Microchip electrophoretic separation for the fast diagnosis of Anaplasma phagocytophilum infection in cattle

We report a diagnostic method for Anaplasma phagocytophilum (A. phagocytophilum) infection in cattle using a nested PCR and microchip electrophoresis (ME). A. phagocytophilum causes human granulocytic anaplasmosis and granulocytic ehrlichiosis, which are emerging tick‐borne zoonotic diseases. Nested PCR was used to amplify genomic DNA samples extracted from cattle blood. The amplified PCR products were analyzed under a sieving gel matrix of 0.7% poly(ethyleneoxide) (M_r=8 000 000) in a conventional glass microchip. In the ME assay, A. phagocytophilum was analyzed within 35 s with a relative standard deviation of 1.30% (n=5) using a programmed field strength gradient (PFSG) as follows: 615.3 V/cm for 0–24 s, 66.7 V/cm for 24–34 s, 615.3 V/cm for 34–100 s. The ME‐PFSG assay was clinically validated by comparing the 16S rRNA gene levels obtained by this method with those measured using conventional slab gel electrophoresis performed with ten cattle blood samples suspected of A. phagocytophilum infection. In contrast to slab gel electrophoresis, the proposed ME‐PFSG methodology had increased sensitivity (200–450 pg/μL), a faster analysis time (<35 s), and required a smaller sample volume (～162 fL).     

Enzymatic transformation of platycosides and one‐step separation of platycodin D by high‐speed countercurrent chromatography

Platycosides, the saponins found in the roots of Platycodon grandiflorum (Platycodi Radix), are typically composed of oleanane triterpenes with two side chains. In platycosides, platycodin D, a glucose unit at C‐3, is a major component, which has several pharmacological activities. Because of the high demand for this compound, we attempted to enzymatically convert platycodin D₃ and platycoside E, having two and three glucose units at C‐3, respectively, into platycodin D. In this study, we tested the ability of several glycosidases to transform platycosides, or more specifically, the ability to transform platycoside E and platycodin D₃ into platycodin D. To obtain pure platycodin D on a preparative scale, high‐speed countercurrent chromatography with a solvent system of ethyl acetate/n‐butanol/water (1.2:1:2, v/v/v) was used for the separation of the enzymatically transformed product. Approximately 39.4 mg of platycodin D (99.8% purity) was obtained from 200 mg of the product in a one‐step separation. The results strongly support the advantage of enzymatic transformation of the platycosides for the efficient enrichment of platycodin D in the complicated extract of the medicinal plant.     

Sensitivity enhancement of CE and CE-MS for the analysis of peptides by a dynamic pH junction

An analytical method of CE-MS and CE with an online preconcentration technique induced by a dynamic pH junction, addition of organic solvent and large volume injection was developed for sensitive determination of peptides in biological samples. Leucine enkephalin, methionine enkephalin, dynorphin A, β-endorphin and angiotensin II were used as model peptides. The optimal online preconcentration conditions were obtained at a sample matrix consisting of 100?mM borate buffer (pH 10.0) with 50% v/v acetonitrile and a BGE containing 1?M formic acid at pH 2.0, along with a 25-cm injection length. Under the optimized conditions, a 4.0×10(3)-1.1×10(4)-fold increase in peak intensity was achieved without degrading the peak shape. This online preconcentration method was applied to analyze the intracellular angiotensin II within the peptides extracted from HL1 cells and approximately increase of 1×10(4)-fold sensitivity was achieved compared to normal condition. Thus, the developed method could be applied to the analysis of various peptides for peptidomics study in biological samples.     

Sulfuretin protects against cytokine-induced ��-cell damage and prevents streptozotocin-induced diabetes

NF-κB activation has been implicated as a key signaling mechanism for pancreatic β-cell damage. Sulfuretin is one of the main flavonoids produced by Rhus verniciflua, which is reported to inhibit the inflammatory response by suppressing the NF-κB pathway. Therefore, we isolated sulfuretin from Rhus verniciflua and evaluated if sulfuretin could inhibit cytokine- or streptozotocin-induced β-cell damage. Rat insulinoma RINm5F cells and isolated rat islets were treated with IL-1β and IFN-γ to induce cytotoxicity. Incubation of cells and islets with sulfuretin resulted in a significant reduction of cytokine-induced NF-κB activation and its downstream events, iNOS expression, and nitric oxide production. The cytotoxic effects of cytokines were completely abolished when cells or islets were pretreated with sulfuretin. The protective effect of sulfuretin was further demonstrated by normal insulin secretion of cytokine-treated islets in response to glucose. Treatment of mice with streptozotocin resulted in hyperglycemia and hypoinsulinemia, which was further evidenced by immunohistochemical staining of islets. However, the diabetogenic effects of streptozotocin were completely prevented when mice were pretreated with sulfuretin. The anti-diabetogenic effects of sulfuretin were also mediated by suppression of NF-κB activation. Collectively, these results indicate that sulfuretin may have therapeutic value in preventing β-cell damage.     

