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Introduction Tobacco smoking remains a leading cause of ill-health, premature mortality and a driver of health inequalities. To support smokers in England, a comprehensive approach to treating tobacco dependence is being implemented. This includes offering support to all people admitted to hospitals, as well as women and pregnant people within NHS settings. We aim to describe the protocol for an evaluation of this tobacco-dependence service.Methods and analysis This is a national evaluation across five regions in England (i.e., South West, West Midlands, Greater Manchester, North East and North Cumbria, Yorkshire and Humber) including 11 NHS Foundation Trusts. It is funded from September 2021 to September 2025. Evaluation settings are acute hospital, maternity and inpatient mental health.Work package (WP) 1 involves qualitative key informant interviews to characterise the policy implementation context. WP 2 involves an online survey to assess the routinisation of the service in practice and staff attitudes regarding tobacco dependence, plus in-depth interviews with relevant practitioners to explore survey findings and interviews with smokers to investigate its usefulness and impact. WP 3 involves descriptive statistical analysis of routinely collected data to assess service uptake and impact on health and care outcomes (e.g., smoking status). WP 4 involves an economic analysis of routinely collected data to determine the financial impact of the service. Qualitative data (WP 1, WP 2) will be analysed using Thematic Analysis and Framework Analysis, respectively. WP 2 survey data will be analysed using descriptive statistics.Ethics and dissemination This evaluation received favourable ethical opinion from Newcastle University (17756/2021) and NHS Wales Research Ethics Committee (22/WA/0203). It has also received Confidentiality Advisory Group support (22CAG0103).

Kilohertz high-frequency spinal cord stimulation (kHF-SCS) is a rapidly advancing neuromodulatory technique in the clinical management of chronic pain. However, the precise cellular mechanisms underlying kHF-SCS-induced paresthesia-free pain relief, as well as the neural responses within spinal pain circuits, remain largely unexplored. In this study, using a novel preparation, we investigated the impact of varying kilohertz frequency SCS on dorsal horn neuron activation. Employing calcium imaging on isolated spinal cord slices, we found that extracellular electric fields at kilohertz frequencies (1, 3, 5, 8, and 10 kHz) induce distinct patterns of activation in dorsal horn neurons. Notably, as the frequency of extracellular electric fields increased, there was a clear and significant monotonic escalation in neuronal activity. This phenomenon was observed not only in superficial dorsal horn neurons, but also in those located deeper within the dorsal horn. Our study demonstrates the unique patterns of dorsal horn neuron activation in response to varying kilohertz frequencies of extracellular electric fields, and we contribute to a deeper understanding of how kHF-SCS induces paresthesia-free pain relief. Furthermore, our study highlights the potential for kHF-SCS to modulate sensory information processing within spinal pain circuits. These insights pave the way for future research aimed at optimizing kHF-SCS parameters and refining its therapeutic applications in the clinical management of chronic pain.

Measurement(s) plant trait Technology Type(s) various Factor Type(s) none Sample Characteristic - Organism Tracheophyta Sample Characteristic - Environment natural environment Sample Characteristic - Location global

Summary: Background: Women are estimated to hold between 70 and 75% of global health positions worldwide yet persistent inequities in power and leadership remain. There is little information on specific enablers and barriers that women working in public health face in India and how those compare with other regions. Methods: We collected and analyzed information from women working in public health in India and East Africa (Kenya, Rwanda, and Uganda) and in global health (Canada and United States), to understand and document the specific enablers and barriers women face in India, compared with other regions. Findings: Several universal themes emerged around factors enabling (mentors, professional networks, leadership based in empathy and team building) or impeding (obvert bias and family responsibilities) women across all contexts. Within this, there are nuances in how women’s leadership growth factors and obstacles play out in India differently than in other contexts. Interpretation: There are important similarities in the enablers and barriers faced by women in India and other geographies and important ways these differs in for women in India. By designing programs and policies at institutional levels to address these factors, we can create a professional ecosystem that works for women in health and beyond. Funding: This research was funded by WomenLift Health, which is funded by the Bill and Melinda Gates Foundation. Representatives from WomenLift Health, listed as authors, participated in the conceptualization of the research to define objectives and core questions, provided commentary and revision to improve the manuscript, and supervised the progress of the research.

Inconsistent yield of subtropical table grape across seasons is often associated with low carbohydrate reserves during flowering. In an attempt to increase TNC and thus yield, sucrose was injected into treated trunks during periods of high carbohydrate demand (i.e., between budburst and flowering). Total non-structural carbohydrate (TNC) concentration dynamics were assessed in the grapevine root and trunk tissues of both control and treated vines. In the control (untreated) vines, the TNC concentration in root and trunk tissues was 13.5% and 7.5% w/dw at leaf fall and 7.2% and 3.7% w/dw at flowering, respectively. This decrease in carbohydrate reserve was estimated at ~500 g/vine and is associated with the re-establishment of the plant canopy in early spring. Carbohydrate reserves remained stable or rose slightly between flowering and harvest and recovered between crop harvest and leaf fall. In treated vines, a constantly pressurised low-pressure in situ trunk injection system (69 kPa) with 5% w/v sucrose solution over 45 days from budburst (to flowering), in each of the two seasons, delivered a widely variable amount of sucrose into each vine with variation ascribed to the amount of internal dead wood in the trunk. In the best circumstances, an average of 150 g sucrose/vine/season was injected, and sucrose-injected vines had higher trunk TNC reserve (4.1% compared to 3.6% w/dw in the control) at flowering. A δ13C (‰) analysis confirmed the presence of injected sucrose in the shoot at flowering. However, the correlation between the amount of loaded sucrose and δ13C in young shoot tissue was poor, indicative of variable partitioning patterns. Inflorescence number per vine and berry yield were markedly higher in sucrose-injected vines, but differences were not significant given the high variation between vines. The addition of KCl to the sucrose solution and use of the healthy vines are recommended to increase sucrose loading using the injection method to address inconsistent yielding of subtropical table grape.

