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Pseudomonas aeruginosa is an opportunistic pathogen that is inherently resistant to many antibiotics and represents an increasing threat due to the emergence of drug-resistant strains. There is a pressing need to develop innovative antimicrobials against this pathogen. In this study, we identified the O-specific antigen (OSA) of P. aeruginosa serotype O6 as a novel target for therapeutic intervention. Binding of monoclonal antibodies and antigen-binding fragments therefrom to O6 OSA leads to rapid outer membrane destabilization and inhibition of cell growth. The antimicrobial effect correlated directly with antibody affinity. Antibody binding to the O antigen of a second lipopolysaccharide (LPS) type present in P. aeruginosa or to the LPS core did not affect cell viability. Atomic force microscopy showed that antibody binding to OSA resulted in early flagellum loss, formation of membrane blebs, and eventually complete outer membrane loss. We hypothesize that antibody binding to OSA disrupts a key interaction in the P. aeruginosa outer membrane.

BACKGROUND The glyphosate-resistant rapid response (GR RR) resistance mechanism in Ambrosia trifida is not due to target-site resistance (TSR) mechanisms. This study explores the physiology of the rapid response and the possibility of reduced translocation and vacuolar sequestration as non-target-site resistance (NTSR) mechanisms. RESULTS GR RR leaf discs accumulated hydrogen peroxide within minutes of glyphosate exposure, but only in mature leaf tissue. The rapid response required energy either as light or exogenous sucrose. The combination of phenylalanine and tyrosine inhibited the rapid response in a dose-dependent manner. Reduced glyphosate translocation was observed in GR RR, but only when associated with tissue death caused by the rapid response. Nuclear magnetic resonance studies indicated that glyphosate enters the cytoplasm and reaches chloroplasts, and it is not moved into the vacuole of GR RR, GR non-rapid response or glyphosate-susceptible A. trifida. CONCLUSION The GR RR mechanism of resistance is not associated with vacuole sequestration of glyphosate, and the observed reduced translocation is likely a consequence of rapid tissue death. Rapid cell death was inhibited by exogenous application of aromatic amino acids phenylalanine and tyrosine. The mechanism by which these amino acids inhibit rapid cell death in the GR RR phenotype remains unknown, and it could involve glyphosate phytotoxicity or other agents generating reactive oxygen species. Implications of these findings are discussed. The GR RR mechanism is distinct from the currently described glyphosate TSR or NTSR mechanisms in other species. © 2017 Society of Chemical Industry

BACKGROUND Glyphosate-resistant (GR) Ambrosia trifida is now present in the midwestern United States and in southwestern Ontario, Canada. Two distinct GR phenotypes are known, including a rapid response (GR RR) phenotype, which exhibits cell death within hours after treatment, and a non-rapid response (GR NRR) phenotype. The mechanisms of resistance in both GR RR and GR NRR remain unknown. Here, we present a description of the RR phenotype and an investigation of target-site mechanisms on multiple A. trifida accessions. RESULTS Glyphosate resistance was confirmed in several accessions, and whole-plant levels of resistance ranged from 2.3- to 7.5-fold compared with glyphosate-susceptible (GS) accessions. The two GR phenotypes displayed similar levels of resistance, despite having dramatically different phenotypic responses to glyphosate. Glyphosate resistance was not associated with mutations in EPSPS sequence, increased EPSPS copy number, EPSPS quantity, or EPSPS activity. CONCLUSION These encompassing results suggest that resistance to glyphosate in these GR RR A. trifida accessions is not conferred by a target-site resistance mechanism. © 2017 Society of Chemical Industry

There is no story in Western literature that epitomizes the dominant Western discourse of individualism as clearly, and its effects as boldly, as Shakespeare's Hamlet. “This above all: to thine own self be true” (I.iii.78) is the cornerstone of Western individualism and the discourse of the individual over the relational. Though Hamlet was written over 500 years ago, the theme of individualism parallels strongly with the present Western construction of madness in mental health. In this article, a narrative case study of Hamlet is presented as a creative vehicle to explain narrative therapy and to challenge the individualized notion of madness by decentering it, unpacking it, and making it visible (Derrida, 1997; White, 1991). It is proposed in Hamlet that self-subjugation related to individualizing discourse, coupled with corresponding discourses concerning duty, honor, and gender, influence Shakespeare's characters’ abilities to communicate and to polyvocally negotiate the meaning of events in their live...

