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The regeneration-capable flatworm Macrostomum lignano is a powerful model organism to study the biology of stem cells in vivo. As a flatworm amenable to transgenesis, it complements the historically used planarian flatworm models, such as Schmidtea mediterranea. However, information on the transcriptome and markers of stem cells in M. lignano is limited. We generated a de novo transcriptome assembly and performed the first comprehensive characterization of gene expression in the proliferating cells of M. lignano, represented by somatic stem cells, called neoblasts, and germline cells. Knockdown of a selected set of neoblast genes, including Mlig-ddx39, Mlig-rrm1, Mlig-rpa3, Mlig-cdk1, and Mlig-h2a, confirmed their crucial role for the functionality of somatic neoblasts during homeostasis and regeneration. The generated M. lignano transcriptome assembly and gene expression signatures of somatic neoblasts and germline cells will be a valuable resource for future molecular studies in M. lignano.

The significance of cardiac stem cell (CSC) populations for cardiac regeneration remains disputed. Here, we apply the most direct definition of stem cell function (the ability to replace lost tissue through cell division) to interrogate the existence of CSCs. By single-cell mRNA sequencing and genetic lineage tracing using two Ki67 knockin mouse models, we map all proliferating cells and their progeny in homoeostatic and regenerating murine hearts. Cycling cardiomyocytes were only robustly observed in the early postnatal growth phase, while cycling cells in homoeostatic and damaged adultmyocardium represented various noncardiomyocyte cell types. Proliferative postdamage fibroblasts expressing follistatin-like protein 1 (FSTL1) closely resemble neonatal cardiac fibroblasts and form the fibrotic scar. Genetic deletion of Fstl1 in cardiac fibroblasts results in postdamage cardiac rupture. We find no evidence for the existence of a quiescent CSC population, for transdifferentiation of other cell types toward cardiomyocytes, or for proliferation of significant numbers of cardiomyocytes in response to cardiac injury.

Developmental gene expression is often controlled by distal regulatory DNA elements called enhancers. Distant enhancer action is restricted to structural chromosomal domains that are flanked by CTCF-associated boundaries and formed through cohesin chromatin loop extrusion. To better understand how enhancers, genes and CTCF boundaries together form structural domains and control expression, we used a bottom-up approach, building series of active regulatory landscapes in inactive chromatin. We demonstrate here that gene transcription levels and activity over time reduce with increased enhancer distance. The enhancer recruits cohesin to stimulate domain formation and engage flanking CTCF sites in loop formation. It requires cohesin exclusively for the activation of distant genes, not of proximal genes, with nearby CTCF boundaries supporting efficient long-range enhancer action. Our work supports a dual activity model for enhancers: its classic role of stimulating transcription initiation and elongation from target gene promoters and a role of recruiting cohesin for the creation of chromosomal domains, the engagement of CTCF sites in chromatin looping and the activation of distal target genes. Here the authors build different regulatory landscapes in an inactive chromatin environment and observe that enhancers can both carry out their classic role of stimulating transcription initiation and elongation at target gene promoters and recruit cohesin to create contact domains, engage CTCF sites and activate distant genes.

Various species of the intestinal microbiota have been associated with the development of colorectal cancer(1,2), but it has not been demonstrated that bacteria have a direct role in the occurrence of oncogenic mutations. Escherichia coli can carry the pathogenicity island pks, which encodes a set of enzymes that synthesize colibactin(3). This compound is believed to alkylate DNA on adenine residues(4,5) and induces double-strand breaks in cultured cells(3). Here we expose human intestinal organoids to genotoxic pks(+)E. coli by repeated luminal injection over five months. Whole-genome sequencing of clonal organoids before and after this exposure revealed a distinct mutational signature that was absent from organoids injected with isogenic pks-mutant bacteria. The same mutational signature was detected in a subset of 5,876 human cancer genomes from two independent cohorts, predominantly in colorectal cancer. Our study describes a distinct mutational signature in colorectal cancer and implies that the underlying mutational process results directly from past exposure to bacteria carrying the colibactin-producing pks pathogenicity island.Organoids derived from human intestinal cells that are co-cultured with bacteria carrying the genotoxic pks(+) island develop a distinct mutational signature associated with colorectal cancer.

