Dietary cystine restriction increases the proliferative capacity of the small intestine of mice.

Currently, over 88 million people are estimated to have adopted a vegan or vegetarian diet. Cysteine is a semi-essential amino acid, which availability is largely dependent on dietary intake of meat, eggs and whole grains. Vegan/vegetarian diets are therefore inherently low in cysteine. Sufficient uptake of cysteine is crucial, as it serves as substrate for protein synthesis and can be converted to taurine and glutathione. We found earlier that intermolecular cystine bridges are essential for the barrier function of the intestinal mucus layer. Therefore, we now investigate the effect of low dietary cystine on the intestine. Mice (8/group) received a high fat diet with a normal or low cystine concentration for 2 weeks. We observed no changes in plasma methionine, cysteine, taurine or glutathione levels or bile acid conjugation after 2 weeks of low cystine feeding. In the colon, dietary cystine restriction results in an increase in goblet cell numbers, and a borderline significant increase mucus layer thickness. Gut microbiome composition and expression of stem cell markers did not change on the low cystine diet. Remarkably, stem cell markers, as well as the proliferation marker Ki67, were increased upon cystine restriction in the small intestine. In line with this, gene set enrichment analysis indicated enrichment of Wnt signaling in the small intestine of mice on the low cystine diet, indicative of increased epithelial proliferation. In conclusion, 2 weeks of cystine restriction did not result in apparent systemic effects, but the low cystine diet increased the proliferative capacity specifically of the small intestine and induced the number of goblet cells in the colon.

Multi-country metabolic signature discovery for chicken health classification.

INTRODUCTION: To decrease antibiotic resistance, their use as growth promoters in the agricultural sector has been largely abandoned. This may lead to decreased health due to infectious disease or microbiome changes leading to gut inflammation. OBJECTIVES: We aimed to generate a m/z signature classifying chicken health in blood, and obtain biological insights from the resulting m/z signature. METHODS: We used direct infusion mass-spectrometry to determine a machine-learned metabolomics signature that classifies chicken health from a blood sample. We then challenged the resulting models by investigating the classification capability of the signature on novel data obtained at poultry houses in previously unseen countries using a Leave-One-Country-Out (LOCO) cross-validation strategy. Additionally, we optimised the number of mass/charge (m/z) values required to maximise the classification capability of Random Forest models, by developing a novel ranking system based on combined univariate t-test and fold-change analyses and building models based on this ranking through forward and reverse feature selection. RESULTS: The multi-country and LOCO models could classify chicken health. Both resulting 25-m/z and 3784-m/z signatures reliably classified chicken health in multiple countries. Through mummichog enrichment analysis on the large m/z signature, we found changes in amino acid metabolism, including branched chain amino acids and polyamines. CONCLUSION: We reliably classified chicken health from blood, independent of genetic-, farm-, feed- and country-specific confounding factors. The 25-m/z signature can be used to aid development of a per-metabolite panel. The extended 3784-m/z version can be used to gain a deeper understanding of the metabolic causes and consequences of low chicken health. Together, they may facilitate future treatment, prevention and intervention.

Human liver organoids: From generation to applications.

In the last decade, research into human hepatology has been revolutionized by the development of mini human livers in a dish. These liver organoids are formed by self-organizing stem cells and resemble their native counterparts in cellular content, multicellular architecture, and functional features. Liver organoids can be derived from the liver tissue or pluripotent stem cells generated from a skin biopsy, blood cells, or renal epithelial cells present in urine. With the development of liver organoids, a large part of previous hurdles in modeling the human liver is likely to be solved, enabling possibilities to better model liver disease, improve (personalized) drug testing, and advance bioengineering options. In this review, we address strategies to generate and use organoids in human liver disease modeling, followed by a discussion of their potential application in drug development and therapeutics, as well as their strengths and limitations.

Rifaximin stimulates nitrogen detoxification by PXR-independent mechanisms in human small intestinal organoids.

