Critical role of cyclic electron transport around photosystem I in the maintenance of photosystem I activity.

In angiosperms, cyclic electron transport around photosystem I (PSI) is mediated by two pathways that depend on the PROTON GRADIENT REGULATION 5 (PGR5) protein and the chloroplast NADH dehydrogenase-like (NDH) complex, respectively. In the Arabidopsis double mutants defective in both pathways, plant growth and photosynthesis are impaired. The pgr5-1 mutant used in the original study is a missense allele and accumulates low levels of PGR5 protein. In this study, we generated two knockout (KO) alleles, designated as pgr5-5 and pgr5-6, using the CRISPR-Cas9 technology. Although both KO alleles showed a severe reduction in P700 similar to the pgr5-1 allele, NPQ induction was less severely impaired in the KO alleles than in the pgr5-1 allele. In the pgr5-1 allele, the second mutation affecting NPQ size was mapped to ~21 cM south of the pgr5-1 locus. Overexpression of the pgr5-1 allele, encoding the glycine130-to-serine change, complemented the pgr5-5 phenotype, suggesting that the pgr5-1 mutation destabilizes PGR5 but that the mutant protein retains partial functionality. Using two KO alleles, we created the double mutants with two chlororespiratory reduction (crr) mutants defective in the NDH complex. The growth of the double mutants was notably impaired. In the double mutant seedlings that survived on the medium containing sucrose, PSI activity evaluated by the P700 oxidation was severely impaired, whereas PSII activity was only mildly impaired. Cyclic electron transport around PSI is required to maintain PSI activity.

Flavodiiron proteins enhance the rate of CO2 assimilation in Arabidopsis under fluctuating light intensity.

The proton concentration gradient (ΔpH) and membrane potential (Δψ) formed across the thylakoid membrane contribute to ATP synthesis in chloroplasts. Additionally, ΔpH downregulates photosynthetic electron transport via the acidification of the thylakoid lumen. K+ exchange antiporter 3 (KEA3) relaxes this downregulation by substituting ΔpH with Δψ in response to fluctuation of light intensity. In the Arabidopsis (Arabidopsis thaliana) line overexpressing KEA3 (KEA3ox), the rate of electron transport is elevated by accelerating the relaxation of ΔpH after a shift from high light (HL) to low light. However, the plant cannot control electron transport toward photosystem I (PSI), resulting in PSI photodamage. In this study, we crossed the KEA3ox line with the line (Flavodiiron [Flv]) expressing the Flv proteins of Physcomitrium patens. In the double transgenic line (Flv-KEA3ox), electrons overloading toward PSI were pumped out by Flv proteins. Consequently, photodamage of PSI was alleviated to the wild-type level. The rate of CO2 fixation was enhanced in Flv and Flv-KEA3ox lines during HL periods of fluctuating light, although CO2 fixation was unaffected in any transgenic lines in constant HL. Upregulation of CO2 fixation was accompanied by elevated stomatal conductance in fluctuating light. Consistent with the results of gas exchange experiments, the growth of Flv and Flv-KEA3ox plants was better than that of WT and KEA3ox plants under fluctuating light.

Intensive perioperative rehabilitation improves surgical outcomes after pancreaticoduodenectomy.

PURPOSE: Although the mortality rate for pancreaticoduodenectomy (PD) has decreased to around 2.8-5% in high-volume centers, postoperative complications are still common in 30-50% of cases. Preoperative exercise, called "prehabilitation," has been recently reported to reduce the frequency of complications after surgery. This study aims to evaluate the impact of the intensive perioperative rehabilitation on improvement of surgical outcomes for patients undergoing PD. METHODS: Between 2003 and 2014, 576 consecutive patients underwent PD in Wakayama Medical University Hospital. Of these, 331 patients received perioperative rehabilitation combined with prehabilitation and postoperative rehabilitation between 2009 and 2014. Previously, 245 patients underwent PD without perioperative rehabilitation between 2003 and 2008. We compared surgical outcomes between the patients undergoing PD with and without perioperative rehabilitation to evaluate the efficacy of our rehabilitation program. RESULTS: The frequency of pulmonary complications was significantly lower in patients undergoing PD with perioperative rehabilitation than those without (0.9% vs. 4.3%, P = 0.011). There were no significant differences in other complication or mortality rates. Length of hospital stay was also shorter in patients receiving perioperative rehabilitation than that of those not receiving it (16 vs. 24 days, P < 0.001). CONCLUSIONS: Intensive perioperative rehabilitation might reduce postoperative pulmonary complications and shorten postoperative hospital stay after PD. Therefore, we suggest that perioperative rehabilitation should be included as part of enhanced recovery after surgery for patients undergoing PD, although further large-scale studies are necessary to confirm our results.

Genome engineering of mammalian haploid embryonic stem cells using the Cas9/RNA system.

Haploid embryonic stem cells (ESCs) are useful for studying mammalian genes because disruption of only one allele can cause loss-of-function phenotypes. Here, we report the use of haploid ESCs and the CRISPR RNA-guided Cas9 nuclease gene-targeting system to manipulate mammalian genes. Co-transfection of haploid ESCs with vectors expressing Cas9 nuclease and single-guide RNAs (sgRNAs) targeting Tet1, Tet2, and Tet3 resulted in the complete disruption of all three genes and caused a loss-of-function phenotype with high efficiency (50%). Co-transfection of cells with vectors expressing Cas9 and sgRNAs targeting two loci on the same chromosome resulted in the creation of a large chromosomal deletion and a large inversion. Thus, the use of the CRISPR system in combination with haploid ESCs provides a powerful platform to manipulate the mammalian genome.