Food and Indoor Air Isolated Bacillus Non-Protein Toxins: : Structures, Physico-Chemical Properties and Mechanisms of Effects on Eukaryotic Cells

We report here the structures and properties of heat-stable, non-protein, and mammalian cell-toxic compounds produced by spore-forming bacilli isolated from indoor air of buildings and from food. Little information is available on the effects and occurrence of heat-stable non-protein toxins produced by bacilli in moisture-damaged buildings. Bacilli emit spores that move in the air and can serve as the carriers of toxins, in a manner similar to that of the spores of toxic fungi found in contaminated indoor air. Bacillus spores in food cause problems because they tolerate the temperatures applied in food manufacture and the spores later initiate growth when food storage conditions are more favorable. Detection of the toxic compounds in Bacillus is based on using the change in mobility of boar spermatozoa as an indicator of toxic exposure. GC, LC, MS, and nuclear magnetic resonance NMR spectroscopy were used for purification, detection, quantitation, and analysis of the properties and structures of the compounds. Toxicity and the mechanisms of toxicity of the compounds were studied using boar spermatozoa, feline lung cells, human neural cells, and mitochondria isolated from rat liver. The ionophoric properties were studied using the BLM (black-lipid membrane) method. One novel toxin, forming ion channels permeant to K+ > Na+ > Ca2+, was found and named amylosin. It is produced by B. amyloliquefaciens isolated from indoor air of moisture-damaged buildings. Amylosin was purified with an RP-HPLC and a monoisotopic mass of 1197 Da was determined with ESI-IT-MS. Furthermore, acid hydrolysis of amylosin followed by analysis of the amino acids with the GS-MS showed that it was a peptide. The presence of a chromophoric polyene group was found using a NMR spectroscopy. The quantification method developed for amylosin based on RP-HPLC-UV, using the macrolactone polyene, amphotericin B (MW 924), as a reference compound. The B. licheniformis strains isolated from a food poisoning case produced a lipopeptide, lichenysin A, that ruptured mammalian cell membranes and was purified with a LC. Lichenysin A was identified by its protonated molecules and sodium- and potassium- cationized molecules with MALDI-TOF-MS. Its protonated forms were observed at m/z 1007, 1021 and 1035. The amino acids of lichenysin A were analyzed with ESI-TQ-MS/MS and, after acid hydrolysis, the stereoisomeric forms of the amino acids with RP-HPLC. The indoor air isolates of the strain of B. amyloliquefaciens produced not only amylosin but also lipopeptides: the cell membrane-damaging surfactin and the fungicidal fengycin. They were identified with ESI-IT-MS observing their protonated molecules, the sodium- and potassium-cationized molecules and analysing the MS/MS spectra. The protonated molecules of surfactin and fengycin showed m/z values of 1009, 1023, and 1037 and 1450, 1463, 1493, and 1506, respectively. Cereulide (MW 1152) was purified with RP-HPLC from a food poisoning strain of B. cereus. Cereulide was identified with ESI-TQ-MS according to the protonated molecule observed at m/z 1154 and the ammonium-, sodium- and potassium-cationized molecules observed at m/z 1171, 1176, and 1192, respectively. The fragment ions of the MS/MS spectrum obtained from the protonated molecule of cereulide at m/z 1154 were also interpreted. We developed a quantification method for cereulide, using RP-HPLC-UV and valinomycin (MW 1110, which structurally resembles cereulide) as the reference compound. Furthermore, we showed empirically, using the BLM method, that the emetic toxin cereulide is a specific and effective potassium ionophore of whose toxicity target is especially the mitochondria.

Hollow sperm syndrome during spermatogenesis in the giant tiger shrimp Penaeus monodon (Fabricius 1798) from eastern Australia

