Intestinal Paneth cell differentiation relies on asymmetric regulation of Wnt signaling by Daam1/2.

The mammalian intestine is one of the most rapidly self-renewing tissues, driven by stem cells residing at the crypt bottom. Paneth cells form a major element of the niche microenvironment providing various growth factors to orchestrate intestinal stem cell homeostasis, such as Wnt3. Different Wnt ligands can selectively activate ß-catenin-dependent (canonical) or -independent (noncanonical) signaling. Here, we report that the Dishevelled-associated activator of morphogenesis 1 (Daam1) and its paralogue Daam2 asymmetrically regulate canonical and noncanonical Wnt (Wnt/PCP) signaling. Daam1/2 interacts with the Wnt inhibitor RNF43, and Daam1/2 double knockout stimulates canonical Wnt signaling by preventing RNF43-dependent degradation of the Wnt receptor, Frizzled (Fzd). Single-cell RNA sequencing analysis revealed that Paneth cell differentiation is impaired by Daam1/2 depletion because of defective Wnt/PCP signaling. Together, we identified Daam1/2 as an unexpected hub molecule coordinating both canonical and noncanonical Wnt, which is fundamental for specifying an adequate number of Paneth cells.

RSPO3 Furin domain-conjugated liposomes for selective drug delivery to LGR5-high cells.

The transmembrane receptor LGR5 potentiates Wnt/ß-catenin signaling by binding both secreted R-spondin (RSPOs) and the Wnt tumor suppressors RNF43/ZNRF3, directing clearance of RNF43/ZNRF3 from the cell surface. Besides being widely used as a stem cell marker in various tissues, LGR5 is overexpressed in many types of malignancies, including colorectal cancer. Its expression characterizes a subpopulation of cancer cells that play a crucial role in tumor initiation, progression and cancer relapse, known as cancer stem cells (CSCs). For this reason, ongoing efforts are aimed at eradicating LGR5-positive CSCs. Here, we engineered liposomes decorated with different RSPO proteins to specifically detect and target LGR5-positive cells. Using fluorescence-loaded liposomes, we show that conjugation of full-length RSPO1 to the liposomal surface mediates aspecific, LGR5-independent cellular uptake, largely mediated by heparan sulfate proteoglycan binding. By contrast, liposomes decorated only with the Furin (FuFu) domains of RSPO3 are taken up by cells in a highly specific, LGR5-dependent manner. Moreover, encapsulating doxorubicin in FuFuRSPO3 liposomes allowed us to selectively inhibit the growth of LGR5-high cells. Thus, FuFuRSPO3-coated liposomes allow for the selective detection and ablation of LGR5-high cells, providing a potential drug delivery system for LGR5-targeted anti-cancer strategies.

Trend of hoverboard related injuries at a pediatric emergency department.

INTRODUCTION: Understanding how the use of hoverboards (HBs) can affect a child's safety is crucial. We describe the characteristics of HB related injuries and provide key messages about child prevention when using these leisure devices. METHODS: This was a retrospective study at an emergency department (ED) of a level-III-trauma center from 2016 to 2019. We tested the differences in children presenting for injury associated with HBs between 2016-2017 and 2018-2019 to better describe the temporal trend of the phenomenon. RESULTS: The rate of Injury associated with HBs / Total injury per 1,000 increased from 0.84 in 2016 to 7.7 in 2017, and then there was a gradual decline. The likelihood of injury was more common in younger children, increasing by 17% with decreasing age in 2018-2019 compared with 2016-2017 (OR: 0.83; 95%CI: 0.71-0.97; p = 0.021). The occurrence of injury in the April-June period was over twice as common in 2018-2019 (OR: 2.05; 95%CI: 1.0-2.05; p = 0.05). Patients were over 4 times more likely to have injured the lower extremity during the 2018-2019 period rather than other body regions (OR: 4.58; 95%CI: 1.23-4.58; p = 0.02). The odds of the indoor injury were more than twice as high in 2018-2019 (OR: 2.04; 95%CI: 1.077-2.04; p = 0.03). CONCLUSION: Despite a decrease in the frequency of HB related injuries after 2017, during the 2018-2019 period, the younger the children, the more they were exposed to injury risk, in addition to a greater occurrence of indoor injuries from HBs compared with 2016-2017. The enhancement of preventive measures is necessary to ensure child safety when using HBs.

