Preparation of Uracil by bacteria isolated from Morchella.

Morchella is one of the most famous rare edible and medicinal fungi over the world. Highly nutritious and immature cultivation techniques led to the high price and the markets have remained tight. The pathogenic bacteria were serious in artificial cultivation of Morchella that affected the growth and yield of Morchella. Isolation of pathogenic bacteria and metabolites were investigated in order to improve the artificial cultivation technology. The isolated strain (YDJZ-01-01C) was identified by Gram staining and sequence of 16S rDNA. Structures of metabolites were confirmed based on NMR spectra and literatures. However, the main products were uracil and thymine that considered as important intermediate of anti-tumor 5-fluorouracil. Interestingly, a new synthetic pathway for preparation of uracil by microorganism was found except for chemical synthesis. The new preparation pathway provided mild, green, sustainable and environment friendly method to produce uracil that meets the needs of modern chemistry.

A low-cost, open-source digital stripchart recorder for chromatographic detectors using a Raspberry Pi.

One of the most critical aspects of chromatographic analysis is effective data acquisition and processing. Typical approaches include software platforms designed for specific instruments or commercial data acquisition hardware boards, both of which require expensive licenses to use and operate. To increase the access and affordability of chromatographic data acquisition, especially for systems in which software control has become obsolete or must be written in-house, an open-source digital stripchart recorder has been developed. This system is built upon a Raspberry Pi single-board computer and a plug-in printed circuit board with the necessary integrated circuits for data acquisition. Using an open-source software called Processing, a complete user interface to control the system was developed that enables the acquisition, filtering, and processing of chromatographic data. The system performs comparably to more expensive platforms, with calculated values for peak area, retention time, and plate count all within 3% of the values calculated by a widely used commercial chromatography data software package.

Dynamic evaluation of a trilobal capillary-channeled polymer fiber shape for reversed phase protein separations and comparison to the eight-channeled form.

A new, trilobal-shaped capillary-channeled polymer fiber is under development to address the issues of poor A-term performance of the previous eight-channeled form. The trilobal geometry should provide better packing homogeneity due to the fewer potential orientations of the symmetric fiber geometry. Comparisons of separation efficiency and peak shape were made between the two fiber shapes through several dynamic parameters. Column hydrodynamics were investigated with two marker compounds, uracil and bovine serum albumin, with van Deemter plots of those two compounds revealing differences in the packing qualities between the different fiber shapes. Parametric fitting to the van Deemter, Knox, and Giddings equations provides insights into the column physical structures. Separation quality for both shapes was evaluated across differences in fiber packing density, gradient rate, and mobile phase linear velocity for the reversed phase separation of a four protein mixture, containing ribonuclease A, cytochrome c, lysozyme, and myoglobin. The results of this study lay the ground work for future efforts in the use of trilobal fibers for the separation of biomacromolecules.

Determination of the Main Nucleosides and Nucleobases in Natural and Cultured Ophiocordyceps xuefengensis.

Ophiocordyceps xuefengensis, a recently described species of Ophiocordycepsthat is associated with the larvae of Phassusnodus (Hepialidae) in the living root or trunk of the medicinal plant Clerodendrumcyrtophyllum, isthe largest known Cordycepsspecies and is recognized as a desirable alternative for natural Ophiocordycepssinensis. This study investigated the main nucleosides and nucleobases in natural and cultured Ophiocordycepsxuefengensis. The contents of the nucleosides and nucleobases in the natural and cultured samples were determined by reverse phase HPLC. The highest concentration of adenosine was found in the natural fruit body and the cultured stroma, with almost no adenosine in the cadaver of Phassusnodus. The contents of adenine, guanosine, uridine and uracil in the cultured mycelium were significantly higher than those in the natural sample. Inosine was only detected in the natural samples. Thymidine and 2-deoxyadenosine were only found in the cadaver of Phassusnodus. Cordycepin was not detected in the five samples examined. These results suggested that the cultured mycelium and cultured stroma of Ophiocordycepsxuefengensis might be a promising substitute for natural O. xuefengensis.

First occurrence of cylindrospermopsin in Portugal: a contribution to its continuous global dispersal.

