Evaluation of measurement uncertainties of an analytical method for the determination of aflatoxin M<Subscript>1</Subscript> in milk and milk powder

The mycotoxin aflatoxin M₁ (AfM₁) is a serious food safety hazard for which the European Commission has already established a maximum permissible level of 0.05 μg/kg AfM₁ in milk and products thereof. For control analysis laboratories are increasingly asked to submit full uncertainties of their analytical results.The evaluation of measurement uncertainties of an analytical method for the determination of AfM₁ in milk and milk powder on the basis of ‘in-house’ validation data in compliance with the ‘Guide to the Expression of Uncertainty in Measurement (GUM)’ [1] and the ‘EURACHEM Guide’ [2] is described. A similar approach will be used to assess the performance of methods employed by laboratories participating in the certification of reference materials for AfM₁ in milk powder.     

An invasive crayfish affects egg survival and the potential recovery of an endangered population of Arctic charr

1. Many fish stocks have declined, because of overharvesting, habitat destruction and introduced species. Despite efforts to rehabilitate some of these stocks, not all are responding or are recovering only slowly. 2. In freshwater systems, introduced crayfish are often problematic, and it has been suggested that their egg predation could reduce recruitment in depleted stocks of native fish. 3. Here, we report the results of a field experiment, using experimental cages, on the extent of predation on eggs of great Arctic charr (Salvelinus umbla) in Lake Vättern, Europe’s fifth largest lake. Here, the great Arctic charr has declined dramatically and is listed as critically endangered. 4. We were able to partition the total loss rate of eggs into background mortality, predation by introduced signal crayfish (Pacifastacus leniusculus) and predation by native fish. The mortality rate of charr eggs because of crayfish was estimated at more than five times that because of native fish. Of the total loss of eggs, 80% is believed to be caused by crayfish and 14% by fish, with 6% being natural background mortality. 5. In a worst case scenario, our data infer that only 25% of the original number of eggs would survive, compared with 75% in the absence of crayfish. This could impair recovery of the stock of the endangered great Arctic charr in Lake Vättern. 6. Contrary to earlier claims that crayfish predation on eggs of great Arctic charr is insignificant, our results indicate that crayfish predation may exceed fish predation and suggest that the abundance of signal crayfish on the spawning sites of great Arctic charr should be managed.     

Bigheaded carps (<Emphasis Type="Italic">Hypophthalmichthys</Emphasis> spp.) at the edge of their invaded range: using hydroacoustics to assess population parameters and the efficacy of harvest as a control strategy in a large North American river

The threat posed by bigheaded carps (Hypophthalmichthys spp.) to novel ecosystems has focused efforts on preventing further range expansion; upstream progression in the Illinois River is a major concern due to its connection with the uninvaded Great Lakes. In addition to an electric barrier system, commercial harvest of silver carp (H. molitrix) and bighead carp (H. nobilis) in the upper river is intended to reduce propagule pressure and prevent range expansion. To quantify demographics and evaluate harvest efficacy, the upper river was sampled between 2012 and 2015 using mobile hydroacoustic methods. Reach-specific densities, size structures and species compositions varied interannually but the advancing population was characterized longitudinally as small-bodied, silver carp-dominated at the highest densities downstream, shifting to large-bodied, bighead carp-dominated at the low-density population front. The use of hydroacoustic sampling for harvest evaluation was validated in backwater lakes; there was a significant positive correlation between density estimates and the corresponding harvest catch-per-unit-effort of bigheaded carps. Localized densities of bigheaded carps were reduced by up to 64.4 % immediately post-harvest but generally rebounded within weeks. However, annual sampling of the entire upper river indicated that density of bigheaded carps decreased by over 40 % (between 2012 and 2013) and subsequently remained stable (between 2013 and 2014). The annual harvest of bigheaded carps increased during this period (from 45,192 to 102,453 individuals), in years of contrasting discharge conditions. At this spatiotemporal scale, harvest appears to have contributed to initial reduction, and subsequent maintenance of, bigheaded carps density levels, but discharge likely plays an important role (e.g., through immigration) in determining the extent of its impact. Mobile hydroacoustic sampling enabled robust quantification of the population over varying spatial scales and density gradients, highlighting the potential of this approach as an assessment tool for invasive fishes in riverine environments.     

