A universal dual-mode hydrogel array based on phage-DNA probe for simultaneous rapid screening and precisely quantitative detection of Escherichia coli O157:H7 in foods by the fluorescent/microfluidic chip electrophoresis methods.

Rapid and specific detection of virulent bacterial strains is a great challenge for food safety regarding large amounts of contaminated samples. Herein, a dual-mode hydrogel array biosensor was constructed to simultaneously rapidly screen and precisely quantitatively detect virulent Escherichia coli O157:H7 (E. coli O157:H7) based on a novel DNA-modified phage probe. First, E. coli O157:H7 was incubated with alginate to form the E. coli O157:H7/hydrogel premix complex. Subsequently, hydrogel formation by cross-linking upon the addition of calcium ions and phages for E. coli O157:H7 modified with a DNA primer (phage-DNA) was added to the alginate hydrogel. The DNA on the complex could trigger rolling circle amplification (RCA) to form a phage probe containing a long-chain DNA skeleton (phage@RCA-DNA). The RCA-DNA was then hybridized with the complementary DNA (cDNA) to form double-stranded DNA fragments (phage@RCA-dsDNA), which could be stained by the SYBR Green dye to emit visual green fluorescence (FL) and determined by a smartphone for rapid screening. Meanwhile, the unreacted cDNA in the supernatant could be quantitatively detected by microfluidic chip electrophoresis (MCE). The signal decrement was also proportional to the bacterial concentration. The detection limit values of E. coli O157:H7 were 50 CFU mL-1 by the FL signal and 6 CFU mL-1 by the MCE signal. The two results could be mutually corrected to decrease the false-positive results. This assay was also employed to detect virulent Salmonella Typhimurium (S. Typhimurium) using the corresponding S. Typhimurium phage@RCA-DNA probe. All these results demonstrated that the universal bioassay was suitable for simultaneous rapid screening and precisely quantitative detection of virulent bacterial strains.

Protective Autophagy in 5-Fluorouracil-Resistant Colorectal Cancer Cells and ERK-RSK-ABCG2 Linkage / Autofagia Protectora en Células de Cáncer Colorrectal Resistentes al 5-Fluorouracilo y Enlace ERK-RSK-ABCG2

SUMMARY: To evaluate the anti-cancer effects of yeast extract on resistant cells, autophagy and necroptosis were investigated in 5-fluorouracil (5-FU)-resistant colorectal cancer cells. Further underlying characteristics on drug resistance were evaluated, focused on ERK-RSK-ABCG2 linkage. SNU-C5 and 5-FU resistant SNU-C5 (SNU-C5/5-FUR) colorectal cancer cells were adopted for cell viability assay and Western blotting to examine the anti-cancer effects of yeast extract. Yeast extract induced autophagy in SNU-C5 cells with increased Atg7, Atg12-5 complex, Atg16L1, and LC3 activation (LC3-II/LC3-I), but little effects in SNU-C5/5-FUR cells with increased Atg12-5 complex and Atg16L1. Both colorectal cancer cells did not show necroptosis after yeast extract treatment. Based on increased ABCG2 and RSK expression after yeast extract treatment, drug resistance mechanisms were further evaluated. As compared to wild type, SNU-C5/5-FUR cells showed more ABCG2 expression, less RSK expression, and less phosphorylation of ERK. ABCG2 inhibitor, Ko143, treatment induces following changes: 1) more sensitivity at 500 mM 5-FU, 2) augmented proliferation, and 3) less phosphorylation of ERK. These results suggest that protective autophagy in SNU-C5/5-FUR cells with increased ABCG2 expression might be candidate mechanisms for drug resistance. As the ERK responses were different from each stimulus, the feasible mechanisms among ERK-RSK-ABCG2 should be further investigated in 5-FU-resistant CRC cells.

