Molecular basis of microhomology-mediated end-joining by purified full-length Polθ

DNA polymerase θ (Polθ) is a unique polymerase-helicase fusion protein that promotes microhomology-mediated end-joining (MMEJ) of DNA double-strand breaks (DSBs). How full-length human Polθ performs MMEJ at the molecular level remains unknown. Using a biochemical approach, we find that the helicase is essential for Polθ MMEJ of long ssDNA overhangs which model resected DSBs. Remarkably, Polθ MMEJ of ssDNA overhangs requires polymerase-helicase attachment, but not the disordered central domain, and occurs independently of helicase ATPase activity. Using single-particle microscopy and biophysical methods, we find that polymerase-helicase attachment promotes multimeric gel-like Polθ complexes that facilitate DNA accumulation, DNA synapsis, and MMEJ. We further find that the central domain regulates Polθ multimerization and governs its DNA substrate requirements for MMEJ. These studies identify unexpected functions for the helicase and central domain and demonstrate the importance of polymerase-helicase tethering in MMEJ and the structural organization of Polθ

Global, regional, and national sex differences in the global burden of tuberculosis by HIV status, 1990–2019: results from the Global Burden of Disease Study 2019

Background Tuberculosis is a major contributor to the global burden of disease, causing more than a million deaths annually. Given an emphasis on equity in access to diagnosis and treatment of tuberculosis in global health targets, evaluations of differences in tuberculosis burden by sex are crucial. We aimed to assess the levels and trends of the global burden of tuberculosis, with an emphasis on investigating differences in sex by HIV status for 204 countries and territories from 1990 to 2019. Methods We used a Bayesian hierarchical Cause of Death Ensemble model (CODEm) platform to analyse 21 505 site-years of vital registration data, 705 site-years of verbal autopsy data, 825 site-years of sample-based vital registration data, and 680 site-years of mortality surveillance data to estimate mortality due to tuberculosis among HIV-negative individuals. We used a population attributable fraction approach to estimate mortality related to HIV and tuberculosis coinfection. A compartmental meta-regression tool (DisMod-MR 2.1) was then used to synthesise all available data sources, including prevalence surveys, annual case notifications, population-based tuberculin surveys, and tuberculosis cause-specific mortality, to produce estimates of incidence, prevalence, and mortality that were internally consistent. We further estimated the fraction of tuberculosis mortality that is attributable to independent effects of risk factors, including smoking, alcohol use, and diabetes, for HIV-negative individuals. For individuals with HIV and tuberculosis coinfection, we assessed mortality attributable to HIV risk factors including unsafe sex, intimate partner violence (only estimated among females), and injection drug use. We present 95% uncertainty intervals for all estimates. Findings Globally, in 2019, among HIV-negative individuals, there were 1.18 million (95% uncertainty interval 1.08-1.29) deaths due to tuberculosis and 8.50 million (7.45-9.73) incident cases of tuberculosis. Among HIV-positive individuals, there were 217 000 (153 000-279 000) deaths due to tuberculosis and 1.15 million (1.01-1.32) incident cases in 2019. More deaths and incident cases occurred in males than in females among HIV-negative individuals globally in 2019, with 342 000 (234 000-425 000) more deaths and 1.01 million (0.82-1.23) more incident cases in males than in females. Among HIV-positive individuals, 6250 (1820-11 400) more deaths and 81 100 (63 300-100 000) more incident cases occurred among females than among males in 2019. Age-standardised mortality rates among HIV-negative males were more than two times greater in 105 countries and age-standardised incidence rates were more than 1.5 times greater in 74 countries than among HIV-negative females in 2019. The fraction of global tuberculosis deaths among HIV-negative individuals attributable to alcohol use, smoking, and diabetes was 4.27 (3.69-5.02), 6.17 (5.48-7.02), and 1.17 (1.07-1.28) times higher, respectively, among males than among females in 2019. Among individuals with HIV and tuberculosis coinfection, the fraction of mortality attributable to injection drug use was 2.23 (2.03-2.44) times greater among males than females, whereas the fraction due to unsafe sex was 1.06 (1.05-1.08) times greater among females than males. Interpretation As countries refine national tuberculosis programmes and strategies to end the tuberculosis epidemic, the excess burden experienced by males is important. Interventions are needed to actively communicate, especially to men, the importance of early diagnosis and treatment. These interventions should occur in parallel with efforts to minimise excess HIV burden among women in the highest HIV burden countries that are contributing to excess HIV and tuberculosis coinfection burden for females. Placing a focus on tuberculosis burden among HIV-negative males and HIV and tuberculosis coinfection among females might help to diminish the overall burden of tuberculosis. This strategy will be crucial in reaching both equity and burden targets outlined by global health milestone

Environment-Dependent Stability and Mechanical Properties of DNA Origami Six-Helix Bundles with Different Crossover Spacings

