Genome-wide chromatin accessibility reveals transcriptional regulation of heterosis in inter-subspecific hybrid rice.

The utilization of rice heterosis is essential for ensuring global food security; however, its molecular mechanism remains unclear. In this study, comprehensive analyses of accessible chromatin regions (ACRs), DNA methylation, and gene expression in inter-subspecific hybrid and its parents were performed to determine the potential role of chromatin accessibility in rice heterosis. The hybrid exhibited abundant ACRs, in which the gene ACRs and proximal ACRs were directly related to transcriptional activation rather than the distal ACRs. Regarding the dynamic accessibility contribution of the parents, paternal ZHF1015 transmitted a greater number of ACRs to the hybrid. Accessible genotype-specific target genes were enriched with overrepresented transcription factors, indicating a unique regulatory network of genes in the hybrid. Compared with its parents, the differentially accessible chromatin regions with upregulated chromatin accessibility were much greater than those with downregulated chromatin accessibility, reflecting a stronger regulation in the hybrid. Furthermore, DNA methylation levels were negatively correlated with ACR intensity, and genes were strongly affected by CHH methylation in the hybrid. Chromatin accessibility positively regulated the overall expression level of each genotype. ACR-related genes with maternal Z04A-bias allele-specific expression tended to be enriched during carotenoid biosynthesis, whereas paternal ZHF1015-bias genes were more active in carbohydrate metabolism. Our findings provide a new perspective on the mechanism of heterosis based on chromatin accessibility in inter-subspecific hybrid rice.

Platelet-derived growth factor AA-modified electrospun fibers promote tendon healing.

Platelet-derived growth factor AA (PDGF-AA) is an important promoter of tissue injury repair and might be a candidate for improving the mechanical properties of repaired tendons. Here, we designed a PDGF-AA-modified poly(lactide-co-glycolide) acid (PLGA) electrospun fibers to promote tendon rehabilitation after injury. In the present study, we grafted PDGF-AA on the surface of PLGA. In structural experiments, we found that the hydrophilicity of PLGA containing PDGF-AA (PLGA-PDGF-AA) increased, but the strength of the material did not change significantly. Moreover, no significant changes in tendon cell proliferation and viability were observed in the PLGA-PDGF-AA treatment compared with the control group. The mouse tendon injury model (n = 9) experiment illustrated that PLGA-PDGF-AA effectively promoted tendon healing, and we confirmed that PLGA-PDGF-AA promoted collagen synthesis and deposition by immunohistochemistry and RT-PCR. Moreover, the mechanical strength of PLGA-PDGF-AA-treated mouse (n = 9) tendon tissue was also higher than that of the PLGA-treated group alone. In conclusion, PLGA-PDGF-AA promoted regeneration after tendon injury and serves as a potential adjuvant material for surgical tendon injury repair.

Design and development of novel fluorescence sensing material for exosome recognition.

Exosomes are extracellular vesicles with relatively specific expression of CD63 transmembrane protein. In this study, We designed and constructed a multisite-targeting polymer which has both fluorescence and targeting recognition. It can bond to the hydrophilic group of CD63 by connecting with hydrogen. The chemical structure and the ability to combine with CD63 of fluorescent monomer and polymer were characterized and confirmed by FTIR and 1H NMR. MTT assay was performed to detect the cytotoxicity and biocompatibility of this polymer. Then we found the cell viability was 80.64% and the hemolysis rate of erythrocyte was only 0.101% even at F concentration of 20 µM. In vitro, the proposed polymer showed better ability to enter cells after linking exosomes via CD63; in vivo, it showed the ability to bind stably to exosomes and target tumor implants.

Radiofrequency Ablation (RFA) Combined with Transcatheter Arterial Chemoembolization (TACE) for Patients with Medium-to-Large Hepatocellular Carcinoma: A Retrospective Analysis of Long-Term Outcome.

