A validated UPLC–MS/MS method for the surveillance of ten aquatic biotoxins in European brackish and freshwater systems

Over the past few decades, there has been an increased frequency and duration of cyanobacterial Harmful Algal Blooms (HABs) in freshwater systems globally. These can produce secondary metabolites called cyanotoxins, many of which are hepatotoxins, raising concerns about repeated exposure through ingestion of contaminated drinking water or food or through recreational activities such as bathing/swimming. An ultra-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) multi-toxin method has been developed and validated for freshwater cyanotoxins; microcystins-LR, -YR, -RR, -LA, -LY and -LF, nodularin, cylindrospermopsin, anatoxin-a and the marine diatom toxin domoic acid. Separation was achieved in around 9 min and dual SPE was incorporated providing detection limits of between 0.3 and 5.6 ng/L of original sample. Intra- and inter-day precision analysis showed relative standard deviations (RSD) of 1.2–9.6% and 1.3–12.0% respectively. The method was applied to the analysis of aquatic samples (n = 206) from six European countries. The main class detected were the hepatotoxins; microcystin-YR (n = 22), cylindrospermopsin (n = 25), microcystin-RR (n = 17), microcystin-LR (n = 12), microcystin-LY (n = 1), microcystin-LF (n = 1) and nodularin (n = 5). For microcystins, the levels detected ranged from 0.001 to 1.51 μg/L, with two samples showing combined levels above the guideline set by the WHO of 1 μg/L for microcystin-LR. Several samples presented with multiple toxins indicating the potential for synergistic effects and possibly enhanced toxicity. This is the first published pan European survey of freshwater bodies for multiple biotoxins, including two identified for the first time; cylindrospermopsin in Ireland and nodularin in Germany, presenting further incentives for improved monitoring and development of strategies to mitigate human exposure.     

Identification of reproduction-related proteins and characterization of the protein disulfide isomerase A6 cDNA in ovaries of the giant tiger shrimp Penaeus monodon

Proteomic analysis was carried out for identification of proteins functionally involved in ovarian development of the giant tiger shrimp (Penaeus monodon). A total of 335 protein spots including 183 spots from vitellogenic (stage II) and 152 spots from mature (stage IV) ovaries of intact P. monodon broodstock were examined. Of these, 75 (40.98%) and 59 (38.82%) spots significantly matched known proteins in the databases, respectively. In addition, 270 protein spots including 167 and 103 spots from respective ovarian stages of eyestalk-ablated broodstock were also characterized. A total of 95 (56.89%) and 62 (60.19%) spots matched known proteins, respectively. Among differentially expressed reproduction-related proteins, the full-length cDNA of protein disulfide isomerase A6 (PmPDIA6) was further characterized by RACE-PCR. PmPDIA6 was 1946 bp in length containing an open reading frame (ORF) of 1293 bp corresponding to a polypeptide of 430 amino acids. PmPDIA6 was up-regulated at stage III ovaries in intact shrimp (P < 0.05). Interestingly, eyestalk ablation resulted in a lower expression level of PmPDIA6 in each stage of ovarian development compared to that of intact broodstock (P < 0.05). Results in this study clearly indicated the potential of cellular proteomic studies and gene expression analysis for identification of proteins/genes differentially expressed during ovarian development of P. monodon.     

Reduction in open field-induced hyperthermia in the rat exposed to chlorpyrifos,an anticholinesterase pesticide

