An Approach to Intersectionally Target Mature Enteroendocrine Cells in the Small Intestine of Mice.

Enteroendocrine cells (EECs) constitute only a small proportion of Villin-1 (Vil1)-expressing intestinal epithelial cells (IECs) of the gastrointestinal tract; yet, in sum, they build the largest endocrine organ of the body, with each of them storing and releasing a distinct set of peptides for the control of feeding behavior, glucose metabolism, and gastrointestinal motility. Like all IEC types, EECs are continuously renewed from intestinal stem cells in the crypt base and terminally differentiate into mature subtypes while moving up the crypt-villus axis. Interestingly, EECs adjust their hormonal secretion according to their migration state as EECs receive altering differentiation signals along the crypt-villus axis and thus undergo functional readaptation. Cell-specific targeting of mature EEC subtypes by specific promoters is challenging because the expression of EEC-derived peptides and their precursors is not limited to EECs but are also found in other organs, such as the brain (e.g., Cck and Sst) as well as in the pancreas (e.g., Sst and Gcg). Here, we describe an intersectional genetic approach that enables cell type-specific targeting of functionally distinct EEC subtypes by combining a newly generated Dre-recombinase expressing mouse line (Vil1-2A-DD-Dre) with multiple existing Cre-recombinase mice and mouse strains with rox and loxP sites flanked stop cassettes for transgene expression. We found that transgene expression in triple-transgenic mice is highly specific in I but not D and L cells in the terminal villi of the small intestine. The targeting of EECs only in terminal villi is due to the integration of a defective 2A separating peptide that, combined with low EEC intrinsic Vil1 expression, restricts our Vil1-2A-DD-Dre mouse line and the intersectional genetic approach described here only applicable for the investigation of mature EEC subpopulations.

Hyperspectral confocal imaging for high-throughput readout and analysis of bio-integrated microlasers.

Integrating micro- and nanolasers into live cells, tissue cultures and small animals is an emerging and rapidly evolving technique that offers noninvasive interrogation and labeling with unprecedented information density. The bright and distinct spectra of such lasers make this approach particularly attractive for high-throughput applications requiring single-cell specificity, such as multiplexed cell tracking and intracellular biosensing. The implementation of these applications requires high-resolution, high-speed spectral readout and advanced analysis routines, which leads to unique technical challenges. Here, we present a modular approach consisting of two separate procedures. The first procedure instructs users on how to efficiently integrate different types of lasers into living cells, and the second procedure presents a workflow for obtaining intracellular lasing spectra with high spectral resolution and up to 125-kHz readout rate and starts from the construction of a custom hyperspectral confocal microscope. We provide guidance on running hyperspectral imaging routines for various experimental designs and recommend specific workflows for processing the resulting large data sets along with an open-source Python library of functions covering the analysis pipeline. We illustrate three applications including the rapid, large-volume mapping of absolute refractive index by using polystyrene microbead lasers, the intracellular sensing of cardiac contractility with polystyrene microbead lasers and long-term cell tracking by using semiconductor nanodisk lasers. Our sample preparation and imaging procedures require 2 days, and setting up the hyperspectral confocal microscope for microlaser characterization requires <2 weeks to complete for users with limited experience in optical and software engineering.

The mitochondrial protease OMA1 acts as a metabolic safeguard upon nuclear DNA damage.

The metabolic plasticity of mitochondria ensures cell development, differentiation, and survival. The peptidase OMA1 regulates mitochondrial morphology via OPA1 and stress signaling via DELE1 and orchestrates tumorigenesis and cell survival in a cell- and tissue-specific manner. Here, we use unbiased systems-based approaches to show that OMA1-dependent cell survival depends on metabolic cues. A metabolism-focused CRISPR screen combined with an integrated analysis of human gene expression data found that OMA1 protects against DNA damage. Nucleotide deficiencies induced by chemotherapeutic agents promote p53-dependent apoptosis of cells lacking OMA1. The protective effect of OMA1 does not depend on OMA1 activation or OMA1-mediated OPA1 and DELE1 processing. OMA1-deficient cells show reduced glycolysis and accumulate oxidative phosphorylation (OXPHOS) proteins upon DNA damage. OXPHOS inhibition restores glycolysis and confers resistance against DNA damage. Thus, OMA1 dictates the balance between cell death and survival through the control of glucose metabolism, shedding light on its role in cancerogenesis.

