Whole lifespan microscopic observation of budding yeast aging through a microfluidic dissection platform

Important insights into aging have been generated with the genetically tractable and short-lived budding yeast. However, it is still impossible today to continuously track cells by high-resolution microscopic imaging (e.g., fluorescent imaging) throughout their entire lifespan. Instead, the field still needs to rely on a 50-y-old laborious and time-consuming method to assess the lifespan of yeast cells and to isolate differentially aged cells for microscopic snapshots via manual dissection of daughter cells from the larger mother cell. Here, we are unique in achieving continuous and high-resolution microscopic imaging of the entire replicative lifespan of single yeast cells. Our microfluidic dissection platform features an optically prealigned single focal plane and an integrated array of soft elastomer-based micropads, used together to allow for trapping of mother cells, removal of daughter cells, monitoring gradual changes in aging, and unprecedented microscopic imaging of the whole aging process. Using the platform, we found remarkable age-associated changes in phenotypes (e.g., that cells can show strikingly differential cell and vacuole morphologies at the moment of their deaths), indicating substantial heterogeneity in cell aging and death. We envision the microfluidic dissection platform to become a major tool in aging research.

COVID-19 alters human microbiomes: a meta-analysis

International audienceIntroduction Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has infected a substantial portion of the world’s population, and novel consequences of COVID-19 on the human body are continuously being uncovered. The human microbiome plays an essential role in host health and well-being, and multiple studies targeting specific populations have reported altered microbiomes in patients infected with SARS-CoV-2. Given the global scale and massive incidence of COVID on the global population, determining whether the effects of COVID-19 on the human microbiome are consistent and generalizable across populations is essential.Methods We performed a synthesis of human microbiome responses to COVID-19. We collected 16S rRNA gene amplicon sequence data from 11 studies sampling the oral and nasopharyngeal or gut microbiome of COVID-19-infected and uninfected subjects. Our synthesis included 1,159 respiratory (oral and nasopharyngeal) microbiome samples and 267 gut microbiome samples from patients in 11 cities across four countries.Results Our reanalyses revealed communitywide alterations in the respiratory and gut microbiomes across human populations. We found significant overall reductions in the gut microbial diversity of COVID-19-infected patients, but not in the respiratory microbiome. Furthermore, we found more consistent community shifts in the gut microbiomes of infected patients than in the respiratory microbiomes, although the microbiomes in both sites exhibited higher host-to-host variation in infected patients. In respiratory microbiomes, COVID-19 infection resulted in an increase in the relative abundance of potentially pathogenic bacteria, including Mycoplasma .Discussion Our findings shed light on the impact of COVID-19 on the human-associated microbiome across populations, and highlight the need for further research into the relationship between long-term effects of COVID-19 and altered microbiota

Design Structural Stability Metrics and Post-Release Defect Density: An Empirical Study

This paper empirically explores the correlations between a suite of structural stability metrics for object-oriented designs and post-release defect density. The investigated stability metrics measure the extent to which the structure of a design is preserved throughout the evolution of the software from one release to the next. As a case study, thirteen successive releases of Apache Ant were analyzed. The results indicate that some of the stability metrics are significantly correlated with post-release defect density. It was possible to construct statistically significant regression models to estimate post-release defect density from subsets of these metrics. The results reveal the practical significance and usefulness of some of the investigated stability metrics as early indicators of one of the important software quality outcomes, which is post-release defect density

Design Structural Stability Metrics and Post-Release Defect Density: An Empirical Study

This paper empirically explores the correlations between a suite of structural stability metrics for object-oriented designs and post-release defect density. The investigated stability metrics measure the extent to which the structure of a design is preserved throughout the evolution of the software from one release to the next. As a case study, thirteen successive releases of Apache Ant were analyzed. The results indicate that some of the stability metrics are significantly correlated with post-release defect density. It was possible to construct statistically significant regression models to estimate post-release defect density from subsets of these metrics. The results reveal the practical significance and usefulness of some of the investigated stability metrics as early indicators of one of the important software quality outcomes, which is post-release defect density

Rationale and design of the CONFIRM2 (Quantitative COroNary CT Angiography Evaluation For Evaluation of Clinical Outcomes: An InteRnational, Multicenter Registry) study.