BACKGROUND The aim of this study was to investigate the prognostic value of radiofrequency ablation (RFA) plus transcatheter arterial chemoembolization (TACE) in hepatocellular carcinoma (HCC) patients with tumor size ranging from 3.0 to 10.0 cm. MATERIAL AND METHODS We retrospectively analyzed data on 201 patients with medium-to-large HCC. According to treatment procedure, the patients were divided into the TACE group (n=124) and the TACE+RFA group (n=77). We recorded data on patient safety, subcapsular hepatic hematoma, large amount of ascites, liver abscess, gallbladder injury, and local skin infection. The overall survival (OS) and progression-free survival (PFS) in the 2 groups were analyzed and compared between groups. RESULTS The median PFS was 4.00 months (3.00-5.00 months) in the TACE group and 9.13 months (6.64-11.62 months) in the TACE+RFA group (P<0.001). Median OS was 12.00 months (8.88-15.13 months) in the TACE group and 27.57 months (20.06-35.08 months) in the TACE+RFA group (P<0.001). In the TACE+RFA group, multivariate Cox regression analysis showed that tumor size ≤5 cm) (HR: 1.952, 95% CI: 1.213-3.143, P=0.006), hepatitis B (HR: 2.323, 95% CI: 1.096-4.923, P=0.028), TACE times (1 or >1) (HR: 1.867, 95% CI: 1.156-3.013, P=0.011), alpha-fetoprotein (AFP) level >200 ng/ml (HR: 2.426, 95% CI: 1.533-3.839, P<0.001), and AST level >40 U/L (HR: 1.946, 95% CI: 1.196-3.166, P=0.007) were independent prognostic factors for overall survival. CONCLUSIONS Combination therapy of TACE with RFA is a safe and effective treatment for patients with medium-to-large HCC, with the long-term beneficial effect of retarding tumor progression and improving PFS and OS.

Recombinant Viral Capsid Protein L2 (rVL2) of HPV 16 Suppresses Cell Proliferation and Glucose Metabolism via ITGB7/C/EBPß Signaling Pathway in Cervical Cancer Cell Lines.

PURPOSE: Capsid protein L2 is the minor capsid protein of human papillomavirus 16 (HPV16). Although L2-based vaccines were developed, the therapeutic effect of recombinant viral capsid protein L2 (rVL2) was still to be illustrated. METHODS: We used glucose uptake and lactate production assay to verify the inhibitory effect of rVL2 on the glucose metabolism in cervical cancer cells. Secondly, we performed gene-chip assay, RT-PCR, and Western blot to determine the role of ITGB7/C/EBPß signaling pathway in rVL2-mediated glucose metabolism in vitro. Finally, we used an animal model to verify the function of rVL2 in cervical cancer. RESULTS: We found that rVL2 reduced glucose uptake and lactate production levels in cervical cancer cells, which caused the inhibition of cell proliferation. rVL2 decreased the expression levels of key metabolic enzymes, including GLUT1, LDHA, and ALDOA, to affect cell metabolism in cervical cancer cells by inhibiting ITGB7/C/EBPß signaling pathway in vitro and in vivo. CONCLUSION: These results demonstrated the vital role of rVL2 in the glycolysis-induced cell growth and proliferation via suppressing ITGB7/C/EBPß signaling axis.

Development and validation of a functional co-dominant SNP marker for the photoperiod thermo-sensitive genic male sterility pms3 (p/tms12-1) gene in rice.

The fertility conversion of photoperiod thermo-sensitive genic male sterility (P/TGMS) lines in rice is mostly regulated by the P/TGMS genes in different environmental conditions. A point mutation with G-C on the pms3 (p/tms12-1) gene regulates the pollen fertility of Nongken58S and a large amount of Nongken58S-derived lines. In this study, we developed and designed a functional co-dominant marker according to the SNP loci for the pms3 (p/tms12-1) gene. We can differentiate the SNP loci in pms3 (p/tms12-1) gene from another TGMS lines and inbred cultivars using the dpms3-54 marker. The results showed that 376 bp band was detected in the homozygous genotype for pms3 (p/tms12-1), while 359 bp band was detected in the homozygous genotype for non-pms3 (p/tms12-1), two bands with 376 and 359 bp were detected in the heterozygous genotype. The dpms3-54 marker can be used to test the purity of two-line hybrid rice seeds and to divide each of F1 plant into homozygous and heterozygous genotypes at the seedling stage. Thus, this study provide a useful functional marker to detect pms3 (p/tms12-1) gene in different genetic resources and populations, which can be applied to the development and breeding of P/TGMS lines in two-line hybrid rice system with marker-assisted selection.

