A portable and miniaturized lab-on-fiber sensor based on a responsive Fabry-Perot resonance cavity for the detection of thiocyanate.

Thiocyanate (SCN-) detection is highly significant because of the toxicity of SCN-. Herein, a portable and miniaturized lab-on-fiber (LOF) sensor is reported for the detection of SCN- through integrating a Fabry-Perot (F-P) optical resonance cavity based on anionic-responsive metal-insulator-metal (MIM) onto an optical fiber tip. The responsive MIM optical resonance cavity is constructed with an intermediate cationic polymer brush layer (poly[2-(methacryloyloxy)ethyl] trimethylammonium chloride, PMETAC) and two silver layers via a facile in situ "layer-by-layer" construction method. When the fabricated LOF sensor was immersed in SCN- solutions, an obvious reflection dip shift can be observed, which is feasible for the quantitative detection of SCN-. What's more, the fabricated LOF sensor exhibits outstanding selectivity and anti-interference against other interfering anions. Furthermore, the fabricated LOF sensor also displays other excellent advantages endowed by the polymer brush film, such as a fast response rate and outstanding reproducibility. Therefore, it is believed that the fabricated miniaturized LOF sensor would show great potential as a portable sensor in future applications, such as environmental monitoring and clinical diagnosis.

Ammonia, thiocyanate, and cyanate removal in an aerobic up-flow submerged attached growth reactor treating gold mine wastewater.

The objective of the study was to investigate the nitrification process, as well as the bio-chemical removal of cyanate and thiocyanate, while treating gold mining wastewater using an aerobic up-flow SAGR. A total of six SAGRs, each packed with locally sourced pea gravel (estimated specific surface area of 297 m-2 m-3), were operated at various HRTs and tested on both low- and high-strength gold mining wastewaters. The two sets of three SAGRs were operated at HRTs of 0.45 days, 1.20 days, and 2.40 days. Nitrification was successfully achieved in all six SAGRs regardless of the wastewater strength or HRT examined. The steady-state, 20 °C surface area loading rate was determined to be 1.2 g-TAN m-2 d-1 in order to comply with an effluent discharge limit at 10 mg-TAN L-1 (i.e., with the wastewater sources examined). At all ammonia loading rates, thiocyanate was successfully removed, and residual concentrations were below 2 mg-SCN-N L-1. Cyanate appeared to be hydrolyzed and subsequently nitrified. Acute toxicity tests conducted on both daphnia and trout revealed the effluent to be safe for direct discharge.

Determination of thiocyanate in exhaled breath condensate.

Thiocyanate is a heme peroxidase substrate that scavenges oxidants produced during inflammation and regulates host defense. In cystic fibrosis (CF) patients, increased airway thiocyanate levels are associated with improved lung function. Research on airway thiocyanate is limited, however, because convenient non-invasive airway sampling methods, such as exhaled breath condensate (EBC), yield low concentrations that are difficult to detect with available assays. In the present study, we developed a method for the determination of thiocyanate in dilute samples using isotope dilution headspace gas chromatography-coupled high-resolution, accurate-mass mass spectrometry (GC-HRMS). The method reliably quantified as little as 4 pmol thiocyanate in EBC and could detect even lower amounts. We successfully measured thiocyanate in EBC from seven healthy donors, with a mean ±â€¯SD of 27 ±â€¯16 nM and a median inter-assay coefficient of variation of 10.4% over six months. The method was applied to other biological fluids (plasma from the same visit as EBC donation; bronchoalveolar lavage fluid [BALF] from infants with CF; and healthy adult mouse BALF), giving reliable quantification of samples ranging from 10 nM to 100 µM. Thiocyanate concentrations in fluids besides EBC were (from lowest to highest): 0.73 ±â€¯0.39 µM in BALF of healthy adult mice (n = 6); 1.4 ±â€¯1.4 µM in BALF from infants with CF (n = 24); 46 ±â€¯22 µM in the plasma of adult volunteers (n = 7). These results demonstrate the utility of this new method for clinical determination of thiocyanate in EBC and other biological fluids.

Simultaneous removal of thiocyanate and nitrogen from wastewater by autotrophic denitritation process.

