Additional file 1 of Reproducible isolation of bovine mammary macrophages for analysis of host pathogen interactions

Supplementary Material

MOESM5 of Streptococcus uberis strains isolated from the bovine mammary gland evade immune recognition by mammary epithelial cells, but not of macrophages

Additional file 5: Other membrane anchored components of the cell envelope from S. uberis strain 233 did not significantly activate NF-κB in pbMEC. pbMEC were transfected with the ELAM driven reporter gene construct (100 ng) and stimulated with 10 µg/mL of the indicated S. uberis component or 30 µg/mL E. coli 1303 for 24 h. The luciferase activity was measured from cell lysates and normalized against their protein concentration. Values are expressed as fold increase above the level of the unstimulated control (ordinate). Each transfection was run in duplicate and assayed from triplicate challenges. (*p < 0.05). Components of the S. uberis cell envelope were prepared by bead disruption of the cells as described for the LTA preparation in the Material and Methods section of the main text. Lipoproteins were obtained by Triton X-114 phase partitioning of the membrane fraction as described [67]. Lipids were extracted according to the method of Bligh and Dyer [68]. Thin Layer Chromatography (TLC) was used to identify glycolipid in the total lipid extract. Samples were developed using a mixture of chloroform/methanol/H2O (65/25/4, v/v/v) and visualized with Hanessian’s and α-naphtol stain. Three glycolipids G1, G2 and G3 were identified. To isolate these glycolipids the crude lipid extract was fractionated on activated Silica Gel 60 and glycolipids were successively eluted with chloroform/methanol in the ratios of 9.5:0.5 (G1), 9:1 (G2), and 1:1 (G3). Those fractions were dried and further purified by preparative TLC to obtain pure specific substances. In the NF-κB assay lipoproteins were used untreated (native), proteinase K (PK) or H2O2 treated (perox). Furthermore NF-κB activation capacity of water and inter phase from the lipid extraction procedure and of the three glycolipids was examined and compared to a challenge with E. coli. The data regarding the lipoproteins show that the slight NF-κB activation is not specifically related to lipoproteins, since both, proteinase K as well as H2O2 treatment destroys the structural integrity of such molecules

Rational Re-engineering of a Transcriptional Silencing PreQ₁ Riboswitch

Re-engineered riboswitches that no longer respond to cellular metabolites, but that instead can be controlled by synthetic molecules, are potentially useful gene regulatory tools for use in synthetic biology and biotechnology fields. Previously, extensive genetic selection and screening approaches were employed to re-engineer a natural adenine riboswitch to create orthogonal ON-switches, enabling translational control of target gene expression in response to synthetic ligands. Here, we describe how a rational targeted approach was used to re-engineer the PreQ₁ riboswitch from Bacillus subtilis into an orthogonal OFF-switch. In this case, the evaluation of just six synthetic compounds with seven riboswitch mutants led to the identification of an orthogonal riboswitch–ligand pairing that effectively repressed the transcription of selected genes in B. subtilis. The streamlining of the re-engineering approach, and its extension to a second class of riboswitches, provides a methodological platform for the creation of new orthogonal regulatory components for biotechnological applications including gene functional analysis and antimicrobial target validation and screening

The global burden of adolescent and young adult cancer in 2019: a systematic analysis for the Global Burden of Disease Study 2019

The global burden of adolescent and young adult cancer in 2019: a systematic analysis for the Global Burden of Disease Study 201

Global, regional, and national comparative risk assessment of 79 behavioural, environmental and occupational, and metabolic risks or clusters of risks in 188 countries, 1990–2013: a systematic analysis for the Global Burden of Disease Study 2013

