Spatial barcoding technologies have the potential to reveal histological details of transcriptomic profiles; however, they are currently limited by their low resolution. Here, we report Seq-Scope, a ...spatial barcoding technology with a resolution comparable to an optical microscope. Seq-Scope is based on a solid-phase amplification of randomly barcoded single-molecule oligonucleotides using an Illumina sequencing platform. The resulting clusters annotated with spatial coordinates are processed to expose RNA-capture moiety. These RNA-capturing barcoded clusters define the pixels of Seq-Scope that are ∼0.5–0.8 μm apart from each other. From tissue sections, Seq-Scope visualizes spatial transcriptome heterogeneity at multiple histological scales, including tissue zonation according to the portal-central (liver), crypt-surface (colon) and inflammation-fibrosis (injured liver) axes, cellular components including single-cell types and subtypes, and subcellular architectures of nucleus and cytoplasm. Seq-Scope is quick, straightforward, precise, and easy-to-implement and makes spatial single-cell analysis accessible to a wide group of biomedical researchers.
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•Seq-Scope repurposes Illumina sequencing platform for spatial transcriptomics•Seq-Scope achieves submicrometer resolution and efficient transcriptome capture•Seq-Scope enabled spatial single cell and subcellular analysis of liver and colon
Seq-Scope uses spatial barcoding and the Illumina sequencing platform to achieve sub-micron resolution spatial transcriptomics, enabling the visualization of transcriptomic heterogeneity at the cellular and subcellular level in various tissues.
Exercise is among the most effective interventions for age-associated mobility decline and metabolic dysregulation. Although long-term endurance exercise promotes insulin sensitivity and expands ...respiratory capacity, genetic components and pathways mediating the metabolic benefits of exercise have remained elusive. Here, we show that Sestrins, a family of evolutionarily conserved exercise-inducible proteins, are critical mediators of exercise benefits. In both fly and mouse models, genetic ablation of Sestrins prevents organisms from acquiring metabolic benefits of exercise and improving their endurance through training. Conversely, Sestrin upregulation mimics both molecular and physiological effects of exercise, suggesting that it could be a major effector of exercise metabolism. Among the various targets modulated by Sestrin in response to exercise, AKT and PGC1α are critical for the Sestrin effects in extending endurance. These results indicate that Sestrin is a key integrating factor that drives the benefits of chronic exercise to metabolism and physical endurance.
Obesity commonly leads to hepatic steatosis, which often provokes lipotoxic injuries to hepatocytes that cause nonalcoholic steatohepatitis (NASH). NASH, in turn, is associated with the accumulation ...of insoluble protein aggregates that are composed of ubiquitinated proteins and ubiquitin adaptor p62/sequestosome 1 (SQSTM1). Formation of p62 inclusions in hepatocytes is the critical marker that distinguishes simple fatty liver from NASH and predicts a poor prognostic outcome for subsequent liver carcinogenesis. However, the molecular mechanism by which lipotoxicity induces protein aggregation is currently unknown. Here, we show that, upon saturated fatty acid‐induced lipotoxicity, TANK binding kinase 1 (TBK1) is activated and phosphorylates p62. TBK1‐mediated p62 phosphorylation is important for lipotoxicity‐induced aggregation of ubiquitinated proteins and formation of large protein inclusions in hepatocytes. In addition, cyclic GMP‐AMP synthase (cGAS) and stimulator of interferon genes (STING), upstream regulators of TBK1, are involved in lipotoxic activation of TBK1 and subsequent p62 phosphorylation in hepatocytes. Furthermore, TBK1 inhibition prevented formation of ubiquitin‐p62 aggregates not only in cultured hepatocytes, but also in mouse models of obesity and NASH. Conclusion: These results suggest that lipotoxic activation of TBK1 and subsequent p62 phosphorylation are critical steps in the NASH pathology of protein inclusion accumulation in hepatocytes. This mechanism can provide an explanation for how hypernutrition and obesity promote the development of severe liver pathologies, such as steatohepatitis and liver cancer, by facilitating the formation of p62 inclusions. (Hepatology 2018).
Excessive accumulation of reactive oxygen species (ROS) and chronic activation of mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) are well-characterized promoters of aging and ...age-associated degenerative pathologies. Sestrins, a family of highly conserved stress-inducible proteins, are important negative regulators of both ROS and mTORC1 signaling pathways; however, the mechanistic basis of how Sestrins suppress these pathways remains elusive. In the past couple of years, breakthrough discoveries about Sestrin signaling and its molecular nature have markedly increased our biochemical understanding of Sestrin function. These discoveries have also uncovered new potential therapeutic strategies that may eventually enable us to attenuate aging and age-associated diseases.
Sestrin suppresses oxidative damage by activation of the antioxidant transcription factor Nrf2, autophagic elimination of damaged mitochondria, and direct detoxification of ROS through its intrinsic peroxidase activity.
The heteropentameric GATOR2 complex is a direct physical target of Sestrin that mediates its mTORC1-regulating functions.
Sestrin activity is regulated by amino acids and other nutrients at multiple levels, including both transcriptional and post-transcriptional mechanisms.
A hSesn2 crystal structure revealed three structural motifs, each responsible for one of the identified functions of hSesn2: ROS detoxification, GATOR2 binding (and subsequently regulating AMPK and mTORC1 signaling), and amino acid (leucine) binding.
