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"Clarke, Margaret"
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F-pili dynamics by live-cell imaging
Bacteria have evolved numerous mechanisms for cell-cell communication, many of which have important consequences for human health. Among these is conjugation, the direct transfer of DNA from one cell to another. For Gram-negative bacteria, conjugation requires thin, flexible filaments (conjugative pili) that are elaborated by DNA donor cells. The structure, function, and especially the dynamics of conjugative pili are poorly understood. Here, we have applied live-cell imaging to characterize the dynamics of F-pili (conjugative pili encoded by the F plasmid of Escherichia coli). We establish that F-pili normally undergo cycles of extension and retraction in the absence of any obvious triggering event, such as contact with a recipient cell. When made, such contacts are able to survive the shear forces felt by bacteria in liquid media. Our data emphasize the role of F-pilus flexibility both in efficiently sampling a large volume surrounding donor cells in liquid culture and in establishing and maintaining cell-cell contact. Additionally and unexpectedly, we infer that extension and retraction are accompanied by rotation about the long axis of the filament.
Journal Article
Retrieval of the Vacuolar H+-ATPase from Phagosomes Revealed by Live Cell Imaging
The vacuolar H+-ATPase, or V-ATPase, is a highly-conserved multi-subunit enzyme that transports protons across membranes at the expense of ATP. The resulting proton gradient serves many essential functions, among them energizing transport of small molecules such as neurotransmitters, and acidifying organelles such as endosomes. The enzyme is not present in the plasma membrane from which a phagosome is formed, but is rapidly delivered by fusion with endosomes that already bear the V-ATPase in their membranes. Similarly, the enzyme is thought to be retrieved from phagosome membranes prior to exocytosis of indigestible material, although that process has not been directly visualized.
To monitor trafficking of the V-ATPase in the phagocytic pathway of Dictyostelium discoideum, we fed the cells yeast, large particles that maintain their shape during trafficking. To track pH changes, we conjugated the yeast with fluorescein isothiocyanate. Cells were labeled with VatM-GFP, a fluorescently-tagged transmembrane subunit of the V-ATPase, in parallel with stage-specific endosomal markers or in combination with mRFP-tagged cytoskeletal proteins.
We find that the V-ATPase is commonly retrieved from the phagosome membrane by vesiculation shortly before exocytosis. However, if the cells are kept in confined spaces, a bulky phagosome may be exocytosed prematurely. In this event, a large V-ATPase-rich vacuole coated with actin typically separates from the acidic phagosome shortly before exocytosis. This vacuole is propelled by an actin tail and soon acquires the properties of an early endosome, revealing an unexpected mechanism for rapid recycling of the V-ATPase. Any V-ATPase that reaches the plasma membrane is also promptly retrieved.
Thus, live cell microscopy has revealed both a usual route and alternative means of recycling the V-ATPase in the endocytic pathway.
Journal Article
Curvature recognition and force generation in phagocytosis
Background
The uptake of particles by actin-powered invagination of the plasma membrane is common to protozoa and to phagocytes involved in the immune response of higher organisms. The question addressed here is how a phagocyte may use geometric cues to optimize force generation for the uptake of a particle. We survey mechanisms that enable a phagocyte to remodel actin organization in response to particles of complex shape.
Results
Using particles that consist of two lobes separated by a neck, we found that
Dictyostelium
cells transmit signals concerning the curvature of a surface to the actin system underlying the plasma membrane. Force applied to a concave region can divide a particle in two, allowing engulfment of the portion first encountered. The phagosome membrane that is bent around the concave region is marked by a protein containing an inverse Bin-Amphiphysin-Rvs (I-BAR) domain in combination with an Src homology (SH3) domain, similar to mammalian insulin receptor tyrosine kinase substrate p53. Regulatory proteins enable the phagocyte to switch activities within seconds in response to particle shape. Ras, an inducer of actin polymerization, is activated along the cup surface. Coronin, which limits the lifetime of actin structures, is reversibly recruited to the cup, reflecting a program of actin depolymerization. The various forms of myosin-I are candidate motor proteins for force generation in particle uptake, whereas myosin-II is engaged only in retracting a phagocytic cup after a switch to particle release. Thus, the constriction of a phagocytic cup differs from the contraction of a cleavage furrow in mitosis.
