Sterigmatocystin and aflatoxin B<Subscript>1</Subscript> contamination of corn,soybean meal,and formula feed in Japan

Sterigmatocystin (STC) and aflatoxin B₁ (AFB₁) were analyzed in 246 corn samples, 126 soybean meal samples, and 861 formula feed samples from the Japanese market between April 2010 and March 2015. The detection rate, the highest concentration, and the mean concentration of STC were respectively 14%, 6.4 μg/kg, and 1.2 μg/kg for corn; 14%, 1.1 μg/kg, and 0.63 μg/kg for soybean meal; and 43%, 9.1 μg/kg, and 0.97 μg/kg for formula feed. The detection rate, the highest concentration, and the mean concentration of AFB₁ were respectively 46%, 24 μg/kg, and 3.9 μg/kg for corn; 30%, 6.7 μg/kg, and 1.1 μg/kg for soybean meal; and 47%, 20 μg/kg, and 1.6 μg/kg for formula feed. A weak negative correlation between the STC and AFB₁ concentrations was observed: there was a high concentration of AFB₁ in samples that contained a lower concentration of STC and vice versa. Spearman’s rank correlation coefficient showed a weak negative correlation of ? 0.30 (p < 0.001, n = 128) for corn and ? 0.23 (p < 0.001, n = 575) for formula feed. In conclusion, no correlation was observed between the mean concentrations of STC contamination in formula feed (0.97 μg/kg) and in corn (1.2 μg/kg) and the blending rate (approximately 50%). The rate of STC contamination in the formula feed (43%) was higher than that in corn (14%). Therefore, it is likely that ingredients other than corn contribute to the contamination of formula feed with STC. In this study, regarding STC, problematic samples were not found.     

Evaluation of an enzyme immunoassay for the detection of the mycotoxin tenuazonic acid in sorghum grains and sorghum-based infant food

An enzyme-linked immunosorbent assay (ELISA) for the Alternaria mycotoxin tenuazonic acid (TeA) was evaluated by comparative analysis of naturally contaminated sorghum grains and sorghum-based infant food, using a stable isotope dilution LC-MS assay (SIDA; limit of detection (LOD) 1.0 μg/kg) as the reference method. LODs of the ELISA were 30 μg/kg in sorghum grains and 220 μg/kg in sorghum-based infant cereals. With SIDA, 100% of the samples (n = 28) had been positive for TeA in a concentration range of 6–584 μg/kg (mean 113 μg/kg). The ELISA consistently detected TeA in all naturally contaminated samples at cut-off levels of 30–60 μg/kg (sorghum) and 200–300 μg/kg (infant cereals), as based on corresponding to SIDA values. Although the ELISA was much less sensitive than the SIDA method, it may be useful as a screening method for sorghum and sorghum-based infant foods and can be employed to identify samples containing elevated concentrations of TeA in food, well below the proposed level of concern (500 μg/kg).     

Survey of moniliformin in wheat- and corn-based products using a straightforward analytical method

A straightforward analytical method was developed and validated to determine the mycotoxin moniliformin in cereal-based foods. Moniliformin is extracted with water and quantified with liquid chromatography tandem mass spectrometry, and its presence confirmed with liquid chromatography-Orbitrap-high-resolution mass spectrometry. The method was validated for flour, bread, pasta and maize samples in terms of linearity, matrix effect, recovery, repeatability and limit of quantification. Quantification was conducted by matrix-matched calibration. Positive samples were confirmed by standard addition. Recovery ranged from 77 to 114% and repeatability from 1 to 14%. The limit of quantification, defined as the lowest concentration tested at which the validation criteria of recovery and repeatability were fulfilled, was 10 μg/kg. The method was applied to 102 cereal-based food samples collected in the Netherlands and Germany. Moniliformin was not detected in bread samples. One of 22 flour samples contained moniliformin at 10.6 μg/kg. Moniliformin occurred in seven out of 25 pasta samples at levels around 10 μg/kg. Moniliformin (MON) was present in eight out of 23 maize products at levels ranging from 12 to 207 μg/kg.     