Pollutants containing sulfur as electron donors will play an important role in the energy-saving denitritation process when organic carbon source was insufficient in wastewater. However, thiocyanate (SCN-), a hazardous pollutant, has not been characterized in denitritation. In this study, the effects of key environmental factors on removal of thiocyanate and nitrogen were investigated in denitritation. The results showed that the maximum removal efficiency of nitrogen was observed in complete removal of thiocyanate and nitrite. The elemental sulfur was observed prior to complete depletion of thiocyanate. The efficiency of denitritation was promoted by NaHCO3 and weakly-alkaline environment. In the sludge containing dominant Thiobacillus genus, nitrite was reduced in the conversion of thiocyanate into elemental sulfur and further into sulfate. The stoichiometric ratio of NO2--N to SCN--N was close to 2.0 when thiocyanate was converted completely into sulfate, which verified complete removal of thiocyanate and nitrite at the NO2--N/SCN--N ratio of 2.0.

Tannins and Tannin-Related Derivatives Enhance the (Pseudo-)Halogenating Activity of Lactoperoxidase.

Several hydrolyzable tannins, proanthocyanidins, tannin derivatives, and a tannin-rich plant extract of tormentil rhizome were tested for their potential to regenerate the (pseudo-)halogenating activity, i.e., the oxidation of SCN- to hypothiocyanite -OSCN, of lactoperoxidase (LPO) after hydrogen peroxide-mediated enzyme inactivation. Measurements were performed using 5-thio-2-nitrobenzoic acid in the presence of tannins and related substances in order to determine kinetic parameters and to trace the LPO-mediated -OSCN formation. The results were combined with docking studies and molecular orbital analysis. The -OSCN-regenerating effect of tannin derivatives relates well with their binding properties toward LPO as well as their occupied molecular orbitals. Especially simple compounds like ellagic acid or methyl gallate and the complex plant extract were found as potent enzyme-regenerating compounds. As the (pseudo-)halogenating activity of LPO contributes to the maintenance of oral bacterial homeostasis, the results provide new insights into the antibacterial mode of action of tannins and related compounds. Furthermore, chemical properties of the tested compounds that are important for efficient enzyme-substrate interaction and regeneration of the -OSCN formation by LPO were identified.

To Analyze the Amelioration of Phenobarbital Induced Oxidative Stress by Erucin, as Indicated by Biochemical and Histological Alterations.

PURPOSE: Phenobarbital is a commonly employed antidepressant and anti-epileptic drug. The cancer promoting activity of this genotoxic xenobiotic is often ignored. It is responsible for oxidative stress leading to modulation in xenobiotic and antioxidative enzymes. Glucosinolates and more specifically their hydrolytic products are known for their antioxidative and anticancer activities. The present study involves the analysis of hepatoprotective effect of erucin (isolated from Eruca sativa (Mill.) Thell.) against phenobarbital mediated hepatic damage in male wistar rats. METHODS: The liver homogenate was analyzed for oxidative stress (superoxide dismutase, catalase, guaiacol peroxidase, ascorbate peroxidase, glutathione reductase and lactate dehydrogenase), other oxidative parameters (thiobarbituric acid reactive species, conjugated dienes and lipid hydroperoxide), phase I enzymes (NADPH-cytochrome P450 reductase, NADH-cytochrome b5 reductase, cytochrome P420, cytochrome P450 and cytochrome b5), phase II enzymes (γ-glutamyl transpeptidase, DT-diaphorase and glutathione-S-transferase), serum parameters (alkaline phosphatase, serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, direct bilirubin and total bilirubin) and certain histological parameters. RESULTS: Erucin accorded protection from phenobarbital induced hepatic damage by normalizing antioxidative enzymes, other oxidative parameters, phase I, II, and serum parameters. CONCLUSIONS: Erucin, an analogue of sulforaphane has the potential to act as an anticancer agent by regulating various biochemical parameters.

Chemical composition and biological activities of Eruca vesicaria subsp. longirostris essential oils.

