Accumulation of HT-2 toxin from contaminated mushroom compost by edible Agaricus bisporus.

Wheat straw is commonly used as a cellulose source in mushroom compost and could be a secondary source of mycotoxin contamination in the food chain. We cultivated edible Agaricus bisporus and Pleurotus ostreatus on T-2/HT-2 artificially-contaminated mushroom compost and developed and in-house validated an UHPLC-MS/MS method for determination of T-2, HT-2, T2-triol and T2-tetraol in mushroom compost and mushroom basidiocarp. A rapid phase I metabolization of T-2 and HT-2 in mushroom compost was observed. In Agaricus bisporus, basidiocarps 8-15 µg kg-1 accumulation of HT-2 calculated on wet weight was measured. No detectable mycotoxins were found in Pleurotus ostreatus basidiocarp.

Comparison of QuEChERS and "dilute and shoot" extraction methods for multi-mycotoxin analysis of samples from button mushroom (Agaricus bisporus) cultivation.

Several components of mushroom compost (wheat straw, chicken manure) can be contaminated with mycotoxins posing food health risks to mushroom consumers. To assess the relevance of such contaminations high-throughput analytical methods are needed. In this study, two sample preparation approaches, dilute & shoot (D&S) and modified citrate buffered Quick, Easy, Cheap, Effective, Rugged, Safe (QuEChERS) were compared in terms of extraction efficiency and matrix effect in case of 13 mycotoxins in complex matrices-wheat straw, the growing media and button mushrooms (Agaricus bisporus)-of mushroom cultivation using high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). D&S method resulted in recoveries of LB medium, button mushroom and compost for ≥60% in case of all investigated mycotoxins except for DON-3G. However, using modified citrate buffered QuEChERS with 2% acidification of the extraction solvent showed the complete loss of strongly polar DON-3G and fumonisin B1 (FB1). The investigated matrices had suppressive effect on ionization in all target mycotoxins except for FB1. Regarding the use of isotopologues to compensate matrix effect, even U-[13C15]-DON and U-[13C24]-T-2 can also be used to quantify their related metabolites in the studied matrices, using internal standard method.

Multi-mycotoxin LC-MS/MS method validation and its application to fifty-four wheat flours in Hungary.

In this study, fifty-four wheat flour and wheat-based products available on the Hungarian market were assessed for twelve mycotoxins. Prior to analysis, a multi-mycotoxin method using high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) was developed and validated for wheat and wheat-based products. A simple extraction with acetonitrile/water/formic acid (79/20/1 v/v%) was used for sample preparation. The limits of quantitation (LOQ) were between 0.02 and 161 µg kg-1. Good linearity (r2 > 0.995) was achieved for all mycotoxins investigated. Recoveries varied between 88 and 120% at three concentration levels. Based on the low relative matrix effect (RSD < 0.15%) of the different wheat flour samples, matrix-matched calibration was used, which also proved its suitability in proficiency testing (z-scores: -0.6 for DON; +1.5 for OTA; -0.5 for ZEA). DON was the predominant mycotoxin, which contaminated 84% of the investigated samples. Metabolised forms of DON were found in spelt, durum flour and some wheat-based products (D3G in 4 samples, 15Ac-DON in 7 samples). T-2 and HT-2 were the second most frequently detected mycotoxins. All investigated samples complied with current European/Hungarian legislation.

A novel variation of the suture suspension facelift.

BACKGROUND: We propose a technical variation of the minimally invasive suture suspension facelift. METHODS: A novel variation of the minimally invasive facelift is proposed. The technique consists of two components. The anterior component addresses aging of the midface by anchoring the SMAS on to the sturdy retroauricular supra-helical deep temporal fascia using approximately 6-8 passes of a 4/0 Mersilene suture. The posterior component addresses cervical aging by securing the posterior edge of the platysma onto the sturdy mastoid fascia, using multiple loops of a 4/0 Mersilene suture. This technique was performed on 100 consecutive patients between 2005 and 2010. RESULTS: The technique was found to be safe due to the plane of dissection remaining superficial to the parotid gland in the anterior component. There were no instances of facial nerve injury or sensory disturbance. None of the patients required a secondary procedure within the first 18 months following the procedure. CONCLUSIONS: This technique offers a safe and effective option for patients who seek a facelift, especially in combination with other procedures. It is envisaged that the use of multiple loops of suture to anchor mobile tissue onto fixed sturdy fascia will contribute to the longevity of the results. The limited skin undermining also makes this procedure a better choice for smokers.

