Biofouling is a major issue in applying nanofiltration and reverse osmosis technologies for wastewater treatment. Biofilm formed on the surface of membranes will severely decline the flux and cause energy waste. In this study, a novel biofouling control method that applies D-amino acids to inhibit biofilm formation was investigated. The D-amino acids previously reported to inhibit biofilm formation and disrupt existing biofilm – D-tyrosine and the mixture of D-tyrosine, D-tryptophan, D-leucine and D-methionine were tested. Pseudomonas aeruginosa and Bacillus subtilis were used as model Gram-negative and Gram-positive bacteria, respectively. D-amino acids have little effect and some effect on inhibition of biofilm formation and disruption of exiting biofilm to Pseudomonas aeruginosa, but have good effect to Bacillus subtilis. A commonly used microtiter plate assay for quantitative biofilm measurement was systematically evaluated and optimized for screening biofilm control agents. The effect of D-tyrosine on inhibition of organic fouling and P. aeruginosa biofouling on NF90 membrane surface in bench scale dead end filtration experiment was examined, which shows that D-tyrosine can effectively inhibit organic fouling and P. aeruginosa biofouling on NF90 membrane surface.

The in situ proximity ligation assay (in situ PLA) is an immunoassay that enables directvisualisation of single protein targets or protein interactions in cell or tissue samples. This project revolves around designing and introducing several novel multiplexable components tobe used in conjunction with Olink Bioscience's Duolink product line. In this report, a novel in silico approach to DNA oligomer interaction design is presented. Using this in silico method, a multiplexed system of DNA oligomers has been designed andevaluated using in situ PLA and fluorescence microscopy.

The diagnosis of cancer today is obsolete, depending upon pattern recognition and non-quantifiable data. The time consuming diagnosis is often performed on biopsies, fixed using non standardised procedures, and leaves room for dubious results. The diagnosis is also invasive, exposing patients to risk of infections and discomfort due to the need of tissue samples. The knowledge about changes in protein expression levels related to cancer can instead be utilized to generate a new diagnostic tool. By adapting the in situ proximity ligation assay (in situ PLA) to cells in solution, it is possible to detect proteins, or protein interactions, within cells without the need for tissue samples. Since the method is both highly sensitive and specific, it delivers reliable results. In this report, the in situ PLA method for cells in solution is combined with flow cytometry readout. Hence, a new and less invasive diagnostic tool for cancer, delivering highly accurate high throughput single cell analysis, may be on the rise.

Certain types of cyanobacteria have the potential to produce toxins including microcystin, a hepatotoxin. Toxic cyanobacterial blooms are becoming increasingly common worldwide. They are a concern in the Great Lakes and surrounding waters. In this study, Lake Ontario’s Bay of Quinte, Lake Erie’s Maumee Bay, and three reservoirs along the Grand River were studied. Environmental variables, cyanobacterial biomass inferred from the Fluoroprobe, and microcystin concentrations were measured. In 2005 the three reservoirs, Belwood Lake, Conestogo Lake, and Guelph Lake were sampled every two weeks from July to September. Belwood Lake was also sampled in October when a cyanobacterial bloom occurred. In 2006 the Bay of Quinte was sampled twice, in July and September, and Maumee Bay was sampled twice, in June and August. Physical variables measured included water transparency and temperature. All species of nitrogen (N) and phosphorus (P) were measured, along with extracted chlorophyll a and particulate carbon (C), N, and P. The distribution of chlorophyll and major algal groups throughout the water column was profiled in situ using a spectral fluorometer (Fluoroprobe).Variable fluorescence of phytoplankton was assessed using Pulse Amplitude Modulated (PAM) fluorometry to measure photosynthetic parameters. Phytoplankton counts were performed on selected samples from the Bay of Quinte and Maumee Bay. Total and dissolved microcystin were measured using the protein phosphatase inhibition assay (PPIA). PPIA was chosen over alternative detection methods because it is a functional assay that measures the level of microcystin in a sample via the amount of protein phosphatase inhibition that it exerts. This yields