BACKGROUND AND AIMS: Recurrent hepatic encephalopathy (HE) is characterized by hyperammonaemia in combination with neuropsychiatric abnormalities and is treated with lactulose and rifaximin. Rifaximin is a pregnane X receptor (PXR) agonist with low systemic and high intestinal bioavailability. The mechanisms by which it alleviates HE are unclear. We used human small intestinal (hSI) organoids to study whether rifaximin, via PXR activation, affects the epithelial biotransformation machinery, and to gain understanding of its low systemic availability. METHODS: We generated PXR knockdown hSI organoids via lentiviral delivery of short hairpin RNAs. Organoids were cultured for 24 h with rifaximin or rifampicin. RNA-sequencing and metabolomics were performed to analyse gene expression and amino acid metabolism. Luminal rifaximin was quantified by photospectrometry. RESULTS: Treatment of wild-type hSI organoids with rifaximin resulted in >twofold differential expression of 131 genes compared to DMSO. These effects were largely PXR independent and related to amino acid metabolism. Rifaximin decreased expression of glutaminase-2 and increased expression of asparagine synthetase and solute carrier 7A11, thereby increasing intracellular glutamine and asparagine concentrations, indicating active ammonia detoxification. Rifaximin was apically excreted into the lumen in an ATP binding cassette B1 (ABCB1)-dependent manner. CONCLUSIONS: Rifaximin-after uptake into enterocytes-stimulates intracellular nitrogen detoxification by PXR-independent mechanisms. Active apical excretion of rifaximin by ABCB1 into the intestinal lumen explains its low systemic bioavailability. Our study implies that rifaximin, next to modulation of the microbiome, has direct effects on ammonia scavenging in the human small intestinal epithelium.

PPARγ lipodystrophy mutants reveal intermolecular interactions required for enhancer activation.

Peroxisome proliferator-activated receptor γ (PPARγ) is the master regulator of adipocyte differentiation, and mutations that interfere with its function cause lipodystrophy. PPARγ is a highly modular protein, and structural studies indicate that PPARγ domains engage in several intra- and inter-molecular interactions. How these interactions modulate PPARγ's ability to activate target genes in a cellular context is currently poorly understood. Here we take advantage of two previously uncharacterized lipodystrophy mutations, R212Q and E379K, that are predicted to interfere with the interaction of the hinge of PPARγ with DNA and with the interaction of PPARγ ligand binding domain (LBD) with the DNA-binding domain (DBD) of the retinoid X receptor, respectively. Using biochemical and genome-wide approaches we show that these mutations impair PPARγ function on an overlapping subset of target enhancers. The hinge region-DNA interaction appears mostly important for binding and remodelling of target enhancers in inaccessible chromatin, whereas the PPARγ-LBD:RXR-DBD interface stabilizes the PPARγ:RXR:DNA ternary complex. Our data demonstrate how in-depth analyses of lipodystrophy mutants can unravel molecular mechanisms of PPARγ function.

High-Level MYCN-Amplified RB1-Proficient Retinoblastoma Tumors Retain Distinct Molecular Signatures.

