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Automated assembly scaffolding elevates a new tomato system for high-throughput genome editing
Get PDFAn autoimmune stem-like CD8 T cell population drives type 1 diabetes
No full textCD8 T cell-mediated autoimmune diseases result from the breakdown of self-tolerance mechanisms in autoreactive CD8 T cells1. How autoimmune T cell populations arise and are sustained and the molecular programs defining the autoimmune T cell state are unknown. In Type 1 diabetes (T1D), beta cell-specific CD8 T cells destroy insulin-producing beta cells. We followed the fate of beta cell-specific CD8 T cells in non-obese diabetic mice throughout the course of T1D. We identified a stem-like autoimmune progenitor (AP) population in the pancreatic draining lymph node (pLN), which self-renews and gives rise to pLN autoimmune mediators (AM). pLN AM migrate to the pancreas, where they differentiate further and destroy beta cells. While transplantation of as few as 20 AP induced T1D, as many as 100,000 pancreatic AM failed to do so. Pancreatic AM are short-lived and stem-like AP must continuously seed the pancreas to sustain beta cell destruction. Single cell RNA-sequencing and clonal analysis revealed that autoimmune CD8 T cells represent unique T cell differentiation states and identified features driving the transition from AP to AM. Strategies aimed at targeting the stem-like AP pool could emerge as novel and powerful immunotherapeutic interventions for T1D
PQN-59 antagonizes microRNA-mediated repression during post-embryonic temporal patterning and modulates translation and stress granule formation in C. elegans
Get PDFmicroRNAs (miRNAs) are potent regulators of gene expression that function in a variety of developmental and physiological processes by dampening the expression of their target genes at a post-transcriptional level. In many gene regulatory networks (GRNs), miRNAs function in a switch-like manner whereby their expression and activity elicit a transition from one stable pattern of gene expression to a distinct, equally stable pattern required to define a nascent cell fate. While the importance of miRNAs that function in this capacity are clear, we have less of an understanding of the cellular factors and mechanisms that ensure the robustness of this form of regulatory bistability. In a screen to identify suppressors of temporal patterning phenotypes that result from ineffective miRNA-mediated target repression, we identified pqn-59, an ortholog of human UBAP2L, as a novel factor that antagonizes the activities of multiple heterochronic miRNAs. Specifically, we find that depletion of pqn-59 can restore normal development in animals with reduced lin-4 and let-7-family miRNA activity. Importantly, inactivation of pqn-59 is not sufficient to bypass the requirement of these regulatory RNAs within the heterochronic GRN. The pqn-59 gene encodes an abundant, cytoplasmically-localized, unstructured protein that harbors three essential "prion-like" domains. These domains exhibit LLPS properties in vitro and normally function to limit PQN-59 diffusion in the cytoplasm in vivo. Like human UBAP2L, PQN-59's localization becomes highly dynamic during stress conditions where it re-distributes to cytoplasmic stress granules and is important for their formation. Proteomic analysis of PQN-59 complexes from embryonic extracts indicates that PQN-59 and human UBAP2L interact with orthologous cellular components involved in RNA metabolism and promoting protein translation and that PQN-59 additionally interacts with proteins involved in transcription and intracellular transport. Finally, we demonstrate that pqn-59 depletion reduces protein translation and also results in the stabilization of several mature miRNAs (including those involved in temporal patterning). These data suggest that PQN-59 may ensure the bistability of some GRNs that require miRNA functions by promoting miRNA turnover and, like UBAP2L, enhancing protein translation
Geometric network analysis provides prognostic information in patients with high grade serous carcinoma of the ovary treated with immune checkpoint inhibitors
Get PDFNetwork analysis methods can potentially quantify cancer aberrations in gene networks without introducing fitted parameters or variable selection. A new network curvature-based method is introduced to provide an integrated measure of variability within cancer gene networks. The method is applied to high-grade serous ovarian cancers (HGSOCs) to predict response to immune checkpoint inhibitors (ICIs) and to rank key genes associated with prognosis. Copy number alterations (CNAs) from targeted and whole-exome sequencing data were extracted for HGSOC patients (n = 45) treated with ICIs. CNAs at a gene level were represented on a protein-protein interaction network to define patient-specific networks with a fixed topology. A version of Ollivier-Ricci curvature was used to identify genes that play a potentially key role in response to immunotherapy and further to stratify patients at high risk of mortality. Overall survival (OS) was defined as the time from the start of ICI treatment to either death or last follow-up. Kaplan-Meier analysis with log-rank test was performed to assess OS between the high and low curvature classified groups. The network curvature analysis stratified patients at high risk of mortality with p = 0.00047 in Kaplan-Meier analysis in HGSOC patients receiving ICI. Genes with high curvature were in accordance with CNAs relevant to ovarian cancer. Network curvature using CNAs has the potential to be a novel predictor for OS in HGSOC patients treated with immunotherapy
The Bur1 cyclin-dependent kinase regulates telomere length in Saccharomyces cerevisiae.
