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BACKGROUND: Despite intensive research, the etiology of acute anterior uveitis (AAU) remains poorly defined. Infection with gram-negative bacteria such as Yersinia, Salmonella, Shigella, and Chlamydia have already been suggested as a possible trigger event for AAU. Helicobacter pylori is also a gram-negative bacterium, shares the lipopolysaccharides, but did not attract the attention of many ophthalmologists until recently. Having in mind the relatively high incidence of H. pylori infection in the population, we propose that H. pylori may also be a trigger factor for AAU. PATIENTS AND METHODS: The presence of anti-H. pylori antibodies in matching serum and aqueous humor samples of 15 idiopathic AAU patients was determined using a commercial Western blot assay. Control serum and aqueous humor were obtained from five patients undergoing cataract surgery. RESULTS: Six out of 15 AAU patients (40%) were serum-positive for H. pylori, and half of these (n = 3) also had anti-H. pylori antibodies in the aqueous humor. All five aqueous humor and sera controls tested negative for H. pylori infection. CONCLUSION: These are the first results demonstrating anti-H. pylori antibodies in the aqueous humor of AAU patients. Further studies are needed to demonstrate whether this antibody is indeed locally produced. Our data may provide first evidence for a causative link between H. pylori infection and AAU. [ABSTRACT FROM AUTHOR]

Introduction: Cervical cancer represents one of the main causes of female, cancer-related mortality worldwide. The majority of cancers are caused by human papillomaviruses such as HPV16 and HPV18. As chemotherapeutic resistance to first-line platinum treatment is still a predominant clinical challenge in advanced cervical cancer, novel treatment options including combinatorial therapies are urgently required to overcome chemotherapeutic resistance. Inhibition of A Disintegrin And Metalloproteinase (ADAM)-family members, heavily involved in tumour progression of a vast range of solid tumours, strongly improved response to chemotherapeutic treatment in other tumour entities including ovarian cancer., Methods: We established two- and three-dimensional models derived from three traditional cervical cancer cell lines and ectocervical cancer-derived organoids. Following characterisation, these models were used to investigate their response to cisplatin treatment in the absence and presence of ADAM inhibitors using viability assays and automated live cell imaging., Results: The pivotal role of the metalloprotease ADAM17 driving chemotherapy resistance was detectable in all ectocervical cultures irrespective of the model system used, whereas ADAM10 inhibition was predominantly effective only in loosely aggregated spheroids. We showed prominent differences regarding treatment responses between 2D monolayers compared to 3D spheroid and 3D organoid model systems. Particularly, the organoid system, regarded as the closest representation of primary tumours, exhibited reliably the combinatorial effect of ADAM17 inhibition and cisplatin in all three individual donors., Discussion: As two- and three-dimensional models of the same cell lines differ in their responses to chemotherapy it is essential to validate treatment strategies in more advanced model systems representing the patient situation more realistically. Ectocervical organoids showed reliable results regarding treatment responses closely mimicking the primary tumours and could therefore serve as an important tool for personalized medicine in cervical cancer. These findings strengthen the role of ADAM17 as a potential novel target for combinatorial treatments to overcome chemoresistance in cervical cancer., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Holthaus, Rogmans, Gursinski, Quevedo-Olmos, Ehsani, Mangler, Flörkemeier, Weimer, Meyer, Maass, Bauerschlag and Hedemann.)

Background: Gastric intestinal metaplasia (GIM) is an essential precancerous lesion. Although the reversal of GIM is challenging, it potentially brings a state-to-art strategy for gastric cancer therapeutics (GC). The lack of the appropriate in vitro model limits studies of GIM pathogenesis, which is the issue this work aims to address for further studies., Method: The air-liquid interface (ALI) model was adopted for the long-term culture of GIM cells in the present work. This study conducted Immunofluorescence (IF), quantitative real-time polymerase chain reaction (qRT-PCR), transcriptomic sequencing, and mucoproteomic sequencing (MS) techniques to identify the pathways for differential expressed genes (DEGs) enrichment among different groups, furthermore, to verify novel biomarkers of GIM cells., Result: Our study suggests that GIM-ALI model is analog to the innate GIM cells, which thus can be used for mucus collection and drug screening. We found genes MUC17, CDA, TRIM15, TBX3, FLVCR2, ONECUT2, ACY3, NMUR2, and MAL2 were highly expressed in GIM cells, while GLDN, SLC5A5, MAL, and MALAT1 showed down-regulated, which can be used as potential biomarkers for GIM cells. In parallel, these genes that highly expressed in GIM samples were mainly involved in cancer-related pathways, such as the MAPK signal pathway and oxidative phosphorylation signal pathway., Conclusion: The ALI model is validated for the first time for the in vitro study of GIM. GIM-ALI model is a novel in vitro model that can mimic the tissue micro-environment in GIM patients and further provide an avenue for studying the characteristics of GIM mucus. Our study identified new markers of GIM as well as pathways associated with GIM, which provides outstanding insight for exploring GIM pathogenesis and potentially other related conditions., (© 2024. The Author(s).)

Introduction: Helicobacter pylori (H. pylori) infection has been associated with gastric carcinogenesis. However, the precise involvement of LRP8, the low-density lipoprotein receptor-related protein 8, in H. pylori pathogenesis and gastric cancer (GC) remains poorly understood., Objectives: To investigate the potential role of LRP8 in H. pylori infection and gastric carcinogenesis., Methods: Three-dimensional human-derived gastric organoids (hGO) and gastric cancer organoids (hGCO) were synthesized from the tissues obtained from human donors. In this work, multi-omics combined with in vivo and in vitro studies were conducted to investigate the potential involvement of LRP8 in H. pylori-induced GC., Results: We found that H. pylori infection significantly upregulated the expression of LRP8 in human GC tissues, cells, organoids, and mouse gastric mucous. In particular, LRP8 exhibited a distinct enrichment in cancer stem cells (CSC). Functionally, silencing of LRP8 affected the formation and proliferation of tumor spheroids, while increased expression of LRP8 was associated with increased proliferation and stemness of GC cells and organoids. Mechanistically, LRP8 promotes the binding of E-cadherin to β-catenin, thereby promoting nuclear translocation and transcriptional activity of β-catenin. Furthermore, LRP8 interacts with the cytotoxin-associated gene A (CagA) to form the CagA/LRP8/β-catenin complex. This complex further amplifies H. pylori-induced β-catenin nuclear translocation, leading to increased transcription of inflammatory factors and CSC markers. Clinical analysis demonstrated that abnormal overexpression of LRP8 is correlated with a poor prognosis and resistance to 5-Fluorouracil in patients with GC., Conclusion: Our findings provide valuable information on the molecular intricacies of H. pylori-induced gastric carcinogenesis, offering potential therapeutic targets and prognostic markers for GC., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Production and hosting by Elsevier B.V.)