Cylindrospermopsin (CYN) was found to occur in Portugal for the first time. In this study CYN values varied from a minimum of 1.4 µg L-1 to a maximum of 12 µg L-1 detected through HPLC technique and confirmed by LC-MS method. Amplification of the cyrC gene was done and was confirmed to be from the genera Aphanizomenon. This study is therefore an important contribution to the knowledge on the dispersal and biogeography of CYN.

Production of uracil from methane by a newly isolated Methylomonas sp. SW1.

Methane is an abundant, inexpensive one-carbon feedstock and one of the most powerful greenhouse gases. Because it does not compete with food demand, it is considered a promising carbon feedstock for the production of valuable products using methanotrophic bacteria. Here, we isolated a novel methanotrophic bacterium, Methylomonas sp. SW1, from a sewage sample obtained from Wonju City Water Supply Drainage Center, Republic of Korea. The conditions for uracil production by Methylomonas sp. SW1, such as Cu2+ concentration and temperature were investigated and optimized. As a result, Methylomonas sp. SW1 produced uracil from methane as a sole carbon source with a titer of 2.1mg/L in 84h without genetic engineering under the optimized condition. The results in this study demonstrate the feasibility of using Methylomonas sp. SW1 for the production of uracil from methane. This is the first report of uracil production from gas feedstock by methanotrophic bacteria.

Peak deconvolution to correctly assess the band broadening of chromatographic columns.

The present study provides experimental evidence for the fact that the peak deconvolution method can be applied to accurately measure the column-only dispersion of the current generation of high speed and high efficiency columns. Unlike the conventional variance difference method, it furthermore preserves any prevailing asymmetry of the column-only peak. This has been demonstrated by testing the same column on three different system configurations, with different extra-column volumes, and showing that, after deconvolution, the resulting column-only peaks coincide very well and produce very similar column-only plate height values (typical relative standard deviation comprising all runs on three different system configurations is 2-2.5%). Extensively studying a large set of theoretically produced peaks (with exactly known variance and asymmetry), it could be shown that the main criterion for the validity of the deconvolution method is that the variance of the system-only peak is minimum 1.5 times smaller than the variance of the column+system peak. The need to add a radial mixer unit to accurately assess the system-only contributions has been demonstrated as well. To illustrate its use and merits, the deconvolution method has been used to establish so-called multiple van Deemter curves, wherein plate height curves relating to different peak width definitions are shown in the same plot. These plots can give new insights in the intrinsic asymmetry of the column-only dispersion.

Is qPCR a Reliable Indicator of Cyanotoxin Risk in Freshwater?

The wide distribution of cyanobacteria in aquatic environments leads to the risk of water contamination by cyanotoxins, which generate environmental and public health issues. Measurements of cell densities or pigment contents allow both the early detection of cellular growth and bloom monitoring, but these methods are not sufficiently accurate to predict actual cyanobacterial risk. To quantify cyanotoxins, analytical methods are considered the gold standards, but they are laborious, expensive, time-consuming and available in a limited number of laboratories. In cyanobacterial species with toxic potential, cyanotoxin production is restricted to some strains, and blooms can contain varying proportions of both toxic and non-toxic cells, which are morphologically indistinguishable. The sequencing of cyanobacterial genomes led to the description of gene clusters responsible for cyanotoxin production, which paved the way for the use of these genes as targets for PCR and then quantitative PCR (qPCR). Thus, the quantification of cyanotoxin genes appeared as a new method for estimating the potential toxicity of blooms. This raises a question concerning whether qPCR-based methods would be a reliable indicator of toxin concentration in the environment. Here, we review studies that report the parallel detection of microcystin genes and microcystin concentrations in natural populations and also a smaller number of studies dedicated to cylindrospermopsin and saxitoxin. We discuss the possible issues associated with the contradictory findings reported to date, present methodological limitations and consider the use of qPCR as an indicator of cyanotoxin risk.

Escherichia coli-Derived Uracil Increases the Antibacterial Activity and Growth Rate of Lactobacillus plantarum.