Purpose: Retinoblastomas are malignant eye tumors diagnosed in young children. Most retinoblastomas are genetically characterized by biallelic inactivation of the RB1 gene. However, 1.5% of tumors demonstrate high-level amplification of the proto-oncogene MYCN. Patients with MYCN-amplified RB1-proficient retinoblastoma receive a diagnosis at an earlier age and show a clinically and histologically more malignant phenotype. This study aimed to identify genome-wide molecular features that distinguish this subtype from other retinoblastomas. Design: Cohort study. Participants: Forty-seven retinoblastoma tumors, comprising 36 RB1 -/-, 4 RB1 +/-, and 7 RB1 +/+ tumors. In total, 5 retinoblastomas displayed high-level MYCN amplification, with 3 being RB1 +/+, 1 being RB1 +/-, and 1 being RB1 -/- . Methods: Integrated analysis, based on gene expression, methylation, and methylation-expression correlations, was performed to identify distinct molecular components of MYCN-amplified RB1-proficient retinoblastomas compared with other retinoblastoma subtypes. The methylation and methylation-expression correlation analysis was initially conducted within a subset of samples (n = 15) for which methylation profiles were available. The significant findings were cross-validated in the entire cohort (n = 47) and in publicly available data. Main Outcome Measures: Differentially expressed genes/pathways, differentially methylated genes, and methylation-driven differential gene expression. Results: A large number of genes (n = 3155) were identified with distinct expression patterns in MYCN-amplified RB1-proficient retinoblastomas. The upregulated and downregulated genes were associated with translation and cell-cycle processes, respectively. Methylation analysis revealed distinct methylated patterns in MYCN-amplified RB1-proficient tumors, many of which showing significant impact on gene expression. Data integration identified a 40-gene expression signature with hypermethylated state resulting in a significant downregulation in MYCN-amplified RB1-proficient retinoblastomas. Cross-validation using the entire cohort and the public domain expression data verified the overall lower expression of these genes not only in retinoblastomas with a MYCN-amplified RB1-proficient background, but also in MYCN-amplified neuroblastomas. These include the metabolism-associated TSTD1 gene and the cyclin-dependent kinase inhibitor gene CDKN2C. Conclusions: MYCN-amplified RB1-proficient retinoblastomas display significantly distinct molecular features compared with other retinoblastomas, including a set of 40 hypermethylation-driven downregulated genes. This gene set can give insight into the biology of MYCN-amplified retinoblastomas and may help us to understand the more aggressive clinical behavior.

Protein Condensation in the Nuclear Receptor Family; Implications for Transcriptional Output.

The nuclear receptor superfamily is a group of transcriptional regulators that orchestrate multiple vital processes such as inflammation, metabolism, and cell proliferation. In recent years, it has become clear that some nuclear receptors form condensates in living cells. These condensates contain high concentrations of proteins and can contain millions of molecules. At these sites, high concentrations of nuclear receptors and co-factors potentially contribute to efficient transcription. While condensate formation has been observed for some nuclear receptors, the majority have unknown condensate formation abilities. Condensate formation abilities for these NRs would implicate an additional layer of regulation for the entire nuclear receptor family. Here, we consider the nuclear receptor superfamily, the current evidence for condensate formation of some of its members and the potential of the whole superfamily to form condensates. Insights into the regulation of assembly or disassembly of nuclear receptor condensates and our considerations for the understudied family members imply that condensate biology might be an important aspect of nuclear receptor-regulated gene transcription.

Post-Translational Modifications of FXR; Implications for Cholestasis and Obesity-Related Disorders.

The Farnesoid X receptor (FXR) is a nuclear receptor which is activated by bile acids. Bile acids function in solubilization of dietary fats and vitamins in the intestine. In addition, bile acids have been increasingly recognized to act as signaling molecules involved in energy metabolism pathways, amongst others via activating FXR. Upon activation by bile acids, FXR controls the expression of many genes involved in bile acid, lipid, glucose and amino acid metabolism. An inability to properly use and store energy substrates may predispose to metabolic disorders, such as obesity, diabetes, cholestasis and non-alcoholic fatty liver disease. These diseases arise through a complex interplay between genetics, environment and nutrition. Due to its function in metabolism, FXR is an attractive treatment target for these disorders. The regulation of FXR expression and activity occurs both at the transcriptional and at the post-transcriptional level. It has been shown that FXR can be phosphorylated, SUMOylated and acetylated, amongst other modifications, and that these modifications have functional consequences for DNA and ligand binding, heterodimerization and subcellular localization of FXR. In addition, these post-translational modifications may selectively increase or decrease transcription of certain target genes. In this review, we provide an overview of the posttranslational modifications of FXR and discuss their potential involvement in cholestatic and metabolic disorders.

Ablation of liver Fxr results in an increased colonic mucus barrier in mice.

