Aberrant expression of HIF3A in plasma of patients with non-small cell lung cancer and its clinical significance.

BACKGROUND: Hypoxia-inducible factors (HIFs) have been evaluated in various cancers and diseases. However, the specific role of hypoxia-inducible factor 3 alpha (HIF3A) in non-small cell lung cancer (NSCLC) remains controversial. MATERIALS AND METHODS: We investigated HIF3A mRNA expression in the plasma and tumor tissues of patients with NSCLC and explored its clinical significance. Plasma samples from 103 cases of lung adenocarcinoma (LUAD) and 96 cases of lung squamous cell carcinoma (LUSC), and tumor-adjacent normal tissues from 58 LUAD and 62 LUSC cases were retrospectively evaluated at the No.8 People's Hospital of Qing Dao. HIF3A expression was explored using RT-qPCR. The clinical significance of HIF3A was evaluated in the plasma and tumor tissues using the receiver operating curve (ROC) and the area under the curve (AUC). RESULTS: Hypoxia-inducible factor 3 alpha expression was notably downregulated in the plasma or tumor tissues of patients with LUAD and LUSC, compared with the healthy control group or adjacent normal tissues. Furthermore, HIF3A expression had a significant positive correlation in the plasma and tumor tissues of LUAD and LUSC patients. Meanwhile, the ROC-AUCs achieved a significantly higher range, from 0.84 to 0.93, with the plasma or tumor tissues of NSCLC patients. Thus, HIF3A expression was not only correlated with plasma and tumor tissues, but also showed potential significance in NSCLC. CONCLUSION: Hypoxia-inducible factor 3 alpha is aberrantly detectable in NSCLC patients in the plasma and tumor tissues. HIF3A may be involved in hypoxic responses during the development and occurrence of NSCLC.

Glycolysis regulates pollen tube polarity via Rho GTPase signaling.

As a universal energy generation pathway utilizing carbon metabolism, glycolysis plays an important housekeeping role in all organisms. Pollen tubes expand rapidly via a mechanism of polarized growth, known as tip growth, to deliver sperm for fertilization. Here, we report a novel and surprising role of glycolysis in the regulation of growth polarity in Arabidopsis pollen tubes via impingement of Rho GTPase-dependent signaling. We identified a cytosolic phosphoglycerate kinase (pgkc-1) mutant with accelerated pollen germination and compromised pollen tube growth polarity. pgkc-1 mutation greatly diminished apical exocytic vesicular distribution of REN1 RopGAP (Rop GTPase activating protein), leading to ROP1 hyper-activation at the apical plasma membrane. Consequently, pgkc-1 pollen tubes contained higher amounts of exocytic vesicles and actin microfilaments in the apical region, and showed reduced sensitivity to Brefeldin A and Latrunculin B, respectively. While inhibition of mitochondrial respiration could not explain the pgkc-1 phenotype, the glycolytic activity is indeed required for PGKc function in pollen tubes. Moreover, the pgkc-1 pollen tube phenotype was mimicked by the inhibition of another glycolytic enzyme. These findings highlight an unconventional regulatory function for a housekeeping metabolic pathway in the spatial control of a fundamental cellular process.

Pooled CRISPR/Cas9 reveals redundant roles of plastidial phosphoglycerate kinases in carbon fixation and metabolism.

Phosphoglycerate kinase (PGK) is a highly conserved reversible enzyme that participates in both glycolysis and photosynthesis. In Arabidopsis thaliana, one cytosolic PGK (PGKc) and two plastidial PGKs (PGKp) are known. It remains debatable whether the two PGKp isozymes are functionally redundant or specialized in plastidial carbon metabolism and fixation. Here, using a pooled clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) strategy, we found that plants with single mutations in pgkp1 or pgkp2 were not significantly affected, whereas a pgkp1pgkp2 double mutation was lethal due to retarded carbon fixation, suggesting that PGKp isozymes play redundant functional roles. Metabolomic analysis demonstrated that the sugar-deficient pgkp1pgkp2 double mutation was partially complemented by exogenous sugar, although respiration intermediates were not rescued. Chloroplast development was defective in pgkp1pgkp2, due to a deficiency in glycolysis-dependent galactoglycerolipid biosynthesis. Ectopic expression of a plastid targeting PGKc did not reverse the pgkp1pgkp2 double-mutant phenotypes. Therefore, PGKp1 and PGKp2 play redundant roles in carbon fixation and metabolism, whereas the molecular function of PGKc is more divergent. Our study demonstrated the functional conservation and divergence of glycolytic enzymes.

Generation of a multiplex mutagenesis population via pooled CRISPR-Cas9 in soya bean.