Quantitative trait locus analysis and fine mapping of the qPL6 locus for panicle length in rice.

KEY MESSAGE: Two QTLs were identified to control panicle length in rice backcross lines, and one QTL qPL6 was finely mapped with potential in high yield breeding. Panicle length (PL) is the key determinant of panicle architecture in rice, and strongly affects yield components, such as grain number per panicle. However, this trait has not been well studied genetically nor its contribution to yield improvement. In this study, we performed quantitative trait locus (QTL) analysis for PL in four backcross populations derived from the cross of Nipponbare (japonica) and WS3 (indica), a new plant type (NPT) variety. Two QTLs were identified on chromosome 6 and 8, designated as qPL6 and qPL8, respectively. Near-isogenic lines (NILs) were developed to evaluate their contribution to important agronomic traits. We found that qPL6 and qPL8 had additive effects on PL trait. For the qPL6 locus, the WS3 allele also increased panicle primary and secondary branches and grain number per panicle. Moreover, this allele conferred wide and strong culms, a character of lodging resistance. By analyzing key recombinants in two steps, the qPL6 locus was finely mapped to a 25-kb interval, and 3 candidate genes were identified. According to the single nucleotide polymorphisms (SNPs) within candidate genes, 5 dCaps markers were designed and used to get haplotypes of 96 modern Chinese varieties, which proved that qPL6 locus is differentiated between indica and temperate japonica varieties. Taken together, the superior qPL6 allele can be applied in rice breeding programs for large sink size, particularly for japonica varieties that originally lack the allele.

Application of 128-slice spiral CT combination scanning in the diagnosis of embolisms in pulmonary arteries and lower extremity veins.

The aim of this study was to evaluate the significance of using multi-row spiral computed tomography (CT) to scan for pulmonary artery thrombosis and lower limb deep vein thrombosis (LVT) in patients with suspected LVT. A total of 110 patients underwent a contrast-enhanced spiral CT inspection of the pulmonary artery and lower extremity veins. Three-dimensional digital image processing, including multi-planar reconstruction (MPR), maximum intensity projection (MIP) and volume rendering (VR), was also conducted; two groups of experienced radiologists analyzed the CT images to evaluate the postprocessing techniques of these CT images. Seventy-five patients were diagnosed with LVT with or without pulmonary embolism (PE); out of these 75, 34 patients were diagnosed with PE and LVT together and 41 patients were diagnosed with LVT alone. A further 31 patients were diagnosed with iliac vein compression syndrome (IVCS), and no embolisms were detected in the remaining four patients. With regard to PE, MPR and MIP demonstrated an accuracy of 100%, while MPR also showed images of LVT with an accuracy of 100%. The follow-up results at 12 months were consistent with the CT scan results. The clinical use of 128-slice spiral CT combination scanning in the detection of PE and LVT has significant potential to improve upon the present methods of diagnosis.

Two Arabidopsis cytochrome P450 monooxygenases, CYP714A1 and CYP714A2, function redundantly in plant development through gibberellin deactivation.

