Amino acid 138 in the HA of a H3N2 subtype influenza A virus increases affinity for the lower respiratory tract and alveolar macrophages in pigs.

Influenza A virus (FLUAV) infects a wide range of hosts and human-to-swine spillover events are frequently reported. However, only a few of these human viruses have become established in pigs and the host barriers and molecular mechanisms driving adaptation to the swine host remain poorly understood. We previously found that infection of pigs with a 2:6 reassortant virus (hVIC/11) containing the hemagglutinin (HA) and neuraminidase (NA) gene segments from the human strain A/Victoria/361/2011 (H3N2) and internal gene segments of an endemic swine strain (sOH/04) resulted in a fixed amino acid substitution in the HA (A138S, mature H3 HA numbering). In silico analysis revealed that S138 became predominant among swine H3N2 virus sequences deposited in public databases, while 138A predominates in human isolates. To understand the role of the HA A138S substitution in the adaptation of a human-origin FLUAV HA to swine, we infected pigs with the hVIC/11A138S mutant and analyzed pathogenesis and transmission compared to hVIC/11 and sOH/04. Our results showed that the hVIC/11A138S virus had an intermediary pathogenesis between hVIC/11 and sOH/04. The hVIC/11A138S infected the upper respiratory tract, right caudal, and both cranial lobes while hVIC/11 was only detected in nose and trachea samples. Viruses induced a distinct expression pattern of various pro-inflammatory cytokines such as IL-8, TNF-α, and IFN-ß. Flow cytometric analysis of lung samples revealed a significant reduction of porcine alveolar macrophages (PAMs) in hVIC/11A138S-infected pigs compared to hVIC/11 while a MHCIIlowCD163neg population was increased. The hVIC/11A138S showed a higher affinity for PAMs than hVIC/11, noted as an increase of infected PAMs in bronchoalveolar lavage fluid (BALF), and showed no differences in the percentage of HA-positive PAMs compared to sOH/04. This increased infection of PAMs led to an increase of granulocyte-monocyte colony-stimulating factor (GM-CSF) stimulation but a reduced expression of peroxisome proliferator-activated receptor gamma (PPARγ) in the sOH/04-infected group. Analysis using the PAM cell line 3D4/21 revealed that the A138S substitution improved replication and apoptosis induction in this cell type compared to hVIC/11 but at lower levels than sOH/04. Overall, our study indicates that adaptation of human viruses to the swine host involves an increased affinity for the lower respiratory tract and alveolar macrophages.

Nucleoprotein reassortment enhanced transmissibility of H3 1990.4.a clade influenza A virus in swine.

The increased detection of H3 C-IVA (1990.4.a) clade influenza A viruses (IAVs) in US swine in 2019 was associated with a reassortment event to acquire an H1N1pdm09 lineage nucleoprotein (pdmNP) gene, replacing a TRIG lineage NP (trigNP). We hypothesized that acquiring the pdmNP conferred a selective advantage over prior circulating H3 viruses with a trigNP. To investigate the role of NP reassortment in transmission, we identified two contemporary 1990.4.a representative strains (NC/19 and MN/18) with different evolutionary origins of the NP gene. A reverse genetics system was used to generate wild-type (wt) strains and swap the pdm and TRIG lineage NP genes, generating four viruses: wtNC/19-pdmNP, NC/19-trigNP, wtMN/18-trigNP, and MN/18-pdmNP. The pathogenicity and transmission of the four viruses were compared in pigs. All four viruses infected 10 primary pigs and transmitted to five indirect contact pigs per group. Pigs infected via contact with MN/18-pdmNP shed virus 2 days earlier than pigs infected with wtMN/18-trigNP. The inverse did not occur for wtNC/19-pdmNP and NC/19-trigNP. This suggests that pdmNP reassortment resulted in a combination of genes that improved transmission efficiency when paired with the 1990.4.a hemagglutinin (HA). This is likely a multigenic trait, as replacing the trigNP gene did not diminish the transmission of a wild-type IAV in swine. This study demonstrates how reassortment and evolutionary change of internal genes can result in more transmissible viruses that influence HA clade detection frequency. Thus, rapidly identifying novel reassortants paired with dominant hemagglutinin/neuraminidase may improve the prediction of strains to include in vaccines.IMPORTANCEInfluenza A viruses (IAVs) are composed of eight non-continuous gene segments that can reassort during coinfection of a host, creating new combinations. Some gene combinations may convey a selective advantage and be paired together preferentially. A reassortment event was detected in swine in the United States that involved the exchange of two lineages of nucleoprotein (NP) genes (trigNP to pdmNP) that became a predominant genotype detected in surveillance. Using a transmission study, we demonstrated that exchanging the trigNP for a pdmNP caused the virus to shed from the nose at higher levels and transmit to other pigs more rapidly. Replacing a pdmNP with a trigNP did not hinder transmission, suggesting that transmission efficiency depends on interactions between multiple genes. This demonstrates how reassortment alters IAV transmission and that reassortment events can provide an explanation for why genetically related viruses with different internal gene combinations experience rapid fluxes in detection frequency.

