Effects of tillage patterns and stover mulching on N2O production, nitrogen cycling genes and microbial dynamics in black soil.

Stover-covered no-tillage (NT) is of great significance to the rational utilization of stover resources and improvement of cultivated land quality, and also has a profound impact on ensuring groundwater, food and ecosystem security. However, the effects of tillage patterns and stover mulching on soil nitrogen turnover remain elusive. Based on the long-term conservation tillage field experiment in the mollisol area of Northeast China since 2007, the shotgun metagenomic sequencing of soils and microcosm incubation were combined with physical and chemical analyses, alkyne inhibition analysis to elucidate the regulatory mechanisms of NT and stover mulching on the farmland soil nitrogen emissions and microbial nitrogen cycling genes. Compared with conventional tillage (CT), NT stover mulching significantly reduced the emission of N2O instead of CO2, especially when 33% mulching was adopted, and correspondingly the nitrate nitrogen of NT33 was higher than that of other mulching amounts. The stover mulching was associated with higher total nitrogen, soil organic carbon and pH. The abundance of AOB (ammonia-oxidizing bacteria)-amoA (ammonia monooxygenase subunit A) was substantially increased by stover mulching, while the abundance of denitrification genes was reduced in most cases. Under alkyne inhibition, the tillage mode, treatment time, gas condition and interactions between them noticeably influenced the N2O emission and nitrogen transformation. In CT, NT0 (no mulching) and NT100 (full mulching), the relative contribution of AOB to N2O production was markedly higher than that of ammonia oxidizing archaea. Different tillage modes were associated with distinct microbial community composition, albeit NT100 was closer to CT than to NT0. Compared with CT, the co-occurrence network of microbial communities was more complex in NT0 and NT100. Our findings suggest that maintaining a low-quantity stover mulching could regulate soil nitrogen turnover toward proficiently enhancing soil health and regenerative agriculture, and coping with global climate change.

Evaluation of fluoride and calcium concentrations in drinking water from public water fountains on a university campus.

Optimal exposure to fluoride and calcium from tap water is beneficial for dental caries prevention. Water fountains may be an important source of drinking water in work and educational settings. The aims of this study were to quantify the fluoride and calcium concentrations of drinking water samples collected from public water fountains on the Indiana University-Purdue University Indianapolis campus; compare the fluoride and calcium concentrations in water collected at 2 different times; and determine whether the presence or absence of a visible external filter affects fluoride and calcium concentrations. Ninety samples were collected from 45 water fountains accessible to the public, and 90 duplicate samples were collected 1 month later. A fluoride ion-selective electrode was used in conjunction with an ion-specific meter to determine fluoride concentration, while atomic absorption spectrometry in an air-acetylene flame was implemented to quantify the calcium concentration. The fluoride and calcium concentration of drinking water samples displayed ranges of 0.62 mg/L to 0.97 mg/L and 56.61 mg/L to 89.11 mg/L, respectively. The concentrations of fluoride and calcium in drinking water were slightly lower at the second collection period (P < 0.001; Wilcoxon signed rank test). No statistically significant differences were observed in the fluoride or calcium concentration of drinking water collected from water fountains with an external filter cartridge in comparison with fountains that did not have a filter. Fluoride concentrations were within the optimal range recommended by the US Public Health Service for fluoridated drinking water, and calcium concentrations were consistent with those reported in previous surveillance studies.

Visualizing in situ translational activity for identifying and sorting slow-growing archaeal-bacterial consortia.