Detection of cyclopiazonic acid (CPA) in maize by immunoassay

Cyclopiazonic acid (α-CPA) is a tremorgenic mycotoxin that is commonly produced by certain species of the aspergilli, in particular Aspergillus flavus, which is more widely known for production of the aflatoxins. Despite the fact that α-CPA may co-occur with aflatoxins, immunoassay-based methods for monitoring for CPA have not been widely developed. We report the development and evaluation of several monoclonal antibodies (mAbs) for α-CPA. Two mAbs in particular were very sensitive, with IC₅₀s of 1.1 and 1 ng/mL (clones 1418 and 1231, respectively). Tolerances to aqueous methanol or acetonitrile were good, which permitted the development of an antigen-immobilized competitive enzyme-linked immunosorbent assay (CI-ELISA) for detection of CPA in maize. Spiked or naturally contaminated maize, extracted with aqueous methanol, was diluted with buffer for analysis. The working range for the assay (IC₂₀ to IC₈₀) was from 5 to 28 μg/kg. Recoveries from maize spiked over the range from 2 to 50 μg/kg averaged 88.6 ± 12.6%. Twenty-eight samples of maize were tested by both the CI-ELISA and a liquid chromatography-fluorescence (LC-FLD) method. For the five samples above the limits of quantitation of both methods, CI-ELISA tended to overestimate CPA content, a difference that we speculate may be due to related metabolites or perhaps “masked” derivatives of CPA. The antibodies developed and the resulting CI-ELISA will be useful tools for further evaluation of the prevalence of this mycotoxin in maize.     

Occurrence of ochratoxin A in spices commercialized in Abidjan (Côte d’Ivoire)

Ochratoxin A (OTA) is a mycotoxin produced mostly by several species of Aspergillus and Penicillium. OTA is nephrotoxic in all animal species in which it has been tested and is cancerogenic in rodents. It is associated with Balkan endemic nephropathy. It is naturally present in many crop products such as cereals (barley, wheat, maize) and dried fruits, spices, coffee, wine, olives, and cocoa. The aim of this study was to assess the contamination of three Ivoirian spices with OTA (ginger, chili, and pepper) widely consumed by the population. A total of 90 spice samples (ginger: n?=?30; chili: n?=?30; pepper n?=?30) was taken from various sales outlets of Abidjan. OTA was quantified using an HPLC apparatus coupled with a fluorimetric detector. The chili and ginger samples were contaminated with OTA at a mean concentration of 57.48?±?174 and 0.12?±?0.15 μg/kg, respectively. No contamination of the pepper samples was detected. Eight (26.67 %) of the chili samples exceeded the maximum limit of 15 μg/kg established by European regulation. These results should serve as an alert on the risk to the consumer population of these products that are highly contaminated with OTA.     

<Emphasis Type="Italic">Alternaria</Emphasis> toxins in South African sunflower seeds: cooperative study

Sunflower seed samples (N = 80) from different sunflower cultivars originating from different localities in South Africa were analyzed for 15 toxins produced by fungi of the genus Alternaria by means of a simple one-step extraction dilute-and-shoot HPLC-MS/MS approach. References for valine-tenuazonic acid (Val-TeA), altenusin (ALTS), and altenuisol (ALTSOH) were isolated from fungal culture extracts and spectroscopically characterized. Additionally, valine-tenuazonic acid was tested regarding its cytotoxicity in comparison with tenuazonic acid (TeA) and showed less activity on HT-29 cells. Furthermore, alternariol monomethyl ether-3-O-ß-D-glucoside (AME-3G) was produced by fermentation of alternariol monomethyl ether (AME) with the fungus Rhizopus oryzae. The seed samples were analyzed both with and without hulls. The method covers the AAL toxins TA₁ and TA₂, altenuene (ALT) and iso-altenuene (iso-ALT), altenuisol, altenusin, altertoxin I (ATX-I) and altertoxin II (ATX-II), alternariol (AOH) and alternariol monomethyl ether, alternariol monomethyl ether-3-O-ß-D-glucoside, tenuazonic acid, allo-tenuazonic acid (allo-TeA) and valine-tenuazonic acid, and tentoxin (TEN). More than 80% of the samples were positive for one or more analytes above the respective limit of detection (0.2–23 μg/kg). Alternariol, its monomethyl ether, tentoxin, tenuazonic acid, altenuisol, and valine-tenuazonic acid were found in quantifiable amounts. The highest prevalences were found for tentoxin (73% positive, mean content 13.2 μg/kg, maximum level 130 ± 0.9 μg/kg) followed by tenuazonic acid (51% positive, mean content 630 μg/kg, maximum level 6300 ± 560 μg/kg). The obtained data were further analyzed statistically to identify quantitative or qualitative relationships between the levels of Alternaria toxin in the samples.     