Reverse-zoonoses of 2009 H1N1 pandemic influenza A viruses and evolution in United States swine results in viruses with zoonotic potential.

The 2009 H1N1 pandemic (pdm09) lineage of influenza A virus (IAV) crosses interspecies barriers with frequent human-to-swine spillovers each year. These spillovers reassort and drift within swine populations, leading to genetically and antigenically novel IAV that represent a zoonotic threat. We quantified interspecies transmission of the pdm09 lineage, persistence in swine, and identified how evolution in swine impacted zoonotic risk. Human and swine pdm09 case counts between 2010 and 2020 were correlated and human pdm09 burden and circulation directly impacted the detection of pdm09 in pigs. However, there was a relative absence of pdm09 circulation in humans during the 2020-21 season that was not reflected in swine. During the 2020-21 season, most swine pdm09 detections originated from human-to-swine spillovers from the 2018-19 and 2019-20 seasons that persisted in swine. We identified contemporary swine pdm09 representatives of each persistent spillover and quantified cross-reactivity between human seasonal H1 vaccine strains and the swine strains using a panel of monovalent ferret antisera in hemagglutination inhibition (HI) assays. The swine pdm09s had variable antigenic reactivity to vaccine antisera, but each swine pdm09 clade exhibited significant reduction in cross-reactivity to one or more of the human seasonal vaccine strains. Further supporting zoonotic risk, we showed phylogenetic evidence for 17 swine-to-human transmission events of pdm09 from 2010 to 2021, 11 of which were not previously classified as variants, with each of the zoonotic cases associated with persistent circulation of pdm09 in pigs. These data demonstrate that reverse-zoonoses and evolution of pdm09 in swine results in viruses that are capable of zoonotic transmission and represent a potential pandemic threat.

Divergent Pathogenesis and Transmission of Highly Pathogenic Avian Influenza A(H5N1) in Swine.

Highly pathogenic avian influenza (HPAI) viruses have potential to cross species barriers and cause pandemics. Since 2022, HPAI A(H5N1) belonging to the goose/Guangdong 2.3.4.4b hemagglutinin phylogenetic clade have infected poultry, wild birds, and mammals across North America. Continued circulation in birds and infection of multiple mammalian species with strains possessing adaptation mutations increase the risk for infection and subsequent reassortment with influenza A viruses endemic in swine. We assessed the susceptibility of swine to avian and mammalian HPAI H5N1 clade 2.3.4.4b strains using a pathogenesis and transmission model. All strains replicated in the lung of pigs and caused lesions consistent with influenza A infection. However, viral replication in the nasal cavity and transmission was only observed with mammalian isolates. Mammalian adaptation and reassortment may increase the risk for incursion and transmission of HPAI viruses in feral, backyard, or commercial swine.

PARNAS: Objectively Selecting the Most Representative Taxa on a Phylogeny.

