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Understanding the molecular and cellular processes involved in lung epithelial regeneration may fuel the development of new therapeutic approaches for lung diseases. We combined new mouse models that allow diphtheria toxin (DTA)-mediated depletion of specific epithelial cell types and GFP-labeling of dividing cells with single-cell transcriptomics to characterize the regeneration of the distal lung. We uncovered new cell types, some of which likely represent epithelial precursors, propose goblet cells as progenitor cells, and provide evidence that adventitial fibroblasts act as supporting cells in epithelial regeneration. We also found that DTA-expressing cells can persist in the lung, express specific inflammatory factors, and resemble a previously undescribed population in the lungs of COVID-19 patients. Our study provides a comprehensive single-cell atlas of the distal lung that characterizes early transcriptional and cellular responses to defined epithelial injury, encompassing proliferation, differentiation, and cell-to-cell interactions.

Transcriptome profiling is a mainstay of translational cancer research and is increasingly finding its way into precision oncology. While bulk RNA sequencing (RNA-seq) is widely available, high costs and long data return time are limiting factors for clinical applications. We investigated a portable nanopore long-read sequencing device (MinION, Oxford Nanopore Technologies) for transcriptome profiling of tumors. In particular, we investigated the impact of lower coverage than that of larger sequencing devices by comparing shallow nanopore RNA-seq data with short-read RNA-seq data generated using reversible dye terminator technology (Illumina) for ten samples representing four cancer types. Coupled with ShaNTi (Shallow Nanopore Sequencing for Transcriptomics), a newly developed data processing pipeline, a turnaround time of five days was achieved. The correlation of normalized gene-level counts between nanopore and Illumina RNA-seq was high for MinION but not for very low-throughput Flongle flow cells (r = 0.89 and r = 0.24, respectively). A cost-saving approach based on multiplexing of four samples per MinION flow cell maintained a high correlation with Illumina data (r = 0.56 – 0.86). In addition, we compared the utility of nanopore and Illumina RNA-seq data for analysis tools commonly applied in translational oncology: (i) Shallow nanopore and Illumina RNA-seq were equally useful for inferring signaling pathway activities with PROGENy. (ii) Highly expressed genes encoding kinases targeted by clinically approved small-molecule inhibitors were reliably identified by shallow nanopore RNA-seq. (iii) In tumor microenvironment composition analysis, quanTIseq performed better than CIBERSORT, likely due to higher average expression of the gene set used for deconvolution. (iv) Shallow nanopore RNA-seq was successfully applied to validate known gene fusions by breakpoint analysis. These findings suggest that shallow nanopore RNA-seq enables rapid, cost-effective, and biologically meaningful transcriptome profiling of tumors and warrants further exploration in precision cancer medicine studies.

BackgroundMetabolic activity alternates between high and low states during different stages of an organism’s life cycle. During the transition from growth to quiescence, a major metabolic shift often occurs from oxidative phosphorylation to glycolysis and gluconeogenesis. We use the entry ofCaenorhabditis elegansinto the dauer larval stage, a developmentally arrested stage formed in response to harsh environmental conditions, as a model to study the global metabolic changes and underlying molecular mechanisms associated with growth to quiescence transition.ResultsHere, we show that the metabolic switch involves the concerted activity of several regulatory pathways. Whereas the steroid hormone receptor DAF-12 controls dauer morphogenesis, the insulin pathway maintains low energy expenditure through DAF-16/FoxO, which also requires AAK-2/AMPKα. DAF-12 and AAK-2 separately promote a shift in the molar ratios between competing enzymes at two key branch points within the central carbon metabolic pathway diverting carbon atoms from the TCA cycle and directing them to gluconeogenesis. When both AAK-2 and DAF-12 are suppressed, the TCA cycle is active and the developmental arrest is bypassed.ConclusionsThe metabolic status of each developmental stage is defined by stoichiometric ratios within the constellation of metabolic enzymes driving metabolic flux and controls the transition between growth and quiescence.