Social work practice focuses specifically on males or females is a more limited area of scholarship when compared to those focused on general models of practice within social work. The majority of practice texts and journals do not differentiate gender when presenting a theory or method of practice. Males are similarly less of a focus than women and other specified groups other than gender. Overall, males are less likely to be a focus of social work practice literature than in other disciplines such as psychology and counseling. Many of the approaches to working with males, in particular early writings, address issues of masculinity as a necessary step in working with males. Social work practice with males can be examined from a policy, population, community or individual perspective, and while all of these areas of practice are important, this article is limited to references addressing clinical practice with individuals and small groups. To that end, while the primary focus is clinical social work practice, references are included from psychology, psychotherapy, and counseling. These references are important, as significant crossover scholarship and research from parallel fields adds to the literature base of social work practice with males. While this article focuses primarily on practice with adult males, a section and selected references addressing practice with male youth and children is included.

Most therapeutic antibodies are produced using either Chinese hamster ovary (CHO) or mouse NS0 (myeloma) cells. Despite their relative ease of use and extensive characterization, the use of mammalian cells for producing mAbs has shortcomings. The first demonstration of the successful expression of an IgG in plants was in 1989; since then, improvements in the production of mAbs by plants have contributed to realistic proposals for the use of plant systems as an alternative platform for the production of therapeutic proteins. Plant-based production of recombinant proteins using N. benthamiana is a safe, low-cost, and highly efficient alternative production platform to the standard CHO or NS0 cell culture systems for the production of high-value therapeutic proteins. This chapter illustrates the developmental stages involved in stable transgenic plant production. Improper glycosylation, such as the attachment of truncated N-glycans, causes mAbs to lose their quaternary open Y-shaped structure. The development of biosimilar drugs in plants requires a humanized N-glycosylation system.

In this article we describe how research demonstrates that the presence of hope and the development and cultivation of hope plays an important role in the client change process. We propose that the concept of hope has significance to outcomes and that this requires enhanced awareness of hope in training and in practice of solution-focused brief therapy (SFBT). As educators and practitioners of SFBT, we have found that hope is mentioned in terms of the implicit nature of hope in SFBT literature (Dolan, 2014), but we rarely find that the essence of the idea of hope is made more explicit in teaching and practice. In this paper we address this gap by linking research findings on the concept of hope from various areas of study to both training in the practice skills and processes of SFBT. Specific focus is placed on the connections among findings in various areas of study and research on hopefulness to demonstrate the explicit means by which hopefulness is created, amplified, and utilized in enhancing a sense of possibility and personal agency during SFBT.

Glyphosate-resistant (GR) Ambrosia trifida is now present in the midwestern United States and in southwestern Ontario, Canada. Two distinct GR phenotypes are known, including a rapid response (GR RR) phenotype, which exhibits cell death within hours after treatment, and a non-rapid response (GR NRR) phenotype. The mechanisms of resistance in both GR RR and GR NRR remain unknown. Here, we present a description of the RR phenotype and an investigation of target-site mechanisms on multiple A. trifida accessions.Glyphosate resistance was confirmed in several accessions, and whole-plant levels of resistance ranged from 2.3- to 7.5-fold compared with glyphosate-susceptible (GS) accessions. The two GR phenotypes displayed similar levels of resistance, despite having dramatically different phenotypic responses to glyphosate. Glyphosate resistance was not associated with mutations in EPSPS sequence, increased EPSPS copy number, EPSPS quantity, or EPSPS activity.These encompassing results suggest that resistance to glyphosate in these GR RR A. trifida accessions is not conferred by a target-site resistance mechanism. © 2017 Society of Chemical Industry.

The glyphosate-resistant rapid response (GR RR) resistance mechanism in Ambrosia trifida is not due to target-site resistance (TSR) mechanisms. This study explores the physiology of the rapid response and the possibility of reduced translocation and vacuolar sequestration as non-target-site resistance (NTSR) mechanisms.GR RR leaf discs accumulated hydrogen peroxide within minutes of glyphosate exposure, but only in mature leaf tissue. The rapid response required energy either as light or exogenous sucrose. The combination of phenylalanine and tyrosine inhibited the rapid response in a dose-dependent manner. Reduced glyphosate translocation was observed in GR RR, but only when associated with tissue death caused by the rapid response. Nuclear magnetic resonance studies indicated that glyphosate enters the cytoplasm and reaches chloroplasts, and it is not moved into the vacuole of GR RR, GR non-rapid response or glyphosate-susceptible A. trifida.The GR RR mechanism of resistance is not associated with vacuole sequestration of glyphosate, and the observed reduced translocation is likely a consequence of rapid tissue death. Rapid cell death was inhibited by exogenous application of aromatic amino acids phenylalanine and tyrosine. The mechanism by which these amino acids inhibit rapid cell death in the GR RR phenotype remains unknown, and it could involve glyphosate phytotoxicity or other agents generating reactive oxygen species. Implications of these findings are discussed. The GR RR mechanism is distinct from the currently described glyphosate TSR or NTSR mechanisms in other species. © 2017 Society of Chemical Industry.

