Title: Low concentrations of glyphosate in water and sediment after direct over-water application to control an invasive aquatic plant.

When an invasive wetland grass degrades a Ramsar wetland and Important Bird Area, decisive management action is called for. To limit the extent and spread of European Phragmites australis, the Ontario government began the first, large-scale application of glyphosate (Roundup CustomⓇ) over standing water to control an invasive species in Canadian history. Between 2016 and 2018, over 1000 ha of marsh were treated. To assess the concentration, movement and longevity of this herbicide in treated marshes, we measured the concentration of glyphosate, its primary breakdown product aminomethylphosphonic acid (AMPA), and the alcohol ethoxylate-based adjuvant AquasurfⓇ in water and sediments in areas of the highest exposure and up to 150 m into adjacent bays. The maximum observed concentration of glyphosate in water was 0.320 mg/L, occurring within 24 hr of application. The maximum glyphosate concentration in sediment was 0.250 mg/kg, occurring within about 30 days of application. AMPA was detectable in water and sediment, indicating microbial breakdown of glyphosate in the marsh, but at low concentrations (maxwater = 0.025 mg/L, maxsed = 0.012 mg/kg). The maximum distance from the point of application that glyphosate was detected in the water was 100 m, while AMPA was detectable only at the edge of where glyphosate was applied (0 m). Concentrations in water returned to pre-treatment levels (0.005 mg/kg) for over one year but less than two years. Concentrations of alcohol ethoxylates were variable in space and time, following a pattern that could not be attributed to AquasurfⓇ use. The direct, over-water application of Roundup CustomⓇ with AquasurfⓇ to control invasive P. australis did not reach concentrations deemed to pose toxicological concern to aquatic biota by the Canadian Council of Ministers of the Environment.

Periphyton bioconcentrates pesticides downstream of catchment dominated by agricultural land use.

Periphyton provides important ecosystem services in aquatic environments, including supporting diverse consumers. We studied pesticide bioconcentration in periphyton in a coastal marsh on Lake Erie. The marsh is within a protected area (Rondeau Provincial Park) but receives discharge from tributaries draining intensively farmed land. Periphyton bioconcentrated 20 pesticide chemicals above levels observed in adjacent water or sediment. Average bioconcentration factors ranged from 12 times for the herbicide dicamba to 6864 times for the fungicide boscalid on a dry-weight basis. Bioconcentration factors were not linearly related to pesticides' log Kow, log Koc, or water solubility (simple linear regressions, p > 0.43). The removal of pesticides from ambient water represents another valuable ecosystem service provided by periphyton. However, we caution that bioconcentration of pesticides in periphyton provides a mechanism through which contemporary and legacy pesticides may enter wetland food webs.

Assessing predicted age-specific breast cancer mortality rates in 27 European countries by 2020.

BACKGROUND: We assessed differences in predicted breast cancer (BC) mortality rates, across Europe, by 2020, taking into account changes in the time trends of BC mortality rates during the period 2000-2010. METHODS: BC mortality data, for 27 European Union (EU) countries, were extracted from the World Health Organization mortality database. First, we compared BC mortality data between time periods 2000-2004 and 2006-2010 through standardized mortality ratios (SMRs) and carrying out a graphical assessment of the age-specific rates. Second, making use of the base period 2006-2012, we predicted BC mortality rates by 2020. Finally, making use of the SMRs and the predicted data, we identified a clustering of countries, assessing differences in the time trends between the areas defined in this clustering. RESULTS: The clustering approach identified two clusters of countries: the first cluster were countries where BC predicted mortality rates, in 2020, might slightly increase among women aged 69 and older compared with 2010 [Greece (SMR 1.01), Croatia (SMR 1.02), Latvia (SMR 1.15), Poland (SMR 1.14), Estonia (SMR 1.16), Bulgaria (SMR 1.13), Lithuania (SMR 1.03), Romania (SMR 1.13) and Slovakia (SMR 1.06)]. The second cluster was those countries where BC mortality rates level off or decrease in all age groups (remaining countries). However, BC mortality rates between these clusters might diminish and converge to similar figures by 2020. CONCLUSIONS: For the year 2020, our predictions have shown a converging pattern of BC mortality rates between European regions. Reducing disparities, in access to screening and treatment, could have a substantial effect in countries where a non-decreasing trend in age-specific BC mortality rates has been predicted.

