Autophagy Contributes to Homeostasis in Esophageal Epithelium Where High Autophagic Vesicle Level Marks Basal Cells With Limited Proliferation and Enhanced Self-Renewal Potential.

BACKGROUND & AIMS: Autophagy plays roles in esophageal pathologies both benign and malignant. Here, we aim to define the role of autophagy in esophageal epithelial homeostasis. METHODS: We generated tamoxifen-inducible, squamous epithelial-specific Atg7 (autophagy related 7) conditional knockout mice to evaluate effects on esophageal homeostasis and response to the carcinogen 4-nitroquinoline 1-oxide (4NQO) using histologic and biochemical analyses. We fluorescence-activated cell sorted esophageal basal cells based on fluorescence of the autophagic vesicle (AV)-identifying dye Cyto-ID and then subjected these cells to transmission electron microscopy, image flow cytometry, three-dimensional organoid assays, RNA sequencing, and cell cycle analysis. Three-dimensional organoids were subjected to passaging, single-cell RNA sequencing, cell cycle analysis, and immunostaining. RESULTS: Genetic autophagy inhibition in squamous epithelium resulted in increased proliferation of esophageal basal cells under homeostatic conditions and also was associated with significant weight loss in mice treated with 4NQO that further displayed perturbed epithelial tissue architecture. Esophageal basal cells with high AV level (Cyto-IDHigh) displayed limited organoid formation capability on initial plating but passaged more efficiently than their counterparts with low AV level (Cyto-IDLow). RNA sequencing suggested increased autophagy in Cyto-IDHigh esophageal basal cells along with decreased cell cycle progression, the latter of which was confirmed by cell cycle analysis. Single-cell RNA sequencing of three-dimensional organoids generated by Cyto-IDLow and Cyto-IDHigh cells identified expansion of 3 cell populations and enrichment of G2/M-associated genes in the Cyto-IDHigh group. Ki67 expression was also increased in organoids generated by Cyto-IDHigh cells, including in basal cells localized beyond the outermost cell layer. CONCLUSIONS: Autophagy contributes to maintenance of the esophageal proliferation-differentiation gradient. Esophageal basal cells with high AV level exhibit limited proliferation and generate three-dimensional organoids with enhanced self-renewal capacity.

Lysyl Oxidase Regulates Epithelial Differentiation and Barrier Integrity in Eosinophilic Esophagitis.

BACKGROUND & AIMS: Epithelial disruption in eosinophilic esophagitis (EoE) encompasses both impaired differentiation and diminished barrier integrity. We have shown that lysyl oxidase (LOX), a collagen cross-linking enzyme, is up-regulated in the esophageal epithelium in EoE. However, the functional roles of LOX in the esophageal epithelium remains unknown. METHODS: We investigated roles for LOX in the human esophageal epithelium using 3-dimensional organoid and air-liquid interface cultures stimulated with interleukin (IL)13 to recapitulate the EoE inflammatory milieu, followed by single-cell RNA sequencing, quantitative reverse-transcription polymerase chain reaction, Western blot, histology, and functional analyses of barrier integrity. RESULTS: Single-cell RNA sequencing analysis on patient-derived organoids revealed that LOX was induced by IL13 in differentiated cells. LOX-overexpressing organoids showed suppressed basal and up-regulated differentiation markers. In addition, LOX overexpression enhanced junctional protein genes and transepithelial electrical resistance. LOX overexpression restored the impaired differentiation and barrier function, including in the setting of IL13 stimulation. Transcriptome analyses on LOX-overexpressing organoids identified an enriched bone morphogenetic protein (BMP) signaling pathway compared with wild-type organoids. In particular, LOX overexpression increased BMP2 and decreased the BMP antagonist follistatin. Finally, we found that BMP2 treatment restored the balance of basal and differentiated cells. CONCLUSIONS: Our data support a model whereby LOX exhibits noncanonical roles as a signaling molecule important for epithelial homeostasis in the setting of inflammation via activation of the BMP pathway in the esophagus. The LOX/BMP axis may be integral in esophageal epithelial differentiation and a promising target for future therapies.

Standardising personalised diabetes care across European health settings: A person-centred outcome set agreed in a multinational Delphi study.