The rice gene ELONGATED UPPERMOST INTERNODE1 (EUI1) encodes a P450 monooxygenase that epoxidizes gibberellins (GAs) in a deactivation reaction. The Arabidopsis genome contains a tandemly duplicated gene pair ELA1 (CYP714A1) and ELA2 (CYP714A2) that encode EUI homologs. In this work, we dissected the functions of the two proteins. ELA1 and ELA2 exhibited overlapping yet distinct gene expression patterns. We showed that while single mutants of ELA1 or ELA2 exhibited no obvious morphological phenotype, simultaneous elimination of ELA1 and ELA2 expression in ELA1-RNAi/ela2 resulted in increased biomass and enlarged organs. By contrast, transgenic plants constitutively expressing either ELA1 or ELA2 were dwarfed, similar to those overexpressing the rice EUI gene. We also discovered that overexpression of ELA1 resulted in a severe dwarf phenotype, while overexpression of ELA2 gave rise to a breeding-favored semi-dwarf phenotype in rice. Consistent with the phenotypes, we found that the ELA1-RNAi/ela2 plants increased amounts of biologically active GAs that were decreased in the internodes of transgenic rice with ELA1 and ELA2 overexpression. In contrast, the precursor GA(12) slightly accumulated in the transgenic rice, and GA(19) highly accumulated in the ELA2 overexpression rice. Taken together, our study strongly suggests that the two Arabidopsis EUI homologs subtly regulate plant growth most likely through catalyzing deactivation of bioactive GAs similar to rice EUI. The two P450s may also function in early stages of the GA biosynthetic pathway. Our results also suggest that ELA2 could be an excellent tool for molecular breeding for high yield potential in cereal crops.

Control of rice grain-filling and yield by a gene with a potential signature of domestication.

Grain-filling, an important trait that contributes greatly to grain weight, is regulated by quantitative trait loci and is associated with crop domestication syndrome. However, the genes and underlying molecular mechanisms controlling crop grain-filling remain elusive. Here we report the isolation and functional analysis of the rice GIF1 (GRAIN INCOMPLETE FILLING 1) gene that encodes a cell-wall invertase required for carbon partitioning during early grain-filling. The cultivated GIF1 gene shows a restricted expression pattern during grain-filling compared to the wild rice allele, probably a result of accumulated mutations in the gene's regulatory sequence through domestication. Fine mapping with introgression lines revealed that the wild rice GIF1 is responsible for grain weight reduction. Ectopic expression of the cultivated GIF1 gene with the 35S or rice Waxy promoter resulted in smaller grains, whereas overexpression of GIF1 driven by its native promoter increased grain production. These findings, together with the domestication signature that we identified by comparing nucleotide diversity of the GIF1 loci between cultivated and wild rice, strongly suggest that GIF1 is a potential domestication gene and that such a domestication-selected gene can be used for further crop improvement.

Gibberellin homeostasis and plant height control by EUI and a role for gibberellin in root gravity responses in rice.

The rice Eui (ELONGATED UPPERMOST INTERNODE) gene encodes a cytochrome P450 monooxygenase that deactivates bioactive gibberellins (GAs). In this study, we investigated controlled expression of the Eui gene and its role in plant development. We found that Eui was differentially induced by exogenous GAs and that the Eui promoter had the highest activity in the vascular bundles. The eui mutant was defective in starch granule development in root caps and Eui overexpression enhanced starch granule generation and gravity responses, revealing a role for GA in root starch granule development and gravity responses. Experiments using embryoless half-seeds revealed that RAmy1A and GAmyb were highly upregulated in eui aleurone cells in the absence of exogenous GA. In addition, the GA biosynthesis genes GA3ox1 and GA20ox2 were downregulated and GA2ox1 was upregulated in eui seedlings. These results indicate that EUI is involved in GA homeostasis, not only in the internodes at the heading stage, but also in the seedling stage, roots and seeds. Disturbing GA homeostasis affected the expression of the GA signaling genes GID1 (GIBBERELLIN INSENSITIVE DWARF 1), GID2 and SLR1. Transgenic RNA interference of the Eui gene effectively increased plant height and improved heading performance. By contrast, the ectopic expression of Eui under the promoters of the rice GA biosynthesis genes GA3ox2 and GA20ox2 significantly reduced plant height. These results demonstrate that a slight increase in Eui expression could dramatically change rice morphology, indicating the practical application of the Eui gene in rice molecular breeding for a high yield potential.

OsRAR1 and OsSGT1 physically interact and function in rice basal disease resistance.