Developmental gene expression is often controlled by distal tissue-specific enhancers. Enhancer action is restricted to topological chromatin domains, typically formed by cohesin-mediated loop extrusion between CTCF-associated boundaries. To better understand how individual regulatory DNA elements form topological domains and control expression, we used a bottom-up approach, building active regulatory landscapes of different sizes in inactive chromatin. We demonstrate that transcriptional output and protection against gene silencing reduces with increased enhancer distance, but that enhancer contact frequencies alone do not dictate transcription activity. The enhancer recruits cohesin to stimulate the formation of local chromatin contact domains and activate flanking CTCF sites for engagement in chromatin looping. Small contact domains can support strong and stable expression of distant genes. The enhancer requires transcription factors and mediator to activate genes over all distance ranges, but relies on cohesin exclusively for the activation of distant genes. Our work supports a model that assigns two functions to enhancers: its classic role to stimulate transcription initiation and elongation from target gene promoters and a role to recruit cohesin for the creation of contact domains, the engagement of flanking CTCF sites in chromatin looping, and the activation of distal target genes. Competing Interest Statement WdL is founder and shareholder of Cergentis B.V.

Introduction As the HIV field evolves to better serve populations which are diverse in risk and access to services, it is crucial to understand and adapt the conceptual tools used to make sense of the HIV pandemic. In this commentary, we discuss the concept of general population. Using a synthetic and historical review, we reflect on the genesis and usage of the general population in HIV research and programme literature, pointing to its moral connotations and its impact on epidemiologic reasoning. Discussion From the early days of the HIV pandemic, the category of general population has carried implicit normative meanings. General population represented those people considered to be undeserving of HIV acquisition, and therefore deserving of a response. Framing the HIV epidemic in sub‐Saharan Africa as a generalized epidemic primarily affecting the general population has contributed to the exclusion of men who have sex with men from epidemic responses. The usage of this category has also masked heterogeneity among those it includes; the increasing focus on the use of interventions such as circumcision and HIV treatment as general population HIV prevention approaches has been marked by a lack of attention to heterogeneity among beneficiaries. Conclusions We recommend that the term general population be retired from the field’s lexicon. HIV programmes should strengthen their capacity to describe the heterogeneity of those they serve and plan their interventions accordingly. To increase the efficiency and impact of the HIV response, it is urgent to stratify the category of general population by risk. Sexual networks are a promising basis for this stratification.

Various species of the intestinal microbiota have been associated with the development of colorectal cancer 1 , 2 , but it has not been demonstrated that bacteria have a direct role in the occurrence of oncogenic mutations. Escherichia coli can carry the pathogenicity island pks , which encodes a set of enzymes that synthesize colibactin 3 . This compound is believed to alkylate DNA on adenine residues 4 , 5 and induces double-strand breaks in cultured cells 3 . Here we expose human intestinal organoids to genotoxic pks + E. coli by repeated luminal injection over five months. Whole-genome sequencing of clonal organoids before and after this exposure revealed a distinct mutational signature that was absent from organoids injected with isogenic pks -mutant bacteria. The same mutational signature was detected in a subset of 5,876 human cancer genomes from two independent cohorts, predominantly in colorectal cancer. Our study describes a distinct mutational signature in colorectal cancer and implies that the underlying mutational process results directly from past exposure to bacteria carrying the colibactin-producing pks pathogenicity island. Organoids derived from human intestinal cells that are co-cultured with bacteria carrying the genotoxic pks + island develop a distinct mutational signature associated with colorectal cancer.

ObjectivesPostoperative delirium is a frequent complication with possible detrimental consequences in older hip fracture patients. Music interventions are promising, with positive effects on risk factors for delirium. This study aimed to assess the impact of perioperative music on postoperative delirium in older hip fracture patients.DesignProspective randomised controlled trial.SettingMulticentre study, performed in six participating hospitals in the Netherlands.ParticipantsEligibility criteria included patients aged ≥65 years with an acute hip fracture requiring surgery and documented informed consent. 449 patients were randomised, with a median age of 81 years (IQR 74–87), including 287 women (63.9%).InterventionsMusic group participants received the intervention preoperatively, intraoperatively, and postoperatively twice a day for 30 min. The control group received standard-of-care, supplemented by headphones without music intraoperatively for equal noise reduction in both groups.Primary and secondary outcome measuresThe primary outcome was delirium diagnosis (Diagnostic and Statistical Manual of Mental Disorders, fifth edition), assessed by a geriatrician. Associations were analysed using regression models. Secondary outcomes included: Delirium Observational Score, anxiety, pain and postoperative complications.ResultsIntention-to-treat analysis showed no statistically significant decrease of delirium in the music group, compared with the control group (OR 0.685 (95% CI 0.378 to 1.242); p=0.21). However, in the modified-intention-to-treat analysis, a significant decrease in postoperative delirium was observed (OR 0.478 (95% CI 0.245 to 0.933); p=0.028), which is substantiated by a logistic regression (OR 0.43 (95 % CI 0.19 to 0.98); p=0.045). Also, more postoperative complications were observed in the control group (93 (43.3%); 66 (32.7); p=0.026) in this analysis. The intervention was associated with high patient satisfaction and no adverse events.ConclusionsThis study suggests a positive effect of music interventions on postoperative delirium, which provides additional evidence for considering the implementation of these interventions in hip fracture care.Trial registration numberInternational Clinical Trial Registry Platform, Dutch Trial Register (www.onderzoekmetmensen.nl/, ID:NTR7036).