During spermatogenesis, giant tiger shrimp (Penaeus monodon) from Queensland, eastern Australia had a high proportion of testicular spermatids that appeared 'hollow' because their nuclei were not visible with the haematoxylin and eosin stain. When examined by transmission electron microscopy, the nuclei of hollow spermatids contained highly decondensed chromatin, with large areas missing fibrillar chromatin. Together with hollow spermatids, testicular pale enlarged (PE) spermatids with weakly staining and marginated chromatin were observed. Degenerate-eosinophilic-clumped (DEC) spermatids that appeared as aggregated clumps were also present in testes tubules. Among 171 sub-adult and adult P. monodon examined from several origins, 43% displayed evidence of hollow spermatids in the testes, 33% displayed PE spermatids and 15% displayed DEC spermatids. These abnormal sperm were also found at lower prevalence in the vas deferens and spermatophore. We propose 'Hollow Sperm Syndrome (HSS)' to describe this abnormal sperm condition as these morphological aberrations have yet to be described in penaeid shrimp. No specific cause of HSS was confirmed by examining either tank or pond cultured shrimp exposed to various stocking densities, temperatures, salinities, dietary and seasonal factors. Compared with wild broodstock, HSS occurred at higher prevalence and severity among sub-adults originating from farms, research ponds and tanks. Further studies are required to establish what physiological, hormonal or metabolic processes may cause HSS and whether it compromises the fertility of male P. monodon.

Hanging drop: An in vitro air toxic exposure model using human lung cells in 2D and 3D structures

Using benzene as a candidate air toxicant and A549 cells as an in vitro cell model, we have developed and validated a hanging drop (HD) air exposure system that mimics an air liquid interface exposure to the lung for periods of 1 h to over 20 days. Dose response curves were highly reproducible for 2D cultures but more variable for 3D cultures. By comparing the HD exposure method with other classically used air exposure systems, we found that the HD exposure method is more sensitive, more reliable and cheaper to run than medium diffusion methods and the CULTEX (R) system. The concentration causing 50% of reduction of cell viability (EC50) for benzene, toluene, p-xylene, m-xylene and o-xylene to A549 cells for 1 h exposure in the HD system were similar to previous in vitro static air exposure. Not only cell viability could be assessed but also sub lethal biological endpoints such as DNA damage and interleukin expressions. An advantage of the HD exposure system is that bioavailability and cell concentrations can be derived from published physicochemical properties using a four compartment mass balance model. The modelled cellular effect concentrations EC50(cell) for 1 h exposure were very similar for benzene, toluene and three xylenes and ranged from 5 to 15 mmol/kg(dry weight) which corresponds to the intracellular concentration of narcotic chemicals in many aquatic species, confirming the high sensitivity of this exposure method. (C) 2013 Elsevier B.V. All rights reserved.

Interactions between Mast Cells, Fibroblasts and Connective Tissue Components

It has long been recognized that mast cells occur throughout connective tissues. Histologic studies have revealed that such cells release their granules into the surrounding environment upon exposure to both immunologic and nonimmunologic stimuli. By microscopy these extracellular granules appeared to be phagocytosed by fibroblasts and by blood-borne phagocytic cells as they entered the site of mast cell degranulation. Such in vivo observations led to the suggestion that mast cells both altered connective tissue components and influenced fibroblast function through these discharged granules. Recent in vitro studies using cultured fibroblasts and isolated mast cells and mast cell granules have confirmed both these hypotheses. In addition, such studies have also documented that fibroblasts degrade ingested mast cell granules. Such studies document that a number of critical interactions may occur between mast cells and connective tissue components.

Circulating tumor cells: Isolation, expansion, characterization and translation to clinic

Circulating tumor cells (CTCs) are the seeds for cancer metastases development, which is responsible for >90% of cancer-related deaths. Accurate quantification of CTCs in human fluids could be an invaluable tool for understanding cancer prognosis, delivering personalized medicine to prevent metastasis and finding cancer therapy effectiveness. Although CTCs were first discovered more than 200 years ago, until now it has been a nightmare for clinical practitioners to capture and diagnose CTCs in clinical settings. Our society needs rapid, sensitive, and reliable assays to identify the CTCs from blood in order to help save millions of lives. Due to the phenotypic EMT transition, CTCs are undetected for more than one-third of metastatic breast cancer patients in clinics. To tackle the above challenges, the first volume in “Circulating Tumor Cells (CTCs): Detection Methods, Health Impact and Emerging Clinical Challenges discusses recent developments of different technologies, which have the capability to target and elucidate the phenotype heterogenity of CTCS. It contains seven chapters written by world leaders in this area, covering basic science to possible device design which can have beneficial applications in society. This book is unique in its design and content, providing an in-depth analysis to elucidate biological mechanisms of cancer disease progression, CTC detection challenges, possible health effects and the latest research on evolving technologies which have the capability to tackle the above challenges. It describes the broad range of coverage on understanding CTCs biology from early predictors of the metastatic spread of cancer, new promising technology for CTC separation and detection in clinical environment and monitoring therapy efficacy via finding the heterogeneous nature of CTCs. (Imprint: Nova Biomedical)