Myxoid liposarcoma (MLS) represents a common subtype of liposarcoma molecularly characterized by a recurrent chromosomal translocation that generates a chimeric FUS-DDIT3 fusion gene. The FUS-DDIT3 oncoprotein has been shown to be crucial in MLS pathogenesis. Acting as a transcriptional dysregulator, FUS-DDIT3 stimulates proliferation and interferes with adipogenic differentiation. As the fusion protein represents a therapeutically challenging target, a profound understanding of MLS biology is elementary to uncover FUS-DDIT3-dependent molecular vulnerabilities. Recently, a specific reliance on the Hippo pathway effector and transcriptional co-regulator YAP1 was detected in MLS; however, details on the molecular mechanism of FUS-DDIT3-dependent YAP1 activation, and YAP1´s precise mode of action remain unclear. In elaborate in vitro studies, employing RNA interference-based approaches, small-molecule inhibitors, and stimulation experiments with IGF-II, we show that FUS-DDIT3-driven IGF-IR/PI3K/AKT signaling promotes stability and nuclear accumulation of YAP1 via deregulation of the Hippo pathway. Co-immunoprecipitation and proximity ligation assays revealed nuclear co-localization of FUS-DDIT3 and YAP1/TEAD in FUS-DDIT3-expressing mesenchymal stem cells and MLS cell lines. Transcriptome sequencing of MLS cells demonstrated that FUS-DDIT3 and YAP1 co-regulate oncogenic gene signatures related to proliferation, cell cycle progression, apoptosis, and adipogenesis. In adipogenic differentiation assays, we show that YAP1 critically contributes to FUS-DDIT3-mediated adipogenic differentiation arrest. Taken together, our study provides mechanistic insights into a complex FUS-DDIT3-driven network involving IGF-IR/PI3K/AKT signals acting on Hippo/YAP1, and uncovers substantial cooperative effects of YAP1 and FUS-DDIT3 in the pathogenesis of MLS.

Introduction: Myxoid liposarcoma (MLS) is molecularly characterized by a recurrent chromosomal translocation which generates a chimeric FUS-DDIT3 fusion gene. The FUS-DDIT3 oncoprotein, acting as a transcriptional dysregulator, has been shown to be essential in MLS pathogenesis, among others through deregulation of IGF-IR/PI3K/AKT signaling, but its exact mode of function remains incompletely understood. Recently, a particular reliance on the Hippo pathway effector and transcriptional co-regulator YAP1 was found in MLS; however, the molecular mechanism of FUS-DDIT3-dependent YAP1 activation and its contribution to MLS pathogenesis remain unclear. Experimental Procedures: The expression of IGF-IR and YAP1 was analyzed in a large cohort of MLS specimens by immunohistochemistry. In vitro analyses were performed employing a human mesenchymal stem cell system stably expressing FUS-DDIT3 and human MLS cell lines. RNA interference-based approaches, experiments with small-molecule kinase inhibitors, co-immunoprecipitation, proximity ligation assays, transcriptome sequencing and adipogenic differentiation assays were performed to determine the interplay of FUS-DDIT3, IGF-IR-dependent signals, and YAP1 in MLS cells. Results: Immunohistochemically, a significant subset of MLS samples showed concurrent expression of IGF-IR and nuclear YAP1. In vitro, FUS-DDIT3-driven IGF-IR signaling was found to promote stability and nuclear accumulation of YAP1 via deregulation of the Hippo pathway. Co-immunoprecipitation and proximity ligation assays revealed nuclear co-localization of FUS-DDIT3 and YAP1 in FUS-DDIT3-expressing mesenchymal stem cells and MLS cell lines. Transcriptome sequencing of MLS cells demonstrated that FUS-DDIT3 and YAP1 co-regulate specific oncogenic gene signatures related to proliferation, cell cycle progression, apoptosis, and adipogenesis. In differentiation assays, FUS-DDIT3 and YAP1 were found to cooperate in adipogenic differentiation arrest. Conclusions: Our study provides molecular insights into a complex FUS-DDIT3-driven network involving IGF-IR signals acting on Hippo/YAP1, and uncovers cooperative effects of YAP1 and FUS-DDIT3 in the pathogenesis of MLS. Citation Format: Ruth Berthold, Ilka Isfort, Cihan Erkut, Lorena Heinst, Inga Grünewald, Eva Wardelmann, Thomas Kindler, Pierre Åman, Thomas G.P. Grünewald, Florencia Cidre-Aranaz, Marcel Trautmann, Stefan Fröhling, Claudia Scholl, Wolfgang Hartmann. Fusion protein-driven IGF-IR signals deregulate hippo pathway promoting oncogenic cooperation of YAP1 and FUS-DDIT3 [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr PR004.

