Fluorescence turn-on detection of protamine based on aggregation-induced emission enhancement characteristics of 4-(6'-carboxyl)hexyloxysalicylaldehyde azine.

A novel fluorescence turn-on method for sensitive and selective detection of protamine was developed based on aggregation-induced emission enhancement (AIEE) characteristics of 4-(6'-carboxyl)hexyloxysalicylaldehyde azine (CHSA) induced by electrostatic interaction between protamine and CHSA(-). Under optimal conditions described, a large Stokes shift of approximately 198 nm could be observed, and the fluorescence enhancement at 538 nm was linearly related to the concentration of protamine in the range of 0.6-18 microg mL(-1) with the relative correlation coefficient of R(2) = 0.9996 (n = 11) and a detection limit as low as 43 ng mL(-1). The relative standard deviation (R.S.D.) was 2.0% (n = 5). The proposed method was successfully utilized in quantifying protamine in diluted horse serum. In addition, due to the special electrostatic association between protamine and heparin, CHSA could also be employed as a probe to study the interaction between them.

Ganoderma polysaccharide and chitosan synergistically ameliorate lipid metabolic disorders and modulate gut microbiota composition in high fat diet-fed golden hamsters.

High-fat diet (HFD) and sucrose intake can lead to hyperlipidemia, hypercholesterolemia, and nonalcoholic fatty liver disease (NAFLD) as well as disturbed gastrointestinal microbiota and dysfunctional intestinal barrier. In the present study, we showed that Ganoderma lucidum polysaccharide and chitosan (PC) significantly mitigated the hyperlipidemia in HFD-fed hamsters via lowering the contents of serum total triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and aspartate aminotransferase (AST). Furthermore, PC changed the composition of gastrointestinal microbiota and elevated the relative abundances of beneficial bacteria, such as Prevotella, Oscillibacter, and SCFA-producers. Interestingly, we also found that the abundances of Prevotella, Alloprevotella, Bifidobacterium, and Alistipes were negatively associated with serum lipid profiles. Collectively, the above-mentioned findings indicated that PC could improve lipid metabolic disorders, at least in part, by modulating gastrointestinal microbiota, suggesting that PC could be used as a potential lipid-lowering ingredient in functional foods. PRACTICAL APPLICATIONS: PC could ameliorate lipid metabolism disorder, at least in part, by regulating specific gut microbiota, suggesting its potential as a novel lipid-lowering ingredient in functional foods. We believed that our findings could be of interest to the readers because they help others further understand the gut microbiota alterations that occurred after PC supplementation in the context of metabolic syndrome (MetS).

Polyunsaturated fatty acids from microalgae Spirulina platensis modulates lipid metabolism disorders and gut microbiota in high-fat diet rats.

Effects of Spirulina platensis 55% ethanol extract (SPL55) on lipid metabolism in high-fat diet-induced hyperlipidaemic rats were investigated. Ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry indicated that SPL55 was enriched with polyunsaturated fatty acids. Meanwhile, serum and liver lipid levels, including total triglyceride, total cholesterol, and low-density-lipoprotein cholesterol, were significantly decreased in hyperlipidaemic rats of SPL55. Analysis of tissue sections showed that SPL55 treatment could markedly inhibit hepatic lipid accumulation and steatosis. Moreover, SPL55 regulated the mRNA and protein expression levels of SREBP-1c, HMG-CoA, PEPCK, ACC, and AMPK genes involved in lipid metabolism. Furthermore, SPL55 led to decrease the abundances of Turicibacter, Clostridium_XlVa, and Romboutsia, which were positive correlation with lipid metabolism indicators, and has also enriched Alloprevotella, Prevotella, Porphyromonadaceae, and Barnesiella. These results provided evidence that SPL55 might be developed as a functional food to ameliorate lipid metabolic disorders and hyperlipidaemia.

The regulation mechanisms of soluble starch and glycerol for production of azaphilone pigments in Monascus purpureus FAFU618 as revealed by comparative proteomic and transcriptional analyses.

