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Membrane localization domain (MLD) was first proposed for a 4-helix-bundle motif in the crystal structure of the C1 domain of Pasteurella multocida toxin (PMT). This structure motif is also found in the crystal structures of several clostridial glycosylating toxins (TcdA, TcdB, TcsL, and TcnA). The Ras/Rap1-specific endopeptidase (RRSP) module of the multifunctional autoprocessing repeats-in-toxins (MARTX) toxin produced by Vibrio vulnificus has sequence homology to the C1-C2 domains of PMT, including a putative MLD. We have determined the solution structure for the MLDs in PMT and in RRSP using solution state NMR. We conclude that the MLDs in these two toxins assume a 4-helix-bundle structure in solution.

In all mammals, growth, development, pregnancy, and lactation increase nutritional demands. Although primate field studies tend to focus on shifts in activity and diet as mechanisms to compensate for these demands, differences in digestive efficiency also are likely to be important. Because the gut microbiota can impact host digestive efficiency, we examined differences in activity budget, diet, and the gut microbial community among adult male (N = 4), adult female (N = 4), and juvenile (N = 5) wild black howler monkeys (Alouatta pigra) across a ten-month period in Palenque National Park, Mexico to determine how adult females and juveniles compensate for increased nutritional demands. Results indicate that adult females and juveniles consumed more protein and energy than adult males. Adult males, adult females, and juveniles also possessed distinct gut microbial communities, unrelated to diet. Juveniles exhibited a gut microbiota characterized by bacteria from the phylum Firmicutes, such as Roseburia and Ruminococcus, and demonstrated high fecal volatile fatty acid content, suggesting increased microbial contributions to host energy balances. Adult females possessed a higher Firmicutes to Bacteroidetes ratio, also suggesting increased energy production, and their gut microbiota was characterized by Lactococcus, which has been associated with folate biosynthesis. On the basis of these patterns, it appears that the gut microbiota differentially contributes to howler monkey nutrition during reproduction and growth. Determining the nutritional and energetic importance of shifts in activity, diet, and the gut microbiota in other nonhuman primate taxa, as well as humans, will transform our understanding of these life history processes and the role of host-microbe relationships in primate evolution. Am J Phys Anthropol 155:652–664, 2014. © 2014 Wiley Periodicals, Inc.

The primate body hosts trillions of microbes. Interactions between primate hosts and these microbes profoundly affect primate physiology, reproduction, health, survival, and ultimately, evolution. It is increasingly clear that primate health cannot be understood fully without knowledge of host-microbial interactions. Our goals here are to review what is known about microbiomes of the female reproductive tract and to explore several factors that influence variation within individuals, as well as within and between primate species. Much of our knowledge of microbial variation derives from studies of humans, and from microbes located in nonreproductive regions (e.g., the gut). We review work suggesting that the vaginal microbiota affects female health, fecundity, and pregnancy outcomes, demonstrating the selective potential for these agents. We explore the factors that correlate with microbial variation within species. Initial colonization by microbes depends on the manner of birth; most microbial variation is structured by estrogen levels that change with age (i.e., at puberty and menopause) and through the menstrual cycle. Microbial communities vary by location within the vagina and can depend on the sampling methods used (e.g., swab, lavage, or pap smear). Interindividual differences also exist, and while this variation is not completely understood, evidence points more to differences in estrogen levels, rather than differences in external physical environment. When comparing across species, reproductive-age humans show distinct microbial communities, generally dominated by Lactobacillus, unlike other primates. We develop evolutionary hypotheses to explain the marked differences in microbial communities. While much remains to be done to test these hypotheses, we argue that the ample variation in primate mating and reproductive behavior offers excellent opportunities to evaluate host-microbe coevolution and adaptation.