Simple Summary The clinical management for aggressive meningiomas remains challenging due to the lack of systemic treatment options. Receptor tyrosine kinases (RTKs) are frequently overexpressed in meningiomas and are associated with poor patient survival. In this study, we evaluated the clinically approved pan-RTK inhibitor ponatinib as a novel candidate for the treatment of aggressive meningiomas. Ponatinib decreased cell viability and proliferation of meningioma cells and subsequently induced programmed cell death. Furthermore, the drug demonstrated a considerable tumor growth inhibition without causing any adverse effects in mice. Mechanistically, this was presumably caused by blocking the PDGFR signaling pathway accompanied by induction of mitochondrial dysfunction. Altogether, the multi-RTKi ponatinib may serve as a promising candidate for targeted therapy for aggressive meningiomas. Abstract To date, there is no standard-of-care systemic therapy for the treatment of aggressive meningiomas. Receptor tyrosine kinases (RTK) are frequently expressed in aggressive meningiomas and are associated with poor survival. Ponatinib is a FDA- and EMA-approved RTK inhibitor and its efficacy in meningioma has not been studied so far. Therefore, we investigated ponatinib as a potential drug candidate against meningioma. Cell viability and cell proliferation of ponatinib-treated meningioma cells were assessed using crystal violet assay, manual counting and BrdU assay. Treated meningioma cell lines were subjected to flow cytometry to evaluate the effects on cell cycle and apoptosis. Meningioma-bearing mice were treated with ponatinib to examine antitumor effects in vivo. qPCR was performed to assess the mRNA levels of tyrosine kinase receptors after ponatinib treatment. Full-length cDNA sequencing was carried out to assess differential gene expression. IC50 values of ponatinib were between 171.2 and 341.9 nM in three meningioma cell lines. Ponatinib induced G0/G1 cell cycle arrest and subsequently led to an accumulation of cells in the subG1-phase. A significant induction of apoptosis was observed in vitro. In vivo, ponatinib inhibited meningioma growth by 72.6%. Mechanistically, this was associated with downregulation of PDGFRA/B and FLT3 mRNA levels, and mitochondrial dysfunction. Taken together, ponatinib is a promising candidate for targeted therapy in the treatment of aggressive meningioma.

Many organisms, including species from all kingdoms of life, can survive desiccation by entering a state with no detectable metabolism. To survive, C. elegans dauer larvae and stationary phase S. cerevisiae require elevated amounts of the disaccharide trehalose. We found that dauer larvae and stationary phase yeast switched into a gluconeogenic mode in which metabolism was reoriented toward production of sugars from non-carbohydrate sources. This mode depended on full activity of the glyoxylate shunt (GS), which enables synthesis of trehalose from acetate. The GS was especially critical during preparation of worms for harsh desiccation (preconditioning) and during the entry of yeast into stationary phase. Loss of the GS dramatically decreased desiccation tolerance in both organisms. Our results reveal a novel physiological role for the GS and elucidate a conserved metabolic rewiring that confers desiccation tolerance on organisms as diverse as worm and yeast. DOI: http://dx.doi.org/10.7554/eLife.13614.001, eLife digest Many organisms can survive losing all the water from their body in periods of severe drought by suspending their life. This ability is called anhydrobiosis (from the Greek for ‘life without water’). When the desiccated organisms encounter water again, they resume life as normal. Two organisms commonly used in research, a roundworm called Caenorhabditis elegans and a yeast called Saccharomyces cerevisiae, are anhydrobiotes. To survive without water, anhydrobiotes alter the chemical reactions that sustain their life, and so change their metabolic state. The organisms also produce molecules that preserve the structure of their cells. One such essential molecule is a sugar called trehalose. However, both worms and yeast can only enter anhydrobiosis during particular stages of life where they do not eat. So where does the trehalose come from? Erkut et al. have now addressed this question by studying the metabolism of C. elegans and S. cerevisiae as these species entered anhydrobiosis. The experiments revealed that while preparing for desiccation, both species change their metabolism to favor creating sugars rather than releasing energy. In this process, the worms and yeast use a biochemical pathway called the glyoxylate shunt, which can convert fat or acetic acid into sugar. Genetic mutations that deactivate this pathway severely reduce the ability of both organisms to produce trehalose and tolerate desiccation. From these findings, Erkut et al. conclude that the source of trehalose in non-feeding worms is their fat deposits, while in yeast it is acetate: a molecule that is derived from ethanol, the end-product of the fermentation process. The glyoxylate shunt had been thought only to be a non-essential biochemical shortcut of another well-known metabolic pathway called the Krebs cycle. Now that Erkut et al. have shown that the glyoxylate shunt has its own specific biological role, further investigation is needed to understand how it is activated to act as a metabolic switch. The molecules that regulate similar metabolic transitions will also need to be identified in future studies. Ultimately, understanding these processes could present new ways of diagnosing and treating metabolic diseases such as diabetes and cancer. DOI: http://dx.doi.org/10.7554/eLife.13614.002