Replacing natural wetlands with stormwater management facilities: Biophysical and perceived social values.

Urban expansion replaces wetlands of natural origin with artificial stormwater management facilities. The literature suggests that efforts to mimic natural wetlands in the design of stormwater facilities can expand the provision of ecosystem services. Policy developments seek to capitalize on these improvements, encouraging developers to build stormwater wetlands in place of stormwater ponds; however, few have compared the biophysical values and social perceptions of these created wetlands to those of the natural wetlands they are replacing. We compared four types of wetlands: natural references sites, natural wetlands impacted by agriculture, created stormwater wetlands, and created stormwater ponds. We anticipated that they would exhibit a gradient in biodiversity, ecological integrity, chemical and hydrologic stress. We further anticipated that perceived values would mirror measured biophysical values. We found higher biophysical values associated with wetlands of natural origin (both reference and agriculturally impacted). The biophysical values of stormwater wetlands and stormwater ponds were lower and indistinguishable from one another. The perceived wetland values assessed by the public differed from the observed biophysical values. This has important policy implications, as the public are not likely to perceive the loss of values associated with the replacement of natural wetlands with created stormwater management facilities. We conclude that 1) agriculturally impacted wetlands provide biophysical values equivalent to those of natural wetlands, meaning that land use alone is not a great predictor of wetland value; 2) stormwater wetlands are not a substantive improvement over stormwater ponds, relative to wetlands of natural origin; 3) stormwater wetlands are poor mimics of natural wetlands, likely due to fundamental distinctions in terms of basin morphology, temporal variation in hydrology, ground water connectivity, and landscape position; 4) these drivers are relatively fixed, thus, once constructed, it may not be possible to modify them to improve provision of biophysical values; 5) these fixed drivers are not well perceived by the public and thus public perception may not capture the true value of natural wetlands, including those impacted by agriculture.

Expression of GABAA α2-, ß1- and ε-receptors are altered significantly in the lateral cerebellum of subjects with schizophrenia, major depression and bipolar disorder.

There is abundant evidence that dysfunction of the γ-aminobutyric acid (GABA)ergic signaling system is implicated in the pathology of schizophrenia and mood disorders. Less is known about the alterations in protein expression of GABA receptor subunits in brains of subjects with schizophrenia and mood disorders. We have previously demonstrated reduced expression of GABA(B) receptor subunits 1 and 2 (GABBR1 and GABBR2) in the lateral cerebella of subjects with schizophrenia, bipolar disorder and major depressive disorder. In the current study, we have expanded these studies to examine the mRNA and protein expression of 12 GABA(A) subunit proteins (α1, α2, α3, α5, α6, ß1, ß2, ß3, δ, ε, γ2 and γ3) in the lateral cerebella from the same set of subjects with schizophrenia (N=9-15), bipolar disorder (N=10-15) and major depression (N=12-15) versus healthy controls (N=10-15). We found significant group effects for protein levels of the α2-, ß1- and ε-subunits across treatment groups. We also found a significant group effect for mRNA levels of the α1-subunit across treatment groups. New avenues for treatment, such as the use of neurosteroids to promote GABA modulation, could potentially ameliorate GABAergic dysfunction in these disorders.

mRNA and protein expression for novel GABAA receptors θ and ρ2 are altered in schizophrenia and mood disorders; relevance to FMRP-mGluR5 signaling pathway.

