Comprehensive Analysis of Hypermutation in Human Cancer.

We present an extensive assessment of mutation burden through sequencing analysis of >81,000 tumors from pediatric and adult patients, including tumors with hypermutation caused by chemotherapy, carcinogens, or germline alterations. Hypermutation was detected in tumor types not previously associated with high mutation burden. Replication repair deficiency was a major contributing factor. We uncovered new driver mutations in the replication-repair-associated DNA polymerases and a distinct impact of microsatellite instability and replication repair deficiency on the scale of mutation load. Unbiased clustering, based on mutational context, revealed clinically relevant subgroups regardless of the tumors' tissue of origin, highlighting similarities in evolutionary dynamics leading to hypermutation. Mutagens, such as UV light, were implicated in unexpected cancers, including sarcomas and lung tumors. The order of mutational signatures identified previous treatment and germline replication repair deficiency, which improved management of patients and families. These data will inform tumor classification, genetic testing, and clinical trial design.

A Sox2 distal enhancer cluster regulates embryonic stem cell differentiation potential.

The Sox2 transcription factor must be robustly transcribed in embryonic stem (ES) cells to maintain pluripotency. Two gene-proximal enhancers, Sox2 regulatory region 1 (SRR1) and SRR2, display activity in reporter assays, but deleting SRR1 has no effect on pluripotency. We identified and functionally validated the sequences required for Sox2 transcription based on a computational model that predicted transcriptional enhancer elements within 130 kb of Sox2. Our reporter assays revealed three novel enhancers--SRR18, SRR107, and SRR111--that, through the formation of chromatin loops, form a chromatin complex with the Sox2 promoter in ES cells. Using the CRISPR/Cas9 system and F1 ES cells (Mus musculus(129) × Mus castaneus), we generated heterozygous deletions of each enhancer region, revealing that only the distal cluster containing SRR107 and SRR111, located >100 kb downstream from Sox2, is required for cis-regulation of Sox2 in ES cells. Furthermore, homozygous deletion of this distal Sox2 control region (SCR) caused significant reduction in Sox2 mRNA and protein levels, loss of ES cell colony morphology, genome-wide changes in gene expression, and impaired neuroectodermal formation upon spontaneous differentiation to embryoid bodies. Together, these data identify a distal control region essential for Sox2 transcription in ES cells.

Inhibition of the ULK1 protein complex suppresses Staphylococcus-induced autophagy and cell death.

Autophagy plays multiple roles in host cells challenged with extracellular pathogens. Here, we aimed to explore whether autophagy inhibition could prevent bacterial infections. We first confirmed widely distinct patterns of autophagy responses in host cells infected with Staphylococcus aureus, as compared with Salmonella Only infection with Staphylococcus produced strong accumulation of lipidated autophagy-related protein LC3B (LC3B-II). Infection with virulent Staphylococcus strains induced formation of p62-positive aggregates, suggestive of accumulated ubiquitinated targets. During Salmonella infection, bacteria remain enclosed by lysosomal-associated membrane protein 2 (LAMP2)-positive lysosomes, whereas virulent Staphylococcus apparently exited from enlarged lysosomes and invaded the cytoplasm. Surprisingly, Staphylococcus appeared to escape from the lysosome without generation of membrane-damage signals as detected by galectin-3 recruitment. In contrast, Salmonella infection produced high levels of lysosomal damage, consistent with a downstream antibacterial xenophagy response. Finally, we studied the Unc-51-like autophagy-activating kinase 1 (ULK1) regulatory complex, including the essential subunit autophagy-related protein 13 (ATG13). Infection of cells with either Staphylococcus or Salmonella led to recruitment of ATG13 to sites of cytosolic bacterial cells to promote autophagosome formation. Of note, genetic targeting of ATG13 suppressed autophagy and the ability of Staphylococcus to infect and kill host cells. Two different ULK1 inhibitors also prevented Staphylococcus intracellular replication and host cell death. Interestingly, inhibition of the ULK1 pathway had the opposite effect on Salmonella, sensitizing cells to the infection. Our results suggest that ULK1 inhibitors may offer a potential strategy to impede cellular infection by S. aureus.

Enhancers and super-enhancers have an equivalent regulatory role in embryonic stem cells through regulation of single or multiple genes.