Soluble immune response suppressor (SIRS) inhibits microtubule function in vivo and microtubule assembly in vitro

Soluble immune response suppressor (SIRS) is a product of concanavalin A-stimulated murine T cells that, when activated or oxidized by macrophages or H2O2 (SIRSox), suppresses in vitro immune responses and inhibits cell division by normal and neoplastic cells. SIRSox is inactivated by a variety of electron donors, which suggests that SIRSox may be an oxidizing agent. Incubation of lymphocytes with SIRSox, but not with SIRS, partially reversed concanavalin A-mediated inhibition of capping of membrane immunoglobulin on B cells, and disrupted the cytoplasmic array of microtubules visualized by fluorescence microscopy. SIRSox also inhibited microtubule assembly in vitro in a concentration-dependent manner. Inactivation of SIRSox by dithiothreitol prevented SIRSox-mediated reversal of inhibition of capping and inhibition of microtubule assembly. These results reveal a pattern of SIRSox activity similar to sulfhydryl-dependent cytoskeletal disrupting agents (e.g., N-ethylmaleimide, cytochalasin A, p-benzoquinone), and suggest that SIRSox-mediated suppression of proliferation may involve interference with sulfhydryl-dependent cytoskeletal events critical for cell division.     

Effect of monoclonal antibodies on the adherence of Bordetella pertussis to Vero cells

Abstract An in vitro assay involving the adherence of Bordetella pertussis to Vero cells was used to quantify the inhibitory effects of monoclonal antibodies (McAb) on adherence. McAbs to agglutinogens (fimbriae), filamentous haemagglutinin (FHA), lymphocytosis promoting factor (LPF) and to an X-mode specific outer membrane protein were tested. X-mode cells adhered to the Vero cells to a greater extent than C-mode cells. McAb to agglutinogen 2 inhibited the adherence of B. pertussis bearing homologous agglutinogen. McAbs to LPF, FHA, agglutinogen 3 and X-mode specific outer membrane protein also partially inhibited the adherence of B. pertussis to Vero cells.     

SPERMATOGONIAL CHALONE(S): EFFECT ON THE PHASES OF THE CELL CYCLE OF TYPE A SPERMATOGONIA IN THE RAT

Adult rats with X-irradiated testes were used to analyze the effect of the spermatogonial chalone(s) on the phases of the cell cycle of type A spermatogonia. Twelve days after irradiation, the animals were used in two experiments designed to test the existence of hypothetical G₂ and S phase chalones. For the G₂ assay, rats injected twice with testicular extract (Group I), liver extract (Group II) or physiological saline (Group III) were killed 10 hr after the initial injection. Mitoses of type A, Intermediate and type B spermatogonia were counted in whole mounts of dissected seminiferous tubules. To test for an S phase inhibitor, two groups of rats were given multiple injections of either testicular extract (Group IV) or saline solution (Group V). Twenty-two hr after the first injection they were injected with [³H]thymidine and killed 2 hr later. Silver grains over labelled type A nuclei were counted in radioautographed sections of testes from these animals. The average grain counts were identical in Groups IV and V, indicating that the testicular extract did not affect type A spermatogonia during the S phase. Counts of type A mitoses in Groups I, II and III revealed that in the animals injected with the testicular extract (Group I) the number of divisions was 50% lower than in the control groups (Groups II and III). In contrast, mitotic activity of differentiating spermatogonia (In + B) was similar in all three groups of animals. This result is attributed to a testicular chalone which specifically inhibits type A spermatogonia during the G₂ phase of the cell cycle. Indirect evidence for a G₁ spermatogonial chalone is also presented, as a result of an analysis of published data (Clermont & Mauger, 1974).     

Microfilaments and tropomyosin of cultured mammalian cells: isolation and characterization

Microfilaments were isolated from cultured mammalian cells, utilizing procedures similar to those for isolation of "native" thin filaments from muscle. Isolated microfilaments from rat embryo, baby hamster kidney (BHK- 21), and Swiss mouse 3T3 cells appeared structurally similar to muscle thin filaments, exhibiting long, 6 nm Diam profiles with a beaded, helical substructure. An arrowhead pattern was observed after reaction of isolated microfilaments with rabbit skeletal muscle myosin subfragment 1. Under appropriate conditions, isolated microfilaments will aggregate into a form that resembles microfilament bundles seen in situ cultured cells. Isolated microfilaments represent a complex of proteins including actin. Some of these components have been tentatively identified, based on coelectrophoresis with purified proteins, as myosin, tropomyosin, and a high molecular weight actin-binding protein. The tropomyosin components of isolated microfilaments were unexpected; polypeptides comigrated on SDS-polyacrylamide gels with both muscle and nonmuscle types of tropomyosin. In order to identify more specifically these subunits, we isolated and partially characterized tropomyosin from three cell types. BHK-21 cell tropomyosin was similar to other nonmuscle tropomyosins, as judged by several criteria. However, tropomyosin isolated from rate embryo and 3T3 cells contained subunits that comigrated with both skeletal muscle and nonmuscle types of myosin, whereas the BHK cell protein consistently contained a minor muscle-like subunit. The array of tropomyosin subunits present in a cell culture was reflected in the polypeptide chain pattern seen on SDS-polyacrylamide gels of microfilaments isolated from that culture. These studies provide a starting point for correlating changes in the ultrastructural organization of microfilaments with alterations in their protein composition.     

