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Abstract Background NS2359 is a potent reuptake blocker of noradrenalin, dopamine, and serotonin. The aim of the study was to investigate the efficacy, safety and cognitive function of NS2359 in adults with a DSM IV diagnosis of ADHD. Methods The study was a multi-centre, double-blind, randomized placebo-controlled, parallel group design in outpatient adults (18–55 years) testing 0.5 mg NS2359 vs. placebo for 8 weeks. Multiple assessments including computerized neuropsychological evaluation were performed. Results There was no significant difference between NS2359 (n = 63) versus placebo (n = 63) on the primary outcome measure reduction in investigator rated ADHD-RS total score (7.8 versus 6.4; p < 0.45). However, in subjects with the inattentive subtype, there were significantly more responders in the NS2359 group compared to placebo (41% versus 7%; p < 0.01). For all secondary variables (ADHD-RS patient rated; The Conners Adult ADHD Scale; The Brown Adult Scale, and CGI-improvement scale) there were no significant differences between the two groups; however, in the inattentive subgroup, the response to treatment was significantly larger than to placebo. NS2359 improved composite factor scores of attention, episodic- and working memory. No serious adverse events were reported with insomnia, headaches and loss of appetite most commonly reported as side effects. Conclusion No overall effect of NS2359 was found on overall symptoms of ADHD. There was also a modest signal of improvement in the inattentive adults with ADHD and cognition warranting further exploration using differing doses.

Background: Troponin I is a blood biomarker of cardiac injury and levels measured using a high-sensitivity assay after pediatric heart transplantation (HT) have not been described. We sought to assess the association between high-sensitivity troponin I (hsTnI) and N-terminal pro-B-type natriuretic peptide (NTproBNP) with treated acute rejection (AR) and graft loss in pediatric heart transplant (HT) recipients., Methods: Serum was collected and banked from pediatric HT recipients prior to cardiac catheterization. Patients with samples drawn within 365 days post-HT were included and followed for up to 5 years. Generalized linear mixed-effect models examined the association between hsTnI and treated AR using a random intercept per patient. Cox proportional hazards models tested the association between maximal hsTnI and NT-proBNP and death/graft loss., Results: HsTnI and NTproBNP values decline in the weeks following HT, after which these biomarkers stabilize. HsTnI was higher in AR versus no AR (6.2 vs. 3.5 ng/L, p < 0.001); doubling of hsTnI increased the odds of AR by 33% (p = 0.004). HsTnI showed moderate discrimination for AR with an AUC of 0.811 (95% CI 0.76, 0.87) and a NPV of 96.4% (95% CI 93.0, 98.1). Elevation in NT-proBNP was not associated with AR. In multivariable Cox modeling, a doubling of maximal NT-proBNP was associated with graft loss (HR 8.96, p = 0.014)., Conclusions: In this pediatric HT cohort, HsTnI was moderately discriminative for AR and higher maximal NT-proBNP was associated with graft loss. HsTnI may add value in pediatric HT non-invasive AR surveillance, and elevated NTproBNP could suggest an increased risk of graft loss., (© 2024 Wiley Periodicals LLC.)

Background: Waitlist mortality (WM) remains elevated in pediatric heart transplantation. Allocation policy is a potential tool to help improve WM. This study aims to identify patients at highest risk for WM to potentially inform future allocation policy changes., Methods: The Pediatric Heart Transplant Society database was queried for patients <18 years of age indicated for heart transplantation between January 1, 2010 to December 31, 2021. Waitlist mortality was defined as death while awaiting transplant or removal from the waitlist due to clinical deterioration. Because WM is low after the first year, analysis was limited to the first 12 months on the heart transplant list. Kaplan-Meier analysis and log-rank testing was conducted to compare unadjusted survival between groups. Cox proportional hazard models were created to determine risk factors for WM. Subgroup analysis was performed for status 1A patients based on body surface area (BSA) at time of listing, cardiac diagnosis, and presence of mechanical circulatory support., Results: In total 5974 children met study criteria of which 3928 were status 1A, 1012 were status 1B, 963 were listed status 2, and 65 were listed status 7. Because of the significant burden of WM experienced by 1A patients, further analysis was performed in only patients indicated as 1A. Within that group of patients, those with smaller size and lower eGFR had higher WM, whereas those patients without congenital heart disease or support from a ventricular assist device (VAD) at time of listing had decreased WM. In the smallest size cohort, cardiac diagnoses other than dilated cardiomyopathy were risk factors for WM. Previous cardiac surgery was a risk factor in the 0.3 to 0.7 m 2 and >0.7 m 2 BSA groups. VAD support was associated with lower WM other than in the single ventricle cohort, where VAD was associated with higher WM. Extracorporeal membrane oxygenation and mechanical ventilation were associated with increased risk of WM in all cohorts., Conclusions: There is significant variability in WM among status-1A patients. Potential refinements to current allocation system should factor in the increased WM risk we identified in patients supported by extracorporeal membrane oxygenation or mechanical ventilation, single ventricle congenital heart disease on VAD support and small children with congenital heart disease, restrictive cardiomyopathy, or hypertrophic cardiomyopathy., Competing Interests: None.

RAD52 is important for the repair of DNA double-stranded breaks 1,2 , mitotic DNA synthesis 3-5 and alternative telomere length maintenance 6,7 . Central to these functions, RAD52 promotes the annealing of complementary single-stranded DNA (ssDNA) 8,9 and provides an alternative to BRCA2/RAD51-dependent homologous recombination repair 10 . Inactivation of RAD52 in homologous-recombination-deficient BRCA1- or BRCA2-defective cells is synthetically lethal 11,12 , and aberrant expression of RAD52 is associated with poor cancer prognosis 13,14 . As a consequence, RAD52 is an attractive therapeutic target against homologous-recombination-deficient breast, ovarian and prostate cancers 15-17 . Here we describe the structure of RAD52 and define the mechanism of annealing. As reported previously 18-20 , RAD52 forms undecameric (11-subunit) ring structures, but these rings do not represent the active form of the enzyme. Instead, cryo-electron microscopy and biochemical analyses revealed that ssDNA annealing is driven by RAD52 open rings in association with replication protein-A (RPA). Atomic models of the RAD52-ssDNA complex show that ssDNA sits in a positively charged channel around the ring. Annealing is driven by the RAD52 N-terminal domains, whereas the C-terminal regions modulate the open-ring conformation and RPA interaction. RPA associates with RAD52 at the site of ring opening with critical interactions occurring between the RPA-interacting domain of RAD52 and the winged helix domain of RPA2. Our studies provide structural snapshots throughout the annealing process and define the molecular mechanism of ssDNA annealing by the RAD52-RPA complex., (© 2024. The Author(s).)

