Mapping clustered mutations in cancer reveals APOBEC3 mutagenesis of ecDNA.

Clustered somatic mutations are common in cancer genomes and previous analyses reveal several types of clustered single-base substitutions, which include doublet- and multi-base substitutions1-5, diffuse hypermutation termed omikli6, and longer strand-coordinated events termed kataegis3,7-9. Here we provide a comprehensive characterization of clustered substitutions and clustered small insertions and deletions (indels) across 2,583 whole-genome-sequenced cancers from 30 types of cancer10. Clustered mutations were highly enriched in driver genes and associated with differential gene expression and changes in overall survival. Several distinct mutational processes gave rise to clustered indels, including signatures that were enriched in tobacco smokers and homologous-recombination-deficient cancers. Doublet-base substitutions were caused by at least 12 mutational processes, whereas most multi-base substitutions were generated by either tobacco smoking or exposure to ultraviolet light. Omikli events, which have previously been attributed to APOBEC3 activity6, accounted for a large proportion of clustered substitutions; however, only 16.2% of omikli matched APOBEC3 patterns. Kataegis was generated by multiple mutational processes, and 76.1% of all kataegic events exhibited mutational patterns that are associated with the activation-induced deaminase (AID) and APOBEC3 family of deaminases. Co-occurrence of APOBEC3 kataegis and extrachromosomal DNA (ecDNA), termed kyklonas (Greek for cyclone), was found in 31% of samples with ecDNA. Multiple distinct kyklonic events were observed on most mutated ecDNA. ecDNA containing known cancer genes exhibited both positive selection and kyklonic hypermutation. Our results reveal the diversity of clustered mutational processes in human cancer and the role of APOBEC3 in recurrently mutating and fuelling the evolution of ecDNA.

Assigning mutational signatures to individual samples and individual somatic mutations with SigProfilerAssignment.

MOTIVATION: Analysis of mutational signatures is a powerful approach for understanding the mutagenic processes that have shaped the evolution of a cancer genome. To evaluate the mutational signatures operative in a cancer genome, one first needs to quantify their activities by estimating the number of mutations imprinted by each signature. RESULTS: Here we present SigProfilerAssignment, a desktop and an online computational framework for assigning all types of mutational signatures to individual samples. SigProfilerAssignment is the first tool that allows both analysis of copy-number signatures and probabilistic assignment of signatures to individual somatic mutations. As its computational engine, the tool uses a custom implementation of the forward stagewise algorithm for sparse regression and nonnegative least squares for numerical optimization. Analysis of 2700 synthetic cancer genomes with and without noise demonstrates that SigProfilerAssignment outperforms four commonly used approaches for assigning mutational signatures. AVAILABILITY AND IMPLEMENTATION: SigProfilerAssignment is available under the BSD 2-clause license at https://github.com/AlexandrovLab/SigProfilerAssignment with a web implementation at https://cancer.sanger.ac.uk/signatures/assignment/.

Visualizing and exploring patterns of large mutational events with SigProfilerMatrixGenerator.

BACKGROUND: All cancers harbor somatic mutations in their genomes. In principle, mutations affecting between one and fifty base pairs are generally classified as small mutational events. Conversely, large mutational events affect more than fifty base pairs, and, in most cases, they encompass copy-number and structural variants affecting many thousands of base pairs. Prior studies have demonstrated that examining patterns of somatic mutations can be leveraged to provide both biological and clinical insights, thus, resulting in an extensive repertoire of tools for evaluating small mutational events. Recently, classification schemas for examining large-scale mutational events have emerged and shown their utility across the spectrum of human cancers. However, there has been no computationally efficient bioinformatics tool that allows visualizing and exploring these large-scale mutational events. RESULTS: Here, we present a new version of SigProfilerMatrixGenerator that now delivers integrated capabilities for examining large mutational events. The tool provides support for examining copy-number variants and structural variants under two previously developed classification schemas and it supports data from numerous algorithms and data modalities. SigProfilerMatrixGenerator is written in Python with an R wrapper package provided for users that prefer working in an R environment. CONCLUSIONS: The new version of SigProfilerMatrixGenerator provides the first standardized bioinformatics tool for optimized exploration and visualization of two previously developed classification schemas for copy number and structural variants. The tool is freely available at https://github.com/AlexandrovLab/SigProfilerMatrixGenerator with an extensive documentation at https://osf.io/s93d5/wiki/home/ .

