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Rates of cannabis initiation among teenagers and young adults are increasing. Further, the use of various forms of cannabis (smoked or vaped) with nicotine (dual use) is increasingly common among young people. The health effects of dual use are lesser known, particularly in the context of high-potency cannabis products and across different routes of administration, which is ominous in terms of predicting future health outcomes. There is a long history of cannabis use being associated with decreased activity and increased snacking, both of which could portend an increased risk of metabolic and cardiovascular disease, particularly when these habits begin during formative years. However, modern forms of cannabis may not have these same effects. Here, we assess whether cannabis use alone and dual use of cannabis with nicotine impact dietary and exercise habits in young people. An anonymous, social media-based survey was designed based on the UC San Diego Inhalant Questionnaire and published diet and exercise questionnaires. A total of 457 surveys were completed. Young sole cannabis users represented 29% of responders, 16% were dual users of cannabis and nicotine, and 55% were non-users of either drug. Although the sole use of cannabis was not associated with dietary or activity differences relative to non-users, dual users of cannabis and nicotine reported higher consumption of unhealthy sugars. This novel finding of dual use being associated with increased sugar intake in young people raises concerns for an increased risk of metabolic syndrome and cardiovascular disease in this population.

The contrast between the disruption of genome topology after cohesin loss and the lack of downstream gene expression changes instigates intense debates regarding the structure-function relationship between genome and gene regulation. Here, by analyzing transcriptome and chromatin accessibility at the single-cell level, we discover that, instead of dictating population-wide gene expression levels, cohesin supplies a general function to neutralize stochastic coexpression tendencies of cis-linked genes in single cells. Notably, cohesin loss induces widespread gene coactivation and chromatin co-opening tens of million bases apart in cis. Spatial genome and protein imaging reveals that cohesin prevents gene co-bursting along the chromosome and blocks spatial mixing of transcriptional hubs. Single-molecule imaging shows that cohesin confines the exploration of diverse enhancer and core promoter binding transcriptional regulators. Together, these results support that cohesin arranges nuclear topology to control gene coexpression in single cells.

Extrachromosomal DNA is a common cause of oncogene amplification in cancer. The non-chromosomal inheritance of ecDNA enables tumors to rapidly evolve, contributing to treatment resistance and poor outcome for patients. The transcriptional context in which ecDNAs arise and progress, including chromosomally-driven transcription, is incompletely understood. We examined gene expression patterns of 870 tumors of varied histological types, to identify transcriptional correlates of ecDNA. Here, we show that ecDNA-containing tumors impact four major biological processes. Specifically, ecDNA-containing tumors up-regulate DNA damage and repair, cell cycle control, and mitotic processes, but down-regulate global immune regulation pathways. Taken together, these results suggest profound alterations in gene regulation in ecDNA-containing tumors, shedding light on molecular processes that give rise to their development and progression.

Household air pollution (HAP) from cooking with solid fuels used during pregnancy has been associated with adverse pregnancy outcomes. The Household Air Pollution Intervention Network (HAPIN) trial was a randomized controlled trial that assessed the impact of a liquefied petroleum gas (LPG) stove and fuel intervention on health in Guatemala, India, Peru, and Rwanda. Here we investigated the effects of the LPG stove and fuel intervention on stillbirth, congenital anomalies and neonatal mortality and characterized exposure-response relationships between personal exposures to fine particulate matter (PM2.5), black carbon (BC) and carbon monoxide (CO) and these outcomes. Pregnant women (18 to

Our previous study identified 52 antiplasmodial peptaibols isolated from fungi. To understand their antiplasmodial mechanism of action, we conducted phenotypic assays, assessed the in vitro evolution of resistance, and performed a transcriptome analysis of the most potent peptaibol, HZ NPDG-I. HZ NPDG-I and 2 additional peptaibols were compared for their killing action and stage dependency, each showing a loss of digestive vacuole (DV) content via ultrastructural analysis. HZ NPDG-I demonstrated a stepwise increase in DV pH, impaired DV membrane permeability, and the ability to form ion channels upon reconstitution in planar membranes. This compound showed no signs of cross resistance to targets of current clinical candidates, and 3 independent lines evolved to resist HZ NPDG-I acquired nonsynonymous changes in the P. falciparum multidrug resistance transporter, pfmdr1. Conditional knockdown of PfMDR1 showed varying effects to other peptaibol analogs, suggesting differing sensitivity.

