Characterizing Mutational Signatures in Human Cancer Cell Lines Reveals Episodic APOBEC Mutagenesis.

Multiple signatures of somatic mutations have been identified in cancer genomes. Exome sequences of 1,001 human cancer cell lines and 577 xenografts revealed most common mutational signatures, indicating past activity of the underlying processes, usually in appropriate cancer types. To investigate ongoing patterns of mutational-signature generation, cell lines were cultured for extended periods and subsequently DNA sequenced. Signatures of discontinued exposures, including tobacco smoke and ultraviolet light, were not generated in vitro. Signatures of normal and defective DNA repair and replication continued to be generated at roughly stable mutation rates. Signatures of APOBEC cytidine deaminase DNA-editing exhibited substantial fluctuations in mutation rate over time with episodic bursts of mutations. The initiating factors for the bursts are unclear, although retrotransposon mobilization may contribute. The examined cell lines constitute a resource of live experimental models of mutational processes, which potentially retain patterns of activity and regulation operative in primary human cancers.

The mutational landscape of human somatic and germline cells.

Over the course of an individual's lifetime, normal human cells accumulate mutations1. Here we compare the mutational landscape in 29 cell types from the soma and germline using multiple samples from the same individuals. Two ubiquitous mutational signatures, SBS1 and SBS5/40, accounted for the majority of acquired mutations in most cell types, but their absolute and relative contributions varied substantially. SBS18, which potentially reflects oxidative damage2, and several additional signatures attributed to exogenous and endogenous exposures contributed mutations to subsets of cell types. The rate of mutation was lowest in spermatogonia, the stem cells from which sperm are generated and from which most genetic variation in the human population is thought to originate. This was due to low rates of ubiquitous mutational processes and may be partially attributable to a low rate of cell division in basal spermatogonia. These results highlight similarities and differences in the maintenance of the germline and soma.

The landscape of somatic mutation in normal colorectal epithelial cells.

The colorectal adenoma-carcinoma sequence has provided a paradigmatic framework for understanding the successive somatic genetic changes and consequent clonal expansions that lead to cancer1. However, our understanding of the earliest phases of colorectal neoplastic changes-which may occur in morphologically normal tissue-is comparatively limited, as for most cancer types. Here we use whole-genome sequencing to analyse hundreds of normal crypts from 42 individuals. Signatures of multiple mutational processes were revealed; some of these were ubiquitous and continuous, whereas others were only found in some individuals, in some crypts or during certain periods of life. Probable driver mutations were present in around 1% of normal colorectal crypts in middle-aged individuals, indicating that adenomas and carcinomas are rare outcomes of a pervasive process of neoplastic change across morphologically normal colorectal epithelium. Colorectal cancers exhibit substantially increased mutational burdens relative to normal cells. Sequencing normal colorectal cells provides quantitative insights into the genomic and clonal evolution of cancer.

Population dynamics of normal human blood inferred from somatic mutations.

Haematopoietic stem cells drive blood production, but their population size and lifetime dynamics have not been quantified directly in humans. Here we identified 129,582 spontaneous, genome-wide somatic mutations in 140 single-cell-derived haematopoietic stem and progenitor colonies from a healthy 59-year-old man and applied population-genetics approaches to reconstruct clonal dynamics. Cell divisions from early embryogenesis were evident in the phylogenetic tree; all blood cells were derived from a common ancestor that preceded gastrulation. The size of the stem cell population grew steadily in early life, reaching a stable plateau by adolescence. We estimate the numbers of haematopoietic stem cells that are actively making white blood cells at any one time to be in the range of 50,000-200,000. We observed adult haematopoietic stem cell clones that generate multilineage outputs, including granulocytes and B lymphocytes. Harnessing naturally occurring mutations to report the clonal architecture of an organ enables the high-resolution reconstruction of somatic cell dynamics in humans.

A multicentre evaluation exploring the impact of an integrated health and social care intervention for the caregivers of ICU survivors.

