Sublethal whole-body irradiation induces permanent loss and dysfunction in pathogen-specific circulating memory CD8 T cell populations.

The increasing use of nuclear energy sources inevitably raises the risk of accidental or deliberate radiation exposure and associated immune dysfunction. However, the extent to which radiation exposure impacts memory CD8 T cells, potent mediators of immunity to recurring intracellular infections and malignancies, remains understudied. Using P14 CD8 T cell chimeric mice (P14 chimeras) with an lymphocytic choriomeningitis virus (LCMV) infection model, we observed that sublethal (5Gy) whole-body irradiation (WBI) induced a rapid decline in the number of naive (TN) and P14 circulating memory CD8 T cells (TCIRCM), with the former being more susceptible to radiation-induced numeric loss. While TN cell numbers rapidly recovered, as previously described, the number of P14 TCIRCM cells remained low at least 9 mo after radiation exposure. Additionally, the remaining P14 TCIRCM in irradiated hosts exhibited an inefficient transition to a central memory (CD62L+) phenotype compared to nonirradiated P14 chimeras. WBI also resulted in long-lasting T cell intrinsic deficits in memory CD8 T cells, including diminished cytokine and chemokine production along with impaired secondary expansion upon cognate Ag reencounter. Irradiated P14 chimeras displayed significantly higher bacterial burden after challenge with Listeria monocytogenes expressing the LCMV GP33-41 epitope relative to nonirradiated controls, likely due to radiation-induced numerical and functional impairments. Taken together, our findings suggest that sublethal radiation exposure caused a long-term numerical, impaired differentiation, and functional dysregulation in preexisting TCIRCM, rendering previously protected hosts susceptible to reinfection.

Simultaneous loss of TSC1 and DEPDC5 in skeletal and cardiac muscles produces early-onset myopathy and cardiac dysfunction associated with oxidative damage and SQSTM1/p62 accumulation.

By promoting anabolism, MTORC1 is critical for muscle growth and maintenance. However, genetic MTORC1 upregulation promotes muscle aging and produces age-associated myopathy. Whether MTORC1 activation is sufficient to produce myopathy or indirectly promotes it by accelerating tissue aging is elusive. Here we examined the effects of muscular MTORC1 hyperactivation, produced by simultaneous depletion of TSC1 and DEPDC5 (CKM-TD). CKM-TD mice produced myopathy, associated with loss of skeletal muscle mass and force, as well as cardiac failure and bradypnea. These pathologies were manifested at eight weeks of age, leading to a highly penetrant fatality at around twelve weeks of age. Transcriptome analysis indicated that genes mediating proteasomal and macroautophagic/autophagic pathways were highly upregulated in CKM-TD skeletal muscle, in addition to inflammation, oxidative stress, and DNA damage signaling pathways. In CKM-TD muscle, autophagosome levels were increased, and the AMPK and ULK1 pathways were activated; in addition, autophagy induction was not completely blocked in CKM-TD myotubes. Despite the upregulation of autolysosomal markers, CKM-TD myofibers exhibited accumulation of autophagy substrates, such as SQSTM1/p62 and ubiquitinated proteins, suggesting that the autophagic activities were insufficient. Administration of a superoxide scavenger, tempol, normalized most of these molecular pathologies and subsequently restored muscle histology and force generation. However, CKM-TD autophagy alterations were not normalized by rapamycin or tempol, suggesting that they may involve non-canonical targets other than MTORC1. These results collectively indicate that the concomitant muscle deficiency of TSC1 and DEPDC5 can produce early-onset myopathy through accumulation of oxidative stress, which dysregulates myocellular homeostasis.Abbreviations: AMPK: AMP-activated protein kinase; CKM: creatine kinase, M-type; COX: cytochrome oxidase; DEPDC5: DEP domain containing 5, GATOR1 subcomplex subunit; DHE: dihydroethidium; EDL: extensor digitorum longus; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; GAP: GTPase-activating protein; GTN: gastrocnemius; MTORC1: mechanistic target of rapamycin kinase complex 1; PLA: plantaris; QUAD: quadriceps; RPS6KB/S6K: ribosomal protein S6 kinase beta; SDH: succinate dehydrogenase; SOL: soleus; SQSTM1: sequestosome 1; TA: tibialis anterior; TSC1: TSC complex subunit 1; ULK1: unc-51 like autophagy activating kinase 1.

