Baicalin Ameliorates Inflammatory Response in a Mouse Model of Rhinosinusitis via Regulating the Treg/Th17 Balance.

OBJECTIVE: Rhinosinusitis is a global health problem affecting millions of people around the world. Baicalin is a bioactive compound isolated from medicinal plant Scutellaria baicalensis Georgi. The present study aims to investigate potential effects of baicalin on clinicopathological changes in nasal/sinus mucosa in a mouse model. METHODS: A mouse model of sinonasal inflammation induced by high dose of ovalbumin was applied to evaluate effects of baicalin. Rhinosinusitis symptoms, histopathological features, levels of histamine, immunoglobulin E (IgE), IL-17A, IL-10, and balance of regulatory T cell (Treg)/T-helper 17 (Th17) responses were examined. RESULTS: Baicalin significantly relieved rhinosinusitis symptoms in mice, reduced histopathological changes, and suppressed serum levels of histamine and IgE in a dose-dependent manner. In lymphocytes of mice, baicalin modulated balance of Treg/Th17 proportions by attenuating Th17 cells and enhancing Treg cells, respectively. The serum IL-17A was decreased and IL-10 was increased in mice treated by baicalin. In addition, baicalin promoted levels of Smad protein 3 (p-Smad3) and forkhead box P3 (FOXP3) to promote Treg cells while suppressed levels of p-Stat3 and retineic-acid-receptor-related orphan nuclear receptor γt (RORγt) to inhibit Th17 cells. CONCLUSIONS: These data demonstrate that baicalin effectively ameliorates sinonasal inflammation in a mouse model by recovering the immunological balance of Treg/Th17 responses. Our finding highlights the potential value of baicalin for the treatment of rhinosinusitis.

New cytotoxic withanolides from Physalis minima.

Phytochemical investigation of Physalis minima led to the isolation of six new withanolides, including physaminilides HK (1-4), two artificial withanolides (5-6), and 19 known ones (7-25). Their structures were elucidated on the basis of spectroscopic analysis, including NMR and electronic circular dichroism (ECD) data. The isolates were evaluated for their cytotoxic activities against A375 human melanoma cells. Compounds 1, 8-9, 12-13, 15-17 and 19 exhibited significant cytotoxic activities with IC50 values in the range of 1.2-7.5 µM.

Berberine represses human gastric cancer cell growth in vitro and in vivo by inducing cytostatic autophagy via inhibition of MAPK/mTOR/p70S6K and Akt signaling pathways.

Berberine, an isoquinoline alkaloid from Coptidis Rhizoma, has been characterized as a potential anticancer drug due to its good anti-tumor effects. However, the molecular mechanisms involved in anti-gastric cancer remain poorly understood. Herein, the role of berberine in gastric cancer suppression by inducing cytostatic autophagy in vitro and in vivo was first investigated. Results showed that berberine induced an obvious growth inhibitory effect on gastric cancer BGC-823 cells without toxicity to human peripheral blood mononuclear cells. Treatment with berberine triggered cell autophagy, as demonstrated by the punctuate distribution of monodansylcadaverine staining and GFP-LC3, as well as the LC3-II, Beclin-1 and p-ULK1 promotion, and p62 degradation. Inhibition of autophagy by 3-MA, CQ, Baf-A1 and BECN1 siRNA obviously increased cell viability of berberine-exposed gastric cancer cells, which confirmed the anti-cancer role of autophagy induced by berberine. Mechanistic studies showed that berberine inhibited mTOR, Akt and MAPK (ERK, JNK and p38) pathways thereby inducing autophagy. Inhibition of above pathways increases berberine induced autophagy and cytotoxicity. Interestingly, mTOR/p70S6K was inhibited by the MAPK but not Akt. Furthermore, inhibition of autophagy reversed berberine down-regulated mTOR, Akt and MAPK. In xenografts, the berberine induced autophagy leads to suppression of tumor proliferation with no side-effect, and western blotting displayed an apparent attenuation of p-mTOR, p-p70S6K, p-Akt, p-ERK, p-JNK and p-p38 in tumors from berberine treated mice. Briefly, these results indicated that berberine repressed human gastric cancer cell growth in vitro and in vivo by inducing cytostatic autophagy via inhibition of MAPK/mTOR/p70S6K and Akt, and provided a molecular basis for the treatment of gastric cancer.