To understand the biogeochemical roles of microorganisms in the environment, it is important to determine when and under which conditions they are metabolically active. Bioorthogonal noncanonical amino acid tagging (BONCAT) can reveal active cells by tracking the incorporation of synthetic amino acids into newly synthesized proteins. The phylogenetic identity of translationally active cells can be determined by combining BONCAT with rRNA-targeted fluorescence in situ hybridization (BONCAT-FISH). In theory, BONCAT-labeled cells could be isolated with fluorescence-activated cell sorting (BONCAT-FACS) for subsequent genetic analyses. Here, in the first application, to our knowledge, of BONCAT-FISH and BONCAT-FACS within an environmental context, we probe the translational activity of microbial consortia catalyzing the anaerobic oxidation of methane (AOM), a dominant sink of methane in the ocean. These consortia, which typically are composed of anaerobic methane-oxidizing archaea (ANME) and sulfate-reducing bacteria, have been difficult to study due to their slow in situ growth rates, and fundamental questions remain about their ecology and diversity of interactions occurring between ANME and associated partners. Our activity-correlated analyses of >16,400 microbial aggregates provide the first evidence, to our knowledge, that AOM consortia affiliated with all five major ANME clades are concurrently active under controlled conditions. Surprisingly, sorting of individual BONCAT-labeled consortia followed by whole-genome amplification and 16S rRNA gene sequencing revealed previously unrecognized interactions of ANME with members of the poorly understood phylum Verrucomicrobia This finding, together with our observation that ANME-associated Verrucomicrobia are found in a variety of geographically distinct methane seep environments, suggests a broader range of symbiotic relationships within AOM consortia than previously thought.

Tandem Mass Spectrometry Imaging and in Situ Characterization of Bioactive Wood Metabolites in Amazonian Tree Species Sextonia rubra.

Driven by a necessity for confident molecular identification at high spatial resolution, a new time-of-flight secondary ion mass spectrometry (TOF-SIMS) tandem mass spectrometry (tandem MS) imaging instrument has been recently developed. In this paper, the superior MS/MS spectrometry and imaging capability of this new tool is shown for natural product study. For the first time, via in situ analysis of the bioactive metabolites rubrynolide and rubrenolide in Amazonian tree species Sextonia rubra (Lauraceae), we were able both to analyze and to image by tandem MS the molecular products of natural biosynthesis. Despite the low abundance of the metabolites in the wood sample(s), efficient MS/MS analysis of these γ-lactone compounds was achieved, providing high confidence in the identification and localization. In addition, tandem MS imaging minimized the mass interferences and revealed specific localization of these metabolites primarily in the ray parenchyma cells but also in certain oil cells and, further, revealed the presence of previously unidentified γ-lactone, paving the way for future studies in biosynthesis.

In situ, accurate, surface-enhanced Raman scattering detection of cancer cell nucleus with synchronous location by an alkyne-labeled biomolecular probe.

A surface-enhanced Raman scattering (SERS) method for in situ detection and analysis of the intranuclear biomolecular information of a cell has been developed based on a small, biocompatible, nuclear-targeting alkyne-tagged deoxyribonucleic acid (DNA) probe (5-ethynyl-2'-deoxyuridine, EDU) that can specially accumulate in the cell nucleus during DNA replications to precisely locate the nuclear region without disturbance in cell biological activities and functions. Since the specific alkyne group shows a Raman peak in the Raman-silent region of cells, it is an interior label to visualize the nuclear location synchronously in real time when measuring the SERS spectra of a cell. Because no fluorescent-labeled dyes were used for locating cell nuclei, this method is simple, nondestructive, non- photobleaching, and valuable for the in situ exploration of vital physiological processes with DNA participation in cell organelles. Graphical abstract A universal strategy was developed to accurately locate the nuclear region and obtain precise molecular information of cell nuclei by SERS.

Detection of Alkynes via Click Chemistry with a Brominated Coumarin Azide by Simultaneous Fluorescence and Isotopic Signatures in Mass Spectrometry.

Alkynes are a key component of click chemistry and used for a wide variety of applications including bioconjugation, selective tagging of protein modifications, and labeling of metabolites and drug targets. However, challenges still exist for detecting alkynes because most 1,2,3-triazole products from alkynes and azides do not possess distinct intrinsic properties that can be used for their facile detection by either fluorescence or mass spectrometry. To address this critical need, a novel brominated coumarin azide was used to tag alkynes and detect alkyne-conjugated biomolecules. This tag has several useful properties: first, it is fluorogenic and the click-chemistry products are highly fluorescent and quantifiable; second, its distinct isotopic pattern facilitates identification by mass spectrometry; and third, its click-chemistry products form a unique pair of reporter ions upon fragmentation that can be used for the quick screening of data. Using a monoclonal antibody conjugated with alkynes, a general workflow has been developed and examined comprehensively.