Fragile X mental retardation protein (FMRP) is an RNA-binding protein that targets ∼5% of all mRNAs expressed in the brain. Previous work by our laboratory demonstrated significantly lower protein levels for FMRP in lateral cerebella of subjects with schizophrenia, bipolar disorder and major depression when compared with controls. Absence of FMRP expression in animal models of fragile X syndrome (FXS) has been shown to reduce expression of gamma-aminobutyric acid A (GABAA) receptor mRNAs. Previous work by our laboratory has found reduced expression of FMRP, as well as multiple GABAA and GABAB receptor subunits in subjects with autism. Less is known about levels for GABAA subunit protein expression in brains of subjects with schizophrenia and mood disorders. In the current study, we have expanded our previous studies to examine the protein and mRNA expression of two novel GABAA receptors, theta (GABRθ) and rho 2 (GABRρ2) as well as FMRP, and metabotropic glutamate receptor 5 (mGluR5) in lateral cerebella of subjects with schizophrenia, bipolar disorder, major depression and healthy controls, and in superior frontal cortex (Brodmann Area 9 (BA9)) of subjects with schizophrenia, bipolar disorder and healthy controls. We observed multiple statistically significant mRNA and protein changes in levels of GABRθ, GABRρ2, mGluR5 and FMRP molecules including concordant reductions in mRNA and proteins for GABRθ and mGluR5 in lateral cerebella of subjects with schizophrenia; for increased mRNA and protein for GABRρ2 in lateral cerebella of subjects with bipolar disorder; and for reduced mRNA and protein for mGluR5 in BA9 of subjects with bipolar disorder. There were no significant effects of confounds on any of the results.

Does the Animal Fun program improve motor performance in children aged 4-6 years?

The Animal Fun program was designed to enhance the motor ability of young children by imitating the movements of animals in a fun, inclusive setting. The efficacy of this program was investigated through a randomized controlled trial using a multivariate nested cohort design. Pre-intervention scores were recorded for 511 children aged 4.83 years to 6.17 years (M=5.42 years, SD=3.58 months). Six control and six intervention schools were compared 6 months later following the intervention, and then again at 18 months after the initial testing when the children were in their first school year. Changes in motor performance were examined using the Bruininks-Oseretsky Test of Motor Proficiency short form. Data were analyzed using multi-level-mixed effects linear regression. A significant Condition×Time interaction was found, F(2,1219)=3.35, p=.035, demonstrating that only the intervention group showed an improvement in motor ability. A significant Sex×Time interaction was also found, F(2,1219)=3.84, p=.022, with boys improving over time, but not girls. These findings have important implications for the efficacy of early intervention of motor skills and understanding the differences in motor performance between boys and girls.

Distinct gene signature revealed in white blood cells, CD4(+) and CD8(+) T cells in (NZBx NZW) F1 lupus mice after tolerization with anti-DNA Ig peptide.

Tolerizing mice polygenically predisposed to lupus-like disease (NZB/NZW F1 females) with a peptide mimicking anti-DNA IgG sequences containing MHC class I and class II T cell determinants (pConsensus, pCons) results in protection from full-blown disease attributable in part to the induction of CD4(+)CD25(+)Foxp3+ and CD8(+)Foxp3+ regulatory T cells. We compared 45 000 murine genes in total white blood cells (WBC), CD4(+) T cells, and CD8(+) T cells from splenocytes of (NZBxNZW) F1 lupus-prone mice tolerized with pCons vs untreated naïve mice and found two-fold or greater differential expression for 448 WBC, 174 CD4, and 60 CD8 genes. We identified differentially expressed genes that played roles in the immune response and apoptosis. Using real-time PCR, we validated differential expression of selected genes (IFI202B, Bcl2, Foxp3, Trp-53, CCR7 and IFNar1) in the CD8(+)T cell microarray and determined expression of selected highly upregulated genes in different immune cell subsets. We also determined Smads expression in different immune cell subsets, including CD4(+) T cells and CD8(+) T cells, to detect the effects of TGF-beta, known to be the major cytokine that accounts for the suppressive capacity of CD8(+) Treg in this system. Silencing of anti-apoptotic gene Bcl2 or interferon genes (IFI202b and IFNar1 in combination) in CD8(+) T cells from tolerized mice did not affect the expression of the other selected genes. However, silencing of Foxp3 reduced expression of Foxp3, Ifi202b and PD1-all of which are involved in the suppressive capacity of CD8(+) Treg in this model.

Lack of horizontal gene transfer of methicillin-resistance genetic determinants from PBP2a-positive, coagulase-negative staphylococci to methicillin-sensitive Staphylococcus aureus using transcutaneous electrical nerve stimulation (TENS).