Adipose Stem Cell Function Maintained with Age: An Intra-Subject Study of Long-Term Cryopreserved Cells.

Background: The progressive decline in tissue mechanical strength that occurs with aging is hypothesized to be due to a loss of resident stem cell number and function. As such, there is concern regarding use of autologous adult stem cell therapy in older patients. To abrogate this, many patients elect to cryopreserve the adipose stromal-vascular fraction (SVF) of lipoaspirate, which contains resident adipose stem cells (ASC). However, it is not clear yet if there is any clinical benefit from banking cells at a younger age. Objectives: We performed a comparative analysis of SVF composition and ASC function from cells obtained under GMP conditions from the same three patients with time gap of 7 to 12 years. Methods: SVF, cryobanked under good manufacturing practice (GMP) conditions, was thawed and cell yield, viability, and cellular composition were assessed. In parallel, ASC proliferation and efficiency of tri-lineage differentiation were evaluated. Results: The results showed no significant differences existed in cell yield and SVF subpopulation composition within the same patient between harvest procedures 7 to 12 years apart. Further, no change in proliferation rates of cultured ASCs was found, and expanded cells from all patients were capable of tri-lineage differentiation. Conclusions: By harvesting fat from the same patient at two time points, we have shown that despite the natural human aging process, the prevalence and functional activity of ASCs in an adult mesenchymal stem cell, is highly preserved. Level of Evidence: 5.

Adipose Derived Stromal Cell (ADSC) Injections for Pain Management of Osteoarthritis in the Human Knee Joint.

BACKGROUND: This safety and feasibility study used autologous adipose-derived stromal vascular cells (the stromal vascular fraction [SVF] of adipose tissue), to treat 8 osteoarthritic knees in 6 patients of grade I to III (K-L scale) with initial pain of 4 or greater on a 10-point Visual Analog Scale (VAS). OBJECTIVES: The primary objective of the study was evaluation of the safety of intra-articular injection of SVF. The secondary objective was to assess initial feasibility for reduction of pain in osteoarthritic knees. METHODS: Adipose-derived SVF cells were obtained through enzymatic disaggregation of lipoaspirate, resuspension in 3 mL of Lactated Ringer's Solution, and injection directly into the intra-articular space of the knee, with a mean of 14.1 million viable, nucleated SVF cells per knee. Metrics included monitoring of adverse events and preoperative to postoperative changes in the Western Ontario and McMaster Universities Arthritis Index (WOMAC), the VAS pain scale, range of motion (ROM), timed up-and-go (TUG), and MRI. RESULTS: No infections, acute pain flares, or other adverse events were reported. At 3-months postoperative, there was a statistically significant improvement in WOMAC and VAS scores (P < .02 and P < .001, respectively), which was maintained at 1 year. Physical therapy measurements for ROM and TUG both improved from preoperative to 3-months postoperative. Standard MRI assessment from preoperative to 3-months postoperative showed no detectable structural differences. All patients attained full activity with decreased knee pain. CONCLUSIONS: Autologous SVF was shown to be safe and to present a new potential therapy for reduction of pain for osteoarthritis of the knee. LEVEL OF EVIDENCE 4: Therapeutic.

Rapid evaluation technique to differentiate mushroom disease-related moulds by detecting microbial volatile organic compounds using HS-SPME-GC-MS.

Headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS) was used to analyse microbial volatile organic compounds (MVOCs) of mushroom disease-related microorganisms. Mycogone perniciosa, Lecanicillum fungicola var. fungicola, and Trichoderma aggressivum f. europaeum species, which are typically harmful in mushroom cultivation, were examined, and Agaricus bisporus (bisporic button mushroom) was also examined as a control. For internal standard, a mixture of alkanes was used; these were introduced as the memory effect of primed septa in the vial seal. Several different marker compounds were found in each sample, which enabled us to distinguish the different moulds and the mushroom mycelium from each other. Monitoring of marker compounds enabled us to investigate the behaviour of moulds. The records of the temporal pattern changes were used to produce partial least squares regression (PLS-R) models that enabled determination of the exact time of contamination (the infection time of the media). Using these evaluation techniques, the presence of mushroom disease-related fungi can be easily detected and monitored via their emitted MVOCs.

Outpatient surgery and sequelae: an analysis of the AAAASF Internet-based quality assurance and peer review database.

Although Internet-based quality assurance and peer review data have demonstrated the safety of procedures performed in the outpatient setting through the analysis of outcomes, the future of patient care will be directed by evidence-based medicine. Large inpatient surgical databases have long existed to provide quality assurance and improvement data for the inpatient cohort of patients. The acquisition of large data sets related to surgical care can best be achieved through the Internet. When outcomes are analyzed in conjunction with the indications for a procedure and the manner that care was delivered, evidence-based medicine is the end product.

Retrospective screening of relevant pesticide metabolites in food using liquid chromatography high resolution mass spectrometry and accurate-mass databases of parent molecules and diagnostic fragment ions.

In recent years, the detection and characterization of relevant pesticide metabolites in food is an important task in order to evaluate their formation, kinetics, stability, and toxicity. In this article, a methodology for the systematic screening of pesticides and their main metabolites in fruit and vegetable samples is described, using LC-HRMS and accurate-mass database search of parent compounds and their diagnostic fragment ions. The approach is based on (i) search for parent pesticide molecules; (ii) search for their metabolites in the positive samples, assuming common fragmentation pathways between the metabolites and parent pesticide molecules; and (iii) search for pesticide conjugates using the data from both parent species and diagnostic fragment ions. An accurate-mass database was constructed consisting of 1396 compounds (850 parent compounds, 447 fragment ions and 99 metabolites). The screening process was performed by the software in an automated fashion. The proposed methodology was evaluated with 29 incurred samples and the output obtained was compared to standard pesticide testing methods (targeted LC-MS/MS). Examples on the application of the proposed approach are shown, including the detection of several pesticide glycosides derivatives, which were found with significantly relevant intensities. Glucose-conjugated forms of parent compounds (e.g., fenhexamid-O-glucoside) and those of metabolites (e.g., despropyl-iprodione-N-glycoside) were detected. Facing the lack of standards for glycosylated pesticides, the study was completed with the synthesis of fenhexamid-O-glucoside for quantification purposes. In some cases the pesticide derivatives were found in a relatively high ratio, drawing the attention to these kinds of metabolites and showing that they should not be neglected in multi-residue methods. The global coverage obtained on the 29 analyzed samples showed the usefulness and benefits of the proposed approach and highlights the practical benefit obtained when the so-called screening methods are used as a complementary tool to standard targeted LC-MS/MS methods.

Study of the effects of operational parameters on multiresidue pesticide analysis by LC-MS/MS.

In this paper, the influence of several operational parameters on a well established multiresidue LC-MS/MS method has been studied in relation to the analysis of 150 pesticides commonly present in vegetable samples. The operational parameters investigated are: (i) the influence of different modifiers (0.1% formic acid; 5mM ammonium formiate; 5mM ammonium acetate in aqueous phase) - both on the retention time and on the analytical response of the studied compounds; (ii) the effect of the analytical column's temperature on the retention time and on the analytical response of the pesticides investigated; (iii) the effects of co-elution in mixture containing 150 pesticides and, additionally, (iv) the carrying out of a study about the common transitions obtained by LC-MS/MS. Various common transitions were found among the 150 pesticides, but there were only two problematic cases, the pairs diuron-fluometuron and prometryn-terbutryn, which have common scanned transitions and have very close retention times. The use of ammonium salts as modifier instead of formic acid reports enhancement or suppression of the response depending on the pesticides. No great influence on the retention time or on the response of the pesticides and commodities studied was observed with relation to the column temperature. Two different columns: an HPLC (5 µm particle size) and an UHPLC analytical column (1.8 µm particle size) have been used. As was expected, shorter run times and lower peak width was achieved with the UHPLC column. In this paper, the effect of the compounds on each other in the MS analysis when the number of co-eluting compounds is quite high is also described. Mainly small suppression or enhancement co-elution effect was observed, but some particular pesticides presented high sensitivity (> ± 60% effect) when they elute together with others. This is an important factor and it has to be taken into account when performing multiresidue pesticide analysis.