The gastroesophageal squamocolumnar junction (GE-SCJ) is a critical tissue interface between the esophagus and stomach, with significant relevance in the pathophysiology of gastrointestinal diseases. Despite this, the molecular mechanisms underlying GE-SCJ development remain unclear. Using single-cell transcriptomics, organoids, and spatial analysis, we examine the cellular heterogeneity and spatiotemporal dynamics of GE-SCJ development from embryonic to adult mice. We identify distinct transcriptional states and signaling pathways in the epithelial and mesenchymal compartments of the esophagus and stomach during development. Fibroblast-epithelial interactions are mediated by various signaling pathways, including WNT, BMP, TGF-β, FGF, EGF, and PDGF. Our results suggest that fibroblasts predominantly send FGF and TGF-β signals to the epithelia, while epithelial cells mainly send PDGF and EGF signals to fibroblasts. We observe differences in the ligands and receptors involved in cell-cell communication between the esophagus and stomach. Our findings provide insights into the molecular mechanisms underlying GE-SCJ development and fibroblast-epithelial crosstalk involved, paving the way to elucidate mechanisms during adaptive metaplasia development and carcinogenesis., (© 2024. The Author(s).)

Copper is an essential trace element for the human body, and its requirement for optimistic immune functions has been recognized for decades. How copper is involved in the innate immune pathway, however, remains to be clarified. Here, we report that copper serves as a signal molecule to regulate the kinase activity of alpha-kinase 1 (ALPK1), a cytosolic pattern-recognition receptor (PRR), and therefore promotes host cell defense against bacterial infection. We show that in response to infection, host cells actively accumulate copper in the cytosol, and the accumulated cytosolic copper enhances host cell defense against evading pathogens, including intracellular and, unexpectedly, extracellular bacteria. Subsequently, we demonstrate that copper activates the innate immune pathway of host cells in an ALPK1-dependent manner. Further mechanistic studies reveal that copper binds to ALPK1 directly and is essential for the kinase activity of this cytosolic PRR. Moreover, the binding of copper to ALPK1 enhances the sensitivity of ALPK1 to the bacterial metabolite ADP-heptose and eventually prompts host cells to elicit an enhanced immune response during bacterial infection. Finally, using a zebrafish in vivo model, we show that a copper-treated host shows an increased production of proinflammatory cytokines, enhanced recruitment of phagosome cells, and promoted bacterial clearance. Our findings uncover a previously unrecognized role of copper in the modulation of host innate immune response against bacterial pathogens and advance our knowledge on the cross talk between cytosolic copper homeostasis and immune system., Competing Interests: Competing interests statement:The authors declare no competing interest.

The mucus serves as a protective barrier in the gastrointestinal tract against microbial attacks. While its role extends beyond merely being a physical barrier, the extent of its active bactericidal properties remains unclear, and the mechanisms regulating these properties are not yet understood. We propose that inflammation induces epithelial cells to secrete antimicrobial peptides, transforming mucus into an active bactericidal agent. To investigate the properties of mucus, we previously developed mucosoid culture models that mimic the healthy human stomach epithelium. Similar to organoids, mucosoids are stem cell-driven cultures; however, the cells are cultivated on transwells at air-liquid interface. The epithelial cells of mucosoids form a polarized monolayer, allowing differentiation into all stomach lineages, including mucus-secreting cells. This setup facilitates the secretion and accumulation of mucus on the apical side of the mucosoids, enabling analysis of its bactericidal effects and protein composition, including antimicrobial peptides. Our findings show that TNFα, IL1β, and IFNγ induce the secretion of antimicrobials such as lactotransferrin, lipocalin2, complement component 3, and CXCL9 into the mucus. This antimicrobial-enriched mucus can partially eliminate Helicobacter pylori , a key stomach pathogen. The bactericidal activity depends on the concentration of each antimicrobial and their gene expression is higher in patients with inflammation and H.pylori -associated chronic gastritis. However, we also find that H. pylori infection can reduce the expression of antimicrobial encoding genes promoted by inflammation. These findings suggest that controlling antimicrobial secretion in the mucus is a critical component of epithelial immunity. However, pathogens like H. pylori can overcome these defenses and survive in the mucosa.