Cookstove Emissions and Performance Evaluation Using a New ISO Protocol and Comparison of Results with Previous Test Protocols.

In 2018, the International Organization for Standardization (ISO) 19867-1 "Harmonized laboratory test protocols" were released for establishing improved quality and comparability for data on cookstove air pollutant emissions, efficiency, safety, and durability. This is the first study that compares emissions [carbon dioxide, carbon monoxide, total hydrocarbons, methane, nitrogen oxides, fine particulate matter (PM2.5), organic carbon, elemental carbon, and ultrafine particles] and efficiency data between the ISO protocol and the Water Boiling Test (WBT). The study examines six stove/fuel combinations [liquefied petroleum gas (LPG), pellet, wood fan, wood rocket, three stone fire, and charcoal] tested in the same US EPA laboratory. Evaluation of the ISO protocol shows improvements over previous test protocols and that results are relatively consistent with former WBT data in terms of tier ratings for emissions and efficiency, as defined by the ISO 19867-3 "Voluntary Performance Targets." Most stove types remain similarly ranked using ISO and WBT protocols, except charcoal and LPG are in higher PM2.5 tiers with the ISO protocol. Additionally, emissions data including polycyclic aromatic hydrocarbons are utilized to compare between the ISO and Firepower Sweep Test (FST) protocols. Compared to the FST, the ISO protocol results in generally higher PM2.5 tier ratings.

Generating realistic null hypothesis of cancer mutational landscapes using SigProfilerSimulator.

BACKGROUND: Performing a statistical test requires a null hypothesis. In cancer genomics, a key challenge is the fast generation of accurate somatic mutational landscapes that can be used as a realistic null hypothesis for making biological discoveries. RESULTS: Here we present SigProfilerSimulator, a powerful tool that is capable of simulating the mutational landscapes of thousands of cancer genomes at different resolutions within seconds. Applying SigProfilerSimulator to 2144 whole-genome sequenced cancers reveals: (i) that most doublet base substitutions are not due to two adjacent single base substitutions but likely occur as single genomic events; (ii) that an extended sequencing context of ± 2 bp is required to more completely capture the patterns of substitution mutational signatures in human cancer; (iii) information on false-positive discovery rate of commonly used bioinformatics tools for detecting driver genes. CONCLUSIONS: SigProfilerSimulator's breadth of features allows one to construct a tailored null hypothesis and use it for evaluating the accuracy of other bioinformatics tools or for downstream statistical analysis for biological discoveries. SigProfilerSimulator is freely available at https://github.com/AlexandrovLab/SigProfilerSimulator with an extensive documentation at https://osf.io/usxjz/wiki/home/ .

Inhalation of Stachybotrys chartarum Fragments Induces Pulmonary Arterial Remodeling.

Stachybotrys chartarum is a fungal contaminant within the built environment and a respiratory health concern in the United States. The objective of this study was to characterize the mechanisms influencing pulmonary immune responses to repeatedly inhaled S. chartarum. Groups of B6C3F1/N mice repeatedly inhaled viable trichothecene-producing S. chartarum conidia (strain A or strain B), heat-inactivated conidia, or high-efficiency particulate absolute-filtered air twice per week for 4 and 13 weeks. Strain A was found to produce higher amounts of respirable fragments than strain B. Lung tissue, serum, and BAL fluid were collected at 24 and 48 hours after final exposure and processed for histology, flow cytometry, and RNA and proteomic analyses. At 4 weeks after exposure, a T-helper cell type 2-mediated response was observed. After 13 weeks, a mixed T-cell response was observed after exposure to strain A compared with a T-helper cell type 2-mediated response after strain B exposure. After exposure, both strains induced pulmonary arterial remodeling at 13 weeks; however, strain A-exposed mice progressed more quickly than strain B-exposed mice. BAL fluid was composed primarily of eosinophils, neutrophils, and macrophages. Both the immune response and the observed pulmonary arterial remodeling were supported by specific cellular, molecular, and proteomic profiles. The immunopathological responses occurred earlier in mice exposed to high fragment-producing strain A. The rather striking induction of pulmonary remodeling by S. chartarum appears to be related to the presence of fungal fragments during exposure.

