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Aims Toxigenic strains of Paenibacillus polymyxa were isolated from buildings connected with the symptoms of ill health. Our aim was to identify the toxic compounds of Paenibacillus polymyxa and to describe their toxic actions. Methods and Results The toxins of Paenibacillus polymyxa were purified and analysed by HPLC and mass spectrometry. Toxic fusaricidins A and B, and LI-F05a with mass ions at m/z 883·7, 897·6 and 897·6, respectively, were found. The cytotoxicity of purified fusaricidins A and B was measured using boar sperm, porcine tubular kidney epithelial cells and murine fibroblasts. The ion channel forming properties of fusaricidins were studied using the black lipid membrane ( BLM) technique. Fusaricidins A and B depolarized the mitochondria of boar sperm, porcine tubular kidney epithelial cells and murine fibroblasts at concentrations of 0·5-1 μg ml−1 and caused nuclear fragmentation and induced apoptosis at concentrations of 2·5-5 μg ml−1. Furthermore, fusaricidins A and B induced K+ permeating single channels. Conclusions It was concluded that fusaricidins were toxic to mitochondria and induced apoptosis in mammalian cells. It was proposed that the observed toxicity of fusaricidins is due their ion channel forming properties. Significance and Impact of the Study This paper revealed, for the first time, the mode of action of Paenibacillus polymyxa fusaricidins toxins towards mammalian cells. Fusaricidins, due to their potassium ionophoricity and mitochondria depolarizing impacts, may have contributed to the health damage observed at sites where the producer strains were isolated at high density. [ABSTRACT FROM AUTHOR]

Abstract: The presence, quantity and origins of potentially toxic airborne substances were searched in moisture damaged indoor environments, where building related ill health symptoms were suspected and reference sites with no health complaints. Boar spermatozoa were used as the toxicity sensor. Indoor aerosols and dusts were collected from kindergartens, schools, offices and residences (n =25) by electrostatic filtering, vacuuming, wiping from elevated surfaces and from the interior of personal computers. Toxicity was measured from the ethanol or methanol extracts of the dusts and aerosols. EC50 was expressed as the lowest concentration of the airborne substance that inhibited motility of >50% of the exposed sperm cells compared to vehicle control, within 30min, 1day or 3–4days of exposure. Remarkably toxic aerosols (EC50 ⩽6μgml−1) were found from 11 sites, all of these were sites with known or suspected for building related ill health. Toxic microbial cultures were obtained from subsamples of the toxic aerosols/dusts. From these cereulide, amylosin, valinomycin and a novel indoor toxin, stephacidin B were identified and toxicities measured. Airborn dispersal of valinomycin from Streptomyces griseus cultures was evaluated using a flow-through chamber. Significant amounts of valinomycin (LC–MS assay) and toxicity (boar sperm motility assay) were carried by air and were after 14days mainly recovered from the interior surfaces of the flow chamber. [ABSTRACT FROM AUTHOR]

A dish of pasta and minced meat caused severe food-poisoning involving both emesis and diarrhoea in two adult persons. Emetic toxin producing strains of Bacillus cereus formed the majority (68% of 122) of strains identified in this food. Haemolytic diarrhoeal toxin was produced by 26% of the strains studied and 6% of the strains produced neither emetic nor haemolytic diarrhoeal toxin. The B. cereus strains isolated from this dish could be divided into four biochemically distinct groups and two different colony morphologies. All emetic toxin producing strains (n=83) were negative for both haemolytic enterotoxin and starch hydrolysis in contrast to haemolytic enterotoxin producers (n=32). Colonies of emetic toxin producing strains were poorly haemolytic, ⩽2 mm zones, in contrast to the diarrhoeal colonies, 4–5 mm zones. This disparity persisted after extended incubation using blood agar supplemented with lithium chloride. Despite the wide diversity of B. cereus biotypes in this single food all emetic toxin producers exhibited narrow haemolysis with negative starch hydrolysis. The findings emphasize that colonies with different properties should be isolated when food-poisoning cases are studied. [Copyright &y& Elsevier]

Sperm motility inhibition assay, earlier shown valuable for the detection of food poisoning non-protein toxins of Bacillus species was developed into an assay useful for specific detection of mitochondria damaging toxins. This was done by assessing the dissipation of the mitochondrial inner membrane transmembrane potential, Δψm under conditions where the plasma membrane permeability barrier remained intact. The Δψm was estimated as the intensity of orange JC-1 fluorescence in the mitochondrial sheath of the exposed spermatozoa. The plasma membrane integrity of the same cells was assessed by observing the exclusion of propidium iodide from the cytoplasm. Three types of mitochondrial toxic responses to microbially made bioactive substances were recognised. Mitochondrial toxicity by gramicidin (A, B, C, D), nigericin, salinomycin, narasin, monensin, calcimycin and antimycin A was characterised by gradual fading of the JC-1 fluorescence in the mitochondria. Dissipation of the Δψm by cereulide, valinomycin and enniatin (A, A1, B, B1) was visible as spotwise quenching of the mitochondrial JC-1 fluorescence. In addition these substances caused hyperpolarisation of the plasma membrane. Oligomycin (A, B, C), ionomycin and staurosporine inhibited the spermatozoan motility, but Δψm was fully preserved. Surfactin and lichenysin A caused mitochondrial damage at concentrations where the plasma membrane was also damaged. [Copyright &y& Elsevier]

