How does the status of errant robot affect our desire for contact? - The moderating effect of team interdependence.

Technological breakthroughs such as artificial intelligence and sensors make human-robot collaboration a reality. Robots with highly reliable, specialised skills gain informal status in collaborative teams, but factors such as unstructured work environments and task requirements make robot error inevitable. So how do status differences of errant robots affect the desire for contact, and do team characteristics also have an impact? This paper describes an intergroup experiment using the Experimental Vignette Method (EVM), based on the Expectation Violation Theory, 214 subjects were invited to test the following hypotheses: (1) Errant robot status has an influence on employees' desire for contact and support for robotics research through negative emotions; (2) Team interdependence is a boundary condition for the effect of errant robot status on negative emotions. This paper contributes to the literature on employee reactions to robot errors in human-robot collaboration and provides suggestions for robot status design.

Complex human-robot collaboration inevitably leads to the phenomenon of robot errors. Based on this, we used an Experimental Vignette Method and found that differences in robot status design and human-robot team design features significantly affect employees' cognitive psychology after robot errors and reduce the negative consequences.

Anti-Biofilm Activity of Chlorogenic Acid against Pseudomonas Using Quorum Sensing System.

Chlorogenic acid is a secondary metabolite produced by many traditional Chinese medicines. Its physiological activities (antibacterial, anti-inflammatory, antioxidant activities, etc.) have been well described. This study aimed to investigate the effects of chlorogenic acid on the biofilm of drinking water bacteria. The effects of chlorogenic acid on the metabolites of the biofilms were also evaluated. Chlorogenic acid was found to have an anti-biofilm effect against Pseudomonas, resulting in biofilm formation in a dose-dependent manner (0.53-25.4 mM CGA). Moreover, the biofilm structure was visibly attenuated. Furthermore, we identified and characterized 23 differential metabolites and associated two metabolic pathways involving beta-alanine metabolism and pyrimidine metabolism that were altered mostly during biofilm formation. A quantitative real-time PCR assay revealed that chlorogenic acid interfered with the signaling molecule synthesis and transcription regulators using the Las, Pqs and Rhl systems. These findings suggest that chlorogenic acid can be a quorum sensing (QS) inhibitor and inhibit biofilm formation. It may be a promising natural product for the prevention of contaminated drinking water.

The Study of a Novel Paeoniflorin-Converting Enzyme from Cunninghamella blakesleeana.

Paeoniflorin is a glycoside compound found in Paeonia lactiflora Pall that is used in traditional herbal medicine and shows various protective effects on the cardio-cerebral vascular system. It has been reported that the pharmacological effects of paeoniflorin might be generated by its metabolites. However, the bioavailability of paeoniflorin by oral administration is low, which greatly limits its clinical application. In this paper, a paeoniflorin-converting enzyme gene (G6046, GenBank accession numbers: OP856858) from Cunninghamella blakesleeana (AS 3.970) was identified by comparative analysis between MS analysis and transcriptomics. The expression, purification, enzyme activity, and structure of the conversion products produced by this paeoniflorin-converting enzyme were studied. The optimal conditions for the enzymatic activity were found to be pH 9, 45 °C, resulting in a specific enzyme activity of 14.56 U/mg. The products were separated and purified by high-performance counter-current chromatography (HPCCC). Two main components were isolated and identified, 2-amino-2-p-hydroxymethyl-methyl alcohol-benzoate (tirs-benzoate) and 1-benzoyloxy-2,3-propanediol (1-benzoyloxypropane-2,3-diol), via UPLC-Q-TOF-MS and NMR. Additionally, paeoniflorin demonstrated the ability to metabolize into benzoic acid via G6046 enzyme, which might exert antidepressant effects through the blood-brain barrier into the brain.

YihE is a novel binding partner of Rho and regulates Rho-dependent transcription termination in the Cpx stress response.