The alpha-adrenergic stimulation of atrial natriuretic factor expression in cardiac myocytes requires calcium influx, protein kinase C, and calmodulin-regulated pathways.

It has been shown recently that alpha-adrenergic agonists can stimulate atrial natriuretic factor (ANF) expression in ventricular cardiac myocytes; however, little is known about the intracellular signals mediating this activation. The present study focused on the potential roles of calcium-regulated kinases and calcium influx in the alpha-adrenergic stimulation of ANF gene expression in ventricular myocardial cell cultures. Myocardial cells maintained for 48 h in serum-free medium supplemented with phenylephrine (PE) possessed up to 15-fold higher levels of ANF peptide and ANF mRNA than control cells. The removal of PE, or the addition of nifedipine, resulted in a rapid decline in ANF expression, suggesting that the sustained elevation of some intracellular messenger (e.g. calcium and/or phospholipid hydrolysis products) was required for the adrenergic response. The calcium channel agonist BAY K 8644 was capable of increasing ANF expression in a nifedipine-sensitive manner; however, unlike PE, it did not stimulate phosphoinositide hydrolysis. The protein kinase C inhibitor, H7, caused an approximate 75% reduction in PE-stimulated ANF expression, but had no effect on BAY K-stimulated expression. W7, a calcium/calmodulin inhibitor, completely blocked the effects of both PE and BAY K 8644. The addition of either H7 or W7 24 h after the PE addition resulted in a decline of ANF expression. These results indicate that alpha-adrenergic agonists augment ANF gene expression through at least two pathways, one that is H7-sensitive, perhaps involving the sustained activation of protein kinase C, and the other that is W7-sensitive, perhaps involving the sustained activation of calmodulin-regulated kinases. Further, it appears that BAY K 8644-mediated increases in ANF expression are independent of protein kinase C activation and dependent on calmodulin-regulated events.     

Assessment of the Microbiological Potential for the Natural Attenuation of Petroleum Hydrocarbons in a Shallow Aquifer System

Abstract A multidisciplinary field study investigating the fate and transport of petroleum hydrocarbons commonly associated with jet-fuel contamination is currently underway at Columbus Air Force Base (AFB), Mississippi. Sixty sediment cores from 12 boreholes were recovered from the study aquifer. The goal of this initial sampling was to characterize the potential microbial activity using 14C-labeled substrates, as well as the presence, abundance, and distribution of specific hydrocarbon degrading genotypes using DNA:DNA hybridization. Enumeration of total microbial abundance using a 16S rDNA universal oligonucleotide probe was compared to traditional enumeration methods. Total culturable populations determined by spread plate analysis ranged from a low of 10(4) to more than 10(6) organisms per gram sediment. Microbial abundance estimated by DNA hybridization studies with 16S rDNA genes ranged from 10(7) to 10(8) organisms per gram sediment. Molecular analysis of aquifer samples using DNA probes targeting genes encoding the degradative enzymes alkane hydroxylase (alkB), catechol 2,3-dioxygenase (nahH), naphthalene dioxygenase (nahA), toluene dioxygenase (todC1C2), toluene monooxygenase (tomA), and xylene monooxygenase (xylA), as well as two probes measuring methanogenic microorganisms, codh (carbon monoxide dehydrogenase) and mcr (methyl coenzyme reductase), revealed that each target gene sequence was present in nearly all 60 samples. The presence of organisms demonstrating the phenotype to degrade BTEX and naphthalene was further supported using mineralization assays with 14C-labeled benzene, toluene, naphthalene, and phenanthrene. Minimal activity occurred during the first 24 hours. After a period of 5-7 days, greater than 40% of the target compounds were mineralized in aquifer sediments.