Morphology and quantification of fungal growth in residential dust and carpets.

Mold growth indoors is associated with negative human health effects, and this growth is limited by moisture availability. Dust deposited in carpet is an important source of human exposure due to potential elevated resuspension compared to hard floors. However, we need an improved understanding of fungal growth in dust and carpet to better estimate human exposure. The goal of this study was to compare fungal growth quantity and morphology in residential carpet under different environmental conditions, including equilibrium relative humidity (ERH) (50%, 85%, 90%, 95%, 100%), carpet fiber material (nylon, olefin, wool) and presence/absence of dust. We analyzed incubated carpet and dust samples from three Ohio homes for total fungal DNA, fungal allergen Alt a 1, and fungal morphology. Dust presence and elevated ERH (≥85%) were the most important variables that increased fungal growth. Elevated ERH increased mean fungal DNA concentration (P < 0.0001), for instance by approximately 1000 times at 100% compared to 50% ERH after two weeks. Microscopy also revealed more fungal growth at higher ERH. Fungal concentrations were up to 100 times higher in samples containing house dust compared to no dust. For fiber type, olefin had the least total fungal growth, and nylon had the most total fungi and A. alternata growth in unaltered dust. Increased ERH conditions were associated with increased Alt a 1 allergen concentration. The results of this study demonstrate that ERH, presence/absence of house dust, and carpet fiber type influence fungal growth and allergen production in residential carpet, which has implications for human exposure.

SigProfilerMatrixGenerator: a tool for visualizing and exploring patterns of small mutational events.

BACKGROUND: Cancer genomes are peppered with somatic mutations imprinted by different mutational processes. The mutational pattern of a cancer genome can be used to identify and understand the etiology of the underlying mutational processes. A plethora of prior research has focused on examining mutational signatures and mutational patterns from single base substitutions and their immediate sequencing context. We recently demonstrated that further classification of small mutational events (including substitutions, insertions, deletions, and doublet substitutions) can be used to provide a deeper understanding of the mutational processes that have molded a cancer genome. However, there has been no standard tool that allows fast, accurate, and comprehensive classification for all types of small mutational events. RESULTS: Here, we present SigProfilerMatrixGenerator, a computational tool designed for optimized exploration and visualization of mutational patterns for all types of small mutational events. SigProfilerMatrixGenerator is written in Python with an R wrapper package provided for users that prefer working in an R environment. SigProfilerMatrixGenerator produces fourteen distinct matrices by considering transcriptional strand bias of individual events and by incorporating distinct classifications for single base substitutions, doublet base substitutions, and small insertions and deletions. While the tool provides a comprehensive classification of mutations, SigProfilerMatrixGenerator is also faster and more memory efficient than existing tools that generate only a single matrix. CONCLUSIONS: SigProfilerMatrixGenerator provides a standardized method for classifying small mutational events that is both efficient and scalable to large datasets. In addition to extending the classification of single base substitutions, the tool is the first to provide support for classifying doublet base substitutions and small insertions and deletions. SigProfilerMatrixGenerator is freely available at https://github.com/AlexandrovLab/SigProfilerMatrixGenerator with an extensive documentation at https://osf.io/s93d5/wiki/home/ .

Cultivation and aerosolization of Stachybotrys chartarum for modeling pulmonary inhalation exposure.