Background: We have developed a new clinical research approach for the quantification of cellular proliferation in human infants to address unanswered questions about tissue renewal and regeneration. The approach consists of oral 15N-thymidine administration to label cells in S-phase, followed by Multi-isotope Imaging Mass Spectrometry for detection of the incorporated label in cell nuclei. To establish the approach, we performed an observational study to examine uptake and elimination of 15N-thymidine. We compared at-home label administration with in-hospital administration in infants with tetralogy of Fallot, a form of congenital heart disease, and infants with heart failure., Methods: We examined urine samples from 18 infants who received 15N-thymidine (50 mg/kg body weight) by mouth for five consecutive days. We used Isotope Ratio Mass Spectrometry to determine enrichment of 15N relative to 14N (%) in urine., Results/findings: 15N-thymidine dose administration produced periodic rises of 15N enrichment in urine. Infants with tetralogy of Fallot had a 3.2-fold increase and infants with heart failure had a 4.3-fold increase in mean peak 15N enrichment over baseline. The mean 15N enrichment was not statistically different between the two patient populations (p = 0.103). The time to peak 15N enrichment in tetralogy of Fallot infants was 6.3 ± 1 hr and in infants with heart failure 7.5 ± 2 hr (mean ± SEM). The duration of significant 15N enrichment after a dose was 18.5 ± 1.7 hr in tetralogy of Fallot and in heart failure 18.2 ± 1.8 hr (mean ± SEM). The time to peak enrichment and duration of enrichment were also not statistically different (p = 0.617 and p = 0.887)., Conclusions: The presented results support two conclusions of significance for future applications: (1) Demonstration that 15N-thymidine label administration at home is equivalent to in-hospital administration. (2) Two different types of heart disease show no differences in 15N-thymidine absorption and elimination. This enables the comparative analysis of cellular proliferation between different types of heart disease., Competing Interests: The authors declare that they have no competing interests., (Copyright: © 2024 Ammanamanchi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

DNA molecules that contain single Holliday junctions have served as model substrates to investigate the pathway in which homologous recombination intermediates are processed. However, the preparation of DNA containing Holliday junctions in high yield remains a challenge. In this work, we used a nicking endonuclease to generate gapped DNA, from which α-structured DNA or figure-8 DNA were created via RecA-mediated reactions. The resulting DNA molecules were found to serve as good substrates for Holliday junction resolvases. The simplified method negates the requirement for radioactive labelling of DNA, making the generation of Holliday junction DNA more accessible to non-experts., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Poly(ADP-ribose) polymerase (PARP) inhibitors are used in the clinic to treat BRCA-deficient breast, ovarian and prostate cancers. As their efficacy is potentiated by loss of the nucleotide salvage factor DNPH1 there is considerable interest in the development of highly specific small molecule DNPH1 inhibitors. Here, we present X-ray crystal structures of dimeric DNPH1 bound to its substrate hydroxymethyl deoxyuridine monophosphate (hmdUMP). Direct interaction with the hydroxymethyl group is important for substrate positioning, while conserved residues surrounding the base facilitate target discrimination. Glycosidic bond cleavage is driven by a conserved catalytic triad and proceeds via a two-step mechanism involving formation and subsequent disruption of a covalent glycosyl-enzyme intermediate. Mutation of a previously uncharacterised yet conserved glutamate traps the intermediate in the active site, demonstrating its role in the hydrolytic step. These observations define the enzyme's catalytic site and mechanism of hydrolysis, and provide important insights for inhibitor discovery., (© 2023. The Author(s).)

Homologous recombination (HR) is essential for error-free repair of DNA double-strand breaks, perturbed replication forks (RFs), and post-replicative single-stranded DNA (ssDNA) gaps. To initiate HR, the recombination mediator and tumor suppressor protein BRCA2 facilitates nucleation of RAD51 on ssDNA prior to stimulation of RAD51 filament growth by RAD51 paralogs. Although ssDNA binding by BRCA2 has been implicated in RAD51 nucleation, the function of double-stranded DNA (dsDNA) binding by BRCA2 remains unclear. Here, we exploit single-molecule (SM) imaging to visualize BRCA2-mediated RAD51 nucleation in real time using purified proteins. We report that BRCA2 nucleates and stabilizes RAD51 on ssDNA either directly or through an unappreciated diffusion-assisted delivery mechanism involving binding to and sliding along dsDNA, which requires the cooperative action of multiple dsDNA-binding modules in BRCA2. Collectively, our work reveals two distinct mechanisms of BRCA2-dependent RAD51 loading onto ssDNA, which we propose are critical for its diverse functions in maintaining genome stability and cancer suppression., Competing Interests: Declaration of interests S.J.B. is a co-founder, VP Science Strategy, and a shareholder of Artios Pharma Ltd. S.J.B. is also a member of the advisory board of Molecular Cell., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Homologous recombination is a fundamental process of life. It is required for the protection and restart of broken replication forks, the repair of chromosome breaks and the exchange of genetic material during meiosis. Individuals with mutations in key recombination genes, such as BRCA2 (also known as FANCD1), or the RAD51 paralogues RAD51B, RAD51C (also known as FANCO), RAD51D, XRCC2 (also known as FANCU) and XRCC3, are predisposed to breast, ovarian and prostate cancers 1-10 and the cancer-prone syndrome Fanconi anaemia 11-13 . The BRCA2 tumour suppressor protein-the product of BRCA2-is well characterized, but the cellular functions of the RAD51 paralogues remain unclear. Genetic knockouts display growth defects, reduced RAD51 focus formation, spontaneous chromosome abnormalities, sensitivity to PARP inhibitors and replication fork defects 14,15 , but the precise molecular roles of RAD51 paralogues in fork stability, DNA repair and cancer avoidance remain unknown. Here we used cryo-electron microscopy, AlphaFold2 modelling and structural proteomics to determine the structure of the RAD51B-RAD51C-RAD51D-XRCC2 complex (BCDX2), revealing that RAD51C-RAD51D-XRCC2 mimics three RAD51 protomers aligned within a nucleoprotein filament, whereas RAD51B is highly dynamic. Biochemical and single-molecule analyses showed that BCDX2 stimulates the nucleation and extension of RAD51 filaments-which are essential for recombinational DNA repair-in reactions that depend on the coupled ATPase activities of RAD51B and RAD51C. Our studies demonstrate that BCDX2 orchestrates RAD51 assembly on single stranded DNA for replication fork protection and double strand break repair, in reactions that are critical for tumour avoidance., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: Endomyocardial biopsy (EMB)-led surveillance is common after pediatric heart transplantation (HT), with some centers performing periodic surveillance EMBs indefinitely after HT. Donor derived cell-free DNA (dd-cfDNA)-led surveillance offers an alternative, but knowledge about its clinical and economic outcomes, both key drivers of potential utilization, are lacking., Methods: Using single-center recipient and center-level data, we describe clinical outcomes prior to and since transition from EMB-led surveillance to dd-cfDNA-led surveillance of pediatric and young adult HT recipients. These data were then used to inform Markov models to compare costs between EMB-led and dd-cfDNA-led surveillance strategies., Results: Over 34.5 months, dd-cfDNA-led surveillance decreased the number of EMBs by 81.8% (95% CI 76.3%-86.5%) among 120 HT recipients (median age 13.3 years). There were no differences in the incidences of graft loss or death among all recipients followed at our center prior to and following implementation of dd-cfDNA-led surveillance (graft loss: 2.9 vs. 1.5 per 100 patient-years; p = .17; mortality: 3.7 vs. 2.2 per 100 patient-years; p = .23). Over 20 years from HT, dd-cfDNA-led surveillance is projected to cost $8545 less than EMB-led surveillance. Model findings were robust in sensitivity and scenario analyses, with cost of EMB, cost of dd-cfDNA testing, and probability of elevated dd-cfDNA most influential on model findings., Conclusions: dd-cfDNA-led surveillance shows promise as a less invasive and cost saving alternative to EMB-led surveillance among pediatric and young adult HT recipients., (© 2023 The Authors. Clinical Transplantation published by John Wiley & Sons Ltd.)