An application of RP-HPLC for determination of the activity of cystathionine ß-synthase and γ-cystathionase in tissue homogenates.

The RP-HPLC-based method of determination of the activity of cystathionine ß-synthase and γ-cystathionase was undertaken in mouse liver, kidney and brain. Products of the reactions, such as cystathionine, α-ketobutyrate, cysteine and glutathione, were measured using the RP-HPLC method. A difference in the cystathionine level between homogenates with totally CTH-inhibiting concentrations of DL-propargylglycine and without the inhibitor was employed to evaluate the activity of cystathionine ß-synthase. Gamma-cystathionase activity was measured using DL-homoserine as a substrate and a sensitive HPLC-based assay to measure α-ketobutyrate. The results confirmed high cystathionine ß-synthase activity and no γ-cystathionase activity in brain, and high γ-cystathionase activity in mouse liver. The method presented here allows for evaluating the relative contribution of CBS and CTH to generation of H2S in tissues. Additionally, it provides results, which reflect the redox status (GSH/GSSG) of a tissue.

Green nondegrading approach to alkyne-functionalized cellulose fibers and biohybrids thereof: synthesis and mapping of the derivatization.

Alkyne-functionalized cellulose fibers have been generated through etherification under basic water or hydroalcoholic conditions (NaOH/H(2)O/isopropanol). For a given NaOH content, the medium of reaction and, more particularly, the water/IPA ratio, were shown to be of crucial importance to derivatize the fibers without altering their integrity and their crystalline nature. It was shown that the degree of substitution (DS) of the fibers increases concomitantly with isopropanol weight ratio and that, contrary to water or water-rich conditions, derivatization of fibers under isopropanol-rich conditions induces an alteration of the fibers. Optimization of etherification conditions in aqueous media afforded functionalized cellulose materials with DS up to 0.20. Raman confocal microscopy on derivatized fibers cross sections stressed that alkyne moieties are incorporated all over the fibers. The resulting fibers were postfunctionalized by molecular probes and macromolecules in aqueous or water-rich conditions. The effectiveness of the grafting was strongly impacted by the nature of the coupling agents.

Preparation of functionalized alkynyl magnetic microspheres for the selective enrichment of cell glycoproteins based on click chemistry.

Functionalized alkynyl polyvinyl alcohol magnetic microspheres (PVA MMs) were developed for the specific enrichment of sialic acid-rich glycoproteins by click chemistry. The capture capability for proteins was evaluated through a novel dual-labeled bovine serum albumin (BSA) that utilizes fluorescence resonance energy transfer (FRET). The PVA MM parameters, including the size and coverage of functionalized groups, were optimized by response surface methodology. The optimal parameters obtained were 1.25-6.31 µm in size and 48.53-73.05% in coverage. Then, the optimal PVA MMs were synthesized, and the morphology and surface chemical properties were characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and Fourier transform infrared spectroscopy (FT-IR). To capture glycoproteins from the cell surface, a bioorthogonal chemical method was applied to metabolically label them with an azide group. The functionalized alkynyl PVA MMs showed a high specificity and strong binding capability for glycoproteins through a [3 + 2] cycloaddition reaction. The results indicated that the functionalized alkynyl PVA MMs could be applied to the enrichment of cell glycoproteins, and the merits of the MMs suggested an attractive and potential way to facilitate glycoprotein research.

Alkyne-tag Raman imaging for visualization of mobile small molecules in live cells.

Alkyne has a unique Raman band that does not overlap with Raman scattering from any endogenous molecule in live cells. Here, we show that alkyne-tag Raman imaging (ATRI) is a promising approach for visualizing nonimmobilized small molecules in live cells. An examination of structure-Raman shift/intensity relationships revealed that alkynes conjugated to an aromatic ring and/or to a second alkyne (conjugated diynes) have strong Raman signals in the cellular silent region and can be excellent tags. Using these design guidelines, we synthesized and imaged a series of alkyne-tagged coenzyme Q (CoQ) analogues in live cells. Cellular concentrations of diyne-tagged CoQ analogues could be semiquantitatively estimated. Finally, simultaneous imaging of two small molecules, 5-ethynyl-2'-deoxyuridine (EdU) and a CoQ analogue, with distinct Raman tags was demonstrated.