New withanolides from Physalis minima and their cytotoxicity against A375 human melanoma cells.

Seven previously undescribed withanolides, namely physaminilide A-G (1-7), and two artificial withanolides (8-9), along with 10 known analogues (10-19) were isolated from Physalis minima. The structures were established by spectroscopic analysis, including NMR and electronic circular dichroism (ECD) data. Cytotoxicity of all the isolates was evaluated against A375 human melanoma cells. Compounds 2, 5, 8, 10, 11 and 15 exhibited significant cytotoxic activities with IC50 values in the range of 1.2-9.4 µM.

Evanescent field response to immunoassay layer thickness on planar waveguides.

The response of a compact photonic immunoassay biosensor based on a planar waveguide to variation in antigen (C-reactive protein) concentration as well as waveguide ridge height has been investigated. Near-field scanning optical microscope measurements indicate 1.7%nm and 3.3%nm top surface optical intensity modulation due to changes in effective adlayer thickness on waveguides with 16.5 and 10 nm ridge heights, respectively. Beam propagation method simulations are in good agreement with the experimental sensitivities as well as the observation of leaky mode interference both within and after the adlayer region.

Microfluidic Protein Patterning on Silicon Nitride Using Solvent Extracted Poly(dimethylsiloxane) Channels.

Biomolecular patterning is essential for the creation of sensing motifs that rely on receptor-ligand binding for selectivity. Microfluidic devices have the potential to aid in the development of simple, robust methods for biomolecular patterning and therefore contribute to the generation of protein, DNA, and cell microarrays. In microfluidic patterning, the choice of both substrate and microfluidic channel material is essential for control of both the receptor binding for maximal signal generation as well as non-specific adsorption that acts as chemical noise. In this study, polystyrene, glass, silicon nitride, and poly(dimethylsiloxane) (PDMS) were evaluated as substrates for protein patterning using two types of PDMS microchannels for patterning, native PDMS and solvent-extracted PDMS (E-PDMS). E-PDMS microfluidic channels resulted in better patterning characteristics than native PDMS channels as determined by a higher fluorescence intensity of immobilized protein on all substrate types tested. Microfluidic patterning was then applied to perform two- and four-layer immunoassays.

Competitive immunoassays for simultaneous detection of metabolites and proteins using micromosaic patterning.

New high-throughput immunoassay methods for rapid point-of-care diagnostic applications represent an unmet need and current focus of numerous innovative methods. We report a new micromosaic competitive immunoassay developed for the analysis of the thyroid hormone thyroxine (T4), inflammation biomarker C-reactive protein (CRP), and the oxidative damage marker 3-nitrotyrosine (BSA-3NT) on a silicon nitride substrate. To demonstrate the versatility of the method, both direct and indirect format competitive immunoassays were developed and could be applied simultaneously for single samples. Signals from standard solutions were fit to a logistic equation, allowing simultaneous detection of T4 (7.7-257.2 nM), CRP (0.3-4.2 microg/mL), and BSA-3NT (0.03-22.3 microg/mL). Total assay time including sample introduction, washing, and fluorescence measurement was less than 45 min. Dissociation constants for affinity pairs in the system have been estimated using regression. This proof-of-concept experiment shows that both small and macromolecular biomarkers can be quantified from a single sample using the method and suggests that groups of clinically related analytes may be analyzed by competitive micromosaic immunoassay techniques.