Lactobacillus plantarum (L. plantarum) is a representative probiotic. In particular, L. plantarum is the first commensal bacterium to colonize the intestine of infants. For this reason, the initial settlement of L. plantarum can play an important role in determining an infant's health as well as their eventual health status as an adult. In addition, L. plantarum combats pathogenic infections (such as Escherichia coli (E. coli), one of the early pathogenic colonizers in an unhealthy infant gut) by secreting antimicrobial substances. The aim of this research was to determine how L. plantarum combats E. coli infection and why it is a representative probiotic in the intestine. Consequently, this research observed that E. coli releases uracil. L. plantarum specifically recognizes E. coli-derived uracil, which increases the growth rate and production of antimicrobial substance of L. plantarum. In addition, through the inhibitory activity test, this study postulates that the antimicrobial substance is a protein and can be considered a bacteriocin-like substance. Therefore, this research assumes that L. plantarum exerts its antibacterial ability by recognizing E. coli and increasing its growth rate as a result, and this phenomenon could be one of the reasons for L. plantarum settling in the intestine of infants as a beneficial bacterium.

The effects of water sample treatment, preparation, and storage prior to cyanotoxin analysis for cylindrospermopsin, microcystin and saxitoxin.

Cyanobacterial harmful algal blooms occur in freshwater lakes, ponds, rivers, and reservoirs, and in brackish waters throughout the world. The wide variety of cyanotoxins and their congeners can lead to frequent exposure of humans through consumption of meat, fish, seafood, blue-green algal products and water, accidental ingestion of contaminated water and cyanobacterial scum during recreational activities, and inhalation of cyanobacterial aerosols. Cyanotoxins can also occur in the drinking water supply. In order to monitor human exposure, sensitive analytical methods such as enzyme linked immunosorbent assay and liquid chromatography-mass spectrometry are often used. Regardless of the analytical method of choice, some problems regularly occur during sample collection, treatment, storage, and preparation which cause toxin loss and therefore underestimation of the true concentration. To evaluate the potential influence of sample treatment, storage and preparation materials on surface and drinking water samples, the effects of different types of materials on toxin recovery were compared. Collection and storage materials included glass and various types of plastics. It was found that microcystin congeners LA and LF adsorbed to polystyrene, polypropylene, high density polyethylene and polycarbonate storage containers, leading to low recoveries (<70%), cylindrospermopsin and saxitoxin did not adsorb to the containers tested. Therefore, this study shows that glass or polyethylene terephthalate glycol containers are the materials of choice for collection and storage of samples containing the cyanotoxins cylindrospermopsin, microcystins, and saxitoxin. This study also demonstrated that after 15 min chlorine decreased the concentration of microcystin LR to <40%, microcystin LA and saxitoxin to <15%, therefore quenching of drinking water samples immediately upon sample collection is critical for accurate analysis. In addition, the effect of various drinking water treatment chemicals on toxin recovery and the behavior of those chemicals in the enzyme linked immunosorbent assays were also studied and are summarized.

Effects of Cylindrospermopsin Producing Cyanobacterium and Its Crude Extracts on a Benthic Green Alga-Competition or Allelopathy?

Cylindrospermopsin (CYN) is a toxic secondary metabolite produced by filamentous cyanobacteria which could work as an allelopathic substance, although its ecological role in cyanobacterial-algal assemblages is mostly unclear. The competition between the CYN-producing cyanobacterium Chrysosporum (Aphanizomenon) ovalisporum, and the benthic green alga Chlorococcum sp. was investigated in mixed cultures, and the effects of CYN-containing cyanobacterial crude extract on Chlorococcum sp. were tested by treatments with crude extracts containing total cell debris, and with cell debris free crude extracts, modelling the collapse of a cyanobacterial water bloom. The growth inhibition of Chlorococcum sp. increased with the increasing ratio of the cyanobacterium in mixed cultures (inhibition ranged from 26% to 87% compared to control). Interestingly, inhibition of the cyanobacterium growth also occurred in mixed cultures, and it was more pronounced than it was expected. The inhibitory effects of cyanobacterial crude extracts on Chlorococcum cultures were concentration-dependent. The presence of C. ovalisporum in mixed cultures did not cause significant differences in nutrient content compared to Chlorococcum control culture, so the growth inhibition of the green alga could be linked to the presence of CYN and/or other bioactive compounds.