Chromatin bridges connecting the two segregating daughter nuclei arise from chromosome fusion or unresolved interchromosomal linkage. Persistent chromatin bridges are trapped in the cleavage plane, triggering cytokinesis delay. The trapped bridges occasionally break during cytokinesis, inducing DNA damage and chromosomal rearrangements. Recently, Caenorhabditis elegans LEM-3 and human TREX1 nucleases have been shown to process chromatin bridges. Here, it is shown that ANKLE1 endonuclease, the human ortholog of LEM-3, accumulates at the bulge-like structure of the midbody via its N-terminal ankyrin repeats. Importantly, ANKLE1 -/- knockout cells display an elevated level of G1-specific 53BP1 nuclear bodies, prolonged activation of the DNA damage response, and replication stress. Increased DNA damage observed in ANKLE1 -/- cells is rescued by inhibiting actin polymerization or reducing actomyosin contractility. ANKLE1 does not act in conjunction with structure-selective endonucleases, GEN1 and MUS81 in resolving recombination intermediates. Instead, ANKLE1 acts on chromatin bridges by priming TREX1 nucleolytic activity and cleaving bridge DNA to prevent the formation of micronuclei and cytosolic dsDNA that activate the cGAS-STING pathway. It is therefore proposed that ANKLE1 prevents DNA damage and autoimmunity by cleaving chromatin bridges to avoid catastrophic breakage mediated by actomyosin contractile forces., (© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Due to medical advances, women with congenital heart disease (CHD) are living longer, healthier lives and many are considering pregnancy. The hemodynamic changes of pregnancy present high risks of morbidity and mortality for many women with CHD. As little is known about these women's reproductive health experiences, this study explores their perceptions of pregnancy and family planning care as related to CHD. Women ages 18-45 years with a diagnosis of CHD associated with a World Health Organization (WHO) classification II-IV for pregnancy morbidity and mortality participated in individual, semi-structured interviews exploring their experiences, attitudes, and preferences toward parenthood, pregnancy, contraception and family planning care provision. Interviews were audio-recorded, transcribed verbatim. Two independent coders performed analysis using deductive and inductive coding approaches. Twenty women with CHD participated in interviews (average age 30.1 years, SD 5.85). Nine women had a prior pregnancy and 14 considered becoming a parent in the future. We identified 5 key themes among the women: (1) CHD impacted their reproductive health goals and decisions; (2) Women with CHD perceived a lack of safe contraceptive methods for their condition; (3) Women desired tailored, disease-specific sexual and reproductive health (SRH) information; (4) Women viewed their cardiologist as the primary source for SRH information and prefer provider-initiated discussions starting in adolescence; and (5) Women desire coordinated pre-pregnancy and intrapartum care between their cardiologists and women's health providers. These results provide a foundation for interventions to improve patient-centered interdisciplinary reproductive healthcare for this population., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Fontan associated liver disease (FALD) potentially impacts Fontan patients undergoing heart transplant. This multi-center study sought to identify pre-transplant risk factors and characterize any post-transplant liver recovery in those patients undergoing heart-alone transplant., Methods: Review of Fontan patients at 12 pediatric institutions who underwent heart transplant between 2001-2019. Radiologists reviewed pre and post-transplant liver imaging for fibrosis. Laboratory, pathology and endoscopy studies were reviewed., Results: 156 patients underwent transplant due to decreased ventricular function (49%), protein losing enteropathy (31%) or plastic bronchitis (10%); median age at transplant was 13.6 years (interquartile range IQR 7.8, 17.2) with a median of 9.3 years (IQR 3.2, 13.4) between the Fontan operation and transplant. Few patients had pre-transplant endoscopy (18%), and liver biopsy (19%). There were 31 deaths (20%). The median time from transplant to death was 0.5 years (95% Confidence Interval CI 0.0, 3.6). The five-year survival was 73% (95% CI 64%, 83%). Deaths were related to cardiac causes in 68% (21/31) and infection in 6 (19%). A pre-transplant elevation in bilirubin was a predictor of death. Higher platelet levels were protective. Immediate post-transplant elevations in creatinine, AST, ALT, and INR were predictive of death. Advanced liver fibrosis identified on ultrasound, computed tomography, or magnetic resonance imaging was not predictive of death. Liver imaging suggested some improvement in liver congestion post-transplant., Conclusions: Elevated bilirubin, but not fibrosis on liver imaging, was associated with post-heart transplant mortality in Fontan patients in this multicenter retrospective study. Additionally, heart transplant may alter the progression of FALD., (© 2022 Wiley Periodicals LLC.)

Our genomes harbor conserved DNA sequences, known as common fragile sites (CFSs), that are difficult to replicate and correspond to regions of genome instability. Following replication stress, CFS loci give rise to breaks or gaps (termed CFS expression) where under-replicated DNA subsequently undergoes mitotic DNA synthesis (MiDAS). We show that loss of the structure-selective endonuclease GEN1 reduces CFS expression, leading to defects in MiDAS, ultrafine anaphase bridge formation, and DNA damage in the ensuing cell cycle due to aberrant chromosome segregation. GEN1 knockout cells also exhibit an elevated frequency of bichromatid constrictions consistent with the presence of unresolved regions of under-replicated DNA. Previously, the role of GEN1 was thought to be restricted to the nucleolytic resolution of recombination intermediates. However, its ability to cleave under-replicated DNA at CFS loci indicates that GEN1 plays a dual role resolving both DNA replication and recombination intermediates before chromosome segregation., Competing Interests: Declaration of interests The authors declare no conflict of interest., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

