, Trypanosoma brucei and T. cruzi, as well as for toxicity against MRC-5 fibroblast cells. Many compounds displayed high general toxicity towards both the protozoa and MRC-5 cells. However, two compounds exhibited more selective inhibitory activity against L. infantum (IC50 <0.5 μg/mL) while two others displayed IC50 <1 μg/mL against T. cruzi in combination with relatively low toxicity against MRC-5 cells. According to criteria set up by the WHO Special Programme for Research & Training in Tropical Diseases (TDR), these compounds could be classified as hits for leishmaniasis and for Chagas disease, respectively. Identification of the hits as well as other SAR data from this initial screening will be valuable for design of more potent and selective potential drugs against these neglected tropical diseases.There is an urgent need for novel and improved drugs against several tropical diseases caused by protozoa. The marine sponge ( Major killers in developing countries include malaria  was 23, and agelasine D (2c) displayed significant inhibitory action also against the other parasites examined. These results encouraged us to examine the antiprotozoal activities of a number of agelasine analogs 1 – 9 in search for more potent and selective compounds. The results are presented in 50 as they may never become relevant hits for further follow-up.The marine sponge metabolite agelasine D (a brucei . To asse0 18 μM) . The sel50 <0.2 μg/mL with SI >20  displayed IC50 values against P. falciparum in the range 0.2 – 1.0 μg/mL, while compounds 3 – 9 were in general only marginally active.For an antimalaria hit, the WHO Special Programme for Research & Training in Tropical Diseases (TDR) defines an activity criterion to be ICh SI >20 . Only co1d was found to be a potent in vitro inhibitor of L. infantum with an IC50 value of 0.093 μg/mL and SI = 22. This compound meets the definition of a hit according to TDR (IC50 <0.5 μg/mL and SI >20) [5 also classifies as a hit. We have previously shown that compound 1d displays a rather broad spectrum of antibacterial activities, incl. inhibition of Mycobacterium tuberculosis [5 were reasonably active against Staphyllococcus aureus, but not against Escherichia coli and M. tuberculosis [1f and 1j appeared also as potent inhibitors of L. infantum, but unfortunately these compounds were equally toxic to MRC-5 cells. Compounds 3 – 4 and 6 – 9 were in general only weakly active against L. infantum.Compound  SI >20) . Compounrculosis ,23, wherT. cruzi growth, as they displayed IC50 values <1 μg/mL [1 also exhibited profound cytotoxicity towards MRC-5 cells, which is also the host cell in the T. cruzi model. Nevertheless, compounds 2a and 5, with SI >50, could be classified as hits according to the definition given above. Agelasine analog 2a is of special interest, since this compound, in contrast to the imidazole 5, displays low inhibitory activity against the other protozoa. In addition, previous studies have revealed the compound 2a is virtually inactive against bacteria  [50 against T. cruzi <1 μg/mL were found also for compounds 1d, 3f and 9.Quite a few of the agelasine analogs inhibited <1 μg/mL , but parculosis) . ModeratT. b. brucei (IC50 <0.2 μg/mL and SI >100) [5 with IC50 <0.11 μg/mL and SI >69.None of the compounds examined qualified as a hit with respect to SI >100) . The maj1 compounds (1a), or sesquiterpene (1b and 1c) derived side-chain in the purine 7-position appear to be more toxic, compared to some of the compounds with longer side-chains. Among the diterpenoids, a phytyl substituent (1j and 1k) and the β-cyclocitral derived side-chain found in 1l and 1m, also results in aspecific activity. The most interesting compounds in this class have a geranylgeranyl side-chain or the same diterpenoid substituent as found in agelasine D (2c). Compounds 1d and 1n, both carrying a methoxy group at N6 and a methyl group at N-9, were identified as antileishmanial hits. A trend seems to be that changing the N6 substituents from methoxy to a benzyloxy-  or tert-butoxy group  generally results in compounds with enhanced selectivity towards Trypanosoma sp., compared to the other parasites, but this modification does not result in significant reduction of MRC-5 cytotoxicity. Similar results were obtained when the N-9 substituent differed from methyl (1g – 1i).From the results in ompounds  generall2 have a primary amino group in the purine 6-position, instead of the alkoxyamino group found in compounds 1. This generally results in reduced cytotoxicity and improved selectivity for antiprotozoal activity. Agelasine analog 2a is a quite selective hit with respect to inhibition