BACKGROUND: Caregivers and family members of Intensive Care Unit (ICU) survivors can face emotional problems following patient discharge from hospital. We aimed to evaluate the impact of a multi-centre integrated health and social care intervention, on caregiver and family member outcomes. METHODS: This study evaluated the impact of the Intensive Care Syndrome: Promoting Independence and Return to Employment (InS:PIRE) programme across 9 sites in Scotland. InS:PIRE is an integrated health and social care intervention. We compared caregivers who attended this programme with a contemporary control group of ICU caregivers (usual care cohort), who did not attend. RESULTS: The primary outcome was anxiety measured via the Hospital Anxiety and Depression Scale at 12 months post-hospital discharge. Secondary outcome measures included depression, carer strain and clinical insomnia. A total of 170 caregivers had data available at 12 months for inclusion in this study; 81 caregivers attended the InS:PIRE intervention and completed outcome measures at 12 months post-hospital discharge. In the usual care cohort of caregivers, 89 completed measures. The two cohorts had similar baseline demographics. After adjustment, those caregivers who attended InS:PIRE demonstrated a significant improvement in symptoms of anxiety (OR: 0.42, 95% CI: 0.20-0.89, p = 0.02), carer strain (OR: 0.39; 95% CI: 0.16-0.98 p = 0.04) and clinical insomnia (OR: 0.40; 95% CI: 0.17-0.77 p < 0.001). There was no significant difference in symptoms of depression at 12 months. CONCLUSIONS: This multicentre evaluation has shown that caregivers who attended an integrated health and social care intervention reported improved emotional health and less symptoms of insomnia, 12 months after the delivery of the intervention.

Modeling the voltage loss mechanisms in lithium-sulfur cells: the importance of electrolyte resistance and precipitation kinetics.

Understanding of the complex electrochemical, transport, and phase-change phenomena in Li-S cells requires experimental characterization in tandem with mechanistic modeling. However, existing Li-S models currently contradict some key features of experimental findings, particularly the evolution of cell resistance during discharge. We demonstrate that, by introducing a concentration-dependent electrolyte conductivity, the correct trends in voltage drop due to electrolyte resistance and activation overpotentials are retrieved. In addition, we reveal the existence of an often overlooked potential drop mechanism in the low voltage-plateau which originates from the limited rate of Li2S precipitation.

Epigenetic characterization of the early embryo with a chromatin immunoprecipitation protocol applicable to small cell populations.

Chromatin immunoprecipitation (ChIP) defines the genomic distribution of proteins and their modifications but is limited by the cell numbers required (ideally >10(7)). Here we describe a protocol that uses carrier chromatin and PCR, 'carrier' ChIP (CChIP), to permit analysis of as few as 100 cells. We assayed histone modifications at key regulator genes (such as Nanog, Pou5f1 (also known as Oct4) and Cdx2) by CChIP in mouse embryonic stem (ES) cells and in inner cell mass (ICM) and trophectoderm of cultured blastocysts. Activating and silencing modifications (H4 acetylation and H3K9 methylation) mark active and silent promoters as predicted, and we find close correlation between values derived from CChIP (1,000 ES cells) and conventional ChIP (5 x 10(7) ES cells). Studies on genes silenced in both ICM and ES cells (Cdx2, Cfc1, Hhex and Nkx2-2, also known as Nkx) show that the intensity of silencing marks is relatively diminished in ES cells, indicating a possible relaxation of some components of silencing on adaptation to culture.

Maternal diet and aging alter the epigenetic control of a promoter-enhancer interaction at the Hnf4a gene in rat pancreatic islets.