A farm management information system (MIS) entails record keeping based on a database management system, typically using a client-server architecture, i.e., an information system, IS, coupled with a variety of tools/methods/models for the support of operational management. The current review adopts a multivocal approach to consider academic and commercial developments in MISs for orchard management, based primarily on the refereed literature but extending to grey literature and interviews of Australian mango orchard managers. Drivers for orchard MIS development include increasing the orchard size and management complexity, including regulatory requirements around labour, chemical spray use and fertilisation. The enablers include improvements in within-orchard communications, distributed (web) delivery systems using desktop and mobile devices, and sensor systems and predictive models, e.g., for pest management. Most orchard MIS-related publications target the commodities of apple, grape, mango and olive in the context of management of plant health (pest and disease), plant development, irrigation and labour management. Harvest forecast and MIS modules are only now beginning to emerge, in contrast to a long history of use in grain production. The commercial systems trend towards an incorporation of financial information, an integration of data from multiple sources and a provision of dashboards that are tailored to the user. Requirements for industry adoption of a MIS are discussed in terms of technical and design features, with a focus on usability and scalability.

The Xi era can be characterised as an ambitious and nationalistic one. Aims such as rejuvenating the Chinese nation and fulfilling the country's historic mission to be a great, rich country are often referred to in elite leadership discourse and state produced propaganda. “China Dream” is amongst the most important slogans used in this language. In terms of the context in which this phrase occurs and the actual deployment, it is one that carries broad connotations and implications about an era where, in ways similar to politics in the West, issues around identity are key, and the important thing is to feel, not just to believe. “China Dream” is a form of language that speaks into the public's deeper instincts and sentiments.

A web mapping XYZ Tile Layer Service, such as Google Earth (GE), provides an amazing resource for the visualization of spatial data against aerial and satellite imagery with global coverage, typically at a resolution finer than 5 m. However, the increasing requirement on spatial accuracy in farm information requires a greater appreciation of the issues involved in the use of such services. Position errors can be created in the georeferencing and orthorectification of images, transformation between reference frames (datums) in map projection, e.g., using a spheroid as compared to an ellipsoid earth model, and tectonic shifts. A review is provided of these issues, and a case study is provided of the horizontal positional accuracy of web map imagery for Australian mango orchards. Positional accuracies varied from 1.804 to 6.131 m across four farms using GE 2021 imagery, between 1.556 and 3.365 m in one farm for the most recent imagery available from each of four web map providers, and from 0.806 m (in 2016) to 10.634 m (in 2003) in one farm for the period of 2003 and 2021 using the historical GE imagery resource. A procedure involving the estimation of four transformation parameters was demonstrated for the alignment of GNSS data with GE imagery. However, as the scale factor was unity and the rotational value was near zero, the use of a simple horizontal mean shift vector was recommended. Further recommendations are provided on (i) the use of web mapping services, with a comparison of the use of UAV survey imagery, and (ii) the need for metadata, particularly the date of collection, on collected position data, in the context of use in farm management information systems.

Early and accurate tomato disease detection using easily available leaf photos is essential for farmers and stakeholders as it help reduce yield loss due to possible disease epidemics. This paper aims to visually identify nine different infectious diseases (bacterial spot, early blight, Septoria leaf spot, late blight, leaf mold, two-spotted spider mite, mosaic virus, target spot, and yellow leaf curl virus) in tomato leaves in addition to healthy leaves. We implemented EfficientNetB5 with a tomato leaf disease (TLD) dataset without any segmentation, and the model achieved an average training accuracy of 99.84% ± 0.10%, average validation accuracy of 98.28% ± 0.20%, and average test accuracy of 99.07% ± 0.38% over 10 cross folds.The use of gradient-weighted class activation mapping (GradCAM) and local interpretable model-agnostic explanations are proposed to provide model interpretability, which is essential to predictive performance, helpful in building trust, and required for integration into agricultural practice.

Neurological constructs are being applied in various fields; within urban studies and built environments, neurourbanism stands out. To understand this concept, this study seeks to conduct a scientometric analysis of the concept of neurourbanism. To do so, we gauged the intellectual structure and clarified the influencers and emerging themes while seeking to identify essential gaps in neurourbanism research in urban studies and the built environment. Data were sorted from Dimensions Artificial Intelligence platform because of its reliability in providing the needed dataset accurately, and the Citespace software was used to analyze the data. Our results suggest plurality in explaining the risk factors in urbanicity research, particularly regarding prevalence, incidence, and the general cause of psychosis in urban living. The study also shows that players in the construction sector, such as engineers, town planners, and developers, have not fully grasped how the built environment assists in improving well-being, reducing stress levels of urbanists, assisting migrants in settling into the community, and the general mental wellness of those who live in the city. The study also identified a correlation between urbanization and mental health and added that the main recipient of rapid urban transformation countries does not show leadership in neurourbanism studies.

Background: In an era of global health security challenges such as the COVID-19 pandemic, there is greater need for strong leadership. Over the past decades, significant investments have been made in global health leadership development programs by governments and philanthropic organizations to address this need. Evaluating the societal impact of these programs remains challenging, despite consensus on the importance of public health leadership. Objective: This article identifies the gaps and highlights the critical role of monitoring and evaluation approaches in assessing the impact of global health leadership programs. Importantly, we also propose the theory of change (TOC) as a common framework and identify a set of tools and indicators that leadership programs can adapt and use. Methods: We carried out an informal review of major global health leadership programs, including a literature review on leadership program evaluation approaches. Current practices in assessing the short- to long-term outcomes of leadership training programs were explored and synthesized. We also examined use of program theory frameworks, such as theory of change to guide the evaluation strategy. We find the TOC approach can be enhanced by integrating evaluation-specific frameworks and establishing broad stakeholder buy-in. We highlight measurement challenges, proposed outcome indicators and evaluation methodologies, and outline the future direction for such efforts. Findings: Most evaluation of current leadership programs is focused on short-term individual-level outcomes, while reports on long-term societal impact were limited. Reciprocal impacts on and benefits for the “host” organizations were not included in evaluation metrics. Most programs had program logic or result chains, but with no well-articulated program theories. Conclusion: Key stakeholders involved in leadership training programs benefit from the evidence of rigorous program evaluations to inform decisions that address barriers in fostering global health leadership and improving population health outcomes. Insight into reciprocal change in host organizations is important. Evaluation of global health leadership training must go beyond the individual trainee and encompass organizational and community-level impacts. Documentation of long-lasting organizational and societal impacts is essential for donors to appreciate the return on their investment. Key Takeaways Evaluation plays an important role in understanding how leadership development takes place and how it contributes to improving public health outcomes. Making the case for investments in leadership development programs requires robust evidence from monitoring and evaluation strategies that link investments beyond the individual-level to longer-term societal impacts. The first critical step towards a strategy for success is for leadership programs to clearly build, articulate, share, and use their program theories or theories of change. Theories of change help identify the pathways (and potential tensions) through which leadership development programs effect change at the individual, organizational and community levels. Evaluation methods that examine outcomes of leadership programs should be multi-method, multi-level, and where possible include counterfactual outcomes. Allocation of funds to evaluate on-going and long-lasting societal impact of leadership programs should be a routine practice.

