Synthesis of functionalized analogs of pyochelin, a siderophore of Pseudomonas aeruginosa and Burkholderia cepacia

Using an improved synthesis of pyochelin, a siderophore common to several pathogenic Pseudomonas species, three functionalized pyochelin analogs were efficiently synthesized starting from appropriate 2-hydroxybenzonitriles.     

Structure and X-ray conformation of pseudodesmins A and B, two new cyclic lipodepsipeptides from Pseudomonas bacteria

Two new cyclic lipodepsipeptides named pseudodesmins A and B have been isolated from Pseudomonas bacteria collected from the mucus layer in the skin of the black belly salamander. Both compounds show moderate antibacterial activity against Gram positive bacteria, including MRSA. Complete ¹H, ¹³C and ¹⁵N NMR assignment of both compounds afforded their covalent structure and served to guide the analysis of LC-MS and X-ray diffraction data from which the final stereochemistry could be established. Both molecules can be categorized as new members of the viscosin group of cyclic lipodepsipeptides.     

Use of non-porous pillar array columns for the separation of Pseudomonas pyoverdine siderophores as an example of a real-world biological sample

We report on the first separation of a complex biomixture in pressure-driven mode using perfectly ordered pillar array columns. The separations were conducted in the reversed-phase mode using a highly aqueous mobile phase, while the outer surface of the non-porous pillars was chemically functionalized with a hydrophobic C8-layer. The samples originated from two different bacterial strains (Pseudomonas aeruginosa PAO1 and Pseudomonas sp. W15Feb38) of fluorescent pseudomonads. These produce fluorescent yellow-green pyoverdines that serve as siderophores to shuttle iron inside the cell. The pyoverdines of both strains were prepared from the supernatant through a crude solid phase extraction without any further purification step. In case of the PAO1 mixture, a separation of 15 components within a column length of 2.5 cm could be observed through the transparent cover glass of the chip. For the W15Feb38 mixture, a separation of eight components could be observed within the same distance. These fast chromatographic separations were compared with those obtained via iso-electrofocusing (IEF), which is the traditionally employed fingerprinting method to characterize pseudomonad strains based on their pyoverdine profiles (siderotyping). With this technique, and despite the injection of a 10,000 times larger sample mass, only nine bands were maximally observed for the PAO1 mixture, whereas maximally six bands were observed in case of the W15Feb38 mixture. The chromatographic pillar array method, yielding a separation in less than 1 min, was also significantly faster than the IEF method, which typically needs 1.5 h. The present system can therefore be considered as a potential alternative fingerprinting tool for the fast identification of different strains of fluorescent pseudomonads, including as diagnostic tool for typing strains of the important opportunistic pathogen P. aeruginosa.     

An easily-automated assay for the physiological state quantification of Pseudomonas sp. M18

In order to foreknow poorly performing cultures before wasting energy to scale them to large cultures, industrial microbial fermentation can greatly benefit from knowledge of the physiological state of cells. The method currently proposed is an easily automated physiological state determination method. We have designed one universal rRNA-specific probe for bacteria and developed novel signal probe hybridization (SPH) assay featuring no RNA extraction and no PCR amplification steps necessary to quantify the physiological state of microbial cells. The microbial cell was lysed with sonication and SDS. Signal probes were applied to hybridize and protect the rRNA target. S1 nuclease was then applied to remove the excessive signal probes, the single-stranded RNA and the mismatch RNA/DNA hybrids. The remaining signal probe was captured with a corresponding capture probe immobilized on a microplate and quantified with a horseradish peroxidase-conjugated color reaction. We then systemically optimized our assay. Results showed that the cell limit of detection (LOD) and the cell limit of quantification (LOQ) were 2.64 × 10⁴ cells and 9.86 × 10⁴ cells per well of microplate, respectively. The limit of detection (LOD) and the limit of quantification (LOQ) of signal probe were 49.0 fM and 344.0 fM respectively. Using this technique, we quantified the 16S rRNA levels during the fermentation process of Pseudomonas sp. M18. Our results indicate that the 16S rRNA levels can directly inform us about the physiological state of microbial cells. This technique has great potential for application to the microbial fermentation industry.     

