Fluorescence identification of arthropathic calcium pyrophosphate single crystals using alizarin red S and a xanthene dipicolylamine ZnII complex.

Calcium pyrophosphate deposition disease, previously known as pseudogout, is a type of chronic and painful joint arthropathy. Accurate identification of calcium pyrophosphate dihydrate (CPPD) single crystals is crucial for determining the best course of treatment. In this study, a two-step method involving alizarin red S (ARS) and a xanthene dipicolylamine ZnII (XDZ) complex was employed for the identification of CPPD single crystals in both triclinic and monoclinic forms using a fluorescence microscope and a microplate reader. The accurate identification method proposed in this study has the potential to advance the diagnosis and treatment of patients suffering from painful gouty arthritis.

Fluorescence Differentiation of ATP-related Multiple Enzymatic Activities in Synovial Fluid as a Marker of Calcium Pyrophosphate Deposition Disease using Kyoto Green.

Calcium pyrophosphate deposition disease (CPPD) is a crystal induced inflammation in joints, and causes severe pain in elderly people. The accumulation of pyrophosphate (PPi) in synovial fluid (SF) results from several enzymatic reactions, especially the highly activated e-NPPs, which catalyze the conversion of ATP to PPi. This study demonstrates the detection of relative catalytic activity of 3 enzymes-ecto-nucleotide pyrophosphatase/phosphodiesterases (e-NPPs), tissue nonspecific alkaline phosphatase (TNAP), and ecto-nucleoside triphosphate diphosphohydrolases (e-NTPDases)-using a single molecular sensor called Kyoto Green. Kyoto Green exhibits excellent performance in sensing the catalytic activity of the commercial representatives of the e-NPPs, TNAP, and e-NTPDases, which are ENPP1, PPase, and apyrase, respectively, in both single-enzyme and multi-enzyme assays. Analysis of SF enzymes in 19 SF samples from human and swine revealed moderate activity of e-NPPs, high activity of e-NTPDases, and low activity of TNAP. Our newly developed method for analysis of multiple enzymatic activities using Kyoto Green in biological SF will assist improvement in accuracy of the CPPD prognosis/diagnosis, which will minimize unnecessary medical procedures.

Fluorescence Detection of Deoxyadenosine in Cordyceps spp. by Indicator Displacement Assay.

A rapid, sensitive and reliable indicator displacement assay (IDA) for specific detection of 2'- and 3'-deoxyadenosine (2'-dAde and 3'-dAde), the latter is also known as cordycepin, was established. The formation of inclusion complex between protonated acridine orange (AOH+) and cucurbit[7]uril (CB7) resulted in the hypochromic shift of fluorescent emission from 530 nm to 512 nm. Addition of cordycepin to the highly fluorescent AOH+/CB7 complex resulted in a unique tripartite AOH+/CB7/dAde complex with diminished fluorescence, and such reduction in emission intensity serves as the basis for our novel sensing system. The detection limits were 11 and 82 µM for 2'- and 3'-deoxyadenosine, respectively. The proposed method also demonstrated high selectivity toward 2'- and 3'-deoxyadenosine, owing to the inability of other deoxynucleosides, nucleosides and nucleotides commonly found in Cordyceps spp. to displace the AOH+ from the AOH+/CB7 complex, which was confirmed by isothermal titration calorimetry (ITC), UV-Visible and proton nuclear magnetic resonance (1H-NMR) spectroscopy. Our method was successfully implemented in the analysis of cordycepin in commercially available Ophiocordyceps and Cordyceps supplements, providing a novel and effective tool for quality assessment of these precious fungi with several health benefits.

Emulsification efficiency of adsorbed chitosan for bacterial cells accumulation at the oil-water interface.