ecologically relevant data as protein phosphatase inhibition is the main mode of microcystin toxicity. The PPIA formulation used in our lab was based on variations in the literature that use unconcentrated water samples directly in the assay. The assay was optimized to employ both a higher and lower standard curve through the use of two enzyme concentrations. The lower enzyme concentration allowed the method detection limit to be decreased to 0.05 µg/L to accommodate our low-microcystin samples. In the Bay of Quinte, microcystin levels were higher in July 2006 (total mean=2.25 μg/L ) than in September 2006 (total mean=0.58 μg/L). In July a cyanobacterial bloom consisting of 97% Microcystis spp. was present. In September 83% of the cyanobacterial biomass was composed of Anabaena spiroides and only 8% was Microcystis spp. In the Bay of Quinte elevated microcystin concentrations were associated with higher soluble reactive P levels, lower seston C:P molar ratios, and lower total N. In Maumee Bay microcystin levels were higher in August 2006 (total mean= 4.45 μg/L) than they were in June 2006 (<0.05 μg/L). In August a cyanobacterial bloom consisting of 22% Microcystis spp. and 48% Aphanizomenon flos-aquae was observed. Higher microcystin concentrations in Maumee Bay were associated with decreased total N: total P molar ratios, increased total P, and decreased water transparency as measured by Secchi depth. Belwood Lake had the highest microcystin levels of the three reservoirs but only once exceeded the recommended World Health Organization concentration of 1.0 μg/L. Belwood Lake’s largest cyanobacterial bloom in October 2005 was accompanied by relatively low microcystin levels (<0.2 μg/L). Conestogo and Guelph lakes always had microcystin levels below 0.2 μg/L and 0.6 μg/L, respectively. In the Grand River reservoirs, increased microcystin concentrations were associated with higher chlorophyll a, higher light attenuation coefficients, lower total N, lower total N: total P molar ratios, higher C:P molar ratios, lower nitrate, higher cyanobacterial biomass, and higher total P. When data from the Bay of Quinte, Maumee Bay, and Grand River reservoirs were pooled, total microcystin had the most significant positive correlation with total P. Total microcystin and water temperature also had a significant positive correlation.

Exopolysaccharides are essential for the establishment of the symbiosis between Sinorhizobium meliloti and Medicago sativa (alfalfa). The ExoS/ChvI two-component regulatory system is known as a regulator of succinoglycan production but the genes that are directly regulated by ChvI have not been determined. Difficulty isolating exoS and chvI null mutants has prompted the suggestion that these genes are essential for S. meliloti viability. We have successfully isolated exoS and chvI null mutants using a merodiploid facilitated strategy. We present evidence that the S. meliloti ExoS/ChvI two-component regulatory system is essential for symbiosis with alfalfa. Phenotypic analyses of exoS and chvI null mutant strains demonstrate that ExoS/ChvI controls both succinoglycan and galactoglucan production and is required for growth on over 21 different carbon sources. These new findings suggest that the ExoS/ChvI regulatory targets might not be the exo genes that are specific for succinoglycan biosynthesis but rather genes that have common influence on both succinoglycan and galactoglucan production. To obtain further insight into the nature of the ChvI regulon, we obtained a purified His•Tag-ChvI and used it to perform modified electrophoretic mobility shift assays. These assays were done using genomic DNA and were followed by cloning of DNA fragments having the highest affinity for ChvI. Sequencing of these fragments revealed that ChvI has a diverse regulon, it affects transcription of genes encoding enzymes that are involved in different pathways. Transcriptional gene fusion assays confirmed that ChvI is important for the activation of the transcription of the msbA2 operon, as well as repression of the transcription of the rhizobactin 1021 operon and genes SMc00262-61. ChvI-regulation of genes that are part of the connected thiamine and histidine biosynthesis pathways suggest that ChvI could act in a concerted manner to avoid limitation of important intermediates in these pathways. This study presents for the first time genes directly regulated by ChvI and this includes none of the exo genes. This work opens new avenues in the understanding of the global regulatory role of the symbiotically important ExoS/ChvI two-component regulatory system.