Previous research shows that approximately half of the coagulase-negative staphylococci (CNS) isolated from patients in the intensive care unit (ICU) at Belfast City Hospital were resistant to methicillin. The presence of this relatively high proportion of methicillin-resistance genetic material gives rise to speculation that these organisms may act as potential reservoirs of methicillin-resistance genetic material to methicillin-sensitive Staphylococcus aureus (MSSA). Mechanisms of horizontal gene transfer from PBP2a-positive CNS to MSSA, potentially transforming MSSA to MRSA, aided by electroporation-type activities such as transcutaneous electrical nerve stimulation (TENS), should be considered. Methicillin-resistant CNS (MR-CNS) isolates are collected over a two-month period from a variety of clinical specimen types, particularly wound swabs. The species of all isolates are confirmed, as well as their resistance to oxacillin by standard disc diffusion assays. In addition, MSSA isolates are collected over the same period and confirmed as PBP2a-negative. Electroporation experiments are designed to mimic the time/voltage combinations used commonly in the clinical application of TENS. No transformed MRSA were isolated and all viable S. aureus cells remained susceptible to oxacillin and PBP2a-negative. Experiments using MSSA pre-exposed to sublethal concentrations of oxacillin (0.25 microg/mL) showed no evidence of methicillin gene transfer and the generation of an MRSA. The study showed no evidence of horizontal transfer of methicillin resistance genetic material from MR-CNS to MSSA. These data support the belief that TENS and the associated time/voltage combinations used do not increase conjugational transposons or facilitate horizontal gene transfer from MR-CNS to MSSA.

Opposing roles of netrin-1 and the dependence receptor DCC in cancer cell invasion, tumor growth and metastasis.

Deleted in colon cancer (DCC) and UNC5 function as netrin dependence receptors by inducing apoptosis in the absence of their ligand and accordingly were recently designated as putative conditional tumor suppressors. Herein, we determined whether netrin-1 and its receptors are implicated in cancer cell invasion and tumor progression. Expression of DCC, UNC5 and adenosine A2B-receptors (A2B-Rs) was investigated by reverse transcription polymerase chain reaction in human colon cancer cells. The impact of DCC restitution and netrin-1 was evaluated on collagen type I invasion, tumor growth and metastasis in nude mice, cancer cell survival and gene expression profiling. Flow cytometry, poly(ADP-ribose)polymerase-1 and caspase-8 activation were used to evaluate the impact of DCC on cell death. Both netrin-1 and A2B-R activation induced the invasive phenotype through the Rho-Rho kinase axis in DCC-deficient human colorectal cancer cells. Restitution of wild-type DCC blocked invasion induced by netrin-1, A2B-R agonist and other agents. Ectopic expression of netrin-1 led to increased growth of human colon tumor xenografts in athymic mice. Conversely, introduction of wt-DCC in kidney MDCKts.src-ggl cells strongly inhibited metastasis in lymph nodes and lungs and increased sensitivity to apoptosis in hypoxia. DNA microarrays revealed that netrin and DCC had common and divergent impacts on gene expression linked to cell cycle, survival, surface signaling and adhesion. Our findings underscore that netrin is a potent invasion and tumor growth-promoting agent and that DCC is a metastasis suppressor gene targeting both proinvasive and survival pathways in a cumulative manner.

Cell cycle attenuation by p120E4F is accompanied by increased mitotic dysfunction.

In addition to their well-established roles at the G1-S checkpoint, recent reports support a role for universal cyclin-dependent kinase (CDK) inhibitors in the control of G2-M and suggest that their induction may stimulate the occurrence of endomitosis or polyploidy in a number of physiological settings. In this report, the stable expression of the p120E4F transcription factor, which attenuates G1-S progression by elevating p21WAF1 and p27KIP1 protein levels, was shown to also interfere with the regulation of G2-M and cytokinesis. Exponentially growing cultures of p120E4F-expressing fibroblast cell lines had reduced levels of CDC2 kinase activity, elevated levels of Cyclin B1 protein, and continuously generated a subpopulation of tetraploid cells and elevated numbers of multinucleated cells. Coexpression of activated Ras, which stimulates Cyclin D1 expression and G1-S-specific cyclin-CDK kinase activities, alleviated these effects without reducing p21WAF1 or p27KIP1 protein levels; p120E4F/ras-expressing cell lines contained reduced levels of Cyclin B1 protein, a restoration of Cyclin B-CDC2 kinase activity to control levels, and exhibited no increase of tetraploid or multinucleated cells. Interestingly, changes in the expression of Cyclin B1 and, to a lesser extent, CDC2 were primarily regulated by post-transcriptional mechanisms. The results indicate that mechanisms which moderately elevate CDK inhibitor levels can reduce CDC2 kinase activity to the point of impeding normal G2-M function and suggest that two molecular determinants commonly associated with the induction of polyploidy in a number of tissues, i.e., elevated levels of universal CDK inhibitors and sustained CDK2 kinase activity, may be solely sufficient to initiate endomitosis.