The conjugative pilus expression (Cpx) stress response system can sense cell envelope stressors, such as misfolded proteins, and upregulate proteases to modify or degrade damaged proteins. YihE is a protein kinase implicated in the Cpx system, and Rho is a transcription termination factor in prokaryotes, but no functional connection between YihE and Rho has been reported. Here, we found that YihE can interact with Rho to form a binary complex with a stoichiometric ratio of 6:1 (Rho:YihE). A low resolution of Rho crystal structure in the presence of YihE was determined. YihE overexpression helped lessen the aberrant effects caused by Rho overexpression, including long cell morphology and other Rho-mediated phenotypes. Overall, YihE is a Rho binding partner that acts as a Rho antagonist in the Cpx stress. YihE binding to Rho would compete RNA from binding to Rho, thereby helping bacteria cope with stress through the regulation of Rho-dependent transcription termination.

In vitro study of the effect of quinoa and quinoa polysaccharides on human gut microbiota.

It has been shown that whole grains and dietary fiber are important for their fermentation characteristics in the large intestine, drawing more and more attention to quinoa and quinoa polysaccharides. In this study, we evaluated the prebiotic effect of quinoa seeds and quinoa polysaccharides after human simulated digestion. The modulatory effect of the quinoa and quinoa polysaccharides (QPs) on the gut microbiota was evaluated by the in vitro fermentation using human fecal microbiota. The yield of polysaccharides extraction was 15.45%. The digestibility of the cooked and uncooked quinoa after simulation of human digestion was 69.04% and 64.09%, respectively. The effect on the microbiota composition and their metabolic products was determined by the assessment of pH, short-chain fatty acids (SCFAs), and changes in the bacterial population. After 24 hr anaerobic incubation, the total SCFAs of cooked, uncooked quinoa, and quinoa polysaccharides were 82.99, 77.11, and 82.73 mM, respectively with a pH decrease. At the phylum, genus, and class level, it has been found that the quinoa substrates enhance the growth of certain beneficial bacteria such as Prevotella and Bacteroides. Quinoa polysaccharides can be considered prebiotic due to their ability to increase Bifidobacterium and Collinsella. Principal component analysis (PCA) showed that there was a distinct modulating effect on the fecal microbiota which represents different distribution. Our research suggests that quinoa and quinoa polysaccharides have a prebiotic potential due to their association with the positive shifts in microbiota composition and short-chain fatty acids production, which highlights the importance of further studies around this topic.

Optimization extraction and purification of biological activity curcumin from Curcuma longa L by high-performance counter-current chromatography.

The extraction condition of curcumin from Curcuma longa L was optimized through four factors and three levels orthogonal experiment based on the results of single factor tests. Under the optimal conditions: the concentration of ethanol  80%, extraction temperature 70°C, the ratio of liquid to material 20, and extraction time 3 h, a crude extract with the yield of curcumin 56.8 mg/g could be obtained. The isolation and purification of curcuminoids from the crude extract was performed on high performance counter current chromatography employing an optimized solvent system n-hexane/ethyl acetate/methanol/water (2/3/3/1, v/v/v/v). From 97 mg crude sample (in which the purity of curmumin was 68.56%), 67 mg curmumin, 18 mg demethoxycurcumin, and 9.7 mg bisdemethoxycurcumin with a high-performance liquid chromatography purity of 98.26, 97.39, and 98.67%, respectively, were obtained within 70 min. The antioxidant activities and cytotoxicity of purified curcumin was comparable to that of the commercial product, indicating that the biological activity of curcumin could be maintained by this method.

The crystal structure of MICU2 provides insight into Ca2+ binding and MICU1-MICU2 heterodimer formation.

The mitochondrial calcium uniporter (MCU) complex mediates the uptake of Ca2+ into mitochondria. Its activity is regulated by a heterodimer of MICU1 and MICU2, two EF-hand-containing proteins that act as the main gatekeeper of the uniporter. Herein we report the crystal structure of human MICU2 at 1.96 Å resolution. Our structure reveals a dimeric architecture of MICU2, in which each monomer adopts the canonical two-lobe structure with a pair of EF-hands in each lobe. Both Ca2+ -bound and Ca2+ -free EF-hands are observed in our structure. Moreover, we characterize the interaction sites within the MICU2 homodimer, as well as the MICU1-MICU2 heterodimer in both Ca2+ -free and Ca2+ -bound conditions. Glu242 in MICU1 and Arg352 in MICU2 are crucial for apo heterodimer formation, while Phe383 in MICU1 and Glu196 in MICU2 significantly contribute to the interaction in the Ca2+ -bound state. Based on our structural and biochemical analyses, we propose a model for MICU1-MICU2 heterodimer formation and its conformational transition from apo to a more compact Ca2+ -bound state, which expands our understanding of this co-regulatory mechanism critical for MCU's mitochondrial calcium uptake function.