Para evaluar los efectos anticancerígenos del extracto de levadura en células resistentes, se investigaron la autofagia y la necroptosis en células de cáncer colorrectal resistentes al 5-fluorouracilo (5-FU). Además se evaluaron otras características subyacentes de la resistencia a los medicamentos centrándose en el enlace ERK-RSK-ABCG2. Se usaron células de cáncer colorrectal SNU-C5 (SNU-C5/5-FUR) resistentes a SNU-C5 y 5- FU para el ensayo de viabilidad celular y la transferencia Western para examinar los efectos anticancerígenos del extracto de levadura. El extracto de levadura indujo autofagia en células SNU-C5 con mayor activación de Atg7, complejo Atg12-5, Atg16L1 y LC3 (LC3-II/LC3-I), pero pocos efectos en células SNU-C5/5-FUR con aumento de Atg12-5 complejo y Atg16L1. Ambas células de cáncer colorrectal no mostraron necroptosis después del tratamiento con extracto de levadura. Se evaluaron los mecanismos de resistencia a los medicamentos. en base al aumento de la expresión de ABCG2 y RSK después del tratamiento con extracto de levadura.En comparación con las de tipo salvaje, las células SNU-C5/5-FUR mostraron más expresión de ABCG2, menos expresión de RSK y menos fosforilación de ERK. El tratamiento con inhibidor de ABCG2, Ko143, induce los siguientes cambios: 1) más sensibilidad a 5-FU 500 mM, 2) proliferación aumentada y 3) menos fosforilación de ERK. Estos resultados sugieren que la autofagia protectora en células SNU-C5/5-FUR con mayor expresión de ABCG2 podría ser un mecanismo candidato para la resistencia a los medicamentos. Como las respuestas de ERK fueron diferentes de cada estímulo, los mecanismos factibles entre ERK-RSK- ABCG2 deberían investigarse más a fondo en células CCR resistentes a 5-FU.

Comparison of the Performance of Bioresonance, Electrophoresis and RT-PCR in the Diagnosis of Feline Infectious Peritonitis.

Feline infectious peritonitis (FIP) continues to be one of the most researched infectious diseases of cats. The diagnosis of FIP is challenging, and diverse techniques have been developed for its accurate diagnosis. However, they have some limitations. The present study was conducted to investigate the efficacy of specific modulation frequency (SMF), compared to other routine diagnostic methods for detecting feline coronavirus. Blood samples were collected from 30 diseased cats suspected of having FIP based on clinical signs. Electrophoresis, polymerase chain reaction (PCR), and SMF tests were performed for each sample. The sensitivity and specificity of each test, as well as the agreement between the tests and the gold standard (the combination of PCR, electrophoresis, and bioresonance results), were calculated using the Kappa coefficient method. The sensitivity and specificity of electrophoresis, PCR, and SMF for the diagnosis of FIP were 70.6%, 70.6%, 100%, and 100%, 72.7%, 81.8%, respectively. According to the findings of the present study, SMF is effective and safe in FIP diagnosis, which is a challenge in veterinary medicine diagnosis.

Polarity-Specific Profiling of Metabolites in Single Cells by Probe Electrophoresis Mass Spectrometry.

Sensitive analysis of metabolites in a single cell is of fundamental significance for the better understanding of biological variability, differential susceptibility in disease therapy, and cell-to-cell heterogeneity as well. Herein, polarity-specific profiling of metabolites in a single cell was implemented by probe electrophoresis mass spectrometry (PEMS), which combined electrophoresis sampling of metabolites from a single cell and nanoelectrospray ionization-mass spectrometry (nanoESI-MS) analysis of the sampled metabolites. Enhanced extraction of either negatively or positively charged metabolites from a single cell was achieved by applying a DC voltage offset of +2.0 and -2.0 V to the probe, respectively. The experimental data demonstrated that PEMS features high throughput (≥200 peaks) and high sensitivity (≥10-times signal enhancement for [Choline + H]+, [Glutamine + H]+, [Arginine + H]+, etc.) in comparison with direct nanoESI-MS analysis. The biological effects of CdSe quantum dots (QDs) and γ-radiation on Allium cepa cells were investigated by PEMS, which revealed that CdSe QDs lead to the increase of intracellular amines while γ-radiation causes the decrease of intracellular acids. Therefore, this work provides an alternative platform to probe novel insights of cells by sensitive analysis of polarity-specific metabolites in a single cell.

Selective enrichment of zein gene of maize from cereal products using magnetic support having pyrrolidinyl peptide nucleic acid probe.

