Promotion of DNA end resection by BRCA1-BARD1 in homologous recombination.

The licensing step of DNA double-strand break repair by homologous recombination entails resection of DNA ends to generate a single-stranded DNA template for assembly of the repair machinery consisting of the RAD51 recombinase and ancillary factors1. DNA end resection is mechanistically intricate and reliant on the tumour suppressor complex BRCA1-BARD1 (ref. 2). Specifically, three distinct nuclease entities-the 5'-3' exonuclease EXO1 and heterodimeric complexes of the DNA endonuclease DNA2, with either the BLM or WRN helicase-act in synergy to execute the end resection process3. A major question concerns whether BRCA1-BARD1 directly regulates end resection. Here, using highly purified protein factors, we provide evidence that BRCA1-BARD1 physically interacts with EXO1, BLM and WRN. Importantly, with reconstituted biochemical systems and a single-molecule analytical tool, we show that BRCA1-BARD1 upregulates the activity of all three resection pathways. We also demonstrate that BRCA1 and BARD1 harbour stand-alone modules that contribute to the overall functionality of BRCA1-BARD1. Moreover, analysis of a BARD1 mutant impaired in DNA binding shows the importance of this BARD1 attribute in end resection, both in vitro and in cells. Thus, BRCA1-BARD1 enhances the efficiency of all three long-range DNA end resection pathways during homologous recombination in human cells.

Plasticity of the Mre11-Rad50-Xrs2-Sae2 nuclease ensemble in the processing of DNA-bound obstacles.

The budding yeast Mre11-Rad50-Xrs2 (MRX) complex and Sae2 function together in DNA end resection during homologous recombination. Here we show that the Ku complex shields DNA ends from exonucleolytic digestion but facilitates endonucleolytic scission by MRX with a dependence on ATP and Sae2. The incision site is enlarged into a DNA gap via the exonuclease activity of MRX, which is stimulated by Sae2 without ATP being present. RPA renders a partially resected or palindromic DNA structure susceptible to MRX-Sae2, and internal protein blocks also trigger DNA cleavage. We present models for how MRX-Sae2 creates entry sites for the long-range resection machinery.

A novel role of the Dna2 translocase function in DNA break resection.

DNA double-strand break repair by homologous recombination entails nucleolytic resection of the 5' strand at break ends. Dna2, a flap endonuclease with 5'-3' helicase activity, is involved in the resection process. The Dna2 helicase activity has been implicated in Okazaki fragment processing during DNA replication but is thought to be dispensable for DNA end resection. Unexpectedly, we found a requirement for the helicase function of Dna2 in end resection in budding yeast cells lacking exonuclease 1. Biochemical analysis reveals that ATP hydrolysis-fueled translocation of Dna2 on ssDNA facilitates 5' flap cleavage near a single-strand-double strand junction while attenuating 3' flap incision. Accordingly, the ATP hydrolysis-defective dna2-K1080E mutant is less able to generate long products in a reconstituted resection system. Our study thus reveals a previously unrecognized role of the Dna2 translocase activity in DNA break end resection and in the imposition of the 5' strand specificity of end resection.

To Cut or Not to Cut: Discovery of a Novel Regulator of DNA Break Resection.

Nucleolytic resection of DNA double-strand breaks is the crucial first step in their repair via homologous recombination. New findings by Tkác et al. (2016) published in this issue of Molecular Cell identify HELB as a novel, cell-cycle-specific negative regulator of DNA end resection.

A conserved Ctp1/CtIP C-terminal peptide stimulates Mre11 endonuclease activity.