Get PDFTelomere length regulation is essential for cell viability in eukaryotes. While many pathways that affect telomere length are known, we do not yet have a complete understanding of the mechanism of length regulation. To identify new pathways that might regulate telomere length, we carried out a genetic screen in yeast and identified the cyclin-dependent kinase complex Bur1/2 as a regulator of telomere length. Mutations in either BUR1 cyclin-dependent kinase or the associated BUR2 cyclin resulted in short telomeres. This regulation did not function through the known role of BUR1 in regulating histone modification as bur1∆ set2∆ and bur2∆ set2∆ double mutants rescued cell growth but did not rescue the telomere shortening effects. We found that both bur1∆ and bur2∆ set2∆ were also defective in de novo telomere addition, and deletion of SET2 did also not rescue this elongation defect. The Bur1/2 cyclin-dependent kinase regulates transcription of many genes. We found that TLC1 RNA levels were reduced in bur2∆ set2∆ mutants; however, overexpression of TLC1 restored the transcript levels but did not restore de novo telomere elongation or telomere length. These data suggest that the Bur1/2 kinase plays a role in telomere elongation separate from its role in transcription of telomerase components. Dissecting the role of the Bur1/2 kinase pathway at telomeres will help complete our understanding of the complex network of telomere length regulation
Aminium cation-radical catalysed selective hydration of (E)-aryl enynes.
No full textThe hydration of carbon-carbon triple bonds is an important and atom economic synthetic transformation. Herein, we report a mild and selective method for the catalytic Markovnikov hydration of (E)-aryl enynes to the corresponding enones, mediated through the bench-stable aminium salt, tris(4-bromophenyl)ammoniumyl hexachloroantimonate (TBPA). The chemoselective and diastereoselective method proceeds under neutral metal-free conditions, delivering excellent product yields from terminal and internal alkyne units. The synthesis of biologically important (E)-3-styrylisocoumarins, including a formal synthesis of the natural product achlisocoumarin III, demonstrates the utility of this novel transformation
Multi-color Flow Cytometry for Comprehensive Analysis of the Tumor Immune Infiltrate in a Murine Model of Breast Cancer.
No full textFlow cytometry is a popular laser-based technology that allows the phenotypic and functional characterization of individual cells in a high-throughput manner. Here, we describe a detailed procedure for preparing a single-cell suspension from mammary tumors of the mouse mammary tumor virus-polyoma middle T (MMTV-PyMT) and analyzing these cells by multi-color flow cytometry. This protocol can be used to study the following tumor-infiltrating immune cell populations, defined by the expression of cell surface molecules: total leukocytes, tumor-associated macrophages (TAMs), conventional dendritic cells (DCs), CD103-expressing DCs, tumor-associated neutrophils, inflammatory monocytes, natural killer (NK) cells, CD4+ T cells, CD8+ T cells, γδT cells, and regulatory T cells
Chromosomal instability accelerates the evolution of resistance to anti-cancer therapies.
No full textAneuploidy is a ubiquitous feature of human tumors, but the acquisition of aneuploidy typically antagonizes cellular fitness. To investigate how aneuploidy could contribute to tumor growth, we triggered periods of chromosomal instability (CIN) in human cells and then exposed them to different culture environments. We discovered that transient CIN reproducibly accelerates the acquisition of resistance to anti-cancer therapies. Single-cell sequencing revealed that these resistant populations develop recurrent aneuploidies, and independently deriving one chromosome-loss event that was frequently observed in paclitaxel-resistant cells was sufficient to decrease paclitaxel sensitivity. Finally, we demonstrated that intrinsic levels of CIN correlate with poor responses to numerous therapies in human tumors. Our results show that, although CIN generally decreases cancer cell fitness, it also provides phenotypic plasticity to cancer cells that can allow them to adapt to diverse stressful environments. Moreover, our findings suggest that aneuploidy may function as an under-explored cause of therapy failure
SuFExable polymers with helical structures derived from thionyl tetrafluoride.
No full textSulfur(VI) fluoride exchange (SuFEx) is a category of click chemistry that enables covalent linking of modular units through sulfur(VI) connective hubs. The efficiency of SuFEx and the stability of the resulting bonds have led to polymer chemistry applications. Now, we report the SuFEx click chemistry synthesis of several structurally diverse SOF4-derived copolymers based on the polymerization of bis(iminosulfur oxydifluorides) and bis(aryl silyl ethers). This polymer class presents two key characteristics. First, the [-N=S(=O)F-O-] polymer backbone linkages are themselves SuFExable and undergo precise SuFEx-based post-modification with phenols or amines to yield branched functional polymers. Second, studies of individual polymer chains of several of these new materials indicate helical polymer structures. The robust nature of SuFEx click chemistry offers the potential for post-polymerization modification, enabling the synthesis of materials with control over composition and conformation