Summary Water is essential for life on Earth. In its absence, however, some organisms can interrupt their life cycle and temporarily enter an ametabolic state, known as anhydrobiosis [1]. It is assumed that sugars (in particular trehalose) are instrumental for survival under anhydrobiotic conditions [2]. However, the role of trehalose remained obscure because the corresponding evidence was purely correlative and based mostly on in vitro studies without any genetic manipulations of trehalose metabolism. In this study, we used C. elegans as a genetic model to investigate molecular mechanisms of anhydrobiosis. We show that the C. elegans dauer larva is a true anhydrobiote: under defined conditions it can survive even after losing 98% of its body water. This ability is correlated with a several fold increase in the amount of trehalose. Mutants unable to synthesize trehalose cannot survive even mild dehydration. Light and electron microscopy indicate that one of the major functions of trehalose is the preservation of membrane organization. Fourier-transform infrared spectroscopy of whole worms suggests that this is achieved by preserving homogeneous and compact packing of lipid acyl chains. By means of infrared spectroscopy, we can now distinguish a "dry, yet alive" larva from a "dry and dead" one.

Under adverse conditions, Caenorhabditis elegans enters a diapause stage called the dauer larva. External cues signal the nuclear hormone receptor DAF-12, the activity of which is regulated by its ligands: dafachronic acids (DAs). DAs are synthesized from cholesterol, with the last synthesis step requiring NADPH, and their absence stimulates dauer formation. Here we show that NADPH levels determine dauer formation in a regulatory mechanism involving key carbohydrate and redox metabolic enzymes. Elevated trehalose biosynthesis diverts glucose-6-phosphate from the pentose phosphate pathway, which is the major source of cellular NADPH. This enhances dauer formation due to the decrease in the DA level. Moreover, DAF-12, in cooperation with DAF-16/FoxO, induces negative feedback of DA synthesis via activation of the trehalose-producing enzymes TPS-1/2 and inhibition of the NADPH-producing enzyme IDH-1. Thus, the dauer developmental decision is controlled by integration of the metabolic flux of carbohydrates and cellular redox potential.

The hypometabolic, stress-resistant dauer larva of Caenorhabditis elegans serves as an excellent model to study the molecular mechanisms of desiccation tolerance, such as maintenance of membrane organization, protein folding, xenobiotic and ROS detoxification in the dry state. Many organisms from diverse taxa of life have the remarkable ability to survive extreme desiccation in the nature by entering an ametabolic state known as anhydrobiosis (life without water). The hallmark of the anhydrobiotic state is the achievement and maintenance of an exceedingly low metabolic rate, as well as preservation of the structural integrity of the cell. Although described more than three centuries ago, the biochemical and biophysical mechanisms underlying this phenomenon are still not fully comprehended. This is mainly due to the fact that anhydrobiosis in animals was studied using non-model organisms, which are very difficult, if not impossible, to manipulate at the molecular level. Recently, we introduced the roundworm (nematode) Caenorhabditis elegans as a model for anhydrobiosis. Taking advantage of powerful genetic, biochemical and biophysical tools, we investigated several aspects of anhydrobiosis in a particular developmental stage (the dauer larva) of this organism. First, our studies allowed confirming the previously suggested role of the disaccharide trehalose in the preservation of lipid membranes. Moreover, in addition to known pathways such as reactive oxygen species defense, heat-shock and intrinsically disordered protein expression, evidence for some novel strategies of anhydrobiosis has been obtained. These are increased glyoxalase activity, polyamine and polyunsaturated fatty acid biosynthesis. All these pathways may constitute a generic toolbox of anhydrobiosis, which is possibly conserved between animals and plants.