Transcriptional enhancers are critical for maintaining cell-type-specific gene expression and driving cell fate changes during development. Highly transcribed genes are often associated with a cluster of individual enhancers such as those found in locus control regions. Recently, these have been termed stretch enhancers or super-enhancers, which have been predicted to regulate critical cell identity genes. We employed a CRISPR/Cas9-mediated deletion approach to study the function of several enhancer clusters (ECs) and isolated enhancers in mouse embryonic stem (ES) cells. Our results reveal that the effect of deleting ECs, also classified as ES cell super-enhancers, is highly variable, resulting in target gene expression reductions ranging from 12% to as much as 92%. Partial deletions of these ECs which removed only one enhancer or a subcluster of enhancers revealed partially redundant control of the regulated gene by multiple enhancers within the larger cluster. Many highly transcribed genes in ES cells are not associated with a super-enhancer; furthermore, super-enhancer predictions ignore 81% of the potentially active regulatory elements predicted by cobinding of five or more pluripotency-associated transcription factors. Deletion of these additional enhancer regions revealed their robust regulatory role in gene transcription. In addition, select super-enhancers and enhancers were identified that regulated clusters of paralogous genes. We conclude that, whereas robust transcriptional output can be achieved by an isolated enhancer, clusters of enhancers acting on a common target gene act in a partially redundant manner to fine tune transcriptional output of their target genes.

The dark side of energy transport along excitonic wires: On-site energy barriers facilitate efficient, vibrationally mediated transport through optically dark subspaces.

We present a novel, counter-intuitive method, based on dark-state protection, for significantly improving exciton transport efficiency through "wires" comprising a chain of molecular sites with an intrinsic energy gradient. Specifically, by introducing "barriers" to the energy landscape at regular intervals along the transport path, we find that undesirable radiative recombination processes are suppressed due to a clear separation of sub-radiant and super-radiant eigenstates in the system. This, in turn, can lead to an improvement in transmitted power by many orders of magnitude, even for very long chains. From there, we analyze the robustness of this phenomenon to changes in both system and environment properties to show that this effect can be beneficial over a range of different thermal and optical environment regimes. Finally, we show that the novel energy landscape presented here may provide a useful foundation for overcoming the short length scales over which exciton diffusion typically occurs in organic photo-voltaics and other nanoscale transport scenarios, thus leading to considerable potential improvements in the efficiency of such devices.

MYCN Amplified Relapse Following Resolution of MYCN Nonamplified 4S Neuroblastoma With Placental Involvement: A Case Report and Review of the Literature.

Congenital neuroblastoma with placental involvement is exceptionally rare, but mortality is high. Detailed examination of placenta including MYCN amplification and segmental chromosomal aberrations should be performed in all suspected cases, as it is noninvasive and readily available. Maternal dissemination has not been reported. In this manuscript, we describe an infant with placental diagnosis of MYCN nonamplified congenital neuroblastoma. This is the first report of a recurrence of congenital 4S neuroblastoma following resolution in which MYCN amplification is only detected in the recurrence. Germline sequencing using a large comprehensive cancer panel did not reveal variants in candidate cancer predisposition genes.

Practical considerations for establishing and maintaining a magnetic resonance imaging safety program in a pediatric practice.

Magnetic resonance imaging is a multipurpose imaging modality that is largely safe, given the lack of ionizing radiation. However there are electromagnetic and biological effects on human tissue when exposed to magnetic environments, and hence there is a risk of adverse events occurring with these exams. It is imperative to understand these risks and develop methods to minimize them and prevent consequent adverse events. Implementing these safety practices in pediatric MR imaging has been somewhat limited because of gaps in information and knowledge among the personnel who are closely involved in the MR environment. The American College of Radiology has provided guidelines on MR safety practices that are helpful in minimizing such adverse events. This article provides an overview of the issues related to MR safety and practical ways to implement them across different health care facilities.

Changes in Therapeutic Intensity Level Following Airway Pressure Release Ventilation in Severe Traumatic Brain Injury.

PURPOSE: Airway pressure release ventilation (APRV) utilizes high levels of airway pressure coupled with brief expiratory release to facilitate open lung ventilation. The aim of our study was to evaluate the effects of APRV-induced elevated airway pressure mean in patients with severe traumatic brain injury. MATERIALS AND METHODS: This was a retrospective cohort study at a 424-bed Level I trauma center. Linear mixed effects models were developed to assess the difference in therapeutic intensity level (TIL), intracranial pressure (ICP), and cerebral perfusion pressure (CPP) over time following the application of APRV. RESULTS: The study included 21 epochs of APRV in 21 patients. In the 6-hour epoch following the application of APRV, the TIL was significantly increased ( P = .002) and the ICP significantly decreased ( P = .041) compared to that before 6 hours. There was no significant change in CPP ( P = .42) over time. The baseline static compliance and time interaction was not significant for TIL (χ2 = 0.2 [ df 1], P = .655), CPP (χ2 = 0 [ df 1], P = 1), or ICP (χ2 = 0.1 [ df 1], P = .752). CONCLUSIONS: Application of APRV in patients with severe traumatic brain injury was associated with significantly, but not clinically meaningful, increased TIL and decreased ICP. No significant change in CPP was observed. No difference was observed based on the baseline pulmonary static compliance.