Here, we describe DNA enrichment of the zein gene from maize using pyrrolidinyl peptide nucleic acid (PNA) immobilized on a magnetic solid support as a capture element. Magnetite nanoparticles (MNP) with a capacity of 373 pmolPNA/mg and coated with poly(N-acryloylglycine) (PNAG) showed a good response to magnetic field. The PNA probe immobilized on the MNP discriminated between non-complementary and complementary DNA using fluorophore-tagged DNA as a model. We applied this system for the enrichment of the zein gene from maize in eight cereal product samples. After DNA desorption from the MNP, and its amplification via polymerase chain reaction (PCR), gel electrophoresis indicated that only cereal samples containing the zein gene from maize yielded positive results, indicating a high binding specificity between the PNA used and the complementary DNA. This PNA-functionalized MNP is potentially useful as an effective nano-solid support for DNA enrichment from other samples.

A Comparative Study of Some Procedures for Isolation of Fruit DNA of Sufficient Quality for PCR-Based Assays.

Food fraud has been and still is a problem in the food industry. It is detectable by several approaches, such as high performance liquid chromatography (HPLC), chemometric assays, or DNA-based techniques, each with its own drawbacks. This work addresses one major drawback of DNA-based methods, in particular their sensitivity to inhibitors contained in particular matrices from which DNA is isolated. We tested five commercial kits and one in-house method characterized by different ways of sample homogenization and DNA capture and purification. Using these methods, DNA was isolated from 10 different fruit species commonly used in plant-based foodstuffs. The quality of the DNA was evaluated by UV-VIS spectrophotometry. Two types of qPCR assays were used for DNA quality testing: (i) Method specific for plant ITS2 region, (ii) methods specific for individual fruit species. Based mainly on the results of real-time PCR assays, we were able to find two column-based kits and one magnetic carrier-based kit, which consistently provided fruit DNA isolates of sufficient quality for PCR-based assays useful for routine analysis and identification of individual fruit species in food products.

[The effect of complex use of nicotinic acid electrophoresis and air bubble baths on microcirculation in patients with diabetic polyneuropathy]. / Vliyanie kompleksnogo primeneniya elektroforeza nikotinovoi kisloty i vozdushno-puzyr'kovykh vann na mikrotsirkulyatsiyu u bol'nykh s diabeticheskoi polineiropatiei.

AIM OF STUDY: To study of the effect of the combined impact of nicotinic acid electrophoresis and air bubble baths on the state of microcirculation (MC) in patients with diabetic polyneuropathy (DPN). MATERIALS AND METHODS: 60 patients with DPN and type 2 diabetes mellitus with a disease duration of 6 to 25 years were examined and treated. All patients were divided into 2 groups: 30 patients of the 1st group received the combined impact of nicotinic acid electrophoresis and air-bubble baths; 30 patients of the 2nd group - only electrophoresis of nicotinic acid according to Vermel. The dynamics of the MC state was evaluated according to laser Doppler flowmetry (LDF). RESULTS: Before treatment, in most patients with DPN (98%), according to LDF, a spastic type of MC was established. After a course of treatment, it was found that the combined use of nicotinic acid electrophoresis and air bubble baths in patients with DPN has a more pronounced corrective effect on the condition of the MC than the use of Vermel's electrophoresis of nicotinic acid only. CONCLUSION: Complex therapy, including electrophoresis of nicotinic acid and air bubble baths, has a pronounced effect on the condition of the MC in patients with DPN and is pathogenetically justified for the treatment of this category of patients.

Methods for the Analysis of Polyphosphate in the Life Sciences.

Inorganic polyphosphate (polyP) is the polymer of orthophosphate and can be found in all living organisms. For polyP characterization, one or more of six parameters are of interest: the molecular structure (linear, cyclic, or branched), the concentration, the average chain length, the chain length distribution, the cellular localization, and the cation composition. Here, the merits, limitations, and critical parameters of the state-of-the-art methods for the analysis of the six parameters from the life sciences are discussed. With this contribution, we aim to lower the entry barrier into the analytics of polyP, a molecule with prominent, yet often incompletely understood, contributions to cellular function.

Electrophoretic deposition of polymethylmethacrylate and composites for biomedical applications.