BACKGROUND & AIMS: The interorgan crosstalk between the liver and the intestine has been the focus of intense research. Key in this crosstalk are bile acids, which are secreted from the liver into the intestine, interact with the microbiome, and upon absorption reach back to the liver. The bile acid-activated farnesoid X receptor (Fxr) is involved in the gut-to-liver axis. However, liver-to-gut communication and the roles of bile acids and Fxr remain elusive. Herein, we aim to get a better understanding of Fxr-mediated liver-to-gut communication, particularly in colon functioning. METHODS: Fxr floxed/floxed mice were crossed with cre-expressing mice to yield Fxr ablation in the intestine (Fxr-intKO), liver (Fxr-livKO), or total body (Fxr-totKO). The effects on colonic gene expression (RNA sequencing), the microbiome (16S sequencing), and mucus barrier function by ex vivo imaging were analysed. RESULTS: Despite relatively small changes in biliary bile acid concentration and composition, more genes were differentially expressed in the colons of Fxr-livKO mice than in those of Fxr-intKO and Fxr-totKO mice (3272, 731, and 1824, respectively). The colons of Fxr-livKO showed increased expression of antimicrobial genes, Toll-like receptors, inflammasome-related genes and genes belonging to the 'Mucin-type O-glycan biosynthesis' pathway. Fxr-livKO mice have a microbiome profile favourable for the protective capacity of the mucus barrier. The thickness of the inner sterile mucus layer was increased and colitis symptoms reduced in Fxr-livKO mice. CONCLUSIONS: Targeting of FXR is at the forefront in the battle against metabolic diseases. We show that ablation of Fxr in the liver greatly impacts colonic gene expression and increased the colonic mucus barrier. Increasing the mucus barrier is of utmost importance to battle intestinal diseases such as inflammatory bowel disease, and we show that this might be done by antagonising FXR in the liver. LAY SUMMARY: This study shows that the communication of the liver to the intestine is crucial for intestinal health. Bile acids are key players in this liver-to-gut communication, and when Fxr, the master regulator of bile acid homoeostasis, is ablated in the liver, colonic gene expression is largely affected, and the protective capacity of the mucus barrier is increased.

Breast milk nutrients driving intestinal epithelial layer maturation via Wnt and Notch signaling: Implications for necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is an often lethal, inflammatory disease of the preterm intestine. The underdeveloped immune system plays an important role; however, the initial trigger for NEC development is likely a damaged intestinal epithelial layer. We hypothesize that due to incomplete maturation of different epithelial cell lineages, nutrients and bacteria are able to damage the epithelial cells and cause the (immature) inflammatory response, food intolerance and malabsorption seen in NEC. Intestinal organoid research has shown that maturation of intestinal epithelial cell lineages is orchestrated by two key signaling pathways: Wnt and Notch. In NEC, these pathways are dysregulated by hyperactivation of Toll-like-receptor-4. Breastfeeding decreases the risk of developing NEC compared to formula milk. Here, we review the intricate link between breast milk components, Wnt and Notch signaling and intestinal epithelial maturation. We argue that (nutritional) interventions regulating these pathways may decrease the risk of NEC development in preterm infants.

Splice variants of metabolic nuclear receptors: Relevance for metabolic disease and therapeutic targeting.

Metabolic nuclear receptors are ligand-activated transcription factors which control a wide range of metabolic processes and signaling pathways in response to nutrients and xenobiotics. Targeting these NRs is at the forefront of our endeavours to generate novel treatment options for diabetes, metabolic syndrome and fatty liver disease. Numerous splice variants have been described for these metabolic receptors. Structural changes, as a result of alternative splicing, lead to functional differences among NR isoforms, resulting in the regulation of different metabolic pathways by these NR splice variants. In this review, we describe known splice variants of FXR, LXRs, PXR, RXR, LRH-1, CAR and PPARs. We discuss their structure and functions, and elaborate on the regulation of splice variant abundance by nutritional signals. We conclude that NR splice variants pose an intriguing new layer of complexity in metabolic signaling, which needs to be taken into account in the development of treatment strategies for metabolic diseases.