Oxidative stress has been associated with many disease conditions in adults and neonates based on clinical and post mortem studies. Trauma is the commonest cause of oxidative stress. However a gap in knowledge of the natural history of oxidative stress in humans was identified as most studies have been post mortem or in animals. <p>The aim of this research is to understand treat and oxidative stress in trauma and surgical patients. The study involved three components including: the development and evaluation of the novel oxistress assay; study of clinical trauma and oxidative stress; and clinical trial of alanyl-glutamine supplementation following major surgery. The novel oxistress assay was used on urine samples in the normal population to determine reference values and subsequently on hospital patients to determine sensitivity and specificity. The study of clinical trauma and oxidative stress evaluated plasma antioxidants (FRAP assay), red cell glutathione (Asensis method), plasma and urine protein carbonyl (Levines method) and total oxidants in plasma and urine (oxistress assay) over 7 day period following trauma. The clinical trial was a double blind study of 69 major surgery patients evaluating biochemical and clinical parameters over 7 day period in comparison with pre-operative status. <p>The novel oxistress assay proves to be a sensitive and accurate bedside diagnostic tool for oxidative stress. It can also be used in the laboratory setting. Oxidative stress is associated with increased trauma severity resulting in antioxidant depletion, strong oxidant production and protein degradation. The presence of pre-morbid medical factors also increased oxidative stress in trauma patients. Oral alanyl-glutamine supplementation (0.3 g/kg) increased plasma glutamine and antioxidant levels while decreasing urine oxidant levels. It significantly reduced hospital stay in non-cancer and higher disease complexity patients. The intervention also reduced the resource intensity weighting (RIW) score. <p>Oxidative stress is a clinical problem in surgery and trauma patients that can now be easily diagnosed at the bedside using the novel oxistress assay. Treatment with alanyl-glutamine is effective in reducing oxidative stress and improving clinical outcome. It is highly recommended probably at a higher dose in order to achieve optimal results.

Studying protein-protein interactions has been vital for understanding how proteins function within the cell, how biological processes are strictly regulated by these interactions, and what molecular mechanisms underlie cellular functions and diseases. Recent biochemical and biophysical studies have provided evidence supporting that G protein-coupled receptors (GPCRs) can and do interact with one another to form dimers or larger oligomeric complexes, which may determine the structure and function of GPCRs, including receptor trafficking, scaffolding and signaling. This may help to understand the physiological roles of GPCRs and mechanisms underlying certain disease pathologies and to provide an alternative approach for drug intervention.<p>Cannabinoid CB1 and dopamine D2 receptors are the most common GPCRs in the brain and exert a mutual regulation in brain functions involved in learning, memory and drug addiction. There is structural and functional evidence supporting the idea that CB1 and D2 receptors physically interact with each other in hippocampal and striatal neurons to modulate their functions. Direct evidence supporting a physical interaction between the CB1 and D2 receptors was obtained from cultured HEK293 cells stably coexpressed with both receptors.<p> This research project was designed to critically test the hypothesis that a specific protein sequence (i.e. motif) in the D2 receptor is responsible for in vitro protein-protein interactions between the CB1 and D2 receptors. To reach this goal, fusion proteins containing various domains and motifs of the CB1 and D2 receptors were prepared and then used first to determine the domains of the CB1 and D2 receptors responsible for in vitro protein-protein interactions between CB1 and D2 receptors, and then to identify the specific motifs in the D2 receptor responsible for in vitro CB1 coupling with the D2 receptors. The major method used in this study is in vitro pull-down assay, which uses a purified and tagged bait protein to generate a specific affinity support that is able to bind and purify a prey protein from a lysate sample. The present study provides the first evidence that CB1 intracellular C-terminal (CB1-CT) and D2 intracellular loop 3 (D2-IL3) can directly interact with each other, and that the specific motifs D2-IL3(Ⅳ1) and D2-IL3(Ⅳ3) in the D2 receptor are likely responsible for their in vitro coupling with the CB1 receptors. <p>The results of the present study are invaluable for future research exploring in vivo protein-protein interaction between the CB1 and D2 receptors in the rat striatum by co-immunoprecipitation. Specifically, future studies will determine whether the identified specific motifs D2-IL3(Ⅳ1) and D2-IL3(Ⅳ3) in the D2 receptor are indeed critical for their in vivo coupling with the CB1 receptors.

