Characterization of nuclear DNA diversity in an individual Leymus chinensis.

Intraorganismal genetic heterogeneity (IGH) exists when an individual organism harbors more than one genotype among its cells. In general, intercellular DNA diversity occurs at a very low frequency and cannot be directly detected by DNA sequencing from bulk tissue. In this study, based on Sanger and high-throughput sequencing, different species, different organs, different DNA segments and a single cell were employed to characterize nucleotide mutations in Leymus chinensis. The results demonstrated that 1) the nuclear DNA showed excessive genetic heterogeneity among cells of an individual leaf or seed but the chloroplast genes remained consistent; 2) a high density of SNPs was found in the variants of the unique DNA sequence, and the similar SNP profile shared between the leaf and seed suggested that nucleotide mutation followed a certain rule and was not random; and 3) the mutation rate decreased from the genomic DNA sequence to the corresponding protein sequence. Our results suggested that Leymus chinensis seemed to consist of a collection of cells with different genetic backgrounds.

Cox-2 Antagonizes the Protective Effect of Sevoflurane on Hypoxia/Reoxygenation-Induced Cardiomyocyte Apoptosis through Inhibiting the Akt Pathway.

OBJECTIVE: To uncover the protective role of sevoflurane on hypoxia/reoxygenation-induced cardiomyocyte apoptosis through the protein kinase B (Akt) pathway. METHODS: An in vitro hypoxia/reoxygenation (H/R) model was established in cardiomyocyte cell line H9c2. Sevoflurane (SEV) was administrated in H9c2 cells during the reoxygenation period. Viability, layered double hydroxide (LDH) release, and apoptosis in H9c2 cells were determined to assess H/R-induced cell damage. Relative levels of apoptosis-associated genes were examined. Moreover, phosphorylation of Akt was determined. RESULTS: H/R injury declined viability and enhanced LDH release and apoptotic rate in H9c2 cells. Cyclooxygenase-2 (Cox-2) was upregulated following H/R injury, which was partially reversed by SEV treatment. In addition, SEV treatment reversed changes in viability and LDH release owing to H/R injury in H9c2 cells, which were further aggravated by overexpression of Cox-2. The Akt pathway was inhibited in H9c2 cells overexpressing Cox-2. CONCLUSIONS: Sevoflurane protects cardiomyocyte damage following H/R via the Akt pathway, and its protective effect was abolished by overexpression of Cox-2.

Rapid amplitude-modulation of a diarylethene photoswitch: en route to contrast-enhanced fluorescence imaging.

A water soluble diarylethene (DAE) derivative that displays exceptionally intense fluorescence from the colorless open form has been synthesized and characterized using UV/vis spectroscopy and fluorescence microscopy. We show that the bright emission from the open form can be rapidly switched using amplitude modulated red light, that is, by light at wavelengths longer than those absorbed by the fluorescent species. This is highly appealing in any context where undesired background fluorescence disturbs the measurement, e.g., the autofluorescence commonly observed in fluorescence microscopy. We show that this scheme is conveniently applicable using lock-in detection, and that robust amplitude modulation of the probe fluorescence is indeed possible also in cell studies using fluorescence microscopy.

A simplicity-guided cocktail approach toward multicolor fluorescent systems.

A molecular cocktail containing two photochromic diarylethene derivatives that displays multicolor emission spanning blue-green to orange in a color-correlated fashion has been devised. The function does not rely on excited state communication such as energy transfer reactions, which is the typical case for similar systems. Instead, harnessing the intrinsic fluorescent and photochromic properties of the two individual diarylethene derivatives run in parallel is enough to realize the color changes. This offers an extremely flexible situation as for the choice of the fluorophores and their respective concentrations. The function is conveniently demonstrated in bulk solution at µM concentrations, where a single light source serves as the color changing stimulus.

Identification of Allergic Epitopes of Soybean ß-Conglycinin in Different Animal Species.