Analysis of the NRAMP1 gene implicated in iron transport: association with multiple sclerosis and age effects.

Multiple sclerosis (MS) is believed to be an autoimmune process occurring in genetically susceptible individuals after an appropriate environmental exposure. We have exploited the homogeneous Afrikaner population of European ancestry to investigate the likelihood that iron dysregulation, in association with infectious and/or autoimmune disease susceptibility, may underlie the MS phenotype in a subgroup of patients. The functional Z-DNA forming repeat polymorphism of the natural resistance-associated macrophage protein-1 (NRAMP1) gene was analyzed in 104 patients diagnosed with MS and 522 Caucasian controls. A family-based control group consisting of 32 parental alleles not transmitted to MS offspring was additionally studied to exclude the likelihood of population substructures. Statistically significant differences in allelic distribution were observed between the patient and control samples drawn from the same population (P < 0.01). Evidence is furthermore provided that alleles considered to be detrimental in relation to autoimmune disease susceptibility may be maintained in the population as a consequence of improved survival to reproductive age following infectious disease challenge. Although it remains to be determined whether the disease phenotype in MS patients with allele 5 of the NRAMP1 promoter polymorphism is directly related to dysregulation of iron or modified susceptibility to viral infection and/or autoimmunity, a combination of these processes most likely underlies the disease phenotype in these patients. In view of the emerging role of polymorphic variants in complex diseases and minimizing of possible confounding factors in this association study, we conclude that allelic variation in the NRAMP1 promoter may contribute significantly to MS susceptibility in the South African Caucasian population.

Detection of biological toxins on an active electronic microchip.

An electric-field-driven assay for fluorescein-labeled staphylococcal enterotoxin B and cholera toxin B was developed on an active electronic microchip. An array of microlocations was transformed into an immunoassay array by electronically biasing electrodes at each microlocation to attract biotinylated capture antibodies. The electric field generated on the array directed the transport, concentration, and binding of biotinylated capture antibodies to streptavidin-coated microlocations. Subsequently, solutions of fluorescein-labeled staphylococcal enterotoxin B and fluorescein-labeled cholera toxin B were electronically addressed to the assay sites by an applied electric field. Each toxin was specifically bound to microlocations containing the appropriate capture antibody with little nonspecific binding to assay sites lacking the appropriate capture antibody. It was possible to detect both toxins from a mixture in a single electronic addressing step; detection was accomplished after a 1-min application of the electric field followed by washing. The ability to perform a rapid, electric field-mediated immunoassay for multiple analytes may provide an advantage over existing approaches.

Application of foodborne disease outbreak data in the development and maintenance of HACCP systems.

Five-hundred and thirty general foodborne outbreaks of food poisoning reported in England and Wales between 1992 and 1996 were reviewed to study their application to the development and maintenance of HACCP systems. Retrospective investigations of foodborne disease outbreaks provided information on aetiological agents, food vehicles and factors that contributed to the outbreaks. Salmonella spp. and foods of animal origin (red meat, poultry and seafood) were most frequently associated with outbreaks during this period. Improper cooking, inadequate storage, cross-contamination and use of raw ingredients in the preparation of food were the most common factors contributing to outbreaks. Classification and cross tabulation of surveillance information relating to aetiological agents, food vehicles and contributory factors facilitates hazard analysis. In forming control measures and their corresponding critical limits, this approach focuses monitoring on those aspects that are critical to the safety of the product. Incorporation of epidemiological data in the documentation of HACCP systems provides assurance that the system is based on the best scientific information available.