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BackgroundCoronavirus disease 2019 (COVID-19) is an immunoinflammatory and hypercoagulable state that contributes to respiratory distress, multi-organ dysfunction, and mortality. Dipyridamole, by increasing extracellular adenosine, has been postulated to be protective for COVID-19 patients through its immunosuppressive, anti-inflammatory, anti-coagulant, vasodilatory, and anti-viral actions. Likewise, low-dose aspirin has also demonstrated protective effects for COVID-19 patients. This study evaluated the effect of these two drugs formulated together as Aggrenox in hospitalized COVID-19 patients.MethodsIn an open-label, single site randomized controlled trial (RCT), hospitalized COVID-19 patients were assigned to adjunctive Aggrenox (Dipyridamole ER 200mg/ Aspirin 25mg orally/enterally) with standard of care treatment compared to standard of care treatment alone. Primary endpoint was illness severity according to changes on the eight-point COVID ordinal scale, with levels of 1 to 8 where higher scores represent worse illness. Secondary endpoints included all-cause mortality and respiratory failure. Outcomes were measured through days 14, 28, and/or hospital discharge.ResultsFrom October 1, 2020 to April 30, 2021, a total of 98 patients, who had a median [IQR] age of 57 [47, 62] years and were 53.1% (n = 52) female, were randomized equally between study groups (n = 49 Aggrenox plus standard of care versus n = 49 standard of care alone). No clinically significant differences were found between those who received adjunctive Aggrenox and the control group in terms of illness severity (COVID ordinal scale) at days 14 and 28. The overall mortality through day 28 was 6.1% (3 patients, n = 49) in the Aggrenox group and 10.2% (5 patients, n = 49) in the control group (OR [95% CI]: 0.40 [0.04, 4.01], p = 0.44). Respiratory failure through day 28 occurred in 4 (8.3%, n = 48) patients in the Aggrenox group and 7 (14.6%, n = 48) patients in the standard of care group (OR [95% CI]: 0.21 [0.02, 2.56], p = 0.22). A larger decrease in the platelet count and blood glucose levels, and larger increase in creatinine and sodium levels within the first 7 days of hospital admission were each independent predictors of 28-day mortality (p < 0.05).ConclusionIn this study of hospitalized patients with COVID-19, while the outcomes of COVID illness severity, odds of mortality, and chance of respiratory failure were better in the Aggrenox group compared to standard of care alone, the data did not reach statistical significance to support the standard use of adjuvant Aggrenox in such patients.

Mycobacterium tuberculosis (M.tb) causes tuberculosis (TB) and remains one of the leading causes of mortality due to an infectious pathogen. Host immune responses have been implicated in driving the progression from infection to severe lung disease. We analyzed longitudinal RNA sequencing (RNAseq) data from the whole blood of 74 TB progressors whose samples were grouped into four six-month intervals preceding diagnosis (the GC6-74 study). We additionally analyzed RNAseq data from an independent cohort of 90 TB patients with positron emission tomography-computed tomography (PET-CT) scan results which were used to categorize them into groups with high and low levels of lung damage (the Catalysis TB Biomarker study). These groups were compared to non-TB controls to obtain a complete whole blood transcriptional profile for individuals spanning from early stages of M.tb infection to TB diagnosis. The results revealed a steady increase in the number of genes that were differentially expressed in progressors at time points closer to diagnosis with 278 genes at 13-18 months, 742 at 7-12 months and 5,131 detected 1-6 months before diagnosis and 9,205 detected in TB patients. A total of 2,144 differentially expressed genes were detected when comparing TB patients with high and low levels of lung damage. There was a large overlap in the genes upregulated in progressors 1-6 months before diagnosis (86%) with those in TB patients. A comprehensive pathway analysis revealed a potent activation of neutrophil and platelet mediated defenses including neutrophil and platelet degranulation, and NET formation at both time points. These pathways were also enriched in TB patients with high levels of lung damage compared to those with low. These findings suggest that neutrophils and platelets play a critical role in TB pathogenesis, and provide details of the timing of specific effector mechanisms that may contribute to TB lung pathology.

ObjectiveRetention to complete follow-up surveys in extensive longitudinal epidemiological cohort studies is vital yet challenging. All of Us developed pilot interventions to improve response rates for follow-up surveys.Study design and settingThe pilot interventions occurred from April 27, 2020, to August 3, 2020. The three arms were: (1) telephone appointment [staff members calling participants offering appointments to complete surveys over phone] (2) postal [mail reminder to complete surveys through U.S. Postal Service], and (3) combination of telephone appointment and postal. Controls received digital-only reminders [program-level digital recontact via email or through the participant portal]. Study sites chose their study arm and participants were not randomized.ResultsA total of 50 sites piloted interventions with 17,593 participants, while 47,832 participants comprised controls during the same period. Of all participants, 6,828 (10.4%) completed any follow-up surveys (1448: telephone; 522: postal; 486: combination; 4372: controls). Follow-up survey completions were 24% higher in the telephone appointment arm than in controls in bivariate analyses. When controlling for confounders, telephone appointment and combination arms increased rates of completion similarly compared to controls, while the postal arm had no significant effect (odds ratio [95% Confidence Interval], telephone appointment:2.01[1.81-2.23]; combination:1.91[1.66-2.20]; postal:0.92[0.79-1.07]). Although the effects of the telephone appointment and combination arms were similar, differential effects were observed across sub-populations.ConclusionTelephone appointments appeared to be the most successful intervention in our study. Lessons learned about retention interventions, and improvement in follow-up survey completion rates provide generalizable knowledge for similar cohort studies and demonstrate the potential value of precision reminders and engagement with sub-populations of a cohort.

In accordance with ancient Egyptian beliefs, the preservation of the body after death was an important prerequisite for the continued existence of the deceased in the afterlife. This involved application of various physical interventions and magical rituals to the corpse. Computed tomography (CT), as the gold-standard technology in the field of paleoradiology, enables deeper insights into details of artificial body preservation. Therefore, CT was applied to investigate age at death, sex, mummification techniques, and the state of soft tissue preservation in 21 child mummies. The specimens are housed in European museums and were found in various archaeological sites. This sample included 12 males, 7 females, and two specimens of unknown sex, all between the ages of one and 14 years. Time periods were determined by radiocarbon dating, and time-related indicators of funerary equipment and decoration. Most of the mummies date from the Late Period to the Roman Period (664 BC-395 AD). Differences were identified concerning techniques of wrapping and embalming and removal of brain and internal organs, depending on age at death, social status and the archaeological site of the individuals. The variety and multifaceted nature of mummification and soft tissue preservation was analyzed in an integrated approach including a large number of specimens. The study highlights the significance of subadult remains as valuable bioarcheological archives to investigate burial customs and religious concepts in past societies.

IntroductionFocused deterrence (FD) is a frequently cited intervention for preventing violence, particularly against violent urban gangs. The Youth Endowment Fund (YEF) believes it could be effective in the UK, based primarily on research conducted in the US. However, we contend that these studies have inadequate methodological designs, lack of rigorous testing, and small sample sizes. Therefore, the evidence supporting focused deterrence as an effective method, particularly outside the US, is inconclusive. The aim of the protocol is to better understand the potential effects of FD in the context of the UK, using a multisite evaluation experimental design to more closely investigate the evidence of its likely impact.MethodsWe planned a realist randomised controlled trial. The design is focused on a multisite trial consisting of two-arm randomised experiments in five locations. Each trial location will test their implementation of a core programme specified by the funder. The multisite nature will allow us to understand differential impacts between locations, improving the external validity of the results. Participants will be randomly selected from a wider pool of eligible individuals for the intervention. We estimate a sample size of approximately N = 1,700 individuals is required. Based on this pooled sample size, a relative reduction of 26% would be detectable in 80% of trials. The trial is coupled with a formative process evaluation of delivery and fidelity. The formative evaluation will use a mixed methods design. The qualitative aspect will include semi-structured cross-sectional and longitudinal interviews with programme leads, programme delivery team, and programme participants, as well as observations of the meetings between the programme delivery team (i.e., community navigators/mentors) and programme participants. The quantitative data for the formative evaluation will be gathered by the sites themselves and consist of routine outcome performance monitoring using administrative data. Sampling for interviews and observations will vary, with the researchers aiming for a higher number of individuals included in the first round of cross-sectional interviews and retaining as many as possible for repeat interviews and observations.DiscussionThis protocol outlines the process and impact evaluation methodology for the most extensive multisite evaluation of focused deterrence to date in the UK. Spanning five distinct sites with seven trials, the evaluation includes a cohort of 2,000 individuals, marking it as the only multisite trial of focused deterrence. Employing an integrated realist evaluation framework, the study uses qualitative and quantitative research methods. The anticipated findings will offer pivotal insights for formulating future violence prevention policies in the UK. They are also expected to contribute significantly to the corpus of literature on violence prevention and intervention evaluation.Trial registrationProtocol registration: ISRCTN: 11650008 4th June 2023.

