Insulin resistance and type 2 diabetes mellitus (T2DM) result from an inability to efficiently store and catabolize surplus energy in adipose tissue. Subcutaneous adipocytes protect against insulin resistance and T2DM by coupling differentiation with the induction of brown fat gene programs for efficient energy metabolism. Mechanisms that disrupt these programs in adipocytes are currently poorly defined, but represent therapeutic targets for the treatment of T2DM. To gain insight into these mechanisms, we performed a high-throughput microscopy screen that identified ubiquitin carrier protein 9 (Ubc9) as a negative regulator of energy storage in human sc adipocytes. Ubc9 depletion enhanced energy storage and induced the brown fat gene program in human sc adipocytes. Induction of adipocyte differentiation resulted in decreased Ubc9 expression commensurate with increased brown fat gene expression. Thiazolidinedione treatment reduced the interaction between Ubc9 and peroxisome proliferator-activated receptor (PPAR)γ, suggesting a mechanism by which Ubc9 represses PPARγ activity. In support of this hypothesis, Ubc9 overexpression remodeled energy metabolism in human sc adipocytes by selectively inhibiting brown adipocyte-specific function. Further, Ubc9 overexpression decreased uncoupling protein 1 expression by disrupting PPARγ binding at a critical uncoupling protein 1 enhancer region. Last, Ubc9 is significantly elevated in sc adipose tissue isolated from mouse models of insulin resistance as well as diabetic and insulin-resistant humans. Taken together, our findings demonstrate a critical role for Ubc9 in the regulation of sc adipocyte energy homeostasis.
Single-cell RNA sequencing offers snapshots of whole transcriptomes but obscures the temporal RNA dynamics. Here we present single-cell metabolically labeled new RNA tagging sequencing (scNT-seq), a method for massively parallel analysis of newly transcribed and pre-existing mRNAs from the same cell. This droplet microfluidics-based method enables high-throughput chemical conversion on barcoded beads, efficiently marking newly transcribed mRNAs with T-to-C substitutions. Using scNT-seq, we jointly profiled new and old transcriptomes in 55,000 single cells. These data revealed time-resolved transcription factor activities and cell-state trajectories at the single-cell level in response to neuronal activation. We further determined rates of RNA biogenesis and decay to uncover RNA regulatory strategies during stepwise conversion between pluripotent and rare totipotent two-cell embryo (2C)-like stem cell states. Finally, integrating scNT-seq with genetic perturbation identifies DNA methylcytosine dioxygenase as an epigenetic barrier into the 2C-like cell state. Time-resolved single-cell transcriptomic analysis thus opens new lines of inquiry regarding cell-type-specific RNA regulatory mechanisms.
T-cell-dependent bispecific antibodies (TDBs) are promising cancer immunotherapies that recruit a patient’s T cells to kill cancer cells. There are increasing numbers of TBDs in clinical trials, demonstrating their widely recognized therapeutic potential. Due to the fact that TDBs engage and activate T cells via an anti-CD3 (aCD3) arm, aCD3 homodimer (aCD3 HD) and high-molecular-weight species (HMWS) are product-related impurities that pose a potential safety risk by triggering off-target T-cell activation through bivalent engagement and dimerization of T-cell receptors (TCRs). To monitor and control the level of unspecific T-cell activation, we developed a sensitive and quantitative T-cell-activation assay, which can detect aCD3 HD in TDB drug product by exploiting its ability to activate T cells in the absence of target cells. This assay provides in-vivo -relevant off-target T-cell-activation readout. Furthermore, we have demonstrated that this assay can serve as a platform assay for detecting T-cell-activating impurities across a broad spectrum of aCD3 bispecific molecules. It therefore has the potential to significantly benefit many T-cell-recruiting bispecific programs.
IntroductionRituximab is an emerging treatment for autoimmune hemolytic anemia. We report the case of a patient with a five-year complete hematologic response to a third course of treatment with rituximab. Cases of response to rituximab re-treatments have been reported, but none to our knowledge that failed multiple prior treatments and achieved as durable a response.Case presentationA 45-year-old Hispanic man presented at age 26 with darkening urine and cold intolerance. His blood tests revealed elevated lactic dehydrogenase and bilirubin, a hemoglobin level of 7.4g/dL, and a positive Coombs test for complement C3 and immunoglobulin G antibody. A diagnosis of autoimmune hemolytic anemia was made. After failing multiple therapies including prednisone, splenectomy, immunoglobulin, cyclosporine, danocrine and azathioprine, our patient was treated with a four-week course of rituximab at a dose of 375mg/m2 weekly, 10 years following initial presentation. He achieved a rapid and complete hematologic response that lasted 25 months. Re-treatment with the same course of rituximab prompted a second response that lasted 18 months. A third re-treatment has achieved an ongoing five-year complete hematologic response.ConclusionsThis is an unusual case of a durable five-year remission of autoimmune hemolytic anemia with rituximab re-treatment following relapse after two prior courses of rituximab and despite the persistence of immunoglobulin G and complement-coated red blood cells. No mechanistic explanations for improved response to rituximab re-treatment in autoimmune hemolytic anemia have been reported in the literature. Future studies of rituximab or other B cell-targeting antibodies in the treatment of autoimmune hemolytic anemia should explore autoantibody immunoglobulin G subclass switching and alterations in complement inhibitory proteins on red blood cell membranes as potential correlates of hematologic response.
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