Environmental factors interact with the genome throughout life to determine gene expression and, consequently, tissue function and disease risk. One such factor that is known to play an important role in determining long-term metabolic health is diet during critical periods of development. Epigenetic regulation of gene expression has been implicated in mediating these programming effects of early diet. The precise epigenetic mechanisms that underlie these effects remain largely unknown. Here, we show that the transcription factor Hnf4a, which has been implicated in the etiology of type 2 diabetes (T2D), is epigenetically regulated by maternal diet and aging in rat islets. Transcriptional activity of Hnf4a in islets is restricted to the distal P2 promoter through its open chromatin configuration and an islet-specific interaction between the P2 promoter and a downstream enhancer. Exposure to suboptimal nutrition during early development leads to epigenetic silencing at the enhancer region, which weakens the P2 promoter-enhancer interaction and results in a permanent reduction in Hnf4a expression. Aging leads to progressive epigenetic silencing of the entire Hnf4a locus in islets, an effect that is more pronounced in rats exposed to a poor maternal diet. Our findings provide evidence for environmentally induced epigenetic changes at the Hnf4a enhancer that alter its interaction with the P2 promoter, and consequently determine T2D risk. We therefore propose that environmentally induced changes in promoter-enhancer interactions represent a fundamental epigenetic mechanism by which nutrition and aging can influence long-term health.

"Can you help me think this through?" How pediatric hospitalists learn from informal peer consultation.

Informal peer consultation (IPC), also called curbside consultation, is a common practice in medicine. Research has shown that physicians use IPC but how this learning occurs during the process has not been studied. This basic qualitative study describes how pediatric hospitalists learn during IPC, framed by Kolb's (2015) Experiential Learning Theory of Growth and Development. Eleven pediatric hospitalists were interviewed. Deidentified transcripts were coded for key themes using inductive methods. The main prompt for informal peer consultation was the perception of uncertainty. Three themes describe the learning process: "Eliciting Perspectives," "Thinking Aloud Together," and "Experiencing Validation. A fourth theme, "Acknowledging Value," described the importance of IPC for modeling how to manage uncertainty with patients' caregivers and medical trainees. By describing the learning process, the results have implications for physicians who engage in IPC and may inform faculty-level professional development initiatives to improve the IPC process.

Recruitment of NCOR1 to VDR target genes is enhanced in prostate cancer cells and associates with altered DNA methylation patterns.

The current study investigated transcriptional distortion in prostate cancer cells using the vitamin D receptor (VDR) as a tool to examine how epigenetic events driven by corepressor binding and CpG methylation lead to aberrant gene expression. These relationships were investigated in the non-malignant RWPE-1 cells that were 1α,25(OH)(2)D(3) responsive (RWPE-1) and malignant cell lines that were 1α,25(OH)(2)D(3) partially responsive (RWPE-2) and resistant (PC-3). These studies revealed that selective attenuation and repression of VDR transcriptional responses in the cancer cell lines reflected their loss of antiproliferative sensitivity. This was evident in VDR target genes including VDR, CDKN1A (encodes p21( (waf1/cip1) )) and GADD45A; NCOR1 knockdown alleviated this malignant transrepression. ChIP assays in RWPE-1 and PC-3 cells revealed that transrepression of CDKN1A was associated with increased NCOR1 enrichment in response to 1α,25(OH)(2)D(3) treatment. These findings supported the concept that retained and increased NCOR1 binding, associated with loss of H3K9ac and increased H3K9me2, may act as a beacon for the initiation and recruitment of DNA methylation. Overexpressed histone methyltransferases (KMTs) were detectable in a wide panel of prostate cancer cell lines compared with RWPE-1 and suggested that generation of H3K9me2 states would be favored. Cotreatment of cells with the KMT inhibitor, chaetocin, increased 1α,25(OH)(2)D(3)-mediated induction of CDKN1A expression supporting a role for this event to disrupt CDKN1A regulation. Parallel surveys in PC-3 cells of CpG methylation around the VDR binding regions on CDKN1A revealed altered basal and VDR-regulated DNA methylation patterns that overlapped with VDR-induced recruitment of NCOR1 and gene transrepression. Taken together, these findings suggest that sustained corepressor interactions with nuclear-resident transcription factors may inappropriately transform transient-repressive histone states into more stable and repressive DNA methylation events.

Ring1B and Suv39h1 delineate distinct chromatin states at bivalent genes during early mouse lineage commitment.