The RAR1 and SGT1 proteins function synergistically or antagonistically in plant innate immune responses. Here, we show that the rice orthologs OsRAR1 and OsSGT1 physically interact in vivo and in yeast. They displayed conserved roles in Arabidopsis disease resistance through ectopic expression in the Arabidopsis rar1 and sgt1 mutants. Overexpression of OsRar1 and OsSGT1 in rice significantly increased basal resistance to a virulent bacterial blight Xanthomonas oryzae pv. oryzae PXO99 but not to another virulent strain DY89031, suggesting race-specific-like basal resistance conferred by OsRar1 and OsSGT1. OsRar1-OE and OsSGT1-OE plants also enhanced resistance to all four virulent blast fungal Magnaporthe oryzae races. Overexpression of the OsSGT1-green fluorescent protein (GFP) fusion most likely caused a dominant negative phenotype which led to race-specific-like basal resistance. Transgenic plants overexpressing OsSGT1-GFP show enhanced resistance to DY89031 but decreased resistance to PXO99, implying that OsSGT1 might be the target of a component required for DY89031 virulence or OsSGT1-GFP might stabilize weak resistance proteins against DY89031. Consistent with the hypothesis of the dominant negative regulation, we observed the reduced sensitivity to auxin of OsSGT1-GFP plants compared with the wild-type ones, and the curling-root phenotype in OsSGT1-OE plants. These results collectively suggest that OsRar1 and OsSGT1 might be differentially required for rice basal disease resistance. Our current study also provides new insight into the roles of OsSGT1 in basal disease resistance.

Functional analysis of rice NPR1-like genes reveals that OsNPR1/NH1 is the rice orthologue conferring disease resistance with enhanced herbivore susceptibility.

The key regulator of salicylic acid (SA)-mediated resistance, NPR1, is functionally conserved in diverse plant species, including rice (Oryza sativa L.). Investigation in depth is needed to provide an understanding of NPR1-mediated resistance and a practical strategy for the improvement of disease resistance in the model crop rice. The rice genome contains five NPR1-like genes. In our study, three rice homologous genes, OsNPR1/NH1, OsNPR2/NH2 and OsNPR3, were found to be induced by rice bacterial blight Xanthomonas oryzae pv. oryzae and rice blast Magnaporthe grisea, and the defence molecules benzothiadiazole, methyl jasmonate and ethylene. We confirmed that OsNPR1 is the rice orthologue by complementing the Arabidopsis npr1 mutant. Over-expression of OsNPR1 conferred disease resistance to bacterial blight, but also enhanced herbivore susceptibility in transgenic plants. The OsNPR1-green fluorescent protein (GFP) fusion protein was localized in the cytoplasm and moved into the nucleus after redox change. Mutations in its conserved cysteine residues led to the constitutive localization of OsNPR1(2CA)-GFP in the nucleus and also abolished herbivore hypersensitivity in transgenic rice. Different subcellular localizations of OsNPR1 antagonistically regulated SA- and jasmonic acid (JA)-responsive genes, but not SA and JA levels, indicating that OsNPR1 might mediate antagonistic cross-talk between the SA- and JA-dependent pathways in rice. This study demonstrates that rice has evolved an SA-mediated systemic acquired resistance similar to that in Arabidopsis, and also provides a practical approach for the improvement of disease resistance without the penalty of decreased herbivore resistance in rice.

[Physiological and genetic analysis of lesion resembling disease mutants (lrd) of Oryza sativa L].

Ten indica rice and eight japonica rice mutants with lesion resembling disease (lrd27-44) were obtained by gamma-ray mutagenesis of the whole genomes. These mutants exhibited diverse lesion mimic phenotypes under different growth environments, could be accordingly classified two types, sensitive and insensitive to environments. Basing on difference in development of lesion mimics, they can be divided into three categories: whole life lesion mimics (WLLM), vegetative initiation lesion mimics (VILM), and reproductive initiation lesion mimics (RILM). Lesion mimics resulted from the programmed cell death and were triggered by light, but not by wounding. The genetic analysis showed that four mutants, lrd32, lrd39, lrd40 and lrd42, were controlled by one or two recessive loci. Among the 18 lrd mutants, lrd37 and lrd40 conferred non-race-specific resistance to Xanthomonas oryzae pv. oryzae. Gene mapping and cloning of Lrd32 and Lrd40 are under way.