The Mre11-Rad50-Nbs1 complex (MRN) is important for repairing DNA double-strand breaks (DSBs) by homologous recombination (HR). The endonuclease activity of MRN is critical for resecting 5'-ended DNA strands at DSB ends, producing 3'-ended single-strand DNA, a prerequisite for HR. This endonuclease activity is stimulated by Ctp1, the Schizosaccharomyces pombe homolog of human CtIP. Here, with purified proteins, we show that Ctp1 phosphorylation stimulates MRN endonuclease activity by inducing the association of Ctp1 with Nbs1. The highly conserved extreme C terminus of Ctp1 is indispensable for MRN activation. Importantly, a polypeptide composed of the conserved 15 amino acids at the C terminus of Ctp1 (CT15) is sufficient to stimulate Mre11 endonuclease activity. Furthermore, the CT15 equivalent from CtIP can stimulate human MRE11 endonuclease activity, arguing for the generality of this stimulatory mechanism. Thus, we propose that Nbs1-mediated recruitment of CT15 plays a pivotal role in the activation of the Mre11 endonuclease by Ctp1/CtIP.

Temperature lowering of liquid nitrogen via injection of helium gas bubbles improves the generation of parahydrogen-enriched gas.

The para spin isomer of hydrogen gas possesses high nuclear spin order that can enhance the NMR signals of a variety of molecular species. Hydrogen is routinely enriched in the para spin state by lowering the gas temperature while flowing through a catalyst. Although parahydrogen enrichments approaching 100% are achievable near the H2 liquefaction temperature of 20 K, many experimentalists operate at liquid nitrogen temperatures (77 K) due to the lower associated costs and overall simplicity of the parahydrogen generator. Parahydrogen that is generated at 77 K provides an enrichment value of ~51% of the para spin isomer; while useful, there are many applications that can benefit from low-cost access to higher parahydrogen enrichments. Here, we introduce a method of improving parahydrogen enrichment values using a liquid nitrogen-cooled generator that operates at temperatures less than 77 K. The boiling temperature of liquid nitrogen is lowered through internal evaporation into helium gas bubbles that are injected into the liquid. Changes to liquid nitrogen temperatures and parahydrogen enrichment values were monitored as a function of helium gas flow rate. The injected helium bubbles lowered the liquid nitrogen temperature to ~65.5 K, and parahydrogen enrichments of up to ~59% were achieved; this represents an ~16% improvement compared with the expected parahydrogen fraction at 77 K. This technique is simple to implement in standard liquid nitrogen-cooled parahydrogen generators and may be of interest to a wide range of scientists that require a cost-effective approach to improving parahydrogen enrichment values.

Prioritising gully remediation in a Great Barrier Reef catchment: An approach using two independent methods of assessing erosion activity in 22,300 gullies.

The strategic reduction and remediation of degraded land is a global environmental priority. This is a particular priority in the Great Barrier Reef catchment area, Australia, where gully erosion a significant contributor to land degradation and water quality deterioration. Urgent action through the prioritisation and remediation of gully erosion sites is imperative to safeguard this UNESCO World Heritage site. In this study, we analyze a comprehensive dataset of 22,311 mapped gullies within a 3480 km2 portion of the lower Burdekin Basin, northeast Australia. Utilizing high-resolution lidar datasets, two independent methods - Minimum Contemporary Estimate (MCE) and Lifetime Average Estimate (LAE) - were developed to derive relative erosion rates. These methods, employing different data processing approaches and addressing different timeframes across the gully lifetime, yield erosion rates varying by up to several orders of magnitude. Despite some expected divergence, both methods exhibit strong, positive correlations with each other and additional validation data. There is a 43% agreement between the methods for the highest yielding 2% of gullies, although 80.5% of high-yielding gullies identified by either method are located within a 1 km proximity of each other. Importantly, distributions from both methods independently reveal that ∼80% of total volume of gully erosion in the study area is produced from only 20% of all gullies. Moreover, the top 2% of gullies generate 30% of the sediment loss and the majority of gullies do not significantly contribute to the overall catchment sediment yield. These results underscore the opportunity to achieve significant environmental outcomes through targeted gully management by prioritising a small cohort of high yielding gullies. Further insights and implications for management frameworks are discussed in the context of the characteristics of this cohort. Overall, this research provides a basis for informed decision-making in addressing gully erosion and advancing environmental conservation efforts.

The nuclease activity of DNA2 promotes exonuclease 1-independent mismatch repair.