Cervical cancer is a leading cause of death among women globally, primarily driven by high-risk papillomaviruses. However, the effectiveness of chemotherapy is limited, underscoring the potential of personalized immunotherapies. Patient-derived organoids, which possess cellular heterogeneity, proper epithelial architecture and functionality, and long-term propagation capabilities offer a promising platform for developing viable strategies. In addition to αβ T cells and natural killer (NK) cells, γδ T cells represent an immune cell population with significant therapeutic potential against both hematologic and solid tumours. To evaluate the efficacy of γδ T cells in cervical cancer treatment, we generated patient-derived healthy and cancer ectocervical organoids. Furthermore, we examined transformed healthy organoids, expressing HPV16 oncogenes E6 and E7. We analysed the effector function of in vitro expanded γδ T cells upon co-culture with organoids. Our findings demonstrated that healthy cervical organoids were less susceptible to γδ T cell-mediated cytotoxicity compared to HPV-transformed organoids and cancerous organoids. To identify the underlying pathways involved in this observed cytotoxicity, we performed bulk-RNA sequencing on the organoid lines, revealing differences in DNA-damage and cell cycle checkpoint pathways, as well as transcription of potential γδ T cell ligands. We validated these results using immunoblotting and flow cytometry. We also demonstrated the involvement of BTN3A1 and BTN2A1, crucial molecules for γδ T cell activation, as well as differential expression of PDL1/CD274 in cancer, E6/E7+ and healthy organoids. Interestingly, we observed a significant reduction in cytotoxicity upon blocking MSH2, a protein involved in DNA mismatch-repair. In summary, we established a co-culture system of γδ T cells with cervical cancer organoids, providing a novel in vitro model to optimize innovative patient-specific immunotherapies for cervical cancer., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Dong, Holthaus, Peters, Koster, Ehsani, Quevedo-Olmos, Berger, Zarobkiewicz, Mangler, Gurumurthy, Hedemann, Chumduri, Kabelitz and Meyer.)

Background: Epigenetic modifications in mammalian DNA are commonly manifested by DNA methylation. In the stomach, altered DNA methylation patterns have been observed following chronic Helicobacter pylori infections and in gastric cancer. In the context of epigenetic regulation, the regional nature of the stomach has been rarely considered in detail., Results: Here, we establish gastric mucosa derived primary cell cultures as a reliable source of native human epithelium. We describe the DNA methylation landscape across the phenotypically different regions of the healthy human stomach, i.e., antrum, corpus, fundus together with the corresponding transcriptomes. We show that stable regional DNA methylation differences translate to a limited extent into regulation of the transcriptomic phenotype, indicating a largely permissive epigenetic regulation. We identify a small number of transcription factors with novel region-specific activity and likely epigenetic impact in the stomach, including GATA4, IRX5, IRX2, PDX1 and CDX2. Detailed analysis of the Wnt pathway reveals differential regulation along the craniocaudal axis, which involves non-canonical Wnt signaling in determining cell fate in the proximal stomach. By extending our analysis to pre-neoplastic lesions and gastric cancers, we conclude that epigenetic dysregulation characterizes intestinal metaplasia as a founding basis for functional changes in gastric cancer. We present insights into the dynamics of DNA methylation across anatomical regions of the healthy stomach and patterns of its change in disease. Finally, our study provides a well-defined resource of regional stomach transcription and epigenetics., (© 2022. The Author(s).)

Crosstalk within the gastric epithelium, which is closely in contact with stromal fibroblasts in the gastric mucosa, has a pivotal impact in proliferation, differentiation and transformation of the gastric epithelium. The human pathogen Helicobacter pylori colonises the gastric epithelium and represents a risk factor for gastric pathophysiology. Infection of H. pylori induces the activation of the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), which is involved in the pro-inflammatory response but also in cell survival. In co-cultures with human gastric fibroblasts (HGF), we found that apoptotic cell death is reduced in the polarised human gastric cancer cell line NCI-N87 or in gastric mucosoids during H. pylori infection. Interestingly, suppression of apoptotic cell death in NCI-N87 cells involved an enhanced A20 expression regulated by NF-κB activity in response to H. pylori infection. Moreover, A20 acts as an important negative regulator of caspase-8 activity, which was suppressed in NCI-N87 cells during co-culture with gastric fibroblasts. Our results provide evidence for NF-κB-dependent regulation of apoptotic cell death in cellular crosstalk and highlight the protective role of gastric fibroblasts in gastric epithelial cell death during H. pylori infection., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Michael Naumann reports financial support was provided by German Research Foundation. Michael Naumann reports financial support was provided by European Union., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Chronic Helicobacter pylori infection is the leading cause of intestinal-type adenocarcinoma, as prolonged Helicobacter colonization triggers chronic active gastritis, which may evolve into adenocarcinoma of the intestinal type. In this environment, cytokines play a significant role in determining the evolution of the infection. In combination with other factors (genetic, environmental and nutritional), the pro-inflammatory response may trigger pro-oncogenic mechanisms that lead to the silencing of tumour-suppressor genes, such as trefoil factor 1 (TFF1). The latter is known to play a protective role by maintaining the gastric mucosa integrity and retaining H. pylori in the mucus layer, preventing the progression of infection and, consequently, the development of gastric cancer (GC). Since TFF1 expression is reduced during chronic Helicobacter infection with a loss of gastric mucosa protection, we investigated the molecular pathways involved in this reduction. Specifically, we evaluated the effect of some pro-inflammatory cytokines on TFF1 regulation in GC and primary gastric cells by RT-qPCR and luciferase reporter assay analyses and the repressor role of the transcription factor C/EBPβ, overexpressed in gastric-intestinal cancer. Our results show that, among several cytokines, IFNγ stimulates C/EBPβ expression, which acts as a negative regulator of TFF1 by binding its promoter at three different sites.

Background: High-content screening (HCS) experiments generate complex data from multiple object features for each cell within a treated population. Usually, these data are analyzed by using population-averaged values of the features of interest, increasing the amount of false positives and the need for intensive follow-up validation. Therefore, there is a strong need for novel approaches with reproducible hit prediction by identifying significantly altered cell populations., Results: Here we describe SOPRA, a workflow for analyzing image-based HCS data based on regression analysis of non-averaged object features from cell populations, which can be run on hundreds of samples using different cell features. Following plate-wise normalization, the values are counted within predetermined binning intervals, generating unique frequency distribution profiles (histograms) for each population, which are then normalized to control populations (control-based normalization). These control-normalized frequency distribution profiles are analyzed using the Bioconductor R-package maSigPro, originally developed to analyze time profiles. However, statistically significant altered frequency distributions are also identified by maSigPro when integrating it into the SOPRA workflow. Finally, significantly changed profiles can be used to generate a heatmap from which altered cell populations with similar phenotypes can be identified, enabling the detection of siRNAs and compounds with the same 'on-target' profile and reducing the number of false positive hits., Conclusions: SOPRA is a novel analysis workflow for the detection of statistically significant normalized frequency distribution profiles of cellular features generated in high-throughput RNAi screens. For the validation of the SOPRA software workflow, a screen for cell cycle progression was used. We were able to identify such profiles for siRNA-mediated gene perturbations and chemical inhibitors of different cell cycle stages. The SOPRA software is freely available from Github., (© 2022. The Author(s).)