Reflections on lipoplasty: history and personal experience.

The author presents a brief history of the development of lipoplasty in the United States over more than 30 years. The chronology includes major clinical advances, along with the author's personal experience and the role of organized plastic surgery in defining and promoting safe practices.

Evaluation of occupational exposure in a slide bearings factory on the basis of urine and blood sample analyses.

The impact of a slide bearings factory on its workers was examined. Urine and blood samples were collected from 42 workers and six people employed in the offices in the same factory (control group). Concentrations of Al, Cu, Pb and Zn in blood and urine samples were measured twice (before and after chelation therapy) by ICP-MS technique using standard addition method. The essential differences in concentrations of elements for workers and control group were evaluated using non-parametric Mann-Whitney U-test. Significant differences between workers and control group were found for Pb in blood and Al in urine samples. The study was also undertaken to indicate correlation between blood and urine element content, workers' ages, their period of work and work section. It was also found that intravenous administration of 1 g of calcium-disodium versanate significantly increased urinary excretion of Pb and Zn, but not Al.

Bioavailability of selenium from selenium-enriched green onions (Allium fistulosum) and chives (Allium schoenoprasum) after 'in vitro' gastrointestinal digestion.

Three sample preparation methods--proteolysis to determine the initial species distribution, and an in vitro gastric and gastrointestinal digestion to assess the bioavailability of selenium--were applied to extract the selenium from selenized green onion and chive samples. Ion exchange chromatography was coupled to a high-performance liquid chromatography-ICP-MS system to analyze the selenium species of Allium samples. The difference in the selenium accumulation capability of green onions and chives was significant. Chive accumulated a one order of magnitude higher amount of selenium than did green onion. After proteolysis of both types of Allium plants, high amounts of organic selenium species such as MeSeCys, SeCys2 and SeMet became accessible. In the case of Se(VI)-enrichment, selenate was the main species in the proteolytic extract. After simulating the human digestion, the organic species were just slightly bioavailable compared with the results from proteolysis. The inorganic selenium content of the selenized samples increased significantly and SeOMet could be detected from the extracts. As an effect of the significant pH change between the gastric and the intestinal tracts, two oxidation processes took place: selenite oxidized to selenate, while SeMet oxidized to SeOMet.

Optimization of GC-ICPMS system parameters for the determination of butyltin compounds in Hungarian freshwater origin sediment and mussel samples.

The optimization and application of gas chromatograph coupled with inductively coupled plasma mass spectrometer (GC-ICPMS) (equipped with a commercially available interface) for the speciation of butyltin compounds in freshwater origin sediment and mussel samples is described. Optimization focused on the system parameters that have the greatest effect on signal intensity such as plasma power, ion lenses and make up gas flow (in the interface). Xenon (Xe) containing argon gas (Ar) was applied as tuning gas providing continuous Xe signal for the optimization of system parameters. It was found that plasma power and make up gas are interrelated variables and provide a set of paired optimum values at each power settings. The absolute optimum values obtained at 800W plasma power and 1.2Lmin(-1) make up gas flow rate when 7mm sample depth was adjusted. The optimum settings obtained were then checked by means of a test solution (tetraethyltin dissolved in hexane). Same optimum conditions were found when tin (Sn) transient signals were monitored. Detection limits were calculated for the three species using the optimized system parameters. Detection limits are the following: for monobutyltin (MBT) 5.6ngSnkg(-1), for dibutyltin (DBT) 6.6ngSnkg(-1) and for tributyltin (TBT) 3.4ngSnkg(-1) obtained. Determination of the butyltin compounds were carried out by means of species-specific isotope dilution analysis. The spike solution contained all species investigated but with altered isotopic composition. Each species were enriched in their (119)Sn isotope. Concentrations found in Hungarian freshwater origin mussel and sediment samples ranged between 19 and 39ngg(-1)for MBT, between 1.2 and 6.3ngg(-1) for DBT and between 1.2 and 3.2ngg(-1) for TBT indicated as Sn in dry weight. Validation of the method was done by means of certified reference materials (BCR CRM 646 and 477). Good agreement was found between certified and experimental values. Normalized deviation (E(n)) was also computed in order to validate the method used. E(n) values obtained ranged between 0.07 and 0.11 for mussel samples and between 0.26 and 0.72 for sediment samples. These values show that isotope dilution-GC-ICPMS methodology is valid for the determination of MBT, DBT and TBT from both types of matrices.