Purification of recombinant monoclonal antibody from transgenic plant extract is technically challenging as it involves the processing of large volume of material, containing low titre of antibody, present along with large quantities of native proteins and other impurities. The conventional approach of capturing antibody from a clarified extract using packed-bed chromatography is therefore not particularly suitable. This study evaluates the suitability of using a combination of ultrafiltration and chromatography for purifying transgenic tobacco-derived human monoclonal antibody. A two-stage cascade ultrafiltration process removed about 97% impurities while ensuring almost complete recovery of antibody, providing 32-fold antibody enrichment in the process. The primary objective of the ultrafiltration step was to reduce the burden on the subsequent chromatographic steps. A two-step chromatographic process was then used to eliminate remaining impurities. Using this approach, recombinant human antibody expressed in tobacco could be purified to greater than 95% purity with 50% overall recovery (ca. 12.5 mg antibody/kg tobacco tissues).

A history and description of narrative therapy is provided including empirical research, theoretical underpinnings, and the clinical process of the practice. Narrative is a postmodern, person-centered practice that promotes change through the exploration of narrative. A narrative is a series of events, linked in sequence, through time, according to a specific plot. In this approach identity is understood to be a narrative, a story of self, and narrative techniques involve exploring the meanings attributed to life events, deconstructing, and reconstructing the meaning of those events in ways that are of benefit to the client. Narrative practice is a practice of liberation in that problems are viewed as not being located inside people but in the social discourses that clients have been recruited into accepting in their lives and by which they may be self-subjugating. Narrative is a respectful, non-blaming approach, which places people as the experts of their lives, and harnesses their innate strengths, skills, and resiliencies to re-story, or re-author themselves in a more positive and enriching way.

To study how the P19 suppressor of gene-silencing protein can be used effectively for the production of therapeutic glycoproteins, the following factors were examined: the genetic elements used for expressing recombinant proteins; the effect of different P19 concentrations; compatibility of P19 with various Nicotiana tabacum cultivars for transgenic expression; the glycan profile of a recombinant therapeutic glycoprotein co-expressed with P19 in an RNAi-based glycomodified Nicotiana benthamiana expression host. The coding sequences for the heavy and light chains of trastuzumab were cloned into five plant expression vectors (102-106) containing different 5' and 3' UTRs, designated as vector sets 102-106 mAb. The P19 protein of Tomato bushy stunt virus (TBSV) was also cloned into vector 103, which contained the Cauliflower mosaic virus (CaMV) 35S promoter and 5'UTR together with the terminator region of the nopaline synthase gene of Agrobacterium. Transient expression of the antibody vectors resulted in different levels of trastuzumab accumulation, the highest being 105 and 106 mAb at about 1% of TSP. P19 increased the concentration of trastuzumab approximately 15-fold (to about 2.3% of TSP) when co-expressed with 103 mAb but did not affect antibody levels with vectors 102 and 106 mAb. When 103 mAb was expressed together with P19 in different N. tabacum cultivars, all except Little Crittenden showed a marked discolouring of the infiltrated areas of the leaf and decreased antibody expression. Co-expression of P19 also abolished antibody accumulation in crosses between N. tabacum cv. I-64 and Little Crittenden, indicating a dominant mode of inheritance for the observed P19-induced responses.

The discovery of auxinic herbicides (e.g., 2,4-D, Dicamba, Picloram) for selective control of broad-leaf weeds in cereal crops revolutionized modern agriculture. These herbicides are inexpensive and do not generally have prolonged residual activity in soil. Although cultivated species of Brassicaceae (e.g., radish and other vegetables) are susceptible to auxinic herbicides, some biotypes of wild mustard (Sinapis arvensis, 2n = 18) were found to be highly resistant to Picloram and Dicamba. Inter-generic hybrids between wild mustard and radish (Raphanus sativus, 2n = 18) were produced by traditional breeding coupled with in vitro embryo rescue/ovule culture. To increase frequency of embryo regeneration and hybrid plant production, several hundred reciprocal crosses were performed between these species. Upon altering cultural conditions and media composition, a high frequency of embryo regeneration and hybrid plant establishment was achieved. A protocol was also optimized for in vitro clonal multiplication of inter-generic hybrids produced by embryo rescue. To evaluate transfer of auxinic herbicide resistance from wild mustard into hybrid plants, several screening tests (involving in vitro, molecular-based as well as whole plant-based tests) were performed. Results indicated that hybrids of R. sativus x S. arevensis were resistant to auxinic herbicides suggesting, that, the resistance trait was transferred to these hybrids from the wild mustard. This research for the first time demonstrates the possibility of transfer of auxinic herbicide resistance from wild mustard to radish.