POLQ is a key effector of DSB repair by microhomology-mediated end-joining (MMEJ) and is overexpressed in many cancers. POLQ inhibitors confer synthetic lethality in HR and Shieldin-deficient cancer cells, which has been proposed to reflect a critical dependence on the DSB repair pathway by MMEJ. Whether POLQ also operates independent of MMEJ remains unexplored. Here, we show that POLQ-deficient cells accumulate post-replicative ssDNA gaps upon BRCA1/2 loss or PARP inhibitor treatment. Biochemically, cooperation between POLQ helicase and polymerase activities promotes RPA displacement and ssDNA-gap fill-in, respectively. POLQ is also capable of microhomology-mediated gap skipping (MMGS), which generates deletions during gap repair that resemble the genomic scars prevalent in POLQ overexpressing cancers. Our findings implicate POLQ in mutagenic post-replicative gap sealing, which could drive genome evolution in cancer and whose loss places a critical dependency on HR for gap protection and repair and cellular viability., Competing Interests: Declaration of interests S.J.B is a co-founder, VP Science Strategy and a shareholder of Artios Pharma Ltd. G.S.H. received consultancy fees from and is a shareholder of Artios Pharma Ltd and is a member of the Molecular Cell advisory board. V.G., J.N., H.M.R.R., and G.C.M.S. are all employees and shareholders of Artios Pharma Ltd. G.C.M.S. is a shareholder of AstraZeneca PLC., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Biofilms are bacterial communities characterized by antibiotic tolerance. Staphylococcus aureus is a leading cause of biofilm infections on medical devices, including prosthetic joints, which represent a significant health care burden. The major leukocyte infiltrate associated with S. aureus prosthetic joint infection (PJI) is granulocytic myeloid-derived suppressor cells (G-MDSCs), which produce IL-10 to promote biofilm persistence by inhibiting monocyte and macrophage proinflammatory activity. To determine how S. aureus biofilm responds to G-MDSCs and macrophages, biofilms were cocultured with either leukocyte population followed by RNA sequencing. Several genes involved in fermentative pathways were significantly upregulated in S. aureus biofilm following G-MDSC coculture, including formate acetyltransferase ( pflB ), which catalyzes the conversion of pyruvate and coenzyme-A into formate and acetyl-CoA. A S. aureus pflB mutant (Δ pflB ) did not exhibit growth defects in vitro . However, Δ pflB formed taller and more diffuse biofilm compared to the wild-type strain as revealed by confocal microscopy. In a mouse model of PJI, the bacterial burden was significantly reduced with Δ pflB during later stages of infection, which coincided with decreased G-MDSC influx and increased neutrophil recruitment, and Δ pflB was more susceptible to macrophage killing. Although formate was significantly reduced in the soft tissue surrounding the joint of Δ pflB -infected mice levels were increased in the femur, suggesting that host-derived formate may also influence bacterial survival. This was supported by the finding that a Δ pflB Δ fdh strain defective in formate production and catabolism displayed a similar phenotype to Δ pflB . These results revealed that S. aureus formate metabolism is important for promoting biofilm persistence.

Background: Acute kidney disease (AKD) is defined as impaired kidney function present for <90 days with or without an acute kidney injury (AKI) event. Adults with AKD have an increased risk for progression to chronic kidney disease (CKD) and mortality. There are no data on the epidemiology of AKD in children after transplant. The aim of this study was to evaluate the incidence and risk factors for AKI, AKD, and CKD in children after transplantation., Methods: This is a retrospective cohort study of all children undergoing non-kidney solid organ transplant between 2011 and 2019 at UPMC Children's Hospital of Pittsburgh. AKI and AKD were defined using the Kidney Disease Improving Global Outcomes criteria. Patients with a new estimated glomerular filtration rate <60 ml/min/1.73m 2 persisting for >3 months met criteria for new CKD. Variables associated with AKI, AKD, and CKD were analyzed., Results: Among 338 patients, 37.9% met criteria for severe AKI, 13% for AKD, and 8% for a new diagnosis of CKD. Stage 3 AKI was independently associated with AKD (OR: 5.35; 95% CI: 2.23-12.86). Severe AKI was not associated with new-onset CKD, whereas AKD was associated with new-onset CKD (OR: 29.74; CI: 11.22-78.82)., Conclusion: AKD may be superior to AKI in predicting risk of CKD in children after non-kidney solid organ transplantation., (© 2021 Wiley Periodicals LLC.)

Background: Third-dose mRNA COVID-19 vaccine is currently recommended in the United States for SOT recipients based in part on data showing diminished immune response, including Ab production, after a two-dose regimen. Data on vaccine response in adolescent and young adult SOT recipients are limited, including no data reported on third-dose responsiveness., Methods: Results of serologic testing in a convenience sample of 28 vaccinated adolescent and young adult HT recipients at a single institution were collected from the medical record and summarized., Results: At a median of 98.5 days (IQR 59-150) after second dose, 17 (61%) had an Ab response. Among 12 who had serology before and after third-dose vaccination, four of seven who were negative prior to third dose became positive at a median of 34 days (IQR 31-39.5) following third dose. No myocarditis, acute rejection, graft dysfunction, graft loss, or deaths were observed., Conclusions: These findings support recommendations for the routine administration of three doses of mRNA vaccines in adolescent and young adult HT recipients and show a potential subpopulation in whom the fourth dose should be contemplated., (© 2022 Wiley Periodicals LLC.)

Cellular proliferation depends on the accurate and timely replication of the genome. Several genetic diseases are caused by mutations in key DNA replication genes; however, it remains unclear whether these genes influence the normal program of DNA replication timing. Similarly, the factors that regulate DNA replication dynamics are poorly understood. To systematically identify trans-acting modulators of replication timing, we profiled replication in 184 cell lines from three cell types, encompassing 60 different gene knockouts or genetic diseases. Through a rigorous approach that considers the background variability of replication timing, we concluded that most samples displayed normal replication timing. However, mutations in two genes showed consistently abnormal replication timing. The first gene was RIF1, a known modulator of replication timing. The second was MCM10, a highly conserved member of the pre-replication complex. Cells from a single patient carrying MCM10 mutations demonstrated replication timing variability comprising 46% of the genome and at different locations than RIF1 knockouts. Replication timing alterations in the mutated MCM10 cells were predominantly comprised of replication delays and initiation site gains and losses. Taken together, this study demonstrates the remarkable robustness of the human replication timing program and reveals MCM10 as a novel candidate modulator of DNA replication timing., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Retransplantation is rare and associated with worse survival and more morbidity. The study aim is to describe an updated cohort of pediatric retransplants, determine if there has been an era effect on outcomes, and understand if identified trends are explained by changes in patient selection., Methods: Pediatric Heart Transplant Society database analysis of retransplantation patients <18 years of age (Era 1: 1993-2001, Era 2: 2002-2010, Era 3: 2011-2018). Multivariate analysis identified risk factors for graft loss. Multiphase parametric hazard modeling was used to depict era and risk factor effect., Results: Survival was lower (p < .0001) for retransplant (n = 222) compared to primary transplant (n = 6548) (median 9.3 vs 20.2 years). Median survival increased from Era 1 to 2 (4.8 vs 9.3 years; p < .0001) with no incremental change in Era 3. Era 2 and 3 retransplants had a longer inter-transplant interval (p < .0001), were less frequently for early graft failure (p = .0004) or acute rejection (p = .007), more frequently from a ventricular assist device (p = .0014), and less frequently from extracorporeal membrane oxygenation (p = .0024). Predictors of graft loss included Era 1 (HR 10.55, p = .001), congenital heart disease (HR 4.42, p = .01), inter-transplant interval <1 year (HR 5.34, p = .002), and mechanical support (ventricular assist device HR 7.47, p = .0042; extracorporeal membrane oxygenation HR 10.09, p < .0001). For each 1-year increase in inter-transplant interval, graft loss risk decreased by 1.15 (p = .0002). Retransplantation was associated with more rejection, infection, and allograft vasculopathy., Conclusions: Graft survival has improved in pediatric retransplants making it a viable option in select patients. Retransplantation should be avoided in the setting of early graft failure especially requiring mechanical support., Competing Interests: Disclosure statement The authors have no conflicts of interest to disclose., (Copyright © 2022 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.)

