Fatty acid oxidation is required for embryonic stem cell survival during metabolic stress.

Metabolic regulation is critical for the maintenance of pluripotency and the survival of embryonic stem cells (ESCs). The transcription factor Tfcp2l1 has emerged as a key factor for the naïve pluripotency of ESCs. Here, we report an unexpected role of Tfcp2l1 in metabolic regulation in ESCs-promoting the survival of ESCs through regulating fatty acid oxidation (FAO) under metabolic stress. Tfcp2l1 directly activates many metabolic genes in ESCs. Deletion of Tfcp2l1 leads to an FAO defect associated with upregulation of glucose uptake, the TCA cycle, and glutamine catabolism. Mechanistically, Tfcp2l1 activates FAO by inducing Cpt1a, a rate-limiting enzyme transporting free fatty acids into the mitochondria. ESCs with defective FAO are sensitive to cell death induced by glycolysis inhibition and glutamine deprivation. Moreover, the Tfcp2l1-Cpt1a-FAO axis promotes the survival of quiescent ESCs and diapause-like blastocysts induced by mTOR inhibition. Thus, our results reveal how ESCs orchestrate pluripotent and metabolic programs to ensure their survival in response to metabolic stress.

Conditional deletion of mTOR discloses its essential role in early B-cell development.

Mechanistic target of rapamycin (mTOR) is a serine-threonine kinase and central regulator of cell growth, differentiation, and survival. mTOR is commonly hyperactivated in a diverse number of cancers and critical roles for mTOR in regulating immune cell differentiation and function have been demonstrated. However, there is little work investigating the roles of mTOR in early B-cell development. Here we demonstrate that conditional disruption of mTOR in developing mouse B cells results in reduced pre-B-cell proliferation and survival, as well as a developmental block at the pre-B-cell stage, with a corresponding lack of peripheral B cells. Upon immunization with NP-CGG antigen, mice with Mtor conditional disruption in early B cells lost their ability to form germinal centers and produce specific antibodies. In competitive BM repopulation assays, donor BM cells from conditional knock-out mice were completely impaired in their ability to reconstitute B cells. Our data reveal the essential role of mTOR in early pre-B-cell development and survival.

Transfer RNA acetylation regulates in vivo mammalian stress signaling.

Transfer RNA (tRNA) modifications are crucial for protein synthesis, but their position-specific physiological roles remain poorly understood. Here we investigate the impact of N4-acetylcytidine (ac4C), a highly conserved tRNA modification, using a Thumpd1 knockout mouse model. We find that loss of Thumpd1-dependent tRNA acetylation leads to reduced levels of tRNALeu, increased ribosome stalling, and activation of eIF2α phosphorylation. Thumpd1 knockout mice exhibit growth defects and sterility. Remarkably, concurrent knockout of Thumpd1 and the stress-sensing kinase Gcn2 causes penetrant postnatal lethality, indicating a critical genetic interaction. Our findings demonstrate that a modification restricted to a single position within type II cytosolic tRNAs can regulate ribosome-mediated stress signaling in mammalian organisms, with implications for our understanding of translation control as well as therapeutic interventions.

Pandemic-associated pernio harbors footprints of an abortive SARS-CoV-2 infection.

Elevated pernio incidence was observed during the COVID-19 pandemic. This prospective study enrolled subjects with pandemic-associated pernio in Wisconsin and Switzerland. Because pernio is a cutaneous manifestation of the interferonopathies, and type I interferon (IFN-I) immunity is critical to COVID-19 recovery, we tested the hypothesis that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-mediated IFN-I signaling might underlie some pernio cases. Tissue-level IFN-I activity and plasmacytoid dendritic cell infiltrates were demonstrated in 100% of the Wisconsin cases. Across both cohorts, sparse SARS-CoV-2 RNA was captured in 25% (6/22) of biopsies, all with high inflammation. Affected patients lacked adaptive immunity to SARS-CoV-2. A hamster model of intranasal SARS-CoV-2 infection was used as a proof-of-principle experiment: RNA was detected in lungs and toes with IFN-I activity at both the sites, while replicating virus was found only in the lung. These data support a viral trigger for some pernio cases, where sustained local IFN-I activity can be triggered in the absence of seroconversion.

A toolbox of IgG subclass-switched recombinant monoclonal antibodies for enhanced multiplex immunolabeling of brain.

Generating recombinant monoclonal antibodies (R-mAbs) from mAb-producing hybridomas offers numerous advantages that increase the effectiveness, reproducibility, and transparent reporting of research. We report here the generation of a novel resource in the form of a library of recombinant R-mAbs validated for neuroscience research. We cloned immunoglobulin G (IgG) variable domains from cryopreserved hybridoma cells and input them into an integrated pipeline for expression and validation of functional R-mAbs. To improve efficiency over standard protocols, we eliminated aberrant Sp2/0-Ag14 hybridoma-derived variable light transcripts using restriction enzyme treatment. Further, we engineered a plasmid backbone that allows for switching of the IgG subclasses without altering target binding specificity to generate R-mAbs useful in simultaneous multiplex labeling experiments not previously possible. The method was also employed to rescue IgG variable sequences and generate functional R-mAbs from a non-viable cryopreserved hybridoma. All R-mAb sequences and plasmids will be archived and disseminated from open source suppliers.

Acoustic Tweezing Cytometry Induces Rapid Initiation of Human Embryonic Stem Cell Differentiation.

Mechanical forces play critical roles in influencing human embryonic stem cell (hESC) fate. However, it remains largely uncharacterized how local mechanical forces influence hESC behavior in vitro. Here, we used an ultrasound (US) technique, acoustic tweezing cytometry (ATC), to apply targeted cyclic subcellular forces to hESCs via integrin-bound microbubbles (MBs). We found that ATC-mediated cyclic forces applied for 30 min to hESCs near the edge of a colony induced immediate global responses throughout the colony, suggesting the importance of cell-cell connection in the mechanoresponsiveness of hESCs to ATC-applied forces. ATC application generated increased contractile force, enhanced calcium activity, as well as decreased expression of pluripotency transcription factors Oct4 and Nanog, leading to rapid initiation of hESC differentiation and characteristic epithelial-mesenchymal transition (EMT) events that depend on focal adhesion kinase (FAK) activation and cytoskeleton (CSK) tension. These results reveal a unique, rapid mechanoresponsiveness and community behavior of hESCs to integrin-targeted cyclic forces.