Agriculture is a valuable economic resource in western Canada, but for decades farmers have focused on intensive production practices while ignoring the long-term health and maintenance of the land. In recent years, the use of conservation agricultural techniques has been encouraged in an effort to conserve prairie landscape while sustaining cropland productivity. Sustainable agricultural practices that promote soil and water conservation and benefit wildlife and prairie biodiversity include conservation tillage and planting of winter cereal crops. Many species of wild birds nest in the ground cover provided by minimum tillage and fall seeded cropland in the spring. Although habitat quality in conservation areas is superior for birds, there is potential for eggs of ground nesting birds to be exposed to herbicides during spring weed control operations. Herbicides commonly used on the prairies to control weed growth in conservational systems include 2,4-D and Buctril-M®. Since the subtlethal effects of exposure to these herbicides may include DNA damage and immunomodulation, the overall goal of this study was to assess whether <i>in ovo</i> exposure to the herbicides 2,4-D and Buctril-M® adversely affects genetic material and/or immune system function in newly hatched domestic chickens (<i>Gallus gallus</i>) and ducks (<i>Anas platyrhynchos</i>), as surrogates for wild bird species.<p>Study design attempted to reproduce actual field exposures by use of an agricultural field spray simulator to apply formulated herbicides (as opposed to pure active ingredients) at recommended crop application rates. In three separate experiments, fertile chicken eggs were sprayed with 2,4-D ester formulation or with Buctril-M® formulation, and fertile duck eggs were sprayed with 2,4-D ester formulation, during either an early (embryonic day 6) or late (embryonic day 15 for chickens or embryonic day 21 for ducks) stage of incubation. Genotoxicity and immune system function were evaluated in the hatchlings as the main toxicological endpoints to assess potential subtle effects from herbicide exposure, but additional measures of general health and development were also evaluated. Two endpoints were used to assess subtle changes to genetic integrity. The comet assay was used to detect structural damage (strand breaks) in avian lymphocyte DNA, as an index of acute genotoxic effects. Flow cytometry was used to examine potential clastogenic effects of the herbicides, by determining if chromosomal changes resulted in variability in the DNA content of avian erythrocytes. Several endpoints were examined to evaluate potential exposure-induced effects on the immune system. Immunopathological assessment of chicks and ducklings included differential lymphocyte counts, as well as immune organ weights and histopathology. The cell-mediated and humoral immune responses in hatchlings were assessed using the delayed-type hypersensitivity test and measurement of systemic antibody production in response to immunization, respectively. Exposure of fertile chicken and duck eggs to Buctril-M® or 2,4-D had no effects on the biomarkers of genetic integrity in this study. Differences in herbicide treatment (high and low concentrations) and times of exposure (early and late incubation stages) did not translate into noticeable factor effects in final model analyses for any of the genotoxicity assay variables evaluated in newly hatched chickens exposed in ovo to 2,4-D. Similarly, comet assay outcomes in chicks exposed to Buctril-M® were not significantly associated with either herbicide treatment or time of exposure as fixed effect factors. Results of the comet assay using peripheral lymphocytes from ducklings provided evidence of potential primary genetic damage associated with the time of spray exposure in ovo. Comet tail DNA content was significantly associated (P = 0.0269) with exposure times, suggesting that ducks may be increasingly sensitive to spray exposure conditions at an early stage of embryological development. Effects of exposure timing were not attributable to herbicide treatment. Although 2,4-D exposure time was associated with DNA strand breakage in ducklings, there was no evidence of chromosomal damage. However, an association between the HPCV values (a measure of DNA content variability) and time of spray exposure was observed in the experiment where 21-day-old chickens were treated in ovo with Buctril-M®. The mean HPCV value for the early exposure group (E6) was significantly greater (P = 0.0210) than that of the group treated later in incubation (E15). However, Buctril-M® the concentration of herbicide did not have any influence on this outcome, and the reason for the difference between exposure times is uncertain, but may be attributed to stress associated with manipulations during spraying. An increase in HPCV, reflecting greater intercellular DNA variability, is indicative of increased incidence of chromosomal damage, which may be an effect of disturbance during early periods of incubation as a result of exposure conditions.