The use of bacterial cell or biocatalyst for industrial synthetic chemistry is on the way of significant growth since the biocatalyst requires low energy input compared to the chemical synthesis and can be considered as a green technology. However, majority of natural bacterial cell surface is hydrophilic which allows poor access to the hydrophobic substrate or product. In this study, Escherichia coli (E. coli) as a representative of hydrophilic bacterial cells were accumulated at the oil-water interface after association with chitosan at a concentration range of 0.75-750 mg/L. After association with negatively charged E coli having a ζ potential of -19.9 mV, a neutralization of positively charged chitosan occurred as evidenced by an increase in the ζ potential value of the mixtures with increasing chitosan concentration up to +3.5 mV at 750 mg/L chitosan. Both emulsification index and droplet size analysis revealed that chitosan-E. coli system is an excellent emulsion stabilizer to date because the threshold concentration was as low as 7.5 mg/L or 0.00075% w/v. A dramatic increase in the surface hydrophobicity of the E. coli as evidenced by an increase in contact angle from 19 to 88° with increasing chitosan concentration from 0 to 750 mg/L, respectively, resulted in an increase in the stability of oil-in-water emulsions stabilized by chitosan-E. coli system. The emulsion was highly stable even the emulsification was performed under 20% salt condition, or temperature ranged between 20 and 50 °C. Emulsification was failed when the oil volume fraction was higher than 0.5, indicating that no phase inversion occurred. The basic investigation presented in this study is a crucial platform for its application in biocatalyst industry and bioremediation of oil spill.

Naturally occurring quercetin and myricetin as potent inhibitors for human ectonucleotide pyrophosphatase/phosphodiesterase 1.

Ecto-nucleotide pyrophosphatases/phosphodiesterases 1 (ENPP1) is a key enzyme in purinergic signaling pathways responsible for cell-to-cell communications and regulation of several fundamental pathophysiological processes. In this study, Kyoto Green, a rapid chemical sensor of pyrophosphate, was employed to screen for effective ENPP1 inhibitors among five representative flavonoids (quercetin, myricetin, morin, kaempferol, and quercetin-3-glucoside), five nucleosides (adenosine, guanosine, inosine, uridine, and cytidine), and five deoxynucleosides (2'- and 3'-deoxyadenosine, 2'-deoxyguanosine, 2'-deoxyinosine, and 2'-deoxyuridine). Conventional colorimetric, fluorescence, and bioluminescence assays revealed that ENPP1 was effectively inhibited by quercetin (Ki ~ 4 nM) and myricetin (Ki ~ 32 nM) when ATP was used as a substrate at pH 7.4. In silico analysis indicated that the presence of a chromone scaffold, particularly one containing a hydroxyl group at the 3' position on the B ring, may promote binding to the active site pocket of ENPP1 and enhance inhibition. This study demonstrated that the naturally derived quercetin and myricetin could effectively inhibit ENPP1 enzymatic activity and may offer health benefits in arthritis management.

Improved polarized light microscopic detection of gouty crystals via dissolution with formalin and ethylenediamine tetraacetic acid.

Conventional polarized light microscopy has been widely used to detect gouty crystals, but its limited sensitivity increases the risk of misidentification. In this study, a number of methods were investigated to improve the sensitivity of polarized light microscopy for the detection of monosodium urate monohydrate (MSUM) and calcium pyrophosphate dihydrate (CPPD) crystals. We found that coating glass slides with poly-L-lysine, a positively charged polymer, improved the attachment of crystals to the glass surface, resulting in clearer crystal images compared to non-coated slides. Additionally, the sensitivity of detection was further enhanced by selective dissolution, in which 40% v/v formalin phosphate buffer was employed to dissolve MSUM crystals but not CPPD while 10% ethylenediamine tetraacetic acid (EDTA) was employed to dissolved CPPD but not MSUM. The other possible interferences were dissolved in both EDTA and formalin solution. These methods were successfully applied to detect gouty crystals in biological milieu, including spiked porcine synovial fluid and inflamed rat subcutaneous air pouch tissues.

Bacteria interface pickering emulsions stabilized by self-assembled bacteria-chitosan network.

An oil-in-water Pickering emulsion stabilized by biobased material based on a bacteria-chitosan network (BCN) was developed for the first time in this study. The formation of self-assembled BCN was possible due to the electrostatic interaction between negatively charged bacterial cells and polycationic chitosan. The BCN was proven to stabilize the tetradecane/water interface, promoting formation of highly stable oil-in-water emulsion (o/w emulsion). We characterized and visualized the BCN stabilized o/w emulsions by scanning electron microscopy (SEM) and laser scanning confocal microscopy (LSCM). Due to the sustainability and low environmental impact of chitosan, the BCN-based emulsions open up opportunities for the development of an environmental friendly new interface material as well as the novel type of microreactor utilizing bacterial cells network.

Bioproduction of vanillin using an organic solvent-tolerant Brevibacillus agri 13.