Loss of FLCN-FNIP1/2 induces a non-canonical interferon response in human renal tubular epithelial cells.

Germline mutations in the Folliculin (FLCN) tumor suppressor gene cause Birt-Hogg-Dubé (BHD) syndrome, a rare autosomal dominant disorder predisposing carriers to kidney tumors. FLCN is a conserved, essential gene linked to diverse cellular processes but the mechanism by which FLCN prevents kidney cancer remains unknown. Here, we show that disrupting FLCN in human renal tubular epithelial cells (RPTEC/TERT1) activates TFE3, upregulating expression of its E-box targets, including RRAGD and GPNMB, without modifying mTORC1 activity. Surprisingly, the absence of FLCN or its binding partners FNIP1/FNIP2 induces interferon response genes independently of interferon. Mechanistically, FLCN loss promotes STAT2 recruitment to chromatin and slows cellular proliferation. Our integrated analysis identifies STAT1/2 signaling as a novel target of FLCN in renal cells and BHD tumors. STAT1/2 activation appears to counterbalance TFE3-directed hyper-proliferation and may influence immune responses. These findings shed light on unique roles of FLCN in human renal tumorigenesis and pinpoint candidate prognostic biomarkers.

MetaboShiny: interactive analysis and metabolite annotation of mass spectrometry-based metabolomics data.

Direct infusion untargeted mass spectrometry-based metabolomics allows for rapid insight into a sample's metabolic activity. However, analysis is often complicated by the large array of detected m/z values and the difficulty to prioritize important m/z and simultaneously annotate their putative identities. To address this challenge, we developed MetaboShiny, a novel R/RShiny-based metabolomics package featuring data analysis, database- and formula-prediction-based annotation and visualization. To demonstrate this, we reproduce and further explore a MetaboLights metabolomics bioinformatics study on lung cancer patient urine samples. MetaboShiny enables rapid and rigorous analysis and interpretation of direct infusion untargeted mass spectrometry-based metabolomics data.

FXR Isoforms Control Different Metabolic Functions in Liver Cells via Binding to Specific DNA Motifs.

BACKGROUND & AIMS: The nuclear receptor subfamily 1 group H member 4 (NR1H4, also called FXR) is a ligand-activated transcription factor that, upon binding of bile acids, regulates the expression of genes involved in bile acid, fat, sugar, and amino acid metabolism. Transcript variants encode the FXR isoforms alpha 1, alpha 2, alpha 3, and alpha 4, which activate different genes that regulate metabolism. Little is known about the mechanisms by which the different isoforms regulate specific genes or how the expression of these genes affects the outcomes of patients given drugs that target FXR. METHODS: We determined genome-wide binding of FXR isoforms in mouse liver organoids that express individual FXR isoforms using chromatin immunoprecipitation, followed by sequencing analysis and DNA motif discovery. We validated regulatory DNA sequences by mobility shift assays and with luciferase reporters using mouse and human FXR isoforms. We analyzed mouse liver organoids and HepG2 cells that expressed the FXR isoforms using chromatin immunoprecipitation, quantitative polymerase chain reaction, and immunoblot assays. Organoids were analyzed for mitochondrial respiration, lipid droplet content, and triglyceride excretion. We used the FXR ligand obeticholic acid to induce FXR activity in organoids, cell lines, and mice. We collected data on the binding of FXR in mouse liver and the expression levels of FXR isoforms and gene targets in human liver tissue and primary human hepatocytes from the Gene Expression Omnibus. RESULTS: In mouse liver cells, 89% of sites that bound FXR were bound by only FXRα2 or FXRα4, via direct interactions with the DNA sequence motif ER-2. Via DNA binding, these isoforms regulated metabolic functions in liver cells, including carbon metabolism and lipogenesis. Incubation with obeticholic acid increased mitochondrial pyruvate transport and reduced insulin-induced lipogenesis in organoids that expressed FXRα2 but not FXRα1. In human liver tissues, levels of FXRα2 varied significantly and correlated with expression of genes predicted to be regulated via an ER-2 motif. CONCLUSIONS: Most metabolic effects regulated by FXR in mouse and human liver cells are regulated by the FXRα2 isoform via specific binding to ER-2 motifs. The expression level of FXRα2 in liver might be used to predict responses of patients to treatment with FXR agonists.