Microscopic examination of spatial transcriptome using Seq-Scope.

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.

Transcriptome and epigenome analyses of vernalization in Arabidopsis thaliana.

Vernalization accelerates flowering after prolonged winter cold. Transcriptional and epigenetic changes are known to be involved in the regulation of the vernalization response. Despite intensive applications of next-generation sequencing in diverse aspects of plant research, genome-wide transcriptome and epigenome profiling during the vernalization response has not been conducted. In this work, to our knowledge, we present the first comprehensive analyses of transcriptomic and epigenomic dynamics during the vernalization process in Arabidopsis thaliana. Six major clusters of genes exhibiting distinctive features were identified. Temporary changes in histone H3K4me3 levels were observed that likely coordinate photosynthesis and prevent oxidative damage during cold exposure. In addition, vernalization induced a stable accumulation of H3K27me3 over genes encoding many development-related transcription factors, which resulted in either inhibition of transcription or a bivalent status of the genes. Lastly, FLC-like and VIN3-like genes were identified that appear to be novel components of the vernalization pathway.

Concurrent activation of growth factor and nutrient arms of mTORC1 induces oxidative liver injury.

mTORC1 is a protein kinase important for metabolism and is regulated by growth factor and nutrient signaling pathways, mediated by the Rheb and Rag GTPases, respectively. Here we provide the first animal model in which both pathways were upregulated through concurrent mutations in their GTPase-activating proteins, Tsc1 and Depdc5. Unlike former models that induced limited mTORC1 upregulation, hepatic deletion of both Tsc1 and Depdc5 (DKO) produced strong, synergistic activation of the mTORC1 pathway and provoked pronounced and widespread hepatocyte damage, leading to externally visible liver failure phenotypes, such as jaundice and systemic growth defects. The transcriptome profile of DKO was different from single knockout mutants but similar to those of diseased human livers with severe hepatitis and mouse livers challenged with oxidative stress-inducing chemicals. In addition, DKO liver cells exhibited prominent molecular pathologies associated with excessive endoplasmic reticulum (ER) stress, oxidative stress, DNA damage and inflammation. Although DKO liver pathologies were ameliorated by mTORC1 inhibition, ER stress suppression unexpectedly aggravated them, suggesting that ER stress signaling is not the major conduit of how hyperactive mTORC1 produces liver damage. Interestingly, superoxide scavengers N-acetylcysteine (NAC) and Tempol, chemicals that reduce oxidative stress, were able to recover liver phenotypes, indicating that mTORC1 hyperactivation induced liver damage mainly through oxidative stress pathways. Our study provides a new model of unregulated mTORC1 activation through concomitant upregulation of growth factor and nutrient signaling axes and shows that mTORC1 hyperactivation alone can provoke oxidative tissue injury.

Mimosine arrests the cell cycle prior to the onset of DNA replication by preventing the binding of human Ctf4/And-1 to chromatin via Hif-1α activation in HeLa cells.

Though the G(1) checkpoint in mammalian cells has been known for decades, the molecular targets that prevent S-phase entry remain unknown. Mimosine is a rare plant amino acid that arrests the cell cycle in the G(1) phase before entry into S phase. Here, we show that mimosine interrupts the binding of Ctf4 to chromatin, which is essential for the initiation of DNA replication in HeLa cells, and this effect is mediated by the Hif-1α-dependent increase in the level of p27. Depletion of Hif-1α results in an increased binding of Ctf4 to chromatin and the entry of cells into S phase even in the presence of mimosine. These results suggest that the binding of Ctf4 to chromatin is the target of the Hif-1α-dependent checkpoint pathway for cell cycle arrest in G(1) phase. Although we observed Hif-1α-dependent arrest in mimosine-treated cells, it is possible that Ctf4 may act as a common target for G(1) arrest in various other checkpoint pathways.