Nowadays, majority of vanillin supplied to the world market is chemically synthesized from a petroleum-based raw material, raising a concern among the consumers regarding the product safety. In this study, an organic solvent-tolerant Brevibacillus agri 13 previously reported for a strong predilectic property was utilized as a whole-cell biocatalyst for bioproduction of vanillin from isoeugenol (IG). B. agri 13 is the first biocatalyst reported for bioproduction of vanillin at a temperature as high as 45°C. Both pH and temperature were found to affect vanillin production significantly. An extreme level of organic solvent tolerance of B. agri 13 allowed us to utilize it in a biphasic system using organic solvents generally considered as highly toxic to most bacteria. With an addition of butyl acetate at 30% (v/v) as an organic second phase, toxicity of IG exerted onto the biocatalyst was reduced dramatically while faster and more efficient vanillin production was obtained (1.7 g/L after 48 h with 27.8% molar conversion).

Development of a whole-cell biocatalyst co-expressing P450 monooxygenase and glucose dehydrogenase for synthesis of epoxyhexane.

Oxygenases-based Escherichia coli whole-cell biocatalyst can be applied for catalysis of various commercially interesting reactions that are difficult to achieve with traditional chemical catalysts. However, substrates and products of interest are often toxic to E. coli, causing a disruption of cell membrane. Therefore, organic solvent-tolerant bacteria became an important tool for heterologous expression of such oxygenases. In this study, the organic solvent-tolerant Bacillus subtilis 3C5N was developed as a whole-cell biocatalyst for epoxidation of a toxic terminal alkene, 1-hexene. Comparing to other hosts tested, high level of tolerance towards 1-hexene and a moderately hydrophobic cell surface of B. subtilis 3C5N were suggested to contribute to its higher 1,2-epoxyhexane production. A systematic optimization of reaction conditions such as biocatalyst and substrate concentration resulted in a 3.3-fold increase in the specific rate. Co-expression of glucose dehydrogenase could partly restored NADPH-regenerating ability of the biocatalyst (up to 38 % of the wild type), resulting in approximately 53 % increase in specific rate representing approximately 22-fold increase in product concentration comparing to that obtained prior to an optimization.

Effect of hydrocolloids on physicochemical properties, stability, and digestibility of Pickering emulsions stabilized by nanofibrillated cellulose.

In this study, the effect of hydrocolloids with different electrostatic characteristics, namely negatively charged xanthan gum (XG), positively charged chitosan (CH), and non-ionic guar gum (GG), on the physicochemical properties, stability, and lipid digestibility of 10% (w/w) soybean oil-in-water Pickering emulsions stabilized by nanofibrillated cellulose (NFC) was investigated. Addition of XG and CH to the NFC-stabilized emulsions significantly increased the oil droplet sizes and apparent viscosity at high shear rates as compared with the addition of GG. The XG added emulsion showed the lowest rate and extent of creaming, whereas the CH added emulsion gave the highest extent of creaming. The addition of XG and CH led to a more pronounced effect on in vitro lipid digestion, i.e. changes in droplet sizes, surface charges, microstructure, and free fatty acid (FFA) release, than the addition of GG. The XG added emulsion showed the lowest rate and extent of lipid digestion possibly due to the high viscosity of the aqueous phase, large oil droplet sizes, and interaction of XG and calcium, resulting in the reduction of lipase activity. The CH added emulsion exhibited the highest extent of lipid digestion possibly due to binding between CH and FFAs and move away from the droplet surfaces, thereby facilitating the lipase activity. In summary, it can be concluded that ionic hydrocolloids exerted more influence on NFC-stabilized Pickering emulsions than non-ionic ones. These results may facilitate the design of highly stable emulsion-based functional food products with added hydrocolloids to promote health and wellness.

A multicolor and ratiometric fluorescent sensing platform for metal ions based on arene-metal-ion contact.

