Reclassification of Olsenella gallinarum as Thermophilibacter gallinarum comb. nov. and description of Thermophilibacter immobilis sp. nov., isolated from the mud in a fermentation cellar used for the production of Chinese Luzhou-flavour Baijiu.

A novel Gram-stain-positive, strictly anaerobic, elliptical, non-motile and non-flagellated bacterium, designed LZLJ-2T, was isolated from the mud in a fermentation cellar used for the production of Chinese Luzhou-flavour Baijiu. Growth occurred at 28-45 °C (optimum, 37 °C), at pH 6.0-7.0 (optimum, pH 6.0) and with concentrations of NaCl up to 2 % (w/v; optimum, 0 %). On the basis of 16S rRNA gene sequence similarity, strain LZLJ-2T belonged to the genus Thermophilibacter and was most closely related to Thermophilibacter mediterraneus Marseille-P3256T (similarity 96.9 %), Olsenella gallinarum ClaCZ62T (similarity 96.6 %) and Thermophilibacter provencensis Marseille-P2912T (similarity 96.4 %). In addition, strain LZLJ-2T had high similarity to the genus Olsenella, including Olsenella profusa DSM 13989T (similarity 94.9 %), Olsenella umbonata DSM 22620T (similarity 94.9 %), Olsenella uli ATCC 49627T (similarity 94.22 %), Tractidigestivibacter scatoligenes DSM 28304T (similarity 93.9 %) and Paratractidigestivibacter faecalis KCTC 15699T (similarity 93.25 %). Comparative genome analysis showed that orthoANI values between strain LZLJ-2T and Thermophilibacter mediterraneus Marseille-P3256T, Olsenella gallinarum ClaCZ62T, Thermophilibacter provencensis Marseille-P2912T, Olsenella profusa DSM 13989T, Olsenella umbonata DSM 22620T, Olsenella uli ATCC 49627T, Tractidigestivibacter scatoligenes DSM 28304T and Paratractidigestivibacter faecalis KCTC 15699T were 78.68, 78.99, 78.29, 73.40, 74.00, 74.30, 75.08 and 77.23 %, and the genome-to-genome distance values were respectively 22.3, 22.5, 22.4, 19.6, 20.5, 19.7, 20.5 and 21.5 %. The genomic DNA G+C content of strain LZLJ-2T was 65.21 mol%. The predominant cellular fatty acids (>10 %) of strain LZLJ-2T were C18 : 1 cis 9 (33.7 %), C14 : 0 (22.0 %) and C18 : 1 cis 9 DMA (13.5 %). d-Glucose, sucrose, mannose, maltose, lactose (weak), salicin, glycerol (weak), cellobiose and trehalose (weak) could be used by strain LZLJ-2T as sole carbon sources. Enzyme activity results showed positive reactions with valine arylamidase, leucine arylamidase, crystine arylamidase, acid phosphatase, alkaline phosphatase, esterase (C4) (weakly positive), naphthol-AS-BI-phosphohydrolase, α-glucosidase and ß-glucosidase. The major end products of glucose fermentation were lactic acid and acetic acid. It produced skatole from indole acetic acid, and produced p-cresol from modified peptone-yeast extract medium with glucose. Based on the 16S rRNA gene trees as well as the genome core gene tree, it is suggested that Olsenella gallinarum are transferred to genus Thermophilibacter as Thermophilibacter gallinarum comb. nov. Based on phenotypic, genotypic and phylogenetic data, strain LZLJ-2T is considered to represent a novel species of the genus Thermophilibacter, for which the name Thermophilibacter immobilis sp. nov. is proposed. The type strain is LZLJ-2T (=KCTC 25162T=JCM 34224T).

Blautia liquoris sp. nov., isolated from the mud in a fermentation cellar used for the production of Chinese strong-flavour liquor.

A novel Gram-positive, non-motile, non-flagellated, strictly anaerobic, non-spore-forming and dumbbell-shaped, coccoid- or chain-shaped bacterium, designated strain LZLJ-3T, was isolated from a mud fermentation cellar which has been used for the production of Chinese strong-flavour liquor for over 100 years. Strain LZLJ-3T grew at 20-40 °C (optimum, 37 °C), at pH 6.0-8.0 (optimum, pH 8.0) and with NaCl concentrations up to 1 % (w/v; optimum, 0 %). Phylogenetic trees established based on 16S rRNA gene sequences showed that strain LZLJ-3T belonged to the genus Blautia of the family Lachnospiraceae, with the highest sequence similarity to Blautia stercoris GAM6-1T (91.7 %) and Blautia faecicola KGMB01111T (91.7 %). Comparative genome analysis showed that the orthologous average nucleotide identity (OrthoANI) and genome-to-genome distance (GGD) values between strain LZLJ-3T and B. stercoris GAM6-1T were respectively 69.1 and 22.9 %; the OrthoANI and GGD values between strain LZLJ-3T and B. faecicola KGMB01111T were respectively 70.86 and 36 % . The DNA G+C content of strain LZLJ-3T genome was 42.1 mol%. The predominant celluar fatty acids (>10 %) of strain LZLJ-3T were C16 : 0 FAME (27.9 %), C14 : 0 FAME (17.6 %) and C16 : 0 DMA (13.0 %). Arabinose, glucose and maltose could be utilized by strain LZLJ-3T as sole carbon sources for growth, with weak utilization of raffinose and l-fucose. API ZYM analysis gave positive reactions with α-galactosidase, ß-galactosidase, α-glucosidase and ß-glucosidase. The major end product of glucose fermentation was acetic acid. Based on the results of phenotypic, genotypic and phylogenetic analyses, strain LZLJ-3T is considered to represent a novel species of Blautia, for which the name Blautia liquoris sp. nov. is proposed. The type strain is LZLJ-3T (=KCTC 25163T=CGMCC 1.5299T=JCM 34225T).

