Assessing DNA Damage Responses Using B Lymphocyte Cultures.

Development of B cells requires the programmed generation and repair of double-stranded DNA breaks in antigen receptor genes. Investigation of the cellular responses to these DNA breaks has established important insights into B cell development and, more broadly, has provided fundamental advances into the molecular mechanisms of DNA damage response pathways. Abelson transformed pre-B cell lines and primary pre-B cell cultures are malleable experimental systems with diverse applications for studying DNA damage responses. This chapter describes methods for generating these cellular systems, inducing and quantifying DSBs, and assessing DNA damage programs.

Mutant U2AF1-induced alternative splicing of H2afy (macroH2A1) regulates B-lymphopoiesis in mice.

Somatic mutations in spliceosome genes are found in ∼50% of patients with myelodysplastic syndromes (MDS), a myeloid malignancy associated with low blood counts. Expression of the mutant splicing factor U2AF1(S34F) alters hematopoiesis and mRNA splicing in mice. Our understanding of the functionally relevant alternatively spliced target genes that cause hematopoietic phenotypes in vivo remains incomplete. Here, we demonstrate that reduced expression of H2afy1.1, an alternatively spliced isoform of the histone H2A variant gene H2afy, is responsible for reduced B cells in U2AF1(S34F) mice. Deletion of H2afy or expression of U2AF1(S34F) reduces expression of Ebf1 (early B cell factor 1), a key transcription factor for B cell development, and mechanistically, H2AFY is enriched at the EBF1 promoter. Induced expression of H2AFY1.1 in U2AF1(S34F) cells rescues reduced EBF1 expression and B cells numbers in vivo. Collectively, our data implicate alternative splicing of H2AFY as a contributor to lymphopenia induced by U2AF1(S34F) in mice and MDS.

Palliative care competencies and education needs of nurses and healthcare assistants involved in the provision of supportive palliative care.

BACKGROUND: This paper investigates the palliative care competencies (knowledge, behaviours, attitudes) and education needs of nurses and healthcare assistants (HCAs) who provide supportive (Level 2) palliative care. METHODS: A mixed-methods study using a sequential exploratory design was used, with findings integrated across sources. Qualitative focus groups were conducted in 2018 with a sample of staff (n=11, all female; nurses=4; HCAs=7) providing supportive palliative care in a single service setting. A quantitative survey, also conducted in 2018, explored the issue with a wider sample within the same setting (n=36; nurses=18; HCAs=18; female=32). RESULTS: Qualitatively, communication was highlighted as an important domain of the competence framework, with many participants acknowledging that the ability to communicate effectively is essential. Quantitatively, participants scored in the lower range for competency variables. A significant difference was observed between HCAs and nurses on measures of knowledge (t= -2.718; df=30; p<.05) and behaviour (t=-3.576; df=30; p<.05), with HCAs scoring significantly higher than nurses. In relation to education, while some participants report being indecisive regarding engaging in education/training, others highlighted the benefit of education, especially its ability to impact on their current practice. CONCLUSION: This research contributes to understanding palliative care competencies among nurses and HCAs working in palliative care, and has important implications for the education and training of nurses and HCAs working in Level 2 palliative care in Ireland.

Cancer-related Fatigue Reported in Online Discussion Groups.

Fatigue is a prevalent cancer-related symptom that is difficult to communicate, define, and treat. To obtain robust descriptions of symptoms, participants were recruited into two online groups that consisted of their dialoguing together in an asynchronous, threaded discussion forum. Participants dialogued for 5 months and completed pre- and post-participation demographic data and symptom ratings. Survey data were described, and change scores were calculated. Excerpts from the dialogue were analyzed using phenomenological techniques. The 28 participants reported low symptoms that did not change significantly from pre- to post-participation. Phenomenological analysis revealed three themes: descriptions of "bone-sucking fatigue," a search for meaning or answers about the fatigue, and attempted remedies. Online support groups provide a venue for sharing symptom experiences, adding to existing knowledge about symptoms in survivors. These descriptions provide information that will aid in developing patient-centered interventions.

The Meaning of Self-efficacy for Symptom Management in the Acute Phase of Hematopoietic Stem Cell Transplantation.