Pluripotent cells develop within the inner cell mass of blastocysts, a mosaic of cells surrounded by an extra-embryonic layer, the trophectoderm. We show that a set of somatic lineage regulators (including Hox, Gata and Sox factors) that carry bivalent chromatin enriched in H3K27me3 and H3K4me2 are selectively targeted by Suv39h1-mediated H3K9me3 and de novo DNA methylation in extra-embryonic versus embryonic (pluripotent) lineages, as assessed both in blastocyst-derived stem cells and in vivo. This stably repressed state is linked with a loss of gene priming for transcription through the exclusion of PRC1 (Ring1B) and RNA polymerase II complexes at bivalent, lineage-inappropriate genes upon trophoblast lineage commitment. Collectively, our results suggest a mutually exclusive role for Ring1B and Suv39h1 in regulating distinct chromatin states at key developmental genes and propose a novel mechanism by which lineage specification can be reinforced during early development.

Epigenetic control of a VDR-governed feed-forward loop that regulates p21(waf1/cip1) expression and function in non-malignant prostate cells.

In non-malignant RWPE-1 prostate epithelial cells signaling by the nuclear receptor Vitamin D Receptor (VDR, NR1I1) induces cell cycle arrest through targets including CDKN1A (encodes p21((waf1/cip1))). VDR dynamically induced individual histone modification patterns at three VDR binding sites (R1, 2, 3) on the CDKN1A promoter. The magnitude of these modifications was specific to each phase of the cell cycle. For example, H3K9ac enrichment occurred rapidly only at R2, whereas parallel accumulation of H3K27me3 occurred at R1; these events were significantly enriched in G(1) and S phase cells, respectively. The epigenetic events appeared to allow VDR actions to combine with p53 to enhance p21((waf1/cip1)) activation further. In parallel, VDR binding to the MCM7 gene induced H3K9ac enrichment associated with rapid mRNA up-regulation to generate miR-106b and consequently regulate p21((waf1/cip1)) expression. We conclude that VDR binding site- and promoter-specific patterns of histone modifications combine with miRNA co-regulation to form a VDR-regulated feed-forward loop to control p21((waf1/cip1)) expression and cell cycle arrest. Dissection of this feed-forward loop in a non-malignant prostate cell system illuminates mechanisms of sensitivity and therefore possible resistance in prostate and other VDR responsive cancers.

Multidisciplinary Initiative to Increase Guideline-concordant Antibiotic Prescription at Discharge for Hospitalized Children with Uncomplicated Community-acquired Pneumonia.

Background: Clinical practice guidelines recommend using narrow-spectrum antibiotics to treat uncomplicated pneumonia in children. This quality improvement (QI) project aimed to evaluate if QI methods could improve guideline-concordant antibiotic prescribing at hospital discharge for children with uncomplicated pneumonia. Methods: For this single-center QI project, we implemented QI interventions in serial plan-do-study-act cycles, focusing on the key drivers targeting general pediatric inpatient resident teams. Interventions included: (1)Small bimonthly group didactic sessions, (2)Visual job aids posted in resident work areas, and (3) A noon conference session. Balancing measures included postdischarge emergency room visits, readmission and adverse drug reactions. Results: To establish a baseline rate, we conducted a chart review of 112 children diagnosed with uncomplicated community-acquired pneumonia during hospitalization from July 2017 through January 2019. The average monthly percentage of children discharged with guideline-concordant antibiotics was 67%. The intervention period was from February 2019 through February 2020, with 118 children meeting the criteria after a review of 262 charts. After our interventions, the average monthly percentage of children discharged with guideline-concordant antibiotics increased to 87%, with the increase persisting for at least 12 months. There were no significant differences in balancing measures pre- and post-interventions. Conclusions: Our QI initiative sustained increased rates of uncomplicated community-acquired pneumonia guideline-concordant antibiotic prescribing at discharge over 12 months without an increase in balancing measures. The enduring changes in prescribing behavior suggest a lasting impact of our interventions.