OBJECTIVE: Standardised person-reported outcomes (PRO) data can contextualise clinical outcomes enabling precision diabetes monitoring and care. Comprehensive outcome sets can guide this process, but their implementation in routine diabetes care has remained challenging and unsuccessful at international level. We aimed to address this by developing a person-centred outcome set for Type 1 and Type 2 diabetes, using a methodology with prospects for increased implementability and sustainability in international health settings. METHODS: We used a three-round questionnaire-based Delphi study to reach consensus on the outcome set. We invited key stakeholders from 19 countries via purposive snowball sampling, namely people with diabetes (N = 94), healthcare professionals (N = 65), industry (N = 22) and health authorities (N = 3), to vote on the relevance and measurement frequency of 64 previously identified clinical and person-reported outcomes. Subsequent consensus meetings concluded the study. RESULTS: The list of preliminary outcomes was shortlisted via the consensus process to 46 outcomes (27 clinical outcomes and 19 PROs). Two main collection times were recommended: (1) linked to a medical visit (e.g. diabetes-specific well-being, symptoms and psychological health) and (2) annually (e.g. clinical data, general well-being and diabetes self management-related outcomes). CONCLUSIONS: PROs are often considered in a non-standardised way in routine diabetes care. We propose a person-centred outcome set for diabetes, specifically considering psychosocial and behavioural aspects, which was agreed by four international key stakeholder groups. It guides standardised collection of meaningful outcomes at scale, supporting individual and population level healthcare decision making. It will be implemented and tested in Europe as part of the H2O project.

IGF2BP1/IMP1 Deletion Enhances a Facultative Stem Cell State via Regulation of MAP1LC3B.

BACKGROUND & AIMS: The intestinal epithelium interfaces with a diverse milieu of luminal contents while maintaining robust digestive and barrier functions. Facultative intestinal stem cells are cells that survive tissue injury and divide to re-establish the epithelium. Prior studies have shown autophagic state as functional marker of facultative intestinal stem cells, but regulatory mechanisms are not known. The current study evaluated a post-transcriptional regulation of autophagy as an important factor for facultative stem cell state and tissue regeneration. METHODS: We evaluated stem cell composition, autophagic vesicle content, organoid formation, and in vivo regeneration in mice with intestinal epithelial deletion of the RNA binding protein IGF2 messenger RNA binding protein 1 (IMP1). The contribution of autophagy to resulting in vitro and in vivo phenotypes was evaluated via genetic inactivation of Atg7. Molecular analyses of IMP1 modulation of autophagy at the protein and transcript localization levels were performed using IMP1 mutant studies and single-molecule fluorescent in situ hybridization. RESULTS: Epithelial Imp1 deletion reduced leucine rich repeat containing G protein coupled receptor 5 cell frequency but enhanced both organoid formation efficiency and in vivo regeneration after irradiation. We confirmed prior studies showing increased autophagy with IMP1 deletion. Deletion of Atg7 reversed the enhanced regeneration observed with Imp1 deletion. IMP1 deletion or mutation of IMP1 phosphorylation sites enhanced expression of essential autophagy protein microtubule-associated protein 1 light chain 3ß. Furthermore, immunofluorescence imaging coupled with single-molecule fluorescent in situ hybridization showed IMP1 colocalization with MAP1LC3B transcripts at homeostasis. Stress induction led to decreased colocalization. CONCLUSIONS: Depletion of IMP1 enhances autophagy, which promotes intestinal regeneration via expansion of facultative intestinal stem cells.

Autophagy contributes to homeostasis in esophageal epithelium where high autophagic vesicle content marks basal cells with limited proliferation and enhanced self-renewal potential.

