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In this review, we describe the structural properties and modification technologies of mRNA, summarize the latest advances in developing mRNA delivery systems, review the preclinical and clinical applications, and put forward our views on the prospect and challenges of developing mRNA into a new class of drug. Botulinum toxin A (BoNT/A) is a potent neurotoxin that acts primarily by silencing synaptic transmission by blocking neurotransmitter release. BoNT/A comprises a light chain (LC/A) intracellular protease and a heavy chain (HC/A) composed of a receptor binding domain (HCC/A) and a translocation domain (HCN/A) that mediates cell entry. Following entry into the neuron, the disulphide bond linking the two peptide chains is reduced to release the LC/A. To gain better insight into the trafficking and fate of BoNT/A before dissociation we have used a catalytically inactive, non-toxic full-length BoNT/A(0) mutant. Our data confirm that BoNT/A(0) enters cortical neurons both in an activity-dependent manner and via a pathway dependent on fibroblast growth factor receptor 3 (Fgfr3) signalling. We demonstrate that both dynamin-dependent endocytosis and lipid rafts are involved in BoNT/A internalisation and that full-length BoNT/A(0) traffics to early endosomes. Furthermore, while a proportion of BoNT/A remains stable in neurons for 3 days, BoNT/A degradation is primarily mediated by the proteasome. Finally, we demonstrate that a fraction of the endocytosed full-length BoNT/A(0) is capable of exiting the cell to intoxicate other neurons. Together, our data shed new light on the entry routes, trafficking and degradation of BoNT/A, and confirm that trafficking properties previously described for the isolated HCC/A receptor binding domain of are also applicable to the intact, full-length toxin. Dihydropyrimidine dehydrogenase (DPD) catalyzes the reduction of uracil and thymine bases with electrons derived from NADPH. The mammalian DPD enzyme is a functional homodimer and has an elaborate cofactor arrangement. Two flavin cofactors (FAD and FMN) reside in two active site cavities that are separated by around 60 Å. The flavins are apparently bridged by four Fe4S4 clusters, two of which are provided by the partner protomer of the dimer. The study of DPD has been hampered by modest yield from both native sources and from heterologous expression in E. coli. In addition, minimal active enzyme is obtained when the DPD gene is fused to an N-terminal 6His-tag. This limitation has dictated the use of traditional purification methods that are made more challenging by apparent over-expression of truncated and/or non-active forms of DPD. Here we detail methods of expression and purification that result in a ~4-fold improvement in the yield of active porcine DPD when expressed in E. coli BL21 DE3 cells via the pET plasmid expression system. The addition of ferrous ions and sulfate during induction provide a small increase in purified active enzyme. However, the addition of FAD and FMN during cell lysis results in a substantial increase in activity that also reduces the relative proportion of non-active, high molecular weight protein contaminants. We also describe methods that permit correlation of the flavin content with the amount of active enzyme and thus permit simple, rapid quantitation and evaluation of purified DPD sample. Diapause is a complex physiological response that allows insects to survive unfavorable environmental conditions, and many signaling pathways participate in regulating this process. However, little is known about TOR signaling in the regulation of diapause. In this study, we found that the TOR pathway-related proteins TOR and Raptor are expressed at low levels in the brains of diapause-destined pupae of Helicoverpa armigera, consistent with a previous report that TOR signaling is associated with development. Interestingly, another TOR signaling-related protein, p-S6K, was increased in the brains of diapause-destined pupae. Our results showed that p-S6K in the brains of diapause-destined pupae can respond to the upstream signals reactive oxygen species (ROS) and AKT and that S6K activates the level of CREB, which binds to the HIF-1α promoter and increases its expression. Previous study has shown that HIF-1α levels elevated by ROS in the brains of diapause-destined pupae cause low mitochondrial activity for insect diapause. MRTX-1257 cost Thus, p-S6K in response to ROS/AKT regulates HIF-1α via activating transcription factor CREB for diapause initiation. BACKGROUND & AIMS Although direct-acting antiviral (DAA) treatment results in a sustained virologic response (SVR) in most patients with chronic hepatitis C virus (HCV) infection, they are at risk of re-infection. Moreover, the immune system is not completely normalized even after SVR (e.g. increased regulatory T (Treg) cell frequency). We developed a DNA vaccine, GLS-6150, to prevent re-infection of patients with DAA-induced SVR and evaluated its safety and immunogenicity in persons with chronic HCV infection (NCT02027116). METHODS GLS-6150 consists of plasmids encoding HCV non-structural proteins (NS3-NS5A) and adjuvant IFNL3. The vaccine was administered four times at 4-week intervals to three groups (1, 3, or 6 mg/vaccination; n=6 per group), followed by a 6 mg boost at 24 weeks (n=14). Peripheral blood T-cell responses were evaluated by IFN-γ enzyme-linked immunospot assays, intracellular cytokine staining, and MHC-I dextramer staining. Treg cell frequency was assessed by flow cytometry. RESULTS Severe adverse events or vaccine discontinuation were not reported. The IFN-γ spot-forming cells specific to NS3-NS5A were increased by GLS-6150. Both CD4+ and CD8+ T cells produced multiple cytokines. However, the frequency and phenotype of HCV-specific MHC-I dextramer+CD8+ T cells were not changed. Interestingly, frequency of Treg cells, particularly activated Treg cells, was decreased by GLS-6150, as expected from previous reports that IFNL3 adjuvants decrease Treg cell frequency. Ex vivo IFN-λ3 treatment reduced Treg frequency in pre-vaccination PBMCs. Finally, Treg cell frequency inversely correlated with HCV-specific, IFN-γ-producing T-cell responses in the study subjects. CONCLUSIONS We demonstrate that GLS-6150 decreases Treg cell frequency and enhances HCV-specific T-cell responses without significant side effects. A phase I clinical trial of GLS-6150 is currently underway in subjects with DAA-induced SVR (NCT03674125).
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