Production of therapeutic monoclonal antibodies using genetically modified plants may provide low cost, high scalability and product safety; however, antibody purification from plants presents a challenge due to the large quantities of biomass that need to be processed. Protein A column chromatography is widely used in the pharmaceutical industry for antibody purification, but its application is limited by cost, scalability and column fouling problems when purifying plant-derived antibodies. Protein A-oleosin oilbodies (Protein A-OB), expressed in transgenic safflower seeds, are relatively inexpensive to produce and provide a new approach for the capture of monoclonal antibodies from plants. When Protein A-OB is mixed with crude extracts from plants engineered to express therapeutic antibodies, the Protein A-OB captures the antibody in the oilbody phase while impurities remain in the aqueous phase. This is followed by repeated partitioning of oilbody phase against an aqueous phase via centrifugation to remove impurities before purified antibody is eluted from the oilbodies. We have developed this purification process to recover trastuzumab, an anti-HER2 monoclonal antibody used for therapy against specific breast-cancers that over express HER2 (human epidermal growth factor receptor 2), from transiently infected Nicotiana benthamiana. Protein A-OB overcomes the fouling problem associated with traditional Protein A chromatography, allowing for the development of an inexpensive, scalable and novel high-resolution method for the capture of antibodies based on simple mixing and phase separation.

The need for rapid and easy technologies for the detection of food-borne and environmental pathogens is essential for safeguarding the health of populations. Furthermore, distribution of tainted food and water can have consequences which can affect whole economies. Antibodies and antibody fragments have been historically used in detection platforms due to their antigen specificity and robust physicochemical properties. In this study, we report the isolation and characterization of antibody fragments from the heavy chain antibody repertoire (VHH) of Camelidae which bind with specificity and high affinity to the Listeria monocytogenes invasin, Internalin B (InlB). To the best of our knowledge, this is the first report of anti-InlB VHHs from camelids. These anti-InlB VHHs were not cross-reactive to the structurally related Listeria invasin Internalin A (InlA) and are potential reagents to be used in the development of detection and medical technologies.

Auxinic herbicides are widely used for control of broadleaf weeds in cereal crops and turfgrass. These herbicides are structurally similar to the natural plant hormone auxin, and induce several of the same physiological and biochemical responses at low concentrations. After several decades of research to understand the auxin signal transduction pathway, the receptors for auxin binding and resultant biochemical and physiological responses have recently been discovered in plants. However, the precise mode of action for the auxinic herbicides is not completely understood despite their extensive use in agriculture for over six decades. Auxinic herbicide-resistant weed biotypes offer excellent model species for uncovering the mode of action as well as resistance to these compounds. Compared with other herbicide families, the incidence of resistance to auxinic herbicides is relatively low, with only 29 auxinic herbicide-resistant weed species discovered to date. The relatively low incidence of resistance to auxinic herbicides has been attributed to the presence of rare alleles imparting resistance in natural weed populations, the potential for fitness penalties due to mutations conferring resistance in weeds, and the complex mode of action of auxinic herbicides in sensitive dicot plants. This review discusses recent advances in the auxin signal transduction pathway and its relation to auxinic herbicide mode of action. Furthermore, comprehensive information about the genetics and inheritance of auxinic herbicide resistance and case studies examining mechanisms of resistance in auxinic herbicide-resistant broadleaf weed biotypes are provided. Within the context of recent findings pertaining to auxin biology and mechanisms of resistance to auxinic herbicides, agronomic implications of the evolution of resistance to these herbicides are discussed in light of new auxinic herbicide-resistant crops that will be commercialized in the near future.

The type and concentration of phenolics found in the cytosol (free), cell wall (bound), and nutrient solution of hydroponic pepper (Capsicum annuum L.) inoculated with Pythium aphanidermatum (Edson) Fitzp. were compared with non-inoculated controls. The quantities and types of free phenolics found in the roots and nutrient solution were greater in inoculated than in control plants. With regard to bound phenolics, while there was no difference between the types of phenolics in inoculated and control roots, quantities were greater in inoculated roots. High Pressure Liquid Chromatography analyses revealed two compounds in root extracts, and one in nutrient solution extracts that were contributors to root discolouration caused by Pythium aphanidermatum colonization. The compounds were subsequently isolated and identified as 4-hydroxybenzoic acid and 3-methoxy-4-hydroxybenzoic acid (vanillic acid) by their High Pressure Liquid Chromatography retention times, UV signature and Nuclear Magnetic Resonance spectroscopic comparisons with pure standards. Addition of various concentrations of 4-hydroxybenzoic acid or vanillic acid to the nutrient solution predisposed healthy plants to infection by zoospores of Pythium aphanidermatum when compared to plants treated with only Pythium aphanidermatum. 4-hydroxybenzoic acid and vanillic acid had little or no effect on colony growth of Pythium aphanidermatum in vitro, but respectively increased and decreased numbers of sporangia produced. These results are the first demonstration of host predisposition to infection by Pythium aphanidermatum caused by phenolic compounds that accumulate in inoculated roots and leach into the nutrient system of hydroponic systems.