NIK/MAP3K14 in hepatocytes orchestrates NASH to hepatocellular carcinoma progression via JAK2/STAT5 inhibition.

OBJECTIVE: Nonalcoholic fatty liver disease (NAFLD) ranges from steatosis to nonalcoholic steatohepatitis (NASH), which often progresses to hepatocellular carcinoma (HCC) through a largely undefined mechanism. NASH and HCC depend on inflammatory signaling, whose master regulator is the NFκB transcription factor family, activated by canonical and non-canonical pathways. METHODS: Here, we investigated non-canonical NFκB-inducing kinase (NIK/MAP3K14) in metabolic NASH, NASH to HCC transition, and DEN-induced HCC. To this end, we performed dietary and chemical interventions in mice that were analyzed via single nucleus sequencing, gene expression and histochemical methods. Ultimately, we verified our mouse results in human patient samples. RESULTS: We revealed that hepatocyte-specific NIK deficiency (NIKLKO) ameliorated metabolic NASH complications and reduced hepatocarcinogenesis, independent of its role in the NFκB pathway. Instead, hepatic NIK attenuated hepatoprotective JAK2/STAT5 signaling that is a prerequisite for NASH and NASH to HCC progression in mice and humans. CONCLUSIONS: Our data suggest NIK-mediated inhibitory JAK2 phosphorylation at serine 633 that might be amenable for future therapeutic interventions in patients.

Hypoxia promotes osteogenesis by facilitating acetyl-CoA-mediated mitochondrial-nuclear communication.

Bone-derived mesenchymal stem cells (MSCs) reside in a hypoxic niche that maintains their differentiation potential. While hypoxia (low oxygen concentration) was reported to critically support stem cell function and osteogenesis, the molecular events triggering changes in stem cell fate decisions in response to normoxia (high oxygen concentration) remain elusive. Here, we study the impact of normoxia on mitochondrial-nuclear communication during stem cell differentiation. We show that normoxia-cultured murine MSCs undergo profound transcriptional alterations which cause irreversible osteogenesis defects. Mechanistically, high oxygen promotes chromatin compaction and histone hypo-acetylation, particularly on promoters and enhancers of osteogenic genes. Although normoxia induces metabolic rewiring resulting in elevated acetyl-CoA levels, histone hypo-acetylation occurs due to the trapping of acetyl-CoA inside mitochondria owing to decreased citrate carrier (CiC) activity. Restoring the cytosolic acetyl-CoA pool remodels the chromatin landscape and rescues the osteogenic defects. Collectively, our results demonstrate that the metabolism-chromatin-osteogenesis axis is perturbed upon exposure to high oxygen levels and identifies CiC as a novel, oxygen-sensitive regulator of the MSC function.

Unraveling Structural Rearrangements of the CFH Gene Cluster in Atypical Hemolytic Uremic Syndrome Patients Using Molecular Combing and Long-Fragment Targeted Sequencing.

Complement factor H (CFH) and its related proteins have an essential role in regulating the alternative pathway of the complement system. Mutations and structural variants (SVs) of the CFH gene cluster, consisting of CFH and its five related genes (CFHR1-5), have been reported in renal pathologies as well as in complex immune diseases like age-related macular degeneration and systemic lupus erythematosus. SV analysis of this cluster is challenging because of its high degree of sequence homology. Following first-line next-generation sequencing gene panel sequencing, we applied Genomic Vision's Molecular Combing Technology to detect and visualize SVs within the CFH gene cluster and resolve its structural haplotypes completely. This approach was tested in three patients with atypical hemolytic uremic syndrome and known SVs and 18 patients with atypical hemolytic uremic syndrome or complement factor 3 glomerulopathy with unknown CFH gene cluster haplotypes. Three SVs, a CFH/CFHR1 hybrid gene in two patients and a rare heterozygous CFHR4/CFHR1 deletion in trans with the common CFHR3/CFHR1 deletion in a third patient, were newly identified. For the latter, the breakpoints were determined using a targeted enrichment approach for long DNA fragments (Samplix Xdrop) in combination with Oxford Nanopore sequencing. Molecular combing in addition to next-generation sequencing was able to improve the molecular genetic yield in this pilot study. This (cost-)effective approach warrants validation in larger cohorts with CFH/CFHR-associated disease.