TGF-��-treated antigen presenting cells suppress collagen-induced arthritis through the promotion of Th2 responses

Collagen-induced arthritis (CIA) is mediated by self-reactive CD4⁺ T cells that produce inflammatory cytokines. TGF-β₂-treated tolerogenic antigen-presenting cells (Tol-APCs) are known to induce tolerance in various autoimmune diseases. In this study, we investigated whether collagen-specific Tol-APCs could induce suppression of CIA. We observed that Tol-APCs could suppress the development and severity of CIA and delay the onset of CIA. Treatment of Tol-APCs reduced the number of IFN-γ- and IL-17-producing CD4⁺ T cells and increased IL-4- and IL-5-producing CD4⁺ T cells upon collagen antigen stimulation in vitro. The suppression of CIA conferred by Tol-APCs correlated with their ability to selectively induce IL-10 production. We also observed that treatment of Tol-APCs inhibited not only cellular immune responses but also humoral immune responses in the process of CIA. Our results suggest that in vitro-generated Tol-APCs have potential therapeutic value for the treatment of rheumatoid arthritis as well as other autoimmune diseases.     

S-Adenosyl-L-methionine ameliorates TNF��-induced insulin resistance in 3T3-L1 adipocytes

An association between inflammatory processes and the pathogenesis of insulin resistance has been increasingly suggested. The IκB kinase-β (IKK-β)/ nuclear factor-κB (NF-κB) pathway is a molecular mediator of insulin resistance. S-Adenosyl-L-methionine (SAM) has both antioxidative and anti-inflammatory properties. We investigated the effects of SAM on the glucose transport and insulin signaling impaired by the tumor necrosis factor α (TNFα) in 3T3-L1 adipocytes. SAM partially reversed the basal and insulin stimulated glucose transport, which was impaired by TNFα. The TNFα-induced suppression of the tyrosine phosphorylation of the insulin receptor substrate-1 (IRS-1) and Akt in 3T3-L1 adipocytes was also reversed by SAM. In addition, SAM significantly attenuated the TNFα-induced degradation of IκB-α and NF-κB activation. Interestingly, SAM directly inhibited the kinase activity of IKK-β in vitro. These results suggest that SAM can alleviate TNFα mediated-insulin resistance by inhibiting the IKK-β/NF-κB pathway and thus can have a beneficial role in the treatment of type 2 diabetes mellitus.     

A near-infrared fluorescent sensor for detection of cyanide in aqueous solution and its application for bioimaging

A new NIR fluorescent sensor based on an amine-substituted heptamethine cyanine dye displayed a highly selective fluorescence enhancement with cyanide in aqueous solutions, and was applied for the imaging of anthropogenic and biogenic cyanide.     

Tunable luminescence and energy transfer process between Tb3+ and Eu3+ in GYAG:Bi3+, Tb3+, Eu3+ phosphors

A series of Eu³⁺ ions co-doped (Gd_0.9Y_0.1)₃Al₅O₁₂:Bi³⁺, Tb³⁺ (GYAG) phosphors have been synthesized by means of solvothermal reaction method. The XRD pattern of GYAG phosphor sintered at 1500 °C confirms their garnet phase. The luminescence properties of these phosphors have been explored by analyzing their excitation and emission spectra along with their decay curves. The excitation spectra of the GYAG:Bi³⁺, Tb³⁺, Eu³⁺ phosphors consists of broad bands in the shorter wavelength region due to 4f⁸ → 4f⁷5d¹ transition of Tb³⁺ ions overlapped with 6s² → 6s¹6p¹ (¹S₀ → ³P₁) transition of Bi³⁺ ions and the charge transfer band of Eu³⁺–O^2?. The present phosphors exhibit green and red colors due to ⁵D₄ → ⁷F₅ transition of Tb³⁺ ions and ⁵D₀ → ⁷F₁ transition of Eu³⁺ ions, respectively. The emission was shifted from green to red color by co-doping with Eu³⁺ ions, which indicate that the energy transfer probability from Tb³⁺ to Eu³⁺ ions are dependent strongly on the concentration of Eu³⁺ ions.