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.

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.

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.

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.

Emergency department initiated resuscitative endovascular balloon occlusion of the aorta (REBOA) for out-of-hospital cardiac arrest is feasible and associated with improvements in end-tidal carbon dioxide.

Objectives: Out-of-hospital cardiac arrest (OHCA) claims the lives of approximately 350,000 people in the United States each year. Resuscitative endovascular balloon occlusion of the aorta (REBOA) when used as an adjunct to advanced cardiac life support may improve cardio-cerebral perfusion. Our primary research objective was to determine the feasibility of emergency department (ED)-initiated REBOA for OHCA patients in an academic urban ED. Methods: This was a single-center, single-arm, early feasibility trial that used REBOA as an adjunct to advanced cardiac life support (ACLS) in OHCA. Subjects under 80 years with witnessed OHCA and who received cardiopulmonary rescuitation (CPR) within 6 minutes were eligible. Results: Five patients were enrolled between February 2020 and April 2021. The procedure was successful in all patients and 4 of 5 (80%) patients had transient return of spontaneous circulation (ROSC) after aortic occlusion. Unfortunately, all patients re-arrested soon after intra-aortic balloon deflation and none survived to hospital admission. At 30 seconds post-aortic occlusion, investigators noted a statistically significant increase in end tidal carbon dioxide of 26% (95% confidence interval, 10%, 44%). Conclusion: Initiating REBOA for OHCA patients in an academic urban ED setting is feasible. Aortic occlusion during chest compressions is temporally associated with improvements in end tidal carbon dioxide 30 seconds after aortic occlusion. Four of 5 patients achieved ROSC after aortic occlusion; however, deflation of the intra-aortic balloon quickly led to re-arrest and death in all patients. Future research should focus on the utilization of partial-REBOA to prevent re-arrest after ROSC, as well as the optimal way to incorporate this technique with other endovascular reperfusion strategies.

65% Parahydrogen from a liquid nitrogen cooled generator.

The isomeric enrichment of parahydrogen (pH2) gas is readily accomplished by lowering the gas temperature in the presence of a catalyst. This enrichment is often pursued at two distinct temperatures: ∼51% pH2 is generated at liquid nitrogen temperatures (77 K), while nearly 100% pH2 can be produced at 20 K. While the liquid nitrogen cooled generator is attractive due to the low cost of entry, there are benefits to having access to greater than 51% pH2 for enhanced NMR applications. In this work, we introduce a low-cost modification to an existing laboratory-constructed liquid nitrogen cooled pH2 generator that provides âˆ¼ 65% pH2. This modification takes advantage of vacuum-mediated boiling point suppression of liquid nitrogen, allowing the temperature of the liquid to be lowered from 77 K to nitrogen's triple point of 63 K. The reduced temperature allowed for the generation of parahydrogen fractions of 63-67% at gas flow rates from 20 to 1000 standard cubic centimeters per minute. We compare this to equivalent experiments that did not utilize the temperature-lowering effects of pressure reduction; these controls generally maintained pH2 fractions of âˆ¼ 50%. All results (experimental and control) agree with the theoretically expected parahydrogen generation at these temperatures. This straightforward modification to an existing pH2 generator may be of interest to a broad range of scientists involved with parahydrogen research by introducing a simple and low-cost entryway to increased pH2 fractions using a conventional liquid nitrogen cooled generator.

Bedside monitoring of hypoxic ischemic brain injury using low-field, portable brain magnetic resonance imaging after cardiac arrest.

