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Technologies capable of programmable translation activation offer strategies to develop therapeutics for diseases caused by insufficient gene expression. Here, we present “translation-activating RNAs” (taRNAs), a bifunctional RNA-based molecular technology that binds to a specific mRNA of interest and directly upregulates its translation. taRNAs are constructed from a variety of viral or mammalian RNA internal ribosome entry sites (IRESs) and upregulate translation for a suite of target mRNAs. We minimize the taRNA scaffold to 94 nucleotides, identify two translation initiation factor proteins responsible for taRNA activity, and validate the technology by amplifying SYNGAP1 expression, a haploinsufficiency disease target, in patient-derived cells. Finally, taRNAs are suitable for delivery as RNA molecules by lipid nanoparticles (LNPs) to cell lines, primary neurons, and mouse liver in vivo. taRNAs provide a general and compact nucleic acid-based technology to upregulate protein production from endogenous mRNAs, and may open up possibilities for therapeutic RNA research. Many diseases are driven by the insufficient expression of critical genes, but few technologies are capable of rescuing these endogenous protein levels. Here, Cao et al. present an RNA-based technology that boosts protein production from endogenous mRNAs by upregulating their translation.

Lead exposure is associated with a wide range of adverse effects on human health. The principal exposure route in the general population is through the diet. In this study, we estimate the dietary lead intake and associated health risks among the residents of Guangzhou, China. Data on lead concentrations were derived from the food safety risk monitoring system, which included 6339 samples from 27 food categories collected in 2014–2017. Food consumption data were taken from a 2011 dietary survey of 2960 Guangzhou residents from 998 households. Dietary lead intake was estimated by age group (3–6, 7–17, 18–59, and ≥60 years), and relevant health risks were assessed using the margin of exposure (MOE) method. The mean and 95th percentiles (P95) of dietary lead intake were respectively 0.7466 and 2.4525 μg/kg body weight per day for preschool children aged 3–6 years; 0.4739 and 1.5522 μg/kg bw/day for school children aged 7–17 years; 0.3759 and 1.1832 μg/kg bw/day for adults aged 18–59 years; and 0.4031 and 1.3589 μg/kg bw/day for adults aged ≥60 years. The MOE value was less than 1 for preschool children at the mean exposure level and for all age groups at the P95 exposure level. Rice and its products, leafy vegetables, and wheat flour and its products were found to be the primary food sources of lead exposure. Our findings suggest that the health risk from dietary lead exposure is low for Guangzhou residents overall, but that young children and consumers of certain foods may be at increased risk. Continued efforts are needed to reduce the dietary lead exposure in Guangzhou.

A novel polystyrene sulfonate sodium (PSS) magnetic material was prepared by surface-initiated atom transfer radical polymerization (SI-ATRP). The starting materials were brominated magnetic material as the carrier and macroinitiator, sodium styrene sulfonate (NaSS) as the monomer, and cuprous bromide/2,2′-dipyridyl as the catalyst system. The PSS material was characterized by Fourier transform infrared spectroscopy (FT-IR), elemental analysis, transmission electron microscope (TEM), thermogravimetric analysis (TGA), scanning electron microscopy (SEM), and a vibrating sample magnetometer (VSM). The adsorption properties of the material were then investigated on sulfa antibiotics. The kinetic and thermodynamic parameters were determined in adsorption of sulfamethazine (the smallest molecular-weight sulfonamide). The adsorption amount of sulfamerazine free acid (SMR) was found to increase with the initial concentration and temperature of SMR in solution. The adsorption effect was maximized at an initial concentration of 0.6 mmol/L. The static saturation adsorption capacity of the material was 33.53 mg/g, Langmuir and Freundlich equations exhibited good fit. The thermodynamic equilibrium equation is calculated as ΔG < 0, ΔH = 38.29 kJ/mol, ΔS > 0, which proves that the adsorption process is a process of spontaneous, endothermic and entropy increase. Kinetic studies show that the quasi-second-order kinetic equation can better fit the kinetic experimental results, which is consistent with the quasi-second-order kinetic model. The experimental results of kinetic studies were well fitted to a quasi-second-order kinetic equation. High performance liquid chromatography (HPLC) of an actual milk sample treated by the PSS magnetic material confirmed the strong adsorption of SMR from milk.