SummaryBackground The Global Burden of Disease, Injuries, and Risk Factor study 2013 (GBD 2013) is the fi rst of a series ofannual updates of the GBD. Risk factor quantifi cation, particularly of modifi able risk factors, can help to identifyemerging threats to population health and opportunities for prevention. The GBD 2013 provides a timely opportunityto update the comparative risk assessment with new data for exposure, relative risks, and evidence on the appropriatecounterfactual risk distribution.Methods Attributable deaths, years of life lost, years lived with disability, and disability-adjusted life-years (DALYs)have been estimated for 79 risks or clusters of risks using the GBD 2010 methods. Risk–outcome pairs meetingexplicit evidence criteria were assessed for 188 countries for the period 1990–2013 by age and sex using three inputs:risk exposure, relative risks, and the theoretical minimum risk exposure level (TMREL). Risks are organised into ahierarchy with blocks of behavioural, environmental and occupational, and metabolic risks at the fi rst level of thehierarchy. The next level in the hierarchy includes nine clusters of related risks and two individual risks, with moredetail provided at levels 3 and 4 of the hierarchy. Compared with GBD 2010, six new risk factors have been added:handwashing practices, occupational exposure to trichloroethylene, childhood wasting, childhood stunting, unsafesex, and low glomerular fi ltration rate. For most risks, data for exposure were synthesised with a Bayesian metaregressionmethod, DisMod-MR 2.0, or spatial-temporal Gaussian process regression. Relative risks were based onmeta-regressions of published cohort and intervention studies. Attributable burden for clusters of risks and all riskscombined took into account evidence on the mediation of some risks such as high body-mass index (BMI) throughother risks such as high systolic blood pressure and high cholesterol.Findings All risks combined account for 57·2% (95% uncertainty interval [UI] 55·8–58·5) of deaths and 41·6%(40·1–43·0) of DALYs. Risks quantifi ed account for 87·9% (86·5?89·3) of cardiovascular disease DALYs, rangingto a low of 0% for neonatal disorders and neglected tropical diseases and malaria. In terms of global DALYs in2013, six risks or clusters of risks each caused more than 5% of DALYs: dietary risks accounting for 11·3 milliondeaths and 241·4 million DALYs, high systolic blood pressure for 10·4 million deaths and 208·1 million DALYs,child and maternal malnutrition for 1·7 million deaths and 176·9 million DALYs, tobacco smoke for 6·1 milliondeaths and 143·5 million DALYs, air pollution for 5·5 million deaths and 141·5 million DALYs, and high BMI for4·4 million deaths and 134·0 million DALYs. Risk factor patterns vary across regions and countries and with time.In sub-Saharan Africa, the leading risk factors are child and maternal malnutrition, unsafe sex, and unsafe water,sanitation, and handwashing. In women, in nearly all countries in the Americas, north Africa, and the MiddleEast, and in many other high-income countries, high BMI is the leading risk factor, with high systolic bloodpressure as the leading risk in most of Central and Eastern Europe and south and east Asia. For men, high systolicblood pressure or tobacco use are the leading risks in nearly all high-income countries, in north Africa and theMiddle East, Europe, and Asia. For men and women, unsafe sex is the leading risk in a corridor from Kenya toSouth Africa.Interpretation Behavioural, environmental and occupational, and metabolic risks can explain half of global mortalityand more than one-third of global DALYs providing many opportunities for prevention. Of the larger risks, theattributable burden of high BMI has increased in the past 23 years. In view of the prominence of behavioural riskfactors, behavioural and social science research on interventions for these risks should be strengthened. Manyprevention and primary care policy options are available now to act on key risks.</p

Global, regional, and national disability-adjusted life-years (DALYs) for 359 diseases and injuries and healthy life expectancy (HALE) for 195 countries and territories, 1990-2017: A systematic analysis for the Global Burden of Disease Study 2017

Global, regional, and national disability-adjusted life-years (DALYs) for 359 diseases and injuries and healthy life expectancy (HALE) for 195 countries and territories, 1990-2017: A systematic analysis for the Global Burden of Disease Study 201

The global burden of adolescent and young adult cancer in 2019: a systematic analysis for the Global Burden of Disease Study 2019

