Rapid detection of the CYP2A6*12 hybrid allele by Pyrosequencing® technology

<p>Abstract</p> <p>Background</p> <p>Identification of <it>CYP2A6 </it>alleles associated with reduced enzyme activity is important in the study of inter-individual differences in drug metabolism. <it>CYP2A6*12 </it>is a hybrid allele that results from unequal crossover between <it>CYP2A6 </it>and <it>CYP2A7 </it>genes. The 5' regulatory region and exons 1–2 are derived from <it>CYP2A7</it>, and exons 3–9 are derived from <it>CYP2A6</it>. Conventional methods for detection of <it>CYP2A6*12 </it>consist of two-step PCR protocols that are laborious and unsuitable for high-throughput genotyping. We developed a rapid and accurate method to detect the <it>CYP2A6*12 </it>allele by Pyrosequencing technology.</p> <p>Methods</p> <p>A single set of PCR primers was designed to specifically amplify both the <it>CYP2A6*1 </it>wild-type allele and the <it>CYP2A6*12 </it>hybrid allele. An internal Pyrosequencing primer was used to generate allele-specific sequence information, which detected homozygous wild-type, heterozygous hybrid, and homozygous hybrid alleles. We first validated the assay on 104 DNA samples that were also genotyped by conventional two-step PCR and by cycle sequencing. <it>CYP2A6*12 </it>allele frequencies were then determined using the Pyrosequencing assay on 181 multi-ethnic DNA samples from subjects of African American, European Caucasian, Pacific Rim, and Hispanic descent. Finally, we streamlined the Pyrosequencing assay by integrating liquid handling robotics into the workflow.</p> <p>Results</p> <p>Pyrosequencing results demonstrated 100% concordance with conventional two-step PCR and cycle sequencing methods. Allele frequency data showed slightly higher prevalence of the <it>CYP2A6*12 </it>allele in European Caucasians and Hispanics.</p> <p>Conclusion</p> <p>This Pyrosequencing assay proved to be a simple, rapid, and accurate alternative to conventional methods, which can be easily adapted to the needs of higher-throughput studies.</p

Global patient outcomes after elective surgery: prospective cohort study in 27 low-, middle- and high-income countries.

BACKGROUND: As global initiatives increase patient access to surgical treatments, there remains a need to understand the adverse effects of surgery and define appropriate levels of perioperative care. METHODS: We designed a prospective international 7-day cohort study of outcomes following elective adult inpatient surgery in 27 countries. The primary outcome was in-hospital complications. Secondary outcomes were death following a complication (failure to rescue) and death in hospital. Process measures were admission to critical care immediately after surgery or to treat a complication and duration of hospital stay. A single definition of critical care was used for all countries. RESULTS: A total of 474 hospitals in 19 high-, 7 middle- and 1 low-income country were included in the primary analysis. Data included 44 814 patients with a median hospital stay of 4 (range 2-7) days. A total of 7508 patients (16.8%) developed one or more postoperative complication and 207 died (0.5%). The overall mortality among patients who developed complications was 2.8%. Mortality following complications ranged from 2.4% for pulmonary embolism to 43.9% for cardiac arrest. A total of 4360 (9.7%) patients were admitted to a critical care unit as routine immediately after surgery, of whom 2198 (50.4%) developed a complication, with 105 (2.4%) deaths. A total of 1233 patients (16.4%) were admitted to a critical care unit to treat complications, with 119 (9.7%) deaths. Despite lower baseline risk, outcomes were similar in low- and middle-income compared with high-income countries. CONCLUSIONS: Poor patient outcomes are common after inpatient surgery. Global initiatives to increase access to surgical treatments should also address the need for safe perioperative care. STUDY REGISTRATION: ISRCTN5181700

Global burden of 288 causes of death and life expectancy decomposition in 204 countries and territories and 811 subnational locations, 1990–2021: a systematic analysis for the Global Burden of Disease Study 2021