BACKGROUND: Assessment of brain injury severity is critically important after survival from cardiac arrest (CA). Recent advances in low-field MRI technology have permitted the acquisition of clinically useful bedside brain imaging. Our objective was to deploy a novel approach for evaluating brain injury after CA in critically ill patients at high risk for adverse neurological outcome. METHODS: This retrospective, single center study involved review of all consecutive portable MRIs performed as part of clinical care for CA patients between September 2020 and January 2022. Portable MR images were retrospectively reviewed by a blinded board-certified neuroradiologist (S.P.). Fluid-inversion recovery (FLAIR) signal intensities were measured in select regions of interest. RESULTS: We performed 22 low-field MRI examinations in 19 patients resuscitated from CA (68.4% male, mean [standard deviation] age, 51.8 [13.1] years). Twelve patients (63.2%) had findings consistent with HIBI on conventional neuroimaging radiology report. Low-field MRI detected findings consistent with HIBI in all of these patients. Low-field MRI was acquired at a median (interquartile range) of 78 (40-136) hours post-arrest. Quantitatively, we measured FLAIR signal intensity in three regions of interest, which were higher amongst patients with confirmed HIBI. Low-field MRI was completed in all patients without disruption of intensive care unit equipment monitoring and no safety events occurred. CONCLUSION: In a critically ill CA population in whom MR imaging is often not feasible, low-field MRI can be deployed at the bedside to identify HIBI. Low-field MRI provides an opportunity to evaluate the time-dependent nature of MRI findings in CA survivors.

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.

Use of a disposable vascular pressure device to guide balloon inflation of resuscitative endovascular balloon occlusion of the aorta: a bench study.

Resuscitative endovascular balloon occlusion of the aorta (REBOA) for rapid hemorrhage control is increasingly being used in trauma management. Its beneficial hemodynamic effects on unstable patients beyond temporal hemostasis has led to growing interest in its use in other patient populations, such as during cardiac arrest from nontraumatic causes. The ability to insert the catheters without fluoroscopic guidance makes the technique available in the prehospital setting. However, in addition to correct positioning, challenges include reliably achieving aortic occlusion while minimizing the risk of balloon rupture. Without fluoroscopic control, inflation of the balloon relies on estimated aortic diameters and on the disappearing pulse in the contralateral femoral artery. In the case of cardiac arrest or absent palpable pulses, balloon inflation is associated with excess risk of overinflation and adverse events (vessel damage, balloon rupture). In this bench study, we examined how the pressure in the balloon is related to the surrounding blood pressure and the balloon's contact with the vessel wall in two sets of experiments, including a pulsatile circulation model. With this data, we developed a rule of thumb to guide balloon inflation of the ER-REBOA catheter with a simple disposable pressure-reading device (COMPASS). We recommend slowly filling the balloon with saline until the measured balloon pressure is 160 mmHg, or 16 mL of saline have been used. If after 16 mL the balloon pressure is still below 160 mmHg, saline should be added in 1-mL increments, which increases the pressure target about 10 mmHg at each step, until the maximum balloon pressure is reached at 240 mmHg (= 24 mL inflation volume). A balloon pressure greater than 250 mmHg indicates overinflation. With this rule and a disposable pressure-reading device (COMPASS), ER-REBOA balloons can be safely filled in austere environments where fluoroscopy is unavailable. Pressure monitoring of the balloon allows for recognition of unintended deflation or rupture of the balloon.

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.

The use of resuscitative endovascular balloon occlusion of the aorta (REBOA) for non-traumatic cardiac arrest: A review.

Resuscitative endovascular balloon occlusion of the aorta (REBOA) has been proposed as a novel approach to managing non-traumatic cardiac arrest (NTCA). During cardiac arrest, cardiac output ceases and perfusion of vital organs is compromised. Traditional advanced cardiac life support (ACLS) measures and cardiopulmonary resuscitation are often unable to achieve return of spontaneous circulation (ROSC). During insertion of REBOA a balloon-tipped catheter is placed into the femoral artery and advanced in a retrograde manner into the aorta while the patient is undergoing cardiopulmonary resuscitation (CPR). The balloon is then inflated to fully occlude the aorta. The literature surrounding the use of aortic occlusion in non-traumatic cardiac arrest is limited to animal studies, case reports and one recent non-controlled feasibility trial. In both human and animal studies, preliminary data show that REBOA may improve coronary and cerebral perfusion pressures and key physiologic parameters during cardiac arrest resuscitation, and animal data have demonstrated improved rates of ROSC. Multiple questions remain before REBOA can be considered as an adjunct to ACLS. If demonstrated to be effective clinically, REBOA represents a potentially cost-effective and generalizable intervention that may improve quality of life for patients with non-traumatic cardiac arrest.