Rodents develop a marked elevation in core temperature (T_c) when they are placed in an open field environment (OFE). Open field-induced hyperthermia (OFH) involves activation of thermoregulatory systems that are quiescent under baseline conditions. Cholinergic stimulation arising from exposure to organophosphates (OP) is expected to modulate OFH; subtle effects of OPs may be observed during OFH that would not occur in the absence of stress. We have found that methyl scopolamine (MS), a peripheral muscarinic antagonist, attenuates stress-induced hyperthermia. To this end, we assessed if a low dose of the OP pesticide chlorpyrifos (CHP) would alter OFH with and without administration of MS. T_c and motor activity (MA) of male, Sprague-Dawley rats were monitored by telemetry while housed in their home cage at an ambient temperature of 22°C. At 9 AM the rats were gavaged with corn oil or 10 mg/kg CHP that resulted in a ∼40% inhibition in plasma cholinesterase activity. T_c and MA were the same in the control and CHP groups prior to OFE. The rats were then dosed IP with saline or 1.0 mg/kg MS at 12 noon and subjected to OFE (61×61×61 cm box) for 1 h while T_c and MA were monitored every 2 min. T_c of the corn oil/saline group increased by 1.2°C during OFE, whereas T_c of the CHP/saline group was significantly attenuated. Administration of MS attenuated the OFH in rats treated with corn oil and CHP. Overall, exposure to OFE exacerbated the effects of a relatively low dose of CHP that had no effect in the unstressed animal. In addition, the OFH response to MS suggests a peripheral (i.e., outside of CNS) cholinergic pathway is operative in the control of OFH.     

Phenotypic and toxicological characterization of toxic Nodularia spumigena from a freshwater lake in Turkey

Cyanobacterial blooms have been occasionally observed in Iznik lake, a freshwater body (salinity = 0.5) located in the western part of Turkey. Nodularia spumigena (Mertens in Juergens) was recorded in the lake in the summer months of 2005. Maximum filament concentration of the species (1.3 × 10⁵ fil L^?1) was measured in August and constituted 60% of total cyanobacteria abundance. Trichomes were solitary, straight and had cells containing gas vesicles. Heterocysts were regularly spaced throughout the filaments. In the isolated filaments nodularin was detected by HPLC, ELISA and PPIA as well as LC–MS. HPLC analysis showed that gravimetric nodularin concentration in cultured N. spumigena cells was 578 μg of nodularin per gram dry weight (d.w.). Apart from nodularin, demethylated nodularin variant was also found in Nodularia cell extract. This is the first report of toxic N. spumigena in a European freshwater lake.     

Antibiofilm activity and post antifungal effect of lemongrass oil on clinical Candida dubliniensis isolate

Candidal infections are often difficult to eradicate due to the resistance of biofilms to antifungal agents. This study aimed at determining the effects of lemongrass (Cymbopogon citratus DC) oil against Candida dubliniensis in both planktonic and biofilms form. The results from broth microdilution method revealed that the minimum inhibitory and minimum fungicidal concentration of lemongrass oil on C. dubliniensis were 0.43 and 0.86 mg/ml, respectively. Employing a formazan salt (XTT tetrazolium) reduction assay for biofilm study, the results showed that the average percentage (mean ± SD) inhibition of lemongrass oil (0.43 mg/ml) on biofilm formation was 91.57 ± 1.31%, while it exhibited more than 80% killing activity against C. dubliniensis in biofilm at concentrations of 1.7 mg/ml. In addition, a significant reduction (P = 0.03) of candidal adhesion to acrylic occurred after a 15 min exposure to 1.7 mg/ml of lemongrass oil. Moreover, limited exposure of yeasts to lemongrass oil at subcidal concentration can suppress growth for more than 24 h. Altogether, the results obtained indicate that lemongrass oil possessed antifungal and antibiofilm activities and could modulate candidal colonization. Therefore, the efficacy of lemongrass oil merits further development of this agent for the therapy of oral candidiasis.     

Deciphering and engineering of the final step halogenase for improved chlortetracycline biosynthesis in industrial Streptomyces aureofaciens