Monascus spp. have been used for thousands of years as a traditional food additive in China. This mold can produce many different types of commercially valuable secondary metabolites of biological activity. Soluble starch and glycerol are the two principal carbon sources universally utilized by Monascus for the production of beneficial metabolites. In this study, the effects and regulation mechanisms of soluble starch and glycerol for M. purpureus FAFU618 on Monascus azaphilone pigments (MonAzPs) were investigated through ultra-performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC-QTOF-MS/MS), comparative proteomics and quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR). The production of intracellular and extracellular pigments was significantly different between the soluble starch group (SSG) and glycerol group (GCG). Additionally, the components of intracellular pigments revealed by UPLC-QTOF-MS/MS showed that Monascin and Ankaflavin increased significantly in the GCG, while Rubropunctatin and Monascorubrin increased in the SSG. Differentially expressed proteins of mycelia between SSG and GCG were analyzed by two-dimensional gel electrophoresis (2-DE) and MALDI-TOF/TOF MS. We identified 27 proteins with statistically altered expression, of which 18 proteins associated with the EMP (glycolytic pathway), translation, energy generation, proteolysis, etc. were up-regulated, and 9 proteins, including ribosomal proteins, heat shock proteins (HSPs) and others, were down-regulated in GCG. Meanwhile, the expression levels of MonAzP biosynthetic genes were also analyzed by RT-qPCR, and the results showed that mppA, mppC, mppR1 and mppR2 were down-regulated, whereas genes MpPKS5, MpFasA2, MpFasB2, mppB, mppD and mppE were up-regulated. Collectively, these findings illustrate that the regulation of MonAzPs is not only closely related to the expression levels of certain proteins in the polyketide synthesis pathway but also closely related to the concentration of primary metabolism-generated molecules that are used as substrates for polyketide synthesis. The present study provides insights into the regulation of different carbon sources on the metabolism of MonAzPs in M. purpureus FAFU618. These results may promote further development of functional foods or medicines from Monascus spp. fermented products.

[Study of derivatives of 2, 6-diaminopyridine as fluorescence probe of transition metal ions].

Three derivatives of 2, 6-diaminopyridine were synthesized with naphthyridine ring and -NH2, -OH and -NHCOCHCH2 groups. They are 2, 4-dimethyl-7-hydroxyl-1, 8-naphthyridine (DMHND), 2, 4-Dimethyl-7-amino-1, 8-naphthyridine (DMAND) and DMAAN. The binding ability of the derivatives of 2, 6-diaminopyridine for metal ions in acetonitrile was investigated using UV-Vis and fluorescence spectrometry. The changes of the dyes spectra were observed in the presence of transition metal ions, but were not observed in the presence of K+ , Na+ , Mg2+ , Ca2+ and Pd2+. Except for DMHND that has weak binding ability for Cu2+, two dyes can bind with Cu2+ at the ratios of DMAAN : Cu2+ = 1 : 1, DMAND : Cu2+ = 1 : 1. The resulting binding curves were analyzed by nonlinear regression methods, giving the association constant for DMAAN to be about 4.4 x 10(5) L x mol(-1), and about 6.3 x 10(6) L x mol(-1) for DMAND. DMAND contains an electron-donating group (amino group) conjugated to an electron-withdrawing group (the pyridine ring), and undergoes intramolecular charge transfer from the donor to the acceptor upon excitation by light. When Cu2+ interacting with the N of the pyridine ring enhances the electron-withdrawing character of this group, the spectrum is thus red-shifted and the intensity is increased. DMAND displayed a high sensitivity among the three derivatives of 2, 6-diaminopyridine as the fluorescence probe of transition metal ions.

[Synthesis and spectral characterization of a novel fluorescent functional monomer 2,4-dimethyl-7-acrylamine-1,8-naphthyridine].

A new fluorescent functional monomer DMAAN has been designed and synthesized for the first time. The monomer was expected to bind with cytosine via complementary hydrogen bonding under certain experimental conditions. The synthetic procedure of DMAAN is as follows. A suspension of 2,6-diaminopyridine in phosphoric acid was added to acetylacetone. The mixture was warmed for thirty minutes on a steam-bath. Then 2,4-dimethyl-7-amino-1,8-naphthyridine(DMAN, mp 216-218 degrees C) was obtained. DMAAN was prepared by the reaction of DMAN with acryloyl chloride in chloroform and triethylether mixed solvent. Acryloyl chloride in anhydrous chloroform was added dropwise over 30 minutes with constant stirring at 4 degrees C. The melting point of the final product was 239-241 degrees C. Mass spectrometry and proton NMR were used to confirm the chemical structure of DMAN and DMAAN. The products were also characterized by UV and fluorescence spectrometry.

Preparation of Z-L-Phe-OH-NBD imprinted microchannel and its molecular recognition study.

An integrated microchip was presented for selective recognition of Z-L-Phe-OH-NBD, using molecular imprinting technique. Molecularly imprinted polymer (MIP) were prepared by copolymerization in the presence of template molecule Z-L-Phe-OH-NBD, in which methacrylic acid and 4-vinylpyridine were used as functional monomers and ethylene dimethacrylate used as crosslinker. Imprinted polymer particles were introduced into a microchannel fabricated with a new material i.e. poly(methylvinylsiloxane) by simply rapid prototyping method. Imprinted effects were evaluated by laser-induced fluorescence (LIF) detection where the results indicated that good selective recognition for Z-L-Phe-OH-NBD in the imprinted polymer was obtained; the adsorption percentage of Z-L-Phe-OH-NBD was 61%. In contrast to conventional molecular imprinting analysis, integration shortened overall analysis time from 4h to 10 min.