Instrumentation for medium-throughput two-dimensional capillary electrophoresis with laser-induced fluorescence detection.

In two-dimensional capillary electrophoresis, a sample undergoes separation in the first dimension capillary by sieving electrophoresis. Fractions are periodically transferred across an interface into a second dimension capillary, where components are further resolved by micellar electrokinetic capillary electrophoresis. Previous instruments employed one pair of capillaries to analyze a single sample. We now report a multiplexed system that allows separation of five samples in parallel. Samples are injected into five first-dimension capillaries, fractions are transferred across an interface to 5 second-dimension capillaries, and analyte is detected by laser-induced fluorescence in a five-capillary sheath-flow cuvette. The instrument produces detection limits of 940 +/- 350 yoctomoles for 3-(2-furoyl)quinoline-2-carboxaldehyde labeled trypsin inhibitor in one-dimensional separation; detection limits degrade by a factor of 3.8 for two-dimensional separations. Two-dimensional capillary electrophoresis expression fingerprints were obtained from homogenates prepared from a lung cancer (A549) cell line, on the basis of capillary sieving electrophoresis (CSE) and micellar electrophoresis capillary chromatography (MECC). An average of 131 spots is resolved with signal-to-noise greater than 10. A Gaussian surface was fit to a set of 20 spots in each electropherogram. The mean spot width, expressed as standard deviation of the Gaussian function, was 2.3 +/- 0.7 transfers in the CSE dimension and 0.46 +/- 0.25 s in the MECC dimension. The standard deviation in spot position was 1.8 +/- 1.2 transfers in the CSE dimension and 0.88 +/- 0.55 s in the MECC dimension. Spot capacity was 300.

Study of interactions between actinomycin D and oligonucleotides by microchip electrophoresis and ESI-MS.

In the present study, the interactions between actinomycin D (ActD) and single stranded DNA (ssDNA) 5'-CGTAACCAACTGCAACGT-3' and a duplex stranded DNA (dsDNA) with this sequence were investigated by microchip-based non-gel sieving electrophoresis and electrospray ionization mass spectrometry (ESI-MS). The ssDNA was designed according to the conserved regions of open reading frame 1b (replicase 1B) following the Tor 2 SARS genome sequence of 15611-15593. The binding constants of the interactions between ActD and ssDNA/dsDNA were (8.3+/-0.32)x10(6)M(-1) (ssDNA) and (2.8+/-0.02)x10(5)M(-1) (dsDNA), respectively, calculated from microchip electrophoresis via Scatchard plot. The binding stoichiometries were 1:1 (single/1ActD molecule) and 1:2 (duplex/2ActD molecules) calculated from microchip electrophoresis, and the results were further verified by ESI-MS. The results obtained by these two methods indicated that ActD bound much more tightly to ssDNA used in this work than dsDNA. Furthermore, this is shown that the microchip-based non-gel sieving electrophoresis method is a rapid, highly sensitive and convenient method for the studies of interactions between DNA and small molecule drugs.

Binding of Ca2+, Mg2+, and heparin by human serum amyloid P component in affinity capillary electrophoresis.

Human serum amyloid P component (SAP) is a glycoprotein circulating in the blood and found in association with all types of amyloid (malfolded potein aggregates) examined so far. Despite uncertainties regarding the precise function of SAP in vivo, the lectin-like properties of this Ca(2+)-activated protein with affinity for anionic saccharides and malfolded proteins are well known. The propensity to form homomeric penta- or decamers in solution and the selfaggregation in the presence of Ca(2+) as well as the tendency of SAP to attach to uncoated fused silica have precluded the analysis of SAP by microelectrophoretic methods. We now work out conditions to characterize the binding of Ca(2+) and Mg(2+) and the binding of heparin to SAP in the presence of divalent metal ions by ACE. The results show a strong binding of heparin (sub-muM apparent dissociation constants) even in the abscence of Ca(2+) at low ionic strength, pH 8.2. Also, a selective interaction with Ca(2+) compared with Mg(2+) is demonstrated. The approach will further the use of microelectrophoretic methods to examine the interactions of SAP with ligands of putative pathophysiological relevance such as lipopolysaccharides and misfolded proteins.