Pasteurella multocida is the causative agent of a number of epizootic and zoonotic diseases. Its major virulence factor associated with atrophic rhinitis in animals and dermonecrosis in bite wounds is P. multocida toxin (PMT). PMT stimulates signal transduction pathways downstream of heterotrimeric G proteins, leading to effects such as mitogenicity, blockade of apoptosis, or inhibition of osteoblast differentiation. On the basis of Gαi2, it was demonstrated that the toxin deamidates an essential glutamine residue of the Gαi2 subunit, leading to constitutive activation of the G protein. Here, we studied the specificity of PMT for its G-protein targets by mass spectrometric analyses and by utilizing a monoclonal antibody, which recognizes specifically G proteins deamidated by PMT. The studies revealed deamidation of 3 of 4 families of heterotrimeric G proteins (Gαq/11, Gαi1,2,3, and Gα12/13 of mouse or human origin) by PMT but not by a catalytic inactive toxin mutant. With the use of G-protein fragments and chimeras of responsive or unresponsive G proteins, the structural basis for the discrimination of heterotrimeric G proteins was studied. Our results elucidate substrate specificity of PMT on the molecular level and provide evidence for the underlying structural reasons of substrate discrimination.—Orth, J. H. C., Fester, I., Siegert, P., Weise, M., Lanner, U., Kamitani, S., Tachibana, T, Wilson, B. A., Schlosser, A., Horiguchi, Y., Aktories, K. Substrate specificity of Pasteurella multocida toxin for α subunits of heterotrimeric G proteins.

The light chain of botulinum neurotoxin A (BoNT/A-LC) is a Zn-dependent protease that specifically cleaves SNAP25 of the SNARE complex, thereby impairing vesicle fusion and neurotransmitter release at neuromuscular junctions. The C-terminus of SNAP25 (residues 141–206) retains full activity for BoNT/A-LC-catalyzed cleavage at P1-P1’ (Gln197-Arg198). Using the structure of a complex between the C-terminus of SNAP25 and BoNT/A-LC as a model to design SNAP25-derived pseudosubstrate inhibitors (SNAPIs) that prevent presentation of the scissile bond to the active site, we introduced multiple His residues to replace Ala-Asn-Gln-Arg (residues 195–198) at the substrate cleavage site, with the intent to identify possible side-chain interactions with the active site Zn. We also introduced multiple Gly residues between the P1-P1’ residues to explore the spatial tolerance within the active-site cleft. Using a FRET substrate YsCsY, we compared a series of SNAPIs for inhibition of BoNT/A-LC. Among the SNAPIs tested, several known cleavage-resistant, single-point mutants of SNAP25 were poor inhibitors, with most of the mutants losing binding affinity. Replacement with His at the active site did not improve inhibition over wildtype substrate. In contrast, Gly-insertion mutants were not only resistant to cleavage, but also surprisingly showed enhanced affinity for BoNT/A-LC. Two of the Gly-insertion mutants exhibited 10-fold lower IC50 values than the wildtype 66-mer SNAP25 peptide. Our findings illustrate a scenario, where the induced fit between enzyme and bound pseudosubstrate fails to produce the strain and distortion required for catalysis to proceed.

Pasteurella multocida toxin (PMT) is an AB toxin that causes pleiotropic effects in targeted host cells. The N-terminus of PMT (PMT-N) is considered to harbor the membrane receptor binding and translocation domains responsible for mediating cellular entry and delivery of the C-terminal catalytic domain into the host cytosol. Previous studies have implicated gangliosides as the host receptors for PMT binding. To gain further insight into the binding interactions involved in PMT binding to cell membranes, we explored the role of various membrane components in PMT binding, utilizing four different approaches: (a) TLC-overlay binding experiments with (125) I-labeled PMT, PMT-N or the C-terminus of PMT; (b) pull-down experiments using reconstituted membrane liposomes with full-length PMT; (c) surface plasmon resonance analysis of PMT-N binding to reconstituted membrane liposomes; (d) and surface plasmon resonance analysis of PMT-N binding to HEK-293T cell membranes without or with sphingomyelinase, phospholipase D or trypsin treatment. The results obtained revealed that, in our experimental system, full-length PMT and PMT-N did not bind to gangliosides, including monoasialogangliosides GM(1) , GM(2) or GM(3) , but instead bound to membrane phospholipids, primarily the abundant sphingophospholipid sphingomyelin or phosphatidylcholine with other lipid components. Collectively, these studies demonstrate the importance of sphingomyelin for PMT binding to membranes and suggest the involvement of a protein co-receptor.