Precise determination of the <Emphasis Type="Italic">Alternaria</Emphasis> mycotoxins alternariol and alternariol monomethyl ether in cereal,fruit and vegetable products using stable isotope dilution assays

Cereal, fruit and vegetable products were analyzed for contamination with the Alternaria mycotoxins alternariol (AOH) and alternariol monomethyl ether (AME) using stable isotope dilution assays (SIDAs). Both toxins were practically not detected in cereals and cereal products: AOH—one out of 13 samples at a content of 4.1 μg/kg; AME—two out of 13 samples at contents ranging between 0.2 and 0.6 μg/kg. However, if cereals for animal nutrition were analyzed, much higher values were found: AOH—five out of six samples (13–250 μg/kg); AME—six out of six samples (3–100 μg/kg). This finding may pose a potential problem concerning animal health. AOH and AME were frequently detected in vegetable products: AOH—5 out of 10 samples (2.6–25 μg/kg); AME—6 out of 10 samples (0.1–5 μg/kg). Tomato products were affected, especially. The highest content of AOH (25 μg/kg) and AME (5 μg/kg) were found in triple concentrated tomato paste. Special wines like “Trockenbeerenauslese” or “Spätlese” (affected by noble rot in the vineyard) contained AOH (4/6 samples; 1.2–4.9 μg/kg) and AME (4/6 samples; 0.1–0.3 μg/kg), but the values did not exceed the values of both toxins that were found generally in wines.     

A mini-survey of moulds and mycotoxins in locally grown and imported wheat grains in Nigeria

A preliminary survey involving limited sample size was conducted to determine the spectrum of moulds and mycotoxins in wheat grains from flour mills and local markets in Nigeria. Fourteen wheat samples were analyzed for moulds using standard mycological methods and for toxic fungal metabolites using a liquid chromatography-tandem mass spectrometric method. Fusarium (range of incidence 12.5–61.7%) dominated in the wheat grains though species of Aspergillus (range of incidence 2.24–3.86%) were also recovered from the samples. The identified fungal species were Aspergillus flavus (7.7%), Aspergillus niger clade (2.6%), Fusarium avenaceum (10.9%), Fusarium culmorum (22.4%) and Fusarium graminearum (56.4%). A total of 54 microbial metabolites were detected in the samples at concentration ranging between 0.01 μg/kg for macrosporin and 2560 μg/kg for deoxynivalenol. Among the four mycotoxins addressed by regulations in the European Union (EU) found in the samples, deoxynivalenol (incidence 100%) dominated in the samples and its levels exceeded the maximum acceptable EU limit (750 μg/kg) in 36% of the samples. This report underscores the need for more robust surveys with larger sample sizes and across several agro-ecologies in the country.     

Occurrence of aflatoxins and its management in diverse cropping systems of central Tanzania