Chlortetracycline (CTC) is an important member from antibiotics tetracycline (TC) family, which inhibits protein synthesis in bacteria and is widely involved in clinical therapy, animal feeds and aquaculture. Previous works have reported intricately the biosynthesis of CTC from the intermediates in random mutants of Streptomyces aureofaciens and the crucial chlorination remained unclear. We have developed the genetic manipulation in an industrial producer, in which about 15.0 g/l CTC predominated along with 1.2 g/l TC, and discovered that chlorination by ctcP (an FADH₂-dependent halogenase gene) is the last inefficient step during CTC biosynthesis. Firstly, the ΔctcP strain accumulated about 18.9 g/l “clean” TC without KBr addition and abolished the production of CTC. Subsequently, CtcP was identified to exhibit a substrate stereo-specificity to absolute TC (4S) rather than TC (4R), with low k_cat of 0.51±0.01 min⁻¹, while it could halogenate several TC analogs. Accordingly, we devised a strategy for overexpression of ctcP in S. aureofaciens and improved CTC production to a final titer of 25.9 g/l. We anticipate that our work will provide a biotechnological potential of enzymatic evolution and strain engineering towards new TC derivatives in microorganisms.     

High serum osteopontin levels in pediatric patients with high risk Langerhans cell histiocytosis

Osteopontin (OPN) acts as an osteoclast activator, a proinflammatory cytokine, and a chemokine attracting histiocytes/monocytes and is abundantly expressed in Langerhans cell histiocytosis (LCH). We investigated whether serum OPN levels are related to disease types in LCH. Fifty-eight newly diagnosed LCH patients were studied; eight with risk organ (liver, spleen and/or hematopoietic) involvements positive multisystem (MS+) disease, 27 with risk organ involvement negative multisystem (MS−) disease and 23 with single system (SS) disease. Pediatric patients with non-inflammatory disease (n = 27) were used as controls. All of patients with MS+ disease were younger than 3 years. Serum OPN levels and 44 kinds of humoral factors were measured by ELISA and Bio-Plex suspension array system, respectively. In the patients younger than 3 years, the median serum OPN level (interquartile range) was 240.3 ng/ml (137.6–456.0) in MS+ (n = 8); 92.7 ng/ml (62.0–213.8) in MS− (n = 14) and 72.5 ng/ml (55.6–94.0) in SS (n = 9) and 74.4 ng/ml (42.2–100.0) in control (n = 12). The OPN values were significantly higher in the MS+ group than the MS−, SS and control groups (p = 0.044, p = 0.001 and p = 0.002, respectively), but not different between the MS−, SS and control groups. In the patients older than 3 years, the median level of serum OPN (IQR) was 56.2 ng/ml (22.9–77.5) in MS− (n = 13), 58.9 ng/ml (31.0–78.7) in SS (n = 14) and 41.9 (28.9–54.1) in control (n = 15). These values did not differ significantly between each group. The serum OPN levels were positively correlated with the serum IL-6, CCL2, IL-18, IL-8 and IL-2 receptor concentration. OPN may be involved in risk organ dissemination and poor prognosis of LCH through the function as inflammatory cytokine/chemokine.     

Celery oil modulates DEHP-induced reproductive toxicity in male rats

The objective of the study was to investigate the protective effect of Apium graveolens (AP) against di-(2-ethylhexyl) phthalate (DEHP)-induced testes injury in rats. Adult rats were divided into nine groups: (1) control group (no treatment); (2) corn oil (60 μg/kg body weight – bwt); (3) AP (50 μg/kg bwt); (4) 300 mg DEHP/kg bwt; (5) 500 mg DEHP/kg bwt; (6) 1000 mg DEHP/kg bwt; (7) 300 mg DEHP/kg bwt + AP; (8) 500 mg DEHP/kg bwt + AP; and (9) 1000 mg DEHP/kg bwt + AP. Oral administration of treatments was performed daily for 6 weeks. DEHP decreased (p < 0.01) body weight, testis weight and serum concentrations of testosterone, cholesterol and total proteins. Moreover, DEHP increased (p < 0.001) total antioxidant capacity in the testis and plasma DEHP level. In addition, DEHP decreased mRNA expression of two testicular steroidogenic enzymes: 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase. DEHP also caused atrophy, vacuolar degeneration and aspermia of the seminiferous tubules. AP administered concurrently with DEHP effectively alleviated most of the DEHP-induced effects. In conclusion, in male rats, DEHP had adverse effects on the testis including inhibition of androgen production. A concurrent administration of A. graveolens (celery oil) protected the testis against DEHP-induced toxicity.     