Commercial Carlinae radix herbal drug: botanical identity, chemical composition and antimicrobial properties.

CONTEXT: Carlinae radix is an herbal drug, commonly used by the locals in southeastern Serbia for the treatment of respiratory and urogenital diseases and, externally, for various skin conditions. There still seems to be no detailed studies correlating the chemical composition of this drug and its ethnopharmacological uses. OBJECTIVE: Chemical composition, antimicrobial activity and mode of action of C. radix essential oil, isolated from commercial samples (confirmation of whose true biological identity was also the aim of this work) were analyzed. Antimicrobial potential of decoctions (extracts prepared by boiling plant material in a given solvent), used in ethnomedicine preferentially to the pure essential oil, was also investigated. MATERIALS AND METHODS: The essential oil obtained by hydrodistillation was screened for antimicrobial activity by disc diffusion and broth microdilution methods. Effects of the oil on the growth of Staphylococcus aureus cells were investigated using turbidimetric measurements and visualized using scanning electron microscopy. Analyses of the chemical composition of the oils were done using gas chromatography and gas chromatography/mass spectrometry. RESULTS AND DISCUSSION: Both the essential oil and the decocts exhibited a very high antimicrobial activity against all tested strains, with S. aureus as the most sensitive one [e.g., for the oil sample the values for minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were 0.02, 0.04 µL/mL, respectively]. Growth curves of S. aureus demonstrated a significant decrease in turbidity (for the MIC concentration this amounted to ca. 70%) showing a concentration-dependent lysis of the cells, confirmed by scanning electron microscopy. Chemical composition, anatomical and morphological features of the sample pointed to Carlina acanthifolia L. (Asteraceae) instead of Carlina acaulis L. (Asteraceae). CONCLUSION: The results showed significant antimicrobial effect of the essential oil and the decoctions and support the use of this plant in ethnomedicine for the treatment of various human infections, especially those caused by S. aureus. Adulteration of the drug would not cause significant differences in its biological activity, since chemical composition of the sample showed high similarity with those containing C. acaulis roots.

[Analysis of a Typical Ozone Pollution Process in Guangzhou in Winter].

This study focused on an ozone pollution event occurring in winter (January) in Guangzhou. Various influencing factors were analyzed, including various atmospheric trace gases, meteorological conditions during the whole pollution process, as well as the characteristics of the main O3 precursor volatile organic compounds (VOCs). The main sources of VOCs and the O3 formation regime were analyzed using an array of tools:the ozone potential formation (OFP), positive matrix factorization (PMF) model, and empirical kinetic modeling approach (EKMA) curve. Feasible strategies for O3 control were suggested. The results showed that O3 and NO2 exceeded the corresponding standards in this winter pollution event, when the concentrations of PM10 and PM2.5 were also high, differing from the air pollution characteristics in summer and autumn. Low boundary layer height (<75 m) and high atmospheric stability at night exacerbated the accumulation of ozone precursors and fine particles. Meteorological conditions such as the increased daytime temperature (5℃), stronger solar radiation (10%), and low horizontal wind speed (<1 m·s-1) favored photochemical reactions and promoted the formation of ozone and fine particles. VOCs were mainly composed of alkanes, and the proportions of alkanes and alkynes in winter were higher than those in the other seasons. Aromatics (xylenes and toluene) and propylene were the key VOCs species leading to O3 formation. The main VOCs sources were vehicle exhaust (22.4%), solvent usage (20.5%), and industrial emissions (17.9%); however, the source with highest OFP was identified as solvent usage. O3 formation in this event was in the VOCs-limited regime, and reducing O3 precursors in the VOCs/NOx ratio of 3:1 was effective and feasible for O3 control. This study explored the causes of an O3 pollution event in winter, which will serve as reference for the synergistic control of O3 and PM2.5 in heavy pollution seasons.

