Function Of Human α3β4α5 Nicotinic Acetylcholine Receptors Is Reduced By The β5(D398N) Variant

Genome-wide studies have strongly associated a non-synonymous polymorphism (rs16969968) that changes the 398th amino acid in the nAChR α5 subunit from aspartic acid to asparagine (D398N), with greater risk for increased nicotine consumption. We have used a pentameric concatemer approach to express defined and consistent populations of α3β4α5 nAChR in Xenopus oocytes. α5(Asn-398; risk) variant incorporation reduces ACh-evoked function compared with inclusion of the common α5(Asp-398) variant without altering agonist or antagonist potencies. Unlinked α3, β4, and α5 subunits assemble to form a uniform nAChR population with pharmacological properties matching those of concatemeric α3β4* nAChRs. α5 subunit incorporation reduces α3β4* nAChR function after coinjection with unlinked α3 and β4 subunits but increases that of α3β4α5 versus α3β4-only concatemers. α5 subunit incorporation into α3β4* nAChR also alters the relative efficacies of competitive agonists and changes the potency of the non-competitive antagonist mecamylamine. Additional observations indicated that in the absence of α5 subunits, free α3 and β4 subunits form at least two further subtypes. The pharmacological profiles of these free subunit α3β4-only subtypes are dissimilar both to each other and to those of α3β4α5 nAChR. The α5 variant-induced change in α3β4α5 nAChR function may underlie some of the phenotypic changes associated with this polymorphism. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc

Differential α4(+)/(-)β2 Agonist-Binding Site Contributions To α4β2 Nicotinic Acetylcholine Receptor Function Within And Between Isoforms

Two α4β2 nicotinic acetylcholine receptor (α4β2-nAChR) isoforms exist with (α4)2(β2)3 and (α4)2(β2)3 subunit stoichiometries and high versus low agonist sensitivities (HS and LS), respectively. Both isoforms contain a pair of α4(+)/(-)β2 agonist- binding sites. The LS isoform also contains a unique α4(+)/(-)α4 site with lower agonist affinity than the α4(+)/(-)β2 sites. However, the relative roles of the conserved α4(+)/(-)β2 agonist-binding sites in and between the isoforms have not been studied. We used a fully linked subunit concatemeric nAChR approach to express pure populations of HS or LS isoform α4β2∗-nAChR. This approach also allowed us to mutate individual subunit interfaces, or combinations thereof, on each isoform background. We used this approach to systematically mutate a triplet of β2 subunit (-)-face E-loop residues to their non-conserved α4 subunit counterparts or vice versa (β2HQT and α4VFL, respectively). Mutant-nAChR constructs (and unmodified controls) were expressed in Xenopus oocytes. Acetylcholine concentration-response curves and maximum function were measured using two-electrode voltage clamp electrophysiology. Surface expression was measured with 125I-mAb 295 binding and was used to define function/nAChR. If the α4(+)/(-)β2 sites contribute equally to function, making identical β2HQT substitutions at either site should produce similar functional outcomes. Instead, highly differential outcomes within the HS isoform, and between the two isoforms, were observed. In contrast, α4VFL mutation effects were very similar in all positions of both isoforms. Our results indicate that the identity of subunits neighboring the otherwise equivalent α4(+)/(-)β2 agonist sites modifies their contributions to nAChR activation and that E-loop residues are an important contributor to this neighbor effect

The Unique a4(1)/(2)a4 Agonist Binding Site in (a4) 3 (b2) 2 Subtype Nicotinic Acetylcholine Receptors Permits Differential Agonist Desensitization Pharmacology versus the (a4) 2 (b2) 3 Subtype s

ABSTRACT Selected nicotinic agonists were used to activate and desensitize high-sensitivity (HS) (a4) 2 (b2) 3 ) or low-sensitivity (LS) (a4) 3 (b2) 2 ) isoforms of human a4b2-nicotinic acetylcholine receptors (nAChRs). Function was assessed using 86 Rb 1 efflux in a stably transfected SH-EP1-ha4b2 human epithelial cell line, and twoelectrode voltage-clamp electrophysiology in Xenopus laevis oocytes expressing concatenated pentameric HS or LS a4b2-nAChR constructs (HSP and LSP). Unlike previously studied agonists, desensitization by the highly selective agonists A-85380 [3-(2(S)-azetidinylmethoxy)pyridine] and sazetidine-A (Saz-A) preferentially reduced a4b2-nAChR HS-phase versus LS-phase responses. The concatenated-nAChR experiments confirmed that approximately 20% of LS-isoform acetylcholine-induced function occurs in an HS-like phase, which is abolished by Saz-A preincubation. Six mutant LSPs were generated, each targeting a conserved agonist binding residue within the LS-isoform-only a4(1)/(2)a4 interface agonist binding site. Every mutation reduced the percentage of LS-phase function, demonstrating that this site underpins LS-phase function. Oocyte-surface expression of the HSP and each of the LSP constructs was statistically indistinguishable, as measured using b2-subunit-specific [ 125 I]mAb295 labeling. However, maximum function is approximately five times greater on a "per-receptor" basis for unmodified LSP versus HSP a4b2-nAChRs. Thus, recruitment of the a4(1)/(2)a4 site at higher agonist concentrations appears to augment otherwisesimilar function mediated by the pair of a4(1)/(2)b2 sites shared by both isoforms. These studies elucidate the receptor-level differences underlying the differential pharmacology of the two a4b2-nAChR isoforms, and demonstrate that HS versus LS a4b2-nAChR activity can be selectively manipulated using pharmacological approaches. Since a4b2 nAChRs are the predominant neuronal subtype, these discoveries likely have significant functional implications, and may provide important insights for drug discovery and development