Selective microextraction of polycyclic aromatic hydrocarbons using a hydrophobic deep eutectic solvent composed with an iron oxide-based nanoferrofluid.

A simple and fast method is described for the extraction of polycyclic aromatic hydrocarbons (PAHs) from complex samples. It is based on the use of a nanoferrofluid modified with a ternary hydrophobic deep eutectic solvent. A predictive model was used for the selection of the optimal eutectic mixture. The entire microextraction only takes a few minutes for completion. Under the optimal extraction conditions (by using menthol, borneol and camphor in a molar ratio of 5:1:4; 80 mg of nanoferrofluid), it offers marked improvements in terms of selectivity and sensitivity. The limits of detection range between 0.31 and 5.9 ng·L-1, and recoveries from spiked samples between 91.3 and 121%. In addition, the strong interactions between PAHs and the extractant were supported by quantum mechanical calculations. This results in a better insight into the microextraction mechanism, providing a fast, environmentally friendly and effective route for the optimization of pretreatment parameters. The method was successfully applied to the determination of the PAHs naphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, chrysene, benzo[b]fluoranthene, benzo[a]pyrene, dibenzo[a,h]anthracene, benzo[g,h,i]perylene and indeno[1,2,3-c,d]pyrene in 12 kinds of coffee samples after different roasting conditions. Graphical abstract Schematic presentation of nanoferrofluid modified with ternary hydrophobic deep eutectic solvent and their application for selective microextraction of ultra-trace polycyclic aromatic hydrocarbons in coffee prior to HPLC analysis.

Preparative and scaled-up separation of high-purity α-linolenic acid from perilla seed oil by conventional and pH-zone refining counter current chromatography.

α-Linolenic acid is an essential omega-3 fatty acid needed for human health. However, the isolation of high-purity α-linolenic acid from plant resources is challenging. The preparative separation methods of α-linolenic acid by both conventional and pH-zone refining counter current chromatography were firstly established in this work. The successful separation of α-linolenic acid by conventional counter current chromatography was achieved by the optimized solvent system n-heptane/methanol/ water/acetic acid (10:9:1:0.04, v/v), producing 466 mg of 98.98% α-linolenic acid from 900 mg free fatty acid sample prepared from perilla seed oil with linoleic acid and oleic acid as by-products. The scaled-up separation in 45× is efficient without loss of resolution and extension of separation time. The separation of α-linolenic acid by pH-zone refining counter current chromatography was also satisfactory by the solvent system n-hexane/methanol/water (10:5:5, v/v) and the optimized concentration of trifluoroacetic acid 30 mM and NH4 OH 10 mM. The separation can be scaled up in 180× producing 9676.7 mg of 92.79% α-linolenic acid from 18 000 mg free fatty acid sample. pH-zone refining counter current chromatography exhibits a great advantage over conventional counter current chromatography with 20× sample loading capacity on the same column.

Separation of high-purity eicosapentaenoic acid and docosahexaenoic acid from fish oil by pH-zone-refining countercurrent chromatography.

The evidence for unique effects of eicosapentaenoic acid and docosahexaenoic acid is growing. Further understanding and exploration of their independent effects in the nutraceutical and pharmaceutical industry is calling for the more efficient separation techniques to overcome the equivalent chain length rule of fatty acids. In this study, free eicosapentaenoic and docosahexaenoic acid were successfully separated by pH-zone-refining countercurrent chromatography for the first time. The different solvent systems and the influence of retainer and eluter concentration on the separation efficiency were investigated. A two-phase solvent system composed of n-heptane/methanol/water (100:55:45, v/v) was selected with 50 mM of trifluoroacetic acid as retainer in the organic phase and 40 mM of ammonium hydroxide as an eluter in the aqueous phase for the separation of 500 mg of free fatty acids from a refined fish oil sample. 79.6 mg of eicosapentaenoic acid and 328.3 mg docosahexaenoic acid were obtained with the purities of 95.5 and 96.9% respectively determined by gas chromatography with mass spectrometry after methyl esterification. The scale-up separation of 1 g of samples from both refined and crude fish oil after urea complexation were also achieved successfully with a markedly increased concentration 150 mM of retainer, producing satisfactory yields and purities of targets.