Four-way DNA intermediates, also known as Holliday junctions (HJs), are formed during homologous recombination and DNA repair, and their resolution is necessary for proper chromosome segregation. To facilitate the biochemical analysis of HJ processing, we developed a method involving DNAzyme self-cleavage to generate 1.8-kb DNA molecules containing either single (sHJ) or double Holliday junctions (dHJs). We show that dHJ DNAs (referred to as HoJo DNAs) are dissolved by the human BLM–TopIIIα–RMI1–RMI2 complex to form two noncrossover products. However, structure-selective endonucleases (human GEN1 and SMX complex) resolve DNA containing single or double HJs to yield a mixture of crossover and noncrossover products. Finally, we demonstrate that chromatin inhibits the resolution of the double HJ by GEN or SMX while allowing BTRR-mediated dissolution.

Abnormal dystrophin production due to mutations in the dystrophin gene causes Duchenne Muscular Dystrophy (DMD). Cases demonstrate considerable genetic and disease progression variability. It is unclear if specific gene mutations are prognostic of outcomes in this population. We conducted a retrospective cohort study of DMD patients followed at 17 centers across the USA and Canada from 2005 to 2015 with goal of understanding the genetic variability of DMD and its impact on clinical outcomes. Cumulative incidence of clinically relevant outcomes was stratified by genetic mutation type, exon mutation location, and extent of exon deletion. Of 436 males with DMD, 324 (74.3%) underwent genetic testing. Deletions were the most common mutation type (256, 79%), followed by point mutations (45, 13.9%) and duplications (23, 7.1%). There were 131 combinations of mutations with most mutations located along exons 45 to 52. The number of exons deleted varied between 1 and 52 with a median of 3 exons deleted (IQR 1-6). Subjects with mutations starting at exon positions 40-54 had a later onset of arrhythmias occurring at median age 25 years (95% CI 18-∞), p = 0.01. Loss of ambulation occurred later at median age of 13 years (95% CI 12-15) in subjects with mutations that started between exons 55-79, p = 0.01. There was no association between mutation type or location and onset of cardiac dysfunction. We report the genetic variability in DMD and its association with timing of clinical outcomes. Genetic modifiers may explain some phenotypic variability., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Endomyocardial biopsy (EMB) is costly and discomforting yet remains a key component of surveillance after pediatric heart transplantation (HT). Donor-derived cell-free DNA (dd-cfDNA) has been histologically validated with high negative predictive value, offering an alternative to surveillance EMB (sEMB)., Methods: We implemented an alternative surveillance protocol using commercially available dd-cfDNA assays in place of sEMB after pediatric HT. Recipients ≧7 months post-HT with reassuring clinical assessment were referred for dd-cfDNA. When not elevated above the manufacturers' threshold, sEMB was deferred. Subsequent clinical status and results of follow-up EMB were analyzed., Results: Over 17 months, 58 recipients [34% female, median age at HT 3.1 years (IQR 0.6-10.6)] had dd-cfDNA assessed per protocol. Median age was 14.8 years (8.4-18.3) and time from HT 6.0 years (2.2-11.2). Forty-seven (81%) had non-elevated dd-cfDNA and 11 (19%) were elevated. During a median of 8.7 months (4.2-15), all are alive without allograft loss/new dysfunction. Among those with non-elevated dd-cfDNA, 24 (51%) had subsequent sEMB at 12.1 months (6.9-12.9) with 23 showing no acute rejection (AR): grade 0R/pAMR0 (n = 16); 1R(1A)/pAMR0 (n = 7). One had AR (grade 2R(3A)/pAMR0) on follow-up sEMB after decreased immunosuppression following a diagnosis of PTLD. All 11 with elevated dd-cfDNA had reflex EMB at 19 days (12-32) with AR in 4: grade 1R(1B-2)/pAMR0 (n = 3); 1R(1B)/pAMR2 (n = 1)., Conclusions: dd-cfDNA assessment in place of selected, per-protocol EMB decreased surveillance EMB by 81% in our pediatric HT recipient cohort with no short-term adverse outcomes. Individual center approach to surveillance EMB will influence the utility of these findings., (© 2021 Wiley Periodicals LLC.)

Mutations in the SETX gene, which encodes Senataxin, are associated with the progressive neurodegenerative diseases ataxia with oculomotor apraxia 2 (AOA2) and amyotrophic lateral sclerosis 4 (ALS4). To identify the causal defect in AOA2, patient-derived cells and SETX knockouts (human and mouse) were analyzed using integrated genomic and transcriptomic approaches. A genome-wide increase in chromosome instability (gains and losses) within genes and at chromosome fragile sites was observed, resulting in changes to gene-expression profiles. Transcription stress near promoters correlated with high GCskew and the accumulation of R-loops at promoter-proximal regions, which localized with chromosomal regions where gains and losses were observed. In the absence of Senataxin, the Cockayne syndrome protein CSB was required for the recruitment of the transcription-coupled repair endonucleases (XPG and XPF) and RAD52 recombination protein to target and resolve transcription bubbles containing R-loops, leading to genomic instability. These results show that transcription stress is an important contributor to SETX mutation-associated chromosome fragility and AOA2., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

Homologous recombination-deficient (HRD) tumours, including those harbouring mutations in the BRCA genes, are hypersensitive to treatment with inhibitors of poly(ADP-ribose) polymerase (PARPis). Despite high response rates, most HRD cancers ultimately develop resistance to PARPi treatment through reversion mutations or genetic/epigenetic alterations to DNA repair pathways. Counteracting these resistance pathways, thereby increasing the potency of PARPi therapy, represents a potential strategy to improve the treatment of HRD cancers. In this review, we discuss recent insights derived from genetic screens that have identified a number of novel genes that can be targeted to improve PARPi treatment of HRD cancers and may provide a means to overcome PARPi resistance., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Freedom from rejection in pediatric heart transplant recipients is highly variable across centers. This study aimed to assess the center variation in methods used to diagnose rejection in the first-year post-transplant and determine the impact of this variation on patient outcomes., Methods: The PHTS registry was queried for all rejection episodes in the first-year post-transplant (2010-2019). The primary method for rejection diagnosis was determined for each event as surveillance biopsy, echo diagnosis, or clinical. The percentage of first-year rejection events diagnosed by surveillance biopsy was used to approximate the surveillance strategy across centers. Methods of rejection diagnosis were described and patient outcomes were assessed based on surveillance biopsy utilization among centers., Results: A total of 3985 patients from 56 centers were included. Of this group, 873 (22%) developed rejection within the first-year post-transplant. Surveillance biopsy was the most common method of rejection diagnosis (71.7%), but practices were highly variable across centers. The majority (73.6%) of first rejection events occurred within 3-months of transplantation. Diagnosis modality in the first-year was not independently associated with freedom from rejection, freedom from rejection with hemodynamic compromise, or overall graft survival., Conclusions: Rejection in the first-year after pediatric heart transplant occurs in 22% of patients and most commonly in the first 3 months post-transplant. Significant variation exists across centers in the methods used to diagnose rejection in pediatric heart transplant recipients, however, these variable strategies are not independently associated with freedom from rejection, rejection with hemodynamic compromise, or overall graft survival., (Copyright © 2021 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.)