BACKGROUND Regular, detailed reporting on population health by underlying cause of death is fundamental for public health decision making. Cause-specific estimates of mortality and the subsequent effects on life expectancy worldwide are valuable metrics to gauge progress in reducing mortality rates. These estimates are particularly important following large-scale mortality spikes, such as the COVID-19 pandemic. When systematically analysed, mortality rates and life expectancy allow comparisons of the consequences of causes of death globally and over time, providing a nuanced understanding of the effect of these causes on global populations. METHODS The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2021 cause-of-death analysis estimated mortality and years of life lost (YLLs) from 288 causes of death by age-sex-location-year in 204 countries and territories and 811 subnational locations for each year from 1990 until 2021. The analysis used 56 604 data sources, including data from vital registration and verbal autopsy as well as surveys, censuses, surveillance systems, and cancer registries, among others. As with previous GBD rounds, cause-specific death rates for most causes were estimated using the Cause of Death Ensemble model-a modelling tool developed for GBD to assess the out-of-sample predictive validity of different statistical models and covariate permutations and combine those results to produce cause-specific mortality estimates-with alternative strategies adapted to model causes with insufficient data, substantial changes in reporting over the study period, or unusual epidemiology. YLLs were computed as the product of the number of deaths for each cause-age-sex-location-year and the standard life expectancy at each age. As part of the modelling process, uncertainty intervals (UIs) were generated using the 2·5th and 97·5th percentiles from a 1000-draw distribution for each metric. We decomposed life expectancy by cause of death, location, and year to show cause-specific effects on life expectancy from 1990 to 2021. We also used the coefficient of variation and the fraction of population affected by 90% of deaths to highlight concentrations of mortality. Findings are reported in counts and age-standardised rates. Methodological improvements for cause-of-death estimates in GBD 2021 include the expansion of under-5-years age group to include four new age groups, enhanced methods to account for stochastic variation of sparse data, and the inclusion of COVID-19 and other pandemic-related mortality-which includes excess mortality associated with the pandemic, excluding COVID-19, lower respiratory infections, measles, malaria, and pertussis. For this analysis, 199 new country-years of vital registration cause-of-death data, 5 country-years of surveillance data, 21 country-years of verbal autopsy data, and 94 country-years of other data types were added to those used in previous GBD rounds. FINDINGS The leading causes of age-standardised deaths globally were the same in 2019 as they were in 1990; in descending order, these were, ischaemic heart disease, stroke, chronic obstructive pulmonary disease, and lower respiratory infections. In 2021, however, COVID-19 replaced stroke as the second-leading age-standardised cause of death, with 94·0 deaths (95% UI 89·2-100·0) per 100 000 population. The COVID-19 pandemic shifted the rankings of the leading five causes, lowering stroke to the third-leading and chronic obstructive pulmonary disease to the fourth-leading position. In 2021, the highest age-standardised death rates from COVID-19 occurred in sub-Saharan Africa (271·0 deaths [250·1-290·7] per 100 000 population) and Latin America and the Caribbean (195·4 deaths [182·1-211·4] per 100 000 population). The lowest age-standardised death rates from COVID-19 were in the high-income super-region (48·1 deaths [47·4-48·8] per 100 000 population) and southeast Asia, east Asia, and Oceania (23·2 deaths [16·3-37·2] per 100 000 population). Globally, life expectancy steadily improved between 1990 and 2019 for 18 of the 22 investigated causes. Decomposition of global and regional life expectancy showed the positive effect that reductions in deaths from enteric infections, lower respiratory infections, stroke, and neonatal deaths, among others have contributed to improved survival over the study period. However, a net reduction of 1·6 years occurred in global life expectancy between 2019 and 2021, primarily due to increased death rates from COVID-19 and other pandemic-related mortality. Life expectancy was highly variable between super-regions over the study period, with southeast Asia, east Asia, and Oceania gaining 8·3 years (6·7-9·9) overall, while having the smallest reduction in life expectancy due to COVID-19 (0·4 years). The largest reduction in life expectancy due to COVID-19 occurred in Latin America and the Caribbean (3·6 years). Additionally, 53 of the 288 causes of death were highly concentrated in locations with less than 50% of the global population as of 2021, and these causes of death became progressively more concentrated since 1990, when only 44 causes showed this pattern. The concentration phenomenon is discussed heuristically with respect to enteric and lower respiratory infections, malaria, HIV/AIDS, neonatal disorders, tuberculosis, and measles. INTERPRETATION Long-standing gains in life expectancy and reductions in many of the leading causes of death have been disrupted by the COVID-19 pandemic, the adverse effects of which were spread unevenly among populations. Despite the pandemic, there has been continued progress in combatting several notable causes of death, leading to improved global life expectancy over the study period. Each of the seven GBD super-regions showed an overall improvement from 1990 and 2021, obscuring the negative effect in the years of the pandemic. Additionally, our findings regarding regional variation in causes of death driving increases in life expectancy hold clear policy utility. Analyses of shifting mortality trends reveal that several causes, once widespread globally, are now increasingly concentrated geographically. These changes in mortality concentration, alongside further investigation of changing risks, interventions, and relevant policy, present an important opportunity to deepen our understanding of mortality-reduction strategies. Examining patterns in mortality concentration might reveal areas where successful public health interventions have been implemented. Translating these successes to locations where certain causes of death remain entrenched can inform policies that work to improve life expectancy for people everywhere. FUNDING Bill & Melinda Gates Foundation