Phytoremediation of lead with green onions (Allium fistulosum) and uptake of arsenic compounds by moonlight ferns (Pteris cretica cv Mayii)

Phytoremediation has been investigated as an alternative to excavation to remediate contamination in soil. In this work, Allium fistulosum (green onions) and Pteris cretica cv Mayii (moonlight ferns) were investigated for phytoremediation. Green onions were planted in lead-spiked soil, and chelating agents were introduced to enhance the uptake of lead by the plants. Lead uptake was low in the absence of chelating reagents. Ethylenediaminetetraacetic acid (EDTA) significantly enhanced the concentration of lead in the stems of green onions, while propylenediaminetetraacetic acid (PDTA) did not induce lead absorption.Moonlight ferns (P. cretica cv Mayii) were planted in a hydroponic system to which arsenic (III), arsenic (V), and monomethylarsenate (MMA) were added with hydroponic solution. Ferns exposed to arsenic (III) showed the highest extraction of arsenic followed by ferns exposed to arsenic (V). The extraction of arsenic by the ferns was higher when arsenic (III) was mixed with arsenic (V) than the combination of arsenic (III) and MMA. These results suggest that inorganic arsenic is phytoextracted preferentially to MMA.     

Substrate specificity and reaction mechanism of recombinant styrene oxide isomerase from Pseudomonas putida S12

To clarify the substrate specificity of the recombinant styrene oxide isomerase, various epoxides were subjected to the reaction. From the substituent effect on the rate of isomerization, the mechanism of the isomerase catalyzed reaction was estimated.     

Makomotines A to D from Makomotake, Zizania latifolia infected with Ustilago esculenta

Four novel compounds, makomotines A to D (1–4), along with two known ones (5, 6) were isolated from Makomotake, Zizania latifolia infected with Ustilago esculenta. The structures were determined or identified by the interpretation of spectroscopic data. Compound 1 suppressed the formation of osteoclast without toxicity.     

Five new isocoumarins from Sonoran desert plant-associated fungal strains Paraphaeosphaeria quadriseptata and Chaetomium chiversii

Five new isocoumarins, paraphaeosphaerins A-C and chaetochiversins A and B, biogenetically related to monocillin I and radicicol, have been isolated from solid agar cultures of Paraphaeosphaeria quadriseptata and Chaetomium chiversii, two fungal strains living in association with the Sonoran desert plants, Opuntia leptocaulis and Ephedra fasciculata, respectively. A new chroman-4-one, aposphaerin C, was also isolated from P. quadriseptata. Their structures and stereochemistry were elucidated using a combination of ¹H and ¹³C homo- and hetero-nuclear 2D NMR techniques, ¹H NMR analysis of Mosher's esters, and chemical correlations.     

Synthesis and photophysical properties of novel succinimidyl benzazole derivatives, evaluated by Candida albicans ATCC 10231 fluorescent staining

New fluorescent succinimidyl benzazole derivatives were synthesised and successfully used to stain Candida albicans ATCC 10231 cells. The dyes were characterised by means of infrared, ¹³C and ¹H NMR spectroscopies and elemental analysis. UV-Vis and steady-state fluorescence in solution were also applied to characterise their photophysical behaviour. The novel dyes were fluorescent in the yellow-green region by a phototautomerism in the excited state (ESIPT) with a large Stokes shift (9065-10962 cm⁻¹). Dual fluorescence could also be observed depending on the solvent polarity. The present dyes were used as new probes by means of culture methodology or direct staining to study the micromorphology of Candida albicans.     

Coordination to copper(II) strongly enhances the in vitro antimicrobial activity of pyridine-derived N(4)-tolyl thiosemicarbazones

Five copper(II) complexes with N(4)-ortho, N(4)-meta and N(4)-para-tolyl thiosemicarbazones derived from 2-formyl and 2-acetylpyridine were obtained and thoroughly characterized. The crystal structure of N(4)-meta-tolyl-2-acetylpyridine thiosemicarbazone (H2Ac4mT) was determined, as well as that of its copper(II) complex [Cu(2Ac4mT)Cl], which contains an anionic ligand and a chloride in the coordination sphere of the metal. The in vitro antimicrobial activities of all thiosemicarbazones and their copper(II) complexes were tested against Salmonella typhimurium and Candida albicans. Upon coordination a substantial decrease in the minimum inhibitory concentration, from 225 to 1478 μmol L⁻¹ for the thiosemicarbazones to 5–30 μmol L⁻¹ for the complexes was observe against the growth of Salmonella typhimurium and from 0.7–26 to 0.3–7 μmol L⁻¹ against the growth of C. albicans, suggesting that complexation to copper(II) could be an interesting strategy of dose reduction.     