The cervix is the gateway to the upper female reproductive tract, connecting the uterus and vagina. It plays crucial roles in fertility and pregnancy maintenance from onset until delivery of the fetus, and prevents pathogen ascension. Compromised functionality of the cervix can lead to disorders, including infertility, chronic infections and cancers. The cervix comprises two regions: columnar epithelium-lined endocervix and stratified squamous epithelium-lined ectocervix, meeting at the squamocolumnar transition zone. So far, two-dimensional cultures of genetically unstable immortalized or cancer cell lines have been primarily used to study cervix biology in vitro. The lack of an in vitro system that reflects the cellular, physiological and functional properties of the two epithelial types has hampered the study of normal physiology, disease development and infection processes. Here we describe a protocol for cell isolation, establishment, long-term culture and expansion of adult epithelial stem cell-derived endocervical and ectocervical organoids from human biopsies and mouse tissue. These two organoid types require unique combinations of growth factors reminiscent of their in vivo tissue niches and different culturing procedures. They recapitulate native three-dimensional tissue architecture and patterning. The protocol to generate these organoids takes 4-6 weeks. We also describe procedures to introduce human papillomavirus oncogenes into the cervical stem cells by genetic manipulation to model cervical cancer and infection of the organoids with the highly prevalent sexually transmitted bacterial pathogen Chlamydia trachomatis. These organoid systems open new possibilities to study cervix biology, infections and cancer evolution, and have potential applications in personalized medicine, drug screening, genome editing and disease modeling., (© 2022. Springer Nature Limited.)

Oncogenic mutations in the small GTPase RAS contribute to ~30% of human cancers. In a Drosophila genetic screen, we identified novel and evolutionary conserved cancer genes that affect Ras-driven tumorigenesis and metastasis in Drosophila including confirmation of the tetraspanin Tsp29Fb. However, it was not known whether the mammalian Tsp29Fb orthologue, TSPAN6, has any role in RAS-driven human epithelial tumors. Here we show that TSPAN6 suppressed tumor growth and metastatic dissemination of human RAS activating mutant pancreatic cancer xenografts. Whole-body knockout as well as tumor cell autonomous inactivation using floxed alleles of Tspan6 in mice enhanced Kras G12D -driven lung tumor initiation and malignant progression. Mechanistically, TSPAN6 binds to the EGFR and blocks EGFR-induced RAS activation. Moreover, we show that inactivation of TSPAN6 induces an epithelial-to-mesenchymal transition and inhibits cell migration in vitro and in vivo. Finally, low TSPAN6 expression correlates with poor prognosis of patients with lung and pancreatic cancers with mesenchymal morphology. Our results uncover TSPAN6 as a novel tumor suppressor receptor that controls epithelial cell identify and restrains RAS-driven epithelial cancer., (© 2022. Crown.)

Helicobacter pylori causes gastric inflammation, gland hyperplasia and is linked to gastric cancer. Here, we studied the interplay between gastric epithelial stem cells and their stromal niche under homeostasis and upon H. pylori infection. We find that gastric epithelial stem cell differentiation is orchestrated by subsets of stromal cells that either produce BMP inhibitors in the gland base, or BMP ligands at the surface. Exposure to BMP ligands promotes a feed-forward loop by inducing Bmp2 expression in the epithelial cells themselves, enforcing rapid lineage commitment to terminally differentiated mucous pit cells. H. pylori leads to a loss of stromal and epithelial Bmp2 expression and increases expression of BMP inhibitors, promoting self-renewal of stem cells and accumulation of gland base cells, which we mechanistically link to IFN-γ signaling. Mice that lack IFN-γ signaling show no alterations of BMP gradient upon infection, while exposure to IFN-γ resembles H. pylori-driven mucosal responses., (© 2022. The Author(s).)

Coinfections with pathogenic microbes continually confront cervical mucosa, yet their implications in pathogenesis remain unclear. Lack of in-vitro models recapitulating cervical epithelium has been a bottleneck to study coinfections. Using patient-derived ectocervical organoids, we systematically modeled individual and coinfection dynamics of Human papillomavirus (HPV)16 E6E7 and Chlamydia, associated with carcinogenesis. The ectocervical stem cells were genetically manipulated to introduce E6E7 oncogenes to mimic HPV16 integration. Organoids from these stem cells develop the characteristics of precancerous lesions while retaining the self-renewal capacity and organize into mature stratified epithelium similar to healthy organoids. HPV16 E6E7 interferes with Chlamydia development and induces persistence. Unique transcriptional and post-translational responses induced by Chlamydia and HPV lead to distinct reprogramming of host cell processes. Strikingly, Chlamydia impedes HPV-induced mechanisms that maintain cellular and genome integrity, including mismatch repair in the stem cells. Together, our study employing organoids demonstrates the hazard of multiple infections and the unique cellular microenvironment they create, potentially contributing to neoplastic progression., (© 2022. The Author(s).)