Mutations in the BRCA1 or BRCA2 tumor suppressor genes predispose individuals to breast and ovarian cancer. In the clinic, these cancers are treated with inhibitors that target poly(ADP-ribose) polymerase (PARP). We show that inhibition of DNPH1, a protein that eliminates cytotoxic nucleotide 5-hydroxymethyl-deoxyuridine (hmdU) monophosphate, potentiates the sensitivity of BRCA -deficient cells to PARP inhibitors (PARPi). Synthetic lethality was mediated by the action of SMUG1 glycosylase on genomic hmdU, leading to PARP trapping, replication fork collapse, DNA break formation, and apoptosis. BRCA1 -deficient cells that acquired resistance to PARPi were resensitized by treatment with hmdU and DNPH1 inhibition. Because genomic hmdU is a key determinant of PARPi sensitivity, targeting DNPH1 provides a promising strategy for the hypersensitization of BRCA -deficient cancers to PARPi therapy., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

SLX4 provides a molecular scaffold for the assembly of multiple protein complexes required for the maintenance of genome stability. It is involved in the repair of DNA crosslinks, the resolution of recombination intermediates, the response to replication stress and the maintenance of telomere length. To carry out these diverse functions, SLX4 interacts with three structure-selective endonucleases, MUS81-EME1, SLX1 and XPF-ERCC1, as well as the telomere binding proteins TRF2, RTEL1 and SLX4IP. Recently, SLX4 was shown to interact with MutSβ, a heterodimeric protein involved in DNA mismatch repair, trinucleotide repeat instability, crosslink repair and recombination. Importantly, MutSβ promotes the pathogenic expansion of CAG/CTG trinucleotide repeats, which is causative of myotonic dystrophy and Huntington's disease. The colocalization and specific interaction of MutSβ with SLX4, together with their apparently overlapping functions, are suggestive of a common role in reactions that promote DNA maintenance and genome stability. This review will focus on the role of SLX4 in DNA repair, the interplay between MutSβ and SLX4, and detail how they cooperate to promote recombinational repair and DNA crosslink repair. Furthermore, we speculate that MutSβ and SLX4 may provide an alternative cellular mechanism that modulates trinucleotide instability.

Chromatin is a barrier to efficient DNA repair, as it hinders access and processing of certain DNA lesions. ALC1/CHD1L is a nucleosome-remodeling enzyme that responds to DNA damage, but its precise function in DNA repair remains unknown. Here we report that loss of ALC1 confers sensitivity to PARP inhibitors, methyl-methanesulfonate, and uracil misincorporation, which reflects the need to remodel nucleosomes following base excision by DNA glycosylases but prior to handover to APEX1. Using CRISPR screens, we establish that ALC1 loss is synthetic lethal with homologous recombination deficiency (HRD), which we attribute to chromosome instability caused by unrepaired DNA gaps at replication forks. In the absence of ALC1 or APEX1, incomplete processing of BER intermediates results in post-replicative DNA gaps and a critical dependence on HR for repair. Hence, targeting ALC1 alone or as a PARP inhibitor sensitizer could be employed to augment existing therapeutic strategies for HRD cancers., Competing Interests: Declaration Of Interests G.H. and S.J.B are inventors on a patent derived from this work. S.J.B. is also scientific co-founder and VP Science Strategy at Artios Pharma Ltd., Babraham Research Campus, Cambridge, UK. The authors declare no other competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Competing Interests: The authors declare no competing interest.

MutSα and MutSβ play important roles in DNA mismatch repair and are linked to inheritable cancers and degenerative disorders. Here, we show that MSH2 and MSH3, the two components of MutSβ, bind SLX4 protein, a scaffold for the assembly of the SLX1-SLX4-MUS81-EME1-XPF-ERCC1 (SMX) trinuclease complex. SMX promotes the resolution of Holliday junctions (HJs), which are intermediates in homologous recombinational repair. We find that MutSβ binds HJs and stimulates their resolution by SLX1-SLX4 or SMX in reactions dependent upon direct interactions between MutSβ and SLX4. In contrast, MutSα does not stimulate HJ resolution. MSH3-depleted cells exhibit reduced sister chromatid exchanges and elevated levels of homologous recombination ultrafine bridges (HR-UFBs) at mitosis, consistent with defects in the processing of recombination intermediates. These results demonstrate a role for MutSβ in addition to its established role in the pathogenic expansion of CAG/CTG trinucleotide repeats, which is causative of myotonic dystrophy and Huntington's disease., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

The RecQ DNA helicase WRN is a synthetic lethal target for cancer cells with microsatellite instability (MSI), a form of genetic hypermutability that arises from impaired mismatch repair 1-4 . Depletion of WRN induces widespread DNA double-strand breaks in MSI cells, leading to cell cycle arrest and/or apoptosis. However, the mechanism by which WRN protects MSI-associated cancers from double-strand breaks remains unclear. Here we show that TA-dinucleotide repeats are highly unstable in MSI cells and undergo large-scale expansions, distinct from previously described insertion or deletion mutations of a few nucleotides 5 . Expanded TA repeats form non-B DNA secondary structures that stall replication forks, activate the ATR checkpoint kinase, and require unwinding by the WRN helicase. In the absence of WRN, the expanded TA-dinucleotide repeats are susceptible to cleavage by the MUS81 nuclease, leading to massive chromosome shattering. These findings identify a distinct biomarker that underlies the synthetic lethal dependence on WRN, and support the development of therapeutic agents that target WRN for MSI-associated cancers.