Soybean can cause allergy in both humans and animals. The herein study aims to identify the antigenic determinants (epitopes) of ß-conglycinin that lead to allergy in different animal species (swine, bovine, and rats). The epitopes of ß-conglycinin were identified through co-immunoprecipitation and mass spectrometry. The binding abilities of seven identified epitope peptides to allergic sera of three animal species were compared by ELISA and dot-blot techniques. Some epitope peptides could be recognized by the three animal allergic sera, while most epitopes showed some differences in binding abilities to the different animal sera. The strongest reaction using swine sera was detected with peptides α2, ß2, and ß3, but the biggest sensitive regions for bovine and rats were peptides α2, ß1, and ß4. Most epitopes of ß-conglycinin exhibited different binding abilities to the three animal sera, in which the biggest sensitive regions were peptides α2, ß2, and ß3 for swine, but peptides α2, ß1, and ß4 were detected for bovine and rats.

An all-photonic full color RGB system based on molecular photoswitches.

On-command changes in the emission color of functional materials is a sought-after property in many contexts. Of particular interest are systems using light as the external trigger to induce the color changes. Here we report on a tri-component cocktail consisting of a fluorescent donor molecule and two photochromic acceptor molecules encapsulated in polymer micelles and we show that the color of the emitted fluorescence can be continuously changed from blue-to-green and from blue-to-red upon selective light-induced isomerization of the photochromic acceptors to the fluorescent forms. Interestingly, isomerization of both acceptors to different degrees allows for the generation of all emission colors within the red-green-blue (RGB) color system. The function relies on orthogonally controlled FRET reactions between the blue emitting donor and the green and red emitting acceptors, respectively.

Investigation of phosphate removal from aqueous solution by both coal gangues.

Equilibrium studies were carried out for the adsorption of phosphate onto newly discharged coal gangue and spontaneous combustion coal gangue, which are industrial solid residues. The experimental data were fitted to the two-parameter equations of Freundlich, Langmuir, Temkin, and Dubinin-Radushkevich and the three-parameter equations of the Redlich-Peterson, Sips and Toth isotherms by non-linear method. All three-parameter isotherm equations have a higher correlation coefficient than the two-parameter isotherm equations. For new discharged coal gangue, the maximum phosphate adsorption capacity is over 2.504 mg/g (as P), and the best two-parameter isotherm is Freundlich, which indicated multilayer adsorption takes place on the surface. For spontaneous combustion coal gangue, the maximum phosphate adsorption capacity is 7.079 mg/g (as P), two times larger than new discharged coal gangue, and the best two-parameter isotherm is Langmuir, suggesting that the adsorption process occurs on a homogenous surface by monolayer adsorption. The three-parameter isotherm model of Redlich-Peterson shows the best fitting in both cases, but parameter g is 0.6138 in new discharged coal gangue (the parameter g is nearly 1, which means that the equilibrium isotherm behaves as the Langmuir, not as the Freundlich isotherm), g approaches to unity in spontaneous combustion coal gangue, suggesting that the two kinds of coal gangues have different adsorption properties.

One-Time Password Generation and Two-Factor Authentication Using Molecules and Light.

Herein, we report the first example of one-time password (OTP) generation and two-factor authentication (2FA) using a molecular approach. OTPs are passwords that are valid for one entry only. For the next login session, a new, different password is generated. This brings the advantage that any undesired recording of a password will not risk the security of the authentication process. Our molecular realization of the OTP generator is based on a photochromic molecular triad where the optical input required to set the triad to the fluorescent form differs depending on the initial isomeric state.

Emergence of a Potent Multidrug Efflux Pump Variant That Enhances Campylobacter Resistance to Multiple Antibiotics.