BACKGROUND: Hematopoietic stem cell transplantation (HSCT) is an intensive treatment that offers the potential for longer life or cure for some types of cancer. Hematopoietic stem cell transplant is associated with decreased quality of life and functional status and distressing symptoms. Self-efficacy for symptom management (SESM) is a person's belief in his/her ability to implement behaviors to manage these symptoms. Presence of SESM can affect symptom distress, healthcare utilization, and posttransplantation outcomes. OBJECTIVE: The aim of this study was to explore the meaning of SESM in adults during the acute phase of HSCT. METHODS: Interviews were conducted before and at 30 days after transplantation. Descriptive thematic analysis was performed on verbatim interview transcripts. RESULTS: Themes of confidence, being responsible, and caring for mind, body, and spirit were identified, with subthemes of self-confidence, confidence in others, confidence and symptom level, vigilance, self-advocacy, and normalcy. Participants reported having high SESM before transplantation and having much less or no SESM when symptom distress was the most severe. CONCLUSIONS: This is the first study to examine the patient's perspective of self-efficacy in the acute phase of HSCT. This contributes to existing literature on the concept of symptom management and expands nursing knowledge of SESM in patients undergoing HSCT. IMPLICATIONS FOR PRACTICE: Nurses can assess SESM before transplantation and implement interventions to enhance SESM when symptoms are at their most distressing after HSCT. The findings from this study can provide the basis for creating behavioral interventions to enhance self-efficacy for symptom management in HSCT patients.

RAG-Mediated DNA Breaks Attenuate PU.1 Activity in Early B Cells through Activation of a SPIC-BCLAF1 Complex.

Early B cell development is regulated by stage-specific transcription factors. PU.1, an ETS-family transcription factor, is essential for coordination of early B cell maturation and immunoglobulin gene (Ig) rearrangement. Here we show that RAG DNA double-strand breaks (DSBs) generated during Ig light chain gene (Igl) rearrangement in pre-B cells induce global changes in PU.1 chromatin binding. RAG DSBs activate a SPIC/BCLAF1 transcription factor complex that displaces PU.1 throughout the genome and regulates broad transcriptional changes. SPIC recruits BCLAF1 to gene-regulatory elements that control expression of key B cell developmental genes. The SPIC/BCLAF1 complex suppresses expression of the SYK tyrosine kinase and enforces the transition from large to small pre-B cells. These studies reveal that RAG DSBs direct genome-wide changes in ETS transcription factor activity to promote early B cell development.

Self-efficacy for symptom management in the acute phase of hematopoietic stem cell transplant: A pilot study.

PURPOSE: Hematopoietic stem cell transplant (HSCT) is an intensive treatment associated with distressing treatment and disease-related symptoms that affect patient outcomes such as functional status and quality of life. Self-efficacy for symptom management (SESM) is a person's belief in their ability to perform behaviors to prevent and relieve symptoms. Presence of SESM can impact symptom distress and functional status. This study describes the changes over time and relationships among SESM, symptom distress, and physical functional status in adults during the acute phase of HSCT. METHODS: Patients (n = 40) completed measures of symptom distress, SESM, and physical function at time points prior to and at days 7, 15 and 30 post-transplant. Clinical outcomes were length of stay and number of readmissions. RESULTS: Symptom distress, physical function, and SESM changed significantly over time. There was a significant negative relationship between symptom distress and physical function and between symptom distress and SESM at all points. The lowest levels of SESM and physical function were at day 7 when symptom distress was highest. Symptom distress was a moderator for the relationship between physical function and SESM at day 15. CONCLUSION: This was the first study to examine SESM in the acute phase of HSCT. Higher SESM was associated with fewer symptoms and increased physical function. Less symptom distress was associated with higher physical function and confidence to manage symptoms. These findings provide the basis for development of patient-centered interventions to enhance SESM when symptoms are at their highest immediately after HSCT.

Self-Efficacy for Management of Symptoms and Symptom Distress in Adults With Cancer: An Integrative Review.