Management of slipped capital femoral epiphysis: What hardware we can use in osteosynthesis in situ?

Epiphysiolysis (or Slipped Capital Femoral Epiphysis, SCFE) is a hip disorder involving children during prepubescence age. Traditionally, it is defined as a postero-medial slippage of the femoral epiphysis on the metaphysis, but, considering that femoral epiphysis is almost "stored" in the acetabulum, it could be better defined as laterally and anterior slippage of femoral metaphysis under the epiphysis.

A phospho-switch controls RNF43-mediated degradation of Wnt receptors to suppress tumorigenesis.

Frequent mutation of the tumour suppressor RNF43 is observed in many cancers, particularly colon malignancies. RNF43, an E3 ubiquitin ligase, negatively regulates Wnt signalling by inducing degradation of the Wnt receptor Frizzled. In this study, we discover that RNF43 activity requires phosphorylation at a triplet of conserved serines. This phospho-regulation of RNF43 is required for zebrafish development and growth of mouse intestinal organoids. Cancer-associated mutations that abrogate RNF43 phosphorylation cooperate with active Ras to promote tumorigenesis by abolishing the inhibitory function of RNF43 in Wnt signalling while maintaining its inhibitory function in p53 signalling. Our data suggest that RNF43 mutations cooperate with KRAS mutations to promote multi-step tumorigenesis via the Wnt-Ras-p53 axis in human colon cancers. Lastly, phosphomimetic substitutions of the serine trio restored the tumour suppressive activity of extracellular oncogenic mutants. Therefore, harnessing phospho-regulation of RNF43 might be a potential therapeutic strategy for tumours with RNF43 mutations.

Norovirus Replication in Human Intestinal Epithelial Cells Is Restricted by the Interferon-Induced JAK/STAT Signaling Pathway and RNA Polymerase II-Mediated Transcriptional Responses.

Human noroviruses (HuNoV) are a leading cause of viral gastroenteritis worldwide and a significant cause of morbidity and mortality in all age groups. The recent finding that HuNoV can be propagated in B cells and mucosa-derived intestinal epithelial organoids (IEOs) has transformed our ability to dissect the life cycle of noroviruses. Using transcriptome sequencing (RNA-Seq) of HuNoV-infected intestinal epithelial cells (IECs), we have found that replication of HuNoV in IECs results in interferon (IFN)-induced transcriptional responses and that HuNoV replication in IECs is sensitive to IFN. This contrasts with previous studies that suggested that the innate immune response may play no role in the restriction of HuNoV replication in immortalized cells. We demonstrated that inhibition of Janus kinase 1 (JAK1)/JAK2 enhanced HuNoV replication in IECs. Surprisingly, targeted inhibition of cellular RNA polymerase II-mediated transcription was not detrimental to HuNoV replication but instead enhanced replication to a greater degree than blocking of JAK signaling directly. Furthermore, we demonstrated for the first time that IECs generated from genetically modified intestinal organoids, engineered to be deficient in the interferon response, were more permissive to HuNoV infection. Taking the results together, our work revealed that IFN-induced transcriptional responses restrict HuNoV replication in IECs and demonstrated that inhibition of these responses mediated by modifications of the culture conditions can greatly enhance the robustness of the norovirus culture system.IMPORTANCE Noroviruses are a major cause of gastroenteritis worldwide, and yet the challenges associated with their growth in culture have greatly hampered the development of therapeutic approaches and have limited our understanding of the cellular pathways that control infection. Here, we show that human intestinal epithelial cells, which represent the first point of entry of human noroviruses into the host, limit virus replication by induction of innate responses. Furthermore, we show that modulating the ability of intestinal epithelial cells to induce transcriptional responses to HuNoV infection can significantly enhance human norovirus replication in culture. Collectively, our findings provide new insights into the biological pathways that control norovirus infection but also identify mechanisms that enhance the robustness of norovirus culture.