QTL Mapping and Marker Development for Tolerance to Sulfur Phytotoxicity in Melon (Cucumis melo).

Elemental sulfur is an effective, inexpensive fungicide for many foliar pathogens, but severe phytotoxicity prohibits its use on many melon varieties. Sulfur phytotoxicity causes chlorosis and necrosis of leaf tissue, leading to plant death in the most sensitive lines, while other varieties have little to no damage. A high-density, genotyping-by-sequencing (GBS)-based genetic map of a recombinant inbred line (RIL) population segregating for sulfur tolerance was used for a quantitative trait loci (QTL) mapping study of sulfur phytotoxicity in melon. One major (qSulf-1) and two minor (qSulf-8 and qSulf-12) QTL were associated with sulfur tolerance in the population. The development of Kompetitive Allele-Specific PCR (KASP) markers developed across qSulf-1 decreased the QTL interval from 239 kb (cotyledons) and 157 kb (leaves) to 97 kb (both tissues). The markers were validated for linkage to sulfur tolerance in a set of melon cultivars. These KASP markers can be incorporated into melon breeding programs for introgression of sulfur tolerance into elite melon germplasm.

Specificity of end resection pathways for double-strand break regions containing ribonucleotides and base lesions.

DNA double-strand break repair by homologous recombination begins with nucleolytic resection of the 5' DNA strand at the break ends. Long-range resection is catalyzed by EXO1 and BLM-DNA2, which likely have to navigate through ribonucleotides and damaged bases. Here, we show that a short stretch of ribonucleotides at the 5' terminus stimulates resection by EXO1. Ribonucleotides within a 5' flap are resistant to cleavage by DNA2, and extended RNA:DNA hybrids inhibit both strand separation by BLM and resection by EXO1. Moreover, 8-oxo-guanine impedes EXO1 but enhances resection by BLM-DNA2, and an apurinic/apyrimidinic site stimulates resection by BLM-DNA2 and DNA strand unwinding by BLM. Accordingly, depletion of OGG1 or APE1 leads to greater dependence of DNA resection on DNA2. Importantly, RNase H2A deficiency impairs resection overall, which we attribute to the accumulation of long RNA:DNA hybrids at DNA ends. Our results help explain why eukaryotic cells possess multiple resection nucleases.

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.

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.

Increased Sensitivity of Focused Cardiac Ultrasound for Pulmonary Embolism in Emergency Department Patients With Abnormal Vital Signs.

BACKGROUND: Focused cardiac ultrasound (FOCUS) is insensitive for pulmonary embolism (PE). Theoretically, when a clot is large enough to cause vital sign abnormalities, it is more likely to show signs of right ventricular dysfunction on FOCUS, although this has not been well quantified. A rapid bedside test that could quickly and reliably exclude PE in patients with abnormal vital signs could be of high utility in emergency department (ED) patients. We hypothesized that in patients with tachycardia or hypotension, the sensitivity of FOCUS for PE would increase substantially. METHODS: We performed a prospective observational multicenter cohort study involving a convenience sample of patients from six urban academic EDs. Patients suspected to have PE with tachycardia (heart rate [HR] ≥ 100 beats/min) or hypotension (systolic blood pressure [sBP] < 90 mm Hg) underwent FOCUS before computed tomography angiography (CTA). FOCUS included assessment for right ventricular dilation, McConnell's sign, septal flattening, tricuspid regurgitation, and tricuspid annular plane systolic excursion. If any of these were abnormal, FOCUS was considered positive, while if all were normal, FOCUS was considered negative. We a priori planned a subgroup analysis of all patients with a HR ≥ 110 beats/min (regardless of their sBP). We then determined the diagnostic test characteristics of FOCUS for PE in the entire patient population and in the predefined subgroup, based on CTA as the criterion standard. Inter-rater reliability of FOCUS was determined by blinded review of images by an emergency physician with fellowship training in ultrasound. RESULTS: A total of 143 subjects were assessed for enrollment and 136 were enrolled; four were excluded because they were non-English-speaking and three because of inability to obtain any FOCUS windows. The mean (±SD) age of enrolled subjects was 56 (±7) years, mean (±SD) HR was 114 (±12) beats/min, and 37 (27.2%) subjects were diagnosed with PE on CTA. In all subjects, FOCUS was 92% (95% confidence interval [CI] = 78% to 98%) sensitive and 64% specific (95% CI = 53% to 73%) for PE. In the subgroup of 98 subjects with a HR ≥ 110 beats/min, FOCUS was 100% sensitive (95% CI = 88% to 100%) and 63% specific (95% CI = 51% to 74%) for PE. There was substantial interobserver agreement for FOCUS (κ = 1.0, 95% CI = 0.31 to 1.0). CONCLUSIONS: A negative FOCUS examination may significantly lower the likelihood of the diagnosis of PE in most patients who are suspected of PE and have abnormal vital signs. This was especially true in those patients with a HR ≥ 110 beats/min. Our results suggest that FOCUS can be an important tool in the initial evaluation of ED patients with suspected PE and abnormal vital signs.