Objective:Stachybotrys chartarum is a hydrophilic fungal species commonly found as a contaminant in water-damaged building materials. Although several studies have suggested that S. chartarum exposure elicits a variety of adverse health effects, the ability to characterize the pulmonary immune responses to exposure is limited by delivery methods that do not replicate environmental exposure. This study aimed to develop a method of S. chartarum aerosolization to better model inhalation exposures. Materials and methods: An acoustical generator system (AGS) was previously developed and utilized to aerosolize and deliver fungal spores to mice housed in a multi-animal nose-only exposure chamber. In this study, methods for cultivating, heat-inactivating, and aerosolizing two macrocyclic trichothecene-producing strains of S. chartartum using the AGS are described. Results and discussion: In addition to conidia, acoustical generation of one strain of S. chartarum resulted in the aerosolization of fungal fragments (<2 µm aerodynamic diameter) derived from conidia, phialides, and hyphae that initially comprised 50% of the total fungal particle count but was reduced to less than 10% over the duration of aerosolization. Acoustical generation of heat-inactivated S. chartarum did not result in a similar level of fragmentation. Delivery of dry, unextracted S. chartarum using these aerosolization methods resulted in pulmonary inflammation and immune cell infiltration in mice inhaling viable, but not heat-inactivated S. chartarum. Conclusions: These methods of S. chartarum growth and aerosolization allow for the delivery of fungal bioaerosols to rodents that may better simulate natural exposure within water-damaged indoor environments.

Proposed Increases in Government Authority Over Research Misconduct Proceedings.

This Viewpoint discusses changes proposed by the US Department of Health and Human Services' Office of Research Integrity that would shift control of research misconduct proceedings from institutional oversight to federal authority.

Macrophage-derived human resistin is induced in multiple helminth infections and promotes inflammatory monocytes and increased parasite burden.

Parasitic helminth infections can be associated with lifelong morbidity such as immune-mediated organ failure. A better understanding of the host immune response to helminths could provide new avenues to promote parasite clearance and/or alleviate infection-associated morbidity. Murine resistin-like molecules (RELM) exhibit pleiotropic functions following helminth infection including modulating the host immune response; however, the relevance of human RELM proteins in helminth infection is unknown. To examine the function of human resistin (hResistin), we utilized transgenic mice expressing the human resistin gene (hRetnTg+). Following infection with the helminth Nippostrongylus brasiliensis (Nb), hResistin expression was significantly upregulated in infected tissue. Compared to control hRetnTg- mice, hRetnTg+ mice suffered from exacerbated Nb-induced inflammation characterized by weight loss and increased infiltration of inflammatory monocytes in the lung, along with elevated Nb egg burdens and delayed parasite expulsion. Genome-wide transcriptional profiling of the infected tissue revealed that hResistin promoted expression of proinflammatory cytokines and genes downstream of toll-like receptor signaling. Moreover, hResistin preferentially bound lung monocytes, and exogenous treatment of mice with recombinant hResistin promoted monocyte recruitment and proinflammatory cytokine expression. In human studies, increased serum resistin was associated with higher parasite load in individuals infected with soil-transmitted helminths or filarial nematode Wuchereria bancrofti, and was positively correlated with proinflammatory cytokines. Together, these studies identify human resistin as a detrimental factor induced by multiple helminth infections, where it promotes proinflammatory cytokines and impedes parasite clearance. Targeting the resistin/proinflammatory cytokine immune axis may provide new diagnostic or treatment strategies for helminth infection and associated immune-mediated pathology.

Molecular insights into the coding region determinant-binding protein-RNA interaction through site-directed mutagenesis in the heterogeneous nuclear ribonucleoprotein-K-homology domains.