Gut colonization by colibactin-producing bacteria is associated with colorectal cancer. A mutational signature of this genotoxin in human cancer indicates causality but only partially accounts for cell transformation. Instead, the failure of adequately resolving DNA damage causes genomic aberrations and chromosomal instability, constituting the main starting point for colibactin-driven cancer., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Helicobacter pylori infection constitutes one of the major risk factors for the development of gastric diseases including gastric cancer. The activation of nuclear factor-kappa-light-chain-enhancer of activated B cells (NF-κB) via classical and alternative pathways is a hallmark of H. pylori infection leading to inflammation in gastric epithelial cells. Tumor necrosis factor receptor-associated factor (TRAF)-interacting protein with forkhead-associated domain (TIFA) was previously suggested to trigger classical NF-κB activation, but its role in alternative NF-κB activation remains unexplored. Here, we identify TRAF6 and TRAF2 as binding partners of TIFA, contributing to the formation of TIFAsomes upon H. pylori infection. Importantly, the TIFA/TRAF6 interaction enables binding of TGFβ-activated kinase 1 (TAK1), leading to the activation of classical NF-κB signaling, while the TIFA/TRAF2 interaction causes the transient displacement of cellular inhibitor of apoptosis 1 (cIAP1) from TRAF2, and proteasomal degradation of cIAP1, to facilitate the activation of the alternative NF-κB pathway. Our findings therefore establish a dual function of TIFA in the activation of classical and alternative NF-κB signaling in H. pylori-infected gastric epithelial cells., (© 2021 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Background & Aims: The homeostasis of the gastrointestinal epithelium relies on cell regeneration and differentiation into distinct lineages organized inside glands and crypts. Regeneration depends on Wnt/β-catenin pathway activation, but to understand homeostasis and its dysregulation in disease, we need to identify the signaling microenvironment governing cell differentiation. By using gastric glands as a model, we have identified the signals inducing differentiation of surface mucus-, zymogen-, and gastric acid-producing cells., Methods: We generated mucosoid cultures from the human stomach and exposed them to different growth factors to obtain cells with features of differentiated foveolar, chief, and parietal cells. We localized the source of the growth factors in the tissue of origin., Results: We show that epidermal growth factor is the major fate determinant distinguishing the surface and inner part of human gastric glands. In combination with bone morphogenetic factor/Noggin signals, epidermal growth factor controls the differentiation of foveolar cells vs parietal or chief cells. We also show that epidermal growth factor is likely to underlie alteration of the gastric mucosa in the precancerous condition atrophic gastritis., Conclusions: Use of our recently established mucosoid cultures in combination with analysis of the tissue of origin provided a robust strategy to understand differentiation and patterning of human tissue and allowed us to draw a new, detailed map of the signaling microenvironment in the human gastric glands., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Viruses manipulate cellular metabolism and macromolecule recycling processes like autophagy. Dysregulated metabolism might lead to excessive inflammatory and autoimmune responses as observed in severe and long COVID-19 patients. Here we show that SARS-CoV-2 modulates cellular metabolism and reduces autophagy. Accordingly, compound-driven induction of autophagy limits SARS-CoV-2 propagation. In detail, SARS-CoV-2-infected cells show accumulation of key metabolites, activation of autophagy inhibitors (AKT1, SKP2) and reduction of proteins responsible for autophagy initiation (AMPK, TSC2, ULK1), membrane nucleation, and phagophore formation (BECN1, VPS34, ATG14), as well as autophagosome-lysosome fusion (BECN1, ATG14 oligomers). Consequently, phagophore-incorporated autophagy markers LC3B-II and P62 accumulate, which we confirm in a hamster model and lung samples of COVID-19 patients. Single-nucleus and single-cell sequencing of patient-derived lung and mucosal samples show differential transcriptional regulation of autophagy and immune genes depending on cell type, disease duration, and SARS-CoV-2 replication levels. Targeting of autophagic pathways by exogenous administration of the polyamines spermidine and spermine, the selective AKT1 inhibitor MK-2206, and the BECN1-stabilizing anthelmintic drug niclosamide inhibit SARS-CoV-2 propagation in vitro with IC 50 values of 136.7, 7.67, 0.11, and 0.13 μM, respectively. Autophagy-inducing compounds reduce SARS-CoV-2 propagation in primary human lung cells and intestinal organoids emphasizing their potential as treatment options against COVID-19.

Emerging mosquito-borne RNA viruses cause massive health complications worldwide. The Zika virus (ZIKV), in particular, has spread dramatically since 2007 and has provoked epidemics in the Americas and the South Pacific. The lack of antiviral therapy and vaccination has focused research on the investigation of ZIKV-host interactions, in order to understand underlying molecular infection mechanisms. We have established an approach for the analysis of ZIKV host dependency factors in a human trophoblast cell line and applied genome-wide CRISPR/Cas9 knockout mutagenesis. The presented method is especially of value for the identification of factors that are essential for placental infection with the potential to serve as targets for antiviral treatment., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Detailed knowledge of the molecular biology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is crucial for understanding of viral replication, host responses, and disease progression. Here, we report gene expression profiles of three SARS-CoV- and SARS-CoV-2-infected human cell lines. SARS-CoV-2 elicited an approximately two-fold higher stimulation of the innate immune response compared to SARS-CoV in the human epithelial cell line Calu-3, including induction of miRNA-155. Single-cell RNA sequencing of infected cells showed that genes induced by virus infections were broadly upregulated, whereas interferon beta/lambda genes, a pro-inflammatory cytokines such as IL-6, were expressed only in small subsets of infected cells. Temporal analysis suggested that transcriptional activities of interferon regulatory factors precede those of nuclear factor κB. Lastly, we identified heat shock protein 90 (HSP90) as a protein relevant for the infection. Inhibition of the HSP90 activity resulted in a reduction of viral replication and pro-inflammatory cytokine expression in primary human airway epithelial cells., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Genotoxic colibactin-producing pks+ Escherichia coli induce DNA double-strand breaks, mutations, and promote tumor development in mouse models of colorectal cancer (CRC). Colibactin's distinct mutational signature is reflected in human CRC, suggesting a causal link. Here, we investigate its transformation potential using organoids from primary murine colon epithelial cells. Organoids recovered from short-term infection with pks+ E. coli show characteristics of CRC cells, e.g., enhanced proliferation, Wnt-independence, and impaired differentiation. Sequence analysis of Wnt-independent organoids reveals an enhanced mutational burden, including chromosomal aberrations typical of genomic instability. Although we do not find classic Wnt-signaling mutations, we identify several mutations in genes related to p53-signaling, including miR-34a. Knockout of Trp53 or miR-34 in organoids results in Wnt-independence, corroborating a functional interplay between the p53 and Wnt pathways. We propose larger chromosomal alterations and aneuploidy as the basis of transformation in these organoids, consistent with the early appearance of chromosomal instability in CRC.