PROBLEM IDENTIFICATION: Self-efficacy for symptom management plays a key role in outcomes, such as quality of life (QOL), functional status, and symptom distress, for adults with cancer. This integrative review identified and assessed evidence regarding self-efficacy for management of symptoms and symptom distress in adults with cancer. LITERATURE SEARCH: The authors performed a search of literature published from 2006-2018, and articles that examined the relationship among self-reported self-efficacy, symptom management, symptom distress or frequency, and severity in adults with cancer were selected for inclusion. DATA EVALUATION: 22 articles met the inclusion criteria. All articles were critically appraised and met standards for methodologic quality. SYNTHESIS: Evidence from this review showed that high self-efficacy was associated with low symptom occurrence and symptom distress and higher general health and QOL. High self-efficacy predicted physical and emotional well-being. Low self-efficacy was associated with higher symptom severity, poorer outcomes, and better overall functioning. IMPLICATIONS FOR RESEARCH: Self-efficacy can be assessed using developed instruments. Presence of a theoretical model and validated instruments to measure self-efficacy for symptom management have set the groundwork for ongoing research.

Perceived Self-Efficacy: A Concept Analysis for Symptom Management in Patients With Cancer
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BACKGROUND: Perceived self-efficacy (PSE) for symptom management plays a key role in outcomes for patients with cancer, such as quality of life, functional status, symptom distress, and healthcare use. Definition of the concept is necessary for use in research and to guide the development of interventions to facilitate PSE for symptom management in patients with cancer.
. OBJECTIVES: This analysis will describe the concept of PSE for symptom management in patients with cancer.
. METHODS: A database search was performed for related publications from 2006-2016. Landmark publications published prior to 2006 that informed the concept analysis were included.
. FINDINGS: Greater PSE for symptom management predicts improved performance outcomes, including functional health status, cognitive function, and disease status. Clarification of the concept of PSE for symptom management will accelerate the progress of self-management research and allow for comparison of research data and intervention development.

RAG-mediated DNA double-strand breaks activate a cell type-specific checkpoint to inhibit pre-B cell receptor signals.

DNA double-strand breaks (DSBs) activate a canonical DNA damage response, including highly conserved cell cycle checkpoint pathways that prevent cells with DSBs from progressing through the cell cycle. In developing B cells, pre-B cell receptor (pre-BCR) signals initiate immunoglobulin light (Igl) chain gene assembly, leading to RAG-mediated DNA DSBs. The pre-BCR also promotes cell cycle entry, which could cause aberrant DSB repair and genome instability in pre-B cells. Here, we show that RAG DSBs inhibit pre-BCR signals through the ATM- and NF-κB2-dependent induction of SPIC, a hematopoietic-specific transcriptional repressor. SPIC inhibits expression of the SYK tyrosine kinase and BLNK adaptor, resulting in suppression of pre-BCR signaling. This regulatory circuit prevents the pre-BCR from inducing additional Igl chain gene rearrangements and driving pre-B cells with RAG DSBs into cycle. We propose that pre-B cells toggle between pre-BCR signals and a RAG DSB-dependent checkpoint to maintain genome stability while iteratively assembling Igl chain genes.

Fasting protects mice from lethal DNA damage by promoting small intestinal epithelial stem cell survival.

Short-term fasting protects mice from lethal doses of chemotherapy through undetermined mechanisms. Herein, we demonstrate that fasting preserves small intestinal (SI) architecture by maintaining SI stem cell viability and SI barrier function following exposure to high-dose etoposide. Nearly all SI stem cells were lost in fed mice, whereas fasting promoted sufficient SI stem cell survival to preserve SI integrity after etoposide treatment. Lineage tracing demonstrated that multiple SI stem cell populations, marked by Lgr5, Bmi1, or HopX expression, contributed to fasting-induced survival. DNA repair and DNA damage response genes were elevated in SI stem/progenitor cells of fasted etoposide-treated mice, which importantly correlated with faster resolution of DNA double-strand breaks and less apoptosis. Thus, fasting preserved SI stem cell viability as well as SI architecture and barrier function suggesting that fasting may reduce host toxicity in patients undergoing dose intensive chemotherapy.

DAPK3 suppresses acini morphogenesis and is required for mouse development.