The ability of its four heterogeneous nuclear RNP-K-homology (KH) domains to physically associate with oncogenic mRNAs is a major criterion for the function of the coding region determinant-binding protein (CRD-BP). However, the particular RNA-binding role of each of the KH domains remains largely unresolved. Here, we mutated the first glycine to an aspartate in the universally conserved GXXG motif of the KH domain as an approach to investigate their role. Our results show that mutation of a single GXXG motif generally had no effect on binding, but the mutation in any two KH domains, with the exception of the combination of KH3 and KH4 domains, completely abrogated RNA binding in vitro and significantly retarded granule formation in zebrafish embryos, suggesting that any combination of at least two KH domains cooperate in tandem to bind RNA efficiently. Interestingly, we found that any single point mutation in one of the four KH domains significantly impacted CRD-BP binding to mRNAs in HeLa cells, suggesting that the dynamics of the CRD-BP-mRNA interaction vary over time in vivo. Furthermore, our results suggest that different mRNAs bind preferentially to distinct CRD-BP KH domains. The novel insights revealed in this study have important implications on the understanding of the oncogenic mechanism of CRD-BP as well as in the future design of inhibitors against CRD-BP function.

Dopamine 2 Receptor Genes Are Associated with Raised Blood Glucose in Schizophrenia.

OBJECTIVE: Type 2 diabetes is commonly found in schizophrenia and is an important contributor to mortality and morbidity in this condition. Dopamine has been implicated in the aetiology of both diabetes and schizophrenia. It is possible that both disorders share a common genetic susceptibility. METHODS: In a cross-sectional study, we examined 2 dopamine D2 receptor (DRD2) single-nucleotide polymorphisms (SNPs) previously associated with schizophrenia (C939 T, rs6275 and C957 T, rs6277) along with fasting blood glucose and body mass index (BMI) in 207 antipsychotic-treated patients with schizophrenia. All participants met DSM-IV criteria for schizophrenia, and those with other psychiatric disorders were excluded. Analysis of covariance was used to compare fasting glucose results by DRD2 genotypes, after controlling for known confounds. For significant associations, follow-up Bonferroni post hoc tests examined differences in fasting glucose levels between genotypes. Specific comparisons were also made using analysis of variance and chi-square (Fisher's exact test). RESULTS: The 2 DRD2 risk genotypes were associated with significant increases in blood glucose, after controlling for BMI, age, sex, dosage and type of antipsychotic medication, number of hospitalisations, and negative symptoms (rs6275, F(2, 182) = 5.901, P = 0.003; rs6277 SNP, F(2, 178) = 3.483, P = 0.033). CONCLUSIONS: These findings support the involvement of DRD2 not only in schizophrenia but also in elevated levels of blood glucose commonly found in antipsychotic-treated patients with schizophrenia. Our data support the notion that diabetes may not merely be a comorbid condition but could be fundamentally associated with the pathogenesis of schizophrenia itself.

Incorporating ethical principles into clinical research protocols: a tool for protocol writers and ethics committees.

A novel Protocol Ethics Tool Kit ('Ethics Tool Kit') has been developed by a multi-stakeholder group of the Multi-Regional Clinical Trials Center of Brigham and Women's Hospital and Harvard. The purpose of the Ethics Tool Kit is to facilitate effective recognition, consideration and deliberation of critical ethical issues in clinical trial protocols. The Ethics Tool Kit may be used by investigators and sponsors to develop a dedicated Ethics Section within a protocol to improve the consistency and transparency between clinical trial protocols and research ethics committee reviews. It may also streamline ethics review and may facilitate and expedite the review process by anticipating the concerns of ethics committee reviewers. Specific attention was given to issues arising in multinational settings. With the use of this Tool Kit, researchers have the opportunity to address critical research ethics issues proactively, potentially speeding the time and easing the process to final protocol approval.

Utilization of palliative radiotherapy for bone metastases near end of life in a population-based cohort.