Due to its highly carcinogenic and mutagenic effect on humans, a maximum tolerable limit of 10 ng/L of benzo[a]pyrene (B[a]P) in drinking water was set by the European Commission (Council Directive 98/83/EC). Although several polyclonal and monoclonal antibodies (mAb) for the detection of B[a]P and other polycyclic aromatic hydrocarbons (PAH) have been developed by others, a traditional enzyme-linked immunosorbent assay (ELISA) with a limit of quantification of 10 ng/L for monitoring B[a]P has not been developed. With this in mind, several single-chain variable fragment (scFv) antibodies were created using existing mAbs against the extremely hydrophobic hapten B[a]P, and their heavy and light chains recombined to make unique variable light (V(L)) and heavy (V(H)) chain combinations. Their binding behaviour was investigated using microtiter plate ELISA and surface plasmon resonance techniques. Specifically, the coding sequences for V(L) and V(H) chains of 10 murine anti-B[a]P antibody producing hybridoma cell lines were isolated by degenerate oligonucleotide primer sets, cloned in phagemid pIT2 and transferred into Escherichia coli HB2151. To systematically investigate the interaction of the V(L) and V(H) domains, three high-affinity B[a]P-specific and one nonspecific clone were selected and recombined to build a set of 16 different V(L) and V(H) combinations. On the basis of our data, it was shown that the V(H) plays the major role for specific binding of B[a]P, whilst the V(L) can, in some cases, increase the final sensitivity of the assay by one order of magnitude. Furthermore, the sequence analysis of scFvs indicates that the complementarity determining region H3 plays a major role in affinity, whilst cross-reactivity to seven other PAHs demonstrates the importance of the V(L) in providing cross-reactivity.

A narrative approach to group work with men who batter is presented with case examples. The approach is based in feminist and constructionist theory and seeks to expose and undermine the effects of patriarchal discourse in the lives and relationships of group members. The approach emphasizes an understanding of gender discourse, taking responsibility for actions, and exploring new definitional possibilities for participants and their relationships. The techniques of radical listening, the principle of least contest, breaking the allure of male entitlement and its effects, and cocreating a new male way of being are discussed with case examples.

Saflufenacil is a PRE herbicide for the control of broadleaf weeds. Field and growth room studies were conducted to explore the tolerance of corn to POST treatments of saflufenacil and BAS 781. Additionally, the potential use of sodium as a safener for saflufenacil was evaluated. Crop injury caused by saflufenacil or BAS 781 was 8 and 38%, respectively, when applied at twice the recommended dose at the spike to two-leaf stage of crop growth. This injury increased to 28 and 65%, respectively, when applied at the three- to four-leaf stage. This level of crop injury resulted in yield loss, particularly when applied at the three- to four-leaf stage. The addition of Na-bentazon to saflufenacil reduced this injury and increased crop dry weight under both field and laboratory conditions. In the field, Na-bentazon also increased corn collar height and yield compared with saflufenacil applied alone. Na-bentazon reduced injury through a reduction in foliar uptake of saflufenacil. Sodium derived from baking soda also provided a safening effect, but only at the lowest dose of saflufenacil tested.

To study the agricultural production of biosimilar antibodies, trastuzumab (Herceptin) was expressed in Nicotiana benthamiana using the magnICON viral-based transient expression system. Immunoblot analyses of crude plant extracts revealed that trastuzumab accumulates within plants mostly in the fully assembled tetrameric form. Purification of trastuzumab from N. benthamiana was achieved using a scheme that combined ammonium sulfate precipitation with affinity chromatography. Following purification, the specificity of the plant-produced trastuzumab for the HER2 receptor was compared with Herceptin and confirmed by western immunoblot. Functional assays revealed that plant-produced trastuzumab and Herceptin have similar in vitro antiproliferative effects on breast cancer cells that overexpress HER2. Results confirm that plants may be developed as an alternative to traditional antibody expression systems for the production of therapeutic mAbs.