The catalytic cysteine of the typical 2-Cys Prx subfamily of peroxiredoxins is occasionally hyperoxidized to cysteine sulfinic acid during the peroxidase catalytic cycle. Sulfinic Prx (Prx–SO ₂H) is ...reduced back to the active form of the enzyme by sulfiredoxin. The abundance of Prx–SO ₂H was recently shown to oscillate with a period of ∼24 h in human red blood cells (RBCs). We have now investigated the molecular mechanism and physiological relevance of such oscillation in mouse RBCs. Poisoning of RBCs with CO abolished Prx–SO ₂H formation, implicating H ₂O ₂ produced from hemoglobin autoxidation in Prx hyperoxidation. RBCs express the closely related PrxI and PrxII isoforms, and analysis of RBCs deficient in either isoform identified PrxII as the hyperoxidized Prx in these cells. Unexpectedly, RBCs from sulfiredoxin-deficient mice also exhibited circadian oscillation of Prx–SO ₂H. Analysis of the effects of protease inhibitors together with the observation that the purified 20S proteasome degraded PrxII–SO ₂H selectively over nonhyperoxidized PrxII suggested that the 20S proteasome is responsible for the decay phase of PrxII–SO ₂H oscillation. About 1% of total PrxII undergoes daily oscillation, resulting in a gradual loss of PrxII during the life span of RBCs. PrxII–SO ₂H was detected in cytosolic and ghost membrane fractions of RBCs, and the amount of membrane-bound PrxII–SO ₂H oscillated in a phase opposite to that of total PrxII–SO ₂H. Our results suggest that membrane association of PrxII–SO ₂H is a tightly controlled process and might play a role in the tuning of RBC function to environmental changes.
During nutritional overload and obesity, hepatocyte function is grossly altered, and a subset of hepatocytes begins to accumulate fat droplets, leading to nonalcoholic fatty liver disease (NAFLD). ...Recent single-cell studies revealed how nonparenchymal cells, such as macrophages, hepatic stellate cells, and endothelial cells, heterogeneously respond to NAFLD. However, it remains to be characterized how hepatocytes, the major constituents of the liver, respond to nutritional overload in NAFLD. Here, using droplet-based, single-cell RNA sequencing (Drop-seq), we characterized how the transcriptomic landscape of individual hepatocytes is altered in response to high-fat diet (HFD) and NAFLD. We showed that the entire hepatocyte population undergoes substantial transcriptome changes upon HFD, although the patterns of alteration were highly heterogeneous, with zonation-dependent and -independent effects. Periportal (zone 1) hepatocytes downregulated many zone 1-specific marker genes, whereas a small number of genes mediating gluconeogenesis were upregulated. Pericentral (zone 3) hepatocytes also downregulated many zone 3-specific genes; however, they upregulated several genes that promote HFD-induced fat droplet formation, consistent with findings that zone 3 hepatocytes accumulate more lipid droplets. Zone 3 hepatocytes also upregulated ketogenic pathways as an adaptive mechanism to HFD. Interestingly, many of the top HFD-induced genes, which encode proteins regulating lipid metabolism, were strongly co-expressed with each other in a subset of hepatocytes, producing a variegated pattern of spatial co-localization that is independent of metabolic zonation. In conclusion, our data set provides a useful resource for understanding hepatocellular alteration during NAFLD at single cell level.
Autophagy is one of the major degradative mechanisms that can eliminate excessive nutrients, toxic protein aggregates, damaged organelles and invading microorganisms. In response to obesity and ...obesity-associated lipotoxic, proteotoxic and oxidative stresses, autophagy plays an essential role in maintaining physiological homeostasis. However, obesity and its associated stress insults can often interfere with the autophagic process through various mechanisms, which result in further aggravation of obesity-related metabolic pathologies in multiple metabolic organs. Paradoxically, inhibition of autophagy, within specific contexts, indirectly produces beneficial effects that can alleviate several detrimental consequences of obesity. In this minireview, we will provide a brief discussion about our current understanding of the impact of obesity on autophagy and the role of autophagy dysregulation in modulating obesity-associated pathological outcomes.
A specific and sensitive fluorescence-based method was developed for the imaging of microbe-induced HOCl production. Furthermore, we demonstrate dual oxidase (DUOX)-mediated HOCl generation in the ...mucosa of live animals providing a novel insight into mucosal innate immunity.
The mTOR complex 1 (mTORC1) and endoplasmic reticulum (ER) stress pathways are critical regulators of intestinal inflammation and colon cancer growth. Sestrins are stress-inducible proteins, which ...suppress both mTORC1 and ER stress; however, the role of Sestrins in colon physiology and tumorigenesis has been elusive due to the lack of studies in human tissues or in appropriate animal models. In this study, we show that human SESN2 expression is elevated in the colon of ulcerative colitis patients but is lost upon p53 inactivation during colon carcinogenesis. In mouse colon, Sestrin2 was critical for limiting ER stress and promoting the recovery of epithelial cells after inflammatory injury. During colitis-promoted tumorigenesis, Sestrin2 was shown to be an important mediator of p53's control over mTORC1 signaling and tumor cell growth. These results highlight Sestrin2 as a novel tumor suppressor, whose downregulation can accelerate both colitis and colon carcinogenesis.
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