Context To date, there are no reports to validate the Tunisian traditional and folklore claims of Eruca vesicaria (L) Cav. subsp. longirostris (Brassicaceae) for the treatment of disease. Objective Investigation of the chemical composition antimicrobial and antioxidant activity of essential oils from Eruca longirostris leaves, stems, roots and fruits. Materials and methods The essential oils of E. longirostris from leaves, stems, roots and fruits were obtained after 4 h of hydrodistillation. Chemical compositions were determined using a combination of GC/FID and GC/MS. The in vitro antimicrobial activity of the volatile constituents of E. longirostris was performed in sterile 96-well microplates against three Gram-positive, four Gram-negative bacteria and one strain as yeast. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration values were reported. Furthermore, the antioxidant activity was evaluated by DPPH and ABTS assays. Results The main compound for fruits, stems and roots was the erucin (96.6%, 85.3% and 83.7%, respectively), while ß-elemene (35.7%), hexahydrofarnesylacetone (23.9%), (E)-ß-damascone (15.4%), erucin (10.6%) and α-longipinene (9.6%) constituted the major compounds in the essential oil of the leaves. The experimental results showed that in all tests, essential oil of fruits showed the better antioxidant activity than the others. On the other hand, the oils of stems, fruits and roots showed significant antimicrobial activity with MIC values ranging from 0.125 to 0.31 mg/mL against Candida species, Gram-positive and Gram-negative bacteria, mainly Salmonella enterica. Conclusions The present results indicate that essential oils of E. longirostris can be used as a source of erucin.

Development of four-stage moving bed biofilm reactor train with a pre-denitrification configuration for the removal of thiocyanate and cyanate.

Two trains (A and B) of four-stage moving bed biofilm reactors (MBBRs) were developed for the degradation of thiocyanate (SCN(-)), cyanate (OCN(-)) and ammonia (NH3). A pre-denitrification configuration was established in the first-stage reactor of the B train using SCN(-) and OCN(-) as the sole carbon source. SCN(-), OCN(-) and NH3 were completely removed in both trains. The highest removal of total nitrogen equivalent (total-N) occurred at a loading rate of 5.6 mg-N L(-1) h(-1). The pre-denitrification configuration resulted in increased total-N removal in the B train (62.6%) compared to the A train (38.5%). Thiobacillus spp. were the predominant bacteria in all MBBRs. Bacteria related to bioprocesses involving anaerobic ammonium oxidation were present in the B train, suggesting that part of nitrogen removal occurs via this pathway. Our results showed that the pre-denitrification configuration increases the efficiency of removal of total-N compounds in the SCN(-)/OCN(-)-degrading MBBR process.

Spatial distribution of perchlorate, iodide and thiocyanate in the aquatic environment of Tianjin, China: environmental source analysis.

Although China is the largest producer of fireworks (perchlorate-containing products) in the world, the pathways through which perchlorate enters the environment have not been characterized completely in this country. In this study, perchlorate, iodide and thiocyanate were measured in 101 water samples, including waste water, surface water, sea water and paired samples of rain water and surface runoff collected in Tianjin, China. The concentrations of the target anions were generally on the order of rain>surface water≈waste water treatment plant (WWTP) influent>WWTP effluent. High concentrations of perchlorate, iodide and thiocyanate were detected in rain samples, ranging from 0.35 to 27.3 (median: 4.05), 0.51 to 8.33 (2.92), and 1.31 to 107 (5.62) ngmL(-)(1), respectively. Furthermore, the concentrations of the target anions in rain samples were significantly (r=0.596-0.750, p<0.01) positively correlated with the concentrations obtained in the paired surface runoff samples. The anions tested showed a clear spatial distribution, and higher concentrations were observed in the upper reaches of rivers, sea waters near the coast, and rain-surface runoff pairs sampled in urban areas. Our results revealed that precipitation may act as an important source of perchlorate, iodide and thiocyanate in surface water. Moreover, iodide concentrations in the Haihe River and Dagu Drainage Canal showed a good correlation with an ideal marker (acesulfame) of domestic waste water, indicating that input from domestic waste water was an important source of iodide in the surface waters of Tianjin.

Algal-bacterial process for the simultaneous detoxification of thiocyanate-containing wastewater and maximized lipid production under photoautotrophic/photoheterotrophic conditions.

