Transcriptome and Metabolome Analyses Reflect the Molecular Mechanism of Drought Tolerance in Sweet Potato.

Sweet potato (Ipomoea batatas (L.) Lam.) is one of the most widely cultivated crops in the world, with outstanding stress tolerance, but drought stress can lead to a significant decrease in its yield. To reveal the response mechanism of sweet potato to drought stress, an integrated physiological, transcriptome and metabolome investigations were conducted in the leaves of two sweet potato varieties, drought-tolerant zhenghong23 (Z23) and a more sensitive variety, jinong432 (J432). The results for the physiological indexes of drought showed that the peroxidase (POD) and superoxide dismutase (SOD) activities of Z23 were 3.68 and 1.21 times higher than those of J432 under severe drought, while Z23 had a higher antioxidant capacity. Transcriptome and metabolome analysis showed the importance of the amino acid metabolism, respiratory metabolism, and antioxidant systems in drought tolerance. In Z23, amino acids such as asparagine participated in energy production during drought by providing substrates for the citrate cycle (TCA cycle) and glycolysis (EMP). A stronger respiratory metabolism ability could better maintain the energy supply level under drought stress. Drought stress also activated the expression of the genes encoding to antioxidant enzymes and the biosynthesis of flavonoids such as rutin, resulting in improved tolerance to drought. This study provides new insights into the molecular mechanisms of drought tolerance in sweet potato.

Integrated Transcriptome and Metabolome Analyses Reveal Details of the Molecular Regulation of Resistance to Stem Nematode in Sweet Potato.

Stem nematode disease can seriously reduce the yield of sweet potato (Ipomoea batatas (L.) Lam). To explore resistance mechanism to stem nematode in sweet potato, transcriptomes and metabolomes were sequenced and compared between two sweet potato cultivars, the resistant Zhenghong 22 and susceptible Longshu 9, at different times after stem nematode infection. In the transcriptional regulatory pathway, mitogen-activated protein kinase signaling was initiated in Zhenghong 22 at the early stage of infection to activate genes related to ethylene production. Stem nematode infection in Zhenghong 22 also triggered fatty acid metabolism and the activity of respiratory burst oxidase in the metabolic pathway, which further stimulated the glycolytic and shikimic pathways to provide raw materials for secondary metabolite biosynthesis. An integrated analysis of the secondary metabolic regulation pathway in the resistant cultivar Zhenghong 22 revealed the accumulation of tryptophan, phenylalanine, and tyrosine, leading to increased biosynthesis of phenylpropanoids and salicylic acid and enhanced activity of the alkaloid pathway. Stem nematode infection also activated the biosynthesis of terpenoids, abscisic acid, zeatin, indole, and brassinosteroid, resulting in improved resistance to stem nematode. Finally, analyses of the resistance regulation pathway and a weighted gene co-expression network analysis highlighted the importance of the genes itf14g17940 and itf12g18840, encoding a leucine-rich receptor-like protein and 1-aminocyclopropane-1-carboxylate synthase, respectively. These are candidate target genes for increasing the strength of the defense response. These results provide new ideas and a theoretical basis for understanding the mechanism of resistance to stem nematode in sweet potato.

Core-Shell Engineering of Conductive Fillers toward Enhanced Dielectric Properties: A Universal Polarization Mechanism in Polymer Conductor Composites.

Flexible dielectric and electronic materials with high dielectric constant (k) and low loss are constantly pursued. Encapsulation of conductive fillers with insulating shells represents a promising approach, and has attracted substantial research efforts. However, progress is greatly impeded due to the lack of a fundamental understanding of the polarization mechanism. In this work, a series of core-shell polymer composites is studied, and the correlation between macroscopic dielectric properties (across entire composites) and microscopic polarization (around single fillers) is investigated. It is revealed that the polarization in polymer conductor composites is determined by electron transport across multiple neighboring conductive fillers-a domain-type polarization. The formation of a core-shell filler structure affects the dielectric properties of tpolymer composites by essentially modifying the filler-cluster size. Based on this understanding, a novel percolative composite is prepared with higher-than-normal filler concentration and optimized shell's electrical resistivity. The developed composite shows both high-k due to enlarged cluster size and low loss due to restrained charge transport simultaneously, which cannot be achieved in traditional percolative composites or via simple core-shell filler design. The revealed polarization mechanism and the optimization strategy for core-shell fillers provide critical guidance and a new paradigm, for developing advanced polymer dielectrics with promising property sets.