Chloramphenicol- (CAP-) restricted access media-molecularly imprinted polymers (CAP-RAM-MIPs) were prepared by precipitation polymerization using CAP as a template molecule, 2-diethylaminoethyl methacrylate (DEAEM) as a functional monomer, ethylene glycol dimethyl acrylate (EDMA) as a crosslinking agent, glycidyl methacrylate (GMA) as an outer hydrophilic functional monomer, and acetonitrile as a pore former and solvent. The CAP-RAM-MIPs were successfully characterized by Fourier-transform infrared spectroscopy, scanning electron microscopy, and thermogravimetric analysis. The adsorption performance was investigated in detail using static, dynamic, and selective adsorption experiments. Adsorption equilibrium could be reached within 11 min. The CAP-RAM-MIPs had a high adsorption rate and good specific adsorption properties. Scatchard fitting curves indicated there were two binding sites for CAP-RAM-MIPs. Adsorption was Freundlich multilayer adsorption and consistent with the quasi-second kinetic model. Using CAP-RAM-MIPs for selective separation and enrichment CAP in bovine serum in combination with high-performance liquid chromatography (HPLC), CAP recovery ranged from 94.1 to 97.9% with relative standard deviations of 0.7–1.5%. This material has broad application prospects in enrichment and separation.

Universally conserved residues (UCRs) are invariable amino acids evolutionarily conserved among members of a protein family across diverse kingdoms of life. UCRs are considered important for stability and/or function of protein families, but it has not been experimentally examined systematically. Cryptochromes are photoreceptors in plants or light-independent components of the circadian clocks in mammals. We experimentally analyzed 51 UCRs of Arabidopsis cryptochrome 2 (CRY2) that are universally conserved in eukaryotic cryptochromes from Arabidopsis to human. Surprisingly, we found that UCRs required for stable protein expression of CRY2 in plants are not similarly required for stable protein expression of human hCRY1 in human cells. Moreover, 74% of the stably expressed CRY2 proteins mutated in UCRs retained wild-type-like activities for at least one photoresponses analyzed. Our finding suggests that the evolutionary mechanisms underlying conservation of UCRs or that distinguish UCRs from non-UCRs determining the same functions of individual cryptochromes remain to be investigated.

Tight and accurate regulation of immunity and thiamine biosynthesis is critical for proper defence mechanisms and several primary metabolic cycles in plants. Although thiamine is known to enhance plant defence by priming, the mechanism by which thiamine biosynthesis responds to immune signals remains poorly understood. Here we identified a novel rice ( Oryza sativa L.) NB-LRR gene via an insertion mutation, this mutant confesses a low seed setting phenotype and the corresponding genetic locus was named OsLSR ( Low seed setting related ). Comparing with wildtype plant, both overexpression and suppression of OsLSR lead to the autoactivation of the rice immune system and accumulation of thiamine, which result in a great fitness cost and yield penalty. Moreover, when fused with eGFP at their C terminus, two fragments, OsLSR1-178 and OsLSR464-546, localized to chloroplasts where thiamine is produced. Our result suggests that OsLSR differs from traditional NB-LRR genes. Its expression is closely related to the immune status and thiamine level in plant cells and should be maintained within a narrow range for rice growth.

How cells respond to light or time is a fundamental question in biology. Cryptochromes (CRYs) are evolutionarily conserved blue light receptors or key components of the circadian oscillator found in major evolutionary lineages, from bacteria to human and have been intensively studied. However, the structure-function relationship of CRYs from evolutionary perspective is unclear. In this thesis, I interrogated the evolutionary roles of universally conserved residues (UCRs) of Arabidopsis thaliana cryptochrome 2 (CRY2) (Chapter 2) and developed optogenetic tools by engineering CRY2 using continuous directed evolution techniques (Chapter 3).UCRs are invariable amino acids evolutionarily conserved among members of a protein family across diverse kingdoms of life. UCRs are considered important for stability and/or function of protein families, but it has not been experimentally examined systematically. In Chapter 2, I experimentally analyzed 51 UCRs of Arabidopsis CRY2 that are universally conserved in eukaryotic cryptochromes from Arabidopsis to human. Surprisingly, I found that UCRs required for stable protein expression of CRY2 in plants are not similarly required for stable protein expression of human hCRY1 in human cells. Moreover, 74% of the stably expressed CRY2 proteins mutated in UCRs retained wild-type-like activities for at least one of the photoresponses I analyzed. My finding suggests that the evolutionary mechanisms underlying conservation of UCRs or that distinguish UCRs from non-UCRs determining the same functions of individual cryptochromes remain to be investigated.CRY2 mainly regulates plant photomorphogenesis through blue-light-specific interactions with numerous protein partners. Such blue-light-specific interactions have been exploited in optogenetics to manipulated biological events in a timely and precisely manner. Chapter 3 focused on the development of a novel pair of blue-light-dependent interacting proteins: CRY2-BIC1 (Blue-light Inhibitor of Cryptochromes 1) and applied PACE (Phage Assisted Continuous Evolution) to increase the dynamic range of CRY2-BIC1 blue-light dependent interaction. I isolated variants of CRY2 with stronger interactions with BIC1 and developed soluble expression and protein-dissociating PACE to facilitate further engineering of CRY2, which could give hints on characteristics of UCRs and non-UCRs.