In this work, a cooperative algal-bacterial system that efficiently degrades thiocyanate (SCN(-)), a toxic contaminant, and exhibits high lipid productivity, was developed. A consortium of mixed bacteria (activated sludge) and microalgae was sequentially cultivated under photoautotrophic and photoheterotrophic modes. The hydrolysis of SCN(-) to ammonium (NH4(+))-nitrogen and subsequent nitrification steps were performed by the initial activated sludge under lithoautotrophic conditions. The NH4(+) and oxidized forms of nitrogen, nitrite (NO2(-)) and nitrate (NO3(-)), were then assimilated and removed by the microalgal cells when light was supplied. After the degradation of SCN(-), the cultivation mode was changed to photoheterotrophic conditions in a sequential manner. Algal-bacterial cultures containing Chlorella protothecoides and Ettlia sp. yielded significantly increased lipid productivity under photoheterotrophic conditions compared to photoautotrophic conditions (28.7- and 17.3-fold higher, respectively). Statistical methodologies were also used to investigate the effects of volatile fatty acids and yeast extract on biomass and lipid production.

Glutamate signalling via a MEKK1 kinase-dependent pathway induces changes in Arabidopsis root architecture.

A chemical genetic approach has been used to investigate the mechanism by which external glutamate (l-Glu) is able to trigger major changes in root architecture in Arabidopsis thaliana L. An initial screen of 80 agonists and antagonists of mammalian glutamate and GABA receptors, using a specially developed 96-well microphenotyping system, found none that replicated the response of the root to l-Glu or antagonized it. However, a larger screen using >1500 molecules bioactive in Saccharomyces cerevisiae (yeast) identified two groups that interfered with the l-Glu response. One of the antagonists, 2-(4-chloro-3-methylphenyl)-2-oxoethyl thiocyanate (CMOT), has been reported to target Ste11, an evolutionarily conserved MAP kinase kinase kinase (MAP3K) in yeast. This led to the discovery that root growth in a triple mekk1 mekk2 mekk3 mutant (mekk1/2/3), defective in a set of three tandemly arranged MAP3Ks, was almost insensitive to l-Glu. However, the sensitivity of mekk1/2/3 roots to inhibition by other amino acids reported to act as agonists of glutamate receptor-like (GLR) channels in Arabidopsis roots (Asn, Cys, Gly and Ser) was unaffected. The l-Glu sensitivity of the mekk1/2/3 mutant was restored by transformation with a construct carrying the intact MEKK1 gene. These results demonstrate that MEKK1 plays a key role in transducing the l-Glu signal that elicits large-scale changes in root architecture, and provide genetic evidence for the existence in plants of an l-Glu signalling pathway analogous to that found in animals.

Apoptotic role of natural isothiocyanate from broccoli (Brassica oleracea italica) in experimental chemical lung carcinogenesis.

CONTEXT: Sulforaphane (SFN) [1-isothiocyanato-4-(methylsulfinyl)butane] is a naturally occurring isothiocyanate found in cruciferous vegetables such as broccoli [Brassica oleracea L. var. italica Plenck. (Brassicaceae)]. Since it is among the most potent bioactive components with antioxidant and antitumor properties, it has received intense attention in the recent years for its chemopreventive properties. OBJECTIVE: The present work determined the rehabilitating role in alleviating the oxidative damage caused by benzo(a)pyrene [B(a)P] to biomolecules and the apoptotic cascade mediated by orally administered isothiocyanate-SFN (9 µmol/mouse/day) against B(a)P (100 mg/kg body weight, i.p.) induced pulmonary carcinogenesis in Swiss albino mice. MATERIALS AND METHODS: Oxidative damage was assessed by measuring lipid peroxidation, 8-hydroxydeoxyguanosine, hydrogen peroxide (H2O2) production, glycoprotein components, protein carbonyl levels and DNA-protein crosslinks. DNA fragmentation by agarose gel electrophoresis and caspase-3 activity by ELISA proved apoptotic induction by SFN along with the protein expression of Bcl-2, Bax and Cyt c. RESULTS: SFN treatment was found to decrease the H2O2 production (p < 0.001) in cancer induced animals, proving its antioxidant potential. Apoptosis was induced by increasing the release of Cyt c (p < 0.001) from mitochondria, decreasing and increasing the expression of Bcl-2 (p < 0.01) and Bax (p < 0.001), respectively. Caspase-3 activity was also enhanced (p < 0.001) which leads to DNA fragmentation in SFN treated groups. CONCLUSION: Our results reflect the rehabilitating role of SFN in B(a)P induced lung carcinogenesis.