Capillary zone electrophoresis characterization of low molecular weight heparin binding to interleukin 2.

A method based on capillary zone electrophoresis (CZE) was used to study the interaction between low molecular weight heparin (LMWH) and interleukin 2 (IL-2). The results showed that the increase of the concentration of LMWH led to the decrease of the peak height and the increase of the peak width of IL-2, but the peak areas were kept constant. The binding constant of IL-2 with LMWH was calculated as 1.2 x 10(6)M(-1) by Scatchard analysis, which is in good agreement with the results found in the references using enzyme-linked immunosorbent assay (ELISA). The results demonstrated that the interaction between IL-2 and LMWH is of fast on-and-off kinetic binding reaction. CZE might be used to study not only slow on-and-off rates interactions, but also fast on-and-off rates ones. The binding constant can be calculated easily, and the method can be applied to study a wide range of heparin-protein interactions.

Capillary zone electrophoresis investigation of the interaction between heparin and granulocyte-colony stimulating factor.

The interaction between standard heparin, low-molecular-weight heparin (LMWH), and granulocyte-colony stimulating factor (G-CSF) was studied by capillary zone electrophoresis. Both qualitative and quantitative characterizations of the heparin-protein binding were determined. The binding constants of the two different groups of heparins with G-CSF, calculated from the Scatchard plot by regression, were 4.805 x 10(5) M(-1) and 4.579 x 10(5) M(-1), respectively. The two binding constants measured are of the same order of magnitude at 10(5) M(-1), indicating that LMWH contains most of the functional groups bound to G-CSF by standard heparin.

Recent advances in the study of biomolecular interactions by capillary electrophoresis.

Capillary electrophoresis (CE) has been abundantly used in the study of molecular interactions owing to such advantages as short analysis time, low sample size requirement, high separation efficiency, and flexible applications. The focus of this paper is to review recent studies and advances (mainly from 1998 to now) in biomolecular interactions using CE. Five CE modes: zone migration CE, affinity CE, frontal analysis (FA), Hummel-Dreyer (HD) and vacancy peak (VP) are cited and compared. Quantitative aspects of the thermodynamics and kinetics of biomolecular interaction are reviewed. Several biomolecular binding systems, including protein-protein (polypeptide), protein-DNA (RNA), protein(polypeptide)-carbohydrate, protein-small molecule, DNA-small molecule, small molecule-small molecule, have been well characterized by CE. CE is shown to be a powerful tool for the determination of the binding parameters of various bioaffinity interactions.

Interaction between netropsin and double-stranded DNA in capillary zone electrophoresis and affinity capillary electrophoresis.

Capillary zone electrophoresis (CZE) and affinity capillary electrophoresis (ACE) were applied to study the interaction between netropsin and a 14mer double-stranded DNA (dsDNA). The use of a polyacrylamide coated capillary can suppress the electroosmotic flow (EOF) and the adsorption of DNA onto the wall. Better analysis of the DNA was achieved in a coated capillary upon Tris-acetate. In CZE, the peak width broadened due to the affinity interaction between dsDNA and netropsin. In ACE, o-toluic acid, a negatively charged molecule was used as the indicator to monitor the changes of EOF when netropsin was added to the running buffer. The 14mer dsDNA showed different mobilities upon various concentrations of netropsin due to the affinity interaction between the dsDNA and netropsin. The binding constants of this interaction were (1.07 +/- 0.10) x 10(5) M(-1) calculated from CZE and (4.75 +/- 0.30) x 10(4) M(-1) from ACE using a Scatchard plot. The binding stoichiometry was 1:1 calculated from CZE which was superior to ACE in this study.