GPC3-targeted CAR-T cells secreting B7H3-targeted BiTE exhibit potent cytotoxicity activity against hepatocellular carcinoma cell in the in vitro assay.

Hepatocellular carcinoma (HCC), the most common primary liver cancer has a high mortality in China, and it is usually diagnosed at a late stage, thereby leaving patients with few effective treatment options. Chimeric antigen receptor-T (CAR-T) cell therapy, a novel immunotherapy that has shown promising results in leukemia, lymphoma and multiple myeloma, is also expected to work well in solid tumors, including HCC. However, the ideal therapeutic efficacy has not yet been achieved, in part due to tumor antigen escape caused by antigen heterogeneity. To overcome such challenge, we screened a panel of biomarkers in HCC cell lines and found that GPC3 and B7H3 were highly expressed on HCC with expression heterogeneity. Then we developed a novel bispecific T cell engagers CAR-T (CAR.T-BiTEs) that drives the expression of a CAR specific for GPC3 and BiTEs against CD3 and B7H3, herein referred to as "GPC3-BiTE CAR." We found that BiTEs promoted the increased activation of untransduced T cells and IFN-γ release. Moreover, BiTEs secreted by GPC3-BiTE CAR-HEK293T cells promoted increased cytotoxicity activity of untransduced T cells against GPC3+/B7H3+ (GPC3 positive/B7H3 positive) and GPC3-/B7H3+(GPC3 negative/B7H3 positive) HCC cell lines. In vitro function assays showed that GPC3-BiTE CAR-T cells exhibited greater cytotoxicity activity against GPC3+/B7H3+ HCC cell lines than GPC3 CAR-T cells (GPC3-targeted CAR-T cells) and B7H3 CAR-T cells (B7H3-targeted CAR-T cells). Furthermore, GPC3-BiTE CAR-T cells exhibited superior cytotoxicity against GPC3 negative HCC cell lines compared with GPC3 CAR T cells. In conclusion, our study showed that GPC3-BiTE CAR T cells exhibited superior antitumor activity than single-target CAR-T cells and can overcome tumor escape induced by antigen heterogeneity, suggesting that this could be a promising therapeutic strategy for HCC.

Chelicerata sDscam isoforms combine homophilic specificities to define unique cell recognition.

Thousands of Down syndrome cell adhesion molecule (Dscam1) isoforms and ∼60 clustered protocadhrein (cPcdh) proteins are required for establishing neural circuits in insects and vertebrates, respectively. The strict homophilic specificity exhibited by these proteins has been extensively studied and is thought to be critical for their function in neuronal self-avoidance. In contrast, significantly less is known about the Dscam1-related family of ∼100 shortened Dscam (sDscam) proteins in Chelicerata. We report that Chelicerata sDscamα and some sDscamß protein trans interactions are strictly homophilic, and that the trans interaction is meditated via the first Ig domain through an antiparallel interface. Additionally, different sDscam isoforms interact promiscuously in cis via membrane proximate fibronectin-type III domains. We report that cell-cell interactions depend on the combined identity of all sDscam isoforms expressed. A single mismatched sDscam isoform can interfere with the interactions of cells that otherwise express an identical set of isoforms. Thus, our data support a model by which sDscam association in cis and trans generates a vast repertoire of combinatorial homophilic recognition specificities. We propose that in Chelicerata, sDscam combinatorial specificity is sufficient to provide each neuron with a unique identity for self-nonself discrimination. Surprisingly, while sDscams are related to Drosophila Dscam1, our results mirror the findings reported for the structurally unrelated vertebrate cPcdh. Thus, our findings suggest a remarkable example of convergent evolution for the process of neuronal self-avoidance and provide insight into the basic principles and evolution of metazoan self-avoidance and self-nonself discrimination.

A chelicerate-specific burst of nonclassical Dscam diversity.