Itches-stimulating compounds from Colocasia esculenta (taro): bioactive-guided screening and LC-MS/MS identification.

Colocasia esculenta (L.) Schoot (taro) is one of the most common crops in the world. Its rhizome was a tonic medicine and accustomed to treat some gastrointestinal disorders in traditional Chinese medicine. Today, the taro was further developed as anticancer prescription in herbal therapy. However, the mucilage of the fresh taro has irritation, and causes itchy feeling. The components in the mucilage were not evident up to now. Two active compounds, uracil and glycol-protein taro lectin (Accession number: A5HMM7), were purified and identified from the fresh taro. The glycol-protein taro lectin showed nerve stimulation activity on dorsal root ganglion (DRG) neurons from GCaMP transgenic mice at the concentration of 1mg/mL.

Degradation of cyanotoxin cylindrospermopsin by TiO2-assisted ozonation in water.

The stable tricyclic structure of the cylindrospermopsin (CYN), a cynotoxin, has presented several challenges to water treatment facilities, as conventional treatment methods have a limited ability to remove it from water. This study examines the effectiveness of titanium dioxide (TiO2) in catalytic ozonation for degrading CYN. The chemical kinetics of the reactions of ozone (O3) and hydroxyl radicals (OH(•)) with CYN were determined. The results reveal that TiO2 significantly increases the rate of degradation of CYN by increasing the rate of production of hydroxyl radicals (OH(•)) by initiating the decomposition of O3 on the surface of the catalyst. At a pH of 7 with 1.0 mg L(-1) O3 and 500 mg L(-1) TiO2; the pseudo-first-order ozone decomposition rate constant (k(D)) increased from 3.04 × 10(-3) to 16.53 × 10(-3) s(-1) and the ratio of OH(•) to O3 concentrations (R(ct)) increased from 1.87 × 10(-8) to 126.4 × 10(-8). The calculated second-order rate constant (k(overall)) of the reaction of CYN with O3 and OH(•) was 3.22 M(-1)s(-1) without TiO2. However, the greatest improvement in k(overall) in this study was observed using 500 mg TiO2 L(-1), which increased koverall by a factor of five. TiO2-catalyzed ozonation is an efficient method of oxidation that reduces the toxic activity of CYN. The results of a Microtox test concerning the toxic activity of CYN during oxidation reveal that catalytic ozonation may either increase or reduce the toxicity of CYN toward test samples. The toxic effects of CYN on the samples are greatly influenced by the TiO2 dosage and reaction time, possibly yielding by-products that may change the mutagenic properties of CYN. Three water samples from a eutrophic lake in Taiwan were examined to evaluate the effect of dissolved organic carbon (DOC) and alkalinity on the oxidation of CYN. DOC had the greatest effect on the oxidation of CYN in the ozonation of eutrophic water. Overall, the degree of CYN oxidation depended on the rate constant of the reaction with ozone and the consumption of ozone by the natural water matrix.

Poly (9-(2-diallylaminoethyl)adenine HCl-co-sulfur dioxide) deposited on silica nanoparticles constructs hierarchically ordered nanocapsules: curcumin conjugated nanocapsules as a novel strategy to amplify guanine selectivity among nucleobases.

Poly (9-(2-diallylaminoethyl)adenine HCl-co-sulfur dioxide) (Poly A) deposited on silica nanoparticles self-assembles to form hierarchically ordered nanocapsules. These nanocapsules can be conjugated with curcumin. The curcumin-conjugated nanocapsules are found to be spherical in size and their size ranges between 200 and 600 nm. We found that curcumin conjugated with silica nanoparticles marginally shows a selectivity (∼20%) for guanine over adenine, cytosine, thymine and uracil, but this selectivity is extraordinarily amplified to more than 500% in curcumin-conjugated nanocapsules prepared from the above procedure. FT-IR spectra along with lifetime measurements suggest that specific interaction between adenine moieties of Poly A nanocapsules and thymine/uracil does not affect the fluorescence of poly A nanocapsules. Thus, the sensitivity and selectivity for guanine estimation is due to hydrophobic interactions, which are assisted by the low water solubility of guanine as compared to the other nucleobases. The present method illustrates a wider linear dynamic range in the higher concentration range as compared to the reported methods. Finally, the degradation study proves that stability of curcumin is improved dramatically in such nanocapsules demonstrating that nanotechnology could be a viable method to improve selectivity of specific analyte and robustness of probe molecule during fluorescence based bio-sensing.

