Efficacy and safety of nanohybrids comprising silver nanoparticles and silicate clay for controlling Salmonella infection

Developing effective and safe drugs is imperative for replacing antibiotics and controlling multidrug-resistant microbes. Nanoscale silicate platelet (NSP) and its nanohybrid, silver nanoparticle/NSP (AgNP/NSP), have been developed, and the nanohybrids show a strong and general antibacterial activity in vitro. Here, their efficacy for protecting Salmonella-infected chicks from fatality and septicemia was evaluated. Both orally administrated NSP and AgNP/NSP, but not AgNPs alone, effectively reduced the systemic Salmonella infection and mortality. In addition, quantitative Ag analyses demonstrated that Ag deposition from AgNP/NSP in the intestines was less than that from conventional AgNPs, indicating that the presence of NSP for immobilizing AgNPs reduced Ag accumulation in tissue and improved the safety of AgNPs. These in vivo results illustrated that both NSP and AgNP/NSP nanohybrid represent potential agents for controlling enteric bacterial infections

Novel Nanohybrids of Silver Particles on Clay Platelets for Inhibiting Silver-Resistant Bacteria

We develop a novel nanohybrid showing a strong antibacterial activity on all of the tested pathogens, including methicillin-resistant Staphylococcus auerus and silver-resistant E. coli. The nanohybrid consists of silver nanoparticles (AgNPs) supported on 1 nm-thick silicate platelets (NSPs). The AgNP/NSP nanohybrid enables to encapsulate bacteria and triggers death signals from the cell membrane. The geographic shape of the NSPs concentrates AgNPs but impedes their penetration into attached cells, mitigating the detrimental effect of silver ion deposition in applied tissues. Moreover, the tightly tethered AgNPs on NSP surface achieve a stronger biocidal effect than silver nitrate, but bypassing Ag+ mechanism, on silver-resistant bacteria. This nanohybrid presents an effective and safe antimicrobial agent in a new perspective

Antibacterial mechanisms of the nanohybrid of the immobilized silver nanoparticles and exfoliated platelet clay

