No abstract

Developing highly active, multivalent ligands as therapeutic agents is challenging because of delivery issues, limited cell permeability, and toxicity. Here, we report intrinsically cell-penetrating multivalent ligands that target the trinucleotide repeat DNA and RNA in myotonic dystrophy type 1 (DM1), interrupting the disease progression in two ways. The oligomeric ligands are designed based on the repetitive structure of the target with recognition moieties alternating with bisamidinium groove binders to provide an amphiphilic and polycationic structure, mimicking cell-penetrating peptides. Multiple biological studies suggested the success of our multivalency strategy. The designed oligomers maintained cell permeability and exhibited no apparent toxicity both in cells and in mice at working concentrations. Furthermore, the oligomers showed important activities in DM1 cells and in a DM1 liver mouse model, reducing or eliminating prominent DM1 features. Phenotypic recovery of the climbing defect in adult DM1 Drosophila was also observed. This design strategy should be applicable to other repeat expansion diseases and more generally to DNA/RNA-targeted therapeutics.

The adult liver has exceptional ability to regenerate, but how it sustains normal metabolic activities during regeneration remains unclear. Here, we use partial hepatectomy (PHx) in tandem with single-cell transcriptomics to track cellular transitions and heterogeneities of ~22,000 liver cells through the initiation, progression, and termination phases of mouse liver regeneration. Our results reveal that following PHx, a subset of hepatocytes transiently reactivates an early-postnatal-like gene expression program to proliferate, while a distinct population of metabolically hyperactive cells appears to compensate for any temporary deficits in liver function. Importantly, through combined analysis of gene regulatory networks and cellcell interaction maps, we find that regenerating hepatocytes redeploy key developmental gene regulons, which are guided by extensive ligand-receptor mediated signaling events between hepatocytes and non-parenchymal cells. Altogether, our study offers a detailed blueprint of the intercellular crosstalk and cellular reprogramming that balances the metabolic and proliferation requirements of a regenerating liver..

Eubacterial translation initiation involves assembly of tRNAfMet, mRNA, initiation factors (IFs) and 30S ribosome in a 30S pre-initiation complex (30S pre-IC), which rearranges and joins 50S ribosome to form 70S IC. Upon releasing IFs, 70S IC becomes elongation-competent 70S. The direct recruitment of initiator tRNA (tRNAfMet) into the ribosomal P-site, crucial in accurate initiation of translation, is attributed to two conserved features of tRNAfMet: (i) formylation of amino acid attached to it and, (ii) the presence of three consecutive G-C base pairs (3GC base pairs) in the anticodon stem. However, the precise roles of these two conserved features of tRNAfMet during the various steps of initiation remain unclear. Using natural and engineered tRNAs, we show that the 3GC pairs license tRNAfMet transitions from 30S to 70S IC and then to elongation-competent 70S by release of IF3. Of the 3GC pairs, the middle GC pair (G30-C40), or merely G30 (in a specific context) suffices in this role and is essential for the sustenance of Escherichia coli. Furthermore, rescue of formylase deficient E. coli by overproduced tRNAfMet reveals that the feature of formylation licenses initial targeting of tRNAfMet to 30S ribosome.

15The adult liver has exceptional ability to regenerate, but how it sustains normal metabolic 16 activities during regeneration remains unclear. Here, we use partial hepatectomy (PHx) in 17 tandem with single-cell transcriptomics to track cellular transitions and heterogeneities of 18 ~22,000 liver cells through the initiation, progression, and termination phases of mouse liver 19 regeneration. Our results reveal that following PHx, a subset of hepatocytes transiently 20 reactivates an early-postnatal-like gene expression program to proliferate, while a distinct 21 population of metabolically hyperactive cells appears to compensate for any temporary deficits 22 in liver function. Importantly, through combined analysis of gene regulatory networks and cell-23 cell interaction maps, we find that regenerating hepatocytes redeploy key developmental gene 24 regulons, which are guided by extensive ligand-receptor mediated signaling events between 25 hepatocytes and non-parenchymal cells. Altogether, our study offers a detailed blueprint of the 26 intercellular crosstalk and cellular reprogramming that balances the metabolic and proliferation 27 requirements of a regenerating liver. 28 RESULTS 77 Cell type composition, heterogeneity and metabolic dynamics of a regenerating liver 78Surgical resection of the adult mouse liver by 2/3 rd PHx induces rapid hyperplasia and 79 hypertrophy in the remnant tissue, such that the liver recovers its original mass and function 80 within seven days (Boyce and Harrison, 2008;Mitchell and Willenbring, 2008) (Figure 1A, 81 Figure 1-figure supplement 1A). Hepatocytes, which constitute the bulk of liver parenchyma 82 are among the first cells to enter cell cycle after PHx, followed by the proliferation of other stromal 83 cells , (Fausto et al., 2006;Su et al., 2002). By labeling new DNA synthesis with 5-ethynyl-2'-84 deoxyuridine (EdU) and combining it with hepatocyte nuclear factor 4-alpha (Hnf4α) 85immunostaining (Bangru et al., 2018), we detected maximal hepatocyte proliferation activity 86 between 24 and 72 hours (h) after PHx, which peaked around 36-48h ( Figure 1B, Figure 1-87 figure supplement 1B). Therefore, to sample the cellular composition and diversity as well as 88 profile their regenerative response at a single-cell resolution, we utilized 10x genomics-based 89 scRNA-seq platform and studied the transcriptomes of all resident cell types isolated from mouse 90 livers at 24, 48, and 96h after PHx or sham surgery ( Figure 1C, Figure 1-figure supplement 91 1C). In parallel, we also collected cells from postnatal day 14 (P14) livers-a midpoint between 92 the neonatal period and weaning-and performed scRNA-seq to analyze the cellular transitions 93 and gene programs associated with normal maturation of the liver. Single cells were isolated by 94 two-step collagenase perfusion (Bhate et al., 2015;Li et al., 2010), followed by magnetic-95 activated cell sorting that allows rapid and easy removal of dead cells. 96After stringent quality control and normalization (Figure 1-figure ...