<p>Among the panel of immunotoxicity tests conducted to evaluate the effects of <i>in ovo</i> exposure to 2,4-D and Buctril-M® on the developing avian immune system, only heterophil/ lymphocyte (H/L) ratios and relative immune organ weights were significantly associated with either herbicide treatment or time of spray exposure in all three experiments. In 21-day-old chicks exposed in ovo to 2,4-D, relative bursa weight was associated with the different herbicide treatments (P = 0.0006). Relative bursa weights were significantly lower in chicks in the low dose group, while the opposite effect was observed in the high dose chicks, compared with the controls. It is unlikely that the observed decrease in bursa weight in the low dose group is causally related to herbicide exposure because a consistent dose-response effect was not observed, but this outcome may be explained by a compensatory immune response. The relative spleen weights of newly hatched chickens exposed in ovo to Buctril-M® exhibited a significant association with herbicide treatment (P = 0.0137). Relative spleen weights for birds in the low dose treatment groups were significantly different than both the control (P = 0.0179) and high dose groups (P = 0.0125). However, there was no significant difference between high dose and control groups, and this outcome reduces the likelihood of a causal relationship between spleen weight and herbicide exposure. In the parallel experiment involving in ovo exposure to 2,4-D to ducklings, relative bursa weight was associated with time of spray exposure (P = 0.0434). Ducklings that hatched from eggs exposed to spray on day 6 of incubation exhibited greater mean relative bursa weights than the birds exposed to spray at a later incubation stage (E21). This result implies that spray exposure during earlier stages of development may result in conditions which affect the humoral immune response, if increased bursal weight is associated with increased B lymphocyte and antibody production. In the same experiment, mean H/L ratios in peripheral blood samples from 21-day-old ducklings were significantly different between the groups treated with the high concentration of 2,4-D and water (control) (P = 0.0395). Although ratios from the birds in the low dose groups were not significantly different from the control groups, changes in H/L ratio values demonstrate a dose dependent relationship with increasing herbicide exposure.<p>Residue analysis of chicken and duck eggs in this study measured transfer of herbicide through the shell and into the embryo 24 hours and up to 5 days (chickens only) after spraying. Mean 2,4-D residue concentrations were higher in both chicken and duck eggs from the high dose (10X) groups than in eggs exposed to the recommended field rate of herbicide application (1X). Embryo residue concentrations in the chicken eggs increased from the day following exposure to 5 days after spraying, in both low and high dose groups. This observation indicates that the risk of contaminant-induced adverse effects may continue to increase for at least several days after exposure, thereby influencing the concentration of herbicide to which the developing embryo is exposed.<p>On the Canadian prairies, wild bird eggs are potentially to be exposed to 2,4-D and Buctril-M® during various stages of embryonic development. The present study examined effects of herbicide exposure at two distinct times during incubation, and demonstrated the potential for subtle impacts on genetic integrity and the immune system. Results indicate that spray exposure during earlier stages of organogenesis may cause more significant adverse effects. Given the possible harmful consequences of the observed changes on the long-term health of wild birds, further research is needed in order to better characterize the risks of in ovo agrochemical exposure in prairie ecosystems.