Aggressive embryonal rhabdomyosarcoma in a 3-month-old boy: A clinical and molecular analysis.

Rhabdomyosarcoma (RMS) represents the most common soft tissue sarcoma in the pediatric age group. While RMS has been traditionally classified on the basis of its histological appearance (with embryonal and alveolar being most common), it is now clear that the PAX-FOXO1 fusion product drives prognosis. We report here a case of pelvic embryonal RMS in a 3-month-old male who was subsequently found to have developed brain metastases during the course of chemotherapy. Cytogenetic analysis of the brain metastases at the time of autopsy as well as next-generation sequencing analysis revealed a reciprocal translocation involving the SH3 domain containing ring finger 3 gene (SH3RF3, on chromosome 2q13) and the Lipase C gene (LIPC, on chromosome 15q21.3). Due to the poor quality of the pretreatment and postresection samples, cytogenetics and NGS analysis looking for the presence of this balanced translocation in these specimens could not be performed. To the authors' knowledge, this translocation has never been described in RMS. Further studies are needed to determine the biological and clinical implications of this novel translocation.

Ten Years of Equine-related Injuries: Severity and Implications for Emergency Physicians.

BACKGROUND: The size, speed, and unpredictable nature of horses present a significant risk for injury in all equine-related activities. OBJECTIVE: We sought to examine the mechanism, severity, frequency, body regions affected, surgical requirements, rehabilitation needs, safety equipment utilization, and outcomes of equine-related injured patients. METHODS: Records of inpatients who sustained an equine-related injury from 2002-2011 with International Classification of Diseases, Ninth Revision codes E828 and E906 were retrospectively reviewed for pertinent data. RESULTS: Ninety patients, 70% female, age (mean ± SD) 37.3 ± 19.4 years, length of stay 3.7 ± 4.5 days, Injury Severity Score 12.9 ± 8.4. Predominant mechanism of injury was fall from horse (46.7%). The chest (23%) was most frequently injured, followed by brain/head (21.5%). Thirty patients (33%) required 57 surgical procedures. Twenty percent of patients required occupational therapy and 33.3% required physical therapy while hospitalized. Only 3% required rehabilitation, with 90% discharged directly home. Safety equipment was not used in 91.9% of patients. One patient sustained a cord injury. Six patients expired, all from extensive head injuries. CONCLUSION: The majority of equine-related injuries occur while pursuing recreational activities and are due to falls. Our patients experienced more severe injuries to the trunk and head and required more surgical intervention for pelvic, facial, and brain injuries than previously reported. Failure to use safety equipment contributes to the risk of severe injury. Education and injury prevention is essential. The need for complex surgical intervention by multiple specialties supports transfer to Level I trauma centers.

Chaotic induced-charge electro-osmosis.

We present direct numerical simulations of the coupled Poisson-Nernst-Planck and Navier-Stokes equations for an electrolyte around a polarizable cylinder subject to an external electric field. For high fields, a novel chaotic flow phenomenon is discovered. Our calculations indicate significant improvement in the prediction of the mean flow relative to standard asymptotic models. These results open possibilities for chaos-enhanced mixing in microdevices and provide insight into barriers to efficient electrokinetic micropumps with broad applications in electrochemical and lab-on-a-chip systems.

Spontaneous expression of the CIC::DUX4 fusion oncoprotein from a conditional allele potently drives sarcoma formation in genetically engineered mice.

CIC::DUX4 sarcoma (CDS) is a rare but highly aggressive undifferentiated small round cell sarcoma driven by a fusion between the tumor suppressor Capicua (CIC) and DUX4. Currently, there are no effective treatments and efforts to identify and translate better therapies are limited by the scarcity of patient tumor samples and cell lines. To address this limitation, we generated three genetically engineered mouse models of CDS (Ch7CDS, Ai9CDS, and TOPCDS). Remarkably, chimeric mice from all three conditional models developed spontaneous soft tissue tumors and disseminated disease in the absence of Cre-recombinase. The penetrance of spontaneous (Cre-independent) tumor formation was complete irrespective of bi-allelic Cic function and the distance between adjacent loxP sites. Characterization of soft tissue and presumed metastatic tumors showed that they consistently expressed the CIC::DUX4 fusion protein and many downstream markers of the disease credentialing the models as CDS. In addition, tumor-derived cell lines were generated and ChIP-seq was preformed to map fusion-gene specific binding using an N-terminal HA epitope tag. These datasets, along with paired H3K27ac ChIP-sequencing maps, validate CIC::DUX4 as a neomorphic transcriptional activator. Moreover, they are consistent with a model where ETS family transcription factors are cooperative and redundant drivers of the core regulatory circuitry in CDS.