Circular extrachromosomal DNA (ecDNA) in patient tumors is an important driver of oncogenic gene expression, evolution of drug resistance and poor patient outcomes. Applying computational methods for the detection and reconstruction of ecDNA across a retrospective cohort of 481 medulloblastoma tumors from 465 patients, we identify circular ecDNA in 82 patients (18%). Patients with ecDNA-positive medulloblastoma were more than twice as likely to relapse and three times as likely to die within 5 years of diagnosis. A subset of tumors harbored multiple ecDNA lineages, each containing distinct amplified oncogenes. Multimodal sequencing, imaging and CRISPR inhibition experiments in medulloblastoma models reveal intratumoral heterogeneity of ecDNA copy number per cell and frequent putative 'enhancer rewiring' events on ecDNA. This study reveals the frequency and diversity of ecDNA in medulloblastoma, stratified into molecular subgroups, and suggests copy number heterogeneity and enhancer rewiring as oncogenic features of ecDNA.

BACKGROUND: There is a long tradition in environmental health of using frameworks for evidence synthesis, such as those of the U.S. Environmental Protection Agency for its Integrated Science Assessments and the International Agency for Research on Cancer Monographs. The framework, Grading of Recommendations Assessment, Development, and Evaluation (GRADE), was developed for evidence synthesis in clinical medicine. The U.S. Office of Health Assessment and Translation (OHAT) elaborated an approach for evidence synthesis in environmental health building on GRADE. METHODS: We applied a modified OHAT approach and a broader narrative assessment to assess the level of confidence in a large systematic review on traffic-related air pollution and health outcomes. DISCUSSION: We discuss several challenges with the OHAT approach and its implementation and suggest improvements for synthesizing evidence from observational studies in environmental health. We consider the determination of confidence using a formal rating scheme of up- and downgrading of certain factors, the treatment of every factor as equally important, and the lower initial confidence rating of observational studies to be fundamental issues in the OHAT approach. We argue that some observational studies can offer high-confidence evidence in environmental health. We note that heterogeneity in magnitude of effect estimates should generally not weaken the confidence in the evidence, and consistency of associations across study designs, populations, and exposure assessment methods may strengthen confidence in the evidence. We mention that publication bias should be explored beyond statistical methods and is likely limited when large and collaborative studies comprise most of the evidence and when accrued over several decades. We propose to identify possible key biases, their most likely direction, and their potential impacts on the results. We think that the OHAT approach and other GRADE-type frameworks require substantial modification to align better with features of environmental health questions and the studies that address them. We emphasize that a broader, narrative evidence assessment based on the systematic review may complement a formal GRADE-type evaluation. https://doi.org/10.1289/EHP11532.

Substance use disorder is a growing public health challenge in the United States. People who use drugs may be more vulnerable to ambient heat due to the effects of drugs on thermoregulation and their risk environment. There have been limited population-based studies of ambient temperature and drug-related morbidity. We examined short-term associations between daily ambient temperature and emergency department (ED) visits for use or overdose of amphetamine, cocaine and opioids in California during the period 2005 to 2019. Daily ZIP code-level maximum, mean, and minimum temperature exposures were derived from 1-km data Daymet products. A time-stratified case-crossover design was used to estimate cumulative non-linear associations of daily temperature for lag days 0 to 3. Stratified analyses by patient sex, race, and ethnicity were also conducted. The study included over 3.4 million drug-related ED visits. We found positive associations between daily temperature and ED visits for all outcomes examined. An increase in daily mean temperature from the 50th to the 95th percentile was associated with ED visits for amphetamine use (OR = 1.072, 95% CI: 1.058, 1.086), cocaine use (OR = 1.044, 95% CI: 1.021, 1.068 and opioid use (OR = 1.041, 95% CI: 1.025, 1.057). Stronger positive associations were also observed for overdose: amphetamine overdose (OR = 1.150, 95% CI: 1.085, 1.218), cocaine overdose (OR = 1.159, 95% CI: 1.053, 1.276), and opioid overdose (OR = 1.079, 95% CI: 1.054, 1.106). In summary, people who use stimulants and opioids may be a subpopulation sensitive to short-term higher ambient temperature. Mitigating heat exposure can be considered in harm reduction strategies in response to the substance use epidemic and global climate change.

