Neurofibromin Deficient Myeloid Cells are Critical Mediators of Aneurysm Formation In Vivo

Background Neurofibromatosis Type 1 (NF1) is a genetic disorder resulting from mutations in the NF1 tumor suppressor gene. Neurofibromin, the protein product of NF1, functions as a negative regulator of Ras activity in circulating hematopoietic and vascular wall cells, which are critical for maintaining vessel wall homeostasis. NF1 patients have evidence of chronic inflammation resulting in development of premature cardiovascular disease, including arterial aneurysms, which may manifest as sudden death. However, the molecular pathogenesis of NF1 aneurysm formation is unknown. Method and Results Utilizing an angiotensin II-induced aneurysm model, we demonstrate that heterozygous inactivation of Nf1 (Nf1+/−) enhanced aneurysm formation with myeloid cell infiltration and increased oxidative stress in the vessel wall. Using lineage-restricted transgenic mice, we show loss of a single Nf1 allele in myeloid cells is sufficient to recapitulate the Nf1+/− aneurysm phenotype in vivo. Finally, oral administration of simvastatin or the antioxidant apocynin, reduced aneurysm formation in Nf1+/− mice. Conclusion These data provide genetic and pharmacologic evidence that Nf1+/− myeloid cells are the cellular triggers for aneurysm formation in a novel model of NF1 vasculopathy and provide a potential therapeutic target

Reduced fire severity offers near-term buffer to climate-driven declines in conifer resilience across the western United States

Increasing fire severity and warmer, drier postfire conditions are making forests in the western United States (West) vulnerable to ecological transformation. Yet, the relative importance of and interactions between these drivers of forest change remain unresolved, particularly over upcoming decades. Here, we assess how the interactive impacts of changing climate and wildfire activity influenced conifer regeneration after 334 wildfires, using a dataset of postfire conifer regeneration from 10,230 field plots. Our findings highlight declining regeneration capacity across the West over the past four decades for the eight dominant conifer species studied. Postfire regeneration is sensitive to high-severity fire, which limits seed availability, and postfire climate, which influences seedling establishment. In the near-term, projected differences in recruitment probability between low- and high-severity fire scenarios were larger than projected climate change impacts for most species, suggesting that reductions in fire severity, and resultant impacts on seed availability, could partially offset expected climate-driven declines in postfire regeneration. Across 40 to 42% of the study area, we project postfire conifer regeneration to be likely following low-severity but not high-severity fire under future climate scenarios (2031 to 2050). However, increasingly warm, dry climate conditions are projected to eventually outweigh the influence of fire severity and seed availability. The percent of the study area considered unlikely to experience conifer regeneration, regardless of fire severity, increased from 5% in 1981 to 2000 to 26 to 31% by mid-century, highlighting a limited time window over which management actions that reduce fire severity may effectively support postfire conifer regeneration. © 2023 the Author(s)