The mitogenic toxin from Pasteurella multocida (PMT) is a member of the dermonecrotic toxin family, which includes toxins from Bordetella, Escherichia coli and Yersinia. Members of the dermonecrotic toxin family modulate G-protein targets in host cells through selective deamidation and/or transglutamination of a critical active site Gln residue in the G-protein target, which results in the activation of intrinsic GTPase activity. Structural and biochemical data point to the uniqueness of PMT among these toxins in its structure and action. Whereas the other dermonecrotic toxins act on small Rho GTPases, PMT acts on the α subunits of heterotrimeric G(q) -, G(i) - and G(12/13) -protein families. To date, experimental evidence supports a model in which PMT potently stimulates various mitogenic and survival pathways through the activation of G(q) and G(12/13) signaling, ultimately leading to cellular proliferation, whilst strongly inhibiting pathways involved in cellular differentiation through the activation of G(i) signaling. The resulting cellular outcomes account for the global physiological effects observed during infection with toxinogenic P. multocida, and hint at potential long-term sequelae that may result from PMT exposure.

In many countries, children with cerebral palsy (CP) receive physical and occupational therapy services to facilitate development and to enhance performance in functional movement, self-care, play, school activities, and leisure. In the past, therapy interventions for children with CP have primarily focused on changing factors within the child at the domain of body function and structure, with the assumption that these changes will improve their abilities in the domains of activity and participation. Therapy approaches are increasing their focus on changing a child's abilities to complete activities. Emerging conceptual frameworks such as a dynamic systems approach to motor development and family-centered services have facilitated the development of alternative treatment approaches that focus on the child and family within their environment.1–7 Dynamic systems theory suggests that the most efficient motor behavior results from the spontaneous self-organization and interaction of many subsystems to achieve a functional goal.3,8 The transactional relationships among the child, the task, and the environment result in efficient solutions for functional motor goals and tasks. Different environments may result in different solutions, and in the same environment, different children may demonstrate different solutions. The concept of self-organization suggests that `pattern and order can emerge from the process of the interaction of the components of a complex subsystem without the need for explicit instructions' (Thelen,9 p79). This perspective of movement makes it more challenging to predict `cause and effect' movement solutions because of the influence of many factors. Dynamic systems theory challenges traditional treatment perspectives, suggesting that typical patterns of movement are not always the optimal solution to a motor goal. Emerging treatment models influenced by dynamic systems theory consider task or activity completion as the goal, with less emphasis on remediation or `normalization' of movement components. Different terms have been used to describe the focus on functional performance: ecological task analysis,10,11 functional therapy,5 goal-directed functional therapy,12 activity focused and goal directed,13,14 activity focused,15 and task-oriented.7 Two before–after studies of young children with CP found significant improvements in function using an activity-focused or functional therapy approach.12,14 In a randomized trial, children receiving task-oriented strength training and practice of functional tasks improved significantly more in gross motor activities than those receiving therapy emphasizing facilitation and normalization of movement patterns.7 Ketelaar et al.5 conducted a randomized trial in which therapists focused on changing the motor skills the child needed to perform a task within their natural environment, based on child- or parent-identified goals. This functional therapy approach was more effective in achieving self-care and motor outcomes than therapy focused on quality of movement. The development and implementation of treatment approaches using a family-centered approach has emphasized that treatment should focus on child and family-identified goals. Wiart et al.16 reported that parents valued involvement in setting therapy goals but often found that subsequent intervention focused less on those functional goals and more on movement quality. Parents believe that setting functional goals facilitates practice of skills and activities in the home.17 Two recent studies found that goal setting leads to significant improvements in achievement of functional movement and activities.14,18 In a recent study, Lowing et al.13 found significant improvements in self-care, mobility, and motor capacity after goal-directed, activity-focused therapy compared with activity-focused therapy where the aims of therapy were more general. An emerging treatment method based on dynamic systems theory and family-centered service focuses on improving a child's motor-based functional activities primarily by changing identified constraints in the task or environment.6 This context-focused approach contrasts with interventions that have focused on facilitating changes within the child's movement abilities or skills. The goal is to change the task or environment to promote functional performance, to allow the use of a child's own movement strategies, and to encourage practice of tasks within the natural environment.6 Pilot studies of the context-focused approach indicated that it was feasible and has potential to facilitate change in motor performance in young children with CP.6,19 What this paper adds This study is the first randomized trial for young children with CP to examine the effects of therapy focused on changing only task or environment, and not the child. Children who received the context-focused therapy made similar improvements to those receiving child-focused therapy. Frequency of therapy may be critical to the success of any intervention. In summary, emerging ideas from dynamic systems theory and family-centered service have stimulated debate about the potential use of a context-focused approach for young children with CP, but evaluation of related intervention protocols is extremely limited. The objective of this multi-site, randomized clinical trial was to evaluate the efficacy of a context-focused approach compared with a child-focused approach in improving performance of functional tasks and mobility, and increasing participation in everyday activities in young children who have CP.