The use of next-generation sequencing technology has enabled phylogenetic studies with hundreds of thousands of taxa. Such large-scale phylogenies have become a critical component in genomic epidemiology in pathogens such as SARS-CoV-2 and influenza A virus. However, detailed phenotypic characterization of pathogens or generating a computationally tractable dataset for detailed phylogenetic analyses requires objective subsampling of taxa. To address this need, we propose parnas, an objective and flexible algorithm to sample and select taxa that best represent observed diversity by solving a generalized k-medoids problem on a phylogenetic tree. parnas solves this problem efficiently and exactly by novel optimizations and adapting algorithms from operations research. For more nuanced selections, taxa can be weighted with metadata or genetic sequence parameters, and the pool of potential representatives can be user-constrained. Motivated by influenza A virus genomic surveillance and vaccine design, parnas can be applied to identify representative taxa that optimally cover the diversity in a phylogeny within a specified distance radius. We demonstrated that parnas is more efficient and flexible than existing approaches. To demonstrate its utility, we applied parnas to 1) quantify SARS-CoV-2 genetic diversity over time, 2) select representative influenza A virus in swine genes derived from over 5 years of genomic surveillance data, and 3) identify gaps in H3N2 human influenza A virus vaccine coverage. We suggest that our method, through the objective selection of representatives in a phylogeny, provides criteria for quantifying genetic diversity that has application in the the rational design of multivalent vaccines and genomic epidemiology. PARNAS is available at https://github.com/flu-crew/parnas.

Place of death in Parkinson's disease and related disorders in England and Wales: post-pandemic trends and implications for care planning.

BACKGROUND: With growing emphasis on palliative care for neurodegenerative conditions, understanding trends in place of death helps improve quality of end-of-life care for people with Parkinson's disease and related disorders (PDRDs), focusing allocation of resources and training and identifying inequalities. OBJECTIVES: Review national and regional place of death trends for people with PDRD including pre- and post-pandemic trends. METHODS: Mortality data for England and Wales (March 2018 and July 2022) were analysed with summary statistics and interrupted time series, exploring place of death for those who died with PDRD, with and without coexisting dementia, with reference to all deaths in England and Wales. RESULTS: Of 2,415,566 adult deaths, 56,790 included mention of PDRD. Hospital deaths were most common in people with PDRD (39.17%), followed by care homes (38.84%). People with PDRD were half as likely to die in hospice compared with the general population (2.03 vs 4.94%). Proportion of care home deaths fell significantly after March 2020 (40.6-37%, P = 0.035). Regionally, London was an outlier with a lower proportion of deaths occurring in care homes with a higher proportion of hospital deaths. CONCLUSION: Place of death for people with PDRD is changing, with more hospice and home deaths. People with PDRD, particularly those with co-existent dementia, are less likely to access inpatient hospice care than the general population. Since the COVID-19 pandemic, the proportion of care home deaths has reduced significantly with an increase in home deaths, with implications for service and resource allocation.

"Why is it not bringing me meaning and purpose?" Former occupational therapists' narratives of leaving the profession.

INTRODUCTION: The demand for occupational therapy services in Australia has experienced considerable growth in the last decade. Despite an increase in occupational therapy numbers, there remains a substantial workforce shortage. One reason for this shortage is difficulty with the retention of occupational therapists and subsequent workforce attrition. The literature attributes planning to leave as being influenced by high workloads, insufficient time, a negative workplace culture, and stress impacting well-being. However, much of this research is specific to a practice area, such as mental health, and does not explore the experiences of those who have already left. Knowing why people leave occupational therapy will inform our understanding of how to support and retain our occupational therapy workforce. METHODS: A qualitative study was undertaken using narrative inquiry in an Australian context. In 2022, stories were gathered from six former occupational therapists who left within the previous 5 years. Data were collected via semi-structured in-depth interviews using Zoom. Interviews were recorded and transcribed verbatim. The data were analysed using narrative and thematic analysis. FINDINGS: Four themes were developed: "I just want to help this person"; "there was no real understanding"; "Trying to find where occupational therapy fits for me"; and "I'm not able to continue … anymore." Participants experienced dissonance between enacting their occupational therapy values and the expectations of their management. They were challenged by unsupportive workplace cultures and leadership styles that undermined their autonomy. They responded with resilience, and trying new ways to continue being occupational therapists, but became increasingly uncomfortable and stressed until they reached a tipping point where they decided to leave. CONCLUSION: Increasing leadership development within the profession is essential, along with creative strategies for meeting funding requirements and fulfilling occupational therapy values. Workplaces that provide autonomy and respect the occupational therapy role are likely to support retention.