CRISPR-Cas9 genome editing has enabled advanced T cell therapies, but occasional loss of the targeted chromosome remains a safety concern. To investigate whether Cas9-induced chromosome loss is a universal phenomenon and evaluate its clinical significance, we conducted a systematic analysis in primary human T cells. Arrayed and pooled CRISPR screens revealed that chromosome loss was generalizable across the genome and resulted in partial and entire loss of the targeted chromosome, including in preclinical chimeric antigen receptor T cells. T cells with chromosome loss persisted for weeks in culture, implying the potential to interfere with clinical use. A modified cell manufacturing process, employed in our first-in-human clinical trial of Cas9-engineered T cells (NCT03399448), reduced chromosome loss while largely preserving genome editing efficacy. Expression of p53 correlated with protection from chromosome loss observed in this protocol, suggesting both a mechanism and strategy for T cell engineering that mitigates this genotoxicity in the clinic.

Household air pollution from solid cooking fuel use during gestation has been associated with adverse pregnancy and birth outcomes. The Household Air Pollution Intervention Network (HAPIN) trial was a randomized controlled trial of free liquefied petroleum gas (LPG) stoves and fuel in Guatemala, Peru, India, and Rwanda. A primary outcome of the main trial was to report the effects of the intervention on infant birth weight. Here we evaluate the effects of a LPG stove and fuel intervention during pregnancy on spontaneous abortion, postpartum hemorrhage, hypertensive disorders of pregnancy, and maternal mortality compared to women who continued to use solid cooking fuels. Pregnant women (18-34 years of age; gestation confirmed by ultrasound at 9-19 weeks) were randomly assigned to an intervention (n = 1593) or control (n = 1607) arm. Intention-to-treat analyses compared outcomes between the two arms using log-binomial models. Among the 3195 pregnant women in the study, there were 10 spontaneous abortions (7 intervention, 3 control), 93 hypertensive disorders of pregnancy (47 intervention, 46 control), 11 post postpartum hemorrhage (5 intervention, 6 control) and 4 maternal deaths (3 intervention, 1 control). Compared to the control arm, the relative risk of spontaneous abortion among women randomized to the intervention was 2.32 (95% confidence interval (CI): 0.60, 8.96), hypertensive disorders of pregnancy 1.02 (95% CI: 0.68, 1.52), postpartum hemorrhage 0.83 (95% CI: 0.25, 2.71) and 2.98 (95% CI: 0.31, 28.66) for maternal mortality. In this study, we found that adverse maternal outcomes did not differ based on randomized stove type across four country research sites.

Passive surveillance systems are widely used to monitor diseases occurrence over wide spatial areas due to their cost-effectiveness and integration into broadly distributed healthcare systems. However, such systems are generally associated with imperfect ascertainment of disease cases and with heterogeneous capture probabilities arising from factors such as differential access to care. Augmenting passive surveillance systems with other surveillance efforts provides a way to estimate the true number of incident cases. We develop a hierarchical modeling framework for analyzing data from multiple surveillance systems that allows for individual-level covariate-dependent heterogeneous capture probabilities, and borrows information across surveillance sites to improve estimation of the true number of incident cases. Inference is carried out via a two-stage Bayesian procedure. Simulation studies illustrated superior performance of the proposed approach with respect to bias, root mean square error, and coverage compared to a model that does not borrow information across sites. We applied the proposed model to data from three surveillance systems reporting pulmonary tuberculosis (PTB) cases in a major center of ongoing transmission in China. The analysis yielded bias-corrected estimates of PTB cases from the passive system and led to the identification of risk factors associated with PTB rates, as well as factors influencing the operating characteristics of the implemented surveillance systems.