Background & Aims: Autophagy has been demonstrated to play roles in esophageal pathologies both benign and malignant. Here, we aim to define the role of autophagy in esophageal epithelium under homeostatic conditions. Methods: We generated tamoxifen-inducible, squamous epithelial-specific Atg7 (autophagy related 7) conditional knockout mice to evaluate effects on esophageal homeostasis and response to the carcinogen 4-nitroquinoline 1-oxide (4NQO) using histological and biochemical analyses. We FACS sorted esophageal basal cells based upon fluorescence of the autophagic vesicle (AV)-identifying dye Cyto-ID, then subjected these cells to transmission electron microscopy, image flow cytometry, 3D organoid assays, RNA-Sequencing (RNA-Seq), and cell cycle analysis. 3D organoids were subjected to passaging, single cell (sc) RNA-Seq, cell cycle analysis, and immunostaining. Results: Genetic autophagy inhibition in squamous epithelium resulted in increased proliferation of esophageal basal cells. Esophageal basal cells with high AV level (Cyto-ID High ) displayed limited organoid formation capability upon initial plating but passaged more efficiently than their counterparts with low AV level (Cyto-ID Low ). RNA-Seq suggested increased autophagy in Cyto- ID High esophageal basal cells along with decreased cell cycle progression, the latter of which was confirmed by cell cycle analysis. scRNA-Seq of 3D organoids generated by Cyto-ID Low and Cyto- ID High cells identified expansion of 3 cell populations, enrichment of G2/M-associated genes, and aberrant localization of cell cycle-associated genes beyond basal cell populations in the Cyto- ID High group. Ki67 expression was also increased in organoids generated by Cyto-ID High cells, including in cells beyond the basal cell layer. Squamous epithelial-specific autophagy inhibition induced significant weight loss in mice treated with 4NQO that further displayed perturbed epithelial tissue architecture. Conclusions: High AV level identifies esophageal epithelium with limited proliferation and enhanced self-renewal capacity that contributes to maintenance of the esophageal proliferation- differentiation gradient in vivo .

Intestinal transit-amplifying cells require METTL3 for growth factor signaling and cell survival.

Intestinal epithelial transit-amplifying cells are essential stem progenitors required for intestinal homeostasis, but their rapid proliferation renders them vulnerable to DNA damage from radiation and chemotherapy. Despite these cells' critical roles in intestinal homeostasis and disease, few studies have described genes that are essential to transit-amplifying cell function. We report that RNA methyltransferase-like 3 (METTL3) is required for survival of transit-amplifying cells in the murine small intestine. Transit-amplifying cell death after METTL3 deletion was associated with crypt and villus atrophy, loss of absorptive enterocytes, and uniform wasting and death in METTL3-depleted mice. Sequencing of polysome-bound and methylated RNAs in enteroids and in vivo demonstrated decreased translation of hundreds of methylated transcripts after METTL3 deletion, particularly transcripts involved in growth factor signal transduction such as Kras. Further investigation verified a relationship between METTL3 and Kras methylation and protein levels in vivo. Our study identifies METTL3 as an essential factor supporting the homeostasis of small intestinal tissue via direct maintenance of transit-amplifying cell survival. We highlight the crucial role of RNA modifications in regulating growth factor signaling in the intestine with important implications for both homeostatic tissue renewal and epithelial regeneration.

Which diabetes specific patient reported outcomes should be measured in routine care? A systematic review to inform a core outcome set for adults with Type 1 and 2 diabetes mellitus: The European Health Outcomes Observatory (H2O) programme.

OBJECTIVES: The objective was to identify candidate patient reported outcomes with potential to inform individual patient care and service development for inclusion in a digital outcome set to be collected in routine care, as part of an international project to enhance care outcomes for people with diabetes. METHODS: PubMed, COSMIN and COMET databases were searched. Published studies were included if they recommended patient reported outcomes that were clinically useful and/or important to people with diabetes. To aid selection decisions, recommended outcomes were considered in terms of the evidence endorsing them and their importance to people with diabetes. RESULTS: Twenty-seven studies recommending 53 diabetes specific outcomes, and patient reported outcome measures, were included. The outcomes reflected the experience of living with diabetes (e.g. psychological well-being, symptom experience, health beliefs and stigma) and behaviours (e.g. self-management). Diabetes distress and self-management behaviours were most endorsed by the evidence. CONCLUSIONS: The review provides a comprehensive list of candidate outcomes endorsed by international evidence and informed by existing outcome sets, and suggestions for measures. PRACTICE IMPLICATIONS: The review offers evidence to guide clinical application. Integrated measurement of these outcomes in care settings holds enormous potential to improve provision of care and outcomes in diabetes.

Patient-derived Colonoids From Disease-spared Tissue Retain Inflammatory Bowel Disease-specific Transcriptomic Signatures.

