Liver zonation, revisited

The concept of hepatocyte functional zonation is well established, with differences in metabolism and xenobiotic processing determined by multiple factors including oxygen and nutrient levels across the hepatic lobule. However, recent advances in single‐cell genomics technologies, including single‐cell and nuclei RNA sequencing, and the rapidly evolving fields of spatial transcriptomic and proteomic profiling have greatly increased our understanding of liver zonation. Here we discuss how these transformative experimental strategies are being leveraged to dissect liver zonation at unprecedented resolution and how this new information should facilitate the emergence of novel precision medicine‐based therapies for patients with liver disease

Novel regulators of cancer stem cell biology in acute myeloid leukaemia

Lifelong haematopoiesis depends critically on a small population of bone marrow resident haematopoietic stem cells (HSCs), which possess unique self-renewal capacity and multilineage differentiation potential, ultimately replenishing all blood lineages. In view of these properties, HSC transplantation (HSCT) is an effective treatment for a wide range of bone marrow disorders. However, owing to the rarity of HSCs in adult haematopoiesis, and our inability to effectively expand HSCs in vitro, the clinical need for HSCT vastly exceeds HSC availability. Acute myeloid leukaemia (AML) is an aggressive clonal disorder of haematopoietic stem and progenitor cells (HSPCs), in which the acquisition of mutations by HSPCs results in a block in their myeloid differentiation, and the generation of self-renewing leukaemic stem cells (LSCs). LSCs initiate and propagate the disease, and given that they are treatment-resistant, often fuel disease relapses. The two major challenges in the field of haematopoiesis currently, are therefore to expand HSCs for transplantation, and to identify novel therapeutic targets to eliminate LSCs. In the last decade, epitranscriptomics has developed as a novel layer of regulation of gene expression. RNA N6-methyladenosine (m6A) is the most abundant internal mRNA modification. m6A is installed on mRNA by methyltransferase complexes known as writers and removed by demethylases defined as erasers. The m6A modification is recognised by reader proteins, including those belonging to the YTH domain family (YTHDF1, YTHDF2, YTHDF3, YTHDC1, YTHDC2). While writers and erasers have been implicated in several cancers, including AML, the roles of m6A readers in normal and malignant haematopoiesis remains unexplored. This thesis describes the study of the YTHDF2 m6A reader in normal haematopoiesis and AML. Using the Vav-iCre system to conditionally delete Ythdf2 from the haematopoietic system of mice, we observed a significant increase in the number of haematopoietic stem and progenitor cells (HSPCs) compared to controls. In primary and secondary long-term competitive transplantation assays, Ythdf2-deficient HSCs reconstituted multilineage haematopoiesis and showed an increased capacity to rapidly repopulate the myeloid lineage. Furthermore, bone marrow analysis 4 months after transplantation revealed an increased contribution of Ythdf2-deficient cells to the HSPC compartments of transplanted mice compared to controls. To study the role of Ythdf2 in AML, we compared the ability of Ythdf2-deficient and control pre-LSCs to generate leukaemia in a murine retroviral AML model. We showed that constitutive (Vav-iCre) and inducible (Mx1-Cre) genetic deletion of Ythdf2 severely compromises the development and propagation of leukaemia driven by a range of oncogenes, including Meis1/Hoxa9, Mll-AF9, PML-RARA and MOZ-TIF2. Mechanistically, we showed that loss of Ythdf2 inhibits degradation of multiple m6A-modified transcripts, including Tnfrsf2, whose upregulation sensitizes Ythdf2-deficient LSCs to TNFinduced apoptosis. In summary, we demonstrated that inhibition of Ythdf2 specifically compromises LSC development and propagation, but simultaneously expands HSCs and enhances their myeloid reconstitution upon transplantation. We therefore propose YTHDF2 as a unique therapeutic target whose inhibition selectively targets LSCs while promoting HSC expansion

Troglitazone Induces Extracellular Matrix and Cytoskeleton Remodeling in Mouse Collecting Duct Cells