The output of genetic mutant screenings in soya bean [Glycine max (L.) Merr.] has been limited by its paleopolypoid genome. CRISPR-Cas9 can generate multiplex mutants in crops with complex genomes. Nevertheless, the transformation efficiency of soya bean remains low and, hence, remains the major obstacle in the application of CRISPR-Cas9 as a mutant screening tool. Here, we report a pooled CRISPR-Cas9 platform to generate soya bean multiplex mutagenesis populations. We optimized the key steps in the screening protocol, including vector construction, sgRNA assessment, pooled transformation, sgRNA identification and gene editing verification. We constructed 70 CRISPR-Cas9 vectors to target 102 candidate genes and their paralogs which were subjected to pooled transformation in 16 batches. A population consisting of 407 T0 lines was obtained containing all sgRNAs at an average mutagenesis frequency of 59.2%, including 35.6% lines carrying multiplex mutations. The mutation frequency in the T1 progeny could be increased further despite obtaining a transgenic chimera. In this population, we characterized gmric1/gmric2 double mutants with increased nodule numbers and gmrdn1-1/1-2/1-3 triple mutant lines with decreased nodulation. Our study provides an advanced strategy for the generation of a targeted multiplex mutant population to overcome the gene redundancy problem in soya bean as well as in other major crops.

Design of A-D-A-Type Organic Third-Order Nonlinear Optical Materials Based on Benzodithiophene: A DFT Study.

The acceptor-donor-acceptor (A-D-A) type conjugated organic molecule has been widely applied in the organic optoelectronics field. A total of Nine compounds (1-9) were designed under the A-D-A framework, with the electron donor benzodithiophene as the core and dicyanomethylene as the acceptor moiety, modifying the benzodithiophene with the phenyl, naphthyl, and difluorinated phenyl groups. The conjugation length can be changed by introducing a thiophene π-conjugated bridge. The geometric structures, electronic structure, excited state properties, aromaticity, and the static- and frequency-dependent second hyperpolarizabilities were investigated by employing high-precision density functional theory (DFT) calculations with an aug-cc-pVDZ basis set. As a result, the three compounds with the longest conjugation length exhibit a smaller energy gap (Egap), larger UV-vis absorption coefficient, and response range, which are the three strongest third-order nonlinear optical (NLO) response properties in this work. This work systematically explored the connection between molecular structure and NLO response, which provides a rational design strategy for high-performance organic NLO materials.

Ultrafast Kinetics of Chlorinated Polymer Donors: A Faster Excitonic Dissociation Path.

Halogen-substituted donor/acceptor materials are widely regarded as a promising strategy toward improved power-conversion efficiencies (PCEs) in polymer solar cells (PSCs). A chlorinated polymer donor, PClBTA-PS, and its non-chlorinated analogue, PBTA-PS, are synthesized. The PClBTA-PS-based devices show significant enhancements in terms of open-circuit voltage (VOC = 0.82 V) and fill factor (FF = 76.20%). In addition, a PCE of 13.20% is obtained, which is significantly higher than that for the PBTA-PS-based devices (PCE = 7.63%). Grazing incident wide-angle X-ray scattering shows that the chlorinated polymer enables better π-π stacking in both pure and blend films. DFT and TD-DFT calculations as well as ultrafast photophysics measurements indicate that chlorinated PClBTA-PS has a smaller bonding energy and a longer spontaneous-emission lifetime. The results also reveal that the charge-transfer-state excitons in PClBTA-PS:IT4Cl blend films split into the charge-separated (CS) state via a faster dissociation path, which produces a higher yield of the CS state. Overall, this study provides a deeper understanding of how a halogen-substituted polymer can improve PSCs in the future.

Elimination of an unfavorable allele conferring pod shattering in an elite soybean cultivar by CRISPR/Cas9.

Pod shattering can lead to devastating yield loss of soybean and has been a negatively selected trait in soybean domestication and breeding. Nevertheless, a significant portion of soybean cultivars are still pod shattering-susceptible, limiting their regional and climatic adaptabilities. Here we performed genetic diagnosis on the shattering-susceptible trait of a national registered cultivar, Huachun6 (HC6), and found that HC6 carries the susceptible genotype of a candidate Pod dehiscence 1 (PDH1) gene, which exists in a significant portion of soybean cultivars. We next performed genome editing on PDH1 gene by clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9). In T2 progenies, several transgene-free lines with pdh1 mutations were characterized without affecting major agronomic traits. The pdh1 mutation significantly improved the pod shattering resistance which is associated with aberrant lignin distribution in inner sclerenchyma. Our work demonstrated that precision breeding by genome editing on PDH1 holds great potential for precisely improving pod shattering resistance and adaptability of soybean cultivars.

Rational Design and Characterization of Symmetry-Breaking Organic Semiconductors in Polymer Solar Cells: A Theory Insight of the Asymmetric Advantage.

Asymmetric molecule strategy is considered an effective method to achieve high power conversion efficiency (PCE) of polymer solar cells (PSCs). In this paper, nine oligomers are designed by combining three new electron-deficient units (unitA)-n1, n2, and n3-and three electron-donating units (unitD)-D, E, and F-with their π-conjugation area extended. The relationships between symmetric/asymmetric molecule structure and the performance of the oligomers are investigated using the density functional theory (DFT) and time-dependent density functional theory (TD-DFT) calculations. The results indicate that asymmetry molecule PEn2 has the minimum dihedral angle in the angle between two planes of unitD and unitA among all the molecules, which exhibited the advantages of asymmetric structures in molecular stacking. The relationship of the values of ionization potentials (IP) and electron affinities (EA) along with the unitD/unitA π-extend are revealed. The calculated reorganization energy results also demonstrate that the asymmetric molecules PDn2 and PEn2 could better charge the extraction of the PSCs than other molecules for their lower reorganization energy of 0.180 eV and 0.181 eV, respectively.