BACKGROUND AND AIMS: A key histopathological feature of inflammatory bowel disease is damage to the mucosa, including breakdown of the epithelial barrier. Human enteroids and colonoids are a critical bench-to-bedside tool for studying the epithelium in inflammatory bowel disease. The goal of the current study was to define transcriptional differences in healthy versus diseased subjects that are sustained in enteroids and colonoids, including from disease-spared tissue. METHODS: Biopsies and matching enteroid or colonoid cultures from pediatric patients with ileal Crohn disease (N = 6) and control subjects (N = 17) were subjected to RNA sequencing followed by bioinformatic and machine learning analyses. Late passage enteroids were exposed to cytokines to assess durable transcriptional differences. RESULTS: We observed substantial overlap of pathways upregulated in Crohn disease in enteroids and ileal biopsies, as well as colonoids and rectal biopsies. KEGG pathways for cytokine-cytokine receptor interaction, chemokine signaling, protein export, and Toll-like receptor signaling were upregulated in both ileal and rectal biopsies, as well as enteroids and colonoids. In vitro cytokine exposure reactivated genes previously increased in biopsies. Machine learning predicted biopsy location (100% accuracy) and donor disease status (83% accuracy). A random forest classifier generated using ileal enteroids identified rectal colonoids from ileal Crohn disease subjects with 80% accuracy. CONCLUSION: We confirmed transcriptional profiles of Crohn disease biopsies are expressed in enteroids and colonoids. Furthermore, transcriptomic data from disease-spared rectal tissue can identify patients with ileal Crohn disease. Our data support the use of patient enteroids and colonoids as critical translational tools for the study of inflammatory bowel disease.

Isolation of Epithelial and Stromal Cells from Colon Tissues in Homeostasis and Under Inflammatory Conditions.

Inflammation of the gastrointestinal tract is a prevalent pathology in diseases such as inflammatory bowel disease (IBD). Currently, there are no therapies to prevent IBD, and available therapies to treat IBD are often sub-optimal. Thus, an unmet need exists to better understand the molecular mechanisms underlying intestinal tissue responses to damage and regeneration. The recent development of single-cell RNA (sc-RNA) sequencing-based techniques offers a unique opportunity to shed light on novel signaling pathways and cellular states that govern tissue adaptation or maladaptation across a broad spectrum of diseases. These approaches require the isolation of high-quality cells from tissues for downstream transcriptomic analyses. In the context of intestinal biology, there is a lack of protocols that ensure the isolation of epithelial and non-epithelial compartments simultaneously with high-quality yield. Here, we report two protocols for the isolation of epithelial and stromal cells from mouse and human colon tissues under inflammatory conditions. Specifically, we tested the feasibility of the protocols in a mouse model of dextran sodium sulfate (DSS)-induced colitis and in human biopsies from Crohn's patients. We performed sc-RNA sequencing analysis and demonstrated that the protocol preserves most of the epithelial and stromal cell types found in the colon. Moreover, the protocol is suitable for immunofluorescence staining of surface markers for epithelial, stromal, and immune cell lineages for flow cytometry analyses. This optimized protocol will provide a new resource for scientists to study complex tissues such as the colon in the context of tissue damage and regeneration. Key features • This protocol allows the isolation of epithelial and stromal cells from colon tissues. • The protocol has been optimized for tissues under inflammatory conditions with compromised cell viability. • This protocol is suitable for experimental mouse models of colon inflammation and human biopsies.

Intestinal transit amplifying cells require METTL3 for growth factor signaling, KRAS expression, and cell survival.

Intestinal epithelial transit amplifying cells are essential stem progenitors required for intestinal homeostasis, but their rapid proliferation renders them vulnerable to DNA damage from radiation and chemotherapy. Despite their critical roles in intestinal homeostasis and disease, few studies have described genes that are essential to transit amplifying cell function. We report that the RNA methyltransferase, METTL3, is required for survival of transit amplifying cells in the murine small intestine. Transit amplifying cell death after METTL3 deletion was associated with crypt and villus atrophy, loss of absorptive enterocytes, and uniform wasting and death in METTL3-depleted mice. Ribosome profiling and sequencing of methylated RNAs in enteroids and in vivo demonstrated decreased translation of hundreds of unique methylated transcripts after METTL3 deletion, particularly transcripts involved in growth factor signal transduction such as Kras. Further investigation confirmed a novel relationship between METTL3 and Kras methylation and protein levels in vivo. Our study identifies METTL3 as an essential factor supporting the homeostasis of small intestinal tissue via direct maintenance of transit amplifying cell survival. We highlight the crucial role of RNA modifications in regulating growth factor signaling in the intestine, with important implications for both homeostatic tissue renewal and epithelial regeneration.