Allele-specific differences in activity of a novel cannabinoid receptor 1 (CNR1) gene intronic enhancer in hypothalamus, dorsal root ganglia, and hippocampus.

Polymorphisms within intron 2 of the CNR1 gene, which encodes cannabinoid receptor 1 (CB(1)), have been associated with addiction, obesity, and brain volume deficits. We used comparative genomics to identify a polymorphic (rs9444584-C/T) sequence (ECR1) in intron 2 of the CNR1 gene that had been conserved for 310 million years. The C-allele of ECR1 (ECR1(C)) acted as an enhancer in hypothalamic and dorsal root ganglia cells and responded to MAPK activation through the MEKK pathway but not in hippocampal cells. However, ECR1(T) was significantly more active in hypothalamic and dorsal root ganglia cells but, significantly, and in contrast to ECR1(C), was highly active in hippocampal cells where it also responded strongly to activation of MAPK. Intriguingly, rs9444584 is in strong linkage disequilibrium with two other SNPs (rs9450898 (r(2) = 0.841) and rs2023239 (r(2) = 0.920)) that have been associated with addiction, obesity (rs2023239), and reduced fronto-temporal white matter volumes in schizophrenia patients as a result of cannabis misuse (rs9450898). Considering their high linkage disequilibrium and the increased response of ECR1(T) to MAPK signaling when compared with ECR1(C), it is possible that the functional effects of the different alleles of rs9444584 may play a role in the conditions associated with rs9450898 and rs2023239. Further analysis of the different alleles of ECR1 may lead to a greater understanding of the role of CNR1 gene misregulation in these conditions as well as chronic inflammatory pain.

Nuclear organization of RNA polymerase II transcription.

Transcription occurs at distinct nuclear compartments termed transcription factories that are specialized for transcription by 1 of the 3 polymerase complexes (I, II, or III). Protein-coding genes appear to move in and out of RNA polymerase II (RNAPII) compartments as they are expressed and silenced. In addition, transcription factories are sites where several transcription units, either from the same chromosome or different chromosomes, are transcribed. Chromosomes occupy distinct territories in the interphase nucleus with active genes preferentially positioned on the periphery or even looped out of the territory. These chromosome territories have been observed to intermingle in the nucleus, and multiple interactions among different chromosomes have been identified in genome-wide studies. Deep sequencing of the transcriptome and RNAPII associated on DNA obtained by chromatin immunoprecipitation have revealed a plethora of noncoding transcription and intergenic accumulations of RNAPII that must also be considered in models of genome function. The organization of transcription into distinct regions of the nucleus has changed the way we view transcription with the evolving model for silencing or activation of gene expression involving physical relocation of the transcription unit to a silencing or activation compartment, thus, highlighting the need to consider the process of transcription in the 3-dimensional nuclear space.

Development of a modified head and neck quality assurance phantom for use in stereotactic radiosurgery trials.

An anthropomorphic head phantom, constructed from a water-equivalent plastic shell with only a spherical target, was modified to include a nonspherical target (pituitary) and an adjacent organ at risk (OAR) (optic chiasm), within 2 mm, simulating the anatomy encountered when treating acromegaly. The target and OAR spatial proximity provided a more realistic treatment planning and dose delivery exercise. A separate dosimetry insert contained two TLD for absolute dosimetry and radiochromic film, in the sagittal and coronal planes, for relative dosimetry. The prescription was 25 Gy to 90% of the GTV, with ≤ 10% of the OAR volume receiving ≥ 8 Gy for the phantom trial. The modified phantom was used to test the rigor of the treatment planning process and phantom reproducibility using a Gamma Knife, CyberKnife, and linear accelerator (linac)-based radiosurgery system. Delivery reproducibility was tested by repeating each irradiation three times. TLD results from three irradiations on a CyberKnife and Gamma Knife agreed with the calculated target dose to within ± 4% with a maximum coefficient of variation of ± 2.1%. Gamma analysis in the coronal and sagittal film planes showed an average passing rate of 99.4% and 99.5% using ± 5%/3 mm criteria, respectively. Results from the linac irradiation were within ± 6.2% for TLD with a coefficient of variation of ± 0.1%. Distance to agreement was calculated to be 1.2 mm and 1.3mm along the inferior and superior edges of the target in the sagittal film plane, and 1.2 mm for both superior and inferior edges in the coronal film plane. A modified, anatomically realistic SRS phantom was developed that provided a realistic clinical planning and delivery challenge that can be used to credential institutions wanting to participate in NCI-funded clinical trials.

