The structure and assembly of rhizobacterial communities are influenced by poplar genotype.

The interaction between plants and microbes dominates plant growth and fitness in specific environments. The study of the relationship between plant genotypes and rhizobacterial community structure would provide a deep insight into the recruitment strategies of plants toward soil bacteria. In this study, three genotypes of 18-year-old mature poplar (H1, H2, and H3) derived from four different parents were selected from a germplasm nursery of Populus deltoides. Rhizosphere soil carbon, nitrogen, and phosphorus properties as well as the 16S rDNA sequences of rhizobacterial communities were analyzed to determine the relationship between poplar genotypes and rhizobacterial communities assembly. The results showed there were significant differences in the diversity (Chao1, ACE index, and Shannon index) of rhizobacterial communities between H1 and H2, as well as between H2 and H3, but no difference between H1 and H3. Principal component analysis also revealed a similar structure of rhizobacterial communities between H1 and H3, whereas the rhizobacterial communities of H2 demonstrated significant differences from H1 and H3. Linear discriminant effect size analysis indicated that there were 11 and 14 different biomarkers in the H1 and H3 genotype, respectively, but 42 in the H2 genotype. Co-occurrence network analysis indicated that the rhizobacterial communities of H2 had a distinct network structure compared to those of the other two genotypes, whereas H1 and H3 had a similar pattern of co-occurrence network. Threshold indicator taxa analysis revealed that 63 genera responded significantly to NO3 --N content and 58 genera to NH4 +-N/NO3 --N ratio. Moreover, the stochastic assembly process was found to be decreased with increasing NO3 --N content and fluctuated with increasing NH4 +-N/NO3 --N ratio. All results indicated that the structure of poplar rhizobacterial communities were influenced by host genotypes, and available nitrogen might play a dominant role in the assembly of rhizobacterial communities. This study would promote the future selection and utilization of rhizobacteria in poplar breeding.

Predicting the effects of radiotherapy based on diffusion kurtosis imaging in a xenograft mouse model of esophageal carcinoma.

The aim of the present study was to assess the predictive value of diffusion kurtosis imaging (DKI) on the effects of radiotherapy in a xenograft model of esophageal cancer. A total of 40 tumor-bearing mice, established by injection of Eca-109 cells in nude mice, were used. The experimental group (n=24) received a single dose of 15 Gy (6 MV by X-ray), and the control group (n=16) did not receive any treatment. Tumor volume, apparent diffusion coefficient (ADC), mean kurtosis (MK) and mean diffusivity (MD) of the two groups were compared, and the expression of aquaporin (AQP) 3 and necrosis ratio at matched time points in xenografts were also observed. There was a significant difference between the two groups from the 7th day of radiotherapy onwards; the xenograft volume of the experimental group was significantly smaller compared with the control group (P<0.05). On the 3rd day, the ADC and MD of the experimental group was significantly higher compared with the control group, and MK was significantly lower compared with the control group (P<0.05). On the 3rd day, AQP3 expression in the experimental group was lower compared with the control group, and the proportion of necrotic cells was higher compared with the control group (P<0.05). Single large fraction dose radiotherapy inhibited the growth of a xenografted esophageal tumor. Changes in ADC, MK and MD were observed prior to morphological changes in the tumor. The change in AQP3 expression and necrosis ratio was in also agreement with the DKI parameters assessed. DKI may thus provide early predictive ability on the effect of radiotherapy in esophageal carcinoma.

Survival Comparision of Three-dimensional Radiotherapy Alone vs. Chemoradiotherapy for Esophageal Squamous Cell Carcinoma.