Lysyl oxidase regulates epithelial differentiation and barrier integrity in eosinophilic esophagitis.

Background & Aims: Epithelial disruption in eosinophilic esophagitis (EoE) encompasses both impaired differentiation and diminished barrier integrity. We have shown that lysyl oxidase (LOX), a collagen cross-linking enzyme, is upregulated in the esophageal epithelium in EoE. However, the functional roles of LOX in the esophageal epithelium remains unknown. Methods: We investigated roles for LOX in the human esophageal epithelium using 3-dimensional organoid and air-liquid interface cultures stimulated with interleukin (IL)-13 to recapitulate the EoE inflammatory milieu, followed by single-cell RNA sequencing, quantitative reverse transcription-polymerase chain reaction, western blot, histology, and functional analyses of barrier integrity. Results: Single-cell RNA sequencing analysis on patient-derived organoids revealed that LOX was induced by IL-13 in differentiated cells. LOX-overexpressing organoids demonstrated suppressed basal and upregulated differentiation markers. Additionally, LOX overexpression enhanced junctional protein genes and transepithelial electrical resistance. LOX overexpression restored the impaired differentiation and barrier function, including in the setting of IL-13 stimulation. Transcriptome analyses on LOX-overexpressing organoids identified enriched bone morphogenetic protein (BMP) signaling pathway compared to wild type organoids. Particularly, LOX overexpression increased BMP2 and decreased BMP antagonist follistatin. Finally, we found that BMP2 treatment restored the balance of basal and differentiated cells. Conclusions: Our data support a model whereby LOX exhibits non-canonical roles as a signaling molecule important for epithelial homeostasis in the setting of inflammation via activation of BMP pathway in esophagus. The LOX/BMP axis may be integral in esophageal epithelial differentiation and a promising target for future therapies.

Intestinal epithelial autophagy is required for the regenerative benefit of calorie restriction.

Calorie restriction can enhance the regenerative capacity of the injured intestinal epithelium. Among other metabolic changes, calorie restriction can activate the autophagy pathway. Although independent studies have attributed the regenerative benefit of calorie restriction to downregulation of mTORC1, it is not known whether autophagy itself is required for the regenerative benefit of calorie restriction. We used mouse and organoid models with autophagy gene deletion to evaluate the contribution of autophagy to intestinal epithelial regeneration following calorie restriction. In the absence of injury, mice with intestinal epithelial-specific deletion of autophagy gene Atg7 (Atg7ΔIEC) exhibit weight loss and histological changes similar to wild-type mice following calorie restriction. Conversely, calorie-restricted Atg7ΔIEC mice displayed a significant reduction in regenerative crypt foci after irradiation compared with calorie-restricted wild-type mice. Targeted analyses of tissue metabolites in calorie-restricted mice revealed an association between calorie restriction and reduced glycocholic acid (GCA) in wild-type mice but not in Atg7ΔIEC mice. To evaluate whether GCA can directly modulate epithelial stem cell self-renewal, we performed enteroid formation assays with or without GCA. Wild-type enteroids exhibited reduced enteroid formation efficiency in response to GCA treatment, suggesting that reduced availability of GCA during calorie restriction may be one mechanism by which calorie restriction favors epithelial regeneration in a manner dependent upon epithelial autophagy. Taken together, our data support the premise that intestinal epithelial Atg7 is required for the regenerative benefit of calorie restriction, due in part to its role in modulating luminal GCA with direct effects on epithelial stem cell self-renewal.NEW & NOTEWORTHY Calorie restriction is associated with enhanced intestinal regeneration after irradiation, but the requirement of autophagy for this process is not known. Our data support the premise that intestinal epithelial autophagy is required for the regenerative benefit of calorie restriction. We also report that luminal levels of primary bile acid glycocholic acid are modulated by epithelial cell autophagy during calorie restriction with direct effects on epithelial stem cell function.

High autophagic vesicle content marks facultative stem cells of the gut.