Syntheses of steroid-based molecularly imprinted polymers and their molecular recognition study with spectrometric detection.

Recognition of five steroid compounds, beta-estradiol, ethynylestradiol, estradiolbenzoate, testosterone and methyltestosterone were studied using a synthesized molecularly imprinted polymer (MIP). When beta-estradiol was used as the template molecule, the polymer was synthesized with methacrylic acid (MAA) as the functional monomer and ethylene glycol dimethacrylate (EGDMA) as the cross linking agent through non-covalent interactions. It is found that the kind of porogen solvent and the polymerization conditions greatly affected the binding ability of a MIP to a certain molecule. Releasing of the template was performed by continuous extraction with methanol containing 10% acetic acid in a Soxhlet extractor. Our results indicated that such carefully synthesized MIP showed specific affinity toward beta-estradiol in the adsorption process.

Study on the binding mode of zinc(II) protoporphyrin and ctDNA in water.

Spectroscopic property of a commercially available luminescent reagent Zinc(II) protoporphyrin (ZnPP) was studied. Dissociation constants of the two protons on the peripheral groups of porphyrin ring of ZnPP were determined as pK(a1)=6.31, pK(a2)=9.37. Binding evidence of ZnPP with ctDNA was found by the phosphorescence intensity change on a filter paper around pH 6.5-9.3 with the association constant being 9.1x10(3) dm(3)/mol. A novel binding mode for ZnPP and calf thymus DNA (ctDNA) suggested that the monomer ZnPP which has no axial coordination, slips into the groove of DNA and interacts with the bases of polynucleotide by zinc coordination and hydrogen bonding between H atom on carboxyl group of ZnPP and O atom on the bases.

Facile, sensitive and selective fluorescence turn-on detection of HSA/BSA in aqueous solution utilizing 2,4-dihydroxyl-3-iodo salicylaldehyde azine.

A novel fluorescence turn-on detection method of human serum albumin (HSA) and bovine serum albumin (BSA) in aqueous solution is investigated using 2,4-dihydroxyl-3-iodo salicylaldehyde azine (DISA). Upon the addition of DISA to HSA/BSA solution, a fluorescence turn-on effect at 529 nm can be observed with a large stokes shift of approximately 129 nm based on hydrophobic binding-mode between protein and dye. Under the optimal condition, the linear ranges of fluorescence intensity for HSA and BSA are 0.1-30 microg mL(-1) with the relative correlation coefficient of R(2)=0.991 (n=10) and 0.3-50 microg mL(-1) with R(2)=0.997 (n=10); and the detection limits for HSA and BSA based on IUPAC (C(DL)=3S(b)/m) are 20 ng mL(-1) and 50 ng mL(-1), respectively.

Insulin is a kinetic but not a thermodynamic inhibitor of amylin aggregation.

One of the most important pathological features of type 2 diabetes is the formation of islet amyloid, of which the major component is amylin peptide. However, the presence of a natural inhibitor such as insulin may keep amylin stable and physiologically functional in healthy individuals. Some previous studies demonstrated that insulin was a potent inhibitor of amylin fibril formation in vitro, but others obtained contradictory results. Hence, it is necessary to elucidate the effects of insulin on amylin aggregation. Here we report that insulin is a kinetic inhibitor of amylin aggregation, only keeping its inhibitory effect for a limited time period. Actually, insulin promotes amylin aggregation after long-term incubation. Furthermore, we found that this promotional effect could be attributed to the copolymerization of insulin and amylin. We also found that insulin copolymerized with amylin monomer or oligomer rather than preformed amylin fibrils. These results suggest that the interaction between insulin and amylin may contribute not only to the inhibition of amylin aggregation but also to the coaggregation of both peptides in type 2 diabetes.

Immunosorbent assay microchip system for analysis of human immunoglobulin G on MagnaBind carboxyl derivatized beads.

An immunosorbent assay system was integrated into a PMVS microchip. MagnaBind carboxyl derivatized beads were introduced into a microchannel, and then human immunoglobulin G (IgG) was bound to the bead surface in the microchannel of the chip. Immunoreaction was conducted in the microchannel for the bead-bounded antigen IgG with the antibody FITC-labelled IgG. On-chip detection was performed using a laser-induced fluorescence (LIF) system. The integration shortened the overall analysis time from 7 h to less than 40 min.