The transition zones of the squamous and columnar epithelia constitute hotspots for the emergence of cancer, often preceded by metaplasia, in which one epithelial type is replaced by another. It remains unclear how the epithelial spatial organization is maintained and how the transition zone niche is remodelled during metaplasia. Here we used single-cell RNA sequencing to characterize epithelial subpopulations and the underlying stromal compartment of endo- and ectocervix, encompassing the transition zone. Mouse lineage tracing, organoid culture and single-molecule RNA in situ hybridizations revealed that the two epithelia derive from separate cervix-resident lineage-specific stem cell populations regulated by opposing Wnt signals from the stroma. Using a mouse model of cervical metaplasia, we further show that the endocervical stroma undergoes remodelling and increases expression of the Wnt inhibitor Dickkopf-2 (DKK2), promoting the outgrowth of ectocervical stem cells. Our data indicate that homeostasis at the transition zone results from divergent stromal signals, driving the differential proliferation of resident epithelial lineages.

Cdc-like kinase 1 (CLK1) is a dual-specificity kinase capable of autophosphorylation on tyrosine residues and Ser/Thr phosphorylation of its substrates. CLK1 belongs to the CLK kinase family that regulates alternative splicing through phosphorylation of serine-arginine rich (SR) proteins. Recent studies have demonstrated that CLK1 has an important role in the replication of influenza A and chikungunya viruses. Furthermore, CLK1 was found to be relevant for the replication of HIV-1 and the West Nile virus, making CLK1 an interesting cellular candidate for the development of a host-directed antiviral therapy that might be efficient for treatment of newly emerging viruses. We describe here our attempts and detailed procedures to obtain the recombinant kinase domain of CLK1 in suitable amounts for crystallization in complex with specific inhibitors. The key solution for the reproducibility of crystals resides in devising and refining expression and purification protocols leading to homogeneous protein. Co-expression of CLK1 with λ-phosphatase and careful purification has yielded crystals of CLK1 complexed with the KH-CB19 inhibitor that diffracted to 1.65 Å. These results paved the path to the screening of more structures of CLK1 complexed compounds, leading to further optimization of their inhibitory activity. Moreover, since kinases are desired targets in numerous pathologies, the approach we report here, the co-expression of kinases with λ-phosphatase, previously used in other kinases, can be adopted as a general protocol in numerous kinase targets for obtaining reproducible and homogenic non-phosphorylated (inactive) forms suitable for biochemical and structural studies thus facilitating the development of novel inhibitors., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Exposure of gastric epithelial cells to the bacterial carcinogen Helicobacter pylori causes DNA double strand breaks. Here, we show that H. pylori-induced DNA damage occurs co-transcriptionally in S-phase cells that activate NF-κB signaling upon innate immune recognition of the lipopolysaccharide biosynthetic intermediate β-ADP-heptose by the ALPK1/TIFA signaling pathway. DNA damage depends on the bi-functional RfaE enzyme and the Cag pathogenicity island of H. pylori, is accompanied by replication fork stalling and can be observed also in primary cells derived from gastric organoids. Importantly, H. pylori-induced replication stress and DNA damage depend on the presence of co-transcriptional RNA/DNA hybrids (R-loops) that form in infected cells during S-phase as a consequence of β-ADP-heptose/ ALPK1/TIFA/NF-κB signaling. H. pylori resides in close proximity to S-phase cells in the gastric mucosa of gastritis patients. Taken together, our results link bacterial infection and NF-κB-driven innate immune responses to R-loop-dependent replication stress and DNA damage.

Carcinoma of the gallbladder (GBC) is the most frequent tumor of the biliary tract. Despite epidemiological studies showing a correlation between chronic infection with Salmonella enterica Typhi/Paratyphi A and GBC, the underlying molecular mechanisms of this fatal connection are still uncertain. The murine serovar Salmonella Typhimurium has been shown to promote transformation of genetically predisposed cells by driving mitogenic signaling. However, insights from this strain remain limited as it lacks the typhoid toxin produced by the human serovars Typhi and Paratyphi A. In particular, the CdtB subunit of the typhoid toxin directly induces DNA breaks in host cells, likely promoting transformation. To assess the underlying principles of transformation, we used gallbladder organoids as an infection model for Salmonella Paratyphi A. In this model, bacteria can invade epithelial cells, and we observed host cell DNA damage. The induction of DNA double-strand breaks after infection depended on the typhoid toxin CdtB subunit and extended to neighboring, non-infected cells. By cultivating the organoid derived cells into polarized monolayers in air-liquid interphase, we could extend the duration of the infection, and we observed an initial arrest of the cell cycle that does not depend on the typhoid toxin. Non-infected intoxicated cells instead continued to proliferate despite the DNA damage. Our study highlights the importance of the typhoid toxin in causing genomic instability and corroborates the epidemiological link between Salmonella infection and GBC. IMPORTANCE Bacterial infections are increasingly being recognized as risk factors for the development of adenocarcinomas. The strong epidemiological evidence linking Helicobacter pylori infection to stomach cancer has paved the way to the demonstration that bacterial infections cause DNA damage in the host cells, initiating transformation. In this regard, the role of bacterial genotoxins has become more relevant. Salmonella enterica serovars Typhi and Paratyphi A have been clinically associated with gallbladder cancer. By harnessing the stem cell potential of cells from healthy human gallbladder explant, we regenerated and propagated the epithelium of this organ in vitro and used these cultures to model S. Paratyphi A infection. This study demonstrates the importance of the typhoid toxin, encoded only by these specific serovars, in causing genomic instability in healthy gallbladder cells, posing intoxicated cells at risk of malignant transformation., (Copyright © 2020 Sepe, Hartl et al.)