UNLABELLED: Bacterial antibiotic efflux pumps are key players in antibiotic resistance. Although their role in conferring multidrug resistance is well documented, the emergence of "super" efflux pump variants that enhance bacterial resistance to multiple drugs has not been reported. Here, we describe the emergence of a resistance-enhancing variant (named RE-CmeABC) of the predominant efflux pump CmeABC in Campylobacter, a major zoonotic pathogen whose resistance to antibiotics is considered a serious antibiotic resistance threat in the United States. Compared to the previously characterized CmeABC transporters, RE-CmeABC is much more potent in conferring Campylobacter resistance to antibiotics, which was shown by increased MICs and reduced intracellular accumulation of antibiotics. Structural modeling suggests that sequence variations in the drug-binding pocket of CmeB possibly contribute to the enhanced efflux function. Additionally, RE-CmeABC expands the mutant selection window of ciprofloxacin, enhances the emergence of antibiotic-resistant mutants, and confers exceedingly high-level resistance to fluoroquinolones, an important class of antibiotics for clinical therapy of campylobacteriosis. Furthermore, RE-CmeABC is horizontally transferable, shifts antibiotic MIC distribution among clinical isolates, and is increasingly prevalent in Campylobacter jejuni isolates, suggesting that it confers a fitness advantage under antimicrobial selection. These findings reveal a new mechanism for enhanced multidrug resistance and an effective strategy utilized by bacteria for adaptation to selection from multiple antibiotics. IMPORTANCE: Bacterial antibiotic efflux pumps are ubiquitously present in bacterial organisms and protect bacteria from the antibacterial effects of antimicrobials and other toxic compounds by extruding them out of cells. Thus, these efflux transporters represent an important mechanism for antibiotic resistance. In this study, we discovered the emergence and increasing prevalence of a unique efflux pump variant that is much more powerful in the efflux of antibiotics and confers multidrug resistance in Campylobacter, which is a major foodborne pathogen transmitted to humans via the food chain. Unlike other specific resistance determinants that only allow bacteria to resist a particular antimicrobial, the acquisition of a functionally enhanced efflux pump will empower bacteria with simultaneous resistance to multiple classes of antibiotics. These findings reveal a previously undescribed mechanism for enhanced multidrug resistance and open a new direction for us to understand how bacteria adapt to antibiotic treatment.

Fluorescent logic systems for sensing and molecular computation: structure-activity relationships in edge-detection.

Molecular logic-based computation continues to throw up new applications in sensing and switching, the newest of which is the edge detection of objects. The scope of this phenomenon is mapped out by the use of structure-activity relationships, where several structures of the molecules and of the objects are examined. The different angles and curvatures of the objects are followed with good fidelity in the visualized edges, even when the objects are in reverse video.

Building pH sensors into paper-based small-molecular logic systems for very simple detection of edges of objects.

Genetically engineered bacteria and reactive DNA networks detect edges of objects, as done in our retinas and as also found within computer vision. We now demonstrate that simple molecular logic systems (a combination of a pH sensor, a photo acid generator, and a pH buffer spread on paper) without any organization can achieve this relatively complex computational goal with good fidelity. This causes a jump in the complexity achievable by molecular logic-based computation and extends its applicability. The molecular species involved in light dose-driven "off-on-off" fluorescence is diverted in the "on" state by proton diffusion from irradiated to unirradiated regions where it escapes a strong quencher, thus visualizing the edge of a mask.

Small molecular logic systems can draw the outlines of objects via edge visualization.

The recently-discovered ability of small logical molecules to recognize edges is exploited to achieve outline drawing from binary templates. Outlines of arbitrary curvature, several colours and thicknesses down to 1 mm are drawn in around 30 min or less by employing a common laboratory two-colour ultraviolet lamp. The outlines and the light dose-driven XOR logic with fluorescence output or 'off-on-off' action which is observed in the irradiated regions are modelled by combining foundational principles of photochemistry, acid-base neutralization and diffusion.

Untargeted metabolomic profiling of amphenicol-resistant Campylobacter jejuni by ultra-high-performance liquid chromatography-mass spectrometry.