Cu(I)-catalyzed tandem benzyldiazoester coupling with terminal alkyne-allene formation-Michael reaction: application to the syntheses of oxa and azacycles.

A simple and practical procedure for the synthesis of aza- and oxacycles, which possess an array of stereogenic functionalities, is described. This protocol relies on tandem Cu-catalyzed coupling of suitably functionalized terminal alkyne with diazoester followed by isomerization and subsequent aza or oxa-Michael reaction, thus generating the required scaffold with high diastereoselectivity.

Comparison of diffusion coefficients for matched pairs of macrocyclic and linear molecules over a drug-like molecular weight range.

The diffusion coefficients of a series of closely matched pairs of macrocyclic and linear molecules have been compared using NMR spectroscopy. The macrocyclic series was designed both to overlap with and extend beyond the molecular weight range typically employed for drug-like molecules. The linear molecules each represent a carbogenic fission of their macrocyclic counterparts, designed to minimize differences in functionality and physicochemical properties. Each series of molecules was prepared using copper catalyzed azide-alkyne cycloaddition (CuAAC) reactions conducted in a flow using a copper tube. The macrocyclic series exhibited consistently higher diffusion across the entire molecular weight range studied. The fold difference in diffusion coefficients between the macrocyclic and linear analogues appeared to be independent of either solvent viscosity or dielectric environment.

HPLC method development and validation of cytotoxic agent phenyl-heptatriyne in Bidens pilosa with ultrasonic-assisted cloud point extraction and preconcentration.

Extraction and pre-concentration of a bioactive marker compound, phenyl-1,3,5-heptatriyne from Bidens pilosa, prior to HPLC has been demonstrated using both organic and ecofriendly solvents. Non-ionic surfactants, viz. Triton X-100, Triton X-114 and Genapol X-80, were used for extraction. No back-extraction or liquid chromatographic steps were required to remove the target phytochemical from the surfactant-rich extractant phase. The optimized cloud point extraction procedure has been shown to be a potentially useful methodology for the preconcentration of the target analyte, with a preconcentration factor of 4-99. Moreover, the method is simple, sensitive, rapid and consumes lesser solvent than traditional methods. An isocratic chromatographic separation and quantitation was accomplished on a C(18) column with acetonitrile-acidified aqueous as mobile phase at a flow rate of 1.0 mL/min, UV detection at 254 nm and specificity with photo diode-array detector (PDA) and MS. Under the optimum experimental conditions recovery was satisfactory (99.18-100.33%) without interference from the surfactant. The method seems to be reliable with intraday precision and interday precision below 2.0%. Good linearity was obtained in the working range from 7.5 to 30 µg/mL with correlation coefficient >0.99. The limits of detection and quantitation were 1.84 and 6.13 µg/mL, respectively. The method was validated following international guidelines and successfully applied for quantitative assays of cytotoxic compound phenyl-1,3,5-heptatriyne in Bidens pilosa.

Effect of polyacetylenic acids from Prunella vulgaris on various plant pathogens.

AIMS: This study is aiming at characterizing antifungal substances from the methanol extract of Prunella vulgaris and at investigating those substances' antifungal and antioomycete activities against various plant pathogens. METHODS AND RESULTS: Two polyacetylenic acids were isolated from P. vulgaris as active principles and identified as octadeca-9,11,13-triynoic acid and trans-octadec-13-ene-9,11-diynoic acid. These two compounds inhibited the growth of Magnaporthe oryzae, Rhizoctonia solani, Phytophthora infestans, Sclerotinia sclerotiorum, Fusarium oxysporum f. sp. raphani, and Phytophthora capsici. In addition, these two compounds and the wettable powder-type formulation of an n-hexane fraction of P. vulgaris significantly suppressed the development of rice blast, tomato late blight, wheat leaf rust, and red pepper anthracnose. CONCLUSIONS: These data show that the extract of P. vulgaris and two polyacetylenic acids possess antifungal and antioomycete activities against a broad spectrum of tested plant pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the occurrence of octadeca-9,11,13-triynoic acid and trans-octadec-13-ene-9,11-diynoic acid in P. vulgaris and their efficacy against plant diseases. The crude extract containing the two polyacetylenic acids can be used as a natural fungicide for the control of various plant diseases.