Farnesoid X receptor and bile acids regulate vitamin A storage.

The nuclear receptor Farnesoid X Receptor (FXR) is activated by bile acids and controls multiple metabolic processes, including bile acid, lipid, carbohydrate, amino acid and energy metabolism. Vitamin A is needed for proper metabolic and immune control and requires bile acids for efficient intestinal absorption and storage in the liver. Here, we analyzed whether FXR regulates vitamin A metabolism. Compared to control animals, FXR-null mice showed strongly reduced (>90%) hepatic levels of retinol and retinyl palmitate and a significant reduction in lecithin retinol acyltransferase (LRAT), the enzyme responsible for hepatic vitamin A storage. Hepatic reintroduction of FXR in FXR-null mice induced vitamin A storage in the liver. Hepatic vitamin A levels were normal in intestine-specific FXR-null mice. Obeticholic acid (OCA, 3 weeks) treatment rapidly reduced (>60%) hepatic retinyl palmitate levels in mice, concurrent with strongly increased retinol levels (>5-fold). Similar, but milder effects were observed in cholic acid (12 weeks)-treated mice. OCA did not change hepatic LRAT protein levels, but strongly reduced all enzymes involved in hepatic retinyl ester hydrolysis, involving mostly post-transcriptional mechanisms. In conclusion, vitamin A metabolism in the mouse liver heavily depends on the FXR and FXR-targeted therapies may be prone to cause vitamin A-related pathologies.

Dissecting the allosteric FXR modulation: a chemical biology approach using guggulsterone as a chemical tool.

Guggulsterone is a promiscuous ligand for endocrine and metabolic lipid receptors traditionally used to treat a number of diseases including diabesity, hyperlipidemia, atherosclerosis, and osteoarthritis. Although relatively weak, its activity at the farnesoid X receptor (FXR) is particularly intriguing as guggulsterone acts as an antagonist with a peculiar ability of gene selective modulation. We report here a chemical biology study with the aim to further characterize the biological action of guggulsterone at the FXR and to obtain further insights into the functional role played by noncanonical FXR binding pockets S2 and S3. Our results suggest that the FXR accessory pockets might act as potential targets for small molecules able to modulate the metabolic activation of the receptor without affecting the anti-inflammatory activity thus revealing a new approach for disclosing selective FXR modulators that might bypass potential side-effects from chronic treatments.

Steroidogenic control of liver metabolism through a nuclear receptor-network.

OBJECTIVE: Coupling metabolic and reproductive pathways is essential for the survival of species. However, the functions of steroidogenic enzymes expressed in metabolic tissues are largely unknown. METHODS AND RESULTS: Here, we show that in the liver, the classical steroidogenic enzyme Cyp17a1 forms an essential nexus for glucose and ketone metabolism during feed-fast cycles. Both gain- and loss-of-function approaches are used to show that hepatic Cyp17a1 is induced by fasting, catalyzes the production of at least one hormone-ligand (DHEA) for the nuclear receptor PPARα, and is ultimately required for maintaining euglycemia and ketogenesis during nutrient deprivation. The feedback-loop that terminates Cyp17a1-PPARα activity, and re-establishes anabolic liver metabolism during re-feeding is mapped to postprandial bile acid-signaling, involving the receptors FXR, SHP and LRH-1. CONCLUSIONS: Together, these findings represent a novel paradigm of homeostatic control in which nutritional cues feed-forward on to metabolic pathways by influencing extragonadal steroidogenesis.

Identification of FDA-approved drugs targeting the Farnesoid X Receptor.