Oncogene amplification on extrachromosomal DNA (ecDNA) drives the evolution of tumours and their resistance to treatment, and is associated with poor outcomes for patients with cancer1-6. At present, it is unclear whether ecDNA is a later manifestation of genomic instability, or whether it can be an early event in the transition from dysplasia to cancer. Here, to better understand the development of ecDNA, we analysed whole-genome sequencing (WGS) data from patients with oesophageal adenocarcinoma (EAC) or Barrett's oesophagus. These data included 206 biopsies in Barrett's oesophagus surveillance and EAC cohorts from Cambridge University. We also analysed WGS and histology data from biopsies that were collected across multiple regions at 2 time points from 80 patients in a case-control study at the Fred Hutchinson Cancer Center. In the Cambridge cohorts, the frequency of ecDNA increased between Barrett's-oesophagus-associated early-stage (24%) and late-stage (43%) EAC, suggesting that ecDNA is formed during cancer progression. In the cohort from the Fred Hutchinson Cancer Center, 33% of patients who developed EAC had at least one oesophageal biopsy with ecDNA before or at the diagnosis of EAC. In biopsies that were collected before cancer diagnosis, higher levels of ecDNA were present in samples from patients who later developed EAC than in samples from those who did not. We found that ecDNAs contained diverse collections of oncogenes and immunomodulatory genes. Furthermore, ecDNAs showed increases in copy number and structural complexity at more advanced stages of disease. Our findings show that ecDNA can develop early in the transition from high-grade dysplasia to cancer, and that ecDNAs progressively form and evolve under positive selection.

Psoriasis vulgaris is a systemic, chronic inflammatory disease affecting 2-3% of the population. Recent advances in the understanding of the pathophysiology of psoriatic disease have facilitated the development of novel therapeutic options with improved safety and efficacy. This article is coauthored by a patient with a lifelong history of psoriasis who experienced multiple treatment failures. He details his diagnosis and treatment experiences, as well as the physical, mental, and social ramifications of his skin condition. He then goes on to elaborate how evolutions in the treatment of psoriatic disease have impacted his life. This case is then discussed from the perspective of a dermatologist specializing in inflammatory skin disorders. We highlight the clinical features of psoriasis, its medical and psychosocial comorbidities, and the current landscape of psoriatic disease treatments.

Objectives: We report results of a systematic review on the health effects of long-term traffic-related air pollution (TRAP) and diabetes in the adult population. Methods: An expert Panel appointed by the Health Effects Institute conducted this systematic review. We searched the PubMed and LUDOK databases for epidemiological studies from 1980 to July 2019. TRAP was defined based on a comprehensive protocol. Random-effects meta-analyses were performed. Confidence assessments were based on a modified Office for Health Assessment and Translation (OHAT) approach, complemented with a broader narrative synthesis. We extended our interpretation to include evidence published up to May 2022. Results: We considered 21 studies on diabetes. All meta-analytic estimates indicated higher diabetes risks with higher exposure. Exposure to NO2 was associated with higher diabetes prevalence (RR 1.09; 95% CI: 1.02; 1.17 per 10 μg/m3), but less pronounced for diabetes incidence (RR 1.04; 95% CI: 0.96; 1.13 per 10 μg/m3). The overall confidence in the evidence was rated moderate, strengthened by the addition of 5 recently published studies. Conclusion: There was moderate evidence for an association of long-term TRAP exposure with diabetes.