Wnt Signaling in 3D: Recent Advances in the Applications of Intestinal Organoids.

Intestinal organoids grown from adult stem cells have emerged as prototype 3D organotypic models for studying tissue renewal and homeostasis. Owing to their strict dependence on Wnt signaling, intestinal organoids offer an unprecedented opportunity to examine Wnt pathway regulation in normal physiology and cancer. We review how alterations in growth factor dependency and organoid morphology can be exploited to identify Wnt signaling mechanisms, characterize mutated pathway components, and predict responses of patient-derived tumors to targeted therapy. We discuss current deficits in the understanding of genotype-phenotype relationships that are to be considered when interpreting mutation-induced changes in organoid morphology.

A Protocol for Multiple Gene Knockout in Mouse Small Intestinal Organoids Using a CRISPR-concatemer.

CRISPR/Cas9 technology has greatly improved the feasibility and speed of loss-of-function studies that are essential in understanding gene function. In higher eukaryotes, paralogous genes can mask a potential phenotype by compensating the loss of a gene, thus limiting the information that can be obtained from genetic studies relying on single gene knockouts. We have developed a novel, rapid cloning method for guide RNA (gRNA) concatemers in order to create multi-gene knockouts following a single round of transfection in mouse small intestinal organoids. Our strategy allows for the concatemerization of up to four individual gRNAs into a single vector by performing a single Golden Gate shuffling reaction with annealed gRNA oligos and a pre-designed retroviral vector. This allows either the simultaneous knockout of up to four different genes, or increased knockout efficiency following the targeting of one gene by multiple gRNAs. In this protocol, we show in detail how to efficiently clone multiple gRNAs into the retroviral CRISPR-concatemer vector and how to achieve highly efficient electroporation in intestinal organoids. As an example, we show that simultaneous knockout of two pairs of genes encoding negative regulators of the Wnt signaling pathway (Axin1/2 and Rnf43/Znrf3) renders intestinal organoids resistant to the withdrawal of key growth factors.

One-step generation of conditional and reversible gene knockouts.

Loss-of-function studies are key for investigating gene function, and CRISPR technology has made genome editing widely accessible in model organisms and cells. However, conditional gene inactivation in diploid cells is still difficult to achieve. Here, we present CRISPR-FLIP, a strategy that provides an efficient, rapid and scalable method for biallelic conditional gene knockouts in diploid or aneuploid cells, such as pluripotent stem cells, 3D organoids and cell lines, by co-delivery of CRISPR-Cas9 and a universal conditional intronic cassette.

Simultaneous paralogue knockout using a CRISPR-concatemer in mouse small intestinal organoids.

Approaches based on genetic modification have been invaluable for investigating a wide array of biological processes, with gain- and loss-of-function approaches frequently used to investigate gene function. However, the presence of paralogues, and hence possible genetic compensation, for many genes necessitates the knockout (KO) of all paralogous genes in order to observe clear phenotypic change. CRISPR technology, the most recently described tool for gene editing, can generate KOs with unprecedented ease and speed and has been used in adult stem cell-derived organoids for single gene knockout, gene knock-in and gene correction. However, the simultaneous targeting of multiple genes in organoids by CRISPR technology has not previously been described. Here we describe a rapid, scalable and cost effective method for generating double knockouts in organoids. By concatemerizing multiple gRNA expression cassettes, we generated a 'gRNA concatemer vector'. Our method allows the rapid assembly of annealed synthetic DNA oligos into the final vector in a single step. This approach facilitates simultaneous delivery of multiple gRNAs to allow up to 4 gene KO in one step, or potentially to increase the efficiency of gene knockout by providing multiple gRNAs targeting one gene. As a proof of concept, we knocked out negative regulators of the Wnt pathway in small intestinal organoids, thereby removing their growth dependence on the exogenous Wnt enhancer, R-spondin1.