Duchenne muscular dystrophy (DMD) is characterized by myocardial fibrosis and left ventricular (LV) dysfunction. Implantable cardioverter defibrillator (ICD) use has not been characterized in this population but is considered for symptomatic patients with severe LV dysfunction (SLVD) receiving guideline-directed medical therapy (GDMT). We evaluated ICD utilization and efficacy in patients with DMD. Retrospective cohort study of DMD patients from 17 centers across North America between January 2, 2005 and December 31, 2015. ICD use and its effect on survival were evaluated in patients with SLVD defined as ejection fraction (EF) < 35% and/ or shortening fraction (SF) < 16% on final echocardiogram. SLVD was present in 57/436 (13.1%) patients, of which 12 (21.1%) died during the study period. Of these 12, (mean EF 20.9 ± 6.2% and SF 13.7 ± 7.2%), 8 received GDMT, 5 received steroids, and none received an ICD. ICDs were placed in 9/57 (15.8%) patients with SLVD (mean EF 31.2 ± 8.5% and SF 10.3 ± 4.9%) at a mean age of 20.4 ± 6.3 years; 8/9 received GDMT, 7 received steroids, and all were alive at study end; mean ICD duration was 36.1 ± 26.2 months. Nine ICDs were implanted at six different institutions, associated with two appropriate shocks for ventricular tachycardia in two patients, no inappropriate shocks, and one lead fracture. ICD use may be associated with improved survival and minimal complications in DMD cardiomyopathy with SLVD. However, inconsistent GDMT utilization may be a significant confounder. Future studies should define optimal indications for ICD implantation in patients with DMD cardiomyopathy.

As survival and neuromuscular function in Duchenne muscular dystrophy (DMD) have improved with glucocorticoid (GC) therapy and ventilatory support, cardiac deaths are increasing. Little is known about risk factors for cardiac and non-cardiac causes of death in DMD. A multi-center retrospective cohort study of 408 males with DMD, followed from January 1, 2005 to December 31, 2015, was conducted to identify risk factors for death. Those dying of cardiac causes were compared to those dying of non-cardiac causes and to those alive at study end. There were 29 (7.1%) deaths at a median age of 19.5 (IQR: 16.9-24.6) years; 8 (27.6%) cardiac, and 21 non-cardiac. Those living were younger [14.9 (IQR: 11.0-19.1) years] than those dying of cardiac [18 (IQR 15.5-24) years, p = 0.03] and non-cardiac [19 (IQR: 16.5-23) years, p = 0.002] causes. GC use was lower for those dying of cardiac causes compared to those living [2/8 (25%) vs. 304/378 (80.4%), p = 0.001]. Last ejection fraction prior to death/study end was lower for those dying of cardiac causes compared to those living (37.5% ± 12.8 vs. 54.5% ± 10.8, p = 0.01) but not compared to those dying of non-cardiac causes (37.5% ± 12.8 vs. 41.2% ± 19.3, p = 0.58). In a large DMD cohort, approximately 30% of deaths were cardiac. Lack of GC use was associated with cardiac causes of death, while systolic dysfunction was associated with death from any cause. Further work is needed to ensure guideline adherence and to define optimal management of systolic dysfunction in males with DMD with hopes of extending survival.

SET and MYND domain-containing protein 1 (SMYD1) has been shown to be responsible for the development of fast twitch and cardiac muscle. Mutations in SMYD1 have been shown to be uniformly fatal in laboratory studies, and not previously described in living humans. We describe here the care of an infant suffering from cardiac failure due to an SMYD1 mutation requiring biventricular assist devices as a bridge to successful heart transplantation. The patient is now doing well 2 years post-transplant and represents a known survivor of a suspected uniformly fatal genetic mutation.

Objectives: To assess the variation in timing of left atrial decompression and its association with clinical outcomes in pediatric patients supported with venoarterial extracorporeal membrane oxygenation across a multicenter cohort., Design: Multicenter retrospective study., Setting: Eleven pediatric hospitals within the United States., Patients: Patients less than 18 years on venoarterial extracorporeal membrane oxygenation who underwent left atrial decompression from 2004 to 2016., Interventions: None., Measurements and Main Results: A total of 137 patients (median age, 4.7 yr) were included. Cardiomyopathy was the most common diagnosis (47%). Cardiac arrest (39%) and low cardiac output (50%) were the most common extracorporeal membrane oxygenation indications. Median time to left atrial decompression was 6.2 hours (interquartile range, 3.8-17.2 hr) with the optimal cut-point of greater than or equal to 18 hours for late decompression determined by receiver operating characteristic curve. In univariate analysis, late decompression was associated with longer extracorporeal membrane oxygenation duration (median 8.5 vs 5 d; p = 0.02). In multivariable analysis taking into account clinical confounder and center effects, late decompression remained significantly associated with prolonged extracorporeal membrane oxygenation duration (adjusted odds ratio, 4.4; p = 0.002). Late decompression was also associated with longer duration of mechanical ventilation (adjusted odds ratio, 4.8; p = 0.002). Timing of decompression was not associated with in-hospital survival (p = 0.36) or overall survival (p = 0.42) with median follow-up of 3.2 years., Conclusions: In this multicenter study of pediatric patients receiving venoarterial extracorporeal membrane oxygenation, late left atrial decompression (≥ 18 hr) was associated with longer duration of extracorporeal membrane oxygenation support and mechanical ventilation. Although no survival benefit was demonstrated, the known morbidities associated with prolonged extracorporeal membrane oxygenation use may justify a recommendation for early left atrial decompression.

Background: As survival and neuromuscular function in Duchenne Muscular Dystrophy (DMD) improve with glucocorticoid therapy and respiratory advances, the proportion of cardiac deaths is increasing. Little is known about the use and outcomes of advanced heart failure (HF) therapies in this population., Methods: A retrospective cohort study of 436 males with DMD was performed, from January 1, 2005-January 1, 2018, with the primary outcome being use of advanced HF therapies including: implantable cardioverter defibrillator (ICD), left ventricular assist device (LVAD), and heart transplantation (HTX)., Results: Nine subjects had an ICD placed, 2 of whom (22.2%) had appropriate shocks for ventricular tachycardia; 1 and 968 days after implant, and all of whom were alive at last follow-up; median 18 (IQR: 12.5-25.5) months from implant. Four subjects had a LVAD implanted with post-LVAD survival of 75% at 1 year; 2 remaining on support and 1 undergoing HTX. One subject was bridged to HTX with ICD and LVAD and was alive at last follow-up, 53 months after HTX., Conclusion: Advanced HF therapies may be used effectively in select subjects with DMD. Further studies are needed to better understand risk stratification for ICD use and optimal candidacy for LVAD implantation and HTX, with hopes of improving cardiac outcomes., Competing Interests: Declaration of interest Carol A. Wittlieb-Weber, MD: None. Chet R. Villa, MD: None. Matthew J. Bock, MD: None. Katheryn E. Gambetta, MD: None. Jonathan N. Johnson, MD: None. Ashwin K. Lal, MD: None. Kurt R. Schumacher, MD: None. Sabrina P. Law, MD: None. Shriprasad R. Deshpande, MD, MS: None. Shawn C. West, MD, MSc: None. Joshua M. Friedland-Little, MD: None. Irene D. Lytrivi, MD: None. Michael A. McCulloch, MD: None. Ryan J. Butts, MD: None. David R. Weber, MD, MSCE: Consulted for Marathon Pharmaceuticals. Kenneth R. Knecht, MD: None.