For the first time, an electrophoretic deposition (EPD) method has been developed for the deposition of polymethylmethacrylate (PMMA) and PMMA-alumina films for biomedical implant applications. The proposed biomimetic approach was based on the use of a bile salt, sodium cholate (NaCh), which served as a multifunctional solubilizing, charging, dispersing and film-forming agent. Investigations revealed PMMA-Ch- and PMMA-alumina interactions, which facilitated the deposition of PMMA and PMMA-alumina films. This approach allows for the use of a non-toxic water-ethanol solvent for PMMA. The proposed deposition strategy can also be used for co-deposition of PMMA with other functional materials. The PMMA and composite films were tested for biomedical implant applications. The PMMA-alumina films showed statistically improved metabolic results compared to both the bare stainless steel substrate and pure PMMA films. Alkaline phosphatase (ALP) activity affirmed the bioactivity and osteoconductive potential of PMMA and composite films. PMMA-alumina films showed greater ALP activity than both the PMMA-coated and uncoated stainless steel.

Impact of Kuntai Capsules on LIF, IGF-1 and EGF expression in the implantation window of endometrium in mice / Impacto de cápsulas de Kuntai en la expresión de LIF, IGF-1 y EGF en la ventana de implantación de endometrio en ratones

To investigate the influence of Kuntai capsules on the expression level of leukemia inhibitory factor (LIF), insulin-like growth factor-I (IGF-1)and epidermal growth factor (EGF) during the mouse's implantation window of superovulation period and controlled ovarian hyperstimulation period. 90 female mice were randomly divided into six groups in control, superovulation and controlled ovarian hyperstimulation (COH) conditions. The RNA expression of EGF, LIF and IGF-1 in the endometrium on the 4th day of pregnancy was detected, and the relative expression was compared. mRNA expression of these three factors in endometrium was significantly lower in superovulation and COH groups than control group (p<0.001). mRNA expression of these three factors in endometrium remained obviously lower in superovulation plus kuntai capsule group and COH plus kuntai capsule group than control group (p<0.01). mRNA expression of these three factors in endometrium was lower in control group than in the NS plus kuntai capsule group (p<0.05). Kuntai capsule cannot completely reverse the endometrial damages caused by superovulation and COH. Thus Kuntai capsule could partially improve a mouse's endometrial receptivity during the implantation window.

Para investigar la influencia de las cápsulas de Kuntai en el nivel de expresión del factor inhibidor de la leucemia (LIF), el factor de crecimiento similar a la insulina I (IGF-1) y el factor de crecimiento epidérmico (EGF) durante la ventana de implantación del ratón del período de superovulación y la hiperestimulación ovárica controlada período, se dividieron aleatoriamente 90 ratones hembra en seis grupos en condiciones de control, superovulación e hiperestimulación ovárica controlada (COH). Se detectó la expresión de ARN de EGF, LIF e IGF-1en el endometrio al cuarto día de embarazo, y se comparó la expresión relativa. La expresión de ARNm de estos tres factores en el endometrio fue significativamente menor en los grupos de superovulación y COH que en el grupo control (p<0,001). La expresión de ARNm de estos tres factores en el endometrio permaneció más baja en el grupo de cápsulas de superovulación más Kuntai y en el grupo de cápsulas de COH más Kuntai respecto del grupo control (p<0,01). La expresión de ARNm de estos tres factores en el endometrio fue menor en el grupo control que en el grupo de cápsula NS más Kuntai (p<0,05). La cápsula de Kuntai no pudo revertir completamente los daños endometriales causados por la superovulación y la COH. Por lo tanto, se sugiere que la cápsula de Kuntai podría mejorar parcialmente la receptividad endometrial de un ratón durante la ventana de implantación.

Carbon-Nanotube-Based Monolithic CMOS Platform for Electrochemical Detection of Neurotransmitter Glutamate.

We present a monolithic biosensor platform, based on carbon-nanotube field-effect transistors (CNTFETs), for the detection of the neurotransmitter glutamate. We used an array of 9'216 CNTFET devices with 96 integrated readout and amplification channels that was realized in complementary metal-oxide semiconductor technology (CMOS). The detection principle is based on amperometry, where electrochemically active hydrogen peroxide, a product of the enzymatic reaction of the target analyte and an enzyme that was covalently bonded to the CNTFET, modulated the conductance of the CNTFET-based sensors. We assessed the performance of the CNTs as enzymatic sensors by evaluating the minimal resolvable concentration change of glutamate in aqueous solutions. The minimal resolvable concentration change amounted to 10 µM of glutamate, which was one of the best values reported for CMOS-based systems so far.