Both the number and rigor of efficacy studies to evaluate intervention techniques used with children with cerebral palsy (CP) have improved in the past decade.1 Whyte and Hart2 identified three important methodological issues that researchers need to consider when designing effective intervention studies: (1) defining the population, (2) choosing appropriate outcome measures, and (3) describing the intervention in enough detail that other researchers and clinicians can replicate it. Researchers are increasingly aware of the importance of characterizing children in terms of their functional abilities and of considering the effect of the intervention in children of differing ability levels.3 The psychometric rigor and evidence of responsiveness of pediatric outcome measures have both improved in the past decade.4 However, descriptions of specific interventions remain the least developed aspect of rehabilitation intervention studies,2,5,6 and systematic reviews of specific interventions report this as a weakness of studies.7–9 Detailed treatment protocols have been used and discussed in the literature on behavioral psychotherapy for over 25 years.10 In the field of psychotherapy, these manual-based therapies or ‘manualized therapies’ require a clinician to match clients with a specific diagnosis to a treatment package that has been developed for that disorder.11 Characteristics of effective treatment protocols or manualized therapies include a theoretical justification for the treatment design, detailed descriptions of the intended patients, the intervention intensity, duration and delivery, and a description of the training and monitoring of persons providing the intervention.2,6 Advocates of manualized therapies claim that they are efficient and reduce the ‘flawed idiosyncratic decision-making of individual therapists’ (Eifert et al.,11 (p500)). For research purposes, a standardized description of an intervention increases the internal validity of outcome studies, allowing systematic evaluation of treatments across settings and sites. Critics of a manualized therapy approach, however, caution that it could encourage a ‘cookbook’ intervention and suggest that adequate flexibility must be included in the instructions to accommodate individual situations.12 Manualized therapy approaches are appearing in pediatric rehabilitation for children with autism13,14 but they are not common practice for the management of children with CP.15 Manualized therapies ensure the specificity of intervention approaches for children with motor disabilities, both in research studies and in the delivery of intervention approaches in clinical practice. This paper describes the ‘context therapy’ intervention approach developed for the clinical trial described in the accompanying paper by Law et al. The theoretical rationale for the context approach, the model of service delivery, and the training procedures are described. Examples of specific intervention solutions are provided and some identified benefits and challenges of introducing this intervention are discussed. Broad concepts of the context therapy approach have been described previously in a published treatment protocol,16 but this paper provides detailed information about the training procedures and intervention guidelines. It identifies the ‘active’ ingredients2,6 of the context therapy approach, allowing clinicians and researchers to replicate it.

Biosecurity is emerging as a major global health priority for which innovative and unprecedented solutions are needed. Biosecurity is a challenging biocomplexity problem involving multifaceted processes such as interactions between humans and nonhuman biota, anthropogenic environmental and ecological factors, and socioeconomic and political pressures. Key to an effective biosecurity strategy will be fundamental understanding of evolutionary, anthropogenic and environmental driving forces at play in transmission and perpetuation of infectious diseases. Biosecurity solutions will depend on increased support of basic biomedical research and public education, enhanced healthcare preparedness, alternative strategies for ensuringsafety, and improved interagency cooperation regarding global health policy. © 2008 Wiley Periodicals, Inc. Complexity, 2008.

The large 1285-amino-acid protein toxin from Pasteurella multocida (PMT) is a multifunctional single-chain polypeptide that binds to and enters eukaryotic cells and acts intracellularly to promote G(q) and G(12/13) protein-dependent calcium and mitogenic signal transduction. Previous studies indicated that the intracellular activity domain responsible for PMT action was located within the C-terminal 600-700 amino acids. In this study, we have exogenously expressed a series of N- and C-terminal PMT fragments directly in mammalian cells and have used the dual luciferase reporter system to assay for toxin-mediated activation of calcium-calcineurin-NFAT signaling (NFAT-luciferase) and mitogenic serum response signaling (SRE-luciferase). Using this approach, we have defined the last 180 amino acids, which encompass the C3 domain in the crystal structure, as the minimum domain sufficient to activate both NFAT and SRE signaling pathways.