IntroductionBreast cancer poses a significant health challenge in Sub-Saharan Africa, particularly in Ghana, where late-stage diagnoses and limited healthcare access contribute to elevated mortality rates. This study focuses on the crucial role of pathology and laboratory medical (PALM) services in the timely diagnosis of breast cancer within Ghana.MethodsA cross-sectional survey of hospitals was completed from November 2020 to October 2021, with 94.8% of identified in-country hospitals participating. Pathology service-related parameters assessed included whether pathology was available for the diagnosis of breast cancer on-site or via external referral, the number of pathology personnel, additional breast cancer diagnostic capabilities including estrogen and progesterone and/or HER2 testing, and the time from biopsy to patients receiving their results. Geospatial mapping was used to identify areas of limited access.ResultsOf the 328 participating hospitals, 136 (41%) reported breast cancer pathology services, with only 6 having on-site capabilities. Pathology personnel, comprising 15 consultants and 15 specialists, were concentrated in major referral centers, particularly in Greater Accra and Kumasi. An assessment of referral patterns suggested that 75% of the population reside within an hour of breast cancer pathology services. Among the 136 hospitals with access to breast cancer pathology, only a limited number reported that results included ER/PR (38%) and HER2 testing (33%).ConclusionGhana has been able to ensure significant pathology service availability through robust referral pathways with centralized labs. Despite this, difficulties persist with the majority of pathology results not including hormone receptor testing which is important in providing tumor specific treatment.

Background Coronavirus disease 2019 (COVID-19) is an immunoinflammatory and hypercoagulable state that contributes to respiratory distress, multi-organ dysfunction, and mortality. Dipyridamole, by increasing extracellular adenosine, has been postulated to be protective for COVID-19 patients through its immunosuppressive, anti-inflammatory, anti-coagulant, vasodilatory, and anti-viral actions. Likewise, low-dose aspirin has also demonstrated protective effects for COVID-19 patients. This study evaluated the effect of these two drugs formulated together as Aggrenox in hospitalized COVID-19 patients. Methods In an open-label, single site randomized controlled trial (RCT), hospitalized COVID-19 patients were assigned to adjunctive Aggrenox (Dipyridamole ER 200mg/ Aspirin 25mg orally/enterally) with standard of care treatment compared to standard of care treatment alone. Primary endpoint was illness severity according to changes on the eight-point COVID ordinal scale, with levels of 1 to 8 where higher scores represent worse illness. Secondary endpoints included all-cause mortality and respiratory failure. Outcomes were measured through days 14, 28, and/or hospital discharge. Results From October 1, 2020 to April 30, 2021, a total of 98 patients, who had a median [IQR] age of 57 [47, 62] years and were 53.1% (n = 52) female, were randomized equally between study groups (n = 49 Aggrenox plus standard of care versus n = 49 standard of care alone). No clinically significant differences were found between those who received adjunctive Aggrenox and the control group in terms of illness severity (COVID ordinal scale) at days 14 and 28. The overall mortality through day 28 was 6.1% (3 patients, n = 49) in the Aggrenox group and 10.2% (5 patients, n = 49) in the control group (OR [95% CI]: 0.40 [0.04, 4.01], p = 0.44). Respiratory failure through day 28 occurred in 4 (8.3%, n = 48) patients in the Aggrenox group and 7 (14.6%, n = 48) patients in the standard of care group (OR [95% CI]: 0.21 [0.02, 2.56], p = 0.22). A larger decrease in the platelet count and blood glucose levels, and larger increase in creatinine and sodium levels within the first 7 days of hospital admission were each independent predictors of 28-day mortality (p < 0.05). Conclusion In this study of hospitalized patients with COVID-19, while the outcomes of COVID illness severity, odds of mortality, and chance of respiratory failure were better in the Aggrenox group compared to standard of care alone, the data did not reach statistical significance to support the standard use of adjuvant Aggrenox in such patients.

Diarrheal diseases are a leading cause of mortality and morbidity, disproportionally affecting persons residing in low and middle-income countries. Accessing high-resolution surveillance data to understand community-level etiology and risk remains challenging, particularly in remote and resource limited populations. A multi-year prospective cohort study was conducted in two rural and two peri-urban villages in Cambodia from 2012 to 2018 to describe the epidemiology and etiology of acute diarrheal diseases within the population. Suspected diarrheal episodes among participants were self-reported or detected via routine weekly household visits. Fresh stool and fecal swabs were tested, and acute-illness and follow-up participant questionnaires collected. Of 5027 enrolled participants, 1450 (28.8%) reported at least one diarrheal incident. A total of 4266 individual diarrhea case events were recorded. Diarrhea incidence rate was calculated to be 281.5 persons per 1000 population per year, with an event rate of 664.3 individual diarrhea events occurring per 1000 population per year. Pathogenic Escherichia coli, Aeromonas spp., and Plesiomonas shigelloides were the most prevalent bacterial infections identified. Hookworm and Strongyloides stercoralis were the predominant helminth species, while Blastocystis hominis and Giardia lamblia were the predominant protozoan species found. Norovirus genotype 2 was the predominant virus identified. Mixed infections of two or more pathogens were detected in 36.2% of positive cases. Risk analyses identified unemployed status increased diarrhea risk by 63% (HR = 1.63 [95% CI 1.46, 1.83]). Individuals without access to protected water sources or sanitation facilities were 59% (HR = 1.59 [95% CI 1.49, 1.69]) and 19% (HR = 1.19 [95% CI 1.12, 1.28]) greater risk of contracting diarrhea, respectively. Patient-level surveillance data captured in this long-term study has generated a unique spatiotemporal profile of diarrheal disease in Cambodia. Understanding etiologies, together with associated epidemiological and community-level risk, provides valuable public health insight to support effective planning and delivery of appropriate local population-targeted interventions.

IntroductionParkinson's disease (PD) is the most common movement disorder, and its prevalence is increasing rapidly worldwide with an ageing population. The UK Biobank is the world's largest and most comprehensive longitudinal study of ageing community volunteers. The cause of the common form of PD is multifactorial, but the degree of causal heterogeneity among patients or the relative importance of one risk factor over another is unclear. This is a major impediment to the discovery of disease-modifying therapies.MethodsWe used an integrated machine learning algorithm (IDEARS) to explore the relative effects of 1,753 measured non-genetic variables in 334,062 eligible UK Biobank participants, including 2,719 who had developed PD since their recruitment into the study.ResultsMale gender was the highest-ranked risk factor, followed by elevated serum insulin-like growth factor 1 (IGF-1), lymphocyte count, and neutrophil/lymphocyte ratio. A group of factors aligned with the symptoms of frailty also ranked highly. IGF-1 and neutrophil/lymphocyte ratio were also elevated in both sexes before PD diagnosis and at the point of diagnosis.DiscussionThe use of machine learning with the UK Biobank provides the best opportunity to explore the multidimensional nature of PD. Our results suggest that novel risk biomarkers, including elevated IGF-1 and NLR, may play a role in, or are indicative of PD pathomechanisms. In particular, our results are consistent with PD being a central manifestation of a systemic inflammatory disease. These biomarkers may be used clinically to predict future PD risk, improve early diagnosis and provide new therapeutic avenues.