Transmission of Human Influenza A Virus in Pigs Selects for Adaptive Mutations on the HA Gene.

Influenza A viruses (FLUAV) cause respiratory diseases in many host species, including humans and pigs. The spillover of FLUAV between swine and humans has been a concern for both public health and the swine industry. With the emergence of the triple reassortant internal gene (TRIG) constellation, establishment of human-origin FLUAVs in pigs has become more common, leading to increased viral diversity. However, little is known about the adaptation processes that are needed for a human-origin FLUAV to transmit and become established in pigs. We generated a reassortant FLUAV (VIC11pTRIG) containing surface gene segments from a human FLUAV strain and internal gene segments from the 2009 pandemic and TRIG FLUAV lineages and demonstrated that it can replicate and transmit in pigs. Sequencing and variant analysis identified three mutants that emerged during replication in pigs, which were mapped near the receptor binding site of the hemagglutinin (HA). The variants replicated more efficiently in differentiated swine tracheal cells compared to the virus containing the wildtype human-origin HA, and one of them was present in all contact pigs. These results show that variants are selected quickly after replication of human-origin HA in pigs, leading to improved fitness in the swine host, likely contributing to transmission. IMPORTANCE Influenza A viruses cause respiratory disease in several species, including humans and pigs. The bidirectional transmission of FLUAV between humans and pigs plays a significant role in the generation of novel viral strains, greatly impacting viral epidemiology. However, little is known about the evolutionary processes that allow human FLUAV to become established in pigs. In this study, we generated reassortant viruses containing human seasonal HA and neuraminidase (NA) on different constellations of internal genes and tested their ability to replicate and transmit in pigs. We demonstrated that a virus containing a common internal gene constellation currently found in U.S. swine was able to transmit efficiently via the respiratory route. We identified a specific amino acid substitution that was fixed in the respiratory contact pigs that was associated with improved replication in primary swine tracheal epithelial cells, suggesting it was crucial for the transmissibility of the human virus in pigs.

Modeling of recovery efficiency of sampling devices used in planetary protection bioburden estimation.

IMPORTANCE: Planetary protection at the National Aeronautics and Space Administration (NASA) requires bioburden on certain spacecraft to be estimated via sampling in order to comply with biological cleanliness requirements. To achieve this, the recovery efficiency of devices used to sample the spacecraft pre-launch must be understood and their uncertainty quantified in order to produce the most reasonable estimates of bioburden. This study brings together experiments performed by NASA and the European Space Agency with approved swab and wipe sampling devices, inoculating steel coupons with laboratory strains of Bacillus spp. spores commonly recovered from spacecraft assembly clean rooms (B. atrophaeus, B. megaterium, B. safensis and B. thuringiensis), with a mathematical model of the assay process to assess recovery efficiency. The statistical treatment developed in this study allows comparison of bioburden estimates made from different devices processed by different methods. This study also gives stakeholders and practitioners a statistically rigorous approach to predict bioburden that can be folded into future modeling efforts.

Geographical variations in long term colorectal cancer outcomes in England: a contemporary population analysis revealing the north-south divide in colorectal cancer survival.

BACKGROUND: Regional variations in healthcare outcomes in England have been historically reported. This study analyses the variations in long term colorectal cancer survival across different regions in England. METHODS: Relative survival analysis of population data obtained from all cancer registries in England between 2010 and 2014. RESULTS: Totally, 167,501 patients were studied. Regions in the southern England had better outcomes with Southwest and Oxford registries having 63.5 and 62.7% 5 year relative survival. In contrast, Trent and Northwest cancer registries had 58.1% relative survival (p < 0.01). The regions in the north fared below the national average. The survival outcomes reflected socio-economic deprivation status, the best performing regions in the south having low levels of deprivation (5.3 and 6.5% having maximum deprivation in Southwest and Oxford, respectively). The regions with worst long term cancer outcomes had high levels of deprivation with 25% and 17% having high levels of deprivation in Northwest and Trent regions. CONCLUSION: There are significant variations in long term colorectal cancer survival between different regions in England, southern England had better relative survival when compared with the northern regions. Disparities in socio-economic depravation status in different regions may be associated with worse colorectal cancer outcomes.