The DNA mismatch repair (MMR) system is a major DNA repair system that corrects DNA replication errors. In eukaryotes, the MMR system functions via mechanisms both dependent on and independent of exonuclease 1 (EXO1), an enzyme that has multiple roles in DNA metabolism. Although the mechanism of EXO1-dependent MMR is well understood, less is known about EXO1-independent MMR. Here, we provide genetic and biochemical evidence that the DNA2 nuclease/helicase has a role in EXO1-independent MMR. Biochemical reactions reconstituted with purified human proteins demonstrated that the nuclease activity of DNA2 promotes an EXO1-independent MMR reaction via a mismatch excision-independent mechanism that involves DNA polymerase δ. We show that DNA polymerase ε is not able to replace DNA polymerase δ in the DNA2-promoted MMR reaction. Unlike its nuclease activity, the helicase activity of DNA2 is dispensable for the ability of the protein to enhance the MMR reaction. Further examination established that DNA2 acts in the EXO1-independent MMR reaction by increasing the strand-displacement activity of DNA polymerase δ. These data reveal a mechanism for EXO1-independent mismatch repair.

TEE guided REBOA deflation following ROSC for non-traumatic cardiac arrest.

Resuscitative endovascular balloon occlusion of the aorta (REBOA) is most commonly used to manage non-compressible torso hemorrhage. It is also emerging as a promising treatment for non-traumatic refractory cardiac arrest. Aortic occlusion during chest compressions increases cardio-cerebral perfusion, increasing the potential for sustained return of spontaneous circulation (ROSC) or serving as a bridge to extracorporeal cardiopulmonary resuscitation (ECPR). Optimal patient selection and post-ROSC management in such cases is uncertain and not well reported in the literature. We present a case of non-traumatic out-of-hospital cardiac arrest in which REBOA was placed in the emergency department with subsequent ROSC. Transesophageal echocardiography was used to guide post-ROSC REBOA management and balloon deflation.

Reconversion of Parahydrogen Gas in Surfactant-Coated Glass NMR Tubes.

The application of parahydrogen gas to enhance the magnetic resonance signals of a diversity of chemical species has increased substantially in the last decade. Parahydrogen is prepared by lowering the temperature of hydrogen gas in the presence of a catalyst; this enriches the para spin isomer beyond its normal abundance of 25% at thermal equilibrium. Indeed, parahydrogen fractions that approach unity can be attained at sufficiently low temperatures. Once enriched, the gas will revert to its normal isomeric ratio over the course of hours or days, depending on the surface chemistry of the storage container. Although parahydrogen enjoys long lifetimes when stored in aluminum cylinders, the reconversion rate is significantly faster in glass containers due to the prevalence of paramagnetic impurities that are present within the glass. This accelerated reconversion is especially relevant for nuclear magnetic resonance (NMR) applications due to the use of glass sample tubes. The work presented here investigates how the parahydrogen reconversion rate is affected by surfactant coatings on the inside surface of valved borosilicate glass NMR sample tubes. Raman spectroscopy was used to monitor changes to the ratio of the (J: 0 → 2) vs. (J: 1 → 3) transitions that are indicative of the para and ortho spin isomers, respectively. Nine different silane and siloxane-based surfactants of varying size and branching structures were examined, and most increased the parahydrogen reconversion time by 1.5×-2× compared with equivalent sample tubes that were not treated with surfactant. This includes expanding the pH2 reconversion time from 280 min in a control sample to 625 min when the same tube is coated with (3-Glycidoxypropyl)trimethoxysilane.

RIF1 in DNA break repair pathway choice.

New findings on the RIF1 protein by four research groups, including Chapman et al. (2013) and Escribano-Díaz et al. (2013) in this issue, provide insights into DNA double-strand break repair pathway choice in mammalian cells.

Single-molecule visualization of human BLM helicase as it acts upon double- and single-stranded DNA substrates.