The Relationship Between Psychological Stress and Anxiety with Gastrointestinal Symptoms Before and During a 56 km Ultramarathon Running Race

Abstract Background This study assessed relationships and sex differences between psychological state (recovery, stress, anxiety, and self-confidence) and gastrointestinal symptoms (GIS) prior to and during a 56 km ultramarathon running race and identified predictive factors of race GIS. Forty-four (26 males, 18 females) ultramarathon competitors completed anxiety, recovery, stress and GIS questionnaires for three days prior to the race and immediately pre-race. Race GIS were assessed immediately post-race. Spearman’s rank order, Mann–Whitney U tests and regression analyses were used to determine correlations and identify sex differences between psychological state and GIS and determine predictors of race GIS. Results Race GIS were significantly correlated with recovery (rs =  − 0.381, p = 0.011), stress (rs = 0.500, p = 0.001) and anxiety (rs = 0.408, p = 0.006), calculated as the mean of the three days preceding the race and on race morning. The correlation between anxiety and GIS was strongest in the 24 h immediately prior to the race (all rs &gt; 0.400, and all p &lt; 0.05), but unclear patterns were identified for stress and recovery. Regression analyses showed 36% and 40% of variation in the severity and number of race GIS was accounted for by body mass and measures of stress, anxiety, and GIS over the three days preceding the race and on race morning (both p &lt; 0.001). There were no sex differences in the number and severity of GIS leading up to or during the race (all p &gt; 0.05), however, females reported greater state anxiety (p = 0.018) and lower self-confidence than males (p = 0.006) over the three days preceding the race and on race morning. Conclusion Endurance athletes that experience GIS during competition should investigate elevated stress and/or anxiety as a potential contributor and identify if management strategies can reduce the occurrence and severity of GIS. </jats:sec

Thigh-length compression stockings and DVT after stroke

Controversy exists as to whether neoadjuvant chemotherapy improves survival in patients with invasive bladder cancer, despite randomised controlled trials of more than 3000 patients. We undertook a systematic review and meta-analysis to assess the effect of such treatment on survival in patients with this disease

Characterization of Tafazzin Splice Variants from Humans and Fruit Flies*

The tafazzin gene encodes a phospholipid-lysophospholipid transacylase involved in cardiolipin metabolism, but it is not known why it forms multiple transcripts as a result of alternative splicing. Here we studied the intracellular localization, enzymatic activity, and metabolic function of four isoforms of human tafazzin and three isoforms of Drosophila tafazzin upon expression in different mammalian and insect systems. When expressed in HeLa cells, all isoforms were localized in mitochondria except for the B-form of Drosophila tafazzin, which was associated with multiple intracellular membranes. Among the human isoforms, only full-length tafazzin (FL) and tafazzin lacking exon 5 (Δ5) had transacylase activity, and only these two isoforms were able to restore a normal cardiolipin pattern, normal respiratory activity of mitochondria, and male fertility in tafazzin-deficient flies. Both FL and Δ5 were associated with large protein complexes in 293T cell mitochondria, but treatment with alkali and proteinase K suggested that the Δ5 isoform was more integrated into the hydrophobic core of the membrane than the FL isoform. Although all Drosophila isoforms showed transacylase activity in vitro, only the A-form supported cardiolipin remodeling in flies. The data suggest that humans express two mitochondrial isoenzymes of tafazzin that have similar transacylase activities but different membrane topologies. Furthermore, the data show that the expression of human tafazzin in flies creates cardiolipin with a Drosophila pattern, suggesting that the characteristic fatty acid profile of cardiolipin is not determined by the substrate specificity of tafazzin

Multiplicity dependence of light (anti-)nuclei production in p–Pb collisions at sNN=5.02 TeV