Understanding living with tracheostomy ventilation for motor neuron disease and the implications for quality of life: a qualitative study protocol.

INTRODUCTION: Home mechanical ventilation can be used to manage symptoms of breathlessness and sustain life for people living with motor neuron disease (plwMND). In the UK, less than 1% of plwMND use tracheostomy ventilation (TV). This contrasts with some other countries, where rates are much higher. Due to a lack of evidence about its feasibility, cost-effectiveness or outcomes, TV is not covered in the UK National Institute for Health and Care Excellence guidance. Most plwMND receiving TV in the UK do so as an unplanned crisis intervention, which can lead to a prolonged hospital stay while a complex care package is arranged. There is insufficient literature addressing the burdens and benefits of TV, how it should be initiated and delivered, and how future care choices for plwMND can be supported. The aim of this research is to provide new understandings of the experiences of plwMND using TV, and those of family members and healthcare professionals (HCPs) involved in their care. METHODS AND ANALYSIS: A UK-wide qualitative study with two workstreams: (1) Patient focused case studies (n=6) including plwMND, family members and HCPs to focus on experiences and tasks of daily living from multiple perspectives. (2) Interviews with plwMND (n=10), family members, including bereaved family members (n=10) and HCPs (n=20) on broader experiences and issues relating to use of TV, such as ethical considerations and decision making. ETHICS AND DISSEMINATION: Ethical approval has been granted by the Leicester South Research Ethics Committee (22/EM/0256). All participants will be asked to provide electronic, written and/or audio recorded informed consent. Study findings will be disseminated in peer-reviewed journals and conference presentations and used to develop new resources for teaching and public information.

Automatic Refractive Error Estimation Using Deep Learning-Based Analysis of Red Reflex Images.

Purpose/Background: We evaluate how a deep learning model can be applied to extract refractive error metrics from pupillary red reflex images taken by a low-cost handheld fundus camera. This could potentially provide a rapid and economical vision-screening method, allowing for early intervention to prevent myopic progression and reduce the socioeconomic burden associated with vision impairment in the later stages of life. Methods: Infrared and color images of pupillary crescents were extracted from eccentric photorefraction images of participants from Choithram Hospital in India and Dargaville Medical Center in New Zealand. The pre-processed images were then used to train different convolutional neural networks to predict refractive error in terms of spherical power and cylindrical power metrics. Results: The best-performing trained model achieved an overall accuracy of 75% for predicting spherical power using infrared images and a multiclass classifier. Conclusions: Even though the model's performance is not superior, the proposed method showed good usability of using red reflex images in estimating refractive error. Such an approach has never been experimented with before and can help guide researchers, especially when the future of eye care is moving towards highly portable and smartphone-based devices.

Myeloproliferative Neoplasm Driven by ETV6-ABL1 in an Adolescent with Recent History of Burkitt Leukemia.

ETV6-ABL1 gene fusion is a rare genetic rearrangement in a variety of malignancies, including myeloproliferative neoplasms (MPN), acute lymphoblastic leukemia (ALL), and acute myeloid leukemia (AML). Here, we report the case of a 16-year-old male diagnosed with a MPN, 7 months post-completion of treatment for Burkitt leukaemia. RNA sequencing analysis confirmed the presence of an ETV6-ABL1 fusion transcript, with an intact, in-frame ABL tyrosine-kinase domain. Of note, secondary ETV6-ABL1-rearranged neoplastic diseases have not been reported to date. The patient was started on a tyrosine kinase inhibitor (TKI; imatinib) and, subsequently, underwent a 10/10 matched unrelated haematopoietic stem cell transplant. He is disease-free five years post-transplant. Definitive evidence of the prognostic influence of the ETV6-ABL1 fusion in haematological neoplasms is lacking; however, overall data suggest that it is a poor prognostic factor, particularly in patients with ALL and AML. The presence of this ETV6-ABL1 fusion should be more routinely investigated, especially in patients with a CML-like picture. More routine use of whole-genome and RNA sequencing analyses in clinical diagnostic care, in conjunction with conventional cytogenetics, will facilitate these investigations.