BACKGROUND AND OBJECTIVE: To compare the survival of esophageal squamous cell carcinoma (ESCC) patients who received chemoradiotherapy (CRT) or radiotherapy (RT) alone. METHODS: A total of 753 well-matched patients were enrolled. A total of 299 patients were treated with CRT, and 454 patients were treated with RT alone. Propensity score matching (PSM) was performed with the R project. The Kaplan-Meier method was used to calculate survival rates, and the log-rank test was used to assess differences in survival. RESULTS: The response rate was 99.0% with CRT and 98.3% with RT alone (p = 0.651). The 1-, 3-, 5- and 10 year overall survival (OS) rates were as follows: 72.2, 40.1, 30.7 and 13.9% with CRT, 68.1, 35.2%, 23.3 and 12.5% with RT alone (p = 0.033); 73.4, 40.1, 31.0 and 16.1% with concurrent chemoradiotherapy (CCRT); and 68.1, 35.2, 23.3 and 12.5% with RT alone (p = 0.028). There was no significant difference in OS between the CCRT group and the sequential chemoradiotherapy (SCRT) group (p = 0.527). Consolidation chemotherapy (CCT) after CCRT led to a significant increase in the OS rate compared with no CCT after CCRT (p = 0.003). Compared with the OS of patients who received 1∼2 cycles of CCT, the OS of patients who received 3∼4 cycles of CCT was significantly improved (p = 0.011). Acute toxic effects were more severe in the CRT, but no significant differences in late reactions. CRT exhibited more appetite loss and fatigue symptoms than RT alone, and dysphagia of CRT relief more obviously. The CRT group had a significantly lower rate of local control failure than the RT alone group (p = 0.019). CONCLUSIONS: For patients with ESCC, CRT led to a significantly improved OS compared to RT alone, and this trend was more obvious with CCRT. CCT after CCRT prolonged OS, especially in patients who received at least 2 cycles of CCT. CRT can reduce the deaths due to local control failure compared to RT alone.

TAp63-Regulated miRNAs Suppress Cutaneous Squamous Cell Carcinoma through Inhibition of a Network of Cell-Cycle Genes.

TAp63 is a p53 family member and potent tumor and metastasis suppressor. Here, we show that TAp63-/- mice exhibit an increased susceptibility to ultraviolet radiation-induced cutaneous squamous cell carcinoma (cuSCC). A human-to-mouse comparison of cuSCC tumors identified miR-30c-2* and miR-497 as underexpressed in TAp63-deficient cuSCC. Reintroduction of these miRNAs significantly inhibited the growth of cuSCC cell lines and tumors. Proteomic profiling of cells expressing either miRNA showed downregulation of cell-cycle progression and mitosis-associated proteins. A mouse to human and cross-platform comparison of RNA-sequencing and proteomics data identified a 7-gene signature, including AURKA, KIF18B, PKMYT1, and ORC1, which were overexpressed in cuSCC. Knockdown of these factors in cuSCC cell lines suppressed tumor cell proliferation and induced apoptosis. In addition, selective inhibition of AURKA suppressed cuSCC cell proliferation, induced apoptosis, and showed antitumor effects in vivo. Finally, treatment with miR-30c-2* or miR-497 miRNA mimics was highly effective in suppressing cuSCC growth in vivo. Our data establish TAp63 as an essential regulator of novel miRNAs that can be therapeutically targeted for potent suppression of cuSCC. SIGNIFICANCE: This study provides preclinical evidence for the use of miR-30c-2*/miR-497 delivery and AURKA inhibition in the treatment of cuSCC, which currently has no FDA-approved targeted therapies.See related commentary by Parrales and Iwakuma, p. 2439.

Genome-wide analysis of day/night DNA methylation differences in Populus nigra.