Towards CMOS Integrated Microfluidics Using Dielectrophoretic Immobilization.

Dielectrophoresis (DEP) is a nondestructive and noninvasive method which is favorable for point-of-care medical diagnostic tests. This technique exhibits prominent relevance in a wide range of medical applications wherein the miniaturized platform for manipulation (immobilization, separation or rotation), and detection of biological particles (cells or molecules) can be conducted. DEP can be performed using advanced planar technologies, such as complementary metal-oxide-semiconductor (CMOS) through interdigitated capacitive biosensors. The dielectrophoretically immobilization of micron and submicron size particles using interdigitated electrode (IDE) arrays is studied by finite element simulations. The CMOS compatible IDEs have been placed into the silicon microfluidic channel. A rigorous study of the DEP force actuation, the IDE's geometrical structure, and the fluid dynamics are crucial for enabling the complete platform for CMOS integrated microfluidics and detection of micron and submicron-sized particle ranges. The design of the IDEs is performed by robust finite element analyses to avoid time-consuming and costly fabrication processes. To analyze the preliminary microfluidic test vehicle, simulations were first performed with non-biological particles. To produce DEP force, an AC field in the range of 1 to 5 V (peak-to-peak) is applied to the IDE. The impact of the effective external and internal properties, such as actuating DEP frequency and voltage, fluid flow velocity, and IDE's geometrical parameters are investigated. The IDE based system will be used to immobilize and sense particles simultaneously while flowing through the microfluidic channel. The sensed particles will be detected using the capacitive sensing feature of the biosensor. The sensing and detecting of the particles are not in the scope of this paper and will be described in details elsewhere. However, to provide a complete overview of this system, the working principles of the sensor, the readout detection circuit, and the integration process of the silicon microfluidic channel are briefly discussed.

Fluorescence and absorption studies of DNA-Pd(II) complex interaction: Synthesis, spectroanalytical investigations and biological activities.

Novel palladium(II) complexes (7a-7e) of substituted quinoline derivatives were synthesized. The complexes were characterized using various techniques such as thermogravimetric analysis (TGA), elemental analysis, conductance measurement, mass, absorption, infra-red (IR), 1 H NMR, 13 C NMR and energy-dispersive X-ray spectroscopy (EDX). Complexes for herring sperm DNA (HS DNA) binding were explored and absorption titration and the binding constant (Kb ) as well as Gibb's free energy were evaluated. Complex 7d exhibited the highest binding constant, therefore the thermodynamic parameters of 7d at different temperatures were evaluated. To support the results of the absorption titration, fluorescence titration, viscosity measurement and molecular docking studies were performed. The fluorescence quenching data as evaluated from Stern-Volmer equation were used to calculate KSV , Kf and the number of binding sites. The results of all these studies were in good agreement with the absorption study. DNA electrophoretic mobility was performed to explore the possible application of metal complexes as artificial metallonucleases. The antibacterial activity of the complexes was accessed against different pathogenic bacteria and cytotoxicity was measured using brine shrimp and S. pombe.

Comparison of the reliability of snap foal Ig test, Gamma-Check E test, refractometry and electrophoresis for determining the immune status of newborn foals in the first hours of life.

Twenty-eight warmblood mares were monitored during their late pregnancy in the Teaching Hospital of Ghent University. The reliability of two commercial assays (enzyme immunoassay and glutaraldehyde coagulation test) used for determining the IgG concentrations of their newborn foals was tested. Mammary secretions were examined at the time of foaling (T0), and then 4 (T1) and 8 (T2) hours after foaling by refractometry and electrophoresis. The foals' blood IgG levels were measured at T1 and T2 as a routine clinical diagnostic examination using two different commercial test kits (SNAP Foal Ig and Gamma-Check E) and T0, T1 and T2 samples were stored (at -18 °C) for immunoglobulin (Ig) determination by electrophoresis. Differences between the results of refractometry and electrophoresis occurred in 27.8% of the colostrum analyses. Some serum IgG could be detected immediately post partum (T0) in 75% of the foals, and 42.82% of the newborn foals acquired a serum concentration of more than 800 mg/dl IgG within 8 h of birth. Compared to the electrophoresis, the glutaraldehyde test scored better (85%) than the enzyme immunoassay (74%), although both are accurate and safe to use since they clearly distinguish between safe and unsafe IgG concentrations.