The farnesoid X receptor (FXR) belongs to the nuclear receptor family and is activated by bile acids. Multiple, chemically rather diverse, FXR agonists have been developed and several of these compounds are currently tested in clinical trials for NAFLD and cholestasis. Here, we investigated possible FXR-agonism or antagonism of existing FDA/EMA-approved drugs. By using our recently developed FRET-sensor, containing the ligand binding domain of FXR (FXR-LBD), 1280 FDA-approved drugs were screened for their ability to activate FXR in living cells using flow cytometry. Fifteen compounds induced the sensor for more than twenty percent above background. Real-time confocal microscopy confirmed that avermectin B1a, gliquidone, nicardipine, bepridil and triclosan activated the FRET sensor within two minutes. These compounds, including fluticasone, increased mRNA expression of FXR target genes OSTα and OSTß in Huh7 cells, and in most cases also of MRP2, SHP and FGF19. Finally, avermectin B1a, gliquidone, nicardipine and bepridil significantly increased IBABP promoter activity in a luciferase reporter assay in a dose-dependent manner. In conclusion, six FDA/EMA-approved drugs currently used in the clinical practice exhibit moderate agonistic FXR activity. This may on the one hand explain (undesired) side-effects, but on the other hand may form an opportunity for polypharmacology.

Effective CRISPR/Cas9-mediated correction of a Fanconi anemia defect by error-prone end joining or templated repair.

Fanconi anemia (FA) is a cancer predisposition syndrome characterized by congenital abnormalities, bone marrow failure, and hypersensitivity to aldehydes and crosslinking agents. For FA patients, gene editing holds promise for therapeutic applications aimed at functionally restoring mutated genes in hematopoietic stem cells. However, intrinsic FA DNA repair defects may obstruct gene editing feasibility. Here, we report on the CRISPR/Cas9-mediated correction of a disruptive mutation in Fancf. Our experiments revealed that gene editing could effectively restore Fancf function via error-prone end joining resulting in a 27% increased survival in the presence of mitomycin C. In addition, templated gene correction could be achieved after double strand or single strand break formation. Although templated gene editing efficiencies were low (≤6%), FA corrected embryonic stem cells acquired a strong proliferative advantage over non-corrected cells, even without imposing genotoxic stress. Notably, Cas9 nickase activity resulted in mono-allelic gene editing and avoidance of undesired mutagenesis. In conclusion: DNA repair defects associated with FANCF deficiency do not prohibit CRISPR/Cas9 gene correction. Our data provide a solid basis for the application of pre-clinical models to further explore the potential of gene editing against FA, with the eventual aim to obtain therapeutic strategies against bone marrow failure.

Progress and challenges of selective Farnesoid X Receptor modulation.

Bile acids are amphipathic molecules that were previously known to serve as fat solubilizers in the intestine in postprandial conditions. In the last two decades, bile acids have been recognized as signaling molecules regulating energy metabolism pathways via, amongst others, the farnesoid X receptor (FXR). Upon bile acid activation, FXR controls expression of genes involved in bile acid, lipid, glucose and amino acid metabolism. In addition, FXR activation has been shown to limit the inflammatory response. The central role of FXR in various aspects of metabolism and inflammation makes FXR an attractive drug target for several diseases, such as obesity, metabolic syndrome, non-alcoholic steatohepatitis, cholestasis and chronic inflammatory diseases of the liver and intestine. However, most of the currently available compounds impact on all discovered FXR-mediated functions and may have, on top of beneficial effects, undesired biological actions depending on the disease. Therefore, research efforts are increasingly focused on the development of selective FXR modulators, i.e. selective bile acid receptor modulators (SBARMs), aimed at limiting the potential side-effects of conventional full FXR agonists upon chronic treatment. Here, we review the rationale for the design of SBARMs comprising dissociation between metabolic and inflammatory signaling, gene-selective and tissue-specific targeting. We discuss the potential structural mechanisms underlying the binding properties of dissociating ligands of FXR in light of ongoing efforts on the generation of dissociated ligands for otxher nuclear receptors, as well as their pharmacological and therapeutic potential.