Examines the isolation of the toxic-metabolite-emitting bacteria and fungus that are contaminating the indoor air of a building where an occupant was suffering from serious building-related ill-health symptoms. Collection of culturable heterotrophic aerobic microorganisms; Epifluorescence micrographs of boar spermatozoa; Nucleotide sequence accession numbers.

Provides information on a study which describes Streptomyces griseus strains that emit a toxin in indoor air and in indoor dust. Materials and methods used; Results and discussion.

Reported is the realisation of a graphene FET microwave amplifier operating at 1 GHz, exhibiting a small-signal power gain of 10 dB and a noise figure of 6.4 dB. The amplifier utilises a matching inductor on the gate yielding a return loss of 20 dB. The design is optimised for maximum gain and the optimum noise figure is extracted by noise modelling and predicted to be close to 1 dB for the intrinsic graphene FET at this frequency. The presented results complement existing graphene FET applications and are promising for future graphene microwave circuits. [ABSTRACT FROM AUTHOR]

Abstract: Salinity increase in the subtropical gyre system may have pre-conditioned the North Atlantic Ocean for a rapid return to stronger overturning circulation and high-latitude warming following meltwater events during the Last Glacial period. Here we investigate the Gulf Stream – subtropical gyre system properties over Dansgaard–Oeschger (DO) cycles 14 to 12, including Heinrich ice-rafting event 5. During the Holocene and Last Glacial Maximum a positive gradient in surface dwelling planktonic foraminifera δ18O (Globigerinoides ruber) can be observed between the Gulf Stream and subtropical gyre, due to decreasing temperature, increasing salinity, and a change from summer to year-round occurrence of G. ruber. We assess whether this gradient was a common feature during stadial-interstadial climate oscillations of Marine Isotope Stage 3, by comparing existing G. ruber δ18O from ODP Site 1060 (subtropical gyre location) and new data from ODP Site 1056 (Gulf Stream location) between 54 and 46 ka. Our results suggest that this gradient was largely absent during the period studied. During the major warm DO interstadials 14 and 12 we infer a more zonal and wider Gulf Stream, influencing both ODP Sites 1056 and 1060. A Gulf Stream presence during these major interstadials is also suggested by the large vertical δ18O gradient between shallow dwelling planktonic foraminifera species, especially G. ruber, and the deep dwelling species Globorotalia inflata at site 1056, which we associate with strong summer stratification and Gulf Stream presence. A major reduction in this vertical δ18O gradient from 51 ka until the end of Heinrich event 5 at 48.5 ka suggests site 1056 was situated within the subtropical gyre in this mainly cold period, from which we infer a migration of the Gulf Stream to a position nearer to the continental shelf, indicative of a narrower Gulf Stream with possibly reduced transport. [Copyright &y& Elsevier]

Factors influencing the production of cereulide, the emetic toxin of Bacillus cereus in food and laboratory media were investigated, using liquid chromatography–ion trap mass spectrometry and sperm motility inhibition bioassay for detection and quantitation. Oxygen was essential for production of the emetic toxin by B. cereus. When beans, rice or tryptic soy broth were inoculated with cereulide producing strains B203, B116 (recent food isolates) or the strain F-4810/72, high amounts (2 to 7 μg ml-1 or g-1 wet wt) of cereulide accumulated during 4-day storage at room temperature. In parallel cultures and foods, stored under nitrogen atmosphere (>99.5% N2), less than 0.05 μg of cereulide ml-1 or g-1 wet wt accumulated. The outcome of the bioassay matched that of the chemical assay, with no indication of interference by substances in the rice or beans. Boiling for 20 to 30 min did not inactivate cereulide or cereulide producing strains in rice or the beans. Adding l-leucine and l-valine (0.3 g l-1) stimulated cereulide production 10- to 20-fold in R2A and in rice water agar. When the B. cereus strains were grown on agar media under permissive conditions (air, room temperature), cereulide was produced overnight with little or no increase when the incubation was extended to 4 days. In broth culture, the production of cereulide started later than 16–24 h. Anoxic storage prevented cereulide production also when the amino acids had been supplied. Packaging with modified atmosphere low in oxygen may thus be used to reduce the risk of cereulide formation during storage of food. [Copyright &y& Elsevier]