Different expression of Fibrinopeptide A and related fragments in serum of type 1 diabetic patients with nephropathy

Type 1 diabetes (insulin-dependent diabetes mellitus, IDDM) is an autoimmune disease affecting about 0.12% of the world's population. Diabetic nephropathy (DN) is a major long-term complication of both types of diabetes and retains a high human, social and economic cost. Thus, the identification of markers for the early detection of DN represents a relevant target of diabetic research.The present work is a pilot study focused on proteomic analysis of serum of controls (n = 9), IDDM patients (n = 10) and DN patients (n = 4) by the ClinProt profiling technology based on mass spectrometry. This approach allowed to identify a pattern of peptides able to differentiate the studied populations with sensitivity and specificity close to 100%. Variance of the results allowed to estimate the sample size needed to keep the expected False Discovery Rate low. Moreover, three peptides differentially expressed in the serum of patients as compared to controls were identified by LC-ESI MS/MS as the whole fibrinopeptide A peptide and two of its fragments, respectively. The two fragments were under-expressed in diabetic patients, while Fibrinopeptide A was over-expressed, suggesting that anomalous turnover of Fibrinopeptide A could be involved in the pathogenesis of DN.     

Essential oil composition of Senecio graciliflorus DC: Comparative analysis of different parts and evaluation of antioxidant and cytotoxic activities

The essential oil of different parts of Senecio graciliflorus DC was obtained by hydrodistillation and analysed by GC-FID and GC–MS for the first time. A total of 17, 20, 19 and 17 constituents were identified comprising 99.90, 95.50, 98.93 and 95.96% of the essential oil of flower, leaf, stem and root parts of Senecio graciliflorus respectively. Monoterpene hydrocarbons predominated in the essential oil with 85.28% in flower, 57.53% in leaf, 67.74% in stem and 64.98% in root oil. α-pinene, cis-ocimene, 1,2,3-trimethylcyclohexane and β-pinene were the major constituents of the essential oil. The flower essential oil exhibited a strong antioxidant potential displaying IC₅₀ values of 21.6 ± 0.6 and 26.0 ± 1.0 μg/ml in DPPH and hydroxyl radical assays respectively. On the other hand the essential oil of flower and root displayed highest cytotoxicity against lung (A-549) cancer cell lines (IC₅₀ = 19.1 ± 0.9 and 21.3 ± 1.1 μg/ml respectively. This study which represents the first report of the essential oil composition and bioevaluation of Senecio graciliflorus, can serve as a new source of cytotoxic and antioxidant activity.     

Roles of adjuvants in aphicidal activity of enzymes from Beauveria bassiana (Ascomycota: Hypocreales) SFB-205 supernatant

Enzymes from Hypocrealean entomopathogenic fungi often encounter unfavorable abiotic and biotic factors during pathogenesis. The present work describes the roles of adjuvants, such as corn oil and polyoxyethylene-(3)-isotridecyl ether (TDE-3), in promoting aphicidal activity of enzyme precipitate from Beauveria bassiana SFB-205 supernatant. Supernatant enzymes including chitinase and proteases were lyophilized by attagel-mediated protein precipitation to produce attagel-mediated enzyme powder (AMEP). Corn oil-based AMEP + TDE-3 suspension showed 96.3% control efficacy against cotton aphids, Aphis gossypii Glover (Hemiptera: Aphididae), in glasshouse conditions at 2 days post-application, whereas water-based AMEP + TDE-3 suspension or TDE-3 alone showed < 20% efficacy. Corn oil-based AMEP + TDE-3 suspension was superior in degrading chitinase-specific substrate (p-nitrophenyl-β-d-acetylglucosaminide) under a drying condition compared to water-based AMEP + TDE-3 suspension. TDE-3 made supernatant, a source material of AMEP, degraded more cotton aphid proteins than supernatant alone in SDS-polyacrylamide gel electrophoresis; supernatant was used to clearly show the degradation without interfering with other proteins such as AMEP. These results suggest that 1) corn oil can slow down the evaporation of the diluted suspension drop and provide more time for enzymes from AMEP to degrade more cuticles at the time of application and 2) TDE-3 can disrupt chitin–protein matrixes within procuticle and facilitate enzymes from AMEP in degrading proteins which increases the exposure of chitin fibers to the attack of chitinases. This approach can provide another strategy in developing biopesticides using entomopathogenic fungi.     