Extrachromosomal DNA (ecDNA) is a common mode of oncogene amplification but is challenging to analyze. Here, we adapt CRISPR-CATCH, in vitro CRISPR-Cas9 treatment and pulsed field gel electrophoresis of agarose-entrapped genomic DNA, previously developed for bacterial chromosome segments, to isolate megabase-sized human ecDNAs. We demonstrate strong enrichment of ecDNA molecules containing EGFR, FGFR2 and MYC from human cancer cells and NRAS ecDNA from human metastatic melanoma with acquired therapeutic resistance. Targeted enrichment of ecDNA versus chromosomal DNA enabled phasing of genetic variants, identified the presence of an EGFRvIII mutation exclusively on ecDNAs and supported an excision model of ecDNA genesis in a glioblastoma model. CRISPR-CATCH followed by nanopore sequencing enabled single-molecule ecDNA methylation profiling and revealed hypomethylation of the EGFR promoter on ecDNAs. We distinguished heterogeneous ecDNA species within the same sample by size and sequence with base-pair resolution and discovered functionally specialized ecDNAs that amplify select enhancers or oncogene-coding sequences.

Oncogene amplification on extrachromosomal DNA (ecDNA) is a common event, driving aggressive tumor growth, drug resistance and shorter survival. Currently, the impact of nonchromosomal oncogene inheritance-random identity by descent-is poorly understood. Also unclear is the impact of ecDNA on somatic variation and selection. Here integrating theoretical models of random segregation, unbiased image analysis, CRISPR-based ecDNA tagging with live-cell imaging and CRISPR-C, we demonstrate that random ecDNA inheritance results in extensive intratumoral ecDNA copy number heterogeneity and rapid adaptation to metabolic stress and targeted treatment. Observed ecDNAs benefit host cell survival or growth and can change within a single cell cycle. ecDNA inheritance can predict, a priori, some of the aggressive features of ecDNA-containing cancers. These properties are facilitated by the ability of ecDNA to rapidly adapt genomes in a way that is not possible through chromosomal oncogene amplification. These results show how the nonchromosomal random inheritance pattern of ecDNA contributes to poor outcomes for patients with cancer.

RNA-binding proteins (RBPs), essential for skeletal muscle regeneration, cause muscle degeneration and neuromuscular disease when mutated. Why mutations in these ubiquitously expressed RBPs orchestrate complex tissue regeneration and direct cell fate decisions in skeletal muscle remains poorly understood. Single-cell RNA-sequencing of regenerating Mus musculus skeletal muscle reveals that RBP expression, including the expression of many neuromuscular disease-associated RBPs, is temporally regulated in skeletal muscle stem cells and correlates with specific stages of myogenic differentiation. By combining machine learning with RBP engagement scoring, we discovered that the neuromuscular disease-associated RBP Hnrnpa2b1 is a differentiation-specifying regulator of myogenesis that controls myogenic cell fate transitions during terminal differentiation in mice. The timing of RBP expression specifies cell fate transitions by providing post-transcriptional regulation of messenger RNAs that coordinate stem cell fate decisions during tissue regeneration.

Mammalian chromosomes are organized into megabase-sized compartments that are further subdivided into topologically associating domains (TADs). While the formation of TADs is dependent on cohesin, the mechanism behind compartmentalization remains enigmatic. Here, we show that the bromodomain and extraterminal (BET) family scaffold protein BRD2 promotes spatial mixing and compartmentalization of active chromatin after cohesin loss. This activity is independent of transcription but requires BRD2 to recognize acetylated targets through its double bromodomain and interact with binding partners with its low-complexity domain. Notably, genome compartmentalization mediated by BRD2 is antagonized on the one hand by cohesin and on the other hand by the BET homolog protein BRD4, both of which inhibit BRD2 binding to chromatin. Polymer simulation of our data supports a BRD2-cohesin interplay model of nuclear topology, in which genome compartmentalization results from a competition between loop extrusion and chromatin-state-specific affinity interactions.

