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.

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.

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.

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.

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.

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.

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.

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.

SPME-GC-pyrolysis-AFS determination of methylmercury in marine fish products by alkaline sample preparation and aqueous phase phenylation derivatization.

Characterization of a cost-efficient analytical method based on alkaline sample digestion with KOH and NaOH, followed by aqueous phase phenylation derivatization with NaBPh4 and solid phase microextraction (SPME) for the determination of methylmercury in typical fish-containing food samples commercially available in Hungary, is reported. The sample preparation procedure along with the applied SPME-GC-pyrolysis-AFS system was validated by measuring certified reference materials (CRM) BCR-464, TORT-2, and a candidate CRM BCR 710. To carry out an estimation of average Hungarian methylmercury exposures via marine fish and/or fish-containing food consumption, 16 commercially available products and 3 pooled representative seafood samples of-according to a previous European survey--the three most consumed fish species in Hungary, herring, sardines, and hake, were analyzed. Methylmercury concentrations of the analyzed samples were in the range 0.016-0.137 microg of MeHg g(-1) dry weight as Hg.

Arsenic speciation in farmed Hungarian freshwater fish.

Arsenic speciation analysis was carried out on freshwater farmed fish collected from an area with elevated groundwater arsenic concentrations in Hungary as well as from outside of the area (control samples). The arsenic species were determined by high-performance liquid chromatography-inductively coupled plasma mass spectrometry on methanol extracts of the muscle tissue from the fish. Catfish (Claries gariepinus) were raised in geothermal water where the average total arsenic concentrations were 167 (contaminated sites) and 15.1 ng As mL(-1) (control); they were all fed an artificial diet containing 2880 microg As kg(-1) total arsenic, mostly present as arsenobetaine. In the catfish, the accumulated total arsenic (2510-4720 microg As kg(-1)) was found mostly in the form of arsenobetaine suggesting that uptake of arsenic was dominated by their diet. Carp (Cyprinus carpio) were cultured in surface lakes with no significant arsenic pollution and had total arsenic concentrations ranging from 62 to 363 microg As kg(-1). The arsenic species found in the carp extracts differed markedly from those in the catfish in that no arsenobetaine was detected. Most samples of carp from the investigated sites contained low concentrations of As(III) (arsenite), As(V) (arsenate), MA (methylarsonate), and DMA (dimethylarsinate), and no other compounds were detected. The four individuals from the control site, however, all contained appreciable levels of oxo-arsenosugar-glycerol and oxo-arsenosugar-phosphate. Indeed, the oxo-arsenosugar-phosphate dominated the speciation pattern for these carp contributing about 75% of the sum of species. The contrast between these two freshwater aquaculture species regarding total arsenic and arsenic species has relevant toxicological aspects in terms of food safety.

Elevating the midface with barbed polypropylene sutures.

Barbed sutures, either used alone in a closed approach or as part of an open face lift procedure, may be combined with malar implants, soft tissue fillers, chemical peeling, and laser resurfacing. Because cephalad cheek repositioning affects adjacent facial areas, results may include shortening of the lower eyelid distance, flattening of the nasolabial fold, elevation of the submalar tissue, improvement of jowling, and decrease in submalar area fullness.

Suction-assisted lipoplasty: physics, optimization, and clinical verification.

BACKGROUND: Although suction-assisted lipoplasty (SAL) has been clinically practiced for more than 25 years, comparatively little investigation into fundamental physics of the instrumentation used in the procedure has been conducted. Moreover, relatively little is known about the clinical impact or merit of the wide variety of instrumentation currently available. OBJECTIVE: In this study, we examined the physics related to the various components of instrumentation used in lipoplasty, and developed means to optimize performance based on quantified bench and clinical data. METHODS: The components used to construct a lipoplasty system (vacuum pump, suction tubing, suction canister, and suction cannula) were first evaluated using methods of bench experimentation. A selected set of components/parameters were then evaluated in a clinical setting, and the results were correlated to the bench data. The following design parameters were analyzed: for cannulas-shaft length, shaft internal diameter, port size/pattern, and venting; for tubing-length, internal diameter, and collapsibility; for canisters-volume, pull-down speed, gradation precision, and splash-related issues; and for vacuum pumps-vacuum level and flow rate. RESULTS: Each of the system components can have a significant impact on the overall performance of the system. A simple calculation is presented that can be used to quantify the relative "resistance" and, therefore, speed of any selected cannula or tube. Port area is shown to be an important aspect of cannula design and clinical performance. Clinical data are shown to correlate reasonably with bench data, which imparts credibility to the bench data and provides a platform from which to extrapolate other bench data to the clinical setting. CONCLUSIONS: With clinical objectives in mind, guidelines and recommendations are presented, based on the data we collected, to optimize a lipoplasty system with regard to choices of the vacuum pump, suction tubing, and canister. With the ideal system in place, the cannula becomes the only remaining variable. Cannula properties and performance were also studied and are discussed in detail.

Secondary lipoplasty.

Although almost all patients who undergo lipoplasty procedures experience satisfactory outcomes with rapid recovery and few complications, misuse of lipoplasty techniques and technology may lead to suboptimal results. In this article, the indications for secondary lipoplasty are outlined, a clinical classification of candidates for secondary lipoplasty is presented, and the benefits and limitations of the approaches to secondary lipoplasty are discussed. (Aesthetic Surg J 2002;22:337-348.).

Clinical application of VASER--assisted lipoplasty: a pilot clinical study.

BACKGROUND: Although lipoplasty is the most frequently performed aesthetic surgical procedure, ultrasound-assisted lipoplasty (UAL) has not been widely adopted because of its increased potential for complications, complex and bulky instrumentation, additional cost, and steep learning curve. OBJECTIVE: We report on the use of the VASER ultrasound device in lipoplasty procedures and compare the clinical outcomes obtained by means of VASER-assisted lipoplasty with those of other UAL devices. METHODS: A superwet technique was used, and the wetting solution was uniformly distributed in the intended treatment area. Skin protection measures included use of specially designed skin ports to protect the incision edges and wet towels adjacent to the port locations. Access incisions were 3 to 4 mm in length. The VASER device was used in VASER (pulsed ultrasound) mode by 2 investigators (P.B.F. and M.L.J.); the continuous ultrasound mode was used by these investigators only if tissue emulsification was not readily achieved by using the VASER mode. A third investigator (E.B.d.S.P.) primarily used the continuous mode. Effective fat fragmentation in either mode was achieved by a maximum of 1 minute of treatment time per 100 mL of infused wetting solution. RESULTS: In a series of 77 patients treated by 3 different clinicians, satisfactory results were obtained with no major complications. This contrasts with an incidence of complications of 7.9% (median, 4.9%) for first- and second-generation UAL devices as determined by statistical analysis of the literature. CONCLUSIONS: The initial clinical experience with VASER-assisted lipoplasty indicates that it is a safe and efficient technique for body-contouring surgery. (Aesthetic Surg J 2002;22:131-146.).

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.