Establishment of a PEG-mediated protoplast transformation system based on DNA and CRISPR/Cas9 ribonucleoprotein complexes for banana.

BACKGROUND: To date, CRISPR/Cas9 RNP editing tools have not been applied to the genetic modification of banana. Here, the establishment of a PEG-mediated banana protoplast transformation system makes it possible to build an efficient DNA-free method for a site-directed mutagenesis system. RESULTS: Protoplasts constitute a versatile platform for transient expression in plant science. In this study, we established a PEG-mediated banana protoplast transformation system. This system was further optimized for successfully delivering CRISPR/Cas9 and CRISPR/Cas12a plasmids and CRISPR/Cas9 ribonucleoproteins (RNPs) for targeted delivery of the PDS gene into banana protoplasts. Specific bands were observed in PCR-Restriction Enzyme Digestion (PCR-RE) assays, and Sanger sequencing of single clones further confirmed the occurrence of indels at target sites. Deep amplicon sequencing results showed that the editing efficiency of the CRISPR/Cas9 system was higher than that of the other two systems. CONCLUSIONS: The PEG-mediated banana protoplast transformation system can serve as a rapid and effective tool for transient expression assays and sgRNA validation in banana. The application of the CRISPR/Cas9 RNP system enables the generation of banana plants engineered by DNA-free gene editing.

High-quality genome assemblies for two Australimusa bananas (Musa spp.) and insights into regulatory mechanisms of superior fiber properties.

Bananas (Musa spp.) are monocotyledonous plants with high genetic diversity in the Musaceae family that are cultivated mainly in tropical and subtropical countries. The fruits are a popular food, and the plants themselves have diverse uses. Four genetic groups (genomes) are thought to have contributed to current banana cultivars: Musa acuminata (A genome), Musa balbisiana (B genome), Musa schizocarpa (S genome), and species of the Australimusa section (T genome). However, the T genome has not been effectively explored. Here, we present the high-quality TT genomes of two representative accessions, Abaca (Musa textilis), with high-quality natural fiber, and Utafun (Musa troglodytarum, Fe'i group), with abundant ß-carotene. Both the Abaca and Utafun assemblies comprise 10 pseudochromosomes, and their total genome sizes are 613 Mb and 619 Mb, respectively. Comparative genome analysis revealed that the larger size of the T genome is likely attributable to rapid expansion and slow removal of transposons. Compared with those of Musa AA or BB accessions or sisal (Agava sisalana), Abaca fibers exhibit superior mechanical properties, mainly because of their thicker cell walls with a higher content of cellulose, lignin, and hemicellulose. Expression of MusaCesA cellulose synthesis genes peaks earlier in Abaca than in AA or BB accessions during plant development, potentially leading to earlier cellulose accumulation during secondary cell wall formation. The Abaca-specific expressed gene MusaMYB26, which is directly regulated by MusaMYB61, may be an important regulator that promotes precocious expression of secondary cell wall MusaCesAs. Furthermore, MusaWRKY2 and MusaNAC68, which appear to be involved in regulating expression of MusaLAC and MusaCAD, may at least partially explain the high accumulation of lignin in Abaca. This work contributes to a better understanding of banana domestication and the diverse genetic resources in the Musaceae family, thus providing resources for Musa genetic improvement.

Stereochemical control of polymorph transitions in nanoscale reactors.

Crystallization of glycine in the cylindrical nanopores of anodic aluminum oxide (AAO) revealed the formation of metastable ß-glycine in pores having diameters less than 200 nm. Two-dimensional X-ray microdiffraction indicated that the [010] axis of the embedded ß-glycine nanocrystals coincided with the pore direction, identical to behavior observed previously in the cylindrical nanopores of polymer monoliths. Whereas the ß-glycine nanocrystals were stable indefinitely in ambient air and persisted upon heating, they transformed to the α polymorph upon standing at room temperature and 90% relative humidity (RH). The α-glycine nanocrystals were oriented with the [010] axis nearly perpendicular to the pore direction, reflecting a nearly 90° rotation of the glycine molecules during the transition. When the ß-glycine nanocrystals were formed in the AAO cylinders in the presence of small amounts of racemic hydrophobic amino acid auxiliaries, which are known to bind selectively to the (010) and (010) faces on the fast-growing end of ß-glycine enantiomorphs, the ß â†’ α phase transition at 90% RH was suppressed. In contrast, ß-glycine nanocrystals grown in the presence of an enantiopure amino acid auxiliary, which binds to the fast-growing end of only one of the enantiomorphs, thus suppressing its formation and leaving the other enantiomorph unperturbed, transformed into the α polymorph under the same conditions. This observation confirms that binding of an amino acid to the {010} faces is stereoselective and that access of water to these faces is essential for the transition to the α polymorph.

Regulating cancer associated fibroblasts with losartan-loaded injectable peptide hydrogel to potentiate chemotherapy in inhibiting growth and lung metastasis of triple negative breast cancer.

Preoperative chemotherapy is effective in improving the prognosis of patients, but its efficacy is impeded by cancer associated fibroblasts (CAFs) that enhance the survival, growth, and metastasis of cancer cells. To inhibit the activity of CAFs, prolonged and localized drug exposure is necessary. Here, we report on the rational design, screening, and evaluation of an injectable peptide hydrogel as a local losartan depot aiming to inhibit CAFs and potentiate chemotherapy. We synthesized a set of peptide derivatives and found that C16-GNNQQNYKD-OH (C16-N) surpassed the others in hydrogel formation and drug encapsulation, due to its flexible hydrocarbon tail and interpeptide hydrogen bonding that allowed supramolecular self-assembly into long filaments with hydrophobic cores. C16-N co-assembled with losartan to form hydrogel from which losartan was sustainably released over 9 days. After intratumoral injection, the hydrogel could be retained in the tumor for more than 9 days, significantly inhibited the CAFs and collagen synthesis in orthotopic 4T1 tumors, and enhanced the efficacy of PEGylated doxorubicin-loaded liposomes (Dox-L) in inhibiting the tumor growth (64% vs. Dox-L alone) and lung metastasis (80% vs. Dox-L alone). These results provide important guiding principles for the rational design of injectable peptide hydrogels aiming to regulate CAFs and improve chemotherapy.

High-performance, low-voltage, and easy-operable bending actuator based on aligned carbon nanotube/polymer composites.

In this work, we show that embedding super-aligned carbon nanotube sheets into a polymer matrix (polydimethylsiloxane) can remarkably reduce the coefficient of thermal expansion of the polymer matrix by two orders of magnitude. Based on this unique phenomenon, we fabricated a new kind of bending actuator through a two-step method. The actuator is easily operable and can generate an exceptionally large bending actuation with controllable motion at very low driving DC voltages (<700 V/m). Furthermore, the actuator can be operated without electrolytes in the air, which is superior to conventional carbon nanotube actuators. Proposed electrothermal mechanism was discussed and confirmed by our experimental results. The exceptional bending actuation performance together with easy fabrication, low-voltage, and controllable motion demonstrates the potential ability of using this kind of actuator in various applicable areas, such as artificial muscles, microrobotics, microsensors, microtransducers, micromanipulation, microcantilever for medical applications, and so on.