Effects of Anacetrapib in Patients with Atherosclerotic Vascular Disease

BACKGROUND: Patients with atherosclerotic vascular disease remain at high risk for cardiovascular events despite effective statin-based treatment of low-density lipoprotein (LDL) cholesterol levels. The inhibition of cholesteryl ester transfer protein (CETP) by anacetrapib reduces LDL cholesterol levels and increases high-density lipoprotein (HDL) cholesterol levels. However, trials of other CETP inhibitors have shown neutral or adverse effects on cardiovascular outcomes. METHODS: We conducted a randomized, double-blind, placebo-controlled trial involving 30,449 adults with atherosclerotic vascular disease who were receiving intensive atorvastatin therapy and who had a mean LDL cholesterol level of 61 mg per deciliter (1.58 mmol per liter), a mean non-HDL cholesterol level of 92 mg per deciliter (2.38 mmol per liter), and a mean HDL cholesterol level of 40 mg per deciliter (1.03 mmol per liter). The patients were assigned to receive either 100 mg of anacetrapib once daily (15,225 patients) or matching placebo (15,224 patients). The primary outcome was the first major coronary event, a composite of coronary death, myocardial infarction, or coronary revascularization. RESULTS: During the median follow-up period of 4.1 years, the primary outcome occurred in significantly fewer patients in the anacetrapib group than in the placebo group (1640 of 15,225 patients [10.8%] vs. 1803 of 15,224 patients [11.8%]; rate ratio, 0.91; 95% confidence interval, 0.85 to 0.97; P=0.004). The relative difference in risk was similar across multiple prespecified subgroups. At the trial midpoint, the mean level of HDL cholesterol was higher by 43 mg per deciliter (1.12 mmol per liter) in the anacetrapib group than in the placebo group (a relative difference of 104%), and the mean level of non-HDL cholesterol was lower by 17 mg per deciliter (0.44 mmol per liter), a relative difference of -18%. There were no significant between-group differences in the risk of death, cancer, or other serious adverse events. CONCLUSIONS: Among patients with atherosclerotic vascular disease who were receiving intensive statin therapy, the use of anacetrapib resulted in a lower incidence of major coronary events than the use of placebo. (Funded by Merck and others; Current Controlled Trials number, ISRCTN48678192 ; ClinicalTrials.gov number, NCT01252953 ; and EudraCT number, 2010-023467-18 .)

Single-strand annealing between inverted DNA repeats: Pathway choice, participating proteins, and genome destabilizing consequences.

Double strand DNA breaks (DSBs) are dangerous events that can result from various causes including environmental assaults or the collapse of DNA replication. While the efficient and precise repair of DSBs is essential for cell survival, faulty repair can lead to genetic instability, making the choice of DSB repair an important step. Here we report that inverted DNA repeats (IRs) placed near a DSB can channel its repair from an accurate pathway that leads to gene conversion to instead a break-induced replication (BIR) pathway that leads to genetic instabilities. The effect of IRs is explained by their ability to form unusual DNA structures when present in ssDNA that is formed by DSB resection. We demonstrate that IRs can form two types of unusual DNA structures, and the choice between these structures depends on the length of the spacer separating IRs. In particular, IRs separated by a long (1-kb) spacer are predominantly involved in inter-molecular single-strand annealing (SSA) leading to the formation of inverted dimers; IRs separated by a short (12-bp) spacer participate in intra-molecular SSA, leading to the formation of fold-back (FB) structures. Both of these structures interfere with an accurate DSB repair by gene conversion and channel DSB repair into BIR, which promotes genomic destabilization. We also report that different protein complexes participate in the processing of FBs containing short (12-bp) versus long (1-kb) ssDNA loops. Specifically, FBs with short loops are processed by the MRX-Sae2 complex, whereas the Rad1-Rad10 complex is responsible for the processing of long loops. Overall, our studies uncover the mechanisms of genomic destabilization resulting from re-routing DSB repair into unusual pathways by IRs. Given the high abundance of IRs in the human genome, our findings may contribute to the understanding of IR-mediated genomic destabilization associated with human disease

Inverted dimers and fold-backs in IR-12.

<p><b>(A)</b> Schematics of AvrII- digested Chr III in IR-12 (OF) and its derivatives: CF, ID, FB (location of probe P-1 is indicated by black box). <b>(B)</b> DSB repair in IR-12 (wt, <i>rad1Δ</i>, <i>sae2Δ</i> and <i>rad1Δsae2Δ)</i> following AvrII digest and hybridization to probe P-1. The respective positions of, CF, ID and FB are indicated. The median efficiencies of FB formation (%) and the range of the median [in brackets] are indicated (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007543#pgen.1007543.s002" target="_blank">S1 Fig</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007543#sec013" target="_blank">Materials and Methods</a> for details). <b>(C)</b> The schematics of BseYI restriction fragments of Chr III in original IR-12 and its derivatives following DSB: OF, CF, ID, FB and Open-FB (formed by denaturation of FB). The location of hybridization probe P-2 is indicated by brown box. <b>(D)</b> Southern blot analysis of DSB repair in <i>sae2Δ</i> derivative of IR-12 using native and denaturing gel electrophoresis (Schematics in Fig 4C) and hybridization to probe P-2. The positions and corresponding sizes of OF, CF, ID, FB and Open-FB following BseYI digestion of DNA isolated before (0-hr) and 6-hr following DSB induction are indicated. <b>(E)</b> Southern blot analysis of DSB repair in <i>rad1Δsae2Δ</i> derivative of IR-12 using native and denaturing gel electrophoresis (schematics in Fig 4C) following hybridization to probe P-2.</p