Funding Information: The authors would like to thank David M. Smith for helpful discussions. Financial support through Emil Aaltonen Foundation, Jane and Aatos Erkko Foundation, Sigrid Jusélius Foundation, Magnus Ehrnrooth Foundation, and Finnish Cultural Foundation (Kalle and Dagmar Välimaa Fund) is gratefully acknowledged. The authors would like to acknowledge the provision of facilities and technical support by Aalto University Bioeconomy Facilities and OtaNano—Nanomicroscopy Center (Aalto‐NMC) and Micronova Nanofabrication Center. Publisher Copyright: © 2022 The Authors. Small published by Wiley-VCH GmbH.The internal design of DNA nanostructures defines how they behave in different environmental conditions, such as endonuclease-rich or low-Mg2+ solutions. Notably, the inter-helical crossovers that form the core of such DNA objects have a major impact on their mechanical properties and stability. Importantly, crossover design can be used to optimize DNA nanostructures for target applications, especially when developing them for biomedical environments. To elucidate this, two otherwise identical DNA origami designs are presented that have a different number of staple crossovers between neighboring helices, spaced at 42- and 21- basepair (bp) intervals, respectively. The behavior of these structures is then compared in various buffer conditions, as well as when they are exposed to enzymatic digestion by DNase I. The results show that an increased number of crossovers significantly improves the nuclease resistance of the DNA origami by making it less accessible to digestion enzymes but simultaneously lowers its stability under Mg2+-free conditions by reducing the malleability of the structures. Therefore, these results represent an important step toward rational, application-specific DNA nanostructure design.Peer reviewe

Publisher Correction: Molecular basis of microhomology-mediated end-joining by purified full-length Polθ (Nature Communications, (2019), 10, 1, (4423), 10.1038/s41467-019-12272-9)

The original version of this Article contained errors in Figure 6. In panel o, the labels incorrectly stated ‘Poleθ’ and “Poleθ + DNA” and should be labelled “Polθ” and “Polθ + DNA”. In the result section, in the sub-section entitled “Polθ Promotes MMEJ of Long ssDNA”, the sentence “Importantly, the ability of Polθ- pol to perform MMEJ on short (≤12 nt) ssDNA (Fig. 1p, left; Supplementary Fig. 3D and 3E), and short (≤15 nt) overhangs, demonstrates it performs interstrand pairing without Polθ-hel”. should read as follow: “Importantly, the ability of Polθ-pol to perform MMEJ on short (≤12 nt) ssDNA (Fig. 1p, left; Supplementary Fig. 3D and 3E), and short (≤15 nt) overhangs, demonstrates that it performs interstrand pairing without Polθ-hel”. In the sub-section entitled “Preventing Intrastrand Pairing Stimulates MMEJ by Polθ-Pol”, the sentence “We predicted that preventing base-pairing opportunities between 3' terminal bases and bases upstream along long the 5' region of long ssDNA substrates would suppress intrastrand pairing and enable interstrand pairing by Polθ-pol (Fig. 3c)”. should read as follows: “We predicted that preventing base-pairing opportunities between 3' terminal bases and bases upstream along the 5' region of long ssDNA substrates would suppress intrastrand pairing and enable interstrand pairing by Polθ-pol (Fig. 3c)”. In the method section, in the “Proteins” sub-section the sentence “Polθ-pol, Polθ-hel and RPA were purified as described”. should read as follows: “Polθ-pol and Polθ-hel were purified as described”. These corrections have now been included in the HTML and pdf of the article. Additionally, a technical problem during the publication process resulted in loss of image quality in Figs. 1, 3 and 4. This has now been corrected in both the PDF and HTML versions of the Article

Radial artery complications occurring after transradial coronary procedures using long hydrophilic-coated introducer sheath: a frequency domain-optical coherence tomography study

This study was performed to analyze the impact of transradial intervention (TRI), performed by long (25-cm) hydrophilic-coated radial introducer sheath (HRS), on radial artery (RA). Both acute damages and chronic intimal modifications, occurring in RA, were assessed using frequency domain-optical coherence tomography (FD-OCT). FD-OCT evaluation of RA was performed in 51 consecutive patients, undergoing TRI by long (25-cm) HRS. FD-OCT was performed from RA ostium to the puncture site. Acute damages such as intimal tears and medial dissections together with chronic intimal modifications, assessed as intimal hyperplasia indexes, were observed and compared between proximal and distal RA segments. Intimal tears were detected in 37\ua0% of patients, especially located in proximal RA segment (p\ua0=\ua00.09). Medial dissections were imaged in 9.8\ua0% of patients with no significant difference between proximal and distal RA segments. Intimal hyperplasia indexes were higher in distal RA segment, with no significant association with a previous history of TRI. In the setting of TRI, performed by long HRS, intimal tears represented the main RA injury occurring in about one-third of patients, while medial dissections only occurred in a small proportion of patients. Distal RA segment was more prone to intimal thickening, although this phenomenon was not associated with repeated transradial procedures