Structure-Activity Studies Of 7-Heteroaryl-3-Azabicyclo[3.3.1]Non-6-Enes: A Novel Class Of Highly Potent Nicotinic Receptor Ligands

The potential for nicotinic ligands with affinity for the α4β2 or α7 subtypes to treat such diverse diseases as nicotine addiction, neuropathic pain, and neurodegenerative and cognitive disorders has been exhibited clinically for several compounds while preclinical activity in relevant in vivo models has been demonstrated for many more. For several therapeutic programs, we sought nicotinic ligands with various combinations of affinity and function across both subtypes, with an emphasis on dual α4β2-α7 ligands, to explore the possibility of synergistic effects. We report here the structure-activity relationships (SAR) for a novel series of 7-heteroaryl-3-azabicyclo[3.3.1]non-6-enes and characterize many of the analogues for activity at multiple nicotinic subtypes. © 2012 American Chemical Society

The first case of Brucella canis in Sweden: background, case report and recommendations from a northern European perspective

Infection with Brucella canis has been diagnosed in Sweden for the first time. It was diagnosed in a three-year-old breeding bitch with reproductive disturbances. Fifteen in-contact dogs were tested repeatedly and all of them were negative for B. canis. The source of infection could not be defined. The present article describes the case and the measures undertaken and gives a short review over B. canis. Recommendations on how to avoid the infection in non-endemic countries are given

Global age-sex-specific fertility, mortality, healthy life expectancy (HALE), and population estimates in 204 countries and territories, 1950-2019 : a comprehensive demographic analysis for the Global Burden of Disease Study 2019

Background: Accurate and up-to-date assessment of demographic metrics is crucial for understanding a wide range of social, economic, and public health issues that affect populations worldwide. The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2019 produced updated and comprehensive demographic assessments of the key indicators of fertility, mortality, migration, and population for 204 countries and territories and selected subnational locations from 1950 to 2019. Methods: 8078 country-years of vital registration and sample registration data, 938 surveys, 349 censuses, and 238 other sources were identified and used to estimate age-specific fertility. Spatiotemporal Gaussian process regression (ST-GPR) was used to generate age-specific fertility rates for 5-year age groups between ages 15 and 49 years. With extensions to age groups 10–14 and 50–54 years, the total fertility rate (TFR) was then aggregated using the estimated age-specific fertility between ages 10 and 54 years. 7417 sources were used for under-5 mortality estimation and 7355 for adult mortality. ST-GPR was used to synthesise data sources after correction for known biases. Adult mortality was measured as the probability of death between ages 15 and 60 years based on vital registration, sample registration, and sibling histories, and was also estimated using ST-GPR. HIV-free life tables were then estimated using estimates of under-5 and adult mortality rates using a relational model life table system created for GBD, which closely tracks observed age-specific mortality rates from complete vital registration when available. Independent estimates of HIV-specific mortality generated by an epidemiological analysis of HIV prevalence surveys and antenatal clinic serosurveillance and other sources were incorporated into the estimates in countries with large epidemics. Annual and single-year age estimates of net migration and population for each country and territory were generated using a Bayesian hierarchical cohort component model that analysed estimated age-specific fertility and mortality rates along with 1250 censuses and 747 population registry years. We classified location-years into seven categories on the basis of the natural rate of increase in population (calculated by subtracting the crude death rate from the crude birth rate) and the net migration rate. We computed healthy life expectancy (HALE) using years lived with disability (YLDs) per capita, life tables, and standard demographic methods. Uncertainty was propagated throughout the demographic estimation process, including fertility, mortality, and population, with 1000 draw-level estimates produced for each metric. Findings: The global TFR decreased from 2·72 (95% uncertainty interval [UI] 2·66–2·79) in 2000 to 2·31 (2·17–2·46) in 2019. Global annual livebirths increased from 134·5 million (131·5–137·8) in 2000 to a peak of 139·6 million (133·0–146·9) in 2016. Global livebirths then declined to 135·3 million (127·2–144·1) in 2019. Of the 204 countries and territories included in this study, in 2019, 102 had a TFR lower than 2·1, which is considered a good approximation of replacement-level fertility. All countries in sub-Saharan Africa had TFRs above replacement level in 2019 and accounted for 27·1% (95% UI 26·4–27·8) of global livebirths. Global life expectancy at birth increased from 67·2 years (95% UI 66·8–67·6) in 2000 to 73·5 years (72·8–74·3) in 2019. The total number of deaths increased from 50·7 million (49·5–51·9) in 2000 to 56·5 million (53·7–59·2) in 2019. Under-5 deaths declined from 9·6 million (9·1–10·3) in 2000 to 5·0 million (4·3–6·0) in 2019. Global population increased by 25·7%, from 6·2 billion (6·0–6·3) in 2000 to 7·7 billion (7·5–8·0) in 2019. In 2019, 34 countries had negative natural rates of increase; in 17 of these, the population declined because immigration was not sufficient to counteract the negative rate of decline. Globally, HALE increased from 58·6 years (56·1–60·8) in 2000 to 63·5 years (60·8–66·1) in 2019. HALE increased in 202 of 204 countries and territories between 2000 and 2019