Anhydrobiotic organisms have the remarkable ability to lose extensive amounts of body water and survive in an ametabolic state. Distributed to various taxa of life, these organisms have developed strategies to efficiently protect their cell membranes and proteins against extreme water loss. Recently, we showed that the dauer larva of the nematode Caenorhabditis elegans is anhydrobiotic and accumulates high amounts of trehalose during preparation to harsh desiccation (preconditioning). Here, we have used this genetic model to study the biophysical manifestations of anhydrobiosis and show that, in addition to trehalose accumulation, dauer larvae dramatically reduce their phosphatidylcholine (PC) content. The chemical composition of the phospholipids (PLs) has key consequences not only for their interaction with trehalose, as we demonstrate with Langmuir−Blodgett monolayers, but also, the kinetic response of PLs to hydration transients is strongly influenced as evidenced by time-resolved FTIR spectroscopy. PLs from preconditioned larvae with reduced PC content exhibit a higher trehalose affinity, a stronger hydration-induced gain in acyl chain free volume, and a wider spread of structural relaxation rates of their lyotropic transitions and sub-headgroup H-bond interactions. The different hydration properties of PC and phosphatidylethanolamine (PE) headgroups are crucial for the hydration-dependent rearrangement of the trehalose-mediated H-bond network. As a consequence, the compressibility modulus of PLs from preconditioned larvae is about 2.6-fold smaller than that from non-preconditioned ones. Thus, the biological relevance of reducing the PC:PE ratio by PL headgroup adaptation should be the preservation of plasma membrane integrity by relieving mechanical strain from desiccated trehalose-containing cells during fast rehydration.

Parkinson's disease is associated with mitochondrial decline in dopaminergic neurons of the substantia nigra. One of the genes linked with the onset of Parkinson's disease, DJ-1/PARK7, belongs to a novel glyoxalase family and influences mitochondrial activity. It has been assumed that glyoxalases fulfill this task by detoxifying aggressive aldehyde by-products of metabolism. Here we show that supplying either D-lactate or glycolate, products of DJ-1, rescues the requirement for the enzyme in maintenance of mitochondrial potential. We further show that glycolic acid and D-lactic acid can elevate lowered mitochondrial membrane potential caused by silencing PINK-1, another Parkinson's related gene, as well as by paraquat, an environmental toxin known to be linked with Parkinson's disease. We propose that DJ-1 and consequently its products are components of a novel pathway that stabilizes mitochondria during cellular stress. We go on to show that survival of cultured mesencephalic dopaminergic neurons, defective in Parkinson's disease, is enhanced by glycolate and D-lactate. Because glycolic and D-lactic acids occur naturally, they are therefore a potential therapeutic route for treatment or prevention of Parkinson's disease., Biology Open, 3 (8), ISSN:2046-6390

The nematode Caenorhabditis elegans can bypass part of its normal larval development by forming a "dauer" state with arrested metabolism. The desiccation-tolerance of this state depends on the synthesis of trehalose during a preconditioning phase, where the worm is first exposed to mildly reduced RH, rendering the dauer state attractive for studying the physiology and genetics of Anhydrobiosis. Here, we correlate physical properties of phospholipids (PLs) with the desiccation tolerance of the larvae from which PLs were extracted. The response of PL structure to transient (seconds) changes in water potential and the role of trehalose in water-mediated structural transitions were addressed. Time-resolved Rapid scan FTIR spectroscopy was used in ATR geometry to record hydration-induced difference spectra. We show by chemical analysis that a reduction in choline content in the PL headgroup composition arises during preconditioning. This leads to a stronger coupling of headgroup hydration to disorder in PL acyl chains based on the time-resolved observation of PO2-, C=O and acyl CH2 stretching modes. Trehalose enhances this effect and leads to more uniform kinetics of hydration transients and lipid transitions. In combination with spectroscopic determination of altered lipid main phase transition temperatures (Tm) in PLs upon preconditioning, the data show that chemical tuning of the kinetics and the extent of coupling of headgroup hydration to acyl chain packing changes is a key process in desiccation tolerance as it may release mechanical stress from membranes during temporal changes of water potential. Additional DSC, ITC and Langmuir-Blodget data show that trehalose interacts more favourably with PLs from preconditioned larvae to support desiccation tolerance. This work highlights that chemical tuning of lyotropic phase transitions plays a fundamental role in the trehalose-dependent desiccation tolerance of C. elegans larvae.