DSB repair pathways.

<p>DSBs can be repaired by either <b>(A)</b> Non Homologous End Joining (NHEJ) that proceeds by ligation of broken ends or <b>(B)</b> Homologous Recombination (HR) where the DSB ends undergo 5’-3’ resection, and repair proceeds via one of the following sub-pathways of HR: (i). SDSA. 3’ end invades and pairs with a homologous donor template producing a displacement loop (D-loop). The 3’ end of the invading strand is used as a primer to initiate new DNA synthesis. The second end of the DSB is resected and pairs with the newly copied strand and initiates a second round of DNA synthesis. (ii) Double Holliday junction (dHJ) pathway; an alternative gene conversion mechanism that synthesizes only a short DNA patch and involves strand invasion step followed by stabilization of a D-loop via “capturing” of the second broken DSB end. Two rounds of synthesis lead to the formation of a dHJ that can be resolved to produce non-crossover or crossover outcomes. (iii) BIR; employed when only one broken end is available for strand invasion, this invasion leads to the initiation of DNA synthesis that proceeds via migrating bubble with asynchronous synthesis of leading and lagging strands and leads to conservative inheritance of newly-synthesized DNA. (iv) SSA between direct DNA repeats. 5’-3’ resection continues until flanking homologous sequences are exposed and annealed to each other. The protruding non-homologous 3’ ends are clipped off and the 3’ ends are used as primers to fill in the gaps. (v) SSA between inverted DNA repeats. (v-a) Inter-molecular SSA between IRs. DSBs occur in two DNA molecules (e.g. sister chromatids), followed by 5’-3’ strand resection, leading to exposure of the IRs as ssDNA, followed by annealing between IRs located on two different DNA molecules. After clipping off the 3’-non-homologous tails, DNA synthesis fills in the gaps leading to the formation of inverted dimers that can also be dicentric. (v-b) Intra-molecular SSA between IRs. 5’-3’ resection exposes the IRs as ssDNA where intra-molecular SSA between IRs occurs. This is followed by clipping off of the non-homologous tails, DNA synthesis and ligation that fills in the gaps ultimately leading to the formation of fold-back (hairpin) structures.</p

The effect of IRs on DSB repair in diploids.

<p><b>(A)</b> Kinetics of DSB repair in diploid strains with IR-1000, IR-12 or No-IR analyzed by CHEF gel electrophoresis followed by Southern hybridization using <i>ADE1</i>-specific probe (hybridized to Chr III and Chr I). A repair product of ~450-kb size (inverted dimer) was detected starting from 2 hours following DSB induction in IR-1000 and IR-12 diploid strains. The fragment labeled CF is the Chr III cut-fragment resulting from HO-induced DSB. An additional (higher) CF band, observed 2-hr after the addition of galactose, corresponds to the processed (partially single-stranded) form of the cut fragment (similar to [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007543#pgen.1007543.ref086" target="_blank">86</a>]). <b>(B)</b> The structure of Chr III in BIR outcomes (Ade⁺ Thr^- Ura⁺) obtained from diploids containing IR-12 by CHEF gel electrophoresis, followed by Southern analysis using <i>ADE1</i>-specific probe. First lane (c): control of DNA before DSB induction. Few examples of outcomes containing GCR (resulting from ectopic BIR) are indicated by asterisks. <b>(C)</b> The number of allelic BIR, ectopic BIR, and long gene conversion (Long-GC) events among representative Ade⁺ Thr^- Ura⁺ outcomes as distinguished by CHEF electrophoresis followed by Southern hybridization using <i>ADE1</i>-specific probe.</p

DSB-induced SSA involving inverted repeats.