Mungbean is susceptible to waterlogging stress; therefore, breeding tolerant varieties would provide Australian growers with management options for wet summer season planting. Selection for waterlogging tolerance could be improved using vegetative indices that correlate to yield. Five mungbean genotypes were exposed to waterlogging stress at the pod-filling stage and characterised for various morphological and physiological traits governing seed yield. Waterlogging during pod filling decreased stomatal conductance (gs) and photosynthetic rate (Asat) to ~27% and 25% compared to control, respectively, resulting in a decline in effective quantum yield of PSII (ФPSII) and maximum efficiency of PSII of dark-adapted leaves (Fv/Fm) and leaf chlorophyll while increasing excitation pressure (1-qP) significantly. Waterlogging at pod filling reduced leaf count (19%), plant height (23%), leaf dry weight (38%), stem dry weight (33%), pod dry weight (36%), above-ground biomass (34%), root biomass (26%), and 100-seed weight (4%). Seed yield was highly positively correlated with Asat (0.86), gs (0.69), chlorophyll content (0.63), and ФPSII (0.59), with a highly negative correlation with 1-qP (−0.87) at 30 days of treatment imposition. A yield penalty of 32% was recorded under waterlogging stress compared to control plants, while the performance of all genotypes was found to be similar in terms of seed yield. Interestingly, genotype AVTMB#3 produced significantly larger seeds under waterlogging stress relative to other genotypes, including the leading Australian mungbean variety, Jade-AU. Based on a robust and significantly strong correlation with seed yield under waterlogging stress, 1-qP and photosynthetic rates (Asat) are recommended as potential indicators for the screening of mungbean genotypes. Thus, the current study presents a framework for screening waterlogging tolerance, which can provide a reasonable basis for the selection of various genotypes in future mungbean breeding programs.

Background Emergency departments (EDs) at level 1 trauma centers are often overcrowded and deny ED-to-ED transfers from lower-tiered centers. Lack of access to timely level 1 care is associated with increased mortality. We evaluated the feasibility of a direct admission (DA) protocol as a method to increase timely access to a level 1 trauma center during periods of ED overcrowding.Methods During periods of ED overcrowding between 1 May and 31 December 2019, we admitted patients from referring EDs directly to the intensive care unit (ICU) or inpatient ward using the DA protocol. In a prospective comparative study design, we compared their outcomes to patients during the same period who were admitted through the ED when the ED was not overcrowded.Results During periods of ED overcrowding, transfer was requested and clinically accepted for 28 patients, of which 23 (82.1%, age 63±20.3 years, men 52.2% men) were successfully admitted via the DA protocol. Five (17.9%) were not successfully transferred due to lack of available inpatient beds. During periods when the ED was not overcrowded, 106 patients (age 62.8±23.1 years, men 52.8%) were admitted via the ED. There were no morbidity or mortality events attributed to the DA process. Time to patient arrival was 2.7 hours (95% CI 2.3 to 3.1) in the DA cohort and 1.9 hours (95% CI 1.5 to 2.4) in the ED-to-ED cohort (p=0.104). Up-triage to the ICU within 24 hours was performed in only one patient (4.3%). In-hospital mortality did not differ (3 (13%) vs. 8 (7.6%), p=0.392).Discussion The DA pathway is a feasible method to safely transfer patients from a referring ED to a higher-care trauma center when its ED is overcrowded.Level of evidence Level III, care management.

This study examined records of 2566 consecutive COVID-19 patients at five Massachusetts hospitals and sought to predict level-of-care requirements based on clinical and laboratory data. Several classification methods were applied and compared against standard pneumonia severity scores. The need for hospitalization, ICU care, and mechanical ventilation were predicted with a validation accuracy of 88%, 87%, and 86%, respectively. Pneumonia severity scores achieve respective accuracies of 73% and 74% for ICU care and ventilation. When predictions are limited to patients with more complex disease, the accuracy of the ICU and ventilation prediction models achieved accuracy of 83% and 82%, respectively. Vital signs, age, BMI, dyspnea, and comorbidities were the most important predictors of hospitalization. Opacities on chest imaging, age, admission vital signs and symptoms, male gender, admission laboratory results, and diabetes were the most important risk factors for ICU admission and mechanical ventilation. The factors identified collectively form a signature of the novel COVID-19 disease.

Eight depth cameras varying in operational principle (stereoscopy: ZED, ZED2, OAK-D; IR active stereoscopy: Real Sense D435; time of flight (ToF): Real Sense L515, Kinect v2, Blaze 101, Azure Kinect) were compared in context of use for in-orchard fruit localization and sizing. For this application, a specification on bias-corrected root mean square error of 20 mm for a camera-to-fruit distance of 2 m and operation under sunlit field conditions was set. The ToF cameras achieved the measurement specification, with a recommendation for use of Blaze 101 or Azure Kinect made in terms of operation in sunlight and in orchard conditions. For a camera-to-fruit distance of 1.5 m in sunlight, the Azure Kinect measurement achieved an RMSE of 6 mm, a bias of 17 mm, an SD of 2 mm and a fill rate of 100% for depth values of a central 50 × 50 pixels group. To enable inter-study comparisons, it is recommended that future assessments of depth cameras for this application should include estimation of a bias-corrected RMSE and estimation of bias on estimated camera-to-fruit distances at 50 cm intervals to 3 m, under both artificial light and sunlight, with characterization of image distortion and estimation of fill rate.