The in vitro boar spermatozoon test was compared with the LC ion trap MS analysis for measuring the cereulide content of a pasta dish, implemented in serious emetic food poisoning caused by Bacillus cereus. Both assays showed that the poisonous food contained approximately 1.6 μg of cereulide g−1 implying the toxic dose in human as ⩽8 μg kg−1 body weight. The threshold concentration of cereulide provoking visible mitochondrial damage in boar sperm exposed in vitro was 2 ng of cereulide ml−1 of extended boar sperm. The same threshold value was found for cereulide extracted from the food and from the cultured bacteria. This shows that other constituents of the food did not enhance or mask the effects of cereulide. Exposure of four human cell lines (HeLa, Caco-2, Calu-3 and Paju) to cereulide showed that the threshold concentration for the loss of mitochondrial membrane potential in human cells was similar to that observed in boar sperm. Human cells and boar sperm were equally sensitive to cereulide. The results show that boar spermatozoan assay is useful for detecting cereulide concentrations toxic to humans. Spermatozoa in commercially available extended fresh boar and cryopreserved bull semen were compared, boar sperms were 100 times more sensitive to cereulide than bull sperms. [Copyright &y& Elsevier]

Presents a study to describe a bioassay in the detection of Bacillus cereus emetic toxin. Methodology used to conduct study; Indication of findings; Discussion on results.

Aims: Emission of toxic metabolites in guttation droplets of common indoor fungi is not well documented. The aims of this study were (i) to compare mycotoxins in biomass and guttation droplets from indoor fungi from a building following health complaints among occupants, (ii) to identify the most toxic strain and to test if mycotoxins in guttation liquids migrated trough air and (iii) to test if toxigenic Penicillium expansum strains grew on gypsum board. Methods and Results: Biomass suspensions and guttation droplets from individual fungal colonies representing Aspergillus, Chaetomium, Penicillium, Stachybotrys and Paecilomyces were screened toxic to mammalian cells. The most toxic strain, RcP61 (CBS 145620), was identified as Pen. expansum Link by sequence analysis of the ITS region and a calmodulin gene fragment, and confirmed by the Westerdijk Institute based on ITS and beta‐tubulin sequences. The strain was isolated from a cork liner, was able to grow on gypsum board and to produce toxic substances in biomass extracts and guttation droplets inhibiting proliferation of somatic cells (PK‐15, MNA, FL) in up to 20 000‐fold dilutions. Toxic compounds in biomass extracts and/or guttation droplets were determined by HPLC and LC‐MS. Strain RcP61 produced communesins A, B and D, and chaetoglobosins in guttation droplets (the liquid emitted from them) and biomass extracts. The toxins of the guttation droplets migrated c. 1 cm through air and condensed on a cool surface. Conclusions: The mycotoxin‐containing guttation liquids emitted by Pen. expansum grown on laboratory medium exhibited airborne migration and were >100 times more toxic in bioassays than guttation droplets produced by indoor isolates of the genera Aspergillus, Chaetomium, Stachybotrys and Paecilomyces. Significance and Impact of the Study: Toxic exudates produced by Pen. expansum containing communesins A, B and D, and chaetoglobosins were transferable by air. This may represent a novel mechanism of mycotoxin dispersal in indoor environment. [ABSTRACT FROM AUTHOR]

Aims: The production of peptaibols, toxic secondary metabolites of Trichoderma, in the indoor environment is not well‐documented. Here, we investigated the toxicity of peptaibols in the guttation droplets and biomass of Trichoderma strains isolated from problematic buildings. Methods and Results: Seven indoor‐isolated strains of T. atroviride, T. trixiae, T. paraviridescens and T. citrinoviride were cultivated on malt extract agar, gypsum boards and paperboards. Their biomass extracts and guttation droplets were highly cytotoxic in resting and motile boar sperm cell assays and in inhibition of somatic cell proliferation assays. The toxins were identified with HPLC/ESI‐MS/MS as trichorzianines, trilongins, trichostrigocins and trichostrigocin‐like peptaibols. They exhibited toxicity profiles similar to the reference peptaibols alamethicin, trilongins, and trichorzianine TA IIIc purified from T. atroviride H1/226. Particular Trichoderma strains emitted the same peptaibols in both their biomasses and exudate droplets. The trilongin‐producing T. citrinoviride SJ40 strain grew at 37°C. Conclusions: To our knowledge, this is the first report of indoor‐isolated Trichoderma strains producing toxic peptaibols in their guttation droplets. Significance and Impact of the Study: This report proves that indoor isolates of Trichoderma release peptaibols in their guttation droplets. The presence of toxins in these types of exudates may serve as a mechanism of aerosol formation for nonvolatile toxins in the indoor air. [ABSTRACT FROM AUTHOR]