Separation and purification of lanosterol, dihydrolanosterol, and cholesterol from lanolin by high-performance counter-current chromatography dual-mode elution method.

Lanosterol is a potential drug for cataracts treatment, which can reverse the aggregation of intracrystalline proteins. The low concentration in lanolin calls for high-performance separation methods. In this study, a counter-current chromatography dual-mode elution method was developed for the first time to separate and purify lanosterol from hexane extract of lanolin after saponification, in which the column was first eluted with the lower phase as mobile phase in head-to-tail mode, followed by the upper phase in the tail-to-head mode. High purity of lanosterol, dihydrolanosterol, and cholesterol can be obtained simultaneously. A solvent system composed of n-heptane/acetonitrile/ethyl acetate (5:5:1, v/v/v) was selected and optimized via partition coefficient determination. Compounds such as 111 mg lanosterol, 84 mg dihydrolanosterol, and 183 mg cholesterol with high purity of 99.77, 95.71, and 91.43%, respectively, analyzed by high-performance liquid chromatography were obtained within 80 min from 700 mg crude extract from 1.78 g lanolin. The method was also used to improve the purity of commercial lanosterol product from 66.97 to above 99%. Counter-current chromatography could serve as a potential and powerful technique for commercial production of highly pure lanosterol.

Crystal Structures of Candida albicans Phosphodiesterase 2 and Implications for Its Biological Functions.

The cAMP signaling system plays important roles in the physiological processes of pathogen yeast Candida albicans, but its functional mechanism has not been well illustrated. Here, we report the enzymatic characterization and crystal structures of C. albicans phosphodiesterase 2 (caPDE2) in the unliganded and 3-isobutyl-1-methylxanthine-complexed forms. caPDE2 is a monomer in liquid and crystal states and specifically hydrolyzes cAMP with a KM of 35 nM. It does not effectively hydrolyze cGMP as shown by the 1.32 × 105-fold specificity of cAMP/cGMP. The crystal structure of caPDE2 shows significant differences from those of human PDEs. First, the N-terminal fragment of caPDE2 (residues 1-201) tightly associates with the catalytic domain to form a rigid molecular entity, implying its stable molecular conformation for C. albicans to resist environmental stresses. Second, the M-loop, a critical fragment for binding of the substrate and inhibitors to human PDEs, is not a part of the caPDE2 active site. This feature of caPDE2 may provide a structural basis for the design of selective inhibitors for the treatment of yeast infection.

Phosphorylation of bacterial L9 and its functional implication in response to starvation stress.

The bacterial L9 (bL9) protein expressed and purified from Escherichia coli is stably phosphorylated. We mapped seven Ser/Thr phosphorylation sites, all of which but one are located at the carboxyl-terminal domain (CTD). When a histidine tag is fused to the C-terminus, bL9 is no longer phosphorylated. Phosphorylation of bL9 causes complete disordering of its CTD and helps cell survival under nutrient-limiting conditions. Previous structural studies of the ribosome have shown that bL9 exhibits two distinct conformations, one of which competes with binding of RelA to the 30s rRNA and prevents RelA activation. Taken together, we suggest that the flexibility of the bL9 CTD enabled by phosphorylation would remove the steric hindrance, serving as a previously unknown mechanism to regulate RelA function and help cell survival under starvation stress.

Separation of the potential G-quadruplex ligands from the butanol extract of Zanthoxylum ailanthoides Sieb. & Zucc. by countercurrent chromatography and preparative high performance liquid chromatography.