Many publications lack sufficient background information (e.g. location) to be interpreted, replicated, or reused for synthesis. This impedes scientific progress and the application of science to practice. Reporting guidelines (e.g. checklists) improve reporting standards. They have been widely taken up in the medical sciences, but not in ecological and agricultural research. Here, we use a community-centred approach to develop a reporting checklist (AgroEcoList 1.0) through surveys and workshops with 23 experts and the wider agroecological community. To put AgroEcoList in context, we also assessed the agroecological community's perception of reporting standards in agroecology. A total of 345 researchers, reviewers, and editors, responded to our survey. Although only 32% of respondents had prior knowledge of reporting guidelines, 76% of those that had said guidelines improved reporting standards. Overall, respondents agreed on the need of AgroEcolist 1.0; only 24% of respondents had used reporting guidelines before, but 78% indicated they would use AgroEcoList 1.0. We updated AgroecoList 1.0 based on respondents' feedback and user-testing. AgroecoList 1.0 consists of 42 variables in seven groups: experimental/sampling set-up, study site, soil, livestock management, crop and grassland management, outputs, and finances. It is presented here, and is also available on github (https://github.com/AgroecoList/Agroecolist). AgroEcoList 1.0 can serve as a guide for authors, reviewers, and editors to improve reporting standards in agricultural ecology. Our community-centred approach is a replicable method that could be adapted to develop reporting checklists in other fields. Reporting guidelines such as AgroEcoList can improve reporting standards and therefore the application of research to practice, and we recommend that they are adopted more widely in agriculture and ecology.

Breast cancer in Ghana is a growing public health problem with increasing incidence and poor outcomes. Lack of access to comprehensive treatment in Ghana may be a contributing factor to its high mortality. The purpose of this study was to evaluate the availability of treatments nationwide and systematically identify high yield areas for targeted expansion. We conducted a cross-sectional, nationwide hospital-based survey from November 2020-October 2021. Surveys were conducted in person with trained research assistants and described hospital availability of all breast cancer treatments and personnel. All individual treatment services were reported, and hospitals were further stratified into levels of multi-modal treatment modeled after the National Comprehensive Cancer Network (NCCN) Framework treatment recommendations for low-resource settings. Level 3 included Tamoxifen and surgery (mastectomy with axillary lymph node sampling); Level 2 included Level 3 plus radiation, aromatase inhibitors, lumpectomy, and sentinel lymph node biopsy; Level 1 included Level 2 plus Her2 therapy and breast reconstruction. Hospitals were identified that could expand to these service levels based on existing services, location and personnel. The distance of the total population from treatment services before and after hypothetical expansion was determined with a geospatial analysis. Of the 328 participating hospitals (95% response rate), 9 hospitals had Level 3 care, 0 had Level 2, and 2 had Level 1. Twelve hospitals could expand to Level 3, 1 could expand to Level 2, and 1 could expand to Level 1. With expansion, the population percentage within 75km of Level 1, 2 and 3 care would increase from 42% to 50%, 0 to 6% and 44% to 67%, respectively. Multi-modal breast cancer treatment is available in Ghana, but it is not accessible to most of the population. Leveraging the knowledge of current resources and population proximity provides an opportunity to identify high-yield areas for targeted expansion.

For adoptive cell transfer (ACT) immunotherapy of tumor-reactive T cells, an effective therapeutic outcome depends upon cell dose, cell expansion in vivo through a minimally differentiated phenotype, long term persistence, and strong cytolytic effector function. An incomplete understanding of the biological coupling between T cell expansion, differentiation, and response to stimulation hinders the co-optimization of these factors. We report on a biophysical investigation of how the short-term kinetics of T cell functional activation, through molecular stimulation and cell-cell interactions, competes with phenotype differentiation. T cells receive molecular stimulation for a few minutes to a few hours in bulk culture. Following this priming period, the cells are then analyzed at the transcriptional level, or isolated as single cells, with continuing molecular stimulation, within microchambers for analysis via 11-plex secreted protein assays. We resolve a rapid feedback mechanism, promoted by T cell-T cell contact interactions, which strongly amplifies T cell functional performance while yielding only minimal phenotype differentiation. When tested in mouse models of ACT, optimally primed T cells lead to complete tumor eradication. A similar kinetic process is identified in CD8+ and CD4+ T cells collected from a patient with metastatic melanoma.

In accordance with ancient Egyptian beliefs, the preservation of the body after death was an important prerequisite for the continued existence of the deceased in the afterlife. This involved application of various physical interventions and magical rituals to the corpse. Computed tomography (CT), as the gold-standard technology in the field of paleoradiology, enables deeper insights into details of artificial body preservation. Therefore, CT was applied to investigate age at death, sex, mummification techniques, and the state of soft tissue preservation in 21 child mummies. The specimens are housed in European museums and were found in various archaeological sites. This sample included 12 males, 7 females, and two specimens of unknown sex, all between the ages of one and 14 years. Time periods were determined by radiocarbon dating, and time-related indicators of funerary equipment and decoration. Most of the mummies date from the Late Period to the Roman Period (664 BC–395 AD). Differences were identified concerning techniques of wrapping and embalming and removal of brain and internal organs, depending on age at death, social status and the archaeological site of the individuals. The variety and multifaceted nature of mummification and soft tissue preservation was analyzed in an integrated approach including a large number of specimens. The study highlights the significance of subadult remains as valuable bioarcheological archives to investigate burial customs and religious concepts in past societies. [ABSTRACT FROM AUTHOR]

Background: The 5-year survival rates for breast and cervical cancers in Ghana are low in comparison to rates in developed countries. This striking disparity is attributed to numerous factors, including limited access and navigability to appropriate services. A one-time cross-sectional, hospital-based survey was performed by the University of Utah in collaboration with Ghana Health Services (GHS) and Health Facilities Regulatory Agency (HeFRA) from November, 2020 to October, 2021 so as to determine existing hospital-based breast and cervical cancer care services capacity and their geographic availability nationwide. This related information remains dynamic in nature and time. The current project employs a public-academic implementation science and research configuration to explore and develop a real-time interface (RTIF) showing the availability of breast and cervical cancer care services at hospital facilities in-country so as to anchor up-to-date data products for the government, private-sector, and patient-centric consumption. Methods and analysis: Multiple methods will be employed to achieve the study objectives between December 2023 to November 2024. The first three objectives shall focus on contextual, needs, and feasibility assessments guided by the domains and constructs within the updated Consolidated Framework for Implementation Research (CFIR) during coding and thematic qualitative analysis. Using purposive sampling, breast and cervical cancer care service stakeholders shall be identified for individual in-depth interviews. The fourth objective will involve creating the RTIF prototype and piloting it in the Eastern Region of Ghana. The final and fifth objective shall employ the systems usability scale (SUS) amongst ten randomly selected individual stakeholders to assess the technical functionality of the interface. A nationwide scale-up shall follow this. [ABSTRACT FROM AUTHOR]

Objective: Retention to complete follow-up surveys in extensive longitudinal epidemiological cohort studies is vital yet challenging. All of Us developed pilot interventions to improve response rates for follow-up surveys. Study design and setting: The pilot interventions occurred from April 27, 2020, to August 3, 2020. The three arms were: (1) telephone appointment [staff members calling participants offering appointments to complete surveys over phone] (2) postal [mail reminder to complete surveys through U.S. Postal Service], and (3) combination of telephone appointment and postal. Controls received digital-only reminders [program-level digital recontact via email or through the participant portal]. Study sites chose their study arm and participants were not randomized. Results: A total of 50 sites piloted interventions with 17,593 participants, while 47,832 participants comprised controls during the same period. Of all participants, 6,828 (10.4%) completed any follow-up surveys (1448: telephone; 522: postal; 486: combination; 4372: controls). Follow-up survey completions were 24% higher in the telephone appointment arm than in controls in bivariate analyses. When controlling for confounders, telephone appointment and combination arms increased rates of completion similarly compared to controls, while the postal arm had no significant effect (odds ratio [95% Confidence Interval], telephone appointment:2.01[1.81–2.23]; combination:1.91[1.66–2.20]; postal:0.92[0.79–1.07]). Although the effects of the telephone appointment and combination arms were similar, differential effects were observed across sub-populations. Conclusion: Telephone appointments appeared to be the most successful intervention in our study. Lessons learned about retention interventions, and improvement in follow-up survey completion rates provide generalizable knowledge for similar cohort studies and demonstrate the potential value of precision reminders and engagement with sub-populations of a cohort. [ABSTRACT FROM AUTHOR]