BACKGROUND: Palliative radiotherapy (PRT) can significantly improve quality of life for patients dying of cancer with bone metastases. However, an aggressive cancer treatment near end of life is an indicator of poor-quality care. But the optimal rate of overall palliative RT use near the end of life is still unknown. We sought to determine the patterns of palliative radiation therapy (RT) utilization in patients with bone metastases towards their end of life in a population-based, publicly funded health care system. METHODS: All consecutive patients with bone metastases treated with RT between 2007 and 2011 were identified in a provincial Canadian cancer registry database. Patients were categorized as receiving RT in the last 2 weeks, 2-4 weeks, or >4 weeks before their death. Associations between RT fractionation utilization by these categories, and patient and provider characteristics were assessed through logistic regression. RESULTS: Of the 16,898 courses 1734 (10.3) and 709 (4.2%) were prescribed to patients in the last 2-4 weeks and <2 weeks of their life, respectively. Primary lung (8%) and gastrointestinal (6.9%) cancers received palliative RT more commonly in the last 2 weeks of life (OR 3.72 [2.86-4.84] & 3.33 [2.42-4.58] respectively, p <0.001). Among the 709 patients who received RT in the last 2 weeks of life, 350 (49), 167 (24), and 127 (18%) were for spine, pelvis, and extremity metastases, respectively. RT was prescribed most frequently to spine (5%) and extremity (4%) metastases p <0.001 in the last two weeks of life, though only varied between 1% (sternum) and 5% (spine) by site of metastases. Single fraction RT was prescribed more commonly in the last 2 weeks of life (64.2%), compared to individuals who received RT 2-4 weeks (54.5), and >4 weeks (47.9%) before death (p <0.001). CONCLUSIONS: This population-based analysis found that only 4% of patients with bone metastases received radiation therapy during the last 2 weeks of their life in our population-based, publicly funded program, though it was significantly higher in patients with lung cancer and those with metastases to the spine or extremity. Appropriately, use of multiple fractions palliative RT was less common in patients closer to death.

Supporting Recognition of Clinical Nurses With the DAISY Award.

What is meaningful recognition? As a nurse leader, are you prepared to answer that question? Understanding the implications and impact of recognition for nursing staff is a powerful tool for nursing leaders. The DAISY Award is used in more than 2,100 organizations around the globe to give meaning to recognition. Here is a glimpse of the power that recognition can bring to an organization, to its leaders, and most importantly to staff.

Putting the "Meaning" in Meaningful Recognition of Nurses: The DAISY Award™.

Healthcare organizations, nurses, patients, and their families benefit from the opportunity to say, "Thank you." The experience of being hospitalized can be devastating. Recognizing compassionate and extraordinary nursing care in a meaningful way is a powerful tool that supports a healthy work environment, engages nursing staff, and impacts the patient care experience. The DAISY Award™ is a legacy of thanks that exemplifies the qualities of "meaningful" recognition.

Reviewing HIV-Related Research in Emerging Economies: The Role of Government Reviewing Agencies.

Little research has explored the possible effects of government institutions in emerging economies on ethical reviews of multinational research. We conducted semi-structured, in-depth telephone interviews with 15 researchers, Research Ethics Committees (RECs) personnel, and a government agency member involved in multinational HIV Prevention Trials Network (HPTN) research in emerging economies. Ministries of Health (MOH) or other government agencies often play pivotal roles as facilitators or barriers in the research ethics approval process. Government agency RECs reviewing protocols may face particular challenges, as they can lack resources, be poorly organized, have inconsistent review processes and limited expertise, and use differing definitions of national interests, including upholding national reputation and avoiding potential exploitation and stigma of the country's population. The MOH/governmental review body may be affected by power dynamics and politics in study reviews; may consider issues both related and unrelated to research ethics as understood elsewhere; and may prioritize particular diseases, treatments, or interventions over other topics/types of research. Poor communication and deeply-rooted tensions may exist between sponsor and host countries, impeding optimal interactions and reviews. Investigators must understand and plan for the potential effects of governmental agencies on multinational collaborative research, including preserving adequate time for agency review, and contacting these agencies beforehand to address issues that may arise. Better understanding of these issues can aid and advance appropriate global scientific collaboration.