In May 2010 the large white butterfly, Pieris brassicae L. (Lepidoptera: Pieridae), was discovered to have established in New Zealand. It is a Palearctic species that-due to its wide host plant range within the Brassicaceae-was regarded as a risk to New Zealand's native brassicas. New Zealand has 83 native species of Brassicaceae including 81 that are endemic, and many are threatened by both habitat loss and herbivory by other organisms. Initially a program was implemented to slow its spread, then an eradication attempt commenced in November 2012. The P. brassicae population was distributed over an area of approximately 100 km2 primarily in urban residential gardens. The eradication attempt involved promoting public engagement and reports of sightings, including offering a bounty for a two week period, systematically searching gardens for P. brassicae and its host plants, removing host plants, ground-based spraying of insecticide to kill eggs and larvae, searching for pupae, capturing adults with nets, and augmenting natural enemy populations. The attempt was supported by research that helped to progressively refine the eradication strategy and evaluate its performance. The last New Zealand detection of P. brassicae occurred on 16 December 2014, the eradication program ceased on 4 June 2016 and P. brassicae was officially declared eradicated from New Zealand on 22 November 2016, 6.5 years after it was first detected and 4 years after the eradication attempt commenced. This is the first species of butterfly ever to have been eradicated worldwide.

Migration is a widespread but highly diverse component of many animal life histories. Fish migrate throughout the world's oceans, within lakes and rivers, and between the two realms, transporting matter, energy, and other species (e.g., microbes) across boundaries. Migration is therefore a process responsible for myriad ecosystem services. Many human populations depend on the presence of predictable migrations of fish for their subsistence and livelihoods. Although much research has focused on fish migration, many questions remain in our rapidly changing world. We assembled a diverse team of fundamental and applied scientists who study fish migrations in marine and freshwater environments to identify pressing unanswered questions. Our exercise revealed questions within themes related to understanding the migrating individual's internal state, navigational mechanisms, locomotor capabilities, external drivers of migration, the threats confronting migratory fish including climate change, and the role of migration. In addition, we identified key requirements for aquatic animal management, restoration, policy, and governance. Lessons revealed included the difficulties in generalizing among species and populations, and in understanding the levels of connectivity facilitated by migrating fishes. We conclude by identifying priority research needed for assuring a sustainable future for migratory fishes.

The performance of a multi-view machine vision method was documented at an orchard level, relative to packhouse count. High repeatability was achieved in night-time imaging, with an absolute percentage error of 2% or less. Canopy architecture impacted performance, with reasonable estimates achieved on hedge, single leader and conventional systems (3.4, 5.0, and 8.2 average percentage error, respectively) while fruit load of trellised orchards was over-estimated (at 25.2 average percentage error). Yield estimations were made for multiple orchards via: (i) human count of fruit load on ~5% of trees (FARM), (ii) human count of 18 trees randomly selected within three NDVI stratifications (CAL), (iii) multi-view counts (MV-Raw) and (iv) multi-view corrected for occluded fruit using manual counts of CAL trees (MV-CAL). Across the nine orchards for which results for all methods were available, the FARM, CAL, MV-Raw and MV-CAL methods achieved an average percentage error on packhouse counts of 26, 13, 11 and 17%, with SD of 11, 8, 11 and 9%, respectively, in the 2019–2020 season. The absolute percentage error of the MV-Raw estimates was 10% or less in 15 of the 20 orchards assessed. Greater error in load estimation occurred in the 2020–2021 season due to the time-spread of flowering. Use cases for the tree level data on fruit load was explored in context of fruit load density maps to inform early harvesting and to interpret crop damage, and tree frequency distributions based on fruit load per tree.

The management and marketing of fruit requires data on expected numbers, size, quality and timing. Current practice estimates orchard fruit load based on the qualitative assessment of fruit number per tree and historical orchard yield, or manually counting a subsample of trees. This review considers technological aids assisting these estimates, in terms of: (i) improving sampling strategies by the number of units to be counted and their selection; (ii) machine vision for the direct measurement of fruit number and size on the canopy; (iii) aerial or satellite imagery for the acquisition of information on tree structural parameters and spectral indices, with the indirect assessment of fruit load; (iv) models extrapolating historical yield data with knowledge of tree management and climate parameters, and (v) technologies relevant to the estimation of harvest timing such as heat units and the proximal sensing of fruit maturity attributes. Machine vision is currently dominating research outputs on fruit load estimation, while the improvement of sampling strategies has potential for a widespread impact. Techniques based on tree parameters and modeling offer scalability, but tree crops are complicated (perennialism). The use of machine vision for flowering estimates, fruit sizing, external quality evaluation is also considered. The potential synergies between technologies are highlighted.

New strategies for spinal cord stimulation (SCS) for chronic pain have emerged in recent years, which may work better via different analgesic mechanisms than traditional low-frequency (e.g., 50 Hz) paresthesia-based SCS. To determine if 10 kHz and burst SCS waveforms might have a similar mechanistic basis, we examined whether these SCS strategies at intensities ostensibly below sensory thresholds would modulate spinal dorsal horn (DH) neuronal function in a neuron type-dependent manner. By using an in vivo electrophysiological approach in rodents, we found that low-intensity 10 kHz SCS, but not burst SCS, selectively activates inhibitory interneurons in the spinal DH. This study suggests that low-intensity 10 kHz SCS may inhibit pain-sensory processing in the spinal DH by activating inhibitory interneurons without activating DC fibers, resulting in paresthesia-free pain relief, whereas burst SCS likely operates via other mechanisms.

Thoroughbred jump racing sits in the spotlight of contemporary welfare and ethical debates about horse racing. In Australia, jump racing comprises hurdle and steeplechase races and has ceased in all but two states, Victoria and South Australia. This paper documents the size, geography, composition, and dynamics of Australian jump racing for the 2012, 2013, and 2014 seasons with a focus on debate about risks to horses. We found that the majority of Australian jump racing is regional, based in Victoria, and involves a small group of experienced trainers and jockeys. Australian jump horses are on average 6.4 years of age. The jump career of the majority of horses involves participating in three or less hurdle races and over one season. Almost one quarter of Australian jump horses race only once. There were ten horse fatalities in races over the study period, with an overall fatality rate of 5.1 fatalities per 1000 horses starting in a jump race (0.51%). There was significant disparity between the fatality rate for hurdles, 0.75 fatalities per 1000 starts (0.075%) and steeplechases, 14 fatalities per 1000 starts (1.4%). Safety initiatives introduced by regulators in 2010 appear to have significantly decreased risks to horses in hurdles but have had little or no effect in steeplechases. Our discussion considers these Animals 2015, 5 1073 data in light of public controversy, political debate, and industry regulation related to jump horse safety.

No abstract available.