銀自古以來即被使用在防腐、抗菌、淨水等用途。銀及其化合物因其傑出的抗菌能力與低細胞毒性亦被廣泛使用在醫療領域，例如創傷敷料、防止燙傷感染等。由銀化合物中解離出的銀離子，其機制也廣泛被研究。例如存在硝酸銀溶液中的銀離子，透過與蛋白質的硫氫基 (Thiol group)作用，破壞細菌蛋白正常功能抑制細菌生長。近年來奈米科技蓬勃發展，奈米是一長度單位，代表10-9m，也就是十億分之一公尺，在此尺度下，許多物質的物理化學性質會出現變化。許多研究者看中銀具抗菌能力且較低細胞毒性的特性，將金屬銀製成奈米等級的奈米銀粒子，對各種病原體的抗菌能力進行廣泛測試，包括格蘭氏陽性菌、格蘭氏陰性菌、真菌甚至是HIV病毒等。單純奈米銀粒子的抗菌能力已經被證實優於銀離子，奈米銀粒子在nanomole等級的濃度即可有效殺菌，而銀離子的有效殺菌濃度則需micromole等級。本實驗的研究材料使用天然奈米脫層黏土(NSP)作為奈米銀粒子的分散劑，將其固定在黏土上，形成奈米銀/奈米脫層黏土(AgNP/NSP)的複合材料。為瞭解此材料的抗菌能力並探討其機制，本研究使用了場發式電子顯微鏡觀察細菌與材料作用的外觀，發現黏土本身即可黏附細菌。過氧化活性物質指示染劑H2DCFDA氧化態為綠螢光形式，透過使用此指示劑，我們進一步發現奈米銀脫層黏土可使細菌產生自由基，而PI/syto9染色顯示經過奈米銀脫層黏土處理後的細菌，細胞膜受損或死亡，因此我們推斷產生的自由基對細菌細胞膜產生氧化壓力特別是細胞膜的脂質過氧化而破壞其完整性最終導致細胞死亡，Glutathione抗氧化劑恢復細胞生存率實驗結果亦能反證活性氧化物質是使細菌死亡的主要因素。細胞營養物質的攝取測試結果也顯示奈米銀脫層黏土降低了細菌攝取碳源的能力，顯著下降的細胞內ATP濃度也與此結果相呼應。在此研究中我們揭露了此新穎奈米複合材料的抗菌機制，希望能為未來更安全更有效率的奈米抗菌材料設計提供參考。Silver is well known for its antimicrobial activity. Due to the extraordinary antibacterial activity, silver and its compound are widely used in medical filed such as wound healing and burns infections. The antibacterial mechanisms of silver ions that dissociated from silver compounds have been studied by many researchers. For instance, silver ions that dissociated from silver nitrate disrupt normal cell function and inhibit cell growth by binding with thiol group in protein. Recent years, nanotechnology developed prosperously. Nano is 10-9 or billionth of one meter. Silver nanoparticles were synthesis for the advantage of its antibacterial activity and low cytotoxicity to eukaryotic cells. The antibacterial activity of silver nanoparticles against broad-spectrum strain of bacteria has been studied, including gram-positive, gram-negative, fungi and even HIV virus. In this study, we have used a novel nanohybrid that consisted of silver nanoparticles and exfoliated nature silicate clay (AgNP/NSP). Silicate clay served as disperse for silver ions in situ reduced into immobilized silver nanoparticeles. To elucidate the antibacterial mechanisms of this nanocomposite, several experiments were applied. FE-SEM observation revealed the morphology of AgNP/NSP treated cells. Reactive oxygen species (ROS) detecting assay indicated the generation of ROS may be the major contributor for the cell membrane damage consistent with the result of Live/Dead assay that AgNP/NSP post-treated cells were dead. And according to the result of free radicals scavengers blocking ROS generation experiment, we can speculate that ROS is mainly result in lipidperoxidation leading to cell membrane damage and cell death. A strong antioxidant glutathione rescued bacteria cells survival rate from AgNP/NSP containing plates also a result consistent with above hypothesis. We also demonstrated that the glucose uptake of cells was diminished by treating AgNP/NSP. The intracellular ATP level of AgNP/NSP post-treated cells also decreased. These results indicat that AgNP/NSP hinder the normal cell physiological function such as metabolism and energy production. In this study, we have elucidated the mechanisms of AgNP/NSP and hope it will be helpful for future design of safer and more sophisticated antibacterial materials.Content Chinese Abstract..............i Abstract.....................ii Content.....................iii Content of Figures...........iv Introduction..................1 Materials and Methods.........7 Results......................14 Discussion...................20 Conclusion...................24 References...................25 Figures......................29 Supplementary Figure.........48 Appendix.....................52 Content of Figures Figure 1. Illustration of Nanosilicate Platelets........29 Figure 2. FE-SEM images of Gram-positive bacteria, Staphylococcus aureus...................................30 Figure 3. FE-SEM images of Gram-negative bacteria, Escherichia coli DH-5α..................................31 Figure 4. FE-SEM images of Salmonella...................32 Figure 5. FE-SEM images, Pseudomonas aeruginosa.........33 Figure 6. AgNP/NSP against silver-resistant strain Escherichia coli J53pMG101..............................34 Figure 7. Live/Dead assay of AgNP/NSP to E.coli J53.....35 Figure 8. Live/Dead assay of AgNP/NSP to E.coli J53pMG101...............................................36 Figure 9. Statistical result of Live/ Dead assay........37 Figure 10. ROS detecting assay fluorescent images.......38 Figure 11. Flow Cytometer result of ROS detecting assay...................................................39 Figure 12. Statistical results of flow cytometer of ROS detecting assay.........................................40 Figure 13. Cell survival rate rescue assay, E.coli J53..41 Figure 14. Protein activity of Luciferase...............42 Figure 15. Protein activity of β-galactosidase..........43 Figure 16. Flow cytometer result of Glucose uptake assay ........................................................44 Figure 17. Statistic results of Glucose uptake assay....45 Figure 18. Fluorescent images of Glucose uptake assay for E.coli J53pMG101........................................46 Figure 19. ATP level determination assay of E.coli J53 and J53pMG101...........................................47 Supplementary Figure 1 The proposed function of silver-resistant gene product..................................48 Supplementary Figure 2 Principle of PI/SYTO9 staining...49 Supplementary figure 3. Chemical structure of H2DCFDA and DCF.....................................................50 Supplementary figure 4. Chemical structure of U83836E and TEMPOL..................................................5

