Replicative aging is associated with loss of genetic heterogeneity from extrachromosomal circular DNA in <i>Saccharomyces cerevisiae</i>

© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research. Circular DNA can arise from all parts of eukaryotic chromosomes. In yeast, circular ribosomal DNA (rDNA) accumulates dramatically as cells age, however little is known about the accumulation of other chromosome-derived circles or the contribution of such circles to genetic variation in aged cells. We profiled circular DNA in Saccharomyces cerevisiae populations sampled when young and after extensive aging. Young cells possessed highly diverse circular DNA populations but 94% of the circular DNA were lost after ∼15 divisions, whereas rDNA circles underwent massive accumulation to >95% of circular DNA. Circles present in both young and old cells were characterized by replication origins including circles from unique regions of the genome and repetitive regions: rDNA and telomeric Y' regions. We further observed that circles can have flexible inheritance patterns: [HXT6/7circle] normally segregates to mother cells but in low glucose is present in up to 50% of cells, the majority of which must have inherited this circle from their mother. Interestingly, [HXT6/7circle] cells are eventually replaced by cells carrying stable chromosomal HXT6 HXT6/7 HXT7 amplifications, suggesting circular DNAs are intermediates in chromosomal amplifications. In conclusion, the heterogeneity of circular DNA offers flexibility in adaptation, but this heterogeneity is remarkably diminished with age.ISSN:1362-4962ISSN:0301-561

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

Hubungan layanan bimbingan dan konseling dengan kedisiplinan peserta didik di Sekolah Menengah Pertama Muhammadiyah 13 Surabaya

Kedisiplinan merupakan bagian penting dalam pendidikan baik konteks pendidikan formal maupun informal. Kedisiplinan hendaknya dipandang sebagai kekuatan positif untuk memebtuk dan mengontrol perilaku siswa. Disiplin merupakan kesadaran akan pentingnya ketertiban dan keberaturan dalam kehidupan. Lemahnya tingkat kedisiplinan akan berdampak pada masalah disiplin dalam bentuk pelanggaran terhadap tata tertib sekolah. Bimbingan dan konseling sebagai bagian integral dari pendidikan mempunyai tanggung jawab mengatasi masalah disiplin siswa di sekolah melalui pelayanan bimbingan. Adanya program bimbingan dan konseling diharapkan memiliki solusi untuk menolong siswa mengontrol hidupnya dalam tingkah laku sesuai norma dan bertanggung jawab. Penelitian ini dilaksanakan di SMP Muhammadiyah 13 Surabaya dengan sampel sebanyak 67 yang diambil dari siswa kelas VII, VIII, IX. Independen variabel dalam penelitian ini adalah layananan bimbinangan dan konseling dengan subvariabel layanan informal, layanan penempatan, layanan pengajaran, layanan penyuluhan, layanan hubungan masyarakat, serta preventif dan kuratif. Sedangkan dependen variabelnya adalah kedisiplinan siswa dengan subvariabel peraturan, penghargaan, hukuman, dan konsistensi. Metode analisis yang digunakan dalam penelitian ini adalah pertama, desckriptive statistics untuk mengetahui distribusi frekuensi temuan data dari lapangan. Kedua, untuk mengetahui signifikansi layanan bimbingan dan konseling dengan tingkat kedisiplinan siswa menggunakan korelasi Product Moment dengan SPSS (Statistical Package For Social Scinces) sebagai alat bantu. Hasil penelitian menunjukkan bahwa terdapat hubungan signifikan antara layanan bimbingan dan konseling dengan kediplinan siswa. Nilai korelasi sebesar 0,879 berada di antara 0,80 s/d 1,00 yang artinya hubungan kedua variabel menunjukkan arah yang sama. Apabila layanan bimbingan dan konseling mengalami kenaikan, maka kedisiplinan siswa juga akan ikut naik. Begitu pula sebaliknya, jika layanan bimbingan dan konseling menglami penurunan, maka kedisiplinan siswa juga akan menurun. Hubungan antara kedua variabel termasuk hubungan yang positif sedang