Post-transplant lymphoproliferative disorder (PTLD) is a significant complication after pediatric heart transplantation (HT), occurring in 5%-15% of patients within 3 years. Data >3 years from HT are limited. We sought to describe the prevalence, risk factors, and outcomes of PTLD occurring late (>3 years) after pediatric HT in the Pediatric Heart Transplant Study from 1993 to 2010. Among 3844 primary HT patients, 110 (3%) developed late, nonrecurrent PTLD. The hazard rate for late PTLD was constant at 0.01 events/year out to 20 years after HT. Risk factors for late PTLD were younger age at HT (HR 1.06, P = 0.003) and Epstein-Barr virus (EBV) naivety (HR 1.65, P = 0.02). Survival after late PTLD was 86% and 68% at 1 and 5 years, with nonwhite race (HR 2.27, P = 0.03) and earlier year of HT (HR 1.03, P = 0.04) independently associated with mortality. Acute rejection and infection were both common after late PTLD, occurring in 26% and 34% of patients. The constant late hazard and contribution of EBV to late PTLD suggest that vigilance for development of PTLD, including for EBV conversion, should persist indefinitely after pediatric HT. The reasons for elevated risk of death for nonwhites after late PTLD are unclear and warrant further investigation., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Mimics should not exist without their models, yet often they do. In the system involving queen and viceroy butterflies, the viceroy is both mimic and co-model depending on the local abundance of the model, the queen. Here, we integrate population surveys, chemical analyses, and predator behavior assays to demonstrate how mimics may persist in locations with low-model abundance. As the queen becomes less locally abundant, the viceroy becomes more chemically defended and unpalatable to predators. However, the observed changes in viceroy chemical defense and palatability are not attributable to differing host plant chemical defense profiles. Our results suggest that mimetic viceroy populations are maintained at localities of low-model abundance through an increase in their toxicity. Sharing the burden of predator education in some places but not others may also lower the fitness cost of warning signals thereby supporting the origin and maintenance of aposematism., Competing Interests: The authors declare no competing interests.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature monocytes and granulocytes. While neutrophils (polymorphonuclear leukocytes [PMNs]) are classically identified as highly differentiated cells specialized for antimicrobial defense, our laboratory has reported minor contributions of PMNs to the immune response during Staphylococcus aureus biofilm infection. However, these two cell types can be difficult to differentiate because of shared surface marker expression. Here we describe a more refined approach to distinguish MDSCs from PMNs utilizing the integrin receptor CD11b combined with conventional Ly6G and Ly6C expression. This approach separated the Ly6G + Ly6C + population that we previously identified in a mouse model of S. aureus orthopedic implant infection into two subsets, namely, CD11b high Ly6G + Ly6C + MDSCs and CD11b low Ly6G + Ly6C + PMNs, which was confirmed by characteristic nuclear morphology using cytospins. CD11b high Ly6G + Ly6C + MDSCs suppressed T cell proliferation throughout the 28-day infection period, whereas CD11b low Ly6G + Ly6C + PMNs had no effect early (day 3 postinfection), although this population acquired suppressive activity at later stages of biofilm development. To further highlight the distinctions between biofilm-associated MDSCs and PMNs versus monocytes, transcriptional profiles were compared by transcriptome sequencing (RNA-Seq). A total of 6,466 genes were significantly differentially expressed in MDSCs versus monocytes, whereas only 297 genes were significantly different between MDSCs and PMNs. A number of genes implicated in cell cycle regulation were identified, and in vivo ethynyldeoxyuridine (EdU) labeling revealed that approximately 50% of MDSCs proliferated locally at the site of S. aureus biofilm infection. Based on their similar transcriptomic profiles to those of PMNs, biofilm-associated MDSCs are of a granulocytic lineage and can be classified as granulocytic MDSCs (G-MDSCs)., (Copyright © 2018 American Society for Microbiology.)

Objective: Our institution uses a valved polytetrafluoroethylene conduit as an alternative to homografts. The objective of this study was to investigate the performance of bicuspid valved polytetrafluoroethylene conduits used for right ventricular outflow tract reconstruction in children aged less than 2 years and to evaluate risk factors for earlier conduit explant., Methods: We performed an Institutional Review Board-approved retrospective chart review of all patients aged less than 2 years who underwent surgical right ventricular outflow tract reconstruction with a bicuspid valved polytetrafluoroethylene conduit or homograft conduit from July 2004 to December 2014. The end points of the study were defined as conduit explant, conduit explant or reintervention, conduit stenosis, and conduit insufficiency., Results: Fifty-four patients underwent 65 right ventricular outflow tract reconstructions with a bicuspid valved polytetrafluoroethylene conduit (n = 39) or a homograft conduit (n = 26, 23 pulmonary, 3 aortic). The majority of diagnoses were truncus arteriosus (n = 28) and tetralogy of Fallot with pulmonary atresia (n = 19). Median age of patients at surgery was 134 (8-323) days and 128 (7-384) days in the PTFE and homograft groups, respectively. There was no difference in demographic data between the 2 groups. Time-to-event analysis demonstrated no difference in time to explant (P = .474) or time to explant or reintervention (P = .206) between the 2 conduit types. Younger age at surgery was the only independent risk factor for conduit explant (subdistribution hazard ratio 1.104 per 30 days younger, P < .001). There was no significant influence of conduit type on the development of moderate conduit stenosis (P = .931) or severe conduit insufficiency (P = .880). Larger conduit z score was protective for the development of moderate conduit stenosis (subdistribution hazard ratio, 0.46; P = .001)., Conclusions: Bicuspid valved polytetrafluoroethylene conduits are a satisfactory choice for right ventricular outflow tract reconstruction in patients aged less than 2 years. Their availability, low cost, and lack of potential sensitization make them an appealing alternative to homograft conduits., (Copyright © 2018 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.)

There is growing acceptance of transplantation across a positive crossmatch for highly allosensitized pediatric HT candidates. While survival may be similar to patients transplanted across a negative crossmatch, costs are unknown. Among 60 HT recipients at our center from 5/07 to 6/12, we analyzed hospital charges and length of stay from the day of HT to discharge and through the first year after transplant. Median age at HT was 6.2 years (15 days-20.5 years). Charges in the first year post-HT were greater for crossmatch-positive patients ($907 678 vs $549 754; P = .017), with a trend toward higher charges for the HT hospitalization ($537 640 vs $407 374; P = .07). Plasmapheresis was more common in crossmatch-positive patients during the HT hospitalization (80% vs 4%, P < .001). In the first year after HT, crossmatch-positive patients had a greater number of endomyocardial biopsies (10 vs 7.5, P = .03) and episodes of treated rejection (2 vs 0, P = .004). Pediatric HT across a positive crossmatch is associated with higher first-year costs, including increased use of plasmapheresis and care around an increased number of rejections. These novel data will help inform decision and policymaking regarding care practices for the growing population of highly sensitized pediatric HT candidates., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)