Mixture Effects of Benzene, Toluene, Ethylbenzene and Xylenes (BTEX) to Lung Carcinoma Cells via a Hanging Drop Air Exposure System

A recently developed hanging drop air exposure system for toxicity studies of volatile chemicals was applied to evaluate the cell viability of lung carcinoma A549 cells after 1 h and 24 h of exposure to benzene, toluene, ethylbenzene and xylenes (BTEX) as individual compounds and mixtures of 4 or 6 components. The cellular chemical concentrations causing 50% reduction of cell viability (EC50) were calculated use a mass balance model and came to 17, 12, 11, 9, 4 and 4 mmol/kg cell dry weight for benzene, toluene, ethylbenzene, m-xylene, o-xylene and p-xylene respectively after 1 h of exposure. The EC50 decreased by a factor of four after 24 h of exposure. All mixture effects were best described by the mixture toxicity model of concentration addition, which is valid for chemicals with the same mode of action. Good agreement with the model predictions were found for benzene, toluene, ethylbenzene and m-xylene at four different representative fixed concentration ratios after 1 h of exposure but lower agreement to mixture prediction was obtained after 24 h of exposure. A recreated car exhaust mixture, which involved the contribution of the more toxic p-xylene and o-xylene, yielded an acceptable but lower quality prediction as well.

Quantitative methods to measure pigmentation variation in farmed Giant Tiger Prawns, Penaeus monodon, and the effects of different harvest methods on cooked colour

Cooked prawn colour is known to be a driver of market price and a visual indicator of product quality for the consumer. Although there is a general understanding that colour variation exists in farmed prawns, there has been no attempt to quantify this variation or identify where this variation is most prevalent. The objectives of this study were threefold: firstly to compare three different quantitative methods to measure prawn colour or pigmentation, two different colorimeters and colour quantification from digital images. Secondly, to quantify the amount of pigmentation variation that exists in farmed prawns within ponds, across ponds and across farms. Lastly, to assess the effects of ice storage or freeze-thawing of raw product prior to cooking. Each method was able to detect quantitative differences in prawn colour, although conversion of image based quantification of prawn colour from RGB to Lab was unreliable. Considerable colour variation was observed between prawns from different ponds and different farms, and this variation potentially affects product value. Different post-harvest methods prior to cooking were also shown to have a profound detrimental effect on prawn colour. Both long periods of ice storage and freeze thawing of raw product were detrimental to prawn colour. However, ice storage immediately after cooking was shown to be beneficial to prawn colour. Results demonstrated that darker prawn colour was preserved by holding harvested prawns alive in chilled seawater, limiting the time between harvesting and cooking, and avoiding long periods of ice storage or freeze thawing of uncooked product.

Who is the South African retail giant behind the David Jones bid?

Yesterday morning came the news South African retailer Woolworths had offered $4 a share to acquire David Jones, a proposal that has the approval of the department store’s board. This offer, worth an estimated A$2.15 billion, represents a 25% premium over the current share price value. An earlier offer, from rival Myer, was rejected earlier this year. But who is Woolworths? Not to be confused with Australia’s largest supermarket, Woolworths SA is one of South Africa’s largest retailers.

Crusted scabies is associated with increased IL-17 secretion by skin T cells

Scabies is an ectoparasitic infestation by the mite Sarcoptes scabiei. Although commonly self-limiting, a fraction of patients develop severely debilitating crusted scabies. The immune mechanisms underlying the development of crusted scabies are unclear, and undertaking longitudinal infection studies in humans is difficult. We utilized a porcine model to compare cellular immune responses in peripheral blood and skin of pigs with different clinical manifestations of scabies (n = 12), and in uninfected controls (n = 6). Although clinical symptoms were not evident until at least 4 weeks post-infestation, the numbers of peripheral IFNγ-secreting CD4+ T cells and γδ T cells increased in infected pigs from week 1 post-infestation. γδ T cells remained increased in the blood at week 15 post-infestation. At week 15, skin cell infiltrates from pigs with crusted scabies had significantly higher CD8+ T cell, γδ T cell and IL-17+ cell numbers than those with ordinary scabies. Peripheral IL-17 levels were not increased, suggesting that localized skin IL-17-secreting T cells may play a critical role in the pathogenesis of crusted scabies development. Given the potential of anti-IL-17 immunotherapy demonstrated for other inflammatory skin diseases, this study may provide a novel therapeutic avenue for patients with recurrent crusted scabies.