Concerns over the occurrence of the veterinary antibiotic monensin (MW 671Da) in animal food products and water have given rise to the need for a sensitive and rapid detection method. In this study, four monensin-specific single chain variable fragments (scFvs) were isolated from a hyperimmunized phage-displayed library originating from splenocytes of a mouse immunized with monensin conjugated to bovine serum albumin (BSA). The coding sequences of the scFvs were engineered in the order 5'-V(L)-linker-V(H)-3', where the linker encodes for Gly(10)Ser(7)Arg. Three rounds of selection were performed against monensin conjugated to chicken ovalbumin (OVA) and keyhole limpet hemocyanin (KLH), alternately. In the third round of selection, two different strategies, which differed in the number of washes and the concentration of the coating conjugates, were used to select for specific binders to monensin. A total of 376 clones from round two and three were screened for their specific binding to monensin conjugates and positive clones were sequenced. It was found that 80% of clones from round three contained a stop codon. After removing the stop codon by site-directed mutagenesis, ten binders with different amino acid sequences were subcloned into the vector pMED2 for soluble expression in Escherichia coli HB2151. Four of these scFvs bound to free monensin as determined using competitive fluorescence polarization assays (C-FPs). IC(50) values ranged from 0.031 and 231 microM. A cross-reactivity assay against salinomycin, lasalocid A, kanamycin and ampicillin revealed that the two best binders were highly specific to monensin.

Buckwheat (Fagropyrum esculentum Moench.), cabbage (Brassica oleracea L), and conventional and glyphosate-resistant varieties of canola (Brassica napus L.) were used to study the bases of saflufenacil and glyphosate interactions. Compared to the addition of Merge (surfactant), the addition of both Transorb (i.e., commercial product, Transorb formulation with glyphosate) and Merge increased the cuticular absorption of [(14)C] saflufenacil in cabbage plants with thick epicuticular wax layers. However, in all cases, the addition of glyphosate reduced the translocation of [(14)C]saflufenacil in glyphosate-susceptible plants, while translocation was not affected in glyphosate-resistant canola. Moreover, the phytotoxicity of saflufenacil reduced the activity of glyphosate, possibly by reducing its translocation in all plant species studied. Increased absorption of saflufenacil by the addition of Transorb (i.e., Transorb formulation with glyphosate) plus Merge appears to increase its contact activity, thus the interaction of saflufenacil and glyphosate involves two separate processes, absorption and translocation.

In addition to Ag recognition, some Abs are capable of killing target organisms in the absence of phagocytes and complement. In this study, we report that an anti-Pseudomonas aeruginosa O6ad LPS IgG1, tobacco-expressed human S20 IgG1 (te-hS20), as well as its recombinant Fab and single-chain variable fragment (scFv) fragments have cellular- and complement-independent bactericidal activity. te-hS20 and its Fab and scFv significantly reduced viability of P. aeruginosa O6ad in dose- and time-dependent manners in vitro and also showed lower levels of bactericidal activity against P. aeruginosa PAO1, but had no activity against P. aeruginosa O10, Escherichia coli TG1, and Streptococcus agalactiae. The H chain and its Fd fragment both had significant Ag-binding and bactericidal activities against P. aeruginosa O6ad. Bactericidal activity was completely inhibited with specific LPS Ag, suggesting that Ag binding is involved in the bactericidal mechanism. Live/dead cell staining and electron microscopic observations indicate that the bactericidal effect was due to disruption of the cell wall and suggest inhibition of cell division. In addition to te-hS20, the Fab and scFv were also protective in vivo, as leukopenic mice had prolonged and improved survival after administration of these Ab fragments followed by challenge with P. aeruginosa O6ad cells at 80–90% lethal dose, supporting a bactericidal mechanism independent of phagocytes and complement. Understanding of the bactericidal mechanism will allow assessment of the potential for therapeutic application of these Abs.

Plants possess enormous potential as factories for the large scale production of therapeutic reagents such as recombinant proteins and antibodies. A major factor limiting commercial advances of plant-derived pharmaceuticals is the cost and inefficiency of purification. As a model system, we have developed a simple yet robust method for immobilizing affinity capture ligands onto solid supports by interfacing the secreted expression and coupling of a chimeric fusion protein in Pichia pastoris to microcrystalline cellulose in a single step. The fusion protein, which consisted of antibody-binding proteins L and G fused to a cellulose-binding domain (LG-CBD), was tethered directly onto cellulose resins added to P. pastoris cultures and subsequently used for antibody purification. Both the antibody-binding protein L and protein G domains were functional, as demonstrated by the ability of cellulose-immobilized LG-CBD to purify both a scFv antibody fragment from yeast and a human IgG1 monoclonal antibody from transgenic tobacco. Furthermore, combining two P. pastoris strains expressing LG-CBD and scFv with CP-102 cellulose in a single culture allowed for easy recovery of biologically active scFv. Direct immobilization of affinity purification ligands, such as LG-CBD, onto inexpensive support matrices such as cellulose is an effective method for the generation of functional, single-use antibody purification reagents. Straightforward preparation of purification reagents will help make antibody purification from genetically modified crop plants feasible and address one of the major bottlenecks facing commercialization of plant-derived pharmaceuticals.