BackgroundIn estimating the global burden of cancer, adolescents and young adults with cancer are often overlooked, despite being a distinct subgroup with unique epidemiology, clinical care needs, and societal impact. Comprehensive estimates of the global cancer burden in adolescents and young adults (aged 15–39 years) are lacking. To address this gap, we analysed results from the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2019, with a focus on the outcome of disability-adjusted life-years (DALYs), to inform global cancer control measures in adolescents and young adults.MethodsUsing the GBD 2019 methodology, international mortality data were collected from vital registration systems, verbal autopsies, and population-based cancer registry inputs modelled with mortality-to-incidence ratios (MIRs). Incidence was computed with mortality estimates and corresponding MIRs. Prevalence estimates were calculated using modelled survival and multiplied by disability weights to obtain years lived with disability (YLDs). Years of life lost (YLLs) were calculated as age-specific cancer deaths multiplied by the standard life expectancy at the age of death. The main outcome was DALYs (the sum of YLLs and YLDs). Estimates were presented globally and by Socio-demographic Index (SDI) quintiles (countries ranked and divided into five equal SDI groups), and all estimates were presented with corresponding 95% uncertainty intervals (UIs). For this analysis, we used the age range of 15–39 years to define adolescents and young adults.FindingsThere were 1·19 million (95% UI 1·11–1·28) incident cancer cases and 396 000 (370 000–425 000) deaths due to cancer among people aged 15–39 years worldwide in 2019. The highest age-standardised incidence rates occurred in high SDI (59·6 [54·5–65·7] per 100 000 person-years) and high-middle SDI countries (53·2 [48·8–57·9] per 100 000 person-years), while the highest age-standardised mortality rates were in low-middle SDI (14·2 [12·9–15·6] per 100 000 person-years) and middle SDI (13·6 [12·6–14·8] per 100 000 person-years) countries. In 2019, adolescent and young adult cancers contributed 23·5 million (21·9–25·2) DALYs to the global burden of disease, of which 2·7% (1·9–3·6) came from YLDs and 97·3% (96·4–98·1) from YLLs. Cancer was the fourth leading cause of death and tenth leading cause of DALYs in adolescents and young adults globally.InterpretationAdolescent and young adult cancers contributed substantially to the overall adolescent and young adult disease burden globally in 2019. These results provide new insights into the distribution and magnitude of the adolescent and young adult cancer burden around the world. With notable differences observed across SDI settings, these estimates can inform global and country-level cancer control efforts.</div

Global, regional, and national mortality among young people aged 10-24 years, 1950-2019: a systematic analysis for the Global Burden of Disease Study 2019

Background: Documentation of patterns and long-term trends in mortality in young people, which reflect huge changes in demographic and social determinants of adolescent health, enables identification of global investment priorities for this age group. We aimed to analyse data on the number of deaths, years of life lost, and mortality rates by sex and age group in people aged 10–24 years in 204 countries and territories from 1950 to 2019 by use of estimates from the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2019. Methods: We report trends in estimated total numbers of deaths and mortality rate per 100 000 population in young people aged 10–24 years by age group (10–14 years, 15–19 years, and 20–24 years) and sex in 204 countries and territories between 1950 and 2019 for all causes, and between 1980 and 2019 by cause of death. We analyse variation in outcomes by region, age group, and sex, and compare annual rate of change in mortality in young people aged 10–24 years with that in children aged 0–9 years from 1990 to 2019. We then analyse the association between mortality in people aged 10–24 years and socioeconomic development using the GBD Socio-demographic Index (SDI), a composite measure based on average national educational attainment in people older than 15 years, total fertility rate in people younger than 25 years, and income per capita. We assess the association between SDI and all-cause mortality in 2019, and analyse the ratio of observed to expected mortality by SDI using the most recent available data release (2017). Findings: In 2019 there were 1·49 million deaths (95% uncertainty interval 1·39–1·59) worldwide in people aged 10–24 years, of which 61% occurred in males. 32·7% of all adolescent deaths were due to transport injuries, unintentional injuries, or interpersonal violence and conflict; 32·1% were due to communicable, nutritional, or maternal causes; 27·0% were due to non-communicable diseases; and 8·2% were due to self-harm. Since 1950, deaths in this age group decreased by 30·0% in females and 15·3% in males, and sex-based differences in mortality rate have widened in most regions of the world. Geographical variation has also increased, particularly in people aged 10–14 years. Since 1980, communicable and maternal causes of death have decreased sharply as a proportion of total deaths in most GBD super-regions, but remain some of the most common causes in sub-Saharan Africa and south Asia, where more than half of all adolescent deaths occur. Annual percentage decrease in all-cause mortality rate since 1990 in adolescents aged 15–19 years was 1·3% in males and 1·6% in females, almost half that of males aged 1–4 years (2·4%), and around a third less than in females aged 1–4 years (2·5%). The proportion of global deaths in people aged 0–24 years that occurred in people aged 10–24 years more than doubled between 1950 and 2019, from 9·5% to 21·6%. Interpretation: Variation in adolescent mortality between countries and by sex is widening, driven by poor progress in reducing deaths in males and older adolescents. Improving global adolescent mortality will require action to address the specific vulnerabilities of this age group, which are being overlooked. Furthermore, indirect effects of the COVID-19 pandemic are likely to jeopardise efforts to improve health outcomes including mortality in young people aged 10–24 years. There is an urgent need to respond to the changing global burden of adolescent mortality, address inequities where they occur, and improve the availability and quality of primary mortality data in this age group. Funding: Bill & Melinda Gates Foundation