BACKGROUND In the last 15 years, large registries and several randomized clinical trials have demonstrated the diagnostic and prognostic value of coronary computed tomography angiography (CCTA). Advances in CT scanner technology and developments of analytic tools now enable accurate quantification of coronary artery disease (CAD), including total coronary plaque volume (TPV) and low attenuation plaque volume (LAP). The primary aim of CONFIRM2, (Quantitative COroNary CT Angiography Evaluation For Evaluation of Clinical Outcomes: An InteRnational, Multicenter Registry) is to perform comprehensive quantification of CCTA findings, including coronary, non-coronary cardiac, non-cardiac vascular, non-cardiac findings, and relate them to clinical variables and cardiovascular clinical outcomes. DESIGN CONFIRM2 is a multicenter, international observational cohort study designed to evaluate multidimensional associations between quantitative phenotype of cardiovascular disease and future adverse clinical outcomes in subjects undergoing clinically indicated CCTA. The targeted population is heterogenous and includes patients undergoing CCTA for atherosclerotic evaluation, valvular heart disease, congenital heart disease or pre-procedural evaluation. Automated software will be utilized for quantification of coronary plaque, stenosis, vascular morphology and cardiac structures for rapid and reproducible tissue characterization. Up to 30,000 patients will be included from up to 50 international multi-continental clinical CCTA sites and followed for 3-4 years. SUMMARY CONFIRM2 is one of the largest CCTA studies to establish the clinical value of a multiparametric approach to quantify the phenotype of cardiovascular disease by CCTA using automated imaging solutions

The Use of Orthologous Sequences to Predict the Impact of Amino Acid Substitutions on Protein Function

Computational predictions of the functional impact of genetic variation play a critical role in human genetics research. For nonsynonymous coding variants, most prediction algorithms make use of patterns of amino acid substitutions observed among homologous proteins at a given site. In particular, substitutions observed in orthologous proteins from other species are often assumed to be tolerated in the human protein as well. We examined this assumption by evaluating a panel of nonsynonymous mutants of a prototypical human enzyme, methylenetetrahydrofolate reductase (MTHFR), in a yeast cell-based functional assay. As expected, substitutions in human MTHFR at sites that are well-conserved across distant orthologs result in an impaired enzyme, while substitutions present in recently diverged sequences (including a 9-site mutant that “resurrects” the human-macaque ancestor) result in a functional enzyme. We also interrogated 30 sites with varying degrees of conservation by creating substitutions in the human enzyme that are accepted in at least one ortholog of MTHFR. Quite surprisingly, most of these substitutions were deleterious to the human enzyme. The results suggest that selective constraints vary between phylogenetic lineages such that inclusion of distant orthologs to infer selective pressures on the human enzyme may be misleading. We propose that homologous proteins are best used to reconstruct ancestral sequences and infer amino acid conservation among only direct lineal ancestors of a particular protein. We show that such an “ancestral site preservation” measure outperforms other prediction methods, not only in our selected set for MTHFR, but also in an exhaustive set of E. coli LacI mutants

Silent but Not Static: Accelerated Base-Pair Substitution in Silenced Chromatin of Budding Yeasts

Subtelomeric DNA in budding yeasts, like metazoan heterochromatin, is gene poor, repetitive, transiently silenced, and highly dynamic. The rapid evolution of subtelomeric regions is commonly thought to arise from transposon activity and increased recombination between repetitive elements. However, we found evidence of an additional factor in this diversification. We observed a surprising level of nucleotide divergence in transcriptionally silenced regions in inter-species comparisons of Saccharomyces yeasts. Likewise, intra-species analysis of polymorphisms also revealed increased SNP frequencies in both intergenic and synonymous coding positions of silenced DNA. This analysis suggested that silenced DNA in Saccharomyces cerevisiae and closely related species had increased single base-pair substitution that was likely due to the effects of the silencing machinery on DNA replication or repair