Weed science has contributed much to agriculture, forestry and natural resource management during its history. However, if it is to remain relevant as a scientific discipline, it is long past time for weed scientists to move beyond a dominating focus on herbicide efficacy testing and address the basic science underlying complex issues in vegetation management at many levels of biological organization currently being solved by others, such as invasion ecologists and molecular biologists. Weed science must not be circumscribed by a narrowly-defined set of tools but rather be seen as an integrating discipline. As a means of assessing current and future research interests and funding trends among weed scientists, the Weed Science Society of America conducted an online survey of its members in summer of 2007. There were 304 respondents out of a membership of 1330 at the time of the survey, a response rate of 23%. The largest group of respondents (41%) reported working on research problems primarily focused on herbicide efficacy and maintenance, funded mainly by private industry sources. Another smaller group of respondents (22%) reported focusing on research topics with a complex systems focus (such as invasion biology, ecosystem restoration, ecological weed management, and the genetics, molecular biology, and physiology of weedy traits), funded primarily by public sources. Increased cooperation between these complementary groups of scientists will be an essential step in making weed science increasingly relevant to the complex vegetation management issues of the 21st century.

Antibody engineering has allowed for the rapid generation of binding agents against virtually any antigen of interest, predominantly for therapeutic applications. Considerably less attention has been given to the development of diagnostic reagents and biosensors using engineered antibodies. Recently, we produced a novel pentavalent bispecific antibody (i.e., decabody) by pentamerizing two single-domain antibodies (sdAbs) through the verotoxin B subunit (VTB) and found both fusion partners to be functional. Using a similar approach, we have engineered a bispecific pentameric fusion protein consisting of five sdAbs and five cellulose-binding modules (CBMs) linked via VTB. To find an optimal design format, we constructed six bispecific pentamers consisting of three different CBMs, fused to the Staphylococcus aureus-specific human sdAb HVHP428, in both orientations. One bispecific pentamer, containing an N-terminal CBM9 and C-terminal HVHP428, was soluble, non-aggregating, and did not degrade upon storage at 4 ºC for over six months. This molecule was dually functional as it bound to cellulose-based filters as well as S. aureus cells. When impregnated in cellulose filters, the bispecific pentamer recognized S. aureus cells in a flow-through detection assay. The ability of pentamerized CBMs to bind cellulose may form the basis of an immobilization platform for multivalent display of high-avidity binding reagents on cellulosic filters for sensing of pathogens, biomarkers and environmental pollutants.

Transgenic plants offer a promising system for large-scale production of therapeutic proteins such as monoclonal antibodies (mAbs). This paper describes a membrane-based process suitable for purification of a humanized mAb expressed in tobacco. Most monoclonal antibody purification schemes rely on the use of Protein A as the affinity ligand for antibody capture. The main objective of our work was to develop non-Protein A-based purification methods to avoid some of the problems and limitations associated with this ligand, e.g. cost, immunotoxicity, and antibody aggregation during elution. Ion exchange membrane chromatography (IEMC) was used for primary capture and preliminary purification of the mAb from tobacco juice. Hydrophobic interaction membrane chromatography (HIMC) was then used for high-resolution purification, followed by ultrafiltration for polishing, desalting and buffer exchange. Using this scheme, both high mAb purity (single peak in size exclusion chromatogram, i.e., ca. 100% purity) and high recovery (77% of mAb spiked into the tobacco extract) were achieved. Membrane chromatography is generally considered unsuitable for resolving bound proteins by gradient elution and is therefore commonly used in the bind and elute mode with a single-step change of mobile phase. We show that the gradient elution process in the HIMC step can be optimized to increase the resolution and thereby obtain product of high purity.

A discussion of the application of evidence-based practice (EBP) in clinical settings is offered from a practitioner's point of view. Questions are raised regarding how the EBP effectiveness client-oriented practical evidence search (COPES) questions and literature review protocol (Gibbs, 2003) guide the operationalization of problems and clients, and suggestions are offered about additional variables to include in this standardized protocol that may enhance the specificity of EBP. An exploration of the EBP collaborative decision-making process is presented from a practice perspective, and a conclusion is drawn about the use of the EBP process in clinical settings.

Growth room studies were conducted to determine the physiological basis of reduced glyphosate efficacy under low soil nitrogen using velvetleaf, common ragweed, and common lambsquarters as model species. Glyphosate dose–response experiments of weeds grown under low (1.5 mM) and high (15 mM) soil N were conducted. Velvetleaf and common lambsquarters grown under low N required 169 g ae ha−1 glyphosate for a significant reduction in biomass, but only 84 g ae ha−1 were required when grown under high N. However, when common ragweed was grown under low or high soil N there was no significant difference in response to glyphosate at all doses tested. The reduced glyphosate efficacy on velvetleaf and common lambsquarters under low N was primarily due to decreased herbicide translocation to the meristem. It appears that low N may decrease the net assimilation of carbon in plants, resulting in a decrease in the net export of sugars and hence glyphosate from mature leaves. Understanding the relationship betw...