Studies on the Components of Crude and Processed Fructus Corni by ESI-MS

The components of crude and processed Fructus Corni were investigated by means of electrospray ionization-tandem mass spectrometry（ESI-MSn） technique in the negative ion mode. Compared with those of crude Fructus Corni, the chemical components of the processed Fructus Corni were changed both in quality and in quantity. From the ESI-MS spectra of the crude and processed Fructus Corni, six peaks were selected to establish the characte-ristic ESI-MS peaks. Several factors in the processing procedure were examined. The experimental results demonstrate that the chemical reactions that occurred in the processing procedure can be used for the elucidation of the processed mechanism of Fructus Corni, which is regularly affected by the processing conditions. The present article provides both the chemistry evidence for the understanding of the processing procedure of Fructus Corni and the specific methodology for the research of the processing procedure and quality identification of traditional Chinese medicine.     

Pressurized fluid extraction of carotenoids from Haematococcus pluvialis and Dunaliella salina and kavalactones from Piper methysticum

Pressurized fluid extraction (PFE) was examined as an alternative technology for the extraction of carotenoids in the green algae Haematococcus pluvialis and Dunaliella salina and kavalactones in Piper methysticum. The extraction process was optimized by varying the key extraction factors of solvent, sample-solvent ratio, temperature, and time. The selectivity and efficiency of extraction parameters were determined with high performance liquid chromatography (LC) and LC-mass spectrometry (LC-MS). Results showed that PFE utilization of conventional solvents under controlled temperature and pressure in an oxygen and light-free environment could result in the use of less solvent in a shorter period of time. PFE showed higher or equal extraction efficiencies as compared with traditional solvent extractions while maintaining the integrity of chemical components. PFE showed high potential for extraction of natural products and nutraceuticals, particularly labile and light sensitive chemicals.     

Calorimetric studies of the growth of Desulfovibrio desulfuricans in the presence of nitrocellulose

Calorimetric results indicate that nitrocellulose (NC)-induced changes in the metabolism of Desulfovibrio desulfuricans 1388 are caused by both chemical (nitrate) and physical (biofilm formation) factors. Nitrate added to lactate-based culture medium with nitrocellulose competed for the electron flux from lactate and suppressed the bacterial sulfidogenesis and growth. The presence of an insoluble compound (carbon backbone of the polymer) induced the creation of a biofilm-like structure with its own metabolism.     

Universal fluorescent tri-probe ligation equipped with capillary electrophoresis for targeting SMN1 and SMN2 genes in diagnosis of spinal muscular atrophy

This is the first ligase chain reaction used for diagnosis of spinal muscular atrophy (SMA). Universal fluorescent tri-probe ligation (UFTPL), a novel strategy used for distinguishing the multi-nucleotide alternations at single base, is developed to quantitatively analyze the SMN1/SMN2 genes in diagnosis of SMA. Ligase chain reaction was performed by adding three probes including universal fluorescent probe, connecting probe and recognizing probe to differentiate single nucleotide polymorphisms in UFTPL. Our approach was based on the two UFTPL products of survival motor neuron 1 (SMN1) and SMN2 genes (the difference of 9 mer) and analyzed by capillary electrophoresis (CE). We successfully determined various gene dosages of SMN1 and SMN2 genes in homologous or heterologous subjects. By using the UFTPL-CE method, the SMN1 and SMN2 genes were fully resolved with the resolution of 2.16 ± 0.37 (n = 3). The r values of SMN1 and SMN2 regression curves over a range of 1–4 copies were above 0.9944. Of the 48 DNA samples, the data of gene dosages were corresponding to that analyzed by conformation sensitive CE and denatured high-performance liquid chromatography (DHPLC). This technique was found to be a good methodology for quantification or determination of the relative genes having multi-nucleotide variants at single base.     