UNLABELLED: Death-associated protein kinase (DAPK3) is a serine/threonine kinase involved in various signaling pathways important to tissue homeostasis and mammalian biology. Considered to be a putative tumor suppressor, the molecular mechanism by which DAPK3 exerts its suppressive function is not fully understood and the field lacks an appropriate mouse model. To address these gaps, an in vitro three-dimensional tumorigenesis model was used and a constitutive DAPK3-knockout mouse was generated. In the 3D morphogenesis model, loss of DAPK3 through lentiviral-mediated knockdown enlarged acinar size by accelerated acini proliferation and apoptosis while maintaining acini polarity. Depletion of DAPK3 enhanced growth factor-dependent mTOR activation and, furthermore, enlarged DAPK3 acini structures were uniquely sensitive to low doses of rapamycin. Simultaneous knockdown of RAPTOR, a key mTORC1 component, reversed the augmented acinar size in DAPK3-depleted structures indicating an epistatic interaction. Using a validated gene trap strategy to generate a constitutive DAPK3-knockout mouse, it was demonstrated that DAPK3 is vital for early mouse development. The Dapk3 promoter exhibits spatiotemporal activity in developing mice and is actively expressed in normal breast epithelia of adult mice. Importantly, reduction of DAPK3 expression correlates with the development of ductal carcinoma in situ (DCIS) and more aggressive breast cancer as observed in the Oncomine database of clinical breast cancer specimens. IMPLICATIONS: Novel cellular and mouse modeling studies of DAPK3 shed light on its tumor-suppressive mechanisms and provide direct evidence that DAPK3 has relevance in early development.

Rituximab with or without bevacizumab for the treatment of patients with relapsed follicular lymphoma.

INTRODUCTION/BACKGROUND: Inhibition of tumor angiogenesis by the interruption of VEGF pathway signaling is of therapeutic value in several solid tumors. Preclinical evidence supports similar importance of the pathway in non-Hodgkin lymphoma. In this randomized phase II trial, we compared the efficacy and toxicity of rituximab with bevacizumab versus single-agent rituximab, in patients with previously-treated follicular lymphoma. PATIENTS AND METHODS: Patients (n = 60) were randomized (1:1) to receive rituximab (375 mg/m(2) intravenously [I.V.] weekly for 4 weeks) either as a single agent or with bevacizumab (10 mg/kg I.V. on days 3 and 15). Patients with an objective response or stable disease at week 12 received 4 additional doses of rituximab (at months 3, 5, 7, and 9); patients who received rituximab/bevacizumab also received bevacizumab 10 mg/kg I.V. every 2 weeks for 16 doses. RESULTS: After a median follow-up of 34 months, PFS was improved in patients who received rituximab/bevacizumab compared with patients who received rituximab alone (median 20.7 vs. 10.4 months respectively; HR, 0.40 (95% confidence interval [CI], 0.20-0.80); P = .007). Overall survival was also improved numerically (73% vs. 53% at 4 years), but did not reach statistical significance (HR, 0.40 (95% CI, 0.15-1.05); P = .055). The addition of bevacizumab increased the toxicity of therapy, but both regimens were well tolerated (no grade 4 toxicity). CONCLUSION: The addition of bevacizumab to rituximab significantly improved PFS. The role of angiogenesis inhibition in the treatment of follicular lymphoma requires further definition in larger clinical trials.

Forkhead box O1 (FOXO1) protein, but not p53, contributes to robust induction of p21 expression in fasted mice.

Reporter mice that enable the activity of the endogenous p21 promoter to be dynamically monitored in real time in vivo and under a variety of experimental conditions revealed ubiquitous p21 expression in mouse organs including the brain. Low light bioluminescence microscopy was employed to localize p21 expression to specific regions of the brain. Interestingly, p21 expression was observed in the paraventricular, arcuate, and dorsomedial nuclei of the hypothalamus, regions that detect nutrient levels in the blood stream and signal metabolic actions throughout the body. These results suggested a link between p21 expression and metabolic regulation. We found that short-term food deprivation (fasting) potently induced p21 expression in tissues involved in metabolic regulation including liver, pancreas and hypothalamic nuclei. Conditional reporter mice were generated that enabled hepatocyte-specific expression of p21 to be monitored in vivo. Bioluminescence imaging demonstrated that fasting induced a 7-fold increase in p21 expression in livers of reporter mice and Western blotting demonstrated an increase in protein levels as well. The ability of fasting to induce p21 expression was found to be independent of p53 but dependent on FOXO1. Finally, occupancy of the endogenous p21 promoter by FOXO1 was observed in the livers of fasted but not fed mice. Thus, fasting promotes loading of FOXO1 onto the p21 promoter to induce p21 expression in hepatocytes.