Simultaneous determination of glucoraphanin and sulforaphane in Brassica oleracea seeds by high-performance liquid chromatography with evaporative light-scattering detector.

In this article, a novel and efficient analysis method is described to simultaneously and quantitatively determine glucoraphanin and its major degradation product sulforaphane by reversed-phase high-performance liquid chromatography (HPLC) coupled with an evaporative light-scattering detector (ELSD). This method was validated according to the regulatory guidelines with respect to precision, accuracy and linearity. The HPLC-ELSD method was successfully used to assess the contents of glucoraphanin and sulforaphane in varieties of Brassica oleracea seeds. The developed analytical method not only avoids the underestimation of total glucoraphanin content, but also provides a reduction in analysis time and good efficiency and sensitivity compared to with conventional HPLC methods.

Sulforaphane is not an effective antagonist of the human pregnane X-receptor in vivo.

Sulforaphane (SFN), is an effective in vitro antagonist of ligand activation of the human pregnane and xenobiotic receptor (PXR). PXR mediated CYP3A4 up-regulation is implicated in adverse drug-drug interactions making identification of small molecule antagonists a desirable therapeutic goal. SFN is not an antagonist to mouse or rat PXR in vitro; thus, normal rodent species are not suitable as in vivo models for human response. To evaluate whether SFN can effectively antagonize ligand activation of human PXR in vivo, a three-armed, randomized, crossover trial was conducted with 24 healthy adults. The potent PXR ligand - rifampicin (300mg/d) was given alone for 7days in arm 1, or in daily combination with 450µmol SFN (Broccoli Sprout extract) in arm 2; SFN was given alone in arm 3. Midazolam as an in vivo phenotype marker of CYP3A was administered before and after each treatment arm. Rifampicin alone decreased midazolam AUC by 70%, indicative of the expected increase in CYP3A4 activity. Co-treatment with SFN did not reduce CYP3A4 induction. Treatment with SFN alone also did not affect CYP3A4 activity in the cohort as a whole, although in the subset with the highest basal CYP3A4 activity there was a statistically significant increase in midazolam AUC (i.e., decrease in CYP3A4 activity). A parallel study in humanized PXR mice yielded similar results. The parallel effects of SFN between humanized PXR mice and human subjects demonstrate the predictive value of humanized mouse models in situations where species differences in ligand-receptor interactions preclude the use of a native mouse model for studying human ligand-receptor pharmacology.

Removement of thiocyanate from industrial wastewater by microwave-Fenton oxidation method.

The microwave radiation oxidation process, Fenton as catalytic agent, was used to remove the thiocyanate from the industrial wastewater. The effects of microwave power, radiation time, pH and the feeding in ways of catalyst on the degradation rate of synthetic wastewater were investigated using the microwave radiation oxidation process by orthogonal experiment. The results show Fenton catalyst ratio was 1:20, the microwave radiation power was 900 W, the microwave radiation time was 7 min and the value of pH was 3. Under the optimum conditions, the removal of KSCN can reach over 90%. The apparent kinetics of removal was studied, which conformed to kinetics first-class reaction. In short, for the thiocyanate from the industrial wastewater, microwave-Fenton oxidation method is feasible and effective.

[Adsorption properties of thiocyanate anion on granular Mg/Al mixed oxides].

Granular Mg/Al mixed oxides (G-Mg3.3 AlO4.8) was successfully synthesized by using Mg/Al layered double hydroxide (Mg/Al-LDH) as the precursor. Its structure was characterized by analytical methods of BET, XRD, SEM and FT-IR. Adsorption properties of thiocyanate anion (SCN-) on this new material were also investigated. Specific surface area and average pore width of G-Mg3.3 AlO4.8 are 269.4 m2 x g(-1) and 13.25 nm respectively. Molding and granulation retains the layer structure and generates no new phase. Freundlich isotherm equation and pseudo-second-order kinetic equation can describe the static adsorption process precisely and adsorption capacity reaches 165.8 mg x g(-1) at 25 degrees C and 500 mg x L(-1). Yoon-Nelson model can predict the breakthrough curves of SCN- on G-Mg3.3 AlO4.8 precisely. The breakthrough adsorption capacity reaches 50.73 mg x g(-1) under the conditions with initial concentration is 100 mg x L(-1), flow rate is 5 mL x min(-1), bed layer altitude is 10 cm and initial pH is 6. The removal rates of SCN- are all above 98% after reusing four times. Consequently, G-Mg3.3 AlO4. 8 is a reusable and high-efficient adsorbent.