Objectives The most perfect example of the mind-body interaction in all of medicine is provided by stress cardiomyopathy. In stress cardiomyopathy, what is initially a purely emotional event may become rapidly fatal. Prolongation of the QT interval is a cardinal feature of the condition, but the mechanism of the prolongation is unknown. We undertook a randomised controlled trial of stress with a cross-over design, comparing the cardiac response of women with a history of stress cardiomyopathy to age-matched controls to explore the mind-body interaction. Our hypothesis is that the hearts of women with a history of stress cardiomyopathy will respond differently to emotional stress than those of the controls. Method This is a randomised cross-over study. Each patient underwent two separate 24-hour Holter monitors performed at least 5 days apart. Baseline recording was followed by either the stress intervention (hyperventilation) or control (diaphragmatic breathing). Our primary endpoint is change in QTc interval over the first hour. Secondary endpoints were change in QTc over 24 hours, and change in SDNN, a measure of heart rate variability. As a secondary stressor, each participant was telephoned four times during their stressed recording and asked to complete a questionnaire. Results Twelve stress cardiomyopathy patients and twelve control patients were recruited. Baseline characteristics did not differ between cases and controls. With hyperventilation, there was a significant initial difference in anxiety (pConclusions QTc response in women with a history of stress cardiomyopathy does not differ from controls. The relevance of QT prolongation and sensitivity in the autonomic response to the pathogenesis of stress cardiomyopathy remains uncertain.

Background Registering a donation decision is fundamental to increasing the number of people who donate the organs and tissues essential for transplantation, but the number of registered organ donors is insufficient to meet this demand. Most people in Australia support organ donation, but only a third have registered their decision on the Australian Organ Donor Register (AODR). We addressed this paradox by investigating how feelings of community, engendered through an ethic of hospitality and care and a non-proselytizing dialogue about organ donation, facilitated the decision to register. Methods An Immediate Registration Opportunity was set up in a large public hospital in NSW, Australia. The public was approached and invited to engage in an open, respectful dialogic interaction that met people where their beliefs were and allowed their concerns and fears about donation to be discussed. This included a survey that measured positive and negative beliefs about organ donation, mood, atmosphere, and feelings of community coupled with an on-the-spot opportunity to register their donation decision. Results Over four days, we interacted with 357 participants; 75.5% (210) of eligible-to-register participants registered on the AODR. Generalized Structural Equation Modelling highlighted that as connection to community increased, so did the salience of positive beliefs about organ donation. Positive beliefs, in turn, were negatively correlated with negative beliefs about donation and, as the strength of negative beliefs decreased, the probability of registration on the AODR increased. Participants who registered on the AODR reported stronger connection to the broader community than participants who did not register. Conclusion A respectful non-judgmental interaction that allows beliefs and concerns about organ donation to be discussed, coupled with an immediate opportunity to register, encouraged registration. Within this framework, feelings of belonging to a community were a key determinant that enabled many to make the decision to register.

The importance of human cell-based in vitro tools to drug development that are robust, accurate, and predictive cannot be understated. There has been significant effort in recent years to develop such platforms, with increased interest in 3D models that can recapitulate key aspects of biology that 2D models might not be able to deliver. We describe the development of a 3D human cell-based in vitro assay for the investigation of nephrotoxicity, using RPTEC-TERT1 cells. These RPTEC-TERT1 proximal tubule organoids ‘tubuloids’ demonstrate marked differences in physiologically relevant morphology compared to 2D monolayer cells, increased sensitivity to nephrotoxins observable via secreted protein, and with a higher degree of similarity to native human kidney tissue. Finally, tubuloids incubated with nephrotoxins demonstrate altered Na+/K+-ATPase signal intensity, a potential avenue for a high-throughput, translatable nephrotoxicity assay.

Mycobacterium tuberculosis (M.tb) causes tuberculosis (TB) and remains one of the leading causes of mortality due to an infectious pathogen. Host immune responses have been implicated in driving the progression from infection to severe lung disease. We analyzed longitudinal RNA sequencing (RNAseq) data from the whole blood of 74 TB progressors whose samples were grouped into four six-month intervals preceding diagnosis (the GC6-74 study). We additionally analyzed RNAseq data from an independent cohort of 90 TB patients with positron emission tomography-computed tomography (PET-CT) scan results which were used to categorize them into groups with high and low levels of lung damage (the Catalysis TB Biomarker study). These groups were compared to non-TB controls to obtain a complete whole blood transcriptional profile for individuals spanning from early stages of M.tb infection to TB diagnosis. The results revealed a steady increase in the number of genes that were differentially expressed in progressors at time points closer to diagnosis with 278 genes at 13–18 months, 742 at 7–12 months and 5,131 detected 1–6 months before diagnosis and 9,205 detected in TB patients. A total of 2,144 differentially expressed genes were detected when comparing TB patients with high and low levels of lung damage. There was a large overlap in the genes upregulated in progressors 1–6 months before diagnosis (86%) with those in TB patients. A comprehensive pathway analysis revealed a potent activation of neutrophil and platelet mediated defenses including neutrophil and platelet degranulation, and NET formation at both time points. These pathways were also enriched in TB patients with high levels of lung damage compared to those with low. These findings suggest that neutrophils and platelets play a critical role in TB pathogenesis, and provide details of the timing of specific effector mechanisms that may contribute to TB lung pathology.

Introduction: Breast cancer poses a significant health challenge in Sub-Saharan Africa, particularly in Ghana, where late-stage diagnoses and limited healthcare access contribute to elevated mortality rates. This study focuses on the crucial role of pathology and laboratory medical (PALM) services in the timely diagnosis of breast cancer within Ghana. Methods: A cross-sectional survey of hospitals was completed from November 2020 to October 2021, with 94.8% of identified in-country hospitals participating. Pathology service-related parameters assessed included whether pathology was available for the diagnosis of breast cancer on-site or via external referral, the number of pathology personnel, additional breast cancer diagnostic capabilities including estrogen and progesterone and/or HER2 testing, and the time from biopsy to patients receiving their results. Geospatial mapping was used to identify areas of limited access. Results: Of the 328 participating hospitals, 136 (41%) reported breast cancer pathology services, with only 6 having on-site capabilities. Pathology personnel, comprising 15 consultants and 15 specialists, were concentrated in major referral centers, particularly in Greater Accra and Kumasi. An assessment of referral patterns suggested that 75% of the population reside within an hour of breast cancer pathology services. Among the 136 hospitals with access to breast cancer pathology, only a limited number reported that results included ER/PR (38%) and HER2 testing (33%). Conclusion: Ghana has been able to ensure significant pathology service availability through robust referral pathways with centralized labs. Despite this, difficulties persist with the majority of pathology results not including hormone receptor testing which is important in providing tumor specific treatment. [ABSTRACT FROM AUTHOR]

A number of antiepileptic medications that modulate GABA(A) mediated synaptic transmission are anxiolytic. The loop diuretics furosemide (Lasix) and bumetanide (Bumex) are thought to have antiepileptic properties. These drugs also modulate GABA(A) mediated signalling through their antagonism of cation-chloride cotransporters. Given that loop diuretics may act as antiepileptic drugs that modulate GABAergic signalling, we sought to investigate whether they also mediate anxiolytic effects. Here we report the first investigation of the anxiolytic effects of these drugs in rat models of anxiety. Furosemide and bumetanide were tested in adult rats for their anxiolytic effects using four standard anxiety models: 1) contextual fear conditioning; 2) fear-potentiated startle; 3) elevated plus maze, and 4) open-field test. Furosemide and bumetanide significantly reduced conditioned anxiety in the contextual fear-conditioning and fear-potentiated startle models. At the tested doses, neither compound had significant anxiolytic effects on unconditioned anxiety in the elevated plus maze and open-field test models. These observations suggest that loop diuretics elicit significant anxiolytic effects in rat models of conditioned anxiety. Since loop diuretics are antagonists of the NKCC1 and KCC2 cotransporters, these results implicate the cation-chloride cotransport system as possible molecular mechanism involved in anxiety, and as novel pharmacological target for the development of anxiolytics. In view of these findings, and since furosemide and bumetanide are safe and well tolerated drugs, the clinical potential of loop diuretics for treating some types of anxiety disorders deserves further investigation.