Evolution of infectious bronchitis virus in Taiwan: Positively selected sites in the nucleocapsid protein and their effects on RNA-binding activity

RNA recombination has been shown to underlie the sporadic emergence of new variants of coronavirus, including the infectious bronchitis virus (IBV), a highly contagious avian pathogen. We have demonstrated that RNA recombination can give rise to a new viral population, supported by the finding that most isolated Taiwanese (TW) IBVs, similar to Chinese (CH) IBVs, exhibit a genetic rearrangement with the American (US) IBV at the 50 end of the nucleocapsid (N) gene. Here, we further show that positive selection has occurred at two sites within the putative crossover region of the N-terminal domain (NTD) of the TW IBV N protein. Based on the crystal structure of the NTD, the stereographic positions of both predicted selected sites do not fall close to the RNA-binding groove. Surprisingly, converting either of the two residues to the amino acid present in most CH IBVs resulted in significantly reduced affinity of the N protein for the synthetic RNA repeats of the viral transcriptional regulatory sequence. These results suggest that modulating the amino acid residue at either selected site may alter the conformation of the N protein and affect the viral RNA–N interaction. This study illustrates that the N protein of the current TW IBV variant has been shaped by both RNA recombination and positive selection and that the latter may promote viral survival and fitness, potentially by increasing the RNAbinding capacity of the N protein

The Antibacterial Efficacy and Mechanism of Plasma-Activated Water Against Salmonella Enteritidis (ATCC 13076) on Shell Eggs

Eggs are one of the most commonly consumed food items. Currently, chlorine washing is the most common method used to sanitize shell eggs. However, chlorine could react with organic matters to form a potential carcinogen, trihalomethanes, which can have a negative impact on human health. Plasma-activated water (PAW) has been demonstrated to inactivate microorganisms e ectively without compromising the sensory qualities of shell eggs. For this study, various amounts (250, 500, 750, or 1000 mL) of PAW were generated by using one or two plasma jet(s) at 60 watts for 20 min with an air flow rate at 6 or 10 standard liters per minute (slm). After being inoculated with 7.0 log CFU Salmonella Enteritidis, one shell egg was placed into PAWfor 30, 60, or 90 s with 1 or 2 acting plasma jet(s). When 2 plasma jets were used in a large amount of water (1000 mL), populations of S. Enteritidis were reduced from 7.92 log CFU/egg to 2.84 CFU/egg after 60 s of treatment. In addition, concentrations of ozone, hydrogen peroxide, nitrate, and nitrite in the PAW were correlated with the levels of antibacterial e cacy. The highest concentrations of ozone (1.22 ppm) and nitrate (55.5 ppm) were obtained with a larger water amount and lower air flow rate. High oxidation reduction potential (ORP) and low pH values were obtained with longer activation time, more plasma jet, and a lower air flow rate. Electron paramagnetic resonance (EPR) analyses demonstrated that reactive oxygen species (ROS) were generated in the PAW. The observation under the scanning electron microscope (SEM) revealed that bacterial cells were swollen, or even erupted after treatment with PAW. These results indicate that the bacterial cells lost control of cell permeability after the PAWtreatment. This study shows that PAW is e ective against S. Enteritidis on shell eggs in a large amount of water. Ozone, nitrate, and ROS could be the main causes for the inactivation of bacterial cells