The staple crops, maize, sorghum, bambara nut, groundnut, and sunflower common in semi-arid agro-pastoral farming systems of central Tanzania are prone to aflatoxin contamination. Consumption of such crop produce, contaminated with high levels of aflatoxin B₁ (AFB₁), affects growth and health. In this paper, aflatoxin contamination in freshly harvested and stored crop produce from central Tanzania was examined, including the efficacy of aflatoxin mitigation technologies on grain/kernal quality. A total of 312 farmers were recruited, trained on aflatoxin mitigation technologies, and allowed to deploy the technologies for 2 years. After 2 years, 188 of the 312 farmers were tracked to determine whether they had adopted and complied with the mitigation practices. Aflatoxigenic Aspergillus flavus and aflatoxin B1 contamination in freshly harvested and stored grains/kernels were assessed. A. flavus frequency and aflatoxin production by fungi were assayed by examining culture characteristics and thin-layer chromatography respectively. AFB₁ was assayed by enzyme-linked immunosorbent assay. The average aflatoxin contamination in freshly harvested samples was 18.8 μg/kg, which is above the acceptable standard of 10 μg/kg. Contamination increased during storage to an average of 57.2 μg/kg, indicating a high exposure risk. Grains and oilseeds from maize, sorghum, and sunflower produced in aboveground reproductive structures had relatively low aflatoxin contamination compared to those produced in geocarpic structures of groundnut and bambara nut. Farmers who adopted recommended post-harvest management practices had considerably lower aflatoxin contamination in their stored kernels/grains. Furthermore, the effects of these factors were quantified by multivariate statistical analyses. Training and behavioral changes by farmers in their post-harvest practice minimize aflatoxin contamination and improve food safety. Moreover, if non-trained farmers receive mitigation training, aflatoxin concentration is predicted to decrease by 28.9 μg/kg on average.     

A Rapid Method with UPLC for the Determination of Fusaric Acid in <Emphasis Type="Italic">Fusarium</Emphasis> Strains and Commercial Food and Feed Products

A rapid, sensitive and validated method for the determination of fusaric acid (FA) in several Fusarium strains and different commercial food and feed products is reported based on ultra-performance liquid chromatography. This method requires only crude sample by a simple extraction with methanol, and requires a very short time of 8 min for completion. Separation of FA was performed at injection volume of 1 μl with a 20:80 (v/v) water/acetonitrile mobile phase containing 0.1 % formic acid at a flow rate of 0.05 ml/min and detected with UV at 220 nm. Nice linearity and good correlation coefficient (R² > 0.99) were obtained in the concentration range of 1–200 μg/ml. Validation was demonstrated using blank samples spiked at three different concentrations with standard solution, and the method yielded more than 98.2 % recovery efficiencies and below 2.56 % R.S.D. when applied in the analysis of FA produced by Fusarium verticillioides and a set of transgenic strains of this fungus. Satisfactory recoveries in the range of 79.1–105.8 % and R.S.D lower than 10 % were also obtained for the tested commercial food and feed products. The concentration FA detection in the transgenic strains ranged from 9.65 to 135 μg/kg (0.29–4.05 μg per gram of biomass). However, FA was not detected in most of the commercial products with the exception of niblet, oatmeal, red kidney bean and soybean, for which the concentrations of FA ranged from 2.5 to 18 μg/kg (below the permitted maximum). These results show that the proposed method has a great potential application to analyze FA from different sources rapidly.     

Serum Level of Zinc and Copper in Sudanese Women with Polycystic Ovarian Syndrome

The paper’s objective was to estimate weekly Hg intake from fish meals based on intervention research. Total Hg (THg) concentrations in blood and hair samples collected from men (n = 67) from an intervention study as well as muscular tissues of fresh and after heat-treating fish were determined using the thermal decomposition amalgamation atomic absorption spectrometry method (TDA-AAS) using direct mercury analyzer (DMA-80). The mean of the estimated weekly intake (EWI) was estimated at 0.62 μg/kg bw/week in the range 0.36–0.96 μg/kg body weight (bw) /week through the consumption of 4 edible marine fish species every day (for 10 days) by the participants from the intervention research in Lodz, Poland. The Hg intake in the volunteers in our intervention study accounted for 38.6% of the provisional tolerable weekly intake (PTWI) (1.6 μg/kg bw, weekly) value. The average Hg concentration in the analyzed fish ranged from 0.018 ± 0.006 mg/kg wet weight (Gadus chalcogrammus) to 0.105 ± 0.015 mg/kg wet weight (Macruronus magellanicus). The results for the average consumers were within PTWI of methylmercury (MeHg). Moreover, the average concentration of Hg in the selected fish after heat treatment did not exceed the maximum permitted concentrations for MeHg (MPCs = 0.5 mg/kg wet weight) in food set by the European Commission Regulation (EC/1881/2006). Hence, the risk of adverse effects of MeHg for the participants is substantially low.     