Despite continuous and active development of fluorescent metal-ion probes, their molecular design for ratiometric detection is restricted by the limited choice of available sensing mechanisms. Here we present a multicolor and ratiometric fluorescent sensing platform for metal ions based on the interaction between the metal ion and the aromatic ring of a fluorophore (arene-metal-ion, AM, coordination). Our molecular design provided the probes possessing a 1,9-bis(2'-pyridyl)-2,5,8-triazanonane as a flexible metal ion binding unit attached to a tricyclic fluorophore. This architecture allows to sense various metal ions, such as Zn(II), Cu(II), Cd(II), Ag(I), and Hg(II) with emission red-shifts. We showed that this probe design is applicable to a series of tricyclic fluorophores, which allow ratiometric detection of the metal ions from the blue to the near-infrared wavelengths. X-ray crystallography and theoretical calculations indicate that the coordinated metal ion has van der Waals contact with the fluorophore, perturbing the dye's electronic structure and ring conformation to induce the emission red-shift. A set of the probes was useful for the differential sensing of eight metal ions in a one-pot single titration via principal component analysis. We also demonstrate that a xanthene fluorophore is applicable to the ratiometric imaging of metal ions under live-cell conditions.

Masking Phosphate with Rare-Earth Elements Enables Selective Detection of Arsenate by Dipycolylamine-ZnII Chemosensor.

Functional reassessment of the phosphate-specific chemosensors revealed their potential as arsenate detectors. A series of dipicolylamine (Dpa)-ZnII chemosensors were screened, among which acridine Dpa-ZnII chemosensor showed the highest capability in sensing arsenate. The presence of excess ZnII improved sensitivity and strengthened the binding between acridine Dpa-ZnII complex to arsenate as well as phosphate. However, due to their response to phosphate, these sensors are not suited for arsenate detection when phosphate is also present. This study demonstrated for the first time that rare-earth elements could effectively mask phosphate, allowing the specific fluorescence detection of arsenate in phosphate-arsenate coexisting systems. In addition, detection of arsenate contamination in the real river water samples and soil samples was performed to prove its practical use. This sensor was further employed for the visualization of arsenate and phosphate uptake in vegetables and flowering plants for the first time, as well as in the evaluation of a potent inhibitor of arsenate/phosphate uptake.

Hydroxypropyl methylcellulose enhances the stability of o/w Pickering emulsions stabilized with chitosan and the whole cells of Lactococcus lactis IO-1.

Hydroxypropyl methylcellulose (HPMC) was used as a co-emulsifier with chitosan and the whole cells of bacteriocin-producing Lactococcus lactis IO-1 (L. lactis IO-1) for the preparation of bacteria interface Pickering emulsion. The obtained emulsion exhibited a high stability against centrifugation force, ionic strength and low temperature, with the whole cells of L. lactis IO-1 located at the oil/water interface. Because L.lactis IO-1 was found to produce peptide lantibiotic against several strains of Gram-positive food pathogen, the highly stable emulsion demonstrated in this study exhibited high potential as an antimicrobial emulsion with several health benefits of chitosan and HPLC useful for food industry.

Fluorescence Sensing of Inorganic Phosphate and Pyrophosphate Using Small Molecular Sensors and Their Applications.

The aim of this contribution is to provide an introduction and a brief summary of the principle of fluorescence molecular sensors specific to inorganic phosphate (Pi) and inorganic pyrophosphate (PPi) as well as their applications. In our introduction we describe the impact of both Pi and PPi in the living organism and in the environment, followed by a description of the principle of fluorescence molecular sensors and the sensing mechanism in solution. We then focus on exciting research which has emerged in recent years on the development of fluorescent sensors specific to Pi and PPi, categorized by chemical interactions between the sensor and the target molecule, such as hydrogen bonding, coordination chemistry, displacement assay, aggregation induced emission or quenching, and chemical reactions.

Turning hydrophilic bacteria into biorenewable hydrophobic material with potential antimicrobial activity via interaction with chitosan.

Alteration of a bacteriocin-producing hydrophilic bacterium, Lactococcus lactis IO-1, into a hydrophobic material with potential antimicrobial activity using chitosan was investigated and compared with five other bacterial species with industrial importance. The negatively charged bacterial cells were neutralized by positively charged chitosan, resulting in a significant increase in the hydrophobicity of the bacterial cell surface. The largest Gram-positive B. megaterium ATCC 14581 showed a moderate response to chitosan while the smaller E. coli DH5α, L. lactis IO-1 and P. putida F1 exhibited a significant response to an increase in chitosan concentration. Because L. lactis IO-1 is a good source for natural peptide lantibiotic that is highly effective against several strains of food spoilage organisms and pathogens, hydrophobic material derived from L. lactis IO-1 and chitosan is a promising novel material with antimicrobial activity for the food and pharmaceutical industries.

