ATPase Inhibitory Factor-1 Disrupts Mitochondrial Ca2+ Handling and Promotes Pathological Cardiac Hypertrophy through CaMKIIδ

ATPase inhibitory factor-1 (IF1) preserves cellular ATP under conditions of respiratory collapse, yet the function of IF1 under normal respiring conditions is unresolved. We tested the hypothesis that IF1 promotes mitochondrial dysfunction and pathological cardiomyocyte hypertrophy in the context of heart failure (HF). Methods and results: Cardiac expression of IF1 was increased in mice and in humans with HF, downstream of neurohumoral signaling pathways and in patterns that resembled the fetal-like gene program. Adenoviral expression of wild-type IF1 in primary cardiomyocytes resulted in pathological hypertrophy and metabolic remodeling as evidenced by enhanced mitochondrial oxidative stress, reduced mitochondrial respiratory capacity, and the augmentation of extramitochondrial glycolysis. Similar perturbations were observed with an IF1 mutant incapable of binding to ATP synthase (E55A mutation), an indication that these effects occurred independent of binding to ATP synthase. Instead, IF1 promoted mitochondrial fragmentation and compromised mitochondrial Ca2+ handling, which resulted in sarcoplasmic reticulum Ca2+ overloading. The effects of IF1 on Ca2+ handling were associated with the cytosolic activation of calcium-calmodulin kinase II (CaMKII) and inhibition of CaMKII or co-expression of catalytically dead CaMKIIδC was sufficient to prevent IF1 induced pathological hypertrophy. Conclusions: IF1 represents a novel member of the fetal-like gene program that contributes to mitochondrial dysfunction and pathological cardiac remodeling in HF. Furthermore, we present evidence for a novel, ATP-synthase-independent, role for IF1 in mitochondrial Ca2+ handling and mitochondrial-to-nuclear crosstalk involving CaMKII

Large scale multifactorial likelihood quantitative analysis of BRCA1 and BRCA2 variants: An ENIGMA resource to support clinical variant classification

The multifactorial likelihood analysis method has demonstrated utility for quantitative assessment of variant pathogenicity for multiple cancer syndrome genes. Independent data types currently incorporated in the model for assessing BRCA1 and BRCA2 variants include clinically calibrated prior probability of pathogenicity based on variant location and bioinformatic prediction of variant effect, co-segregation, family cancer history profile, co-occurrence with a pathogenic variant in the same gene, breast tumor pathology, and case-control information. Research and clinical data for multifactorial likelihood analysis were collated for 1,395 BRCA1/2 predominantly intronic and missense variants, enabling classification based on posterior probability of pathogenicity for 734 variants: 447 variants were classified as (likely) benign, and 94 as (likely) pathogenic; and 248 classifications were new or considerably altered relative to ClinVar submissions. Classifications were compared with information not yet included in the likelihood model, and evidence strengths aligned to those recommended for ACMG/AMP classification codes. Altered mRNA splicing or function relative to known nonpathogenic variant controls were moderately to strongly predictive of variant pathogenicity. Variant absence in population datasets provided supporting evidence for variant pathogenicity. These findings have direct relevance for BRCA1 and BRCA2 variant evaluation, and justify the need for gene-specific calibration of evidence types used for variant classification

Tropical forest light regimes in a human-modified landscape

Light is the key energy input for all vegetated systems. Forest light regimes are complex, with the vertical pattern of light within canopies influenced by forest structure. Human disturbances in tropical forests impact forest structure and hence may influence the light environment and thus competitiveness of different trees. In this study, we measured vertical diffuse light profiles along a gradient of anthropogenic disturbance, sampling intact, logged, secondary, and fragmented sites in the biodiversity hot spot of the Atlantic forest, southeast Brazil, using photosynthetically active radiation sensors and a novel approach with estimations of vertical light profiles from hemispherical photographs. Our results show clear differences in vertical light profiles with disturbance: Fragmented forests are characterized by rapid light extinction within their low canopies, while the profiles in logged forests show high heterogeneity and high light in the mid-canopy despite decades of recovery. The secondary forest showed similar light profiles to intact forest, but with a lower canopy height. We also show that in some cases the upper canopy layer and heavy liana infestations can severely limit light penetration. Light extinction with height above the ground and depth below the canopy top was highest in fragmented forest and negatively correlated with canopy height. The novel, inexpensive, and rapid methods described here can be applied to other sites to quantify rarely measured vertical light profiles

Large scale multifactorial likelihood quantitative analysis of BRCA1 and BRCA2 variants: An ENIGMA resource to support clinical variant classification

Abstract The multifactorial likelihood analysis method has demonstrated utility for quantitative assessment of variant pathogenicity for multiple cancer syndrome genes. Independent data types currently incorporated in the model for assessing BRCA1 and BRCA2 variants include clinically calibrated prior probability of pathogenicity based on variant location and bioinformatic prediction of variant effect, co-segregation, family cancer history profile, co-occurrence with a pathogenic variant in the same gene, breast tumor pathology, and case-control information. Research and clinical data for multifactorial likelihood analysis were collated for 1395 BRCA1/2 predominantly intronic and missense variants, enabling classification based on posterior probability of pathogenicity for 734 variants: 447 variants were classified as (likely) benign, and 94 as (likely) pathogenic; 248 classifications were new or considerably altered relative to ClinVar submissions. Classifications were compared to information not yet included in the likelihood model, and evidence strengths aligned to those recommended for ACMG/AMP classification codes. Altered mRNA splicing or function relative to known non-pathogenic variant controls were moderately to strongly predictive of variant pathogenicity. Variant absence in population datasets provided supporting evidence for variant pathogenicity. These findings have direct relevance for BRCA1 and BRCA2 variant evaluation, and justify the need for gene-specific calibration of evidence types used for variant classification. This article is protected by copyright. All rights reserved.Peer reviewe