Development of a liquid chromatography/electrospray selected reaction monitoring method for the determination of organoarsenic species in marine and freshwater samples.

Cation- and anion-exchange high-performance liquid chromatography/electrospray selected reaction monitoring (HPLC/ES-SRM) methods were developed for the determination of 15 organoarsenic compounds in marine and freshwater samples. The results demonstrate that the developed HPLC/ES-SRM methods are powerful approaches for the identification of organoarsenic species in crude sample extracts. The detection limits, linearity as well as reproducibility for most of the species are comparable or even better than those measured by the HPLC/inductively coupled plasma mass spectrometry (ICPMS) technique. The qualitative analysis of the extracts shows that the developed methods allow for the identification of arsenicals which were not detectable by ICPMS. It was also demonstrated that the signal suppression caused by matrix effects means a significant limitation in the quantification of arsenicals by ES-SRM detection. This drawback is manifested especially in the case of the slightly retained species. The three sample-cleanup chromatographic methods including off-line size-exclusion, on-line reversed-phase and on-line oppositely charged ion-exchange approaches proved to be ineffective for separation of the signal-suppressive matrix from the analytes. The standard addition calibration seems to be a suitable solution for such problems.

Selenium species in aqueous extracts of alfalfa sprouts by two-dimensional liquid chromatography coupled to inductively coupled plasma mass spectrometry and electrospray mass spectrometry detection.

The complementary use of two different liquid chromatographic mechanisms coupled to inductively coupled plasma mass spectrometry (ICP-MS) for selenium (Se) specific detection has permitted the screening of the most abundant Se-containing fractions in selenized alfalfa sprouts (Medicago sativa). Aqueous extracts of the sprouts were fractionated first by size exclusion chromatography (SEC) using a Superdex Peptide column and a mobile phase containing an ammonium acetate buffer (pH 7). Further purification of the individual SEC Se-containing fractions was carried out using two different chromatographic systems: a Shodex Ashaipack column, with a mixed mechanism of size exclusion and ion exchange, and a conventional reversed phase C8 using ion-pairing reagents. In both cases, the columns were coupled to an inductively coupled plasma mass spectrometer equipped with an octapole reaction system for Se specific detection. This system allowed the on-line monitoring of the most abundant Se isotopes (78Se, 80Se) by reducing the possible polytomic interferences affecting these ions by adding hydrogen (2 mL min(-1)) to the octapole reaction cell. The results obtained by both separation mechanisms were highly comparable, revealing the presence of Se-methionine and Se-methyl selenocysteine. Both compounds were then confirmed by analyzing the corresponding fractions by electrospray quadrupole-time-of-flight (ESI-Q-TOF) mass spectrometry. Finally, an additional Se-containing species showing Se isotope distribution was detected at a molecular ion m/z 239 in the ESI-Q-TOF. The collision-induced dissociation of the m/z 239 and 237 ions (corresponding to 80Se and 78Se isotopes, respectively) revealed the possible presence as well of a derivative of the Se-2-propenyl selenocysteine.

Certification of a new selenized yeast reference material (SELM-1) for methionine, selenomethinone and total selenium content and its use in an intercomparison exercise for quantifying these analytes.