Peroxisome proliferator-activated receptor (PPARγ) has been shown to have a protective role in the nephron through its ability to inhibit a transforming growth factor- (TGF-β) mediated fibrotic response. In contrast, PPARγ was also shown to induce a mesenchymal transformation in epithelial intestinal cells. A fibrotic response in the collecting duct has only recently been established; however, the entire collecting duct has not been fully examined. Inner medullary collecting duct cells (IMCD-K2) and mouse cortical collecting duct cells (M1), representing the cortical and medullary collecting duct, were exposed to 5–10 μM troglitazone for 24 hours. Troglitazone resulted in an elongated morphology, 60% decreases in E-cadherin and β-catenin, a 35% decrease in α-catenin, and a 1.5-fold increase in fibronectin. These effects were not reversed with PPARγ antagonists or affected with PPARγ overexpression. Our results indicate that troglitazone induced a mesenchymal-like transformation in M1 and IMCD-K2 epithelial cells independently of PPARγ

Troglitazone Induces Extracellular Matrix and Cytoskeleton Remodeling in Mouse Collecting Duct Cells

Peroxisome proliferator-activated receptor (PPARγ) has been shown to have a protective role in the nephron through its ability to inhibit a transforming growth factor- (TGF-β) mediated fibrotic response. In contrast, PPARγ was also shown to induce a mesenchymal transformation in epithelial intestinal cells. A fibrotic response in the collecting duct has only recently been established; however, the entire collecting duct has not been fully examined. Inner medullary collecting duct cells (IMCD-K2) and mouse cortical collecting duct cells (M1), representing the cortical and medullary collecting duct, were exposed to 5–10 μM troglitazone for 24 hours. Troglitazone resulted in an elongated morphology, 60% decreases in E-cadherin and β-catenin, a 35% decrease in α-catenin, and a 1.5-fold increase in fibronectin. These effects were not reversed with PPARγ antagonists or affected with PPARγ overexpression. Our results indicate that troglitazone induced a mesenchymal-like transformation in M1 and IMCD-K2 epithelial cells independently of PPARγ

Enteropathogen co-infection in UK cats with diarrhoea

BACKGROUND: Individual enteropathogen infections in healthy and clinically ill cats are well described, but prevalence and patterns of enteropathogen co-infection have only been reported on a limited basis. We studied enteropathogen co-infection in diarrhoeic UK cats using results of a real time PCR assay for 8 enteropathogenic species; feline coronavirus (Co), feline panleukopenia virus (Pa), Clostridium perfringens (Cl), Salmonella enterica (Sa), Giardia spp. (Gi), Tritrichomonas foetus (Tr), Cryptosporidium spp. (Cr), and Toxoplasma gondii (To). Age, gender, breed and history were recorded. PCR panels from 1088 diarrhoeic cats were available for analysis. RESULTS: Overall enteropathogen prevalence was 56.9% (Co), 22.1% (Pa), 56.6% (Cl), 0.8% (Sa), 20.6% (Gi), 18.8% (Tr), 24.4% (Cr) and 1.0% (To). Prevalence of Co, Gi and Tr was higher in pedigree cats compared to non-pedigree cats (DSH) and prevalence decreased with increasing age for Co, Pa, Gi, Cr and Tr. Co-infection was common: ≥2 enteropathogens were detected in 62.5% of cats, and 13.3% of cats had ≥4 enteropathogens. Mean ( [Formula: see text]) enteropathogen co-infection 2.01 (±1.3 SD), was significantly higher in pedigree cats ( [Formula: see text] =2.51) compared to DSH ( [Formula: see text] =1.68) and decreased with age ( [Formula: see text] =2.64 <6 months, [Formula: see text] =1.68 for >1 yr). More cats were negative for all 8 enteropathogens tested (12.7%) than expected. When exact combinations of co-infection were examined, Tr tended to be found in combinations with Co, Cl, and Gi. CONCLUSIONS: Multiple infections should be considered the most likely result of faecal testing in cats, and case management needs to take this into account. In contrast, the relatively high percentage of cats negative for all 8 enteropathogens tested could indicate an innate resistance to infection. Alternatively it could indicate a lack of exposure to these 8 enteropathogens or the presence of other enteropathogens not assessed by this assay