Our understanding of the molecular mechanisms of many neurological disorders has been greatly enhanced by the discovery of mutations in genes linked to familial forms of these diseases. These have facilitated the generation of cell and animal models that can be used to understand the underlying molecular pathology. Recently, there has been a surge of interest in the use of patient-derived cells, due to the development of induced pluripotent stem cells and their subsequent differentiation into neurons and glia. Access to patient cell lines carrying the relevant mutations is a limiting factor for many centres wishing to pursue this research. We have therefore generated an open-access collection of fibroblast lines from patients carrying mutations linked to neurological disease. These cell lines have been deposited in the National Institute for Neurological Disorders and Stroke (NINDS) Repository at the Coriell Institute for Medical Research and can be requested by any research group for use in in vitro disease modelling. There are currently 71 mutation-defined cell lines available for request from a wide range of neurological disorders and this collection will be continually expanded. This represents a significant resource that will advance the use of patient cells as disease models by the scientific community.

The biguanide, metformin, is the first-choice therapeutic agent for type-2 diabetes, although the mechanisms that underpin metformin clinical efficacy remain the subject of much debate, partly due to the considerable variation in patient response to metformin. Identification of poor responders by genotype could avoid unnecessary treatment and provide clues to the underlying mechanism of action. GWAS identified SNPs associated with metformin treatment success at a locus containing the NPAT (nuclear protein, ataxia-telangiectasia locus) and ATM (ataxia-telangiectasia mutated) genes. This implies that gene sequence dictates a subsequent biological function to influence metformin action. Hence, we modified expression of NPAT in immortalized cell lines, primary mouse hepatocytes and mouse tissues, and analysed the outcomes on metformin action using confocal microscopy, immunoblotting and immunocytochemistry. In addition, we characterised the metabolic phenotype of npat heterozygous knockout mice and established the metformin response following development of insulin resistance. NPAT protein was localised in the nucleus at discrete loci in several cell types, but over-expression or depletion of NPAT in immortalised cell models did not change cellular responses to biguanides. In contrast, metformin regulation of respiratory exchange ratio (RER) was completely lost in animals lacking one allele of npat. There was also a reduction in metformin correction of impaired glucose tolerance, however no other metabolic abnormalities, or response to metformin, were found in the npat heterozygous mice. In summary, we provide methodological advancements for the detection of NPAT, demonstrate that minor reductions in NPAT mRNA levels (20-40%) influence metformin regulation of RER, and propose that the association between NPAT SNPs and metformin response observed in GWAS, could be due to loss of metformin modification of cellular fuel usage.

Ecological processes are strongly shaped by human landscape modification, and understanding the reciprocal relationship between ecosystems and modified landscapes is critical for informed conservation. Single axis measures of spatial heterogeneity proliferate in the contemporary gradient ecology literature, though they are unlikely to capture the complexity of ecological responses. Here, we develop a standardized approach for defining multi-dimensional gradients of human influence in heterogeneous landscapes and demonstrate this approach to analyze landscape characteristics of ten ecologically distinct US cities. Using occupancy data of a common human-adaptive songbird collected in each of the cities, we then use our dual-axis gradients to evaluate the utility of our approach. Spatial analysis of landscapes surrounding ten US cities revealed two important axes of variation that are intuitively consistent with the characteristics of multi-use landscapes, but are often confounded in single axis gradients. These were, a hard-to-soft gradient, representing transition from developed areas to non-structural soft areas; and brown-to-green, differentiating between two dominant types of soft landscapes: agriculture (brown) and natural areas (green). Analysis of American robin occurrence data demonstrated that occupancy responds to both hard-to-soft (decreasing with development intensity) and brown-to-green gradient (increasing with more natural area). Overall, our results reveal striking consistency in the dominant sources of variation across ten geographically distinct cities and suggests that our approach advances how we relate variation in ecological responses to human influence. Our case study demonstrates this: robins show a remarkably consistent response to a gradient differentiating agricultural and natural areas, but city-specific responses to the more traditional gradient of development intensity, which would be overlooked with a single gradient approach. Managing ecological communities in human dominated landscapes is extremely challenging due to a lack of standardized approaches and a general understanding of how socio-ecological systems function, and our approach offers promising solutions.

ObjectiveThe experiences of frontline healthcare professionals are essential in identifying strategies to mitigate the disruption to healthcare services caused by the COVID-19 pandemic.MethodsWe conducted a cross-sectional study of TB and HIV professionals in low and middle-income countries (LMIC). Between May 12 and August 6, 2020, we collected qualitative and quantitative data using an online survey in 11 languages. We used descriptive statistics and thematic analysis to analyse responses.Findings669 respondents from 64 countries completed the survey. Over 40% stated that it was either impossible or much harder for TB and HIV patients to reach healthcare facilities since COVID-19. The most common barriers reported to affect patients were: fear of getting infected with SARS-CoV-2, transport disruptions and movement restrictions. 37% and 28% of responses about TB and HIV stated that healthcare provider access to facilities was also severely impacted. Strategies to address reduced transport needs and costs-including proactive coordination between the health and transport sector and cards that facilitate lower cost or easier travel-were presented in qualitative responses. Access to non-medical support for patients, such as food supplementation or counselling, was severely disrupted according to 36% and 31% of HIV and TB respondents respectively; qualitative data suggested that the need for such services was exacerbated.ConclusionPatients and healthcare providers across numerous LMIC faced substantial challenges in accessing healthcare facilities, and non-medical support for patients was particularly impacted. Synthesising recommendations of frontline professionals should be prioritised for informing policymakers and healthcare service delivery organisations.

Multiple national and international trends and drivers are radically changing what biological security means for the United Kingdom (UK). New technologies present novel opportunities and challenges, and globalisation has created new pathways and increased the speed, volume and routes by which organisms can spread. The UK Biological Security Strategy (2018) acknowledges the importance of research on biological security in the UK. Given the breadth of potential research, a targeted agenda identifying the questions most critical to effective and coordinated progress in different disciplines of biological security is required. We used expert elicitation to generate 80 policy-relevant research questions considered by participants to have the greatest impact on UK biological security. Drawing on a collaboratively-developed set of 450 questions, proposed by 41 experts from academia, industry and the UK government (consulting 168 additional experts) we subdivided the final 80 questions into six categories: bioengineering; communication and behaviour; disease threats (including pandemics); governance and policy; invasive alien species; and securing biological materials and securing against misuse. Initially, the questions were ranked through a voting process and then reduced and refined to 80 during a one-day workshop with 35 participants from a variety of disciplines. Consistently emerging themes included: the nature of current and potential biological security threats, the efficacy of existing management actions, and the most appropriate future options. The resulting questions offer a research agenda for biological security in the UK that can assist the targeting of research resources and inform the implementation of the UK Biological Security Strategy. These questions include research that could aid with the mitigation of Covid-19, and preparation for the next pandemic. We hope that our structured and rigorous approach to creating a biological security research agenda will be replicated in other countries and regions. The world, not just the UK, is in need of a thoughtful approach to directing biological security research to tackle the emerging issues.