Flow rate dependent extra-column variance from injection in capillary liquid chromatography.

Efficiency and resolution in capillary liquid chromatography (LC) can be significantly affected by extra-column band broadening, especially for isocratic separations. This is particularly a concern in evaluating column bed structure using non-retained test compounds. The band broadening due to an injector supplied with a commercially available capillary LC system was characterized from experimental measurements. The extra-column variance from the injection valve was found to have an extra-column contribution independent of the injection volume, showing an exponential dependence on flow rate. The overall extra-column variance from the injection valve was found to vary from 34 to 23 nL. A new mathematical model was derived that explains this exponential contribution of extra-column variance on chromatographic performance. The chromatographic efficiency was compromised by ∼130% for a non-retained analyte because of injection valve dead volume. The measured chromatographic efficiency was greatly improved when a new nano-flow pumping system with integrated injection valve was used.

Multi-detection method for five common microalgal toxins based on the use of microspheres coupled to a flow-cytometry system.

Freshwater and brackish microalgal toxins, such as microcystins, cylindrospermopsins, paralytic toxins, anatoxins or other neurotoxins are produced during the overgrowth of certain phytoplankton and benthic cyanobacteria, which includes either prokaryotic or eukaryotic microalgae. Although, further studies are necessary to define the biological role of these toxins, at least some of them are known to be poisonous to humans and wildlife due to their occurrence in these aquatic systems. The World Health Organization (WHO) has established as provisional recommended limit 1µg of microcystin-LR per liter of drinking water. In this work we present a microsphere-based multi-detection method for five classes of freshwater and brackish toxins: microcystin-LR (MC-LR), cylindrospermopsin (CYN), anatoxin-a (ANA-a), saxitoxin (STX) and domoic acid (DA). Five inhibition assays were developed using different binding proteins and microsphere classes coupled to a flow-cytometry Luminex system. Then, assays were combined in one method for the simultaneous detection of the toxins. The IC50's using this method were 1.9±0.1µg L(-1) MC-LR, 1.3±0.1µg L(-1) CYN, 61±4µg L(-1) ANA-a, 5.4±0.4µg L(-1) STX and 4.9±0.9µg L(-1) DA. Lyophilized cyanobacterial culture samples were extracted using a simple procedure and analyzed by the Luminex method and by UPLC-IT-TOF-MS. Similar quantification was obtained by both methods for all toxins except for ANA-a, whereby the estimated content was lower when using UPLC-IT-TOF-MS. Therefore, this newly developed multiplexed detection method provides a rapid, simple, semi-quantitative screening tool for the simultaneous detection of five environmentally important freshwater and brackish toxins, in buffer and cyanobacterial extracts.

First report of cylindrospermopsin production by two cyanobacteria (Dolichospermum mendotae and Chrysosporum ovalisporum) in Lake Iznik, Turkey.

Cylindrospermopsin (CYN) is a cytotoxic alkaloid produced by cyanobacteria. The distribution of this toxin is expanding around the world and the number of cyanobacteria species producing this toxin is also increasing. CYN was detected for the first time in Turkey during the summer months of 2013. The responsible species were identified as Dolichospermum (Anabaena) mendotae and Chrysosporum (Aphanizomenon) ovalisporum. The D. mendotae increased in May, however, C. ovalisporum formed a prolonged bloom in August. CYN concentrations were measured by LC-MS/MS and ranged from 0.12 µg·mg⁻¹ to 4.92 µg·mg⁻¹ as dry weight, respectively. Both species were the only cyanobacteria actively growing and CYN production was attributed solely to these species. Despite CYN production by C. ovalisporum being a well-known phenomenon, to our knowledge, this is the first report of CYN found in D. mendotae bloom.

[Alkaloids from cervi cornu pantotrichum and its effect on murine splenocytes proliferation].