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

Effects of chlorophenol / hydrogen peroxide versus xylitol or chlorhexidine as chewing gum on salivary flow rate, pH, buffer capacity and salivary Streptococcus mutans scores

Objectives: A medicated chewing gum is a solid, single-dose preparation intended to be chewed for a certain period of time and deliver the drug. It may contain one or more than one active pharmaceutical ingredient. Whithin this context, we formulated a medicated gum with three active pharmaceutical ingredients: Camphor, p-chlorophenol and hydrogen peroxide, to be evaluated as therapeutic agents in dental caries. The aim of this study was to compare the effects on salivary streptococcus mutans, pH, buffer capacity and secretion rate of three medicated gums containing chlorophenol / hydrogen peroxide, xylitol or chlorhexidine. Methods: Double-blind, randomized crossover treatment in 24 subjects. The patients were randomized and subjected to six different treatment sequences. The subjects used, 1 gum tablet three times a day for 20 minutes, morning, noon and night. At the beginning and end of the three experimental phases saliva samples were obtained for determining the pH, buffer capacity, salivary flow rate and quantitative enumeration of S. mutans. Results: The use of medicated gum with camphor/p-chlorophenol and hydrogen peroxide did not modify the salivary chemical parameters measured, and not significantly reduced the number of S. mutans, after 7 days. Chewing gums with chlorhexidine significantly reduced the quantitative counting of S. mutans salivary and flow rate in a period of 7 days. Conclusions: The use of medicated chewing gums based on camphor/p-chlorophenol and hydrogen peroxide does not alter the chemical salivary parameters and does not significantly reduce the number of S. mutans, after a use over a period of 7 days

Caries and fluorosis in the Santiago metropolitan region in Chile: The impact of the fluoridation of the water

Purpose: To assess the damage and the prevalence of caries and fluorosis in children and adolescents in the metropolitan area after 8 years of drinking water fluoridation and to compare them with the baseline study. Methods: This was a prevalence study. The sample was selected using two-step probability sampling and stratified according to socioeconomic level. Subjects included 2,323 schoolchildren aged 6 to 8 years and 12 years living within the metropolitan region. The parents of all students provided consent and were previously included in the study. The subjects were clinically examined by calibrated dentists, who used World Health Organization (WHO) screening criteria and indicators to determine the presence of caries and dental fluorosis. Results: No cavity damage was found in 23.68% of the children. The average dmft was 3.18 for children aged 6 to 8 years. The DMFT was 0.59 in children aged 6 to 8 years and 2.6 in children 12 years compared with the baseline study. These differences were statistically significant. There were also significant differences in the DMFT index for each socioeconomic status group. The average number of dams was higher among children of low socioeconomic status. Of the studied children, 14.3% of children had dental fluorosis. Fluorosis was very mild in 12.35% of the cases, mild in 1.98% and moderate in 0.26%. There were no cases of severe fluorosis (classified according to Dean’s index). Conclusion: We conclude that after 8 years drinking water fluoridation in the metropolitan area, the number of children with no history of caries has increased by approximately 100%. The number of cases significantly affected by caries has also decreased significantly. The incidence of dental fluorosis has increased, but to milder degrees

A Flow Cytometric Clonogenic Assay Reveals the Single-Cell Potency of Doxorubicin

Standard cell proliferation assays use bulk media drug concentration to ascertain the potency of chemotherapeutic drugs; however, the relevant quantity is clearly the amount of drug actually taken up by the cell. To address this discrepancy, we have developed a flow cytometric clonogenic assay to correlate the amount of drug in a single cell with the cell’s ability to proliferate using a cell tracing dye and doxorubicin, a naturally fluorescent chemotherapeutic drug. By varying doxorubicin concentration in the media, length of treatment time, and treatment with verapamil, an efflux pump inhibitor, we introduced 10[superscript 5]–10[superscript 10] doxorubicin molecules per cell; then used a dye-dilution assay to simultaneously assess the number of cell divisions. We find that a cell’s ability to proliferate is a surprisingly conserved function of the number of intracellular doxorubicin molecules, resulting in single-cell IC[subscript 50] values of 4–12 million intracellular doxorubicin molecules. The developed assay is a straightforward method for understanding a drug’s single-cell potency and can be used for any fluorescent or fluorescently labeled drug, including nanoparticles or antibody–drug conjugates.Hertz Foundation (Fellowship)National Science Foundation (U.S.). Graduate Research Fellowship ProgramPfizer Inc.National Cancer Institute (U.S.) (David H. Koch Institute for Integrative Cancer Research at MIT. Support (Core) Grant P30-CA14051