Global burden of 369 diseases and injuries in 204 countries and territories, 1990–2019: a systematic analysis for the Global Burden of Disease Study 2019

Background: In an era of shifting global agendas and expanded emphasis on non-communicable diseases and injuries along with communicable diseases, sound evidence on trends by cause at the national level is essential. The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) provides a systematic scientific assessment of published, publicly available, and contributed data on incidence, prevalence, and mortality for a mutually exclusive and collectively exhaustive list of diseases and injuries. Methods: GBD estimates incidence, prevalence, mortality, years of life lost (YLLs), years lived with disability (YLDs), and disability-adjusted life-years (DALYs) due to 369 diseases and injuries, for two sexes, and for 204 countries and territories. Input data were extracted from censuses, household surveys, civil registration and vital statistics, disease registries, health service use, air pollution monitors, satellite imaging, disease notifications, and other sources. Cause-specific death rates and cause fractions were calculated using the Cause of Death Ensemble model and spatiotemporal Gaussian process regression. Cause-specific deaths were adjusted to match the total all-cause deaths calculated as part of the GBD population, fertility, and mortality estimates. Deaths were multiplied by standard life expectancy at each age to calculate YLLs. A Bayesian meta-regression modelling tool, DisMod-MR 2.1, was used to ensure consistency between incidence, prevalence, remission, excess mortality, and cause-specific mortality for most causes. Prevalence estimates were multiplied by disability weights for mutually exclusive sequelae of diseases and injuries to calculate YLDs. We considered results in the context of the Socio-demographic Index (SDI), a composite indicator of income per capita, years of schooling, and fertility rate in females younger than 25 years. Uncertainty intervals (UIs) were generated for every metric using the 25th and 975th ordered 1000 draw values of the posterior distribution. Findings: Global health has steadily improved over the past 30 years as measured by age-standardised DALY rates. After taking into account population growth and ageing, the absolute number of DALYs has remained stable. Since 2010, the pace of decline in global age-standardised DALY rates has accelerated in age groups younger than 50 years compared with the 1990–2010 time period, with the greatest annualised rate of decline occurring in the 0–9-year age group. Six infectious diseases were among the top ten causes of DALYs in children younger than 10 years in 2019: lower respiratory infections (ranked second), diarrhoeal diseases (third), malaria (fifth), meningitis (sixth), whooping cough (ninth), and sexually transmitted infections (which, in this age group, is fully accounted for by congenital syphilis; ranked tenth). In adolescents aged 10–24 years, three injury causes were among the top causes of DALYs: road injuries (ranked first), self-harm (third), and interpersonal violence (fifth). Five of the causes that were in the top ten for ages 10–24 years were also in the top ten in the 25–49-year age group: road injuries (ranked first), HIV/AIDS (second), low back pain (fourth), headache disorders (fifth), and depressive disorders (sixth). In 2019, ischaemic heart disease and stroke were the top-ranked causes of DALYs in both the 50–74-year and 75-years-and-older age groups. Since 1990, there has been a marked shift towards a greater proportion of burden due to YLDs from non-communicable diseases and injuries. In 2019, there were 11 countries where non-communicable disease and injury YLDs constituted more than half of all disease burden. Decreases in age-standardised DALY rates have accelerated over the past decade in countries at the lower end of the SDI range, while improvements have started to stagnate or even reverse in countries with higher SDI. Interpretation: As disability becomes an increasingly large component of disease burden and a larger component of health expenditure, greater research and developm nt investment is needed to identify new, more effective intervention strategies. With a rapidly ageing global population, the demands on health services to deal with disabling outcomes, which increase with age, will require policy makers to anticipate these changes. The mix of universal and more geographically specific influences on health reinforces the need for regular reporting on population health in detail and by underlying cause to help decision makers to identify success stories of disease control to emulate, as well as opportunities to improve. Funding: Bill & Melinda Gates Foundation. © 2020 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 licens