Conclusions
Phagocytes scan a particle surface for convex and concave regions. By modulating the spatiotemporal pattern of actin organization, they are capable of switching between different modes of interaction with a particle, either arresting at a concave region and applying force in an attempt to sever the particle there, or extending the cup along the particle surface to identify the very end of the object to be ingested. Our data illustrate the flexibility of regulatory mechanisms that are at the phagocyte's disposal in exploring an environment of irregular geometry.
Journal Article
Secondary transfer of adult mantled howlers (Alouatta palliata) on Hacienda La Pacifica, Costa Rica: 1975-2009
Natal emigration by male and female mantled howlers (Alouatta palliata), and subsequent immigration into breeding groups, is well documented for the free-ranging population on Hacienda La Pacifica, Costa Rica, but secondary transfer was considered rare (Glander in Int J Primatol 3:415-436, 1992). Population surveys in 1998 and 2006 caused us to question our assumptions and to re-evaluate our long-term data set from a post hoc perspective. We first identified all animals observed or captured as adults in more than one non-natal group anywhere in the population. We then systematically analyzed joining or leaving by adults in seven groups tracked for various times from 1975 to 2005 for patterns suggesting secondary transfer. Fourteen adults (nine females, five males) were found in two different non-natal groups as adults. In addition, one male and one female that became dominant and reproduced in their natal group later transferred to a second group, and one female was known to be a tertiary transfer. Data from the seven tracked social groups indicate that 35% of all the males and 29% of all the females were potential secondary transfers. In these groups, males leaving or joining was not associated with group size or absolute number of females. Females leaving or joining was not associated with group size or absolute number of males, but females left groups with more females and joined groups with fewer females. Both sexes left groups with unfavorable sex ratios for their sex and joined groups with sex ratios more favorable for their sex. Since a favorable sex ratio is associated with reproductive success in other howler populations, this suggests secondary transfer as a reproductive strategy. Other factors could also influence secondary transfer.
Journal Article
An Ecosystem Approach to Employment and Autism Spectrum Disorder
Relatively little is yet known about employment readiness and elements that promote access to, and the retention of, employment for adults with autism spectrum disorder (ASD). This paper posits elements within the ecosystem of employment and ASD. The ecosystem approach locates employment among persons with ASD as inextricably linked with broader community resources, family support, workplace capacity building (e.g., employer, co-workers) and policy. Application of the approach is offered through process evaluation data yielded from an ecosystem-informed job readiness program entitled, ‘EmploymentWorks Canada’. Findings illustrate job readiness in the context of the broader ecosystem that envelopes salient components in the aim of community engagement and quality of life. Recommendations are offered for community-based applications and for program and research development.
Journal Article
Geographic Deregulation of Banking and Economic Growth
State-level U.S. data for 1965 through 1994 are used to test for the effect of bank deregulation on economic growth. In particular, this study tests whether intrastate branch and interstate bank deregulation, measured by the activities of banks and bank holding companies, expedites growth in real per capita income through its effect on the size of banking markets. The empirical analysis supports the hypothesis that such deregulation enhances short-run economic growth.