Contamination with ergot bodies (<Emphasis Type="Italic">Claviceps purpurea sensu</Emphasis><Emphasis Type="Italic">lato</Emphasis>) of two horse pastures in Northern Germany

Because the occurrence of Claviceps in European pastures may have been overlooked to cause serious health problem for grazing animals, we documented the degree of Claviceps contamination in two horse pastures and estimated whether the horses could have ingested a critical quantity of alkaloids. We counted the Claviceps sclerotia and determined alkaloid levels using high performance liquid chromatography with fluorescence detection. Depending on the location, the number of sclerotia varied from 0.09 to 0.19 per square meter (central area) and from 0.23 to 55.8 per square meter (border strips). Alkaloid levels in individual sclerotia also varied in different genera of grasses, ranging from 0.98 ± 0.17 μg/kg in Agrostis sp. to 25.82 ± 9.73 μg/kg in Dactylis sp., equivalent to 0.98 μg/kg and 7.26 mg/kg. Sclerotia from Dactylis contained high levels of ergosine (0.209 % ± 0.100 %) and ergocristine (0.374 % ± 0.070 %). Depending on the localization in pastures, alkaloid levels in forage (dry matter, DM) ranged from 16.1 to 45.4 μg/kg in central areas and from 23.9 to 722 μg/kg in border strips. The amount of alkaloids that a horse could have ingested depended on its daily DM uptake, which was higher in the central areas (5.85 kg/day) than in the border strips (2.73 or 0.78 kg/day). In the central areas, this amount of alkaloids ranged from 94.2 to 265.9 μg/day; and in the border strips, from 65.3 (in 2.73 kg DM/day) to as much as 563.8 μg/day (in 0.78 kg DM/day). All these amounts are higher than the European averages for alkaloids ingested by horses via feedstuffs.     

Pre-harvest aflatoxins and <Emphasis Type="Italic">Aspergillus flavus</Emphasis> contamination in variable germplasms of red chillies from Kunri,Pakistan

Various cultivars of red chilli were collected from a small town named Kunri, located in the province Sindh, Pakistan. This town is a hub of red chilli production in Asia. A total of 69 samples belonging to 6 cultivars were obtained and analysed for the occurrence of aflatoxins and Aspergillus flavus, to explore the potential of resistant and susceptible germplasm. Aflatoxins were detected by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC), while A. flavus was isolated and identified using agar plate, blotter paper, deep freezing and dilution techniques. Molecular characterization using internal transcribed spacer (ITS) 1/4 and A. flavus specific FL1-F/R primers confirmed the identity of A. flavus. The data revealed that 67 and 75% samples contaminated with aflatoxin B₁ (AFB₁) and with A. flavus, respectively. A highly susceptible chilli cultivar was ‘Nagina’, showing 78.8% frequency of total aflatoxins (1.2–600 μg/kg) and a mean of 87.7 μg/kg for AFB₁ and 121.9 μg/kg for total aflatoxins. A. flavus was detected with 93% frequency and 2.14 × 10⁴ colony forming units. In contrast, cultivars ‘Kunri’ and ‘Drooping Type’ were found to be resistant, with low levels of aflatoxins and fungal counts. The study was conducted for the first time to explore two potential cultivars that were less susceptible towards A. flavus and aflatoxin contamination. These cultivars could be preferably cultivated and thereby boost Pakistan’s chilli production.     

Aflatoxin B<Subscript>1</Subscript> contamination in feed from Puglia and Basilicata regions (Italy): 5 years monitoring data

During a 5-year period from 2010 to 2014, n = 919 samples of feed and raw materials were analyzed for aflatoxin B₁ (AFB₁) contamination using accredited ELISA screening methods. Only 0.76 % of these samples were non-compliant with maximum levels set by the European Union Regulation 32/2002. Non-compliant samples were mainly from the province of Bari (n = 3 samples, mean AFB₁ value 7.03 μg/kg), although the highest AFB₁ levels were found in two samples from the provinces of Foggia and Brindisi, at 32.6 ± 3.6 μg/kg and 31.0 ± 4.0 μg/kg, respectively. Mean AFB₁ levels in samples contaminated but compliant with the limits ranged from 1.4 to 2.2 μg/kg. Considering the great importance of climate conditions in mycotoxins production, during crops production and during the critical phases of materials storage and/or transport, to better understand the variability in contamination levels, the analytical results were reviewed in term of temperature and relative environmental humidity in the sampling areas. Correlations between aflatoxin B₁ levels in feed and these climate factors might explain seasonal and annual variations in contamination levels. The data from the present study provide useful suggestions for the organization of targeted monitoring plans and the protection of consumers, as well as for improvement in the quality standards of zootechnological activities and feed industry.     