OBJECTIVE: To study the alkaloids of Cervi Cornu Pantotrichum and its effect on murine splenocytes proliferation. METHODS: The constituents isolation and purification from Cervi Cornu Pantotrichum was carried out by reported column chromatography including Sephadex LH-20 and MCI (CHP20P) and their structures were elucidated on the basis of spectral compounds. The method of MTT was used to examine the effects of eight alkaloids and total alkaloids content (TAC) of Cervi Cornu Pantotrichum on murine splenocytes proliferation. RESULTS: Eleven compounds were isolated from Cervi Cornu Pantotrichum, and their structures were identified as follows: uracil (1), hypoxanthine (2), uridine (3) inosine (4), guanosine (5), 2'-deoxyguanosine (6), guanine (7), thymidine (8), thymine (9), cytidine (10) and adenosine (11). By the experiment of murine splenocytes proliferation activity in vitro, the results showed that the total alkaloids, uracil and adenosine had significantly promoted the proliferation of mouse spleen cells. CONCLUSION: Compounds 4 - 11 are isolated from Cervi Cornu Pantotrichum for the first time. The total alkaloids is one of the material basis of immunomodulatory effects of Cervi Cornu Pantotrichum, and uracil and adenosine are the most active.

Real-time analysis of self-assembled nucleobases by Venturi easy ambient sonic-spray ionization mass spectrometry.

In this study, the real-time analysis of self-assembled nucleobases was employed by Venturi easy ambient sonic-spray ionization mass spectrometry (V-EASI-MS). With the analysis of three nucleobases including 6-methyluracil (6MU), uracil (U) and thymine (T) as examples, different orders of clusters centered with different metal ions were recorded in both positive and negative modes. Compared with the results obtained by traditional electrospray ionization mass spectrometry (ESI-MS) under the same condition, more clusters with high orders, such as [6MU7+Na](+), [6MU15+2NH4](2+), [6MU10+Na](+), [T7+Na](+), and [T15+2NH4](2+) were detected by V-EASI-MS, which demonstrated the soft ionization ability of V-EASI for studying the non-covalent interaction in a self-assembly process. Furthermore, with the injection of K(+) to the system by a syringe pumping, the real-time monitoring of the formation of nucleobases clusters was achieved by the direct extraction of samples from the system under the Venturi effect. Therefore, the effect of cations on the formation of clusters during self-assembly of nucleobases was demonstrated, which was in accordance with the reports. Free of high voltage, heating or radiation during the ionization, this technique is much soft and suitable for obtaining the real-time information of the self-assembly system, which also makes it quite convenient for extraction samples from the reaction system. This "easy and soft" ionization technique has provided a potential pathway for monitoring and controlling the self-assembly processes.

Separation of alogliptin enantiomers in cyclodextrin-modified capillary electrophoresis: a validated method.

The single enantiomer drug, alogliptin (Alo, Nesina®) is a novel, orally available and selective dipeptidyl peptidase-4 inhibitor used for the treatment of type II diabetes. Following its pKa determination by CE-pH titration, a validated chiral CE method has been developed to separate Alo enantiomers. Preliminary screening of the native CDs and their ten derivatives revealed that sulfopropylated-γ-CD, sulfopropylated-ß-CD and sulfopropylated-γ-CD, sulfobutyl-ether-ß-CD (SBE-ß-CD) and sulfobutyl-ether-γ-CD enabled enantioresolution. Furthermore, cavity size dependent enantiomer migration order reversal was observed between γ- and ß-CD derivatives. To improve enantioseparation, buffer composition and pH, CD concentration, applied voltage, temperature, and injection parameters were optimized for the Alo/ SBE-ß-CD system, yielding a resolution of 8.34. RSD percentage of the resolution value, migration times, and corrected peak areas were below 3 and 5% during testing repeatability and intermediate precision. LOD and LOQ values were found to be 2 and 6 µg/mL, respectively, for each enantiomer. Calibration lines ranging from 6 to 250 µg/mL were constructed with r(2) > 0.9997. Robustness could be successfully verified by using the Plackett-Burman statistical experimental design. The optimized system containing 5 mM SBE-ß-CD in a 25 mM acetate buffer at pH 4.75 was found promising to detect 0.1% distomer in the presence of the API.