Understanding how macroautophagy/autophagy contributes to tissue homeostasis is essential for understanding organismal health. The intestinal epithelium is an ideal model to define mechanisms that regulate tissue homeostasis because it houses well-defined populations of intestinal stem cells. Active intestinal stem cells (a-ISCs) are defined by their active cycling and self-renewal during homeostasis, which supports continual tissue turnover in vivo. In vitro, this is observed as long-term organoid formation capacity. A second population of stem cells, called "facultative intestinal stem cells" (f-ISCs), are defined by their ability to 1) survive tissue damage that depletes the injury-sensitive a-ISCs and 2) reenter the cell cycle to repopulate the a-ISC compartment and regenerate the epithelium. The prospective identification of f-ISCs has been challenging, as cells expressing markers of multiple differentiated lineages, particularly secretory lineages, appear to function as f-ISCs in diverse injury contexts. We evaluated cell age (defined as time elapsed after cell cycle exit) and autophagic state (marked by autophagic vesicle content) as molecular features that may be related to f-ISC capacity. We found that autophagic state, but not cell age, prospectively identifies f-ISCs within multiple lineages. As such, we describe autophagy as a lineage-agnostic marker of f-ISC capacity in the mammalian intestine.

Spatial transcriptomics add a new dimension to our understanding of the gut.

The profound complexity of the intestinal mucosa demands a spatial approach to the study of gut transcriptomics. Although single-cell RNA sequencing has revolutionized our ability to survey the diverse cell types of the intestine, knowledge of cell type alone cannot fully describe the cells that make up the intestinal mucosa. During homeostasis and disease, dramatic gradients of oxygen, nutrients, extracellular matrix proteins, morphogens, and microbiota collectively dictate intestinal cell state, and only spatial techniques can articulate differences in cellular transcriptomes at this level. Spatial transcriptomic techniques assign transcriptomic data to precise regions in a tissue of interest. In recent years, these protocols have become increasingly accessible, and their application in the intestinal mucosa has exploded in popularity. In the gut, spatial transcriptomics typically involve the application of tissue sections to spatially barcoded RNA sequencing or laser capture microdissection followed by RNA sequencing. In combination with single-cell RNA sequencing, these spatial sequencing approaches allow for the construction of spatial transcriptional maps at pseudosingle-cell resolution. In this review, we describe the spatial transcriptomic technologies recently applied in the gut and the previously unattainable discoveries that they have produced.

Geometric engineering of organoid culture for enhanced organogenesis in a dish.

Here, we introduce a facile, scalable engineering approach to enable long-term development and maturation of organoids. We have redesigned the configuration of conventional organoid culture to develop a platform that converts single injections of stem cell suspensions to radial arrays of organoids that can be maintained for extended periods without the need for passaging. Using this system, we demonstrate accelerated production of intestinal organoids with significantly enhanced structural and functional maturity, and their continuous development for over 4 weeks. Furthermore, we present a patient-derived organoid model of inflammatory bowel disease (IBD) and its interrogation using single-cell RNA sequencing to demonstrate its ability to reproduce key pathological features of IBD. Finally, we describe the extension of our approach to engineer vascularized, perfusable human enteroids, which can be used to model innate immune responses in IBD. This work provides an immediately deployable platform technology toward engineering more realistic organ-like structures in a dish.

IMP1/IGF2BP1 in human colorectal cancer extracellular vesicles.

Colorectal cancer (CRC) is a leading cause of cancer-related death. There is an urgent need for new methods of early CRC detection and monitoring to improve patient outcomes. Extracellular vesicles (EVs) are secreted, lipid-bilayer bound, nanoparticles that carry biological cargo throughout the body and in turn exhibit cancer-related biomarker potential. RNA binding proteins (RBPs) are posttranscriptional regulators of gene expression that may provide a link between host cell gene expression and EV phenotypes. Insulin-like growth factor 2 RNA binding protein 1 (IGF2BP1/IMP1) is an RBP that is highly expressed in CRC with higher levels of expression correlating with poor prognosis. IMP1 binds and potently regulates tumor-associated transcripts that may impact CRC EV phenotypes. Our objective was to test whether IMP1 expression levels impact EV secretion and/or cargo. We used RNA sequencing, in vitro CRC cell lines, ex vivo colonoid models, and xenograft mice to test the hypothesis that IMP1 influences EV secretion and/or cargo in human CRC. Our data demonstrate that IMP1 modulates the RNA expression of transcripts associated with extracellular vesicle pathway regulation, but it has no effect on EV secretion levels in vitro or in vivo. Rather, IMP1 appears to affect EV regulation by directly entering EVs in a transformation-dependent manner. These findings suggest that IMP1 has the ability to shape EV cargo in human CRC, which could serve as a diagnostic/prognostic circulating tumor biomarker.NEW & NOTEWORTHY This work demonstrates that the RNA binding protein IGF2BP1/IMP1 alters the transcript profile of colorectal cancer cell (CRC) mRNAs from extracellular vesicle (EV) pathways. IMP1 does not alter EV production or secretion in vitro or in vivo, but rather enters CRC cells where it may further impact EV cargo. Our work shows that IMP1 has the ability to shape EV cargo in human CRC, which could serve as a diagnostic/prognostic circulating tumor biomarker.