Using Ad-Related Network Behavior to Distinguish Ad Libraries

Mobile app ads pose a far greater security threat to users than adverts on computer browsers. This is because app developers must embed a Software Development Kit (SDK), called an ad library or ad lib for short, provided by ad networks (i.e., ad companies) into their app program, and then merge and compile it into an Android PacKage (APK) execution file. The ad lib thus becomes a part of the entire app, and shares the whole permissions granted to the app. Unfortunately, this also resulted in many security issues, such as ad libs abusing the permissions to collect and leak private data, ad servers redirecting ad requests to download malicious JavaScript from unknown servers to execute it in the background of the mobile operating system without the user’s consent. The more well-known an embedded ad lib, the safer the app may be, and vice versa. Importantly, while decompiling an APK to inspect its source code may not identify the ad lib(s), executing the app on a simulator can reveal the network behavior of the embedded ad lib(s). Ad libs exhibit different behavior patterns when communicating with ad servers. This study uses a dynamic analysis method to inspect an executing app, and plots the ad lib behavior patterns related to the advertisement into a graph. It is then determined whether or not the ad lib is from a trusted ad network using comparisons of graph similarities

Convenient Synthesis of (E)-5-Aryl(halo)methylenebicyclo- [2.2.2]oct-2-enes and -[2.2.1]hept-2-enes via Lewis Acid- Promoted Carbohalogenation of Cyclic 2,6-Enynols

Abstract: An efficient synthesis of (E)-5-aryl(halo)-methylenebicyclo[2.2.2]oct-2-enes is reported. Lewis acid-promoted carbohalogenation of 4-(3-arylprop-2-ynyl)-cyclohex-2-enols in dichloromethane proceeds rapidly to afford the exo-methylenebridged bicycles in good yields. This method also provides an easy access to (E)-5-aryl(halo)methyl-A C H T U N G T R E N N U N G enebicyclo[2.2.1]hept-2-enes from the five-membered ring 2,6-enynols. The reactions are procedurally simple and high yielding, producing the aryl(halo)methylene-bridged bicycles in minutes under air and mild conditions

Overexpression of <i>OsPR10a</i> in rice enhanced resistance to <i>Xoo</i> infection.

<p>(A) Schematic diagram of the <i>Ubi</i>::<i>OsPR10a</i> overexpression construct. <i>Ubip</i>: the maize <i>ubiquitin</i> promoter; <i>OsPR10a</i>: the coding sequence of <i>OsPR10a</i>; <i>NosT</i>: nopaline synthase terminator. (B) The expression of <i>OsPR10a</i> mRNA in four independent transgenic lines determined by northern-blot analysis using <i>OsPR10a</i> coding region as a probe. (C) Ectopic expression of <i>OsPR10a</i> in rice enhanced resistance to <i>Xoo</i> infection. 4-week-old plants (4–5 plants per pot) were wounded in leaf tips and sprayed with <i>Xoo</i> (1.0×10⁸ CFU/mL). Photographs were taken at 0, 10, 12 and 16 days after inoculation. Bars = 10 cm. After 16 DAI, all WT plants (100%) were dead and collapsed, therefore they were not photographed. (D) Quantification of the plants that survived after <i>Xoo</i> infection. Four-week-old rice plants were sprayed with <i>Xoo</i> (1.0×10⁸ CFU/mL) as described above. The experiments were repeated three times. Groups that share the same letter are not significantly different estimated by ANOVA (P 0.05). Data are shown as means ±SD (n = 10). DAI: days after inoculation.</p

Phosphate starvation induced <i>OsPR10a</i> gene expression in rice suspension-cultured cells.

<p>Rice suspension cells were cultured in MS medium for 3 days and transferred to MS medium supplemented with (+P_i) or without (-P_i) phosphate. Total RNA was isolated from cells and subjected to northern blot analysis using the <i>OsPR10a</i> coding region as a probe. The rRNA served as a total RNA loading control.</p