Insecticidal properties of Pimpinella anisum essential oils against the Culex quinquefasciatus and the non-target organism Daphnia magna

The efficacy of an essential oil obtained from Pimpinella anisum fruits and its major compound, trans-Anethole, was tested on the eggs, larvae and adults of Culex quinquefasciatus. While causing no significant mortality on eggs, other tested stages were very sensitive to the essential oil and trans-Anethole. LC₅₀ for the 2nd to 4th instar larvae was estimated as 26–27 μL·L^− 1 and 15–19 μL·L^− 1 for the essential oil and trans-Anethole, respectively. As for the essential oil applied on adults, LC(LD)₅₀ was estimated as 9.3 μL mL^− 1 (spray test), 1.9 μL L^− 1 (fumigation test) and 0.6 μg cm^− 2 (tarsal test), and for trans-Anethole as 8.1 μL mL^− 1 (spray test), 2.1 μL L^− 1 (fumigation test) and 0.4 μg cm^− 2 (tarsal test). The time needed to achieve 50% mortality after application of LC(LD)₉₉ of the essential oil was significantly different; for example, in larvicidal assays it ranged from 15 to 235 min depending on the larval instar, and from 9 to 180 min when applied to adults, depending on the mode of application. It was also found that temperature had an important effect on the larvicidal efficacy of the essential oil, and oviposition deterrent activity was studied.The essential oil and trans-Anethole were toxic for Daphnia magna (62–92% mortality) and significantly reduced its fertility at high concentrations (35–50 μL mL^− 1) and long exposure (48 h). However, no negative effect on Daphnia mortality or fertility was found at shorter exposure times (6 h) and/or lower concentrations (20 μL mL^− 1).Based on the results of this study, we can recommend the essential oil from P. anisum as a suitable active substance for potential botanical insecticides.     

Differential proteomics of the plasma of individuals with sepsis caused by Acinetobacter baumannii

This study examines alterations in the plasma proteome in ten adults affected by sepsis caused by Acinetobacter baumannii as compared to paired healthy controls. 2-DE profiles of plasma from patients and paired healthy donors, depleted of the six most abundant proteins, were analysed by the DIGE technique. Protein spot detection and quantification were performed with the Differential In-gel Analysis and Biological Variation Analysis modules of the DeCyder^™ software. Differentially expressed proteins were identified by mass spectrometry (MALDI-TOF/TOF) after colloidal Coomassie blue staining.Almost 900 spots were detected on a unique 2-D gel by the DIGE technique. A total of 269 protein spots of differential abundance were shown to be statistically significant (2.5-fold) with p values of p ≤ 0.01 (135 spots) and p ≤ 0.05 (134 spots) as determined by the t test. Seventy-one spots were submitted to mass spectrometry and about 30% could be successfully identified.This multiplex approach significantly reduced experimental variability, allowing for the confident detection of small differences in protein levels. Results include differentially expressed lipoproteins as well as proteins belonging to inflammatory/coagulation pathways and the kallikrein–kinin system. These data improves the knowledge for future developments in sepsis diagnosis, staging and therapy.     