Successes, challenges, and opportunities in providing evidence-based teletherapy to children who have experienced trauma as a response to Covid-19: A national survey of clinicians.

While teletherapy is not a new phenomenon, most clinicians have not been trained and do not routinely practice it. The current study was designed to ascertain challenges and opportunities presented by the widescale usage of teletherapy especially for traumatized children, which was necessitated by the COVID-19 pandemic. Two hundred and fifty clinicians across the United States providing teletherapy to traumatized children completed an online survey. Results revealed that many logistical aspects of treatment were perceived to be easier when implemented remotely. Some clinical aspects of care were also perceived to be easier, notably engagement with caregivers. Developing rapport, assessing emotions, and keeping children's attention, however, were perceived as more challenging. Child characteristics such as age, attention span, and screen fatigue were viewed as creating challenges. Most clinicians had not received training in relevant topics for teletherapy and were eager to receive such training. These results suggest many avenues for refining and fine-tuning remote mental health services especially for children.

Young-onset colorectal cancer: Insights from an English population-based study.

AIM: Young colorectal cancer (CRC) patients are reported to have more aggressive disease, an advanced stage at diagnosis and conflicting survival outcomes. The aim of this study was to analyse the demographics, clinicopathological features and prognosis of young CRC at a population-based level in England. METHOD: This is a retrospective review of all CRC patients using data from Public Health England collated from regional cancer registries in England between 2010 and 2014. Those aged 40 years and below were classified as young and those over 40 were classified as older. RESULTS: Overall, 167,501 patients had CRC. Of these, 3757 patients (2.2%) were young. Right-sided cancers were more common in younger patients (48.2% vs. 32.9%, p < 0.001). Favourable histological grade (well or moderately differentiated) was present in 83.1% and 73.5% of young and older patients, respectively. The percentage of young and older patients being diagnosed at an early stage (Stages 1 and 2) was similar at 40.6% vs. 42.9%. The 5-year age- and gender-adjusted relative survival (cancer specific) was significantly better for young patients when compared with older patients diagnosed with CRC. Additionally, overall 5-year survival was better for younger patients (71.6% and 47.2%, p < 0.001 in young and older CRC patients respectively). CONCLUSION: The increased right-sided colon cancer in young CRC patients in England warrants attention. Contrary to previous reports, they do not present at later stage. Young CRC patients have better overall and relative survival than older patients with CRC.

Suicide deaths following police contact: A review of coronial inquest findings.

We analyzed 10 years of Australian coronial inquest documents on people who died by suicide or suspected suicide following recent contact with the police. We identified 149 cases of suicide or suspected suicides via coronial websites, 34 of which contained recommendations. We developed four themes from these recommendations, the most common of which was for police to change current practices or policies and modify police officer training. Coroners considered effective working relationships with external stakeholders able to assist police an important means of reducing risk of suicide.

Multigenerational memory and adaptive adhesion in early bacterial biofilm communities.

Using multigenerational, single-cell tracking we explore the earliest events of biofilm formation by Pseudomonas aeruginosa During initial stages of surface engagement (≤20 h), the surface cell population of this microbe comprises overwhelmingly cells that attach poorly (∼95% stay <30 s, well below the ∼1-h division time) with little increase in surface population. If we harvest cells previously exposed to a surface and direct them to a virgin surface, we find that these surface-exposed cells and their descendants attach strongly and then rapidly increase the surface cell population. This "adaptive," time-delayed adhesion requires determinants we showed previously are critical for surface sensing: type IV pili (TFP) and cAMP signaling via the Pil-Chp-TFP system. We show that these surface-adapted cells exhibit damped, coupled out-of-phase oscillations of intracellular cAMP levels and associated TFP activity that persist for multiple generations, whereas surface-naïve cells show uncorrelated cAMP and TFP activity. These correlated cAMP-TFP oscillations, which effectively impart intergenerational memory to cells in a lineage, can be understood in terms of a Turing stochastic model based on the Pil-Chp-TFP framework. Importantly, these cAMP-TFP oscillations create a state characterized by a suppression of TFP motility coordinated across entire lineages and lead to a drastic increase in the number of surface-associated cells with near-zero translational motion. The appearance of this surface-adapted state, which can serve to define the historical classification of "irreversibly attached" cells, correlates with family tree architectures that facilitate exponential increases in surface cell populations necessary for biofilm formation.