Cytorhabdovirus phosphoprotein shows RNA silencing suppressor activity in plants, but not in insect cells

RNA silencing in plants and insects provides an antiviral defense and as a countermeasure most viruses encode RNA silencing suppressors (RSS). For the family Rhabdoviridae, no detailed functional RSS studies have been reported in plant hosts and insect vectors. In agroinfiltrated Nicotiana benthamiana leaves we show for the first time for a cytorhabdovirus, lettuce necrotic yellows virus (LNYV), that one of the nucleocapsid core proteins, phosphoprotein (P) has relatively weak local RSS activity and delays systemic silencing of a GFP reporter. Analysis of GFP small RNAs indicated that the P protein did not prevent siRNA accumulation. To explore RSS activity in insects, we used a Flock House virus replicon system in Drosophila S2 cells. In contrast to the plant host, LNYV P protein did not exhibit RSS activity in the insect cells. Taken together our results suggest that P protein may target plant-specific components of RNA silencing post siRNA biogenesis.

Polyphenolic contents and the effects of methanol extracts from mango varieties on breast cancer cells

Bioactivities of peel and flesh extracts of 3 genetically diverse mango (Mangifera indica L.) varieties were studied. Nam Doc Mai peel extracts, containing the largest amounts of polyphenols, were associated with an effect on MCF-7 viable cell numbers with an IC50 (dose required for 50% inhibition of cell viability) of 56 μg/mL and significantly (p<0.01) induced cell death in MDA-MB-231 cells, compared with other varieties. Hydrophilic fractions of Nam Doc Mai peel extracts had the highest bioactivity values against both MCF-7 and MDA-MB-231 cells. Soluble polyphenols were present in the largest amounts in most hydrophilic fractions. The Nam Doc Mai mango variety contains high levels of fruit peel bioactivity, which appears to be related to the nature of the polyphenol composition.

Performance and physiological responses of combined t-bar and PIT tagged giant mud crabs (Scylla serrata)

Mud crabs (Scylla spp.) are intensively caught throughout South-East Asia and support a very substantial commercial, recreational fishing and aquaculture industry. Identification of individual animals is important to improve understanding and management of this species. However, tagging of crustaceans is difficult as they frequently molt and internal tags can pose a hazard to consumers. In this pilot study we tested a new method combining passive integrated transponder tags and t-bar tags externally. 45 giant mud crabs (Scylla serrata) were captured from the wild and kept in tanks for a maximum of 10 months. We inserted tags into the abdomen of 35 giant mud crabs and tested a modified method where the combined t-bar/PIT-tag was inserted into the muscle tissue of the rear leg between the dorsal carapace plate and the top of the abdominal flap. Tagged crabs with the modified method showed 85% tag retention for molting crabs. We tested the same method in the field where 852 individuals were tagged with combined t-bar/PIT-tags of which 82 were recaptured showing 100% tag retention but without any evidence of molting having occurred. The tested method of combined t-bar/PIT-tags in giant mud crabs can further improve monitoring for wild and aquaculture populations and can be deployed widely with low cost.

Performance and physiological responses of combined t-bar and PIT tagged giant mud crabs (Scylla serrata)

Mud crabs (Scylla spp.) are intensively caught throughout South-East Asia and support a very substantial commercial, recreational fishing and aquaculture industry. Identification of individual animals is important to improve understanding and management of this species. However, tagging of crustaceans is difficult as they frequently molt and internal tags can pose a hazard to consumers. In this pilot study we tested a new method combining passive integrated transponder tags and t-bar tags externally. 45 giant mud crabs (Scylla serrata) were captured from the wild and kept in tanks for a maximum of 10 months. We inserted tags into the abdomen of 35 giant mud crabs and tested a modified method where the combined t-bar/PIT-tag was inserted into the muscle tissue of the rear leg between the dorsal carapace plate and the top of the abdominal flap. Tagged crabs with the modified method showed 85% tag retention for molting crabs. We tested the same method in the field where 852 individuals were tagged with combined t-bar/PIT-tags of which 82 were recaptured showing 100% tag retention but without any evidence of molting having occurred. The tested method of combined t-bar/PIT-tags in giant mud crabs can further improve monitoring for wild and aquaculture populations and can be deployed widely with low cost.