Evaluation of insecticidal activity of the essential oil of Allium chinense G. Don and its major constituents against Liposcelis bostrychophila Badonnel

Water-distilled essential oil from the dried bulbs of Allium chinense (Liliaceae) was analyzed by gas chromatography–mass spectrometry (GC–MS). Eighteen compounds, accounting for 98.4% of the total oil, were identified and the main components of the essential oil of A. chinense were methyl allyl trisulfide (30.7%), dimethyl trisulfide (24.1%), methyl propyl disulfide (12.8%) and dimethyl disulfide (9.6%) followed by methyl allyl disulfide (3.4%) and methyl propyl trisulfide (3.6%). The essential oil exhibited contact toxicity against the booklice (Liposcelis bostrychophila) with an LC₅₀ value of 441.8 μg/cm² while the two major constituents, dimethyl trisulfide and methyl propyl disulfide had LC₅₀ values of 153.0 μg/cm² and 738.0 μg/cm² against the booklice, respectively. The essential oil of A. chinense possessed strong fumigant toxicity against the booklice with an LC₅₀ value of 186.5 μg/l while methyl allyl trisulfide (LC₅₀ = 90.4 μg/l) and dimethyl trisulfide (LC₅₀ = 114.2 μg/l) exhibited stronger fumigant toxicity than methyl propyl disulfide (LC₅₀ = 243.4 μg/l) and dimethyl disulfide (LC₅₀ = 340.8 μg/l) against the booklice. The results indicated that the essential oil and its major constituents have potential for development into natural insecticides or fumigants for control of insects in stored grains.     

Glycosylated Alpha-1-acid glycoprotein 1 as a potential lung cancer serum biomarker

Presently existing screening approaches for lung cancer are not being proving sufficient and sensitive, so a study was conducted to identify disease related biomarker proteins for diagnostic applications. A total of 100 lung cancer patients (88 non-small cell lung cancer and 12 small cell lung cancer) and 50 healthy controls were included in this study. Serum samples of patients and healthy controls were subjected to a series of proteomic approaches and as a result of two dimensional gel electrophoresis, a ∼43 kDa protein was found to be differentially expressed compared to healthy controls. Quantitative profiling of two dimensional gels by Dymension software analysis displayed 3.58 fold increased expression of ∼43 kDa protein in squamous cell carcinoma and 2.92 fold in case of adenocarcinoma. Mass spectrometric analysis resulted in identification of 8 differentially expressed proteins, out of which human Alpha-1-acid glycoprotein 1 was targeted for further validations. This candidate protein exhibited N-linked glycosylation at five amino acid residues; 33, 56, 72, 93, and 103 with significant score of 0.66, 0.78, 0.78, 0.53 and 0.66, respectively. Sandwich ELISA quantified high serum levels of Alpha-1-acid glycoprotein 1 in squamous cell carcinoma (2.93 g/l ± 1.22) and adenocarcinoma (2.39 g/l ± 1.13) when compared with healthy controls (0.83 g/l ± 0.21). One-way ANOVA analysis predicted highly significant variation of Alpha-1-acid glycoprotein 1, among all the study types (F-value 65.37, p-value 0.000). This study may prove as a non-invasive, cost effective and sensitive scheme for diagnosis of lung cancer, by passing the expensive and painful screening procedures.     

Identification of Agrostis tenuis leaf proteins in response to As(V) and As(III) induced stress using a proteomics approach

Agrostis tenuis is known to be able to metabolise arsenate (As(V)) and arsenite (As(III)) which are toxic salts for most plants. A proteomic approach was developed to identify proteins expressed in response to treatments with these salts. A. tenuis plants were grown hydroponically in the presence of 134 and 668 μM As(V) or As(III) for 8 days at pH 7. During arsenic treatments, leaves showed chlorotic symptoms but fresh and dry leaf weights were not reduced, except in the presence of 668 μM As(III). On the contrary, a slight increase in biomass was observed with high As(V) concentrations. Thus, A. tenuis was more sensitive to As(III) than to As(V) and biomass was affected. Proteomic analysis enabled identification of a set of A. tenuis leaf proteins differentially expressed in response to arsenic exposure including a major functionally homogeneous group of enzymes such as oxygen-evolving enhancer protein, RuBisCO small and large subunits, RuBisCO activase and ATP synthase involved in the Calvin or Krebs cycle. The adaptative response to treatments resulted in partial disruption of the photosynthetic processes with prominent fragmentation of the RubisCO. Other proteins expressed differently from controls were identified and are possibly involved in the tolerance mechanisms of A. tenuis to arsenic treatments.