Alzheimer’s disease is the leading cause of dementia in the elderly. Pathologically it is characterized by the presence of amyloid plaques and neuronal loss within the brain tissue of affected individuals. It is now widely hypothesised that fibrillar structures represent an inert structure. Biophysical and toxicity assays attempting to characterize the formation of both the fibrillar and the intermediate oligomeric structures of Aβ typically involves preparing samples which are largely monomeric; the most common method by which this is achieved is to use the fluorinated organic solvent 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP). Recent evidence has suggested that this method is not 100% effective in producing an aggregate free solution. We show, using dynamic light scattering, size exclusion chromatography and small angle X-ray scattering that this is indeed the case, with HFIP pretreated Aβ peptide solutions displaying an increased proportion of oligomeric and aggregated material and an increased propensity to aggregate. Furthermore we show that an alternative technique, involving treatment with strong alkali results in a much more homogenous solution that is largely monomeric. These techniques for solubilising and controlling the oligomeric state of Aβ are valuable starting points for future biophysical and toxicity assays.

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The scale-up of HIV services in sub-Saharan Africa has catalyzed the development of highly effective chronic care systems. The strategies, systems, and tools developed to support life-long HIV care and treatment are locally owned contextually appropriate resources, many of which could be adapted to support continuity care for noncommunicable chronic diseases (NCD), such as diabetes mellitus (DM). We conducted two proof-of-concept studies to further the understanding of the status of NCD programs and the feasibility and effectiveness of adapting HIV program-related tools and systems for patients with DM. In Swaziland, a rapid assessment illustrated gaps in the approaches used to support DM services at 15 health facilities, despite the existence of chronic care systems at HIV clinics in the same hospitals, health centers, and clinics. In Ethiopia, a pilot study found similar gaps in DM services at baseline and illustrated the potential to rapidly improve the quality of care and treatment for DM by adapting HIV-specific policies, systems, and tools.

By 2050, Africa's population is projected to exceed 2 billion. Africa will have to increase food production more than 50% in the coming 50 years to meet the nutritional requirements of its growing population. Nowhere is the need to increase agricultural productivity more pertinent than in much of Sub-Saharan Africa, where it is currently static or declining. Optimal pest management will be essential, because intensification of any system creates heightened selection pressures for pests. Plant-parasitic nematodes and their damage potential are intertwined with intensified systems and can be an indicator of unsustainable practices. As soil pests, nematodes are commonly overlooked or misdiagnosed, particularly where appropriate expertise and knowledge transfer systems are meager or inadequately funded. Nematode damage to roots results in less efficient root systems that are less able to access nutrients and water, which can produce symptoms typical of water or nutrient deficiency, leading to misdiagnosis of the underlying cause. Damage in subsistence agriculture is exacerbated by growing crops on degraded soils and in areas of low water retention where strong root growth is vital. This review focuses on the current knowledge of economically important nematode pests affecting key crops, nematode control methods and the research and development needs for sustainable management, stakeholder involvement and capacity building in the context of crop security in East and Southern Africa, especially Kenya, Tanzania, Uganda and Zimbabwe., (© 2015 Society of Chemical Industry.)

Root-knot nematodes are obligate parasites that invade roots and induce the formation of specialized feeding structures. Although physiological and molecular changes inside the root leading to feeding site formation have been studied, very little is known about the molecular events preceding root penetration by nematodes. In order to investigate the influence of root exudates on nematode gene expression before plant invasion and to identify new genes potentially involved in parasitism, sterile root exudates from the model plant Arabidopsis thaliana were produced and used to treat Meloidogyne incognita pre-parasitic second-stage juveniles. After confirming the activity of A. thaliana root exudates (ARE) on M. incognita stylet thrusting, six new candidate genes identified by cDNA-AFLP were confirmed by qRT-PCR as being differentially expressed after incubation for one hour with ARE. Using an in vitro inoculation method that focuses on the events preceding the root penetration, we show that five of these genes are differentially expressed within hours of nematode exposure to A. thaliana roots. We also show that these genes are up-regulated post nematode penetration during migration and feeding site initiation. This study demonstrates that preceding root invasion plant-parasitic nematodes are able to perceive root signals and to respond by changing their behaviour and gene expression.

European foredunes are almost exclusively colonised by Ammophila arenaria, and both the natural succession and the die-out of this plant have been linked to populations of plant-parasitic nematodes (PPN). The overarching aim of this study was to investigate top-down control processes of PPN in these natural ecosystems through comparative analyses of the diversity and dynamics of PPN and their microbial enemies. Our specific aims were, first, to identify and quantify PPN microbial enemies in European sand dunes; second, to assess their life history traits, their spatial and temporal variation in these ecosystems, and third, to evaluate their control potential of PPN populations. This was done by seasonal sampling of a range of sites and making observations on both the nematode and the microbial enemy communities in rhizosphere sand. Nine different nematode microbial enemies belonging to different functional groups were detected in European sand dunes. Their high diversity in these low productivity ecosystems could both result from or lead to the lack of dominance of a particular nematode genus. The distribution of microbial enemies was spatially and temporally variable, both among and within sampling sites. Obligate parasites, either with low host-specificity or having the ability to form an environmentally resistant propagule, are favoured in these ecosystems and are more frequent and abundant than facultative parasites. Three microbial enemies correlated, either positively or negatively, with PPN population size: Catenaria spp., Hirsutella rhossiliensis and Pasteuria penetrans. Microbial-enemy supported links in the food-web may be involved in the control of PPN populations through indirect effects. The endospore-forming P. penetrans was the most successful top-down control agent, and was implicated in the direct control of Meloidogyne spp. and indirect facilitation of Pratylenchus spp. Overall, our findings suggest strong and diverse top-down control effects on the nematode community in these natural ecosystems.

The alkaline serine protease VCP1 of the fungus Pochonia chlamydosporia belongs to a family of subtilisin-like enzymes that are involved in infection of nematode and insect hosts. It is involved early in the infection process, removing the outer proteinaceous vitelline membrane of nematode eggs. Little is known about the regulation of this gene, even though an understanding of how nutrients and other factors affect its expression is critical for ensuring its efficacy as a biocontrol agent. This paper provides new information on the regulation of vcp1 expression. Sequence analysis of the upstream regulatory region of this gene in 30 isolates revealed that it was highly conserved and contained sequence motifs characteristic of genes that are subject to carbon, nitrogen and pH-regulation. Expression studies, monitoring enzyme activity and mRNA, confirmed that these factors affect VCP1 production. As expected, glucose reduced VCP1 expression and for a few hours so did ammonium chloride. Surprisingly, however, by 24 h VCP1 levels were increased in the presence of ammonium chloride for most isolates. Ambient pH also regulated VCP1 expression, with most isolates producing more VCP1 under alkaline conditions. There were some differences in the response of one isolate with a distinctive upstream sequence including a variant regulatory-motif profile. Cryo-scanning electron microscopy studies indicated that the presence of nematode eggs stimulates VCP1 production by P. chlamydosporia, but only where the two are in close contact. Overall, the results indicate that readily-metabolisable carbon sources and unfavourable pH in the rhizosphere/egg-mass environment may compromise nematode parasitism by P. chlamydosporia. However, contrary to previous indications using other nematophagous and entomopathogenic fungi, ammonium nitrate (e.g. from fertilizers) may enhance biocontrol potential in some circumstances.