G-quadruplex DNA structure is considered to be a very attractive target for antitumor drug design due to its unique role in maintaining telomerase activities. Therefore, discovering ligands with high stability of G-quadruplex structure is of great interest. In this paper, pH-zone refining counter current chromatography (CCC) and preparative high performance liquid chromatography (HPLC) were employed for the separation of potent G-quadruplex ligands from the n-butanol fraction of the crude extract of Zanthoxylum ailanthoides, which is a traditional Chinese medicine recently found to display high inhibitory activity against several human cancer cells. The 75% aqueous ethanol extract of the stem bark of Z. ailanthoides and its fractions with petroleum ether, ethyl acetate and n-butanol displayed almost the same G-quadruplex stabilization ability. Here, pH-zone refining CCC was used for the separation of the alkaloids from the n-butanol fraction by a seldom used solvent system composed of dichloromethane-methanol-water (4:1:2.5) with 10mM TEA in the organic stationary phase as retainer and 10mM HCl in the aqueous mobile phase as eluter. Compounds I, II and III were obtained, with purity greater than 95%, in the quantities of 31.2, 94.0, and 26.4mg respectively from 300mg of lipophilic fraction within 80min, which were identified as three tetrahydroprotoberberines isolated for the first time in this plant. In addition, a phenylpropanoid glycoside compound IV (Syringin), an isoquinoline (Magnoflorine, V), and two lignin isomers (+)-lyoniresiol-3α-O-ß-d-glucopyranoside (VI) and (-)-lyoniresinol -3α-O-ß-D -glucopyranoside (VII) were isolated by traditional CCC together with preparative HPLC. Compounds IV, V, VI and VII were obtained, with purity greater than 95%, in the quantities of 4.0, 13.2, 6.7, and 6.5mg respectively from 960mg of hydrophilic fraction. Among the seven isolated compounds, tetrahydroprotoberberine I, II and III were found to display remarkable stabilization effects on G-quadruplex by increasing G-quadruplex's Tm approximately 10°C, which may be the most potent G-quadruplex ligands in Z. ailanthoides.

Characterization of DicB by partially masking its potent inhibitory activity of cell division.

DicB, a protein encoded by the Kim (Qin) prophage in Escherichia coli, inhibits cell division through interaction with MinC. Thus far, characterization of DicB has been severely hampered owing to its potent activity which ceases cell division and leads to cell death. In this work, through fusing maltose-binding protein to the N-terminus of DicB (MBP-DicB), we successfully expressed and purified recombinant DicB that enabled in vitro analysis for the first time. More importantly, taking advantage of the reduced inhibitory activity of MBP-DicB, we were able to study its effects on cell growth and morphology. Inhibition of cell growth by MBP-DicB was systematically evaluated using various DicB constructs, and their corresponding effects on cell morphology were also investigated. Our results revealed that the N-terminal segment of DicB plays an essential functional role, in contrast to its C-terminal tail. The N-terminus of DicB is of critical importance as even the first amino acid (following the initial Met) could not be removed, although it could be mutated. This study provides the first glimpse of the molecular determinants underlying DicB's function.

Expression and preliminary characterization of human MICU2.

MICU2 has been reported to interact with MICU1 and participate in the regulation of mitochondrial Ca(2+) uptake, although the molecular determinants underlying the function of MICU2 is unknown. In order to characterize MICU2 we screened a series of N-terminal and C-terminal truncations and obtained constructs which can be expressed in abundance, giving rise to soluble samples to enable subsequent characterizations. Size exclusion chromatography (SEC) and multi-angle laser light scattering (MALLS) revealed that MICU2 exists as a monomer in Ca(2+)-free conditions but forms a dimer in Ca(2+)-bound conditions. Unlike MICU1, the C-helix domain of MICU2 exhibits no influence on protein conformation in both Ca(2+)-free and Ca(2+)-bound forms. Furthermore, mutation of the first EF-hand abolishes the ability of MICU2 to switch to a dimer in the presence of Ca(2+), indicating that the first EF-hand is not only involved in Ca(2+) binding but also in conformational change. Our pull-down and co-immunoprecipitation assays suggest that, in addition to disulfide bonds, salt bridges also contribute to MICU1-MICU2 heterodimer formation.