Colorimetric Detection of Carcinogenic Aromatic Amine Using Layer-by-Layer Graphene Oxide/Cytochrome c Composite.

Graphene and its derivatives were found to be efficient modulators of enzymes in various systems. However, the modulating mechanism was not well discussed for long time. Inspired by the artificial enzyme-enhancing property of graphene oxide (GO) toward cytochrome c (cyt. c), we have successfully fabricated a protein/GO hybrid structure via a layer-by-layer (LbL) strategy. The obtained LbL assemblies showed great enhancement in peroxidase activity of cyt. c, as well as excellent stability, resistance to extreme environment change, and also possibility for recycling by simple centrifugation without any obvious activity loss. The LbL cyt. c/GO hybrids were expanded to a colorimetric sensing system for the detection of carcinogenic aromatic amines. The probe showed high sensitivity and selectivity for aromatic amines over various competing soluble aromatic compounds and could be determined by the naked eye or portable devices. The working mechanism was well studied through kinetic evaluation, experimental characterization, and molecular dynamics simulations. This work does not only introduce a new graphene/protein hybrid material or a rapid and sensitive visualization of carcinogenic aromatic amines, but also spread the practical application of biomolecule-graphene interface strategy and further give a better understanding of the interaction of graphene and protein.

An integrated logic system for time-resolved fluorescent "turn-on" detection of cysteine and histidine base on terbium (III) coordination polymer-copper (II) ensemble.

Cysteine (Cys) and histidine (His) both play indispensable roles in many important biological activities. An enhanced Cys level can result in Alzheimer's and cardiovascular diseases. Likewise, His plays a significant role in the growth and repair of tissues as well as in controlling the transmission of metal elements in biological bases. Therefore, it is meaningful to detect Cys and His simultaneously. In this work, a novel terbium (III) coordination polymer-Cu (II) ensemble (Tb(3+)/GMP-Cu(2+)) was proposed. Guanosine monophosphate (GMP) can self-assemble with Tb(3+) to form a supramolecular Tb(3+) coordination polymer (Tb(3+)/GMP), which can be suited as a time-resolved probe. The fluorescence of Tb(3+)/GMP would be quenched upon the addition of Cu(2+), and then the fluorescence of the as-prepared Tb(3+)/GMP-Cu(2+) ensemble would be restored again in the presence of Cys or His. By incorporating N-Ethylmaleimide and Ni(2+) as masking agents, Tb(3+)/GMP-Cu(2+) was further exploited as an integrated logic system and a specific time-resolved fluorescent "turn-on" assay for simultaneously sensing His and Cys was designed. Meanwhile it can also be used in plasma samples, showing great potential to meet the need of practical application.

"Molecular beacon"-hosted thioflavin T: Applications for label-free fluorescent detection of iodide and logic operations.

In this work, we presented a simple, label-free and rapid-responsive fluorescence assay for iodide (I(-)) detection based on "molecular beacon (MB)"-hosted thioflavin T (ThT), achieving a limit of detection as low as 158 nM. The proposed method exhibited very good selectivity to I(-) ions over other anions interference due to the strong binding force between I(-) ions with Hg(2+). Upon the addition of I(-) ions, it would capture Hg(2+) from a T-Hg(2+)-T complex belonging to the MB-like DNA hairpin structure, which eventually quenched the initial fluorescence as output. In addition, it was successfully applied for operation of an integrated DNA logic gate system and to the determination of I(-) in real samples such as human urine.

Ratiometric fluorescence detection of silver ions using thioflavin T-based organic/inorganic hybrid supraparticles.

In this work, we present a new type of functional organic/inorganic hybrid supraparticle that spontaneously assembles from silver ions (Ag(+)), iodide ions (I(-)) and thioflavin T (ThT) under aqueous solution conditions. ThT alone in aqueous solution was weakly fluorescent with an emission band at 494 nm, which was related to the monomer. However, in the above-mentioned hybrid supraparticle (i.e., ThT@AgI SP) structure, the ThT monomer can form a dimer with a new emission band. The new band shifted to 546 nm and the emission intensity increased. We further present a facile strategy of reversible fluorescence switching of ThT by a simple cation (Ag(+)) and anions (I(-) and S(2-)), which can be employed for the ratiometric fluorescence detection of Ag(+) with high sensitivity and selectivity. The linear range of detecting Ag(+) was from 100 nM to 10 µM, with a limit of detection as low as approximately 50 nM. Moreover, it can be successfully applied for the operation of a logic gate system and to the sensing of Ag(+) in real water samples.