Clonal selection of hematopoietic stem cells after gene therapy for sickle cell disease.

Gene therapy (GT) provides a potentially curative treatment option for patients with sickle cell disease (SCD); however, the occurrence of myeloid malignancies in GT clinical trials has prompted concern, with several postulated mechanisms. Here, we used whole-genome sequencing to track hematopoietic stem cells (HSCs) from six patients with SCD at pre- and post-GT time points to map the somatic mutation and clonal landscape of gene-modified and unmodified HSCs. Pre-GT, phylogenetic trees were highly polyclonal and mutation burdens per cell were elevated in some, but not all, patients. Post-GT, no clonal expansions were identified among gene-modified or unmodified cells; however, an increased frequency of potential driver mutations associated with myeloid neoplasms or clonal hematopoiesis (DNMT3A- and EZH2-mutated clones in particular) was observed in both genetically modified and unmodified cells, suggesting positive selection of mutant clones during GT. This work sheds light on HSC clonal dynamics and the mutational landscape after GT in SCD, highlighting the enhanced fitness of some HSCs harboring pre-existing driver mutations. Future studies should define the long-term fate of mutant clones, including any contribution to expansions associated with myeloid neoplasms.

Diagnostic utility of whole genome sequencing in adults with B-other acute lymphoblastic leukemia.

Genomic profiling during the diagnosis of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) in adults is used to guide disease classification, risk stratification, and treatment decisions. Patients for whom diagnostic screening fails to identify disease-defining or risk-stratifying lesions are classified as having B-other ALL. We screened a cohort of 652 BCP-ALL cases enrolled in UKALL14 to identify and perform whole genome sequencing (WGS) of paired tumor-normal samples. For 52 patients with B-other, we compared the WGS findings with data from clinical and research cytogenetics. WGS identified a cancer-associated event in 51 of 52 patients, including an established subtype defining genetic alterations that were previously missed with standard-of-care (SoC) genetics in 5 of them. Of the 47 true B-other ALL, we identified a recurrent driver in 87% (41). A complex karyotype via cytogenetics emerges as a heterogeneous group, including distinct genetic alterations associated with either favorable (DUX4-r) or poor outcomes (MEF2D-r and IGK::BCL2). For a subset of 31 cases, we integrated the findings from RNA sequencing (RNA-seq) analysis to include fusion gene detection and classification based on gene expression. Compared with RNA-seq, WGS was sufficient to detect and resolve recurrent genetic subtypes; however, RNA-seq can provide orthogonal validation of findings. In conclusion, we demonstrated that WGS can identify clinically relevant genetic abnormalities missed with SoC testing as well as identify leukemia driver events in virtually all cases of B-other ALL.

A systems biology approach identifies molecular networks defining skeletal muscle abnormalities in chronic obstructive pulmonary disease.

Chronic Obstructive Pulmonary Disease (COPD) is an inflammatory process of the lung inducing persistent airflow limitation. Extensive systemic effects, such as skeletal muscle dysfunction, often characterize these patients and severely limit life expectancy. Despite considerable research efforts, the molecular basis of muscle degeneration in COPD is still a matter of intense debate. In this study, we have applied a network biology approach to model the relationship between muscle molecular and physiological response to training and systemic inflammatory mediators. Our model shows that failure to co-ordinately activate expression of several tissue remodelling and bioenergetics pathways is a specific landmark of COPD diseased muscles. Our findings also suggest that this phenomenon may be linked to an abnormal expression of a number of histone modifiers, which we discovered correlate with oxygen utilization. These observations raised the interesting possibility that cell hypoxia may be a key factor driving skeletal muscle degeneration in COPD patients.

Cricopharyngeal Achalasia Presenting as Acute Dysphagia in a Pediatric Patient.