Protein-encoding and 16S rRNA genes of Pasteuria penetrans populations from a wide range of geographic locations were examined. Most interpopulation single nucleotide polymorphisms (SNPs) were detected in the 16S rRNA gene. However, in order to fully resolve all populations, these were supplemented with SNPs from protein-encoding genes in a multilocus SNP typing approach. Examination of individual 16S rRNA gene sequences revealed the occurrence of "cryptic" SNPs which were not present in the consensus sequences of any P. penetrans population. Additionally, hierarchical cluster analysis separated P. penetrans 16S rRNA gene clones into four groups, and one of which contained sequences from the most highly passaged population, demonstrating that it is possible to manipulate the population structure of this fastidious bacterium. The other groups were made from representatives of the other populations in various proportions. Comparison of sequences among three Pasteuria species, namely, P. penetrans, P. hartismeri, and P. ramosa, showed that the protein-encoding genes provided greater discrimination than the 16S rRNA gene. From these findings, we have developed a toolbox for the discrimination of Pasteuria at both the inter- and intraspecies levels. We also provide a model to monitor genetic variation in other obligate hyperparasites and difficult-to-culture microorganisms.

Expression profiles were identified in the fungus Pochonia chlamydosporia, a biological control agent of plant parasitic nematodes, through a cDNA-amplified fragment length polymorphism approach. Two isolates with different host ranges, IMI 380407 and IMI 331547, were assayed in conditions of saprotrophic-to-parasitic transition, through in vitro assays. Gene expression profiles from three different nutritional conditions and four sampling times were generated, with eggs of host nematodes Globodera pallida and Meloidogyne incognita. Expression of transcripts changed in RNA fingerprints obtained under different nutritional stresses (starvation in presence/absence of eggs, or rich growth media). Transcript derived fragments (TDFs) obtained from the expression profiles corresponded to 6,800 products. A subset was sequenced and their expression profile confirmed through RT PCR. A total of 57 TDFs were selected for further analysis, based on similarities to translated or annotated sequences. Genes expressed during egg parasitism for both IMI 380407 and IMI 331547 were involved in metabolic functions, cellular signal regulation, cellular transport, regulation of gene expression, DNA repair, and other unknown functions. Multivariate analysis of TDF expression showed three groups for IMI 380407 and one for IMI 331547, each characterized by expression of genes related to eggs parasitism. Common amplification profiles among TDF clusters from both isolates also reflected a pool of constitutive genes, not affected by the nutritional conditions and nematode associations, related to general metabolic functions. The differential expression of parasitism related genes suggest a network of induced/repressed products, playing a role in fungal signaling and infection, with partial overlaps in host infection and parasitism traits.

It has long been recognized that chemotaxis is the primary means by which nematodes locate host plants. Nonetheless, chemotaxis has received scant attention. We show that chemotaxis is predicted to take nematodes to a source of a chemo-attractant via the shortest possible routes through the labyrinth of air-filled or water-filled channels within a soil through which the attractant diffuses. There are just two provisos: (i) all of the channels through which the attractant diffuses are accessible to the nematodes and (ii) nematodes can resolve all chemical gradients no matter how small. Previously, this remarkable consequence of chemotaxis had gone unnoticed. The predictions are supported by experimental studies of the movement patterns of the root-knot nematodes Meloidogyne incognita and Meloidogyne graminicola in modified Y-chamber olfactometers filled with Pluronic gel. By providing two routes to a source of the attractant, one long and one short, our experiments, the first to demonstrate the routes taken by nematodes to plant roots, serve to test our predictions. Our data show that nematodes take the most direct route to their preferred hosts (as predicted) but often take the longest route towards poor hosts. We hypothesize that a complex of repellent and attractant chemicals influences the interaction between nematodes and their hosts.

Aims: To establish a reliable protocol to extract DNA from Pasteuria penetrans endospores for use as template in multiple strand amplification, thus providing sufficient material for genetic analyses. To develop a highly sensitive PCR-based diagnostic tool for P. penetrans., Methods and Results: An optimized method to decontaminate endospores, release and purify DNA enabled multiple strand amplification. DNA purity was assessed by cloning and sequencing gyrB and 16S rRNA gene fragments obtained from PCR using generic primers. Samples indicated to be 100%P. penetrans by the gyrB assay were estimated at 46% using the 16S rRNA gene. No bias was detected on cloning and sequencing 12 housekeeping and sporulation gene fragments from amplified DNA. The detection limit by PCR with Pasteuria-specific 16S rRNA gene primers following multiple strand amplification of DNA extracted using the method was a single endospore., Conclusions: Generation of large quantities DNA will facilitate genomic sequencing of P. penetrans. Apparent differences in sample purity are explained by variations in 16S rRNA gene copy number in Eubacteria leading to exaggerated estimations of sample contamination. Detection of single endospores will facilitate investigations of P. penetrans molecular ecology., Significance and Impact of the Study: These methods will advance studies on P. penetrans and facilitate research on other obligate and fastidious micro-organisms where it is currently impractical to obtain DNA in sufficient quantity and quality.