Campylobacter jejuni, an important foodborne microorganism, poses severe and emergent threats to human health as antibiotic resistance becomes increasingly prevalent. The mechanisms of drug resistance are hard to decipher, and little is known at the metabolic level. Here we apply metabolomic profiling to discover metabolic changes associated with amphenicol (chloramphenicol and florfenicol) resistance mutations of Campylobacter jejuni. An optimized sample preparation method was combined with ultra-high-performance liquid chromatography-time-of-flight mass spectrometry (UHPLC-TOF/MS) and pattern recognition for the analysis of small-molecule biomarkers of drug resistance. UHPLC-triple quadrupole MS operated in multiple reaction monitoring mode was used for quantitative analysis of metabolic features from UHPLC-TOF/MS profiling. Up to 41 differential metabolites involved in glycerophospholipid metabolism, sphingolipid metabolism, and fatty acid metabolism were observed in a chloramphenicol-resistant mutant strain of Campylobacter jejuni. A panel of 40 features was identified in florfenicol-resistant mutants, demonstrating changes in glycerophospholipid metabolism, sphingolipid metabolism, and tryptophan metabolism. This study shows that the UHPLC-MS-based metabolomics platform is a promising and valuable tool to generate new insights into the drug-resistant mechanism of Campylobacter jejuni.

Identification of a novel G2073A mutation in 23S rRNA in amphenicol-selected mutants of Campylobacter jejuni.

OBJECTIVES: This study was conducted to examine the development and molecular mechanisms of amphenicol resistance in Campylobacter jejuni by using in vitro selection with chloramphenicol and florfenicol. The impact of the resistance development on growth rates was also determined using in vitro culture. METHODS: Chloramphenicol and florfenicol were used as selection agents to perform in vitro stepwise selection. Mutants resistant to the selective agents were obtained from the selection process. The mutant strains were compared with the parent strain for changes in MICs and growth rates. The 23S rRNA gene and the L4 and L22 ribosomal protein genes in the mutant strains and the parent strain were amplified and sequenced to identify potential resistance-associated mutations. RESULTS: C. jejuni strains that were highly resistant to chloramphenicol and florfenicol were obtained from in vitro selection. A novel G2073A mutation in all three copies of the 23S rRNA gene was identified in all the resistant mutants examined, which showed resistance to both chloramphenicol and florfenicol. In addition, all the mutants selected by chloramphenicol also exhibited the G74D modification in ribosomal protein L4, which was previously shown to confer a low-level erythromycin resistance in Campylobacter species. The mutants selected by florfenicol did not have the G74D mutation in L4. Notably, the amphenicol-resistant mutants also exhibited reduced susceptibility to erythromycin, suggesting that the selection resulted in cross resistance to macrolides. CONCLUSIONS: This study identifies a novel point mutation (G2073A) in 23S rRNA in amphenicol-selected mutants of C. jejuni. Development of amphenicol resistance in Campylobacter likely incurs a fitness cost as the mutant strains showed slower growth rates in antibiotic-free media.

[Effect of geniposide on LPS-induced activation of TLR4-NF-κB pathway in RAW264.7 macrophage cell line].

OBJECTIVE: To study the anti-inflammatory mechanism of geniposide and observe the effect of geniposide on the expression of Toll-like receptor 4 (TLR4), the activity of NF-κB, and the release of pro-inflammatory cytokines- TNF-α, IL-1, and IL-6-in the RAW264.7 macrophages treated with lipopolysaccharide (LPS). METHODS: There were three experimental groups, including the control group, LPS group and LPS combined with geniposide group in this study. RAW264.7 macrophage cells were treated with LPS to induce cellular inflammation. Cell proliferation was measured by CCK-8. The concentrations of TNF-α, IL-1, and IL-6 in cell culture media were measured by ELISA. mRNA levels of TLR4 and P65 were examined by real-time PCR. The protein levels of p-IκB, P65, p-P65 and TLR4 were detected by Western blotting. RESULTS: Geniposide had no effect on cell proliferation. However, geniposide down-regulated the expression of TNF-α, IL-1, and IL-6, and also inhibited the expression of TLR4 and the activity of NF-κB. CONCLUSION: Geniposide exerts its anti-inflammatory effect through inhibiting the activity of NF-κB in the TLR4-NF-κB pathway in macrophages.