Antifungal Activity of Chitosan-Coated Poly(lactic-co-glycolic) Acid Nanoparticles Containing Amphotericin B

Amphotericin B (AmB) is one of the most used drugs for the treatment of systemic fungal infections; however, the treatment causes several toxic manifestations, including nephrotoxicity and hemolytic anemia. Chitosan-coated poly(lactide-co-glycolide) (PLGA) nanoparticles containing AmB were developed with the aim to decrease AmB toxicity and propose the oral route for AmB delivery. In this work, the antifungal efficacy of chitosan-coated PLGA nanoparticles containing AmB was evaluated in 20 strains of fungus isolates from patients with vulvovaginal candidiasis (01 Candida glabrata and 03 Candida albicans), bloodstream infections (04 C. albicans and 01 C. tropicalis) and patients with urinary tract infection (04 Candida albicans, 02 Trichosporon asahii, 01 C. guilhermondii, 03 C. glabrata) and 01 Candida albicans ATCC 90028. Moreover, the cytotoxicity over erythrocytes was evaluated. The single-emulsion solvent evaporation method was suitable for obtaining chitosan-coated PGLA nanoparticles containing AmB. Nanoparticles were spherical in shape, presented mean particle size about 460 nm, positive zeta potential and encapsulation efficiency of 42%. Moreover, nanoparticles prolonged the AmB release. All the strains were susceptible to plain AmB and nanostructured AmB, according to EUCAST breakpoint version 8.1 (resistant > 1 μg/mL), using broth microdilution method. In C. albicans (urine, blood, and vulvovaginal secretion isolates, and 1 ATCC), the MIC value of AmB-loaded nanoparticles varied from 0.25 to 0.5 μg/mL and EUCAST varied from 0.03 to 0.5 μg/mL. In urine and vulvovaginal secretion isolates of C. glabrata, the MIC value of AmB-loaded nanoparticles varied from 0.25 to 0.5 μg/mL and EUCAST varied from 0.03 to 0.015 μg/mL. In urine isolates of C. guilhermondii, the MIC value of AmB-loaded nanoparticles was 0.12 μg/mL and EUCAST was 0.06 μg/mL. In blood isolates of C. tropicalis, the MIC value of AmB-loaded nanoparticles was 0.5 μg/mL and EUCAST was 0.25 μg/mL. Finally, in urine isolates of T asahii, the MIC value of AmB-loaded nanoparticles was 1 μg/mL and EUCAST varied from 0.5 to 1 μg/mL. In the cytotoxicity assay, plain AmB was highly hemolytic (100% in 24 h) while AmB-loaded chitosan/PLGA nanoparticles presented negligible hemolysis.     

Efficient protoplast isolation and transient gene expression system for <Emphasis Type="Italic">Phalaenopsis</Emphasis> hybrid cultivar ‘Ruili Beauty’

With the release of the Phalaenopsis equestris (Schauer) Rchb.f. genome database, more in-depth studies of Phalaenopsis spp. will be carried out in the future. Transient gene expression in protoplasts is a useful system for gene function analysis, which is especially true for Phalaenopsis, whose stable genetic transformation is difficult and extremely time-consuming. In this study, juvenile leaves from aseptic Phalaenopsis seedlings were used as the starting material for protoplast isolation. After protocol refinement, the highest yield of viable protoplasts [5.94 × 10⁶ protoplasts g^?1 fresh weight (FW)] was achieved with 1.0% (w/v) Cellulase Onozuka R-10, 0.7% (w/v) Macerozyme R-10, and 0.4 M D-mannitol, with an enzymolysis duration of 6 h. As indicated by transient expression of green fluorescent protein (GFP), a transformation efficiency of 41.7% was achieved with 20% (w/v) polyethylene glycol (PEG-4000), 20 μg plasmid DNA, 2 × 10⁵ mL^?1 protoplasts, and a transfection duration of 30 min. The protocol established here will be valuable for functional studies of Phalaenopsis genes.     