Purification, Punishment, and Control: Eating Disorders, Self-Harm, and Child Sexual Abuse.

Eating disorders continue to be viewed as curable diseases, forcing people into predetermined narratives of pathology that shape how they are viewed and treated. Situated in a feminist application of Bakhtin's sociological linguistics, we were concerned with how participants understood eating disorders, the nature of their experiences, and the causes of their distress. Following a dialogical method, multiple in-depth interviews were conducted with seven women who experienced an eating disorder and who had been sexually abused previously, and participants' own drawings and poetry were obtained to gain deeper insights into meanings and emotions. We found an eating disorder offered a perception of cleanliness and renewal that was attractive to participants who experienced overwhelming shame. It is critical that researchers use a range of visual and sensory methods to move eating disorder understandings and treatment beyond illness and pathology.

Barriers and enablers to implementing mental well-being programs through Australian rural football clubs-A qualitative descriptive study.

ISSUE ADDRESSED: Suicide rates in rural Australia are almost twice as high as those in urban areas. In rural communities, football clubs are often the 'hub' of the community and are being explored as an avenue to deliver mental health and well-being promotion. The aim of this study was to explore barriers and enablers for programs promoting mental health and well-being through rural Australian football clubs. METHOD: This qualitative descriptive study included 12 individuals of 10 rural clubs affiliated with the South Australian National Football League. Recruitment occurred via emails to club secretaries/presidents. Semi-structured telephone interviews explored mental health and well-being issues experienced in clubs, previous involvement with mental well-being programs and potential barriers/enablers for future programs. Interviews were transcribed verbatim and analysed thematically. RESULTS: Thematic analysis identified three themes encompassing barriers and enablers: (1) more than a football club, (2) attitudes towards mental health and (3) what is needed to implement a program. The third theme had subthemes of resources, importance of timing, mental health initiatives and components of a program. CONCLUSION: Key barriers included difficulty getting people involved due to individual attitudes towards mental health, and not having the finances/resources to implement a program. Major enablers included the important role the football club serves in the community, the inclusion of speakers with credibility and making the program engaging. SO WHAT?: This study identifies key factors which may impact on community engagement and program effectiveness for mental health and well-being programs delivered via rural football clubs.

Evidence-based prescribing for post-traumatic stress disorder.

There is strong research evidence to support the pharmacological treatment of post-traumatic stress disorder (PTSD) as a second line to trauma-focused psychological interventions. Fluoxetine, paroxetine, sertraline and venlafaxine are the best-evidenced drugs, with lower-level evidence for other medications. It is important that prescribing for PTSD is evidence-based.

Drosophila DNA polymerase theta utilizes both helicase-like and polymerase domains during microhomology-mediated end joining and interstrand crosslink repair.