Fluorescence detection of total count of Escherichia coli and Staphylococcus aureus on water-soluble CdSe quantum dots coupled with bacteria

The aim of this paper was to demonstrate a fluorescence measurement method for rapid detection of two bacterial count by using water-soluble quantum dots (QDs) as a fluorescence marker, and spectrofluorometer acted as detection apparatus, while Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were as detection target bacteria. Highly luminescent water-soluble CdSe QDs were first prepared by using thioglycolic acid (TGA) as a ligand, and were then covalently coupled with target bacteria. The bacterial cell images were obtained using fluorescence microscopy. Our results showed that CdSe QDs prepared in water phase were highly luminescent, stable, and successfully conjugated with E. coli and S. aureus. The fluorescence method could detect 10²-10⁷ CFU/mL total count of E. coli and S. aureus in 1-2 h and the low detection limit is 10² CFU/mL. A linear relationship of the fluorescence peak intensity and log total count of E. coli and S. aureus have been established using the equation Y = 118.68X − 141.75 (r = 0.9907).     

Synthesis and characterization of novel fluorescent N-glycoconjugates

Novel fluorescent N-glycoconjugates containing d-glucose, glycine and coumarin or naphthalenetriazole derivatives were prepared by peptide synthesis type methods. The fluorescence properties (spectra, quantum yields) of the compounds were evaluated.     

New cytotoxic sesquiterpenes from the red algae Laurencia obtusa and Laurencia microcladia

Three new sesquiterpenes (1-3), along with five known (5-9), were isolated from the organic extract of the red alga Laurencia obtusa, collected from the coastal rocks of Serifos in the Aegean Sea. A new dimeric sesquiterpene of the cyclolaurane-type (4) along with four previously reported (7, 10-12) metabolites, were isolated from the extract of Laurencia microcladia, collected at Chios island in the North Aegean Sea. The structures and the relative stereochemistry of the compounds are proposed on the basis of their spectral data. The cytotoxicity of the isolated metabolites was evaluated against several cell lines including human tumor cell lines.     

Synthesis of fluorescent water-soluble functionalised benzo[a]phenoxazinium salts

In order to develop long-wavelength functionalised oxazine dyes with good water solubility and high photostability for biological applications, a series of novel side-chain functionalised benzo[a]phenoxazinium salts were synthesised and characterised. These polycyclic cationic compounds showed strong fluorescence in ethanol and water (pH 7.4), with an emission wavelengths higher than 637 nm, as well as high quantum yields and moderate Stokes’ shifts. The photostability of the fluorophores synthesised, under irradiation at 419 nm, was good to excellent in ethanol and moderate in water at physiological pH.     

Capisterones A and B from the tropical green alga Penicillus capitatus: unexpected anti-fungal defenses targeting the marine pathogen Lindra thallasiae

The mechanism by which marine algae show resistance to pathogenic microorganisms remains poorly understood. To examine the possible role that algal secondary metabolites play in the prevention of infection, we examined the abundant green alga Penicillus capitatus, one of the major shallow water algae found in the Tropical Atlantic Ocean. Both aqueous and EtOAc extracts of this alga were found to be potent inhibitors of the well-known marine algal pathogen Lindra thallasiae. Using L. thallasiae in bioassay-guided fractionation, we isolated two new triterpene sulfate esters, capisterones A (1) and B (2). The capisterones are potent inhibitors of L. thallasiae at natural and below natural concentrations. The structures of the capisterones, with relative stereochemistry only, were assigned by comprehensive spectral analyses that relied heavily on 2D NMR methods.     

2,6-Cyclo-xenicanes from the brown algae Dilophus fasciola and Dilophus spiralis

Seven new diterpenes, featuring the rare 2,6-cyclo-xenicane skeleton, along with eleven previously reported metabolites were isolated from the organic extracts of the brown algae Dilophus fasciola and Dilophus spiralis. The structure elucidation of the isolated natural products was based on detailed analyses of their spectroscopic data (NMR, MS, IR, UV), whereas the assignment of their relative configurations was assisted by molecular modelling studies.