Plant-type RNA editing transferred into Escherichia coli: Expected and surprising new insights for editing factors and how they recognize their targets

Plant-type C-to-U RNA editing is a post-transcriptional process that converts cytidines to uridines in mitochondrial and chloroplast transcripts. This process is mediated by pentatricopeptide repeat (PPR) proteins, which recognize specific targets via their PLS repeats and perform cytidine deamination via their C-terminal extension, the DYW domain. In the model moss Physcomitrium patens, all 13 editing sites are fully assigned to 9 DYW-type PPR proteins, making it an ideal model for studying RNA editing mechanisms. Additionally, plant-type RNA editing has been successfully established in other heterologous systems, such as Escherichia coli. The faithful editing efficiency in comparison to plants makes it a powerful system for further investigating RNA editing mechanisms. The mitochondrial editing factor PPR56 from P. patens efficiently edits its two targets, nad3eU230SL and nad4eU272SL, in both plant and E. coli systems. However, when compared to other PPR proteins such as PPR65 and PPR78, PPR56 exhibits over 100 off-targets in the E. coli transcriptome. Detailed mechanistic studies in the bacterial system have shown that: (i) single amino acid modifications on the PLS motif can redirect PPR proteins to new targets, (ii) the RNA editing activity of PPR proteins is influenced by the target context, and (iii) the editing efficiency is enhanced by tandem targets. A new candidate site, cox3eU290SF, was found to be influenced by PPR56, which suggests a scanning mechanism of PPR proteins along transcripts. Although C-to-U RNA editing is mainly restricted to land plants, one of the exceptions is the heterolobosean protist Naegleria gruberi, which stores 10 DYW-type PPR proteins in its nuclear genome and has two editing sites in its mitochondrial transcriptome. In this study, NgPPR45 was rearranged, resulting in a DYW-type PPR protein with a mitochondrial signal peptide that nicely fits the two editing sites according to the PPR-RNA binding code, making it an ideal candidate. Since knockout studies could not be performed in N. gruberi, different chimeras of PpPPR78 with NgPPR45 were tested in the ppr78 knockout P. patens. The chimera with the PLS stretch of PpPPR78 and the complete C-terminal extensions of NgPPR45 could edit one of the targets for PpPPR78, cox1eU755SL, up to 82%. This proves that the C-terminal extensions of NgPPR45 have a functional cytidine deaminase and can act in fusion with the independently evolved moss PPR stretch. The failure of other chimeras points out that complete C-terminal extensions might be important for fusion proteins from different systems. PPR proteins, with their target-specific cytidine deamination process, make them an ideal molecular tool for transcriptomic engineering. However, chimeras of PPR proteins do not always work. The PLS stretch of PPR65 is not competitive with the C-terminal extensions of PPR56, while the opposite is true. Changing the DYW domains of PPR56 from different heterologous sources results in restricted or widened off-target numbers in the E. coli transcriptome. The experiments show that the preference of the DYW domains does not influence the immediate sequence of the editing site environment, but rather has a long-range impact on the upstream PPR stretch. To further investigate the mechanism of RNA editing, PPR proteins can be overexpressed in the cytosol of plants along with a supplied target in knockout plants. Additionally, off-targets in the nuclear transcriptome can be examined to provide a wider range of options for transcriptomic engineering. Due to its high activity and flexibility, PPR56 is an excellent editing factor and could serve as a solid foundation for a molecular tool

Solar energy as a renewable resource for cooling

The thesis contains mainly two parts: 1) the solar energy resource assessment through measurements and comparison of solar irradiance models and analysis of atmospheric turbidity factors; 2) Sensitivity analysis of solar cooling system, monitoring on solar cooling system and pre-design of a solar cooling system test rig in Politecnico di Torino. Firstly, the solar energy resource assessment is based on the measurements of solar beam normal irradiance and solar global horizontal irradiance in Turin during the whole 2010 with available data from end of January until end of December except two thirds of August when the Politecnico di Torino was closed since the campaign was carried on inside the department of Energy. Measured data have been compared with clear-sky models and the atmospheric turbidity factors have been calculated and compared with that in 1975-6 to evaluate the air-quality variation during the last three decades. Secondly, a series of sensitivity analysis on solar cooling system with various configurations has been undertaken through simulation software Polysun. Field data of solar cooling system in IPLA (Istituto per le Piante da Legno e l’Ambiente) have been monitored and analyzed. The main conclusions are: The ASHRAE clear-sky model mostly fits the solar beam normal irradiance while it has been found that the solar diffuse radiation factor ‘C’ (C=Gdh/Gbn) actually not constant but varies all through the day in a typical way; The turbidity factors are lower in 2010 during winter time due to usage of cleaner fuels and district heating while during summer they increase due to heavier traffic; Solar factor (SF) increases as solar collector area is larger, but its increasing ratio is decreasing and SF decreases as volume size of storage tank is bigger after the demand side is satisfied; the three-week monitoring campaign has shown a satisfactory performance of this solar cooling system in PUEEL, although the solar installation appears oversized

Mandatory Retirement and the Consumption Puzzle: Prices Decline or Quantities Decline?

This paper investigates household consumption changes at retirement by utilizing a comprehensive, diary-based household survey from China. The survey contains both consumption quantity and price information, which permits separating quantity changes from price changes. The mandatory retirement policy in China provides a quasi-experimental setting for identification of the true causal effects of fully anticipated retirement. Using regression discontinuity models, we show that food expenditure declines at retirement, particularly among the low-education group, and that the decline is driven by price declines instead of quantity declines. Shopping time for food increases at retirement, consistent with the price and quantity changes

Flat bands promoted by Hund's rule coupling in the candidate double-layer high-temperature superconductor La3_3Ni2_2O7_7

We report strongly correlated electronic band structure calculations of the recently discovered double-layer high-temperature superconductor La$_3$Ni$_2$O$_7$ under pressure. Our calculations reveal dual nature of Ni-$d$ electrons with almost localized $d_{z^2}$ orbitals due to onsite Coulomb repulsion and very flat hybridization bands of Ni-$d_{x^2-y^2}$ and Ni-$d_{z^2}$ quasiparticles near the Fermi energy. We find that the quasiparticle effective mass are greatly enhanced by the Hund's rule coupling and their lifetimes are inversely proportional to the temperature, which explains the experimentally observed strange metal behavior in the normal state. We also find strong antiferromagnetic spin correlations of Ni-$d$ electrons, which may provide the pairing force of quasiparticles for the high-temperature superconductivity. These give a potential explanation of two key observations in experiment and connect the superconducting La$_3$Ni$_2$O$_7$ with cuprate high-temperature superconductors. The presence of flat bands and the interplay of orbital-selective Mott, Hund, and Kondo physics make La$_3$Ni$_2$O$_7$ a unique platform for exploring rich emergent quantum many-body phenomena in the future.Comment: 6 pages, 4 figure