High plasma adenosine levels in overweight/obese pregnant women

We aim to investigate whether overweight/obese pregnant women have elevated plasma levels of adenosine associated with increased consumption of high-calorie food. Sixty women were included. They were divided into lean (n = 23 and n = 12) or overweight/obese (n = 7 and n = 18) non-pregnant and pregnant women, respectively. Clinical records and maternal blood samples were collected after informed consent. A self-reported dietary questionnaire was also completed. Plasma adenosine levels were determined with high-performance liquid chromatography. Biochemical parameters, including glucose, total protein, and lipid profile, were determined using standard colorimetric assays. Adenosine levels were higher in pregnant women than in non-pregnant women (18.7 ± 1.6 vs 10.8 ± 1.3 nM/μg protein, respectively, p < 0.0001). Overweight/obese pregnant women (21.9 ± 2.5 nM/μg protein) exhibited higher adenosine levels than lean pregnant (14.5 ± 1.0 nM/μg protein, p = 0.04) or non-pregnant women (11.7 ± 1.5 nM/μg protein, p = 0.0005). Also, pregnant women with elevated weight gain exhibited higher (26.2 ± 3.7 nM/μg protein) adenosine levels than those with adequate weight gain (14.9 ± 1.4 nM/μg protein, p = 0.03). These differences were not statistically significant compared with those of pregnant women with reduced weight gain (17.4 ± 2.1 nM/μg protein, p = 0.053). Body mass index and adenosine only in pregnant women were positively correlated (r = 0.39, p = 0.02). While, polyunsaturated fatty acid (PUFA) consumption was negatively correlated with plasma adenosine levels only in non-pregnant women (r = ?0.33, p = 0.03). Pregnancy is associated with high plasma adenosine levels, which are further elevated in pregnant women who are overweight/obese. High PUFA intake might reduce plasma adenosine levels in non-pregnant women.     

Evaluation of Mineral Concentrations in Maternal Serum Before and After Birth and in Newborn Cord Blood Postpartum—Preliminary Study

The mineral levels in maternal serum change during pregnancy and may be correlated with those of newborn cord blood. The aim of this study was to evaluate the concentrations of calcium (Ca), magnesium (Mg), zinc (Zn), iron (Fe), and copper (Cu) in maternal blood before and after delivery and in umbilical cord vein and artery serum. The study was carried out in 64 Caucasian pregnant women who delivered in a district hospital in Greater Poland region, aged 28.1 ± 5.4 years, with a mean gestational age of 39.2 ± 1.3 weeks. Blood samples were taken from women 2–8 h before delivery and immediately after childbirth. The umbilical cord artery and vein blood of newborns was obtained immediately after childbirth. The levels of minerals in serum were determined by flame atomic absorption spectrometry. A significant drop in the concentrations of Mg (17.71 ± 1.51 vs 17.07 ± 1.61 μg/ml; p < 0.007), Fe (1.08 ± 0.46 vs 0.82 ± 0.35 μg/ml; p < 0.0004), and Zn (0.63 ± 0.17 vs 0.46 ± 0.16; p < 0.0001) in maternal serum was found after delivery. Moreover, higher levels of Ca, Fe, and Zn and lower levels of Cu were observed in the umbilical vein (Ca: 102.80 ± 7.80 μg/ml; p < 0.0001, Fe: 1.96 ± 0.43 μg/ml; p < 0.0001, Zn: 0.65 ± 0.16 μg/ml; p < 0.0001, Cu: 0.36 ± 0.09 μg/ml; p < 0.0001) and in the umbilical artery cord blood (Ca: 98.07 ± 8.18 μg/ml; p < 0.0001, Fe: 1.63 ± 0.30 μg/ml; p < 0.0001, Zn: 0.65 ± 0.15 μg/ml; p < 0.0001, and Cu: 0.36 ± 0.10 μg/ml; p < 0.0001) compared to the maternal serum (Ca: 85.05 ± 10.76 μg/ml, Fe: 0.82 ± 0.35 μg/ml, Zn: 0.46 ± 0.16 μg/ml, and Cu: 1.90 ± 0.35 μg/ml). Fe levels in the cord artery serum negatively correlated with blood loss during delivery (R = ?0.48; p = 0.01), while the Ca concentration in the maternal serum after birth decreased with the age of the women (R = ?0.25; p = 0.03). In conclusion, it seems that the process of birth alters the mineral levels in pregnant women’s blood. Moreover, it was found that blood loss and the age of the mother are associated with mineral concentrations in the maternal serum and cord artery blood.     