Mammals are serially infected with a variety of microorganisms, including bacteria and parasites. Each infection reprograms the immune system's responses to re-exposure and potentially alters responses to first-time infection by different microorganisms. To examine whether infection with a metazoan parasite modulates host responses to subsequent bacterial infection, mice were infected with the hookworm-like intestinal nematode Nippostrongylus brasiliensis, followed in 2-4 weeks by peritoneal injection of the pathogenic bacterium Klebsiella pneumoniae. Survival from Klebsiella peritonitis two weeks after parasite infection was better in Nippostrongylus-infected animals than in unparasitized mice, with Nippostrongylus-infected mice having fewer peritoneal bacteria, more neutrophils, and higher levels of protective interleukin 6. The improved survival of Nippostrongylus-infected mice depends on IL-4 because the survival benefit is lost in mice lacking IL-4. Because mast cells protect mice from Klebsiella peritonitis, we examined responses in mast cell-deficient Kit(W-sh)/Kit(W-sh) mice, in which parasitosis failed to improve survival from Klebsiella peritonitis. However, adoptive transfer of cultured mast cells to Kit(W-sh)/Kit(W-sh) mice restored survival benefits of parasitosis. These results show that recent infection with Nippostrongylus brasiliensis protects mice from Klebsiella peritonitis by modulating mast cell contributions to host defense, and suggest more generally that parasitosis can yield survival advantages to a bacterially infected host.

Pancreatic ductal adenocarcinoma (PDA) is an extremely aggressive malignancy, which carries a dismal prognosis. Activating mutations of the Kras gene are common to the vast majority of human PDA. In addition, recent studies have demonstrated that embryonic signaling pathway such as Hedgehog and Notch are inappropriately upregulated in this disease. The role of another embryonic signaling pathway, namely the canonical Wnt cascade, is still controversial. Here, we use gene array analysis as a platform to demonstrate general activation of the canonical arm of the Wnt pathway in human PDA. Furthermore, we provide evidence for Wnt activation in mouse models of pancreatic cancer. Our results also indicate that Wnt signaling might be activated downstream of Hedgehog signaling, which is an early event in PDA evolution. Wnt inhibition blocked proliferation and induced apoptosis of cultured adenocarcinoma cells, thereby providing evidence to support the development of novel therapeutical strategies for Wnt inhibition in pancreatic adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDA) is an extremely aggressive malignancy, which carries a dismal prognosis. Activating mutations of the Kras gene are common to the vast majority of human PDA. In addition, recent studies have demonstrated that embryonic signaling pathway such as Hedgehog and Notch are inappropriately upregulated in this disease. The role of another embryonic signaling pathway, namely the canonical Wnt cascade, is still controversial. Here, we use gene array analysis as a platform to demonstrate general activation of the canonical arm of the Wnt pathway in human PDA. Furthermore, we provide evidence for Wnt activation in mouse models of pancreatic cancer. Our results also indicate that Wnt signaling might be activated downstream of Hedgehog signaling, which is an early event in PDA evolution. Wnt inhibition blocked proliferation and induced apoptosis of cultured adenocarcinoma cells, thereby providing evidence to support the development of novel therapeutical strategies for Wnt inhibition in pancreatic adenocarcinoma.

This study focuses on the multidisciplinary investigation of three stucco-shrouded mummies with mummy portrait from Egypt dating from the late 3rd to the middle of the 4th century AD, corresponding to the late Roman Period. These three mummies were excavated in the early 17th and late 19th centuries in the Saqqara necropolis near the ancient Egyptian capital of Memphis. Two of them experienced an interesting collection history, when they became part of the collection of the Elector of Saxony and King of Poland August II in Dresden, Germany, in 1728. The investigation includes information about the mummies' discovery, collection history and shroud decoration obtained through Egyptological expertise. In addition, information on the state of preservation, technique of artificial mummification, age at death, sex, body height and health of the deceased was achieved through computed tomography (CT) analysis. Research yielded an adult male, a middle-aged female and a young female. Due to the rather poorly preserved bodies of the male and middle-aged female, a specific technique of artificial mummification could not be ascertained. Brain and several internal organs of the well-preserved young female were identified. Wooden boards, beads of necklaces, a hairpin, and metal dense items, such as lead seals, nails and two coins or medallions were discovered. Paleopathological findings included carious lesions, Schmorl's nodes, evidence of arthritis and a vertebral hemangioma. The study revealed insights on the decoration and burial preparation of individuals of upper socioeconomic status living in the late Roman Period, as well as comprehensive bioanthropological information of the deceased.

BACKGROUND:Understanding how the conservation of nature can lead to improvement in human conditions is a research area with significant growth and attention. Progress towards effective conservation requires understanding mechanisms for achieving impact within complex social-ecological systems. Causal models are useful tools for defining plausible pathways from conservation actions to impacts on nature and people. Evaluating the potential of different strategies for delivering co-benefits for nature and people will require the use and testing of clear causal models that explicitly define the logic and assumptions behind cause and effect relationships. OBJECTIVES AND METHODS:In this study, we outline criteria for credible causal models and systematically evaluated their use in a broad base of literature (~1,000 peer-reviewed and grey literature articles from a published systematic evidence map) on links between nature-based conservation actions and human well-being impacts. RESULTS:Out of 1,027 publications identified, only ~20% of articles used any type of causal models to guide their work, and only 14 total articles fulfilled all criteria for credibility. Articles rarely tested the validity of models with empirical data. IMPLICATIONS:Not using causal models risks poorly defined strategies, misunderstanding of potential mechanisms for affecting change, inefficient use of resources, and focusing on implausible efforts for achieving sustainability.

PurposeThe 14-item Resilience Scale (RS14) is a tool designed to measure psychological resilience. It has been used effectively in diverse populations. However, its applicability is largely unknown for Sub-Saharan adolescent populations and completely unknown for orphaned and separated adolescents and youths (OSAY), a highly vulnerable population for whom resilience may be critical. This study assesses the RS14's psychometric properties for OSAY in Uasin Gishu County, Kenya.MethodsSurvey responses from a representative sample of 1016 OSAY (51.3% female) aged 10-25 (mean = 16; SD = 3.5) living in institutional and home-based environments in Uasin Gishu County were analyzed. The RS14's psychometric properties were assessed by examining internal consistency reliability, confirmatory factor analyses, and convergent validity using correlations between resilience and each of social support and depression. Sub-analyses were conducted by age and sex.ResultsResilience scores ranged from 14-98 (mean = 66; SD = 19) with no sex-based significant difference. Resilience was higher for those aged ≥18 (mean = 69; range = 14-98) versus age ConclusionThis study demonstrates reasonable evidence that the RS14 is both valid and reliable for measuring psychological resilience in the population of OSAY in western Kenya.

Whitebark pine (Pinus albicaulis), a keystone species and an obligate mutualist of the Clark's nutcracker (Nucifraga columbiana), is rapidly declining throughout its range. Evidence suggests this decline is leading to a downward trend in local nutcracker populations, which would in-turn decrease whitebark pine regeneration. Our objectives were to (1) evaluate temporal variation in nutcracker habitat use as a function of whitebark pine and Douglas-fir (Pseudotsuga menziesii) habitat, at local and landscape scales, (2) develop metrics for predicting when whitebark pine communities require intervention to sustain nutcracker visitation, and (3) test McKinney et al. (2009) and Barringer et al.'s (2012) models predicting nutcracker occurrence. Between 2009 and 2013, we carried out 3,135 audio-visual Clark's nutcracker surveys at 238 random points in the southern Greater Yellowstone Ecosystem. Using Bayesian occupancy models and cross-product model selection, we evaluated the association between nutcracker occurrence and habitat variables during five stages of the nutcracker annual cycle, while accounting for imperfect detection. Nutcracker occurrence was most strongly associated with the presence of cone-bearing whitebark pine trees (rather than cone density) and the area of whitebark pine on the landscape. To promote a high, >75%, probability of occurrence at a site within the study area, we recommend a management plan that achieves a landscape composed of a minimum of 12,500-25,000 ha of cone-bearing whitebark pine habitat within a 32.6 km radius. Additionally, an optimal habitat mosaic includes moderate levels of Douglas-fir habitat. Models currently used to guide whitebark pine management strategies underpredicted nutcracker occurrence in our study area, suggesting these strategies may not be appropriate in the region. We cannot predict how this mutualistic relationship will change as the population density of each species shifts. We therefore suggest conducting periodic surveys to re-evaluate the relationship as the environment changes and management strategies are implemented.