Double strand breaks (DSBs) and interstrand crosslinks (ICLs) are toxic DNA lesions that can be repaired through multiple pathways, some of which involve shared proteins. One of these proteins, DNA Polymerase θ (Pol θ), coordinates a mutagenic DSB repair pathway named microhomology-mediated end joining (MMEJ) and is also a critical component for bypass or repair of ICLs in several organisms. Pol θ contains both polymerase and helicase-like domains that are tethered by an unstructured central region. While the role of the polymerase domain in promoting MMEJ has been studied extensively both in vitro and in vivo, a function for the helicase-like domain, which possesses DNA-dependent ATPase activity, remains unclear. Here, we utilize genetic and biochemical analyses to examine the roles of the helicase-like and polymerase domains of Drosophila Pol θ. We demonstrate an absolute requirement for both polymerase and ATPase activities during ICL repair in vivo. However, similar to mammalian systems, polymerase activity, but not ATPase activity, is required for ionizing radiation-induced DSB repair. Using a site-specific break repair assay, we show that overall end-joining efficiency is not affected in ATPase-dead mutants, but there is a significant decrease in templated insertion events. In vitro, Pol θ can efficiently bypass a model unhooked nitrogen mustard crosslink and promote DNA synthesis following microhomology annealing, although ATPase activity is not required for these functions. Together, our data illustrate the functional importance of the helicase-like domain of Pol θ and suggest that its tethering to the polymerase domain is important for its multiple functions in DNA repair and damage tolerance.

Development of an evidence-based Evaluation Toolkit for suicide prevention networks in regional Australia.

PROBLEM: Suicide is a global public health concern, particularly for those living in rural, regional and remote areas. Despite numerous prevention initiatives, suicide continues to impact individuals, families and communities. Suicide prevention networks are an innovative, community-based approach to addressing suicide within local government areas. Anecdotally, suicide prevention networks make an important contribution to suicide prevention, yet frameworks for evaluating their impact do not exist. The purpose of this project was to develop an Evaluation Toolkit to be used by suicide prevention networks throughout South Australia to monitor their impact. DESIGN: Participatory action research was used to work with one suicide prevention network in regional South Australia to co-design and produce the Evaluation Toolkit. SETTING: One regional South Australian town, with an established suicide prevention network. KEY MEASURES FOR IMPROVEMENT: The development of an Evaluation Toolkit, to allow all South Australian suicide prevention networks to measure their impact in a consistent manner. STRATEGIES FOR CHANGE: The Evaluation Toolkit has been made freely available in electronic form and as a hard copy booklet for use by all South Australian suicide prevention networks. EFFECTS OF CHANGE: The purpose of this project was to develop the Evaluation Toolkit. Formal assessment of its usability and effectiveness is yet to be undertaken, but preliminary anecdotal evidence indicates aspects of the Evaluation Toolkit are useful, especially the questionnaire templates that can be adapted and used to evaluate various community events. LESSONS LEARNT: Participatory action research is a useful method for working with community-based suicide prevention groups.

Flagellar Stators Stimulate c-di-GMP Production by Pseudomonas aeruginosa.

Flagellar motility is critical for surface attachment and biofilm formation in many bacteria. A key regulator of flagellar motility in Pseudomonas aeruginosa and other microbes is cyclic diguanylate (c-di-GMP). High levels of this second messenger repress motility and stimulate biofilm formation. c-di-GMP levels regulate motility in P. aeruginosa in part by influencing the localization of its two flagellar stator sets, MotAB and MotCD. Here, we show that while c-di-GMP can influence stator localization, stators can in turn impact c-di-GMP levels. We demonstrate that the swarming motility-driving stator MotC physically interacts with the transmembrane region of the diguanylate cyclase SadC. Furthermore, we demonstrate that this interaction is capable of stimulating SadC activity. We propose a model by which the MotCD stator set interacts with SadC to stimulate c-di-GMP production under conditions not permissive to motility. This regulation implies a positive-feedback loop in which c-di-GMP signaling events cause MotCD stators to disengage from the motor; then disengaged stators stimulate c-di-GMP production to reinforce a biofilm mode of growth. Our studies help to define the bidirectional interactions between c-di-GMP and the flagellar machinery.IMPORTANCE The ability of bacterial cells to control motility during early steps in biofilm formation is critical for the transition to a nonmotile, biofilm lifestyle. Recent studies have clearly demonstrated the ability of c-di-GMP to control motility via a number of mechanisms, including through controlling transcription of motility-related genes and modulating motor function. Here, we provide evidence that motor components can in turn impact c-di-GMP levels. We propose that communication between motor components and the c-di-GMP synthesis machinery allows the cell to have a robust and sensitive switching mechanism to control motility during early events in biofilm formation.