Recovery of avocado (<Emphasis Type="Italic">Persea americana</Emphasis> Mill.) plants transformed with the antifungal plant defensin gene PDF1.2

Embryogenic avocado cultures derived from ‘Hass’ protoplasts were genetically transformed with the plant defensin gene (pdf1.2) driven by the CaMV 35S promoter in pGPTV with uidA as a reporter gene and bar, the gene for resistance to phosphinothricin, the active ingredient of the herbicide Finale® (Basta) (Bayer Environmental Science, Research Triangle Park, Durham, NC ). Transformation was mediated by Agrobacterium tumefaciens strain EHA105. Transformed cultures were selected in the presence of 3.0 mg l^?1 phosphinothricin in liquid maintenance medium for 3–4 mo. Liquid maintenance medium consisted of modified MS medium containing (per liter) 12 mg NH₄NO₃ and 30.3 mg KNO₃ and supplemented with 0.1 mg l^?1 thiamine HCl, 100 mg l^?1 myo-inositol, 30 g l^?1 sucrose, 3.0 mg l^?1 phosphinothricin, and 0.41 μM picloram. Somatic embryo development from transformed cultures was initiated on MS medium supplemented with 45 g l^?1 sucrose, 4 mg l^?1 thiamine HCl, 100 mg l^?1 myo-inositol, 10% (v/v) filter-sterilized coconut water, 3.0 mg l^?1 phosphinothricin, and 6.0 g l^?1 gellan gum. Limited plant recovery occurred from somatic embryos on semi-solid MS medium supplemented with 3.0 mg l^?1 phosphinothricin, 4.44 μM 6-benzylaminopurine (BA), and 2.89 μM GA₃; transformed shoots were micrografted on in vitro-grown seedling rootstocks. Approximately 1 yr after acclimatization in the greenhouse, transformed shoots were air-layered to recover transformed roots. Genetic transformation of embryogenic cultures, somatic embryos, and regenerated plants was confirmed by polymerase chain reaction (PCR), Southern blot hybridization, the XGLUC reaction for uidA, and application of the herbicide Finale® to regenerated plants.     

Sensitive Determination of Fentanyl in Low-Volume Serum Samples by LC-MS/MS

Fentanyl is a widely used drug in the management of pain. Present LC-MS/MS methods for analysis of fentanyl require a large volume of serum, but yet the sensitivity was at about 50 pg/mL. Here, we report a modified liquid-liquid extraction method for the analysis of fentanyl in serum. The method is very sensitive with a LLOQ of 5 pg/mL while using only 0.175 mL of serum for analysis. The separation was performed on a Zorbax XDB-C18 column (4.6?×?50 mm, 1.8 μm, 600 bar) using a mobile phase of water: acetonitrile (70:30 v/v) with 0.1% formic acid that was pumped isocratically at a flow rate of 0.5 mL per minute. The calibration curve was found to be linear over a range of 5–10,000 pg/mL. The inter-day and intra-day accuracy and precision were tested using low (20 pg/mL), medium (1000 pg/mL), and high (5000 pg/mL) quality control samples of fentanyl prepared in blank human serum and were within ±?15% of the nominal value. Fentanyl was also found to be stable in various storage and sample preparation conditions, including short-term bench-top storage (for 5 h), freeze-thaw cycling (three cycles), long-term frozen condition (4.5 months at -?70°C), and post-preparative storage (for 48 h).