Bloom helicase (BLM) and its orthologs are essential for the maintenance of genome integrity. BLM defects represent the underlying cause of Bloom Syndrome, a rare genetic disorder that is marked by strong cancer predisposition. BLM deficient cells accumulate extensive chromosomal aberrations stemming from dysfunctions in homologous recombination (HR). BLM participates in several HR stages and helps dismantle potentially harmful HR intermediates. However, much remains to be learned about the molecular mechanisms of these BLM-mediated regulatory effects. Here, we use DNA curtains to directly visualize the activity of BLM helicase on single molecules of DNA. Our data show that BLM is a robust helicase capable of rapidly (∼70-80 base pairs per second) unwinding extensive tracts (∼8-10 kilobases) of double-stranded DNA (dsDNA). Importantly, we find no evidence for BLM activity on single-stranded DNA (ssDNA) that is bound by replication protein A (RPA). Likewise, our results show that BLM can neither associate with nor translocate on ssDNA that is bound by the recombinase protein RAD51. Moreover, our data reveal that the presence of RAD51 also blocks BLM translocation on dsDNA substrates. We discuss our findings within the context of potential regulator roles for BLM helicase during DNA replication and repair.

Guiding Cardiopulmonary Resuscitation with Focused Echocardiography: A Report of Five Cases.

Background: Focused transthoracic echocardiography has been used to determine etiologies of cardiac arrest and evaluate utility of continuing resuscitation after cardiac arrest. Few guidelines exist advising ultrasound timing within the advanced cardiac life support algorithm. Natural timing of echocardiography occurs during the pulse check, when views are unencumbered by stabilization equipment or vigorous movements. However, recent studies suggest that ultrasound performance during pulse checks prolongs the pause duration of cardiopulmonary resuscitation. Transesophageal echocardiography studies have demonstrated benefits in this regard, but there have been no transthoracic echocardiography studies assessing the physical performance of compressions during cardiopulmonary resuscitation. Objective: The purpose of this study was to describe cases where echocardiography performed at the beginning of the cardiac arrest algorithm offers actionable information to cardiopulmonary resuscitation itself without delaying provision of compressions. Conclusion: Providers using focused echocardiography to evaluate cardiac arrest patients should consider initiating scans at the start of compressions to identify the optimal location for compression delivery and to detect inadequate compressions. Subsequent visualization of full left ventricular compression may be seen after a location change, and combined with end tidal carbon dioxide values, gives indication for improved forward circulatory flow. Although it is not possible in all patients, doing so hastens provision of quality compressions that affect hemodynamic parameters without causing prolongations to the pulse check pause. Further research is needed to determine patient outcomes from both out-of-hospital and in-hospital cardiac arrest when cardiopulmonary resuscitation is visually guided by focused echocardiography.

A DNA nick at Ku-blocked double-strand break ends serves as an entry site for exonuclease 1 (Exo1) or Sgs1-Dna2 in long-range DNA end resection.

The repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) is initiated by nucleolytic resection of the DNA break ends. The current model, being based primarily on genetic analyses in Saccharomyces cerevisiae and companion biochemical reconstitution studies, posits that end resection proceeds in two distinct stages. Specifically, the initiation of resection is mediated by the nuclease activity of the Mre11-Rad50-Xrs2 (MRX) complex in conjunction with its cofactor Sae2, and long-range resection is carried out by exonuclease 1 (Exo1) or the Sgs1-Top3-Rmi1-Dna2 ensemble. Using fully reconstituted systems, we show here that DNA with ends occluded by the DNA end-joining factor Ku70-Ku80 becomes a suitable substrate for long-range 5'-3' resection when a nick is introduced at a locale proximal to one of the Ku-bound DNA ends. We also show that Sgs1 can unwind duplex DNA harboring a nick, in a manner dependent on a species-specific interaction with the ssDNA-binding factor replication protein A (RPA). These biochemical systems and results will be valuable for guiding future endeavors directed at delineating the mechanistic intricacy of DNA end resection in eukaryotes.

Mapping Resistance to Alternaria cucumerina in Cucumis melo.