DNA methylation is an important mechanism of epigenetic modification. Methylation changes during stress responses and developmental processes have been well studied; however, their role in plant adaptation to the day/night cycle is poorly understood. In this study, we detected global methylation patterns in leaves of the black poplar Populus nigra 'N46' at 8:00 and 24:00 by methylated DNA immunoprecipitation sequencing (MeDIP-seq). We found 10,027 and 10,242 genes to be methylated in the 8:00 and 24:00 samples, respectively. The methylated genes appeared to be involved in multiple biological processes, molecular functions, and cellular components, suggesting important roles for DNA methylation in poplar cells. Comparing the 8:00 and 24:00 samples, only 440 differentially methylated regions (DMRs) overlapped with genic regions, including 193 hyper- and 247 hypo-methylated DMRs, and may influence the expression of 137 downstream genes. Most hyper-methylated genes were associated with transferase activity, kinase activity, and phosphotransferase activity, whereas most hypo-methylated genes were associated with protein binding, ATP binding, and adenyl ribonucleotide binding, suggesting that different biological processes were activated during the day and night. Our results indicated that methylated genes were prevalent in the poplar genome, but that only a few of these participated in diurnal gene expression regulation.

Involvement of microRNA-mediated gene expression regulation in the pathological development of stem canker disease in Populus trichocarpa.

MicroRNAs (miRNAs), a type of short (21-23 nucleotides), non-coding RNA molecule, mediate repressive gene regulation through RNA silencing at the post-transcriptional level, and play an important role in defense and response to abiotic and biotic stresses. In the present study, Affymetrix® miRNA Array, real-time quantitative PCR (qPCR) for miRNAs and their targets, and miRNA promoter analysis were used to validate the gene expression patterns of miRNAs in Populus trichocarpa plantlets induced with the poplar stem canker pathogen, Botryosphaeria dothidea. Twelve miRNAs (miR156, miR159, miR160, miR164, miR166, miR168, miR172, miR319, miR398, miR408, miR1448, and miR1450) were upregulated in the stem bark of P. trichocarpa, but no downregulated miRNAs were found. Based on analysis of the miRNAs and their targets, a potential co-regulatory network was developed to describe post-transcriptional regulation in the pathological development of poplar stem canker. There was highly complex cross-talk between diverse miRNA pathway responses to biotic and abiotic stresses. The results suggest that miR156 is probably an integral component of the miRNA response to all environmental stresses in plants. Cis-regulatory elements were binding sites for the transcription factors (TFs) on DNA. Promoter analysis revealed that TC-rich repeats and a W1-box motif were both tightly related disease response motifs in Populus. Promoter analysis and target analysis of miRNAs also revealed that some TFs regulate their activation/repression. Furthermore, a feedback regulatory network in the pathological development of poplar stem canker is provided. The results confirm that miRNA pathways regulate gene expression during the pathological development of plant disease, and provide new insights into understanding the onset and development of poplar stem canker.

Sequencing the genome of Marssonina brunnea reveals fungus-poplar co-evolution.

BACKGROUND: The fungus Marssonina brunnea is a causal pathogen of Marssonina leaf spot that devastates poplar plantations by defoliating susceptible trees before normal fall leaf drop. RESULTS: We sequence the genome of M. brunnea with a size of 52 Mb assembled into 89 scaffolds, representing the first sequenced Dermateaceae genome. By inoculating this fungus onto a poplar hybrid clone, we investigate how M. brunnea interacts and co-evolves with its host to colonize poplar leaves. While a handful of virulence genes in M. brunnea, mostly from the LysM family, are detected to up-regulate during infection, the poplar down-regulates its resistance genes, such as nucleotide binding site domains and leucine rich repeats, in response to infection. From 10,027 predicted proteins of M. brunnea in a comparison with those from poplar, we identify four poplar transferases that stimulate the host to resist M. brunnea. These transferas-encoding genes may have driven the co-evolution of M. brunnea and Populus during the process of infection and anti-infection. CONCLUSIONS: Our results from the draft sequence of the M. brunnea genome provide evidence for genome-genome interactions that play an important role in poplar-pathogen co-evolution. This knowledge could help to design effective strategies for controlling Marssonina leaf spot in poplar.

Enhanced tolerance to NaCl and LiCl stresses by over-expressing Caragana korshinskii sodium/proton exchanger 1 (CkNHX1) and the hydrophilic C terminus is required for the activity of CkNHX1 in Atsos3-1 mutant and yeast.