Quantitative profiling of glucosinolates by LC-MS analysis reveals several cultivars of cabbage and kale as promising sources of sulforaphane.

Sulforaphane is an isothiocyanate well known for its potential health benefits. With the aim of finding sulforaphane supply sources, its precursor, glucoraphanin, was widely searched for among Brassica oleracea varieties. Quantitative profiling of seven glucosinolates by LC-MS analysis was performed on 6 cultivars of broccoli, 32 of cabbage and 24 cultivars of kale. The glucoraphanin levels found in three cultivars of cabbage and six cultivars of kale were comparable with, or even higher than, the highest of broccoli (119.4 mg/100g FW). The most promising group belonged to the black kale, Cavolo nero. Use of a C30 column and an ammonium formate buffer in LC-MS and a micro plate solid phase extraction technique was highly effective.

Natural sulforaphane as a functional chemopreventive agent: including a review of isolation, purification and analysis methods.

Epidemiological data show that a diet rich in fruits and vegetables can reduce the risk from a number of cancers and chronic diseases. Sulforaphane (SF), a phytochemical constituent of cruciferous vegetables, has been widely researched in recent decades as a potential chemopreventive compound. Nonexistent in intact vegetables, natural SF, is formed from glucoraphanin hydrolyzed by myrosinase. This review summarizes and compares different analysis, isolation and purification methods engaged in SF research. Major important chemopreventive properties of SF investigated in existing research are reviewed and discussed, including antioxidant, anticarcinogenic and anti-inflammatory functions. Considering the potential applications of SF in the future, metabolism, stability and formulation developments of SF are also discussed. Research opportunities are identified based on the review of existing studies to facilitate future explorations on SF, a promising natural compound in chemopreventive therapy.

Nonionic surfactant enhanced semipermanent coatings for capillary electrophoresis of inorganic anions without use of organic additives.

Separation of inorganic anions by capillary electrophoresis (CE) is usually conducted in co-electroosmotic mode due to the large electrophoretic mobilities of inorganic anions. Semipermanent surfactant coatings have been shown to be effective for CE of inorganic anions due to their strong capability of electroosmotic flow (EOF) manipulation. However, semipermanent coatings often suffer from their unsatisfactory stability. In addition, organic solvent additives are usually required to adjust the selectivity, which also aggravate the degradation of coating. In this work, a novel semipermanent coating consisting of cationic Gemini surfactant 18-10-18 and nonionic surfactant Tween 20 was developed to separate inorganic anions in CE. This coating is easy to prepare and more stable than pure Gemini coating. The introduction of nonionic surfactant in the coating not only suppresses the reversed EOF but can also adjust the selectivity of separation. Good separations of six model anions were achieved, the separation efficiency was as high as 65040-169700 plates/m and the RSDs of the migration times were less than 0.5 and 2.5% for run-to-run and day-to-day assays, respectively. Calibration curves were linear in the range of 0.05-5.0 mM; the detection limits ranged from 20 to 50 µM. More importantly, no organic solvents are required in the background buffer to achieve the satisfactory separations. This guarantees the coating stability and makes the method greener than most of other methods for CE of inorganic anions.

Pseudomonas sax genes overcome aliphatic isothiocyanate-mediated non-host resistance in Arabidopsis.

Most plant-microbe interactions do not result in disease; natural products restrict non-host pathogens. We found that sulforaphane (4-methylsulfinylbutyl isothiocyanate), a natural product derived from aliphatic glucosinolates, inhibits growth in Arabidopsis of non-host Pseudomonas bacteria in planta. Multiple sax genes (saxCAB/F/D/G) were identified in Pseudomonas species virulent on Arabidopsis. These sax genes are required to overwhelm isothiocyanate-based defenses and facilitate a disease outcome, especially in the young leaves critical for plant survival. Introduction of saxCAB genes into non-host strains enabled them to overcome these Arabidopsis defenses. Our study shows that aliphatic isothiocyanates, previously shown to limit damage by herbivores, are also crucial, robust, and developmentally regulated defenses that underpin non-host resistance in the Arabidopsis-Pseudomonas pathosystem.