European canker, caused by the necrotrophic fungal phytopathogen Neonectria ditissima, is one of the most damaging apple diseases worldwide. An understanding of the molecular basis of N. ditissima virulence is currently lacking. Identification of genes with an up-regulation of expression during infection, which are therefore probably involved in virulence, is a first step towards this understanding. Reverse transcription quantitative real-time PCR (RT-qPCR) can be used to identify these candidate virulence genes, but relies on the use of reference genes for relative gene expression data normalisation. However, no report that addresses selecting appropriate fungal reference genes for use in the N. ditissima-apple pathosystem has been published to date. In this study, eight N. ditissima genes were selected as candidate RT-qPCR reference genes for gene expression analysis. A subset of the primers (six) designed to amplify regions from these genes were specific for N. ditissima, failing to amplify PCR products with template from other fungal pathogens present in the apple orchard. The efficiency of amplification of these six primer sets was satisfactory, ranging from 81.8 to 107.53%. Analysis of expression stability when a highly pathogenic N. ditissima isolate was cultured under 10 regimes, using the statistical algorithms geNorm, NormFinder and BestKeeper, indicated that actin and myo-inositol-1-phosphate synthase (mips), or their combination, could be utilised as the most suitable reference genes for normalisation of N. ditissima gene expression. As a test case, these reference genes were used to study expression of three candidate virulence genes during a time course of infection. All three, which shared traits with fungal effector genes, had up-regulated expression in planta compared to in vitro with expression peaking between five and six weeks post inoculation (wpi). Thus, these three genes may well be involved in N. ditissima pathogenicity and are priority candidates for further functional characterization.

Eye temperature measured using infrared thermography (IRT) can be used as a non-invasive measure of autonomic nervous system (ANS) activity in cattle. The objective of this study was to evaluate if changes in eye temperature (measured using IRT) can be used to non-invasively measure ANS activity in sheep. Twenty, 2 to 4-year-old, Romney ewes were randomly assigned to receive either epinephrine (EPI) or physiological saline (SAL) for 5 min administered via jugular catheter (n = 10 ewes/treatment). Eye temperature (°C) was recorded continuously using IRT for approximately 25 min before and 20 min after the start of infusion. Heart rate and heart rate variability, measured using the root mean square of successive differences (RMSSD) and the standard deviation of all inter-beat intervals (SDNN), were recorded for 5 min before and up to 10 min after the start of infusion. Blood samples were taken before and after the infusion period to measure plasma epinephrine, norepinephrine, cortisol and packed cell volume (PCV) concentrations. During the infusion period, maximum eye temperature was on average higher (P0.05) concentrations in ewes compared to an infusion of saline. PCV concentrations were higher (P

ObjectiveSuicide is a leading cause of death among Aboriginal and Torres Strait Islander people. Friends, family and frontline workers (for example, teachers, youth workers) are often best positioned to provide initial assistance if someone is at risk of suicide. We developed culturally appropriate expert consensus guidelines on how to provide mental health first aid to Australian Aboriginal and Torres Strait Islander people experiencing suicidal thoughts or behaviour and used this as the basis for a 5-hour suicide gatekeeper training course called Talking About Suicide. This paper describes the outcomes for participants in an uncontrolled trial of this training course.MethodsWe undertook an uncontrolled trial of the Talking About Suicide course, delivered by Aboriginal and Torres Strait Islander Mental Health First Aid instructors to 192 adult (i.e. 18 years of age or older) Aboriginal and Torres Strait Islander (n = 110) and non-Indigenous (n = 82) participants. Questionnaires capturing self-report outcomes were self-administered immediately before (n = 192) and after attending the training course (n = 188), and at four-months follow-up (n = 98). Outcome measures were beliefs about suicide, stigmatising attitudes, confidence in ability to assist, and intended and actual actions to assist a suicidal person.ResultsDespite a high level of suicide literacy among participants at pre-course measurement, improvements at post-course were observed in beliefs about suicide, stigmatising attitudes, confidence in ability to assist and intended assisting actions. While attrition at follow-up decreased statistical power, some improvements in beliefs about suicide, stigmatising attitudes and intended assisting actions remained statistically significant at follow-up. Importantly, actual assisting actions taken showed dramatic improvements between pre-course and follow-up. Participants reported feeling more confident to assist a suicidal person after the course and this was maintained at follow-up. The course was judged to be culturally appropriate by those participants who identified as Aboriginal and/or Torres Strait Islanders.ImplicationsThe results of this uncontrolled trial were encouraging, suggesting that the Talking About Suicide course was able to improve participants' knowledge, attitudes, and intended assisting actions as well as actual actions taken.

BackgroundResistance among bacterial infections is increasingly well-documented in high-income countries; however, relatively little is known about bacterial antimicrobial resistance in low-income countries, where the burden of infections is high.MethodsWe prospectively screened all adult inpatients at a referral hospital in Rwanda for suspected infection for seven months. Blood, urine, wound and sputum samples were cultured and tested for antibiotic susceptibility. We examined factors associated with resistance and compared hospital outcomes for participants with and without resistant isolates.ResultsWe screened 19,178 patient-days, and enrolled 647 unique participants with suspected infection. We obtained 942 culture specimens, of which 357 were culture-positive specimens. Of these positive specimens, 155 (43.4%) were wound, 83 (23.2%) urine, 64 (17.9%) blood, and 55 (15.4%) sputum. Gram-negative bacteria comprised 323 (88.7%) of all isolates. Of 241 Gram-negative isolates tested for ceftriaxone, 183 (75.9%) were resistant. Of 92 Gram-negative isolates tested for the extended spectrum beta-lactamase (ESBL) positive phenotype, 66 (71.7%) were ESBL positive phenotype. Transfer from another facility, recent surgery or antibiotic exposure, and hospital-acquired infection were each associated with resistance. Mortality was 19.6% for all enrolled participants.ConclusionsThis is the first published prospective hospital-wide antibiogram of multiple specimen types from East Africa with ESBL testing. Our study suggests that low-resource settings with limited and inconsistent access to the full range of antibiotic classes may bear the highest burden of resistant infections. Hospital-acquired infections and recent antibiotic exposure are associated with a high proportion of resistant infections. Efforts to slow the development of resistance and supply effective antibiotics are urgently needed.

IntroductionBritish Columbia (BC), Canada declared a public health emergency in April 2016 for opioid overdose. Comprehensive data was needed to identify risk factors, inform interventions, and evaluate response actions. We describe the development of an overdose cohort, including linkage strategy, case definitions, and data governance model, and present the resulting characteristics, including data linkage yields and case overlap among data sources.MethodsOverdose events from hospital admissions, physician visits, poison centre and ambulance calls, emergency department visits, and coroner's data were grouped into episodes if records were present in multiple sources. A minimum of five years of universal health care records (all prescription dispensations, fee-for-service physician billings, emergency department visits and hospitalizations) were appended for each individual. A 20% random sample of BC residents and a 1:5 matched case-control set were generated. Consultation and prioritization ensured analysts worked to address questions to directly inform public health actions.Results10,456 individuals suffered 14,292 overdoses from January 1, 2015 to Nov 30, 2016. Only 28% of overdose events were found in more than one dataset with the unique contribution of cases highest from ambulance records (32%). Compared with fatal overdoses, non-fatal events more often involved females, younger individuals (20 to 29 years) and those 60 or older. In 78% of illegal drug deaths, there was no associated ambulance response. In the year prior to first recorded overdose, 60% of individuals had at least one ED visit, 31% at least one hospital admission, 80% at least one physician visit, and 87% had filled at least one prescription in a community pharmacy.ConclusionWhile resource-intensive to establish, a linked cohort is useful for characterizing the full extent of the epidemic, defining sub-populations at risk, and patterns of contact with the health system. Overdose studies in other jurisdictions should consider the inclusion of multiple data sources.