Aims: To develop a simple, rapid, reliable protocol producing consistent polymerase chain reaction (PCR) fingerprints of Pochonia chlamydosporia var. chlamydosporia biotypes for analysing different fungal isolates during co-infection of plants and nematodes., Methods and Results: DNA extracted from different P. chlamydosporia biotypes was fingerprinted using enterobacterial repetitive intragenic consensus (ERIC)-PCR. Four extraction methods (rapid alkaline lysis; microLYSIS-PLUS; DNeasy; FTA cards) gave consistent results within each protocol but these varied between protocols. Reproducible fingerprints were obtained only if DNA was extracted from fresh fungal cultures that were free of agar. Some DNA degradation occurred during storage, except with the FTA cards, used with this fungus for the first time, which provide a method for long-term archiving. Rapid alkaline lysis and ERIC-PCR identified fungal isolates from root and nematode egg surfaces when plants were treated with different combinations of fungal biotypes; the dominant biotype isolated from the rhizosphere was not always the most abundant in eggs., Conclusions: ERIC-PCR fingerprinting can reliably detect and identify different P. chlamydosporia biotypes. It is important to use fresh mycelium and the same DNA isolation method throughout each study., Significance and Impact of the Study: This evaluation of methods to assess genetic diversity and identify specific P. chlamydosporia biotypes is relevant to other mycelial fungi.

Cysteine proteinases from the fruit and latex of plants, such as papaya, pineapple and fig, have previously been shown to have substantial anthelmintic efficacy, in vitro and in vivo, against a range of animal parasitic nematodes. In this paper, we describe the in vitro effects of these plant extracts against 2 sedentary plant parasitic nematodes of the genera Meloidogyne and Globodera. All the plant extracts examined caused digestion of the cuticle and decreased the activity of the tested nematodes. The specific inhibitor of cysteine proteinases, E-64, blocked this activity completely, indicating that it was essentially mediated by cysteine proteinases. In vitro, plant cysteine proteinases are active against second-stage juveniles of M. incognita and M. javanica, and some cysteine proteinases also affect the second-stage juveniles of Globodera rostochiensis. It is not known yet whether these plant extracts will interfere with, or prevent invasion of, host plants.

The Pasteuria group of endospore-forming bacteria has been studied as a biocontrol agent of plant-parasitic nematodes. Techniques have been developed for its detection and quantification in soil samples, and these mainly focus on observations of endospore attachment to nematodes. Characterization of Pasteuria populations has recently been performed with DNA-based techniques, which usually require the extraction of large numbers of spores. We describe a simple immunological method for the quantification and characterization of Pasteuria populations. Bayesian statistics were used to determine an extraction efficiency of 43% and a threshold of detection of 210 endospores g(-1) sand. This provided a robust means of estimating numbers of endospores in small-volume samples from a natural system. Based on visual assessment of endospore fluorescence, a quantitative method was developed to characterize endospore populations, which were shown to vary according to their host.

Pasteuria penetrans is a gram-positive, endospore-forming eubacterium that apparently is a member of the Bacillus-Clostridium clade. It is an obligate parasite of root knot nematodes (Meloidogyne spp.) and preferentially grows on the developing ovaries, inhibiting reproduction. Root knot nematodes are devastating root pests of economically important crop plants and are difficult to control. Consequently, P. penetrans has long been recognized as a potential biocontrol agent for root knot nematodes, but the fastidious life cycle and the obligate nature of parasitism have inhibited progress on mass culture and deployment. We are currently sequencing the genome of the Pasteuria bacterium and have performed amino acid level analyses of 33 bacterial species (including P. penetrans) using concatenation of 40 housekeeping genes, with and without insertions/deletions (indels) removed, and using each gene individually. By application of maximum-likelihood, maximum-parsimony, and Bayesian methods to the resulting data sets, P. penetrans was found to cluster tightly, with a high level of confidence, in the Bacillus class of the gram-positive, low-G+C-content eubacteria. Strikingly, our analyses identified P. penetrans as ancestral to Bacillus spp. Additionally, all analyses revealed that P. penetrans is surprisingly more closely related to the saprophytic extremophile Bacillus haladurans and Bacillus subtilis than to the pathogenic species Bacillus anthracis and Bacillus cereus. Collectively, these findings strongly imply that P. penetrans is an ancient member of the Bacillus group. We suggest that P. penetrans may have evolved from an ancient symbiotic bacterial associate of nematodes, possibly as the root knot nematode evolved to be a highly specialized parasite of plants.

The microbial and nematode populations associated with two plants (tomato and cabbage) inoculated with the nematophagous fungus, Pochonia chlamydosporia var. chlamydosporia or root knot nematode (Meloidogyne incognita), or both, were compared with those in unplanted controls. The dominant factor affecting culturable microbial populations was found to be the presence or absence of tomato plants. Generally microbial colony counts were lowest in unplanted soil, small increases were associated with cabbage and significantly greater numbers with tomato plants. Differences in microbial diversity (estimated from community profiles of carbon substrate utlisation, using Biolog) were observed between planted and unplanted soils, however, there were few differences between soils with either of the two plants. The presence of P. chlamydosporia was associated with a reduction in the numbers of plant parasitic nematodes (51%-78%) including the migratory ectoparasites, whereas free-living nematodes, culturable bacteria and bacterial populations assessed by Biolog were unaffected by the application of fungus.

The Paecilomyces lilacinus is the most widely tested fungus for the control of root-knot and cyst nematodes. The fungus has also been implicated in a number of human and animal infections, difficulties in diagnosis often result in misdiagnosis or delays in identification leading to a delay in treatment. Here, we report the development of species-specific primers for the identification of P. lilacinus based on sequence information from the ITS gene, and their use in identifying P. lilacinus isolates, including clinical isolates of the fungus. The primer set generated a single PCR fragment of 130 bp in length that was specific to P. lilacinus and was also used to detect the presence of P. lilacinus from soil, roots and nematode eggs. Real-time PCR primers and a TaqMan probe were also developed and provided quantitative data on the population size of the fungus in two field sites. PCR, bait and culture methods were combined to investigate the presence and abundance of the fungus from two field sites in the United Kingdom where potato cyst nematode populations were naturally declining, and results demonstrated the importance of using a combination of methods to investigate population size and activity of fungi.