Automated Laser‐Transfer Synthesis of High‐Density Microarrays for Infectious Disease Screening

Laser-induced forward transfer (LIFT) is a rapid laser-patterning technique for high-throughput combinatorial synthesis directly on glass slides. A lack of automation and precision limits LIFT applications to simple proof-of-concept syntheses of fewer than 100 compounds. Here, an automated synthesis instrument is reported that combines laser transfer and robotics for parallel synthesis in a microarray format with up to 10 000 individual reactions cm−2. An optimized pipeline for amide bond formation is the basis for preparing complex peptide microarrays with thousands of different sequences in high yield with high reproducibility. The resulting peptide arrays are of higher quality than commercial peptide arrays. More than 4800 15-residue peptides resembling the entire Ebola virus proteome on a microarray are synthesized to study the antibody response of an Ebola virus infection survivor. Known and unknown epitopes that serve now as a basis for Ebola diagnostic development are identified. The versatility and precision of the synthesizer is demonstrated by in situ synthesis of fluorescent molecules via Schiff base reaction and multi-step patterning of precisely definable amounts of fluorophores. This automated laser transfer synthesis approach opens new avenues for high-throughput chemical synthesis and biological screening

Adult haematopoietic stem cells lacking Hif-1α self-renew normally

The haematopoietic stem cell (HSC) pool is maintained under hypoxic conditions within the bone marrow (BM) microenvironment. Cellular responses to hypoxia are largely mediated by hypoxia-inducible factors, Hif-1 and Hif-2. The oxygen-regulated alpha subunits of Hif-1 and Hif-2 (namely, Hif-1α and Hif-2α) form dimers with their stably expressed beta subunits, and control the transcription of downstream hypoxia-responsive genes to facilitate adaptation to low oxygen tension. An initial study concluded that Hif-1α is essential for HSC maintenance, whereby Hif-1α-deficient HSCs lost their ability to self-renew in serial transplantation assays. In another study, we demonstrated that Hif-2α is dispensable for cell-autonomous HSC maintenance, both under steady-state conditions and following transplantation. Given these unexpected findings, we set out to revisit the role of Hif-1α in cell-autonomous HSC functions. Here we demonstrate that inducible acute deletion of Hif-1α has no impact on HSC survival. Notably, unstressed HSCs lacking Hif-1α efficiently self-renew and sustain long-term multilineage haematopoiesis upon serial transplantation. Finally, Hif-1α-deficient HSCs recover normally after hematopoietic injury induced by serial administration of 5-fluorouracil. We therefore conclude that despite the hypoxic nature of the BM microenvironment, Hif-1α is dispensable for cell-autonomous HSC maintenance

The mRNA m6A reader YTHDF2 suppresses proinflammatory pathways and sustains hematopoietic stem cell function

The mRNA N6-methyladenosine (m6A) modification has emerged as an essential regulator of normal and malignant hematopoiesis. Inactivation of the m6A mRNA reader YTHDF2, which recognizes m6A-modified transcripts to promote m6A-mRNA degradation, results in hematopoietic stem cell (HSC) expansion and compromises acute myeloid leukemia. Here we investigate the long-term impact of YTHDF2 deletion on HSC maintenance and multilineage hematopoiesis. We demonstrate that Ythdf2-deficient HSCs from young mice fail upon serial transplantation, display increased abundance of multiple m6A-modified inflammation-related transcripts, and chronically activate proinflammatory pathways. Consistent with the detrimental consequences of chronic activation of inflammatory pathways in HSCs, hematopoiesis-specific Ythdf2 deficiency results in a progressive myeloid bias, loss of lymphoid potential, HSC expansion, and failure of aged Ythdf2-deficient HSCs to reconstitute multilineage hematopoiesis. Experimentally induced inflammation increases YTHDF2 expression, and YTHDF2 is required to protect HSCs from this insult. Thus, our study positions YTHDF2 as a repressor of inflammatory pathways in HSCs and highlights the significance of m6A in long-term HSC maintenance

Targeting the RNA m⁶A reader YTHDF2 selectively compromises cancer stem cells in acute myeloid leukemia

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