Drug Distribution in Living Cells via Label-Free Molecular Fingerprint.

A new application of stimulated Raman scattering (SRS) uses the benefit of a label-free molecular fingerprint to image the uptake and distribution of an alkyne-based drug in living cells. This method delivers information on cellular molecular composition and drug-cell interaction, showing the potential of SRS in drug development.

Separation of triacylglycerols containing allenic and acetylenic fatty acids by enantiomeric liquid chromatography-mass spectrometry.

Triacylglycerols (TAGs) containing less common fatty acids (FAs) were isolated from the seeds of three plants (Santalum album, Crepis foetida, and Leucas aspera). These FAs had allenic (laballenic acid, Lb) and acetylenic (crepenynic, C; ximenynic acids, Xi) bonds. TAGs were analyzed on reversed-phase and chiral columns. High-resolution tandem mass spectrometry identified TAGs by positive electrospray ionization (ESI+). Twenty-two molecular species of TAGs isolated from the seed oil of Santalum album were separated by RP-HPLC and chiral HPLC methods and identified by positive electrospray ionization tandem MS detection (ESI+-MS). Two major enantiomers, i.e., sn-OOLb and sn-LLLb (O represents oleic acid; and L represents linoleic acid), were synthesized from the appropriate phosphatidylcholines. This allowed the identification of enantiomers after separation by chiral chromatography by tandem mass spectrometry. Similarly, TAGs from the seeds of Crepis foetida, and Leucas aspera were analyzed by reversed-phase chromatography and identified by mass spectrometry. Four enantiomers (sn-OOC, sn-LLC, sn-OOXi, and sn-LLXi) were synthesized. A total of six and three enantiomers of TAGs containing crepenynic and ximenynic acids, respectively, were identified by chiral column analysis. The retention times of TAGs containing allenic and acetylenic bonds were always greater on the reversed-phase column than TAGs with the same number of carbon atoms and the same unsaturation (e.g., LLL versus LLLb). From the chiral column, the regioisomers and enantiomers were eluted in the order of symmetric-asymmetric-asymmetric (i.e., sn-OCO, sn-COO, and sn-OOC). Through tandem mass spectrometry, we were able to identify and distinguish regioisomer [DAG]+-type ions, i.e., [MNH4NH3RCOOH]+, that can be considered diagnostic. Unfortunately, enantiomers and TAGs with the same numbers of carbon atoms and the same unsaturation levels have identical mass spectra, such as LLL and LLLb.

Dihydromatricaria acid: acetylenic acid secreted by soldier beetle.

The aposematic cantharid beetle Chauliognathus lecontei produces a defensive secretion, from glands in its thorax and abdomen, containing 8-cis-dihydromatricaria acid. Similar acetylenic compounds are known only from certain fungi and flowering plants.

Ternary hybrid TiO2-PANI-AuNPs for photocatalytic A3-coupling of aldehydes, amines and alkynes: First photochemical synthesis of propargyl amines.

The present paper describes the successful synthesis and application of a ternary hybrid consists of gold nanoparticles decorated on titania-polyaniline (TiO2-PANI-AuNPs) for A3-coupling between aldehydes, terminal alkynes and amines under visible irradiation. The synthesis of ternary hybrid involved the coating of PANI on TiO2 via an oxidative polymerization method followed by the deposition of AuNPs on TiO2-PANI using trisodium citrate as a reducing agent. The synthesized photocatalyst was characterized by various techniques like FT-IR, SEM, HR-TEM, XRD, UV-VIS, ICP-AES, TGA, XPS and PL spectroscopy. The synthesized photocatalyst exhibited higher efficiency which was presumed due to the suppressed electron-hole recombination and better electron mobility at the surface of the photocatalyst. In the hybrid, polyaniline was found to enhance the activity as well as stability of the photocatalyst owing to its chemical interaction with titania and gold. Furthermore, after the reaction, the photocatalyst could readily be recovered and recycled for several runs with consistent photoactivity.