BACKGROUND: The immunoglobulin (Ig) superfamily receptor Down syndrome cell adhesion molecule (Dscam) gene can generate tens of thousands of isoforms via alternative splicing, which is essential for both nervous and immune systems in insects. However, further information is required to develop a comprehensive view of Dscam diversification across the broad spectrum of Chelicerata clades, a basal branch of arthropods and the second largest group of terrestrial animals. RESULTS: In this study, a genome-wide comprehensive analysis of Dscam genes across Chelicerata species revealed a burst of nonclassical Dscams, categorised into four types-mDscam, sDscamα, sDscamß, and sDscamγ-based on their size and structure. Although the mDscam gene class includes the highest number of Dscam genes, the sDscam genes utilise alternative promoters to expand protein diversity. Furthermore, we indicated that the 5' cassette duplicate is inversely correlated with the sDscam gene duplicate. We showed differential and sDscam- biased expression of nonclassical Dscam isoforms. Thus, the Dscam isoform repertoire across Chelicerata is entirely dominated by the number and expression levels of nonclassical Dscams. Taken together, these data show that Chelicerata evolved a large conserved and lineage-specific repertoire of nonclassical Dscams. CONCLUSIONS: This study showed that arthropods have a large diversified Chelicerata-specific repertoire of nonclassical Dscam isoforms, which are structurally and mechanistically distinct from those of insects. These findings provide a global framework for the evolution of Dscam diversity in arthropods and offer mechanistic insights into the diversification of the clade-specific Ig superfamily repertoire.

Role and convergent evolution of competing RNA secondary structures in mutually exclusive splicing.

Exon or cassette duplication is an important means of expanding protein and functional diversity through mutually exclusive splicing. However, the mechanistic basis of this process in non-arthropod species remains poorly understood. Here, we demonstrate that MRP1 genes underwent tandem exon duplication in Nematoda, Platyhelminthes, Annelida, Mollusca, Arthropoda, Echinodermata, and early-diverging Chordata but not in late-diverging vertebrates. Interestingly, these events were of independent origin in different phyla, suggesting convergent evolution of alternative splicing. Furthermore, we showed that multiple sets of clade-conserved RNA pairings evolved to guide species-specific mutually exclusive splicing in Arthropoda. Importantly, we also identified a similar structural code in MRP exon clusters of the annelid, Capitella teleta, and chordate, Branchiostoma belcheri, suggesting an evolutionarily conserved competing pairing-guided mechanism in bilaterians. Taken together, these data reveal the molecular determinants and RNA pairing-guided evolution of species-specific mutually exclusive splicing spanning more than 600 million years of bilaterian evolution. These findings have a significant impact on our understanding of the evolution of and mechanism underpinning isoform diversity and complex gene structure.

A large family of Dscam genes with tandemly arrayed 5' cassettes in Chelicerata.

Drosophila Dscam1 (Down Syndrome Cell Adhesion Molecules) and vertebrate clustered protocadherins (Pcdhs) are two classic examples of the extraordinary isoform diversity from a single genomic locus. Dscam1 encodes 38,016 distinct isoforms via mutually exclusive splicing in D. melanogaster, while the vertebrate clustered Pcdhs utilize alternative promoters to generate isoform diversity. Here we reveal a shortened Dscam gene family with tandemly arrayed 5' cassettes in Chelicerata. These cassette repeats generally comprise two or four exons, corresponding to variable Immunoglobulin 7 (Ig7) or Ig7-8 domains of Drosophila Dscam1. Furthermore, extraordinary isoform diversity has been generated through a combination of alternating promoter and alternative splicing. These sDscams have a high sequence similarity with Drosophila Dscam1, and share striking organizational resemblance to the 5' variable regions of vertebrate clustered Pcdhs. Hence, our findings have important implications for understanding the functional similarities between Drosophila Dscam1 and vertebrate Pcdhs, and may provide further mechanistic insights into the regulation of isoform diversity.

Long-range RNA pairings contribute to mutually exclusive splicing.

Mutually exclusive splicing is an important means of increasing the protein repertoire, by which the Down's syndrome cell adhesion molecule (Dscam) gene potentially generates 38,016 different isoforms in Drosophila melanogaster. However, the regulatory mechanisms remain obscure due to the complexity of the Dscam exon cluster. Here, we reveal a molecular model for the regulation of the mutually exclusive splicing of the serpent pre-mRNA based on competition between upstream and downstream RNA pairings. Such dual RNA pairings confer fine tuning of the inclusion of alternative exons. Moreover, we demonstrate that the splicing outcome of alternative exons is mediated in relative pairing strength-correlated mode. Combined comparative genomics analysis and experimental evidence revealed similar bidirectional structural architectures in exon clusters 4 and 9 of the Dscam gene. Our findings provide a novel mechanistic framework for the regulation of mutually exclusive splicing and may offer potentially applicable insights into long-range RNA-RNA interactions in gene regulatory networks.