Visual fluorescence detection of H2O2 and glucose based on "molecular beacon"-hosted Hoechst dyes.

In this work, a label-free molecular beacon (MB)-like biosensor is designed for the determination of H2O2 and glucose based on the fluorescence regulation of Hoechst dyes hosted by the designed AT-rich single-stranded DNA (ssDNA), in which Hg(2+) and cysteine (Cys) act as activators. The designed AT-rich ssDNA (ATprobe) can be directed to form a hairpin with an Hg(2+)-induced T-Hg(2+)-T complex, which provides a medium for enhancing the fluorescence of Hoechst dyes significantly. On the other hand, Cys can effectively grab Hg(2+) from the T-Hg(2+)-T complex by thiol-Hg(2+) interactions, destructing the hairpin and then switching the Hoechst dyes to the fluorescence "off" state. Combined with these properties, we have demonstrated its application for label-free fluorescence "turn on" detection of H2O2. The sensing mechanism is based on the specific reaction between H2O2 and Cys catalyzed by I(-), the resulting disulfide reverses the Cys-mediated fluorescence decrease of the MB-hosted Hoechst dyes. The approach achieves a low detection limit of 0.1 µM for H2O2. Moreover, this method is further applied to the noninvasive detection of glucose in artificial saliva and urine samples, combining with glucose oxidase (GOx) for the oxidation of glucose and formation of H2O2. Compared to traditional methods, the proposed design is cost-effective, simple to prepare and manipulate without fluorescence labeling or chemical modification.

Time-resolved probes and oxidase-based biosensors using terbium(III)-guanosine monophosphate-mercury(II) coordination polymer nanoparticles.

A novel lanthanide coordination polymer nanoparticle (LCPN)-based ternary complex was synthesized via the self-assembly of a terbium ion (Tb(3+)) with a nucleotide (GMP) and a mercury ion (Hg(2+)) in aqueous solution. The as-prepared LCPN-based ternary complex (Tb-GMP-Hg) can be applied to the development of time-resolved luminescence assays and oxidase-based biosensors.

[Using green fluorescent protein as a reporter to monitor elimination of selectable marker genes from transgenic plants].

In genetic modification of plants, once the transformants are obtained, selection markers are no longer required in mature plants. At present, the Cre/lox site-specific recombination system is most widely used to eliminate the selectable marker genes from the transgenic plants. In this study, attempt was made to favour the selection of marker-free plants in the re-transformation method. Green fluorescent protein (GFP) can be directly visualized in living cells, tissues or organisms under UV illumination. This advantage of GFP is exploited in the development of a practical approach in which GFP is used as a visual marker to monitor the removal of the selectable marker gene from transgenic plants. For that purpose, the pGNG binary vector was constructed, in which the GFP gene (gfp) was linked to the expression cassette Nos P-nptII-NosT and the two units were cloned between two directly-orientated lox sites. The CaMV 35S promoter was placed before the first lox site and used to drive GFP expression. The beta-glucuronidase gene (gus) of Escherichia coli was cloned behind the second lox site without a promoter, thus would not be expressed in this position. Tobacco plants were first transformed with pGNG and selected on kanamycin (Kan)-containing media. Regenerated transgenic shoots were readily singled out by GFP fluorescence. The GFP-expressing plants were then re-transformed with pCambia1300-Cre containing hygromycin phosphotransferase gene (hpt) as a selectable marker gene. The Cre-mediated recombination resulted in the elimination of lox-flanked genes, herein gfp and nptII, from the plant genome and brought the GUS gene next to the 35S promoter. Our data demonstrated that transgenic plants free of nptII were easily selected by monitoring the loss of green fluorescence, and at the same time, GUS (here as a target protein) was expressed in the nptII-free plants. Finally, hpt and cre were removed from the progenies of the nptII-free plants by gene segregation.

[The effect of changing body posture on thrombin activity and concentration].

AIM: To provide proof for Evidence-based Medicine as well quality control, our laboratory detected the thrombin activity on various body position. METHODS: By autogenous contrast and cross matched survey, 105 volunteers divided into 3 season patches of winter, spring and summer, blood samples were drawn from the same part in both standing and lying position. Both samples and the quality control were detected to investigate the effect of the body position to thrombin activity's changing. The data were analyzed by SPSS 10.0. RESULTS: Taking the lying's data as baseline, the average changing on all those the "5" index was 7.07% and the highest changing reached 9.33%. This kind changing had great significant differences (P < 0.01). According to the t value, sequences ranged: FIB > TT > PT > INR > APTT. FIB, TT and APTT's values slowly raised, adversely PT and INR slowly went down. While sitting for 15 min after lying, these indices returned to 95.2% of the original value in sitting position in addition. Season, age and device had no relationship with body position. CONCLUSION: Changing body position can result in obvious physiological variation of thrombin activity.