Global, regional, and national progress towards Sustainable Development Goal 3.2 for neonatal and child health: all-cause and cause-specific mortality findings from the Global Burden of Disease Study 2019

Background: Sustainable Development Goal 3.2 has targeted elimination of preventable child mortality, reduction of neonatal death to less than 12 per 1000 livebirths, and reduction of death of children younger than 5 years to less than 25 per 1000 livebirths, for each country by 2030. To understand current rates, recent trends, and potential trajectories of child mortality for the next decade, we present the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2019 findings for all-cause mortality and cause-specific mortality in children younger than 5 years of age, with multiple scenarios for child mortality in 2030 that include the consideration of potential effects of COVID-19, and a novel framework for quantifying optimal child survival. Methods: We completed all-cause mortality and cause-specific mortality analyses from 204 countries and territories for detailed age groups separately, with aggregated mortality probabilities per 1000 livebirths computed for neonatal mortality rate (NMR) and under-5 mortality rate (U5MR). Scenarios for 2030 represent different potential trajectories, notably including potential effects of the COVID-19 pandemic and the potential impact of improvements preferentially targeting neonatal survival. Optimal child survival metrics were developed by age, sex, and cause of death across all GBD location-years. The first metric is a global optimum and is based on the lowest observed mortality, and the second is a survival potential frontier that is based on stochastic frontier analysis of observed mortality and Healthcare Access and Quality Index. Findings: Global U5MR decreased from 71·2 deaths per 1000 livebirths (95% uncertainty interval [UI] 68·3–74·0) in 2000 to 37·1 (33·2–41·7) in 2019 while global NMR correspondingly declined more slowly from 28·0 deaths per 1000 live births (26·8–29·5) in 2000 to 17·9 (16·3–19·8) in 2019. In 2019, 136 (67%) of 204 countries had a U5MR at or below the SDG 3.2 threshold and 133 (65%) had an NMR at or below the SDG 3.2 threshold, and the reference scenario suggests that by 2030, 154 (75%) of all countries could meet the U5MR targets, and 139 (68%) could meet the NMR targets. Deaths of children younger than 5 years totalled 9·65 million (95% UI 9·05–10·30) in 2000 and 5·05 million (4·27–6·02) in 2019, with the neonatal fraction of these deaths increasing from 39% (3·76 million [95% UI 3·53–4·02]) in 2000 to 48% (2·42 million; 2·06–2·86) in 2019. NMR and U5MR were generally higher in males than in females, although there was no statistically significant difference at the global level. Neonatal disorders remained the leading cause of death in children younger than 5 years in 2019, followed by lower respiratory infections, diarrhoeal diseases, congenital birth defects, and malaria. The global optimum analysis suggests NMR could be reduced to as low as 0·80 (95% UI 0·71–0·86) deaths per 1000 livebirths and U5MR to 1·44 (95% UI 1·27–1·58) deaths per 1000 livebirths, and in 2019, there were as many as 1·87 million (95% UI 1·35–2·58; 37% [95% UI 32–43]) of 5·05 million more deaths of children younger than 5 years than the survival potential frontier. Interpretation: Global child mortality declined by almost half between 2000 and 2019, but progress remains slower in neonates and 65 (32%) of 204 countries, mostly in sub-Saharan Africa and south Asia, are not on track to meet either SDG 3.2 target by 2030. Focused improvements in perinatal and newborn care, continued and expanded delivery of essential interventions such as vaccination and infection prevention, an enhanced focus on equity, continued focus on poverty reduction and education, and investment in strengthening health systems across the development spectrum have the potential to substantially improve U5MR. Given the widespread effects of COVID-19, considerable effort will be required to maintain and accelerate progress