Advanced Insulin Synthesis by One-pot/Stepwise Disulfide Bond Formation Enabled by S-Protected Cysteine Sulfoxide.

An advanced insulin synthesis is presented that utilizes one-pot/stepwise disulfide bond formation enabled by acid-activated S-protected cysteine sulfoxides in the presence of chloride anion. S-chlorocysteine generated from cysteine sulfoxides reacts with an S-protected cysteine to afford S-sulfenylsulfonium cation, which then furnishes the disulfide or reversely returns to the starting materials depending on the S-protection employed and the reaction conditions. Use of S-acetamidomethyl cysteine (Cys(Acm)) and its sulfoxide (Cys(Acm)(O)) selectively give the disulfide under weak acid conditions in the presence of MgCl2 even if S-p-methoxybenzyl cysteine (Cys(MBzl)) and its sulfoxide (Cys(MBzl)(O)) are also present. In contrast, the S-MBzl pair yields the disulfide under more acidic conditions in the presence of a chloride anion source. These reaction conditions allowed a one-pot insulin synthesis. Additionally, lipidated insulin was prepared by a one-pot disulfide-bonding/lipidation sequence.

Eco-friendly strategy for CO2 enrichment performance in commercial greenhouses based on the CO2 spatial distribution and photosynthesis.

CO2 enrichment is an essential environmental control technology due to its significantly enhancing effect on crop production capacity. Despite being a key energy consumer in protected agriculture (i.e. greenhouse systems), CO2 enrichment remains at a low energy use efficiency level, highlighting the need for developing more energy-efficiency strategies for CO2 enrichment. Therefore, this study employed the computational fluid dynamics (CFD) simulation method to replicate the CO2 diffusion process resulting from CO2 enrichment in three commercial strawberry greenhouses with varying geometric characteristics. Based on the CFD-simulated CO2 concentration distributions, the leaf photosynthetic rate was calculated using a mathematical model group. The CO2 enrichment efficiency was then analysed by calculating the ratio of increased photosynthesis across the cultivation area to the amount of energy (in CO2 equivalent) used. The efficiency peaked when the average CO2 concentration was approximately 500 µmol mol-1, thereby providing guidance for determining the target concentration of CO2 enrichment in production. Although this study is limited as the CFD simulation only considered a typical short-period CO2 enrichment event, future research will provide a broader analysis by considering changes throughout the day.

Short-Chain Guide RNA for Site-Directed A-to-I RNA Editing.

Site-directed RNA editing is a promising genetic modification technology for therapeutic and pharmaceutical applications. We previously constructed adenosine deaminases acting on RNA (ADAR)-guiding RNAs (AD-gRNAs) that direct A-to-I RNA editing activity of native human ADAR2 into a programmable target site. In this study, we developed the short-chain AD-gRNA (shAD-gRNA) as a potential basic framework for practical RNA-editing oligonucleotides. Based on knowledge of previous AD-gRNA, shAD-gRNAs were designed to have the shortest possible sequence for the induction of editing activity. In vitro, compared to the original AD-gRNA, the shAD-gRNAs showed similar or superior editing induction activity, depending on the target RNA sequence, and had lower off-target editing activity around the target site, which is predicted to be a hotspot for off-target editing. Moreover, shAD-gRNAs achieved target RNA editing with both exogenous and endogenous human ADARs in cultured cells. Our results present shAD-gRNA as a short basic framework that would be applicable to further development for practical RNA-editing oligonucleotides.

Non-invasive 11C-Imaging Revealed the Spatiotemporal Variability in the Translocation of Photosynthates Into Strawberry Fruits in Response to Increasing Daylight Integrals at Leaf Surface.

The efficiency of photosynthate translocation from leaves to fruits directly affects dry matter partitioning. Therefore, controlling photosynthate translocation dynamics is critical for high-yield and high-quality fruit production. Accordingly, photosynthate translocation changes must be characterized using data obtained at a higher spatiotemporal resolution than those provided by conventional methods. In this study, 11C-photosynthate translocation into strawberry (Fragaria × ananassa Duch.) fruits in individual plants was visualized non-invasively and repeatedly using a positron emission tracer imaging system (PETIS) to assess the spatiotemporal variability in the translocation dynamics in response to increasing daylight integrals (i.e., 0.5-, 4.5-, and 9-h exposures to 400 µmol m-2 s-1 at the leaf surface). Serial images of photosynthate translocation into strawberry fruits obtained from the PETIS confirmed that 11C-photosynthates were translocated heterogeneously into each fruit on the same inflorescence. The amount of translocated 11C-photosynthates and the translocation rate into each fruit significantly increased as the integrated light intensity at the leaf surface increased. An analysis of the pedicel of each fruit also confirmed that the photosynthate translocation rate increased. The cumulated photosynthesis in leaves increased almost linearly during the light period, suggesting that an increase in the amount of photosynthates in leaves promotes the translocation of photosynthates from leaves, resulting in an increase in the photosynthate translocation rate in pedicels and enhanced photosynthate accumulation in fruits. Additionally, the distribution pattern of photosynthate translocated to fruits did not change during the light period, nor did the order of the sink activity (11C radioactivity/fruit dry weight), which is the driving force for the prioritization of the 11C-partitioning between competing organs, among fruits. Thus, this is the first study to use 11C-radioisotopes to clarify the spatiotemporal variability in photosynthate translocation from source leaves to individual sink fruits in vivo in response to increasing daylight integrals at a high spatiotemporal resolution.

Dynamic Analysis of Photosynthate Translocation Into Strawberry Fruits Using Non-invasive 11C-Labeling Supported With Conventional Destructive Measurements Using 13C-Labeling.

In protected strawberry (Fragaria × ananassa Duch.) cultivation, environmental control based on the process of photosynthate translocation is essential for optimizing fruit quality and yield, because the process of photosynthate translocation directly affects dry matter partitioning. We visualized photosynthate translocation to strawberry fruits non-invasively with 11CO2 and a positron-emitting tracer imaging system (PETIS). We used PETIS to evaluate real-time dynamics of 11C-labeled photosynthate translocation from a 11CO2-fed leaf, which was immediately below the inflorescence, to individual fruits on an inflorescence in intact plant. Serial photosynthate translocation images and animations obtained by PETIS verified that the 11C-photosynthates from the source leaf reached the sink fruit within 1 h but did not accumulate homogeneously within a fruit. The quantity of photosynthate translocation as represented by 11C radioactivity varied among individual fruits and their positions on the inflorescence. Photosynthate translocation rates to secondary fruit were faster than those to primary or tertiary fruits, even though the translocation pathway from leaf to fruit was the longest for the secondary fruit. Moreover, the secondary fruit was 25% smaller than the primary fruit. Sink activity (11C radioactivity/dry weight [DW]) of the secondary fruit was higher than those of the primary and tertiary fruits. These relative differences in sink activity levels among the three fruit positions were also confirmed by 13C tracer measurement. Photosynthate translocation rates in the pedicels might be dependent on the sink strength of the adjoining fruits. The present study established 11C-photosynthate arrival times to the sink fruits and demonstrated that the translocated material does not uniformly accumulate within a fruit. The actual quantities of translocated photosynthates from a specific leaf differed among individual fruits on the same inflorescence. To the best of our knowledge, this is the first reported observation of real-time translocation to individual fruits in an intact strawberry plant using 11C-radioactive- and 13C-stable-isotope analyses.