Autophagic state prospectively identifies facultative stem cells in the intestinal epithelium.

The intestinal epithelium exhibits a rapid and efficient regenerative response to injury. Emerging evidence supports a model where plasticity of differentiated cells, particularly those in the secretory lineages, contributes to epithelial regeneration upon ablation of injury-sensitive stem cells. However, such facultative stem cell activity is rare within secretory populations. Here, we ask whether specific functional properties predict facultative stem cell activity. We utilize in vivo labeling combined with ex vivo organoid formation assays to evaluate how cell age and autophagic state contribute to facultative stem cell activity within secretory lineages. Strikingly, we find that cell age (time elapsed since cell cycle exit) does not correlate with secretory cell plasticity. Instead, high autophagic vesicle content predicts plasticity and resistance to DNA damaging injury independently of cell lineage. Our findings indicate that autophagic status prior to injury serves as a lineage-agnostic marker for the prospective identification of facultative stem cells.

The phagosomal solute transporter SLC15A4 promotes inflammasome activity via mTORC1 signaling and autophagy restraint in dendritic cells.

Phagocytosis is the necessary first step to sense foreign microbes or particles and enables activation of innate immune pathways such as inflammasomes. However, the molecular mechanisms underlying how phagosomes modulate inflammasome activity are not fully understood. We show that in murine dendritic cells (DCs), the lysosomal histidine/peptide solute carrier transporter SLC15A4, associated with human inflammatory disorders, is recruited to phagosomes and is required for optimal inflammasome activity after infectious or sterile stimuli. Dextran sodium sulfate-treated SLC15A4-deficient mice exhibit decreased colon inflammation, reduced IL-1ß production by intestinal DCs, and increased autophagy. Similarly, SLC15A4-deficient DCs infected with Salmonella typhimurium show reduced caspase-1 cleavage and IL-1ß production. This correlates with peripheral NLRC4 inflammasome assembly and increased autophagy. Overexpression of constitutively active mTORC1 rescues decreased IL-1ß levels and caspase1 cleavage, and restores perinuclear inflammasome positioning. Our findings support that SLC15A4 couples phagocytosis with inflammasome perinuclear assembly and inhibition of autophagy through phagosomal content sensing. Our data also reveal the previously unappreciated importance of mTORC1 signaling pathways to promote and sustain inflammasome activity.

Leaving No One Behind: Interventions and Outcomes of the COVID-19 Vaccine Maximising Uptake Programme.

The devastating impact of COVID-19 on individuals and communities has accelerated the development of vaccines and the deployment of ambitious vaccination programmes to reduce the risks of infection, infection transmission and symptom severity. However, many people delay or refuse to get vaccinated against COVID-19, for many complex reasons. Vaccination programmes that are tailored to address individual and communities' COVID-19 concerns can improve vaccine uptake rates and help achieve the required herd-immunity threshold. The Maximising Uptake Programme has led to the vaccination of 7979 people from February-August 2021 in the South West of England, UK, who are at high risk of severe illness from COVID-19 and/or may not access the COVID-19 vaccines through mass vaccination centres and general practices. These include: people experiencing homelessness; non-English-speaking people; people from minority ethnic groups; refugees and asylum seekers; Gypsy, Roma, Travelers and boat people; and those who are less able to access vaccination centres, such as people with learning difficulties, serious mental illness, drug and alcohol dependence, people with physical and sensory impairment, and people with dementia. Outreach work coupled with a targeted communication and engagement campaign, co-designed with community leaders and influencers, have led to significant engagement and COVID-19 vaccine uptake among the target populations.