Aims The aim of this study was to elucidate the prevalence of lichenysin production in Bacillus licheniformis and to see whether this feature was restricted to certain genotypes. Secondly, we wanted to see whether cytotoxicity reflected the measured levels of lichenysin. Methods and Results Fifty-three genotyped strains of B. licheniformis, representing a wide variety of sources, were included. lch AA gene fragments were detected in all strains by polymerase chain reaction ( PCR). All 53 strains produced lichenysins with four molecular masses as confirmed by LC- MS/ MS (liquid chromatography-tandem mass spectrometry) analysis. The amounts of lichenysin varied more than two orders of magnitude between strains and were irrespective of genotype. Finally, there was a strong association between lichenysin concentrations and toxicity towards boar spermatozoa, erythrocytes and Vero cells. Conclusions Lichenysin synthesis was universal among the 53 B. licheniformis strains examined. The quantities varied considerably between strains, but were not specifically associated with genotype. Cytotoxicity was evident at lichenysin concentrations above 10 μg ml−1, which is in accordance with previous studies. Significance and Impact of Study This study might be of interest to those working on B. licheniformis for commercial use as well as for authorities who make risk assessments of B. licheniformis when used as a food and feed additive. [ABSTRACT FROM AUTHOR]

In vitro toxicological tests have been proposed as an approach to complement the chemical safety assessment of food contact materials, particularly those with a complex or unknown chemical composition such as paper and board. Among the concerns raised regarding the applicability of in vitro tests are the effects of interference of the extractables on the outcome of the cytotoxicity and genotoxicity tests applied and the role of known compounds present in chemically complex materials, such as paper and board, either as constituents or contaminants. To answer these questions, a series of experiments were performed to assess the role of natural substances (wood extracts, resin acids), some additives (diisopropylnaphthalene, phthalates, acrylamide, fluorescent whitening agents) and contaminants (2,4-diaminotoluene, benzo[a]pyrene) in the toxicological profile of paper and board. These substances were individually tested or used to spike actual paper and board extracts. The toxic concentrations of diisopropylnaphthalenes and phthalates were compared with those actually detected in paper and board extracts showing conspicuous toxicity. According to the results of the spiking experiments, the extracts did not affect the toxicity of tested chemicals nor was there any significant metabolic interference in the cases where two compounds were used in tests involving xenobiotic metabolism by the target cells. While the identified substances apparently have a role in the cytotoxicity of some of the project samples, their presence does not explain the total toxicological profile of the extracts. In conclusion, in vitro toxicological testing can have a role in the safety assessment of chemically complex materials in detecting potentially harmful activities not predictable by chemical analysis alone. [ABSTRACT FROM AUTHOR]

Abstract: Nineteen food contact papers and boards and one non-food contact board were extracted following test protocols developed within European Union funded project BIOSAFEPAPER. The extraction media were either hot or cold water, 95% ethanol or Tenax, according to the end use of the sample. The extractable dry matter content of the samples varied from 1200 to 11,800mg/kg (0.8–35.5mg/dm2). According to GC–MS the main substances extracted into water were pulp-derived natural products such as fatty acids, resin acids, natural wood sterols and alkanols. Substances extracted into ethanol particularly, were diisopropylnaphthalenes, alkanes and phthalic acid esters. The non-food contact board showed the greatest number and highest concentrations of GC–MS detectable compounds. The extracts were subjected to a battery of in vitro toxicity tests measuring both acute and sublethal cytotoxicity and genotoxic effects. None of the water or Tenax extracts was positive in cytotoxicity or genotoxicity assays. The ethanol extract of the non-food contact board gave a positive response in the genotoxicity assays, and all four ethanol extracts gave positive response(s) in the cytotoxicity assays to some extent. These responses could not be pinpointed to any specific compound, although there appeared a correlation between the total amount of extractables and toxicity. [Copyright &y& Elsevier]

The daily quality control of semen at a Finnish artificial insemination (AI) bull station is based on subjective motility and sperm morphology of young bulls entering the semen collection program. Semen quality dropped suddenly in autumn 1998. During 5 consecutive months, the number of rejected ejaculates and discarded frozen semen batches due to poor motility increased, and the number of all forms of abnormal spermatozoa increased. However, for the accepted ejaculates, a 60 day nonreturn rate was normal.The summer of 1998 in Finland was rainy, and the hay used in the AI station was visibly moldy. Immunoassay and gas chromatography–mass spectrometry (GC–MS) detected Fusarium mycotoxins HT-2 and T-2, but no zearalenone in the hay. Occurrence of mycotoxins such as T-2 and HT-2 in the moldy hay coincided with, and may have been responsible for the impaired semen quality in AI bulls. This case report will draw the attention to the possible hazards when feeding moldy hay. [Copyright &y& Elsevier]