<p><b>(A)</b> In strains with IRs separated by long (1-kb) spacers (IR-1000), inter-molecular SSA (involving IRs in different sister chromatids) is the predominant SSA mechanism and led to the formation of inverted dicentric dimers (ID-1000). <b>(B)</b> In a case of IR separated by small (~12-bps) spacers (IR-12), intra-molecular SSA leading to the formation of fold-backs (FBs) predominates. The preferred pathway in each case is indicated by a thicker black arrow. Rad1 is involved in clipping of 3’-flaps during the ID and FB formation. Breakage of dicentrics in mitotic anaphase leads to the repair by BIR or chromosome-loss leading to genomic instability. FB-12 are processed by MRX-Sae2 complex, while FB-1000 are processed by Rad1. Processing of FBs can also lead to GC, BIR or chromosome loss and eventually to genomic instability.</p

Formation of ID and FB structures in strains with 100-bp and 500-bp spacer between IRs.

<p><b>(A)</b> The structures of AvrII restriction fragments of Chr III (OF) in IR-100 strain (2-kb IR separated by 100-bp spacer) and its derivatives following DSB: CF, ID, FB. <b>(B)</b> Southern blot analysis of DSB repair in wt, <i>rad1Δ</i>, <i>sae2Δ</i> and <i>rad1Δsae2Δ</i> derivatives of IR-100 following hybridization to probe P-1 (depicted by black box in Fig 5A) <b>(C)</b> The schematics of AvrII digest of Chr III (OF) in IR-500 strain (2-kb IR separated by 500-bp spacer) and its derivatives following DSB: CF, ID, FB. <b>(D)</b> Southern blot analysis of DSB repair in wt, <i>rad1Δ</i>, <i>sae2Δ</i> and <i>rad1Δsae2Δ</i> derivatives of IR-500 strain following hybridization to probe P-1. The median efficiencies of FB formation (%) and the range of the median [in brackets] are indicated (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007543#pgen.1007543.s002" target="_blank">S1 Fig</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007543#sec013" target="_blank">Materials and Methods</a> for details).</p

Genetic control of FB formation.

<p><b>(A)</b> Schematics of AvrII digest of Chr III (OF) in IR-1000 and its DSB-induced derivatives including: CF, ID, FB. <b>(B)</b> Analysis of ID and FB formation in IR-1000 containing one of the following deletions: <i>rad1Δ</i>, <i>rad10Δ</i>, <i>saw1Δ</i>, <i>slx4Δ</i>, <i>rad14Δ</i>, <i>slx1Δ</i>, <i>mus81Δ</i>, <i>yen1Δ</i>, <i>rad2Δ</i>, <i>rad1Δslx1Δ</i>, <i>rad1Δmus81Δ</i>, <i>rad1Δyen1Δ or rad1Δrad2Δ</i> using AvrII digest of DNA, gel electrophoresis and hybridization with probe P-1 (indicated in Fig 6A by black box). <b>(C)</b> The role of <i>RAD52</i> and <i>RAD51</i> in FB formation in IR-1000 derivatives analyzed by AvrII digest followed by hybridization with probe P-1. <b>(D)</b> The schematics of AvrII digest of Chr III in IR-12 strain (OF) and its DSB-induced derivatives: CF, ID, FB. <b>(E).</b> Analysis of ID and FB formation in <i>sae2Δ</i>, <i>sae2Δrad52Δ</i>, and <i>sae2Δrad51Δ</i> derivatives of IR-12 following AvrII digestion and hybridization with probe P-1.</p