A new selenized yeast reference material (SELM-1) produced by the Institute for National Measurement Standards, National Research Council of Canada (INMS, NRC) certified for total selenium (2,059+/-64 mg kg(-1)), methionine (Met, 5,758+/-277 mg kg(-1)) and selenomethionine (SeMet, 3,431+/-157 mg kg(-1)) content is described. The +/-value represents an expanded uncertainty with a coverage factor of 2. SeMet and Met amount contents were established following a methanesulfonic acid digestion of the yeast using GC-MS and LC-MS quantitation. Isotope dilution (ID) calibration was used for both compounds, using 13C-labelled SeMet and Met. Total Se was determined after complete microwave acid digestion based on ID ICP-MS using a 82Se spike or ICP-OES spectrometry using external calibration. An international intercomparison exercise was piloted by NRC to assess the state-of-the-art of measurement of selenomethione in SELM-1. Determination of total Se and methionine was also attempted. Seven laboratories submitted results (2 National Metrology Institutes (NMIs) and 5 university/government laboratories). For SeMet, ten independent mean values were generated. Various acid digestion and enzymatic procedures followed by LC ICP-MS, LC AFS or GC-MS quantitation were used. Four values were based on species-specific ID calibration, one on non-species-specific ID with the remainder using standard addition (SA) or external calibration (EC). For total selenium, laboratories employed various acid digestion procedures followed by ICP-MS, AFS or GC-MS quantitation. Four laboratories employed ID calibration, the remaining used SA or EC. A total of seven independent results were submitted. Results for methionine were reported by only three laboratories, all of which used various acid digestion protocols combined with determination by GC-MS and LC UV. The majority of participants submitted values within the certified range for SeMet and total Se, whereas the intercomparison was judged unsuccessful for Met because only two external laboratories provided values, both of which were outside the certified range.

Relationships and bioaccumulation of chemical elements in the Baikal seal (Phoca sibirica).

Concentrations of Al, Ba, Cd, Cu, Fe, Mn, Mo, Si, Sr, Zn, Ca, K, Mg, Na and P in the livers of Baikal seal, plankton, zoobenthos, and fish, constituting the food sources for the seals, were determined by ICP-MS and ICP-AES. The accumulation of elements in the liver of seals, affected by internal and external (environmental) factors, was assessed by multidimensional (ANOVA, FA) and correlation analyses. FA has enabled identification of abiotic and biotic factors responsible for the accumulation of elements in the livers of Baikal seals. Significant influence of sex and development stage of the seals analysed on hepatic concentrations of some elements was found. The observed differences in element concentrations between pups, males and females could be attributable to the reproductive cycle of this species. ANOVA showed differences in concentrations of Fe, Zn, Cu and Cd in seals from the three separate basins of the lake. BMFs suggest biomagnification of Fe and Zn in the fish-seal trophic link.

Selenium speciation in Agaricus bisporus and Lentinula edodes mushroom proteins using multi-dimensional chromatography coupled to inductively coupled plasma mass spectrometry.

In this study, selenium species from Se containing proteins in mushrooms (Agaricus bisporus and Lentinula edodes) were investigated with size-exclusion liquid chromatography coupled to UV and inductively coupled plasma mass spectrometry (ICP-MS). Different protein extraction protocols were investigated. Variability of the fractionation patterns with three extraction media (0.1M NaOH, 30 mM Tris-HCl, and enzymatic digestions) was evaluated for both mushroom types. A 24 h Tris-HCl extraction followed by acetone addition was found to be optimal for protein precipitation. Presumably protein bound selenoamino acids were released using enzymes (proteinase K, protease XIV and trypsin). The selenium speciation of the proteolytic extract of the water soluble proteins fraction was carried out by using reversed-phase ion-pairing high performance liquid chromatography (RP-HPIPC) coupled on-line to ICP-MS for selenium specific detection. Selenocystine, selenomethionine, methylselenocysteine and inorganic selenium were established in both samples utilizing retention time standards and standard additions to the sample.