Multiple studies have demonstrated rapid bacterial genome evolution during chronic infection with Helicobacter pylori In contrast, little was known about genetic changes during the first stages of infection, when selective pressure is likely to be highest. Using single-molecule, real-time (SMRT) and Illumina sequencing technologies, we analyzed genome and methylome evolution during the first 10 weeks of infection by comparing the cag pathogenicity island ( cag PAI)-negative H. pylori challenge strain BCS 100 with pairs of H. pylori reisolates from gastric antrum and corpus biopsy specimens of 10 human volunteers who had been infected with this strain as part of a vaccine trial. Most genetic changes detected in the reisolates affected genes with a surface-related role or a predicted function in peptide uptake. Apart from phenotypic changes of the bacterial envelope, a duplication of the catalase gene was observed in one reisolate, which resulted in higher catalase activity and improved survival under oxidative stress conditions. The methylomes also varied in some of the reisolates, mostly by activity switching of phase-variable methyltransferase (MTase) genes. The observed in vivo mutation spectrum was remarkable for a very high proportion of nonsynonymous mutations. Although the data showed substantial within-strain genome diversity in the challenge strain, most antrum and corpus reisolates from the same volunteers were highly similar to each other, indicating that the challenge infection represents a major selective bottleneck shaping the transmitted population. Our findings suggest rapid in vivo s election of H. pylori during early-phase infection providing adaptation to different individuals by common mechanisms of genetic and epigenetic alterations. IMPORTANCE Exceptional genetic diversity and variability are hallmarks of Helicobacter pylori , but the biological role of this plasticity remains incompletely understood. Here, we had the rare opportunity to investigate the molecular evolution during the first weeks of H. pylori infection by comparing the genomes and epigenomes of H. pylori strain BCS 100 used to challenge human volunteers in a vaccine trial with those of bacteria reisolated from the volunteers 10 weeks after the challenge. The data provide molecular insights into the process of establishment of this highly versatile pathogen in 10 different human individual hosts, showing, for example, selection for changes in host-interaction molecules as well as changes in epigenetic methylation patterns. The data provide important clues to the early adaptation of H. pylori to new host niches after transmission, which we believe is vital to understand its success as a chronic pathogen and develop more efficient treatments and vaccines., (Copyright © 2020 Estibariz et al.)

The mucosal epithelium is a common target of damage by chronic bacterial infections and the accompanying toxins, and most cancers originate from this tissue. We investigated whether colibactin, a potent genotoxin 1 associated with certain strains of Escherichia coli 2 , creates a specific DNA-damage signature in infected human colorectal cells. Notably, the genomic contexts of colibactin-induced DNA double-strand breaks were enriched for an AT-rich hexameric sequence motif, associated with distinct DNA-shape characteristics. A survey of somatic mutations at colibactin target sites of several thousand cancer genomes revealed notable enrichment of this motif in colorectal cancers. Moreover, the exact double-strand-break loci corresponded with mutational hot spots in cancer genomes, reminiscent of a trinucleotide signature previously identified in healthy colorectal epithelial cells 3 . The present study provides evidence for the etiological role of colibactin in human cancer.

Human guanylate binding protein 1 (hGBP1) belongs to the dynamin superfamily of GTPases and conveys host defense against intracellular bacteria and parasites. During infection, hGBP1 is recruited to pathogen-containing vacuoles, such as Chlamydia trachomatis inclusions, restricts pathogenic growth, and induces the activation of the inflammasome pathway. hGBP1 has a unique catalytic activity to hydrolyze guanosine triphosphate (GTP) to guanosine monophosphate (GMP) in two consecutive cleavage steps. However, the functional significance of this activity in host defense remains elusive. Here, we generate a structure-guided mutant that specifically abrogates GMP production, while maintaining fast cooperative GTP hydrolysis. Complementation experiments in human monocytes/macrophages show that hGBP1-mediated GMP production is dispensable for restricting Chlamydia trachomatis growth but is necessary for inflammasome activation. Mechanistically, GMP is catabolized to uric acid, which in turn activates the NLRP3 inflammasome. Our study demonstrates that the unique enzymology of hGBP1 coordinates bacterial growth restriction and inflammasome signaling., Competing Interests: Declaration of Interests We declare no conflict of interest., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

High-grade serous ovarian cancer (HGSOC) likely originates from the fallopian tube (FT) epithelium. Here, we established 15 organoid lines from HGSOC primary tumor deposits that closely match the mutational profile and phenotype of the parental tumor. We found that Wnt pathway activation leads to growth arrest of these cancer organoids. Moreover, active BMP signaling is almost always required for the generation of HGSOC organoids, while healthy fallopian tube organoids depend on BMP suppression by Noggin. Fallopian tube organoids modified by stable shRNA knockdown of p53, PTEN, and retinoblastoma protein (RB) also require a low-Wnt environment for long-term growth, while fallopian tube organoid medium triggers growth arrest. Thus, early changes in the stem cell niche environment are needed to support outgrowth of these genetically altered cells. Indeed, comparative analysis of gene expression pattern and phenotypes of normal vs. loss-of-function organoids confirmed that depletion of tumor suppressors triggers changes in the regulation of stemness and differentiation., (© 2020 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

The Hippo pathway has been initially discovered by screening genes that regulate organ size in Drosophila . Recent studies have highlighted the role of the Hippo pathway in controlling organ size, tissue homeostasis and regeneration, and signaling dysregulation, especially the overactivation of the transcriptional coactivator YAP/TAZ, which leads to uncontrolled cell growth and malignant transformation. The core components of the Hippo pathway may initiate tumorigenesis by inducing tumor stem cells and proliferation, ultimately leading to metastasis and drug resistance, which occurs extensively in gynecological malignancies, including cervical cancer, ovarian cancer, and endometrial cancer. In this review, we attempt to systematically summarize recent progress in our understanding of the mechanism of Hippo pathway regulation in tumorigenesis and the mechanisms that underlie alterations during gynecological malignancies, as well as new therapeutic strategies., Competing Interests: None., (AJCR Copyright © 2020.)