Infection with Alternaria cucumerina causes Alternaria leaf blight (ALB), a disease characterized by lesion formation on leaves, leading to substantial yield and quality losses in Cucumis melo (melon). Although fungicides are effective against ALB, reduction in the frequency of application would be economically and environmentally beneficial. Resistant melon lines have been identified but the genetic basis of this resistance has not been determined. A saturated melon genetic map was constructed with markers developed through genotyping by sequencing of a recombinant inbred line population (F6 to F10; n = 82) derived from single-seed descent of a F2 population from a cross between the ALB-resistant parent MR-1 and the ALB-susceptible parent Ananas Yokneum. The population was evaluated for A. cucumerina resistance with an augmented block greenhouse study using inoculation with the wounded-leaf method. Multiple quantitative trait loci (QTL) mapping identified two QTL that explained 33.9% of variation in lesion area. Several candidate genes within range of these QTL were identified using the C. melo v3.5 genome. Markers linked to these QTL will be used to accelerate efforts to breed melon cultivars resistant to ALB.

The role of resuscitative endovascular balloon occlusion of the aorta (REBOA) as an adjunct to ACLS in non-traumatic cardiac arrest.

Non-traumatic cardiac arrest is a major public health problem that carries an extremely high mortality rate. If we hope to increase the survivability of this condition, it is imperative that alternative methods of treatment are given due consideration. Balloon occlusion of the aorta can be used as a method of circulatory support in the critically ill patient. Intra-aortic balloon pumps have been used to temporize patients in cardiogenic shock for decades. More recently, resuscitative endovascular balloon occlusion of the aorta (REBOA) has been utilized in the patient in hemorrhagic shock or cardiac arrest secondary to trauma. Aortic occlusion in non-traumatic cardiac arrest has the effect of reducing the vascular volume that the generated cardiac output is distributed across. This augments myocardial and cerebral perfusion, increasing the probability of a return to a good quality of life for the patient. This phenomenon has been the subject of numerous animal studies dating back to the early 1980s; however, the human evidence is limited to several small case series. Animal research has demonstrated improvements in cerebral and coronary perfusion pressure during ACLS that lead to statistically significant differences in mortality. Several case series in humans have replicated these findings, suggesting the efficacy of this procedure. The objectives of this review are to: 1) introduce the reader to REBOA 2) review the physiology of NTCA and examine the current limitations of traditional ACLS 3) summarize the literature regarding the efficacy and feasibility of aortic balloon occlusion to support traditional ACLS.

Emergency physician performed tricuspid annular plane systolic excursion in the evaluation of suspected pulmonary embolism.

OBJECTIVES: The primary objectives were to describe the diagnostic characteristics tricuspid annular plane systolic excursion (TAPSE) for pulmonary embolism (PE) and to optimize the measurement cutoff of TAPSE for the diagnosis of PE. Secondary objectives included assessment of interrater reliability and the quantitative visual estimation of TAPSE. METHODS: This is a prospective observational cohort study involving a convenience sample of patients at an urban academic emergency department. Patients underwent focused right heart echocardiogram (FOCUS) before computed tomographic angiography (CTA) for suspected PE. RESULTS: A total of 150 patients were enrolled, 32 of whom (21.3%) were diagnosed as having a PE. A receiver operating characteristic curve analysis yielded 2.0 cm as the optimal cutoff for TAPSE in the diagnosis of PE, with a sensitivity of 72% (95% confidence interval [CI], 53-86), a specificity of 66% (95% CI, 57-75), and an area under the curve of 0.73 (95% CI, 0.65-0.83). In patients with tachycardia or hypotension, post hoc analysis demonstrated that FOCUS is 100% (95% CI, 80-100) sensitive for PE, whereas TAPSE is 94% (95% CI, 71-99) sensitive for PE. The intraclass correlation coefficient was 0.87 (95% CI, 0.79-0.93). Emergency physicians with training in echocardiography accurately visually estimated TAPSE, with a κ statistic of 0.94 (95% CI, 0.87-0.98). CONCLUSIONS: Emergency physicians with training in echocardiography can reliably measure TAPSE and are able to accurately visually estimate TAPSE as either normal or abnormal. When using an abnormal cutoff of less than 2.0 cm, TAPSE has moderate diagnostic value in patients with suspected PE. On post hoc analysis, TAPSE and FOCUS appear to be highly sensitive for PE in patients with tachycardia or hypotension.