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.

Enhancement of BLM-DNA2-Mediated Long-Range DNA End Resection by CtIP.

DNA double-strand break repair by homologous recombination entails the resection of DNA ends to reveal ssDNA tails, which are used to invade a homologous DNA template. CtIP and its yeast ortholog Sae2 regulate the nuclease activity of MRE11 in the initial stage of resection. Deletion of CtIP in the mouse or SAE2 in yeast engenders a more severe phenotype than MRE11 nuclease inactivation, indicative of a broader role of CtIP/Sae2. Here, we provide biochemical evidence that CtIP promotes long-range resection via the BLM-DNA2 pathway. Specifically, CtIP interacts with BLM and enhances its helicase activity, and it enhances DNA cleavage by DNA2. Thus, CtIP influences multiple aspects of end resection beyond MRE11 regulation.

Ultrastructural Characterization of the Glomerulopathy in Alport Mice by Helium Ion Scanning Microscopy (HIM).

The glomerulus exercises its filtration barrier function by establishing a complex filtration apparatus consisting of podocyte foot processes, glomerular basement membrane and endothelial cells. Disruption of any component of the glomerular filtration barrier leads to glomerular dysfunction, frequently manifested as proteinuria. Ultrastructural studies of the glomerulus by transmission electron microscopy (TEM) and conventional scanning electron microscopy (SEM) have been routinely used to identify and classify various glomerular diseases. Here we report the application of newly developed helium ion scanning microscopy (HIM) to examine the glomerulopathy in a Col4a3 mutant/Alport syndrome mouse model. Our study revealed unprecedented details of glomerular abnormalities in Col4a3 mutants including distorted podocyte cell bodies and disorganized primary processes. Strikingly, we observed abundant filamentous microprojections arising from podocyte cell bodies and processes, and presence of unique bridging processes that connect the primary processes and foot processes in Alport mice. Furthermore, we detected an altered glomerular endothelium with disrupted sub-endothelial integrity. More importantly, we were able to clearly visualize the complex, three-dimensional podocyte and endothelial interface by HIM. Our study demonstrates that HIM provides nanometer resolution to uncover and rediscover critical ultrastructural characteristics of the glomerulopathy in Col4a3 mutant mice.

Reconstituted System for the Examination of Repair DNA Synthesis in Homologous Recombination.

In homologous recombination (HR), DNA polymerase δ-mediated DNA synthesis occurs within the displacement loop (D-loop) that is made by the recombinase Rad51 in conjunction with accessory factors. We describe in this chapter the reconstitution of the D-loop and repair DNA synthesis reactions using purified Saccharomyces cerevisiae HR (Rad51, RPA, and Rad54) and DNA replication (PCNA, RFC, and DNA polymerase δ) proteins and document the role of the Pif1 helicase in DNA synthesis via a migrating DNA bubble intermediate. These reconstituted systems are particularly valuable for understanding the conserved mechanism of repair DNA synthesis dependent on DNA polymerase δ and its cognate helicase in eukaryotic organisms.