BACKGROUND:Malaria reduction and future elimination in China is made more difficult by the importation of cases from neighboring endemic countries, particularly Myanmar, Laos, and Vietnam, and increased travel to Africa by Chinese nationals. The increasing prevalence of artemisinin resistant parasites across Southeast Asia highlights the importance of monitoring the parasite importation into China. Artemisinin resistance in the Mekong region is associated with variants of genes encoding the K13 kelch domain protein (pf13k), found in specific genetic backgrounds, including certain alleles of genes encoding the chloroquine resistance transporter (pfcrt) and multidrug resistance transporter PgH1 (pfmdr1). METHODS:In this study we investigated the prevalence of drug resistance markers in 72 P. falciparum samples from uncomplicated malaria infections in Tengchong and Yingjiang, counties on the Yunnan-Myanmar border. Variants of pf13k, pfcrt and pfmdr1 are described. RESULTS:Almost all parasites harboured chloroquine-resistant alleles of pfcrt, whereas pfmdr1 was more diverse. Major mutations in the K13 propeller domain associated with artemisinin resistance in the Mekong region (C580Y, R539T and Y493H) were absent, but F446I and two previously undescribed mutations (V603E and V454I) were identified. Protein structural modelling was carried out in silico on each of these K13 variants, based on recently published crystal structures for the K13 propeller domain. Whereas F446I was predicted to elicit a moderate destabilisation of the propeller structure, the V603E substitution is likely to lead to relatively high protein instability. We plotted these stability estimates, and those for all previously described variants, against published values for in vivo parasitaemia half-life, and found that quadratic regression generates a useful predictive algorithm. CONCLUSION:This study provides a baseline of P. falciparum resistance-associated mutations prevalent at the China-Myanmar border. We also show that protein modelling can be used to generate testable predictions as to the impact of pfk13 mutations on in vivo (and potentially in vitro) artemisinin susceptibility.

Type 1, or autoimmune, diabetes is caused by the T-cell mediated destruction of the insulin-producing pancreatic beta cells. Non-obese diabetic (NOD) mice spontaneously develop autoimmune diabetes akin to human type 1 diabetes. For this reason, the NOD mouse has been the preeminent murine model for human type 1 diabetes research for several decades. However, humanized mouse models are highly sought after because they offer both the experimental tractability of a mouse model and the clinical relevance of human-based research. Autoimmune T-cell responses against insulin, and its precursor proinsulin, play central roles in the autoimmune responses against pancreatic beta cells in both humans and NOD mice. As a first step towards developing a murine model of the human autoimmune response against pancreatic beta cells we set out to replace the murine insulin 1 gene (Ins1) with the human insulin gene (Ins) using CRISPR/Cas9. Here we describe a NOD mouse strain that expresses human insulin in place of murine insulin 1, referred to as HuPI. HuPI mice express human insulin, and C-peptide, in their serum and pancreata and have normal glucose tolerance. Compared with wild type NOD mice, the incidence of diabetes is much lower in HuPI mice. Only 15-20% of HuPI mice developed diabetes after 300 days, compared to more than 60% of unmodified NOD mice. Immune-cell infiltration into the pancreatic islets of HuPI mice was not detectable at 100 days but was clearly evident by 300 days. This work highlights the feasibility of using CRISPR/Cas9 to create mouse models of human diseases that express proteins pivotal to the human disease. Furthermore, it reveals that even subtle changes in proinsulin protect NOD mice from diabetes.

Myosin regulatory light chain (LC20) phosphorylation plays an important role in vascular smooth muscle contraction and cell migration. Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) phosphorylates LC20 (its only known substrate) exclusively at S19. Rho-associated kinase (ROCK) and zipper-interacting protein kinase (ZIPK) have been implicated in the regulation of LC20 phosphorylation via direct phosphorylation of LC20 at T18 and S19 and indirectly via phosphorylation of MYPT1 (the myosin targeting subunit of myosin light chain phosphatase, MLCP) and Par-4 (prostate-apoptosis response-4). Phosphorylation of MYPT1 at T696 and T853 inhibits MLCP activity whereas phosphorylation of Par-4 at T163 disrupts its interaction with MYPT1, exposing the sites of phosphorylation in MYPT1 and leading to MLCP inhibition. To evaluate the roles of MLCK, ROCK and ZIPK in these phosphorylation events, we investigated the time courses of phosphorylation of LC20, MYPT1 and Par-4 in serum-stimulated human vascular smooth muscle cells (from coronary and umbilical arteries), and examined the effects of siRNA-mediated MLCK, ROCK and ZIPK knockdown and pharmacological inhibition on these phosphorylation events. Serum stimulation induced rapid phosphorylation of LC20 at T18 and S19, MYPT1 at T696 and T853, and Par-4 at T163, peaking within 30-120 s. MLCK knockdown or inhibition, or Ca2+ chelation with EGTA, had no effect on serum-induced LC20 phosphorylation. ROCK knockdown decreased the levels of phosphorylation of LC20 at T18 and S19, of MYPT1 at T696 and T853, and of Par-4 at T163, whereas ZIPK knockdown decreased LC20 diphosphorylation, but increased phosphorylation of MYPT1 at T696 and T853 and of Par-4 at T163. ROCK inhibition with GSK429286A reduced serum-induced phosphorylation of LC20 at T18 and S19, MYPT1 at T853 and Par-4 at T163, while ZIPK inhibition by HS38 reduced only LC20 diphosphorylation. We also demonstrated that serum stimulation induced phosphorylation (activation) of ZIPK, which was inhibited by ROCK and ZIPK down-regulation and inhibition. Finally, basal phosphorylation of LC20 in the absence of serum stimulation was unaffected by MLCK, ROCK or ZIPK knockdown or inhibition. We conclude that: (i) serum stimulation of cultured human arterial smooth muscle cells results in rapid phosphorylation of LC20, MYPT1, Par-4 and ZIPK, in contrast to the slower phosphorylation of kinases and other proteins involved in other signaling pathways (Akt, ERK1/2, p38 MAPK and HSP27), (ii) ROCK and ZIPK, but not MLCK, are involved in serum-induced phosphorylation of LC20, (iii) ROCK, but not ZIPK, directly phosphorylates MYPT1 at T853 and Par-4 at T163 in response to serum stimulation, (iv) ZIPK phosphorylation is enhanced by serum stimulation and involves phosphorylation by ROCK and autophosphorylation, and (v) basal phosphorylation of LC20 under serum-free conditions is not attributable to MLCK, ROCK or ZIPK.

The implementation and popularity of next generation sequencing (NGS) has led to the development of various rapid whole mitochondrial genome sequencing techniques. We summarise an efficient and cost-effective NGS approach for mitochondrial genomic DNA in humans using the Ion Torrent platform, and further discuss our bioinformatics pipeline for streamlined variant calling. Ion 316 chips were utilised with the Ion Torrent semi-conductor platform Personal Genome Machine (PGM) to perform tandem sequencing of mitochondrial genomes from the core pedigree (n = 315) of the Norfolk Island Health Study. Key improvements from commercial methods focus on the initial PCR step, which currently requires extensive optimisation to ensure the accurate and reproducible elongation of each section of the complete mitochondrial genome. Dual-platform barcodes were incorporated into our protocol thereby extending its potential application onto Illumina-based systems. Our bioinformatics pipeline consists of a modified version of GATK best practices tailored for mitochondrial genomic data. When compared with current commercial methods, our method, termed high throughput mitochondrial genome sequencing (HTMGS), allows high multiplexing of samples and the use of alternate library preparation reagents at a lower cost per sample (~1.7 times) when compared to current commercial methodologies. Our HTMGS methodology also provides robust mitochondrial sequencing data (>450X average coverage) that can be applied and modified to suit various study designs. On average, we were able to identify ~30 variants per sample with 572 variants observed across 315 samples. We have developed a high throughput sequencing and analysis method targeting complete mitochondrial genomes; with the potential to be platform agnostic with analysis options that adhere to current best practices.