A primer for achieving glycemic control in the cardiac surgical patient.

Maintaining glycemic control (blood glucose <180 mg/dL) has been shown to reduce morbidity and enhance long-term survival in patients with diabetes mellitus following coronary artery bypass graft (CABG) surgery. In this review we present a management strategy to achieve perioperative glycemic control in all patients undergoing CABG surgery, with and without diabetes mellitus, designed to achieve compliance with current Surgical Care Improvement Project (SCIP) and Society of Thoracic Surgeons (STS) guidelines.

Prehospital factors associated with mortality in injured air medical patients.

BACKGROUND: Air medical transport provides rapid transport to definitive care. Overtriage and the expense and risk of transport may offset survival benefits. OBJECTIVE: We assessed the ability of prehospital factors to predict resource need for helicopter-transported patients. METHODS: We performed a prospective, observational cohort analysis of injured scene patients taken to one of two level I trauma centers from October 2009 to September 2010. Variables analyzed included patient demographics, diagnoses, and clinical outcomes (in-hospital mortality, emergent surgery within 24 hours, blood transfusion within 24 hours, and intensive care unit [ICU] admission ≥24 hours, as well as a combined outcome of all clinical outcomes). Prehospital variables were prospectively obtained from air medical providers at the time of transport and included past medical history, mechanism of injury, and clinical factors. We compared those variables with and without the outcomes of interest via χ(2) analysis and the Kruskal-Wallis test, where appropriate. Multivariate logistic regression identified factors associated with outcomes of interest with the intent of developing a clinical prediction tool. RESULTS: Five hundred fifty-seven patients were transported during the study period. The majority of the patients were male (67%) and white (95%) and had an injury that occurred in a rural location (58%). Most injuries were blunt (97%), and patients had a median Injury Severity Score (ISS) of 9. The overall mortality was 4%; 48% of the patients had one of the four outcomes. The most common reasons for requesting air transport were motor vehicle collision (MVC) with high-risk mechanism (18%), MVC at a speed greater than 20 mph (18%), Glasgow Coma Scale score (GCS) less than 14 (15%), and loss of consciousness (LOC) greater than 5 minutes (15%). Factors associated with mortality were age greater than 44 years, GCS less than 14, systolic blood pressure (SBP) less than 90 mmHg, and flail chest. This model had 100% sensitivity and 50% specificity and missed no deaths. The combined endpoint of all four outcomes (death, receipt of blood, surgery, ICU admission) included intubation by emergency medical services, two or more fractures of the humerus/femur, presence of a neurovascular injury, a crush injury to the head, failure to localize to pain on examination, GCS less than 14, or the presence of a penetrating head injury. This model had a sensitivity of 57% (53%-61%) and a specificity of 78% (75%-87%). CONCLUSIONS: Very few prehospital criteria were associated with clinically important outcomes in helicopter-transported patients. Evidence-based guidelines for the most appropriate utilization of air medical transport need to be further evaluated and developed for injured patients.

Bioluminescence imaging captures the expression and dynamics of endogenous p21 promoter activity in living mice and intact cells.

To interrogate endogenous p21(WAF1/CIP1) (p21) promoter activity under basal conditions and in response to various forms of stress, knock-in imaging reporter mice in which expression of firefly luciferase (FLuc) was placed under the control of the endogenous p21 promoter within the Cdkn1a gene locus were generated. Bioluminescence imaging (BLI) of p21 promoter activity was performed noninvasively and repetitively in mice and in cells derived from these mice. We demonstrated that expression of FLuc accurately reported endogenous p21 expression at baseline and under conditions of genotoxic stress and that photon flux correlated with mRNA abundance and, therefore, bioluminescence provided a direct readout of p21 promoter activity in vivo. BLI confirmed that p53 was required for activation of the p21 promoter in vivo in response to ionizing radiation. Interestingly, imaging of reporter cells demonstrated that p53 prevents the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway from activating p21 expression when quiescent cells are stimulated with serum to reenter the cell cycle. In addition, low-light BLI identified p21 expression in specific regions of individual organs that had not been observed previously. This inducible p21(FLuc) knock-in reporter strain will facilitate imaging studies of p53-dependent and -independent stress responses within the physiological context of the whole animal.