Lecithin suspensions for electrophoretic deposition on stainless steel coatings.

Lecithin is a mixture of phospholipids (PLs) that are found in living organisms. It gained the interest as a bio- and hemocompatible modifying agent for biomaterials. In this paper, we focused on the elaboration of a simple and well-described technology of metals coating with low-cost substance that could be useful in biomaterials industry. We studied the utility of lecithin suspension for stainless steel coating by electrophoretic deposition method. Our goal was to find a relationship between the conditions of lecithin suspension preparation, obtained suspension properties (vesicles size and structure, zeta potential, electrophoretic mobility) and lecithin coating features (topography, roughness). We found that final pH value, zeta potential and electrophoretic mobility of lecithin suspensions were not altered by initial solution pH value. However, the presence of hydrated Na+ ions forced forming of large multi-layered vesicles. We obtained uniform lecithin coatings with the use of electrophoretic deposition, which has a great potential to be used in a large scale.

Evolution of 1, 3, 5-trisubstituted bipyrazole scaffold based platinum(II) complexes as a biological active agent.

Square planar mononuclear platinum(II) complexes having general formula [Pt(Ln)Cl2], (where, Ln = L1-4) were synthesized with neutral bidentate heterocyclic 1,3,5-trisubstituted bipyrazole based ligands. The synthesized compounds were characterized by physicochemical method such as TGA, molar conductance, micro-elemental analysis and magnetic moment, and spectroscopic method such as, FT-IR, UV-vis, 1H NMR, 13C NMR and mass spectrometry. Biological applications of the compounds were carried out using in vitro brine shrimp lethality bioassay, in vitro antimicrobial study against five different pathogens, and cellular level cytotoxicity against Schizosaccharomyces pombe (S. Pombe) cells. Pt(II) complexes were tested for DNA interaction activities using electronic absorption titration, viscosity measurements study, fluorescence quenching technique and molecular docking assay. Binding constants (Kb) of ligands and complexes were observed in the range of 0.23-1.07 × 105 M-1 and 0.51-3.13 × 105 M-1, respectively. Pt(II) complexes (I-IV) display an excellent binding tendency to biomolecule (DNA) and possess comparatively high binding constant (Kb) values than the ligands. The DNA binding study indicate partial intercalative mode of binding in complex-DNA. The gel electrophoresis activity was carried out to examine DNA nuclease property of pUC19 plasmid DNA.

Type C feruloyl esterase from Aspergillus ochraceus: a butanol specific biocatalyst for the synthesis of hydroxycinnamates in a ternary solvent system

Background: Aspergillus ochraceus was isolated from coffee pulp and selected as an interesting hydroxycinnamoyl esterase strain producer, using an activity microplate high-throughput screening method. In this work, we purified and characterized a new type C A. ochraceus feruloyl esterase (AocFaeC), which synthesized specifically butyl hydroxycinnamates in a ternary solvent system. Results: AocFaeC was produced by solid state fermentation, reaching its maximal activity (1.1 U/g) after 48 h of culture. After purification, the monomeric protein (34 kDa) showed a specific activity of 57.9 U/mg towards methyl ferulate. AocFaeC biochemical characterization confirmed its identity as a type C feruloyl esterase and suggested the presence of a catalytic serine in the active site. Its maximum hydrolytic activity was achieved at 40°C and pH 6.5 and increased by 109 and 77% with Ca2+ and Mg2+, but decreased by 90 and 45% with Hg2+ and Cu2+, respectively. The initial butyl ferulate synthesis rate increased from 0.8 to 23.7 nmol/min after transesterification condition improvement, using an isooctane:butanol:water ternary solvent system, surprisingly the synthesis activity using other alcohols was negligible. At these conditions, the synthesis specific activities for butyl p-coumarate, sinapinate, ferulate, and caffeate were 87.3, 97.6, 168.2, and 234 U/µmol, respectively. Remarkably, AocFaeC showed 5 folds higher butyl caffeate synthesis rate compared to type B Aspergillus niger feruloyl esterase, a well-known enzyme for its elevated activity towards caffeic acid esters. Conclusions: Type C feruloyl esterase from A. ochraceus is a butanol specific biocatalyst for the synthesis of hydroxycinnamates in a ternary solvent system

Effects of manganese toxicity on the protein profile of tomato (Solanum lycopersicum) roots as revealed by two complementary proteomic approaches, two-dimensional electrophoresis and shotgun analysis.