Journal Article
The Well-Being and Resilience of Canadian Christian Clergy
Clergy play significant leadership, educational, and caregiving roles in society. However, burnout is a concern for the clergy profession, those they serve, and their families. Effects include decreased ministry effectiveness, lower sense of personal accomplishment in their role, and negative impacts on quality of family life and relationships. Given these risks, knowledge of the nature of Christian clergy’s current resilience and well-being in Canada may provide valuable intelligence to mitigate these challenges. In summary, the purpose of this research was to describe and analyze the status of clergy resilience and well-being in Canada, together with offering focused insights. Resilience and well-being surveys used by the co-authors with educators and nurses were adapted for use in this study. This instrument was developed to gain insight into baseline patterns of resilience and well-being and included questions across seven sections: (1) demographic information. (2) health status, (3) professional quality of life, (4) Cantril Well-Being Scale, (5) Ego-Resiliency Scale, (6) Grit Scale, and (7) open-ended questions. The findings provided valuable insights into clergy well-being and resilience that can benefit individual clerics, educational institutions, denominations, and congregations. The participants’ current resilience and well-being included high levels of resiliency, moderate grit, and satisfaction with health and wellness. Other significant findings included the impact of congregational flourishing and age. This study found that clergy well-being and resilience was doing well despite the increased adversity of the COVID-19 pandemic. Implications of this study are that clerics may need unique supports based on their age and also whether they serve in a congregation they perceive as flourishing.
Journal Article
Rationale and design of the balANZ trial: A randomised controlled trial of low GDP, neutral pH versus standard peritoneal dialysis solution for the preservation of residual renal function
Background
The main hypothesis of this study is that neutral pH, low glucose degradation product (GDP) peritoneal dialysis (PD) fluid better preserves residual renal function in PD patients over time compared with conventional dialysate.
Methods/Design
Inclusion criteria are adult PD patients (CAPD or APD) aged 18-81 years whose first dialysis was within 90 days prior to or following enrolment and who have a residual GFR ≥ 5 ml/min/1.73 m
2
, a urine output ≥ 400 ml/day and an ability to understand the nature and requirements of this trial. Pregnant or lactating patients or individuals with an active infection at the time of enrolment, a contra-indication to PD or participation in any other clinical trial where an intervention is designed to moderate rate of change of residual renal function are excluded. Patients will be randomized 1:1 to receive either neutral pH, low GDP dialysis solution (Balance
®
) or conventional dialysis solution (Stay.safe
®
) for a period of 2 years. During this 2 year study period, urinary urea and clearance measurements will be performed at 0, 3, 6, 9, 12, 18 and 24 months. The primary outcome measure will be the slope of residual renal function decline, adjusted for centre and presence of diabetic nephropathy. Secondary outcome measures will include time from initiation of peritoneal dialysis to anuria, peritoneal small solute clearance, peritoneal transport status, peritoneal ultrafiltration, technique survival, patient survival, peritonitis rates and adverse events. A total of 185 patients has been recruited into the trial.
Discussion
This investigator-initiated study has been designed to provide evidence to help nephrologists determine the optimal dialysis solution for preserving residual renal function in PD patients.
Trial Registration
Australian New Zealand Clinical Trials Registry Number: ACTRN12606000044527
Journal Article
Neural Dynamics of the Motor System in Speech Perception
The role of the motor system in speech perception has provoked considerable debate over the past 50 years. Motor and sensory cortices are traditionally thought to be functionally separate systems. However, many studies have shown their roles in both action and perception to be highly integrated. In particular, this has been observed in regard to speech, where listening to speech sounds elicits neural activity in motor regions of the brain in both adults and infants. The primary goal of the present study was to investigate the role of motor and motor planning brain regions in speech perception throughout various stages of language development. Three datasets containing magnetoencephalography (MEG) data from 2-, 6-, 7-, and 11-month-old infants and adults were used to address four experimental questions related to the role that motor brain systems play in the auditory perception of speech. The four experimental questions examine the relationship between activation in auditory sensory and motor regions of the brain with respect to: 1) the temporal structure of activation in sensory as opposed to motor brain regions, 2) the development of neural responses with increasing age, 3) the role of language experience, and 4) potential differences between speech as opposed to nonspeech auditory signals. Results showed that motor and motor planning regions are activated during speech perception across all ages. At 2 months of age, infants show activity in both motor and motor planning regions in response to speech, but not to nonspeech acoustic stimuli. This provides evidence that infants’ activation of sensory and motor brain regions in response to speech does not require experience producing speech.
Dissertation