Application of counter-current chromatography as a new pretreatment method for the determination of polycyclic aromatic hydrocarbons in environmental water.

Counter-current chromatography (CCC) was investigated as a new sample pretreatment method for the determination of trace polycyclic aromatic hydrocarbons (PAHs) in water environmental samples. The experiment was performed with a non-aqueous binary two-phase solvent system composed of n-heptane and acetonitrile. The CCC column was first filled with the upper stationary phase, and then a large volume of water sample was pumped into the column while the CCC column was rotated at 1600 rpm. Finally, the trace amounts of PAHs extracted and enriched in the stationary phase were eluted out by the lower mobile phase and determined by gas chromatography-flame ionization detector (GC-FID) or gas chromatography-mass spectrometry (GC-MS). The enrichment and cleanup of PAHs can be fulfilled online by this method with high recoveries (84.1-103.2%) and good reproducibility (RSDs: 4.9-12.2%) for 16 EPA PAHs under the optimized CCC pretreatment conditions. This method has been successfully applied to determine PAHs in lake water where 8 PAHs were detected in the concentration of 40.9-89.9 ng/L. The present method is extremely suitable for the preparation of large volume of environmental water sample for the determination of trace amounts of organic pollutants including PAHs as studied in this paper.

Development and evaluation of a spiral tube column for counter-current chromatography.

An improved type-J counter-current chromatography (CCC) planet centrifuge with two spiral tube columns (volume 2×15 mL, ß value 0.3-0.7, tubing 0.8 mm id) was developed and evaluated for its retention ability of four typical different solvent systems including heptane-methanol (1:1, v/v) (A), hexane-ethyl acetate-methanol-water (1:1:1:1, v/v) (B), n-butanol-acetic acid-water (4:1:5, v/v) (C), PEG1000-K(2)HPO(4)-water (12.5:12.5:75, w/w) (D) under eight different operation modes. The results indicated that the spiral tube column could significantly increase the retention of four typical solvent systems compared with a traditional multilayer coil column with similar parameters (volume 35 mL, ß value 0.3-0.7, tubing 0.8 mm id). The retention of stationary phase (S(f)) for the less polar system (A) and moderately polar solvent system (B) can be increased by about 10%, and for the polar system (C) and aqueous two-phase system (ATPS) (D) by 30-40%. The preliminary applications of this spiral tube column to the separation of small molecular compounds such as moderately polar theaflavins, polar anthocyanins and dipeptides were successful. Acceptable resolution can be obtained between cytochrome c and myoglobin, lysozyme and myoglobin when it was applied on protein separation; however, it still needs to be improved with regard to its column efficiency.

Separation and identification of polyphenols in apple pomace by high-speed counter-current chromatography and high-performance liquid chromatography coupled with mass spectrometry.

Apple pomace, a by-product in the processing of apple juice, was investigated as a potential source of polyphenols. Two methods of separation and purification of polyphenols from apple pomace extract were established by combination of gel chromatography with high-speed counter-current chromatography (HSCCC) and solvent extraction with HSCCC, respectively. The optimal separation was performed on a Sephadex LH-20 column using gradient aqueous ethanol as eluting solvent from 0% to 100% in increments of 10%. HPLC analysis indicated that main polyphenols existed in fractions eluted between 40% and 50% aqueous ethanol. The fractions of interest from column were separated by HSCCC with the solvent system hexane-ethyl acetate-1% aqueous acetic acid (0.5:9.5:10, v/v/v). Ethyl acetate fractionation of the apple pomace extract followed by direct HSCCC separation by the same solvent system in the volume ratio of 1:9:10 also produced a good separation of the main polyphenols of interest. Six high-purity polyphenols were achieved tentatively and identified by HPLC/MS: chlorogenic acid (1, m/z 354), quercetin-3-glucoside/quercetin-3-glacaside (2, m/z 464), quercetin-3-xyloside (3, m/z 434), phloridzin (4, m/z 436), quercetin-3-arabinoside (5, m/z 434), and quercetin-3-rhamnoside (6, m/z 448). These results provided a preliminary foundation for further development and exploration of apple pomace.