The type II secretion system is used by many pathogenic and non-pathogenic bacteria for the extracellular secretion of enzymes and toxins. <i>Aeromonas hydrophila</i> is a Gram-negative pathogen that secretes proteins via the type II secretion system.<p> In the studies described here, a series of yeast two-hybrid assays was performed to identify protein-protein interactions in the type II secretion system of <i>A. hydrophila</i>. The periplasmic domains of ExeA and ExeB were assayed for interactions with the periplasmic domains of Exe A, B, C, D, K, L, M, and N. Interactions were observed for both ExeA and ExeB with the secretin ExeD in one orientation. In addition, a previously identified interaction between ExeC and ExeD was observed. In order to further examine and map these interactions, a series of eight two-codon insertion mutations in the amino terminal domain of ExeD was screened against the periplasmic domains of ExeA and ExeB. As a result, the interactions were verified and mapped to subdomains of the ExeD periplasmic domain. To positively identify the region of ExeD involved in the interactions with ExeA, B, C and D, deletion mutants of ExeD were constructed based on the two-codon insertion mutation mapping of subdomains of the ExeD periplasmic domain, and yeast two-hybrid assays were carried out. The results showed that a fragment of the periplasmic domain of ExeD, from amino acid residue 26 to 200 of ExeD, was involved in the interactions with ExeA, B and C. As an independent assay for interactions between ExeAB and the secretin, His-tagged derivatives of the periplasmic domains of ExeA and ExeB were constructed and co-purification on Ni-NTA agarose columns was used to test for interactions with untagged ExeD. These experiments confirmed the interaction between ExeA and ExeD, although there was background in the co-purification test.<p> These results provide support for the hypothesis that the ExeAB complex functions to organize the assembly of the secretin through interactions between both peptidoglycan and the secretin that result in its multimerization into the peptidoglycan and outer membrane layers of the envelope.

Reagents that bind proteins are applicable in biology for detection of molecules, perturbation of signaling pathways and development of small-molecule pharmaceuticals. Protein ligands interact with proteins, inhibiting or altering their function. They are isolated from combinatorial libraries to interact with a specific target, using selection techniques such as phage display or yeast-two-hybrid assay. For the latter, one inconvenience is the detection of false positives, which can be solved by screening pools containing the samples to be tested, instead of individual samples. Samples are distributed in the pools following a pooling design. The PI-deconvolution pooling design was developed to screen cDNA libraries using the yeast-two-hybrid assay, which are smaller in size than protein ligand combinatorial libraries. Modifications to the PI-deconvolution screening technique were developed to adapt it to the screening of protein ligand combinatorial libraries using the yeast-two-hybrid assay. Every spot of the array containing the combinatorial library was randomly pooled. However, the yeast-two-hybrid assay loses sensitivity when strains are pooled. As PI-deconvolution requires detecting every interaction, we determined the optimal amount of library members that can be pooled in a spot, and the optimal number of replicates to ensure the detection of an interaction.<p> The yeast-two-hybrid assay was used to perform a screening of a combinatorial library with seven domains of BCR-ABL, which were pooled according to PI-deconvolution. BCR-ABL is a chimeric protein with unregulated kinase activity that is responsible for chronic myelogenous leukemia. The scaffold used in the combinatorial library was an engineered intein that forms lariat peptides. After a screening of this library was performed, positive interactions were detected in 775 spots of the arrays that contained 1432 positive hits. Only 53 spots were deconvoluted. The coding sequences of the lariat peptides were determined for 23 lariat peptides interacted with the GEF domain of BCR, and for ABL, two with the FABD domain, one with the SH1 domain, and one with the SH3 domain. Finally, a &beta;-galactosidase assay was performed to assess the affinity of the lariat peptides for their target.<p> The isolated lariat peptides are potential inhibitors of BCR-ABL that can have therapeutic potential. This study will improve other screenings of combinatorial libraries with the yeast-two-hybrid assay.