Interplay between Ku and Replication Protein A in the Restriction of Exo1-mediated DNA Break End Resection.

DNA double-strand breaks can be eliminated via non-homologous end joining or homologous recombination. Non-homologous end joining is initiated by the association of Ku with DNA ends. In contrast, homologous recombination entails nucleolytic resection of the 5'-strands, forming 3'-ssDNA tails that become coated with replication protein A (RPA). Ku restricts end access by the resection nuclease Exo1. It is unclear how partial resection might affect Ku engagement and Exo1 restriction. Here, we addressed these questions in a reconstituted system with yeast proteins. With blunt-ended DNA, Ku protected against Exo1 in a manner that required its DNA end-binding activity. Despite binding poorly to ssDNA, Ku could nonetheless engage a 5'-recessed DNA end with a 40-nucleotide (nt) ssDNA overhang, where it localized to the ssDNA-dsDNA junction and efficiently blocked resection by Exo1. Interestingly, RPA could exclude Ku from a partially resected structure with a 22-nt ssDNA tail and thus restored processing by Exo1. However, at a 40-nt tail, Ku remained stably associated at the ssDNA-dsDNA junction, and RPA simultaneously engaged the ssDNA region. We discuss a model in which the dynamic equilibrium between Ku and RPA binding to a partially resected DNA end influences the timing and efficiency of the resection process.

Multifaceted role of the Topo IIIα-RMI1-RMI2 complex and DNA2 in the BLM-dependent pathway of DNA break end resection.

BLM, a RecQ family DNA helicase mutated in Bloom's Syndrome, participates in homologous recombination at two stages: 5' DNA end resection and double Holliday junction dissolution. BLM exists in a complex with Topo IIIα, RMI1 and RMI2. Herein, we address the role of Topo IIIα and RMI1-RMI2 in resection using a reconstituted system with purified human proteins. We show that Topo IIIα stimulates DNA unwinding by BLM in a manner that is potentiated by RMI1-RMI2, and that the processivity of resection is reliant on the Topo IIIα-RMI1-RMI2 complex. Topo IIIα localizes to the ends of double-strand breaks, thus implicating it in the recruitment of resection factors. While the single-stranded DNA binding protein RPA plays a major role in imposing the 5' to 3' polarity of resection, Topo IIIα also makes a contribution in this regard. Moreover, we show that DNA2 stimulates the helicase activity of BLM. Our results thus uncover a multifaceted role of the Topo IIIα-RMI1-RMI2 ensemble and of DNA2 in the DNA resection reaction.

Who Would Pay for State Alcohol Tax Increases in the United States?

INTRODUCTION: Despite strong evidence that increasing alcohol taxes reduces alcohol-related harm, state alcohol taxes have declined in real terms during the past 3 decades. Opponents of tax increases argue that they are unfair to "responsible" drinkers and those who are financially disadvantaged. The objectives of this study were to assess the impact of hypothetical state alcohol tax increases on the cost of alcohol for adults in the United States on the basis of alcohol consumption and sociodemographic characteristics. METHODS: The increased net cost of alcohol (ie, product plus tax) from a series of hypothetical state alcohol tax increases was modeled for all 50 states using data from the 2011 Behavioral Risk Factor Surveillance System, IMPACT Databank, and the Alcohol Policy Information System. Costs were assessed by drinking pattern (excessive vs nonexcessive) and by sociodemographic characteristics. RESULTS: Among states, excessive drinkers would pay 4.8 to 6.8 times as much as nonexcessive drinkers on a per capita basis and would pay at least 72% of aggregate costs. For nonexcessive drinkers, the annual cost from even the largest hypothetical tax increase ($0.25 per drink) would average less than $10.00. Drinkers with higher household incomes and non-Hispanic white drinkers would pay higher per capita costs than people with lower incomes and racial/ethnic minorities. CONCLUSION: State-specific tax increases would cost more for excessive drinkers, those with higher incomes, and non-Hispanic whites. Costs to nonexcessive drinkers would be modest. Findings are relevant to developing evidence-based public health practice for a leading preventable cause of death.