Breast cancer is the most common cancer in women worldwide. Despite recent developments in breast cancer detection and treatment, 1.38 million women each year are still affected. Breast cancer heterogeneity at the population and single-cell level, complexity and developing different metastases are setting several challenges to develop efficient breast cancer therapies. RNA interference (RNAi) represents an opportunity to silence gene expression and inhibit specific pathways in cancer cells. In order to reap the full advantages of RNAi-based therapy, different pathways that sustain cancer cells growth have been targeted using specific siRNAs. The present study investigated the ability of a set of cytotoxic siRNAs to inhibit growth of breast cancer cells. These siRNAs are targeting eukaryotic elongation factor 2 (EEF2), polo-like kinase 1 (PLK1), G protein-coupled receptor kinase 4 (GRK4) and sphingosine kinase interacting protein (SKIP5). To facilitate their targeted delivery, the human epidermal growth factor receptor-3 (HER3)-specific aptamer A30 was used. The in vitro results described in this work indicate that combining the highly specific HER3 aptamer with cytotoxic siRNAs targeting (EEF2, PLK1, GRK4 and SKIP5) can inhibit its activity and ultimately suppress proliferation of HER3 positive breast cancer cells.

The colonic epithelial turnover is driven by crypt-base stem cells that express the R-spondin receptor Lgr5. Signals that regulate epithelial regeneration upon stem cell injury are largely unknown. Here, we explore the dynamics of Wnt signaling in the colon. We identify two populations of cells with active Wnt signaling: highly proliferative Lgr5 + /Axin2 + cells, as well as secretory Lgr5 - /Axin2 + cells. Upon Lgr5 + cell depletion, these cells are recruited to contribute to crypt regeneration. Chemical injury induced by DSS leads to a loss of both Lgr5 + cells and Axin2 + cells and epithelial regeneration is driven by Axin2 -  cells, including differentiated Krt20 + surface enterocytes. Regeneration requires stromal Rspo3, which is present at increased levels upon injury and reprograms Lgr5 - but Lgr4 + differentiated cells. In contrast, depletion of stromal Rspo3 impairs crypt regeneration, even upon mild injury. We demonstrate that Rspo3 is essential for epithelial repair via induction of Wnt signaling in differentiated cells.

Influenza A virus carries eight negative single-stranded RNAs and uses spliced mRNAs to increase the number of proteins produced from them. Several genome-wide screens for essential host factors for influenza A virus replication revealed a necessity for splicing and splicing-related factors, including Cdc-like kinase 1 (CLK1). This CLK family kinase plays a role in alternative splicing regulation through phosphorylation of serine-arginine rich (SR) proteins. To examine the influence that modulation of splicing regulation has on influenza infection, we analyzed the effect of CLK1 knockdown and inhibition. CLK1 knockdown in A549 cells reduced influenza A/WSN/33 virus replication and increased the level of splicing of segment 7, which encodes the viral M1 and M2 proteins. CLK1-/- mice infected with influenza A/England/195/2009 (H1N1pdm09) virus supported lower levels of virus replication than wild-type mice. Screening of newly developed CLK inhibitors revealed several compounds that have an effect on the level of splicing of influenza A gene segment M in different models and decrease influenza A/WSN/33 virus replication in A549 cells. The promising inhibitor KH-CB19, an indole-based enaminonitrile with unique binding mode for CLK1, and its even more selective analogue NIH39 showed high specificity towards CLK1 and had a similar effect on influenza mRNA splicing regulation. Taken together, our findings indicate that targeting host factors that regulate splicing of influenza mRNAs may represent a novel therapeutic approach., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

The gastric pathogen Helicobacter pylori activates the NF-κB pathway in human epithelial cells via the recently discovered α-kinase 1 TRAF-interacting protein with forkhead-associated domain (TIFA) axis. We and others showed that this pathway can be triggered by heptose 1,7-bisphosphate (HBP), an LPS intermediate produced in gram-negative bacteria that represents a new pathogen-associated molecular pattern (PAMP). Here, we report that our attempts to identify HBP in lysates of H. pylori revealed surprisingly low amounts, failing to explain NF-κB activation. Instead, we identified ADP- glycero -β-D- manno -heptose (ADP heptose), a derivative of HBP, as the predominant PAMP in lysates of H. pylori and other gram-negative bacteria. ADP heptose exhibits significantly higher activity than HBP, and cells specifically sensed the presence of the β-form, even when the compound was added extracellularly. The data lead us to conclude that ADP heptose not only constitutes the key PAMP responsible for H. pylori -induced NF-κB activation in epithelial cells, but it acts as a general gram-negative bacterial PAMP.-Pfannkuch, L., Hurwitz, R., Traulsen, J., Sigulla, J., Poeschke, M., Matzner, L., Kosma, P., Schmid, M., Meyer, T. F. ADP heptose, a novel pathogen-associated molecular pattern identified in Helicobacter pylori .

Wnt signalling stimulated by binding of R-spondin (Rspo) to Lgr-family members is crucial for gastrointestinal stem cell renewal. Infection of the stomach with Helicobacter pylori stimulates increased secretion of Rspo by myofibroblasts, leading to an increase in proliferation of Wnt-responsive Axin2 + Lgr5 - stem cells in the isthmus of the gastric gland and finally gastric gland hyperplasia. Basal Lgr5 + cells are also exposed to Rspo3, but their response remains unclear. Here, we demonstrate that-in contrast to its known mitogenic activity-Rspo3 induces differentiation of basal Lgr5 + cells into secretory cells that express and secrete antimicrobial factors, such as intelectin-1, into the lumen. The depletion of Lgr5 + cells or the knockout of Rspo3 in myofibroblasts leads to hypercolonization of the gastric glands with H. pylori, including the stem cell compartment. By contrast, systemic administration or overexpression of Rspo3 in the stroma clears H. pylori from the gastric glands. Thus, the Rspo3-Lgr5 axis simultaneously regulates both antimicrobial defence and mucosal regeneration.