Analyzing BRCA1/2 tumor pathogenic variants (TPVs) in epithelial tubal/ovarian cancers (EOCs) has become an essential part of the diagnostic workflow in many centers to guide treatment options and genetic cascade testing. However, there is no standardization of testing procedures, including techniques, gene assays, or sequencers used, and data on the execution of tumor tests remains scarce. Therefore, we evaluated characteristics of BRCA1/2 tumor testing in advanced-stage EOC with real-world national data. Pathology reports of patients diagnosed with EOC in 2019 in the Netherlands were obtained from the Dutch Pathology Registry (PALGA), and data regarding histological subtype and BRCA1/2 tumor tests were extracted. A total of 999 patients with advanced-stage EOC were included. Tumor tests were performed for 502 patients (50.2%) and BRCA1/2 TPVs were detected in 14.7%. Of all tests, 48.6% used hybrid capture techniques and 26.5% used PCR-based techniques. More than half of the tests (55.0%) analyzed other genes in addition to BRCA1/2. Overall, this study highlights the heterogeneity in the execution of BRCA1/2 tumor tests. Despite a lack of evidence of quality differences, we emphasize that adequate reporting and internal and external quality monitors are essential for the high-quality implementation and execution of reliable BRCA1/2 tumor testing, which is crucial for identifying all patients with BRCA1/2 TPVs.

Nowadays, tumor tests to analyze DNA in tumor cells from epithelial tubal/ovarian cancers (EOCs) are performed in many centers to detect tumor pathogenic variants (TPVs) in the BRCA1/2 genes. Information on the presence of these TPVs guides treatment options and further genetic testing in patients and relatives. However, there is no standardization of testing procedures, and information about how testing is performed is limited. Therefore, we described how BRCA1/2 tumor testing is performed in 999 EOC patients in the Netherlands in 2019 using real-world clinical data. Tumor tests were performed for 502 patients (50.2%) and TPVs were detected in 14.7% of the tests. This study shows that there is variability in the execution of BRCA1/2 tumor tests, but there were no indications for quality differences. Adequate reporting and quality monitors are essential to ensure that all centers perform reliable tumor tests to ultimately identify all patients with BRCA1/2 TPVs. Analyzing BRCA1/2 tumor pathogenic variants (TPVs) in epithelial tubal/ovarian cancers (EOCs) has become an essential part of the diagnostic workflow in many centers to guide treatment options and genetic cascade testing. However, there is no standardization of testing procedures, including techniques, gene assays, or sequencers used, and data on the execution of tumor tests remains scarce. Therefore, we evaluated characteristics of BRCA1/2 tumor testing in advanced-stage EOC with real-world national data. Pathology reports of patients diagnosed with EOC in 2019 in the Netherlands were obtained from the Dutch Pathology Registry (PALGA), and data regarding histological subtype and BRCA1/2 tumor tests were extracted. A total of 999 patients with advanced-stage EOC were included. Tumor tests were performed for 502 patients (50.2%) and BRCA1/2 TPVs were detected in 14.7%. Of all tests, 48.6% used hybrid capture techniques and 26.5% used PCR-based techniques. More than half of the tests (55.0%) analyzed other genes in addition to BRCA1/2. Overall, this study highlights the heterogeneity in the execution of BRCA1/2 tumor tests. Despite a lack of evidence of quality differences, we emphasize that adequate reporting and internal and external quality monitors are essential for the high-quality implementation and execution of reliable BRCA1/2 tumor testing, which is crucial for identifying all patients with BRCA1/2 TPVs.