The measurement of the deuteron and anti-deuteron production in the rapidity range −1 < y < 0 as a function of transverse momentum and event multiplicity in p–Pb collisions at √sNN = 5.02 TeV is presented. (Anti-)deuterons are identified via their specific energy loss dE/dx and via their time-of- flight. Their production in p–Pb collisions is compared to pp and Pb–Pb collisions and is discussed within the context of thermal and coalescence models. The ratio of integrated yields of deuterons to protons (d/p) shows a significant increase as a function of the charged-particle multiplicity of the event starting from values similar to those observed in pp collisions at low multiplicities and approaching those observed in Pb–Pb collisions at high multiplicities. The mean transverse particle momenta are extracted from the deuteron spectra and the values are similar to those obtained for p and particles. Thus, deuteron spectra do not follow mass ordering. This behaviour is in contrast to the trend observed for non-composite particles in p–Pb collisions. In addition, the production of the rare 3He and 3He nuclei has been studied. The spectrum corresponding to all non-single diffractive p-Pb collisions is obtained in the rapidity window −1 < y < 0 and the pT-integrated yield dN/dy is extracted. It is found that the yields of protons, deuterons, and 3He, normalised by the spin degeneracy factor, follow an exponential decrease with mass number

Jet fragmentation transverse momentum distributions in pp and p-Pb collisions at s \sqrt{s} , sNN \sqrt{s_{\mathrm{NN}}} = 5.02 TeV

Jet fragmentation transverse momentum (j$_{T}$) distributions are measured in proton-proton (pp) and proton-lead (p-Pb) collisions at $\sqrt{s_{\mathrm{NN}}}$ = 5.02 TeV with the ALICE experiment at the LHC. Jets are reconstructed with the ALICE tracking detectors and electromagnetic calorimeter using the anti-k$_{T}$ algorithm with resolution parameter R = 0.4 in the pseudorapidity range |η| < 0.25. The j$_{T}$ values are calculated for charged particles inside a fixed cone with a radius R = 0.4 around the reconstructed jet axis. The measured j$_{T}$ distributions are compared with a variety of parton-shower models. Herwig and Pythia 8 based models describe the data well for the higher j$_{T}$ region, while they underestimate the lower j$_{T}$ region. The j$_{T}$ distributions are further characterised by fitting them with a function composed of an inverse gamma function for higher j$_{T}$ values (called the “wide component”), related to the perturbative component of the fragmentation process, and with a Gaussian for lower j$_{T}$ values (called the “narrow component”), predominantly connected to the hadronisation process. The width of the Gaussian has only a weak dependence on jet transverse momentum, while that of the inverse gamma function increases with increasing jet transverse momentum. For the narrow component, the measured trends are successfully described by all models except for Herwig. For the wide component, Herwig and PYTHIA 8 based models slightly underestimate the data for the higher jet transverse momentum region. These measurements set constraints on models of jet fragmentation and hadronisation

Measurements of the groomed and ungroomed jet angularities in pp collisions at s \sqrt{s} = 5.02 TeV

International audienceThe jet angularities are a class of jet substructure observables which characterize the angular and momentum distribution of particles within jets. These observables are sensitive to momentum scales ranging from perturbative hard scatterings to nonperturbative fragmentation into final-state hadrons. We report measurements of several groomed and ungroomed jet angularities in pp collisions at $\sqrt{s}$ = 5.02 TeV with the ALICE detector. Jets are reconstructed using charged particle tracks at midrapidity (|η| < 0.9). The anti-k$_{T}$ algorithm is used with jet resolution parameters R = 0.2 and R = 0.4 for several transverse momentum {p}_{\mathrm{T}}^{\mathrm{ch}} ^{jet} intervals in the 20–100 GeV/c range. Using the jet grooming algorithm Soft Drop, the sensitivity to softer, wide-angle processes, as well as the underlying event, can be reduced in a way which is well-controlled in theoretical calculations. We report the ungroomed jet angularities, λ$_{α}$, and groomed jet angularities, λ$_{α,g}$, to investigate the interplay between perturbative and nonperturbative effects at low jet momenta. Various angular exponent parameters α = 1, 1.5, 2, and 3 are used to systematically vary the sensitivity of the observable to collinear and soft radiation. Results are compared to analytical predictions at next-to-leading-logarithmic accuracy, which provide a generally good description of the data in the perturbative regime but exhibit discrepancies in the nonperturbative regime. Moreover, these measurements serve as a baseline for future ones in heavy-ion collisions by providing new insight into the interplay between perturbative and nonperturbative effects in the angular and momentum substructure of jets. They supply crucial guidance on the selection of jet resolution parameter, jet transverse momentum, and angular scaling variable for jet quenching studies.[graphic not available: see fulltext