Coordination ligand exchange of a xanthene probe-Ce(III) complex for selective fluorescence sensing of inorganic pyrophosphate.

A fluorescence sensing system for inorganic pyrophosphate based on ligand exchange of the Ce(III) complex of a xanthene-type probe is developed. This sensing system is successfully applied to the fluorescence detection of polymerase-catalyzed DNA amplification using loop-mediated isothermal amplification.

Lipase-catalyzed synthesis of hydrophobically modified dextrans: activity and regioselectivity of lipase from Candida rugosa.

Vinyl decanoate-modified dextran macromolecules (DexT40-VD) were synthesized in dimethyl sulfoxide at 50°C using lipase AY from Candida rugosa for catalyzing transesterification between polysaccharide and vinyl fatty esters. The extent of dextran modification (quantified by the molar ratio of attached alkyl tails to sugar repeat units) with native-, pH-adjusted-, 18-crown-6 ether pretreated pH-adjusted-, and stepwise addition of pretreated lipase AY yielded <3%, 49%, 64% and 96% modified dextran respectively. Lipase AY accelerated the transesterification of DexT40 from 2- to 63-fold higher than the non-catalyzed system. This procedure was extended to other acyl donors showing that modification pattern exhibited regioselectivity depending on acyl donor structure. Regioselectivity equaled between 2- and 3-OH with saturated fatty acyl donors. The 2-OH was favored for unsaturated fatty acyl donors, while sterically hindered acyl donors oriented modification toward 3-OH position. DexT40-VD at 96% modification was a water-insoluble polymer forming 150nm diameter nanoparticles in water which can be used as drug carrier systems.

Design of dual-emission chemosensors for ratiometric detection of ATP derivatives.

Nucleoside pyrophosphate (nucleoside PP) derivatives are widespread in living cells and play pivotal roles in various biological events. We report novel fluorescence chemosensors for nucleoside PPs that make use of coordination chemistry. The chemosensors, which contain two Zn(II)-dipicolylamine units, bind strongly to nucleoside PPs (K(app)>10(6) M(-1)) in aqueous solution and sense them by a dual-emission change. Detailed fluorescence and UV/Vis spectral studies revealed that the emission changes of the chemosensors upon binding to nucleoside PPs can be ascribed to the loss of coordination between Zn(II) and the acridine fluorophore. This is a unique sensing system based on the anion-induced rearrangement of the coordination. Furthermore, we demonstrated the utility of these chemosensors in real-time monitoring of two important biological processes involving nucleoside PP conversion: the apyrase-catalyzed hydrolysis of nucleoside PPs and the glycosyl transfer catalyzed by beta-1,4-galactosyltransferase.

Arsenic uptake by native fern species in Thailand: effect of chelating agents on hyperaccumulation of arsenic by Pityrogramma calomelanos.

Nineteen native fern species collected from an area in Thailand with high arsenic concentration in soil and in ground water as a result of tin mining was screened for elevated arsenic concentration in fronds. Two species of fern were found to contain elevated arsenic in their fronds in nature: Pityrogramma calomelanos (108-1156 microg g(-1) dried weight) and Pteris vittata (79 microg g(-1) dried weight). Under hydroponic culture Pityrogramma calomelanos (a silver back fern) accumulated arsenic in its shoot at rate of 4616 microg(-1) (dried weight). The accumulation of arsenic in Pityrogramma calomelanos shoot doubled with the addition of an EDTA (Ethelenediamine tetraacetic acid) chelating agent. The highest accumulation occurred 6 weeks after exposure to 10 mg L(-1) arsenic as disodium hydrogen arsenate. The addition of another chelating agent, DMS (Dimercaptosuccinic acid), resulted in a 5-fold decrease in arsenic concentration in the Pityrogramma calomelanos shoot compared to control after 6 weeks of exposure to arsenic. The contrasting effect of the EDTA and DMSA chelating agent was attributed to the strong binding of the thiol group to arsenic ion. This study indicated that Pityrogramma calomelanos uptake and translocate arsenic in the form of arsenate and arsenite rather than the As-DMSA complex. Using phytoextraction efficiency calculations, it was determined that Pityrogramma calomelanos gave the highest arsenic phytoextraction efficiency at 6 weeks after exposure to arsenic in the EDTA treatment, with an efficiency of 77.8 mg As based on whole plant biomass.