A discussion of the ethical requirements of male social workers to actively support women in the struggle for gender equality is presented. Specifically, two questions are addressed: (1) Are males in the social work profession, who take no active position on sexism and equality, practicing unethically according to the National Association of Social Worker (NASW) Code of Ethics (1999) by supporting the patriarchal structure via inaction, while accepting the privileges it affords, including benefiting financially and professionally through the analysis and treatment of its effects? And, if so, (2) do male social work practitioners have a moral and ethical duty to actively support social change which would enhance equality between women and men for the benefit of their clients, co-workers, and selves? The cultural perception of social need is discussed as being inseparable from the perception of moral obligation and the two concepts are explored through the lenses of four paradigms: (a) structural functional...

The genetic basis for auxinic herbicide resistance of a wild mustard biotype is unknown, although the physiological basis of its resistance is well characterized. Since the auxinic herbicide-resistant (R) biotype and normally susceptible (S) wild mustard plants are genetically diverse, development of completely homozygous R and S lines is a prerequisite for isolation and subsequent characterization of the resistance gene. Completely homozygous lines can be produced using either in vitro microspore culture or by conventional breeding methods (i.e., several generations of selfing); the former being a less time consuming method. With this in mind, microspore culture was used to produce homozygous wild mustard plants. However, despite altering many variables, only a few cotyledonary stage embryos and haploid plantlets could be produced. Upon transfer to soil only one doubled haploid plant established. This plant was self-sterile, cross compatible and resistant to auxinic herbicides. This is the first report describing production of a microspore-derived doubled haploid of wild mustard. However, because only one R doubled haploid plant could be produced, conventional breeding methods (i.e., repeated selfing) will be required for production of both R and S wild mustard plants that are completely homozygous.

White bean (Phaseolus vulgaris L.) was used to study the antagonism caused by Na-bentazon on the phytotoxic action of the sulfonylurea (SU) herbicide tritosulfuron. After 168 h, uptake and translocation of [14C]tritosulfuron were reduced by 60 and 89%, respectively, when Na-bentazon was added to the mixture. Addition of (NH4)2SO4 or replacement of Na-bentazon with NH4-bentazon completely eliminated the negative effects on [14C]tritosulfuron uptake but not on its translocation. Scanning electron microscopy revealed that a mixture of Na-bentazon plus tritosulfuron plus DASH HC (0.156%) formed a rough layer of grain-like crystals on the leaf surface, whereas the addition of (NH4)2SO4 or replacement of Na-bentazon with NH4-bentazon resulted in amorphous deposits that may be more easily absorbed. The antagonism of tritosulfuron's phytotoxicity by Na-bentazon involves two separate processes, chemical (uptake effect) and biochemical (translocation effect). Keywords: Bentazon; tritosulfuron; white bean (Phaseolus...

The production of recombinant vaccines in plants may help to reduce the burden of veterinary diseases, which cause major economic losses and in some cases can affect human health. While there is abundant research in this area, a knowledge gap exists between the ability to create and evaluate plant-based products in the laboratory, and the ability to take these products on a path to commercialization. The current report, arising from a workshop sponsored by an Organisation for Economic Co-operation and Development (OECD) Co-operative Research Programme, addresses this gap by providing guidance in planning for the commercialization of plant-made vaccines for animal use. It includes relevant information on developing business plans, assessing market opportunities, manufacturing scale-up, financing, protecting and using intellectual property, and regulatory approval with a focus on Canadian regulations.

The physiological basis for MCPA resistance in a hemp-nettle (Galeopsis tetrahit L.) biotype, obtained from a MCPA-resistant field population, was investigated. Dose-response studies revealed that the resistance factor for MCPA, based on GR50 comparisons of total dry weight of resistant (R) and susceptible (S) plants, was 3.3. Resistance factors for fluroxypyr, dicamba, 2,4-D, glyphosate, and chlorsulfuron were 8.2, 1.7, 1.6, 0.7, and 0.6, respectively. MCPA resistance was not due to differences in absorption, because both R and S biotypes absorbed 54% of applied [14C]MCPA 72 h after treatment. However, R plants exported less (45 vs 58% S) recovered 14C out of treated leaves to the apical meristem (6 vs 13% S) and root (32 vs 38% S). In both biotypes, approximately 20% of the 14C recovered in planta was detected as MCPA metabolites. However, less of the 14C recovered in the roots of R plants was MCPA. Therefore, two different mechanisms protect R hemp-nettle from MCPA phytotoxicity: a lower rate of MCPA translocation and a higher rate of MCPA metabolism in the roots. In support of these results, genetic studies indicated that the inheritance of MCPA resistance is governed by at least two nuclear genes with additive effects.