The aim of this work was to assess the effects of manganese (Mn) toxicity on the proteome of tomato roots using two proteomic approaches, shotgun and two-dimensional electrophoresis. The shotgun approach yielded 367 reliable proteins, whereas the 2-DE approach detected 340 consistent spots. The 2-DE method found 54 proteins changing in relative abundance in the excess Mn treatment, whereas the shotgun detected changes in 118 proteins. Only 7% of the differential proteins were found by both methods, illustrating their complementary nature. Metabolic pathways most affected were protein metabolism, oxido-reductases and signaling. Results support that Mn toxicity alters the protein turnover and impairs energy production in roots, leading to changes in glycolysis, pyruvate metabolism, TCA and oxidative phosphorylation. Excess Mn also induced changes in peroxidases and hydrolases participating in cell wall lignification and suberization and activated plant defense mechanisms, with changes occurring via pathogenesis-related proteins as well as peroxidases. Finally, Mn toxicity elicited regulatory mechanisms and affected the abundance of root nutrient reservoir proteins. The overall analysis of the differential root proteome upon Mn toxicity suggests a general slowdown of metabolic activities, especially energy production, cell wall integrity and protein turnover, which occurs in parallel with increases in stress related proteins.

A powerful on line ABTS+-CE-DAD method to screen and quantify major antioxidants for quality control of Shuxuening Injection.

A novel method of on-line 2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate)-Capillary Electrophoresis-Diode Array Detector (on-line ABTS+-CE-DAD) was developed to screen the major antioxidants from complex herbal medicines. ABTS+, one of well-known oxygen free radicals was firstly integrated into the capillary. For simultaneously detecting and separating ABTS+ and chemical components of herb medicines, some conditions were optimized. The on-line ABTS+-CE-DAD method has successfully been used to screen the main antioxidants from Shuxuening injection (SI), an herbal medicines injection. Under the optimum conditions, nine ingredients of SI including clitorin, rutin, isoquercitrin, Quercetin-3-O-D-glucosyl]-(1-2)-L-rhamnoside, kaempferol-3-O-rutinoside, kaempferol-7-O-ß-D-glucopyranoside, apigenin-7-O-Glucoside, quercetin-3-O-[2-O-(6-O-p-hydroxyl-E-coumaroyl)-D-glucosyl]-(1-2)-L-rhamnoside, 3-O-{2-O-[6-O-(p-hydroxyl-E-coumaroyl)-glucosyl]}-(1-2) rhamnosyl kaempfero were separated and identified as the major antioxidants. There is a linear relationship between the total amount of major antioxidants and total antioxidative activity of SI with a linear correlation coefficient of 0.9456. All the Relative standard deviations of recovery, precision and stability were below 7.5%. Based on these results, these nine ingredients could be selected as combinatorial markers to evaluate quality control of SI. It was concluded that on-line ABTS+-CE-DAD method was a simple, reliable and powerful tool to screen and quantify active ingredients for evaluating quality of herbal medicines.

Antioxidative Activity of Methanolic and Water Extracts from the Hyperthermophilic Archaeon Aeropyrum pernix K1.

The hyperthermophilic archaeon Aeropyrum pernix has adapted to optimal growth under high temperatures in saline environments and under oxidizing conditions. In the present study, we focused on the antioxidative activity of proteins from A. pernix K1. Following high temperature methanol and water extractions of the protein from the biomass of A. pernix K1, the total sulphydryl groups and radical scavenging activities were investigated. The total protein in the methanolic extract was 36% lower and showed 10% fewer sulphydryl groups than that from the water extract. However, the radical scavenging activity of the water extract was four-fold greater than for the methanolic extract. The proteins of both of these extracts were separated by two-dimensional electrophoresis, and selected proteins were identified using mass spectrometry. The majority of these identified proteins were intracellular proteins, such as those involved in oxidative stress responses and osmotic stress responses, and proteins with hydrolase and dehydrogenase activities. These proteins are also common to most organisms, and included putative uncharacterized proteins.