Methods for the purification and detection of single nucleotide KRAS mutations on extrachromosomal circular DNA in human plasma

Abstract Backgrounds Despite recent advances, many cancers are still detected too late for curative treatment. There is, therefore, a need for the development of new diagnostic methods and biomarkers. One approach may arise from the detection of extrachromosomal circular DNA (eccDNA), which is part of cell‐free DNA in human plasma. Aims First, we assessed and compared two methods for the purification of eccDNA from plasma. Second, we tested for an easy diagnostic application of eccDNA liquid biopsy‐based assays. Materials & Methods For the comparison we tested a solid‐phase silica purification method and a phenol/chloroform method with salt precipitation. For the diagnostic application of eccDNA we developed and tested a qPCR primer‐based SNP detection system, for the detection of two well‐established cancer‐causing KRAS mutations (G12V and G12R) on circular DNA. This investigation was supported by purifying, sequencing, and analysing clinical plasma samples for eccDNAs containing KRAS mutant alleles in 0.5 mL plasma from 16 pancreatic ductal adenocarcinoma patients and 19 healthy controls. Results In our method comparison we observed, that following exonuclease treatment a lower eccDNA yield was found for the phenol/chloroform method (15.7%–26.7%) compared with the solid‐phase purification approach (47.8%–65.9%). For the diagnostic application of eccDNA tests, the sensitivity of the tested qPCR assay only reached ~10−3 in a background of 105 wild type (wt) KRAS circular entities, which was not improved by general amplification or primer‐based inhibition of wt KRAS amplification. Furthermore, we did not detect eccDNA containing KRAS in any of the clinical samples. Discussion A potential explanation for our inability to detect any KRAS mutations in the clinical samples may be related to the general low abundance of eccDNA in plasma. Conclusion Taken together our results provide a benchmark for eccDNA purification methods while raising the question of what is required for the optimal fast and sensitive detection of SNP mutations on eccDNA with greater sensitivity than primer‐based qPCR detection

Dip5 interacts with Gnp1 and Mep2.

<p>Physical interaction between Dip5 and transporters Hxt1, Mep1, Mep2, Dip5, Gnp1 was tested with the membrane protein-based split-ubiquitin system in the CEN.PK background. Ade^- His^- cells (THY.AP4) expressing the C-terminal part of ubiquitin fused to Dip5 and the transcriptional activator PLV were mated to Ade^- His^- cells (THY.AP5) expressing the N-terminal part of ubiquitin fused to either Hxt1, Mep1, Mep2, Dip5 or Gnp1. Interacting protein-pairs formed a functional ubiquitin that released PLV for induction of the HIS3 and ADE2 genes, enabling growth on synthetic complete medium without adenine and histidine.</p

<i>S. cerevisiae</i> strains and populations used in this study.

<p><i>S. cerevisiae</i> strains and populations used in this study.</p

Peptide modifications in six clonal isolates from prolonged nitrogen-limited populations.

<p>Denoted are the protein modifications resulting from mutations in the clonal isolates. The corresponding mutations are described in Material and Methods.</p

Gap1-dependent <i>FLO11</i> expression and invasive growth in Ptr3^647::C…N.

<p>Amino acid permease gene product in invasive growth ability in Ptr3^647::C…N was tested in a ura3 PTR3^647::C…N background (RB428) by introducing gene deletions dip5 (RB468), gnp1 (RB469) or gap1 (RB547). Cells were grown on rich complex medium (YPD) for 2 day at 30°C (A, upper panel) and flushed with water to expose invasive growth (A, lower panel). Northern blot of FLO11 mRNA for strains in A are shown in B (upper panel). RNA for ribosomal subunits 18S and 28S served as loading controls (B, lower panel). Genes of modulating proteins in invasive growth were deleted in the RB428 mutant: flo11 (RB457), tec1 (RB460), tpk2 (RB461), cyr1 (RB465), gcn4 (RB472), mss11 (RB474), ssy1 (RB477) and ptr3 (RB492). Cells were grown on YPD for 3 days at 30°C (C). The plate was flushed with water to test for invasive growth (D). Distribution of deletion strains in C and D are in E.</p