Contributions made by CDC25 phosphatases to proliferation of intestinal epithelial stem and progenitor cells.

The CDC25 protein phosphatases drive cell cycle advancement by activating cyclin-dependent protein kinases (CDKs). Humans and mice encode three family members denoted CDC25A, -B and -C and genes encoding these family members can be disrupted individually with minimal phenotypic consequences in adult mice. However, adult mice globally deleted for all three phosphatases die within one week after Cdc25 disruption. A severe loss of absorptive villi due to a failure of crypt epithelial cells to proliferate was observed in the small intestines of these mice. Because the Cdc25s were globally deleted, the small intestinal phenotype and loss of animal viability could not be solely attributed to an intrinsic defect in the inability of small intestinal stem and progenitor cells to divide. Here, we report the consequences of deleting different combinations of Cdc25s specifically in intestinal epithelial cells. The phenotypes arising in these mice were then compared with those arising in mice globally deleted for the Cdc25s and in mice treated with irinotecan, a chemotherapeutic agent commonly used to treat colorectal cancer. We report that the phenotypes arising in mice globally deleted for the Cdc25s are due to the failure of small intestinal stem and progenitor cells to proliferate and that blocking cell division by inhibiting the cell cycle engine (through Cdc25 loss) versus by inducing DNA damage (via irinotecan) provokes a markedly different response of small intestinal epithelial cells. Finally, we demonstrate that CDC25A and CDC25B but not CDC25C compensate for each other to maintain the proliferative capacity of intestinal epithelial stem and progenitor cells.

Response of small intestinal epithelial cells to acute disruption of cell division through CDC25 deletion.

The CDC25 protein phosphatases (CDC25A, B, and C) drive cell cycle transitions by activating key components of the cell cycle engine. CDC25A and CDC25B are frequently overproduced in human cancers. Disruption of Cdc25B or Cdc25C individually or in combination has no effect on mouse viability. Here we report that CDC25A is the only family member to provide an essential function during early embryonic development, and that other family members compensate for its loss in adult mice. In contrast, conditional disruption of the entire family is lethal in adults due to a loss of small intestinal epithelial cell proliferation in crypts of Lieberkühn. Cdc25 loss induced Wnt signaling, and overall crypt structures were preserved. In the face of continuous Wnt signaling, nearly all crypt epithelial progenitors differentiated into multiple cell lineages, including crypt base columnar cells, a proposed stem cell. A small population of Musashi/Dcamkl-1/nuclear beta-catenin-positive epithelial cells was retained in these crypts. These findings have implications for the development of novel, less cytotoxic cancer chemotherapeutic drugs that specifically target the cell cycle.

Normal cell cycle and checkpoint responses in mice and cells lacking Cdc25B and Cdc25C protein phosphatases.

The Cdc25 family of protein phosphatases positively regulates cell division by activating cyclin-dependent protein kinases (CDKs). In humans and rodents, there are three Cdc25 family members--denoted Cdc25A, Cdc25B, and Cdc25C--that can be distinguished based on their subcellular compartmentalizations, their abundances and/or activities throughout the cell cycle, the CDKs that they target for activation, and whether they are overexpressed in human cancers. In addition, murine forms of Cdc25 exhibit distinct patterns of expression throughout development and in adult tissues. These properties suggest that individual Cdc25 family members contribute distinct biological functions in embryonic and adult cell cycles of mammals. Interestingly, mice with Cdc25C disrupted are healthy, and cells derived from these mice exhibit normal cell cycles and checkpoint responses. Cdc25B-/- mice are also generally normal (although females are sterile), and cells derived from Cdc25B-/- mice have normal cell cycles. Here we report that mice lacking both Cdc25B and Cdc25C are obtained at the expected Mendelian ratios, indicating that Cdc25B and Cdc25C are not required for mouse development or mitotic entry. Furthermore, cell cycles, DNA damage responses, and Cdc25A regulation are normal in cells lacking Cdc25B and Cdc25C. These findings indicate that Cdc25A, or possibly other phosphatases, is able to functionally compensate for the loss of Cdc25B and Cdc25C in mice.