Adsorption kinetics of silicic acid on akaganeite.

As part of a series of studies on the interaction between ferric ions and silicic acid in the hydrosphere, the adsorption of silicic acid on akaganeite was investigated kinetically at various pH values. The adsorption of silicic acid increased with increasing pH over an initial pH range of 4-11.5. In the kinetic experiment, the Cl(-) was released from akaganeite much faster than silicic acid was adsorbed. From this result, we concluded that chloride ions bound on the surface of akaganeite are released and Fe-OH or Fe-O(-) sites are formed, which then acts as an adsorption site for silicic acid. The uptake mechanism of silicic acid by akaganeite is significantly different from that by schwertmannite, despite the presence of the same tunnel structure.

Silica deposition induced by isolated aluminum ions bound on chelate resin as a model compound of the surface of microbes.

To elucidate the mechanism of silica biodeposition in hot spring water, which is induced by Al(3+) ions bound to the surface of microbes, a chelate resin (Chelex 100) was used as a model compound of the surface of microbes. No silicic acid was adsorbed on the Na type Chelex 100, whereas silicic acids were significantly adsorbed to the Al type Chelex 100. In the Al type Chelex 100, the Al(3+) ions were present as 1:1 tridentate complex with iminodiacetate (IDA) group. After adsorption of silicic acid to Al type Chelex 100, a IDA-Al-O-Si-(OH)(3) site formed. The site acted as a template for the successive adsorption of silicic acids to form silica sheets around Al type Chelex 100 particles. In conclusion, Al(3+) ions bound to the surface of microbes play a key role as a trigger for the biodeposition of silica in hot spring water.

Formation of a silicato complex of zinc in aqueous solution and its accelerating effect on the formation of silica scales in cooling water systems.

This study elucidates the effect of zinc (Zn), which is an anticorrosive water additive, on the formation of silica scales from cooling water. In these experiments, the silica scales were analyzed by EPMA, and the results indicate that Zn is sorbed into the silica scales during formation. Measurements of the solubility of Zn(OH)(2) at various concentrations of silicic acid demonstrate that Zn is present as a silicato complex of Zn (SCZ) in cooling water. From adsorption experiments of the SCZ on silica and alumina, which are major components of the silica scales, it can be concluded that the SCZ accelerates the formation of silica scales from cooling water.

Prevalence and antimicrobial resistance of Campylobacter isolates in broilers from China.

The prevalence and antimicrobial resistance of Campylobacter spp. in broiler chickens were determined in Shandong Province, China. In total, 275 Campylobacter isolates were obtained from 767 broiler cecal samples, including 208 Campylobacter jejuni, 53 Campylobacter coli, and 14 unidentified Campylobacter isolates. Minimal inhibitory concentrations of 11 antimicrobial agents were determined using the agar dilution method recommended by CLSI. More than 98% of the tested Campylobacter isolates were resistant to quinolones (nalidixic acid, ciprofloxacin and enrofloxacin) and tetracyclines (tetracycline and doxycycline). The C. jejuni isolates also exhibited a high rate of resistance to phenicol antibiotics and a moderate rate of resistance to macrolides and gentamicin. On the contrary, the C. coli isolates showed a high-level resistance to macrolides and gentamicin and little resistance to phenicol antibiotics. The vast majority of the Campylobacter isolates were classified as multidrug resistant. These findings reveal a broad extent of antimicrobial resistance in Campylobacter isolates from poultry in China and underline the need for prudent use of antibiotics in poultry production to minimize the spread of antibiotic resistant Campylobacter.