The adsorption potential of FMBO, FeOOH, MnO2 for the removal of Cd^2＋, Cu^2＋ and Pb^2＋ in aqueous systems was investigated in this study. Comparing to FMBO and FeOOH, MnO2 offered a much higher removal capacity towards the three metal ions. The maximal adsorption capacity of MnO2 for Cd^2＋, Cu^2＋ and Pb^2＋ were 1.23, 2.25 and 2.60 mmol· g^-1, respectively. And that for FMBO were 0.37, 1.13, and 1.18mmol·g^-1 and for FeOOH were 0.11, 0.86 and 0.48 mmol·g^-1, respectively. The adsorption behaviors of the three metal ions on the three adsorbents were all significantly affected by pH values and heavy metal removal efficiency increased with pH increased. The Langmuir and Freundlieh adsorption models were used to describe the adsorption equilibrium of the three metal ions onto the three adsorbents. Results showed that the adsorption equilibrium data fitted well to Langmuir isotherm and this indicated that adsorption of metal ions occurred on the three metal oxides adsorbents limited to the formation of a monolayer. More negative charged of MnOa surface than that of FMBO and FeOOH could be ascribed by lower pHiep of MnO2 than that of FMBO and FeOOH and this could contribute to more binding sites on MnO2 surface than that of FMBO and FeOOH. The higher metal ions uptake by MnO2 than FMBO and FeOOH could be well explained by the surface charge mechanism.

Augmented glycolysis due to metabolic reprogramming in lung myofibroblasts is critical to their profibrotic phenotype. The primary glycolysis byproduct, lactate, is also secreted into the extracellular milieu, together with which myofibroblasts and macrophages form a spatially restricted site usually described as fibrotic niche. Therefore, we hypothesized that myofibroblast glycolysis might have a non-cell autonomous effect through lactate regulating the pathogenic phenotype of alveolar macrophages. Here, we demonstrated that there was a markedly increased lactate in the conditioned media of TGF-β1 (transforming growth factor-β1)-induced lung myofibroblasts and in the BAL fluids (BALFs) from mice with TGF-β1- or bleomycin-induced lung fibrosis. Importantly, the media and BALFs promoted profibrotic mediator expression in macrophages. Mechanistically, lactate induced histone lactylation in the promoters of the profibrotic genes in macrophages, consistent with the upregulation of this epigenetic modification in these cells in the fibrotic lungs. The lactate inductions of the histone lactylation and profibrotic gene expression were mediated by p300, as evidenced by their diminished concentrations in p300-knockdown macrophages. Collectively, our study establishes that in addition to protein, lipid, and nucleic acid molecules, a metabolite can also mediate intercellular regulations in the setting of lung fibrosis. Our findings shed new light on the mechanism underlying the key contribution of myofibroblast glycolysis to the pathogenesis of lung fibrosis.

Dysregulation of cellular metabolism has been shown to participate in several pathologic processes. However, the role of metabolic reprogramming is not well appreciated in the pathogenesis of organ fibrosis. To determine if glycolytic reprogramming participates in the pathogenesis of lung fibrosis and assess the therapeutic potential of glycolytic inhibition in treating lung fibrosis. A cell metabolism assay was performed to determine glycolytic flux and mitochondrial respiration. Lactate levels were measured to assess glycolysis in fibroblasts and lungs. Glycolytic inhibition by genetic and pharmacologic approaches was used to demonstrate the critical role of glycolysis in lung fibrosis. Augmentation of glycolysis is an early and sustained event during myofibroblast differentiation, which is dependent on the increased expression of critical glycolytic enzymes, in particular, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3). Augmented glycolysis contributes to the stabilization of hypoxia-inducible factor 1-α, a master regulator of glycolytic enzymes implicated in organ fibrosis, by increasing cellular levels of tricarboxylic acid cycle intermediate succinate in lung myofibroblasts. Inhibition of glycolysis by the PFKFB3 inhibitor 3PO or genomic disruption of the PFKFB3 gene blunted the differentiation of lung fibroblasts into myofibroblasts, and attenuated profibrotic phenotypes in myofibroblasts isolated from the lungs of patients with idiopathic pulmonary fibrosis. Inhibition of glycolysis by 3PO demonstrates therapeutic benefit in bleomycin-induced and transforming growth factor-β1-induced lung fibrosis in mice. Our data support the novel concept of glycolytic reprogramming in the pathogenesis of lung fibrosis and provide proof-of-concept that targeting this pathway may be efficacious in treating fibrotic disorders, such as idiopathic pulmonary fibrosis.