In-hospital mortality risk stratification in children aged under 5 years with pneumonia with or without pulse oximetry: A secondary analysis of the Pneumonia REsearch Partnership to Assess WHO REcommendations (PREPARE) dataset

Objectives We determined the pulse oximetry benefit in pediatric pneumonia mortality risk stratification and chest-indrawing pneumonia in-hospital mortality risk factors. Methods We report the characteristics and in-hospital pneumonia-related mortality of children aged 2-59 months who were included in the Pneumonia Research Partnership to Assess WHO Recommendations dataset. We developed multivariable logistic regression models of chest-indrawing pneumonia to identify mortality risk factors. Results Among 285,839 children, 164,244 (57.5%) from hospital-based studies were included. Pneumonia case fatality risk (CFR) without pulse oximetry measurement was higher than with measurement (5.8%, 95% confidence interval [CI] 5.6-5.9% vs 2.1%, 95% CI 1.9-2.4%). One in five children with chest-indrawing pneumonia was hypoxemic (19.7%, 95% CI 19.0-20.4%), and the hypoxemic CFR was 10.3% (95% CI 9.1-11.5%). Other mortality risk factors were younger age (either 2-5 months [adjusted odds ratio (aOR) 9.94, 95% CI 6.67-14.84] or 6-11 months [aOR 2.67, 95% CI 1.71-4.16]), moderate malnutrition (aOR 2.41, 95% CI 1.87-3.09), and female sex (aOR 1.82, 95% CI 1.43-2.32). Conclusion Children with a pulse oximetry measurement had a lower CFR. Many children hospitalized with chest-indrawing pneumonia were hypoxemic and one in 10 died. Young age and moderate malnutrition were risk factors for in-hospital chest-indrawing pneumonia-related mortality. Pulse oximetry should be integrated in pneumonia hospital care for children under 5 years

Global investments in pandemic preparedness and COVID-19: development assistance and domestic spending on health between 1990 and 2026

Background The COVID-19 pandemic highlighted gaps in health surveillance systems, disease prevention, and treatment globally. Among the many factors that might have led to these gaps is the issue of the financing of national health systems, especially in low-income and middle-income countries (LMICs), as well as a robust global system for pandemic preparedness. We aimed to provide a comparative assessment of global health spending at the onset of the pandemic; characterise the amount of development assistance for pandemic preparedness and response disbursed in the first 2 years of the COVID-19 pandemic; and examine expectations for future health spending and put into context the expected need for investment in pandemic preparedness. Methods In this analysis of global health spending between 1990 and 2021, and prediction from 2021 to 2026, we estimated four sources of health spending: development assistance for health (DAH), government spending, out-of-pocket spending, and prepaid private spending across 204 countries and territories. We used the Organisation for Economic Co-operation and Development (OECD)'s Creditor Reporting System (CRS) and the WHO Global Health Expenditure Database (GHED) to estimate spending. We estimated development assistance for general health, COVID-19 response, and pandemic preparedness and response using a keyword search. Health spending estimates were combined with estimates of resources needed for pandemic prevention and preparedness to analyse future health spending patterns, relative to need. Findings In 2019, at the onset of the COVID-19 pandemic, US$9·2 trillion (95$7·3 trillion (95% UI 7·2–7·4) in 2019; 293·7 times the $24·8 billion (95$43·1 billion in development assistance was provided to maintain or improve health. The pandemic led to an unprecedented increase in development assistance targeted towards health; in 2020 and 2021, $1·8 billion in DAH contributions was provided towards pandemic preparedness in LMICs, and$37·8 billion was provided for the health-related COVID-19 response. Although the support for pandemic preparedness is 12·2% of the recommended target by the High-Level Independent Panel (HLIP), the support provided for the health-related COVID-19 response is 252·2% of the recommended target. Additionally, projected spending estimates suggest that between 2022 and 2026, governments in 17 (95% UI 11–21) of the 137 LMICs will observe an increase in national government health spending equivalent to an addition of 1% of GDP, as recommended by the HLIP. Interpretation There was an unprecedented scale-up in DAH in 2020 and 2021. We have a unique opportunity at this time to sustain funding for crucial global health functions, including pandemic preparedness. However, historical patterns of underfunding of pandemic preparedness suggest that deliberate effort must be made to ensure funding is maintained