of T. cruzi. Since only three compounds of this class (2a – 2c) were included in this initial screening, few other conclusions regarding SAR can be drawn at this point.Agelasine D and analogs 3a – 3e are neutral isomers of compounds 1. In general, they exhibit rather low antiprotozoal activities, with exception of compound 3c, which is a potent but non-selective inhibitor of Trypanosoma sp. 9-Geranylgeranyladenine 3f was a quite selective T. cruzi inhibitor with only moderate toxicity.Compounds N-9 Dealkylation of compounds 1 and 2 results in derivatives with the general structure 4. This modification was particularly detrimental for antiplasmodial and antileishmania activity, while antitrypanosomal activities were also somewhat reduced and toxicity towards MRC-5 cells was virtually unchanged or even increased (cf. 4c with 1e).5 – 9 is the imidazole derivative 5. Compared to the agelasine analogs 1d – 1f, the whole pyrimidine ring is removed. This modification results in a less toxic compound with a broad antiprotozoal spectrum. It is worth noting that the benzimidazole derivative 6 is more toxic (MRC-5) and less active against L. infantum compared to the corresponding imidazole 5.The more interesting compound among the miscellaneous structures P. falciparum, L. infantum, T. b. brucei and T. cruzi, as well as for cytotoxicity on MRC-5 fibroblast cells. Many compounds displayed high general toxicity. Nevertheless, two compounds (1d and 5) were identified as hits for leishmaniasis and two (2a and 5) for Chagas disease. Identification of the hits as well as other SAR data from this initial screening will be valuable for design of more potent and selective potential drugs against these neglected tropical diseases.Agelasine D and several agelasine analogs and related structures were screened for inhibitory activity against 1a, 1d, and 3b [1b-1c, 1e-1k, 2a-2b, 3a, 3c-3g, 4-8 [1l-1m, 9 [1n-1o, 2c [All compounds studied were synthesized as described before; , and 3b , 1b-1c, -3g, 4-8 , 1l-1m, 1l-1m, 9 , 1n-1o, n-1o, 2c . Stock sThe experiments were performed in 96-well plates (Greiner) at four-fold dilutions in a dose-titration range of 64 μg/mL to 0.25 μg/mL. Dilutions were carried out by a programmable precision robotic station . Each plate also contained medium-controls (blanks: 0% growth), infected untreated controls (negative control: 100% growth) and reference controls (positive control). Tests were run in duplicate in two independent experiments.50.The integrated panel of microbial screens for the present study and the standard screening methodologies were adopted as have been described before . CompounP. falciparum GHA-strain was used. Parasites were cultured in human erythrocytes A+ at 37 °C under a low oxygen atmosphere  in a modular incubation chamber [The chloroquine-suceptible  chamber . The cul chamber . One hunT. b. brucei: Trypomastigotes of T. b. brucei Squib-427 strain (suramin-sensitive) were cultured at 37 °C and 5% CO2 in Hirumi-9 medium [4 trypomastigotes/well. After 72 h incubation, parasite growth was assessed fluorimetrically by adding resazurin [9 medium , supplemesazurin  for 24 hT. cruzi: Tulahuen CL2 strain (nifurtimox-sensitive) was maintained on MRC-5 cells in minimal essential medium (MEM) supplemented with 20 mM L-Glutamine, 16.5 mM sodium hydrogen carbonate and FCS (5%) at 37 °C and 5% CO2. To determine in vitro anti-trypanosomal activity, 4×103 MRC-5 cells and 4×104 parasites were added to each well of test plate with compound. After incubation at 37 °C for 7 days, parasite growth was assessed by adding of β-galactosidase substrate, chlorophenol red β-D-galactopyranoside [ranoside  for 4 h Leishmania infantum amastigotes (MHOM/ET 67) were collected from an infected donor hamster and used to infect primary peritoneal mouse macrophages. To determine in vitro antileishmanial activity, 3×104 macrophages were seeded in each well of a 96-well plate. After 48 h outgrowth, 5×104 amastigotes/well were added and incubated for 2 h at 37 °C. Pre-diluted compounds were subsequently added and the plates were further incubated for 120 h at 37 °C and 5% CO2. Parasite burdens were determined microscopically after Giemsa staining and expressed as a percentage of the blank controls without compound.2 cells, human fetal lung fibroblast, were cultivated in MEM, supplemented with L-glutamine (20 mM), 16.5 mM sodium hydrogen carbonate and 5% FCS at 37 °C and 5% CO2. For the assay, 104 MRC-5 cells/well were seeded onto the test plates containing the pre-diluted compounds and incubated at 37 °C and 5% CO2 for 72 h. Cell viability was determined after addition of resazurin.MRC-5 SV