OBJECTIVE: To describe a case of idiopathic cricopharyngeal achalasia (CPA) in a pediatric patient with acute onset of dysphagia managed conservatively with supportive care. METHODS: Sixteen-month-old boy presented with acute onset of gagging and coughing with feeding. His exam was notable for a well-appearing child with pooling of oral secretions and coarse breath sounds. Plain film series did not show radio-opaque foreign body (FB) and an esophagram demonstrated an endoluminal filling defect of the cervical esophagus and aspiration of contrast. He was taken to the operating room for urgent endoscopy but no FB or food impaction was observed. He had persistent symptoms that required further evaluation and a multidisciplinary team approach. Bedside laryngoscopy did not reveal any abnormalities. Modified barium swallow (MBS) study revealed upper esophageal sphincter (UES) dysfunction, consistent with cricopharyngeal achalasia. Repeat upper endoscopy with biopsies demonstrated mucosal irritation overlying the UES but histologic studies were negative for infectious causes. RESULTS: He was treated with supportive care, including nasogastric feedings for nutrition supplementation as he was unable to tolerate oral feedings without aspiration. Over the course of 3 months after discharge, his symptoms resolved and repeat MBS was normal. CONCLUSION: CPA is a rare cause of dysphagia in the pediatric population. Conservative management with supportive care is a reasonable approach in cases with acute onset in otherwise healthy children without underlying medical problems.

Development and Implementation of a Peer Curbside Consult Service for Pediatric Hospitalists.

OBJECTIVE: To describe the development and implementation of a Peer Curbside Consult Service (PCCS) for a pediatric hospital medicine (PHM) division. METHODS: We developed a pilot intervention with hospitalists at a freestanding children's hospital to provide peer consultation services for challenging clinical cases. Postconsultation surveys collected from both the requesting and consulting hospitalists provided feedback about the program. The 12-point Template for Intervention Description and Replication (TIDieR) checklist is used to describe the process for program creation and implementation. RESULTS: The PCCS has provided 60 consultations in the first 2 years since implementation in April 2020 and supports a large PHM division with >75 members who practice at a tertiary care, freestanding children's hospital and 7 affiliate sites. Hospitalists request peer consultation for challenging clinical cases. The consultations were typically conducted in person or via telephone. Currently, 11 PHM faculty members within the division volunteer as consultants, with 2 assigned per week. Electronic postconsultation experience surveys were received from 70% of requesting and 89% of consultant hospitalists. We also provide preliminary data from this pilot intervention in the Supplemental Information. CONCLUSIONS: We successfully established a peer consult service that provided just-in-time clinical decision support across the various practice sites. Through transparent reporting using the TIDieR checklist, other divisions may be able to replicate and adapt their own peer consult program.

Undifferentiated embryonic cell transcription factor 1 regulates ESC chromatin organization and gene expression.

Previous reports showed that embryonic stem (ES) cells contain hyperdynamic and globally transcribed chromatin-properties that are important for ES cell pluripotency and differentiation. Here, we demonstrate a role for undifferentiated embryonic cell transcription factor 1 (UTF1) in regulating ES cell chromatin structure. Using chromatin immunoprecipitation-on-chip analysis, we identified >1,700 UTF1 target genes that significantly overlap with previously identified Nanog, Oct4, Klf-4, c-Myc, and Rex1 targets. Gene expression profiling showed that UTF1 knock down results in increased expression of a large set of genes, including a significant number of UTF1 targets. UTF1 knock down (KD) ES cells are, irrespective of the increased expression of several self-renewal genes, Leukemia inhibitory factor (LIF) dependent. However, UTF1 KD ES cells are perturbed in their differentiation in response to dimethyl sulfoxide (DMSO) or after LIF withdrawal and display increased colony formation. UTF1 KD ES cells display extensive chromatin decondensation, reflected by a dramatic increase in nucleosome release on micrococcal nuclease (MNase) treatment and enhanced MNase sensitivity of UTF1 target genes in UTF1 KD ES cells. Summarizing, our data show that UTF1 is a key chromatin component in ES cells, preventing ES cell chromatin decondensation, and aberrant gene expression; both essential for proper initiation of lineage-specific differentiation of ES cells.