With the aid of laboratory typing techniques, infectious disease surveillance networks have the opportunity to obtain powerful information on the emergence, circulation, and evolution of multiple genotypes, serotypes or other subtypes of pathogens, informing understanding of transmission dynamics and strategies for prevention and control. The volume of typing performed on clinical isolates is typically limited by its ability to inform clinical care, cost and logistical constraints, especially in comparison with the capacity to monitor clinical reports of disease occurrence, which remains the most widespread form of public health surveillance. Viewing clinical disease reports as arising from a latent mixture of pathogen subtypes, laboratory typing of a subset of clinical cases can provide inference on the proportion of clinical cases attributable to each subtype (i.e., the mixture components). Optimizing protocols for the selection of isolates for typing by weighting specific subpopulations, locations, time periods, or case characteristics (e.g., disease severity), may improve inference of the frequency and distribution of pathogen subtypes within and between populations. Here, we apply the Disease Surveillance Informatics Optimization and Simulation (DIOS) framework to simulate and optimize hand foot and mouth disease (HFMD) surveillance in a high-burden region of western China. We identify laboratory surveillance designs that significantly outperform the existing network: the optimal network reduced mean absolute error in estimated serotype-specific incidence rates by 14.1%; similarly, the optimal network for monitoring severe cases reduced mean absolute error in serotype-specific incidence rates by 13.3%. In both cases, the optimal network designs achieved improved inference without increasing subtyping effort. We demonstrate how the DIOS framework can be used to optimize surveillance networks by augmenting clinical diagnostic data with limited laboratory typing resources, while adapting to specific, local surveillance objectives and constraints.

Extrachromosomal DNA (ecDNA) is prevalent in human cancers and mediates high expression of oncogenes through gene amplification and altered gene regulation1. Gene induction typically involves cis-regulatory elements that contact and activate genes on the same chromosome2,3. Here we show that ecDNA hubs-clusters of around 10-100 ecDNAs within the nucleus-enable intermolecular enhancer-gene interactions to promote oncogene overexpression. ecDNAs that encode multiple distinct oncogenes form hubs in diverse cancer cell types and primary tumours. Each ecDNA is more likely to transcribe the oncogene when spatially clustered with additional ecDNAs. ecDNA hubs are tethered by the bromodomain and extraterminal domain (BET) protein BRD4 in a MYC-amplified colorectal cancer cell line. The BET inhibitor JQ1 disperses ecDNA hubs and preferentially inhibits ecDNA-derived-oncogene transcription. The BRD4-bound PVT1 promoter is ectopically fused to MYC and duplicated in ecDNA, receiving promiscuous enhancer input to drive potent expression of MYC. Furthermore, the PVT1 promoter on an exogenous episome suffices to mediate gene activation in trans by ecDNA hubs in a JQ1-sensitive manner. Systematic silencing of ecDNA enhancers by CRISPR interference reveals intermolecular enhancer-gene activation among multiple oncogene loci that are amplified on distinct ecDNAs. Thus, protein-tethered ecDNA hubs enable intermolecular transcriptional regulation and may serve as units of oncogene function and cooperative evolution and as potential targets for cancer therapy.

The human transcriptome contains many types of noncoding RNAs, which rival the number of protein-coding species. From long noncoding RNAs (lncRNAs) that are over 200 nucleotides long to piwi-interacting RNAs (piRNAs) of only 20 nucleotides, noncoding RNAs play important roles in regulating transcription, epigenetic modifications, translation, and cell signaling. Roles for noncoding RNAs in disease mechanisms are also being uncovered, and several species have been identified as potential drug targets. On May 11-14, 2021, the Keystone eSymposium Noncoding RNAs: Biology and Applications brought together researchers working in RNA biology, structure, and technologies to accelerate both the understanding of RNA basic biology and the translation of those findings into clinical applications.

We appreciate the emphasis on the flexibility of the OHAT approach, including a narrative assessment. We respectfully disagree that we incorrectly portrayed the OHAT approach, and we clarify below with [...]