Reliability and validity of the Developmental Coordination Disorder Questionnaire (DCD-Q) was assessed using a population-based sample of 608 children (311 males, 297 females; mean age 7 y 8 mo [SD 2 y 4 mo]), a sample of 55 children with DCD referred to a rehabilitation clinic, and a control sample of 55 children matched for age and sex (48 males, seven females in each sample; mean age 8 y 3 mo [SD 2 y]). The DCD-Q is reliable and valid in the age range for which the questionnaire was developed(8 y-14 y 7 mo) and in a younger age range (4-8 y). Sensitivity and specificity of the DCD-Q was assessed using the Movement Assessment Battery for Children as the criterion standard. The DCD-Q met the standard for sensitivity (80%) in the clinic-referred sample (81.6%), but not in the population-based sample (28.9%). Specificity almost reached the standard of 90%: 89% in the population-based sample and 84% in the clinic-referred sample.

Raf-1 is a key protein kinase in the mitogen-activated protein kinase cascade. We have subcloned the catalytic domain of Raf-1 into the bacterial expression vectors, pTrcHisB and pGEX-6P-1, denoted as His6-ΔNRaf and GST-RafBXB, respectively. Chromatography of the recombinant proteins using Ni-NTA agarose, Sephacryl S-300, and glutathione-sepharose revealed association of Raf-1 catalytic domain in a high molecular weight complex with a 57 kDa protein. Microsequencing of this 57 kDa protein identified it as GroEL, a heat shock protein in E. coli important for protein folding. GroEL association with the Raf-1 catalytic domain is specific, as evidenced by its association with both Raf-1 constructs. Native-PAGE gels and Western analysis of gel filtration fractions revealed association of the catalytic domain with a large molecular weight complex consistent with the tetradecameric complex of GroEL. A peptide library of 384 do-decapeptides corresponding to the entire catalytic domain of Raf-1 was constructed by the spot synthesis method. Binding of GroEL and ELISA analysis revealed a preferential GroEL binding site in the β1 sheet region of the Raf-1 kinase domain.

The bacterial population of the primate vaginal canal is an infant primate's first exposure to the microbial population inhabiting the outside world. Yet, little is known about this population and the effect it might have on the development and survival of the infant primate. As a first step toward characterizing the vaginal microbiota of a nonhuman primate, we used denaturing gradient gel electrophoresis to evaluate variations in the vaginal microbiota of a group of 35 baboons (Papio hamadryas), which were housed in a facility where they shared the same diet and the same environmental conditions. We found that, despite the uniform environment, there were appreciable differences in the composition of the microbiota from one individual to another. Our results also indicate that a simple swab test is sufficient for sampling the vaginal microbiota in the field, a finding that should help make more detailed characterization of the microbiota of wild primates feasible in the future. Am. J. Primatol. 72:467–474, 2010. © 2010 Wiley-Liss, Inc.

Silver sulfide, silver thiosulfate complex and silver chloride complexes were tested and compared with free silver ion for their toxic effects in 96 h flow through acute toxicity tests using fathead minnows (Pimephales promelas). Thirty day fathead minnow embryo larval tests were conducted with silver sulfide and silver thiosulfate complex. Extensive chemical analyses were performed to quantify actual exposures. Compared with free silver ion, silver chloride complexes were about 300 times less toxic acutely silver sulfide was at least 15,000 times less toxic acutely and silver thiosulfate complex was more than 17,500 times less toxic acutely. The estimated maximum acceptable toxicant concentrations (MATCs) determined from the embryo larval tests with silver sulfide and silver thiosulfate complex were greater than 11 mg/L (as total silver) and greater than 16 but less than 35 mg/L (as total silver), respectively. The MATC previously reported for free silver ion from tests with rainbow trout (Salmo gairdneri) was greater than 0 00009 but less than 0 00017 mg/L. These differences in acute toxicities and the differences of five orders of magnitude in the MATC values are attributed to the influence of chemical speciation on the effects of silver in the aquatic environment. Thus, the speciation of silver is an essential factor in its potential to affect fish and, presumably, other aquatic life, and therefore should be considered in environmental risk assessment.