The nematophagous fungus Pochonia chlamydosporia is a potential biocontrol agent against root knot and cyst nematodes. Genetic transformation of the fungus to introduce visual marker genes, novel traits, or changes in expression levels of endogenous genes, would greatly enhance understanding of its behaviour on nematode-infested roots and of its interactions with other soil and rhizosphere microorganisms. A transformation system for the introduction of novel genes into P. chlamydosporia has been developed. Methods to generate protoplasts, introduce DNA and regenerate transformed viable fungal mycelium have been optimised, using plasmids carrying the green fluorescent protein marker gene gfp and the hygromycin resistance gene hph. Cultures of P. chlamydosporia were resistant to high levels of a range of fungal inhibitors, including hygromycin, that are commonly used with dominant selectable marker genes in the transformation of other fungi. However, regenerating protoplasts transformed with hph could be selected by their ability to grow through an agar overlay containing 1 mg ml(-1) hygromycin. Green fluorescence was observed in protoplasts and regenerating mycelium after transformation with gfp, but the GFP phenotype was lost on subculture. Maintenance of introduced genes was not stable, and during subculture, PCR assays indicated that the transformants lost both hph and gfp. When these genes were introduced on the same plasmid, segregation of hph and gfp was observed prior to their loss. It was unclear whether the introduced plasmids were able to replicate autonomously in P. chlamydosporia, or if they integrated transiently into the fungal genome. Possible reasons for the instability of the transformants are discussed.

A RAPD-PCR assay was developed and used to test for competitive variability in growth of the nematode biological control fungus Pochonia chlamydosporia. Saprophytic competence in soil with or without tomato plants was examined in three isolates of the fungus: RES 280 (J), originally isolated from potato cyst nematode (PCN) cysts; RES 200 (I) and RES 279 (S), both originally isolated from root knot nematode (RKN) eggs. Viable counts taken at 70 d indicated that I was the best saprophyte followed by S, with J the poorest. RAPD-PCR analysis of colonies from mixed treatments revealed that there was a cumulative effect of adding isolates to the system. This suggested that the isolates did not interact and that they may occupy separate niches in soil and the rhizosphere. To investigate parasitic ability, soils were seeded with two isolates of the fungus: J and S, singly or in combination. Tomato or potato plants were grown in these soils: free of nematodes, or inoculated with PCN or RKN, and incubated for 77 d. The abundance of the PCN isolate J in PCN cysts was significantly greater than that of the RKN isolate S but in RKN egg masses, S was significantly more abundant than J. RAPD-PCR analysis of colonies from mixed treatments confirmed that J was more abundant than S in PCN cysts whereas the converse was observed on RKN egg masses. This substantiates the phenomenon of nematode host preference at the infraspecific level of P. chlamydosporia and highlights its relevance for biological control of plant parasitic nematodes.

Potato cyst nematodes (PCN) are serious pests in commercial potato production, causing yield losses valued at approximately $300 million in the European Community. The nematophagous fungus Plectosphaerella cucumerina has demonstrated its potential as a biological control agent against PCN populations by reducing field populations by up to 60% in trials. The use of biological control agents in the field requires the development of specific techniques to monitor the release, population size, spread or decline, and pathogenicity against its host. A range of methods have therefore been developed to monitor P. cucumerina. A species-specific PCR primer set (PcCF1-PcCR1) was designed that was able to detect the presence of P. cucumerina in soil, root, and nematode samples. PCR was combined with a bait method to identify P. cucumerina from infected nematode eggs, confirming the parasitic ability of the fungus. A selective medium was adapted to isolate the fungus from root and soil samples and was used to quantify the fungus from field sites. A second P. cucumerina-specific primer set (PcRTF1-PcRTR1) and a Taqman probe (PcRTP1) were designed for real-time PCR quantification of the fungus and provided a very sensitive means of detecting the fungus from soil. PCR, bait, and culture methods were combined to investigate the presence and abundance of P. cucumerina from two field sites in the United Kingdom where PCN populations were naturally declining. All methods enabled differences in the activity of P. cucumerina to be detected, and the results demonstrated the importance of using a combination of methods to investigate population size and activity of fungi.

The nematophagous fungus, Pochonia chlamydosporia (Goddard) Zare & Gams, has been investigated as a potential biological control agent for use in integrated pest management strategies for Meloidogyne incognita (Kof & White) Chitwood in vegetable crops. The release of the fungus as a biological control agent requires a diagnostic method to monitor its spread in the environment and to gain knowledge of its ecology. Only molecular methods are sufficiently discriminating to enable the detection of specific isolates of fungi in soil. A method to extract DNA from soil was developed to increase the efficacy of PCR-based diagnostic tests that use specific primers. A selected isolate of P chlamydosporia var catenulata was applied at densities similar to those that occur naturally in nematode-suppressive soils. The fungus significantly reduced nematode infestations in soil following a tomato crop, in a strategy that combined the use of the fungus with crop rotation. The survival of the fungus in soil was also examined in controlled conditions in which it remained in soil in densities significantly greater than its original application rate for at least 5 months. Hence, it seems that populations of this fungus may be built up in soil and have significant effects on the regulation of root-knot nematode populations.

Pochonia chlamydosporia var. catenulata is a potential biocontrol agent against root-knot nematodes. Diagnosis of isolates has relied on morphological identification, and is both time-consuming and difficult. beta-tubulin primers have been developed for the identification of this fungus that were specific enough to distinguish between varieties of the fungus within the same species. Separate primers have been developed for the specific detection of P. chlamydosporia var. catenulata based on ITS sequences, which were able to detect the fungus in soil from various sites in Cuba where the biocontrol agent had been added. When the PCR diagnosis was combined with serial dilution of soil samples on selective medium, colonies were rapidly identified. The fungus was still present, albeit at low densities, in soils inoculated five years previously. The development of a baiting method allowed quick in situ screening of the isolates' ability to infect nematode eggs, and when combined with PCR diagnosis both varieties of the fungus could be detected in infected eggs. RFLP analysis of ITS sequences from P. chlamydosporia provided an extra level of discrimination between isolates.

The mitosporic fungus Pochonia chlamydosporia is a potential biocontrol agent for cyst (Heterodera spp. and Globodera spp.) and root knot (Meloidogyne spp.) nematodes, which are important agricultural plant pests. 54 isolates from diverse geographical regions and several nematode hosts were used in this study. Genetic variation was examined using enterobacterial repetitive intergenic consensus (ERIC) primed PCR and sequences from the internal transcribed spacer (ITS) rRNA region. ERIC PCR yielded 35 scorable binary characters from all the fungi tested and cluster analysis of the data showed that isolates from cyst nematodes were more genetically variable than those from root knot nematodes. The ITS regions were highly conserved, the only significant difference being an extra thymidine in isolates from Meloidogyne spp. Assays with nematode eggs indicated that isolates differ in their ability to infect different nematode genera. The results indicate host related variation in P. chlamydosporia. This finding has significant implications for the application of P. chlamydosporia as a biocontrol agent.