Sodium/proton exchangers (NHX antiporters) play important roles in plant responses to salt stress. Previous research showed that hydrophilic C-terminal region of Arabidopsis AtNHX1 negatively regulates the Na(+)/H(+) transporting activity. In this study, CkNHX1 were isolated from Caragana korshinskii, a pea shrub with high tolerance to salt, drought, and cold stresses. Transcripts of CkNHX1 were detected predominantly in roots, and were significantly induced by NaCl stress in stems. Transgenic yeast and Arabidopsisthalianasos3-1 (Atsos3-1) mutant over-expressing CkNHX1 and its hydrophilic C terminus-truncated derivative, CkNHX1-ΔC, were generated and subjected to NaCl and LiCl stresses. Expression of CkNHX1 significantly enhanced the resistance to NaCl and LiCl stresses in yeast and Atsos3-1 mutant. Whereas, compared with expression of CkNHX1, the expression of CkNHX1-ΔC had much less effect on NaCl tolerance in Atsos3-1 and LiCl tolerance in yeast and Atsos3-1. All together, these results suggest that the predominant expression of CkNHX1 in roots might contribute to keep C. korshinskii adapting to the high salt condition in this plant's living environment; CkNHX1 could recover the phenotype of Atsos3-1 mutant; and the hydrophilic C-terminal region of CkNHX1 should be required for Na(+)/H(+) and Li(+)/H(+) exchanging activity of CkNHX1.

Dynamic analysis of Ca²+ level during bovine oocytes maturation and early embryonic development.

Mammalian oocyte maturation and early embryo development processes are Ca(2+)-dependent. In this study, we used confocal microscopy to investigate the distribution pattern of Ca(2+) and its dynamic changes in the processes of bovine oocytes maturation, in vitro fertilization (IVF), parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) embryo development. During the germinal vesicle (GV) and GV breakdown stage, Ca(2+) was distributed in the cortical ooplasm and throughout the oocytes from the MI to MII stage. In IVF embryos, Ca(2+) was distributed in the cortical ooplasm before the formation of the pronucleus. In 4-8 cell embryos and morulas, Ca(2+) was present throughout the blastomere. In PA embryos, Ca(2+) was distributed throughout the blastomere at 48 h, similar to in the 4-cell and 8-cell phase and the morula. At 6 h after activation, there was almost no distribution of Ca(2+) in the SCNT embryos. However, Ca(2+) was distributed in the donor nucleus at 10 h and it was distributed throughout the blastomere in the 2-8 cell embryos. In this study, Ca(2+) showed significant fluctuations with regularity of IVF and SCNT groups, but PA did not. Systematic investigation of the Ca(2+) location and distribution changes during oocyte maturation and early embryo development processes should facilitate a better understanding of the mechanisms involved in oocyte maturation, reconstructed embryo activation and development, ultimately improving the reconstructed embryo development rate.

Patterns of molecular evolution and predicted function in thaumatin-like proteins of Populus trichocarpa.

Some pathogenesis-related proteins (PR proteins) are subject to positive selection, while others are under negative selection. Here, we report the patterns of molecular evolution in thaumatin-like protein (TLP, PR5 protein) genes of Populus trichocarpa. Signs of positive selection were found in 20 out of 55 Populus TLPs using the likelihood ratio test and ML-based Bayesian methods. Due to the connection between the acidic cleft and the antifungal activity, the secondary structure and three-dimensional structure analyses predicted antifungal activity beta-1,3-glucanase activities in these TLPs. Moreover, the coincidence with variable basic sites in the acidic cleft and positively selected sites suggested that fungal diseases may have been the main environmental stress that drove rapid adaptive evolution in Populus.

Imbalance of matrix metalloproteinases/tissue inhibitor of metalloproteinase-1 and loss of fibronectin expression in patients with congestive heart failure.

OBJECTIVE: Previous studies have demonstrated that an imbalance of matrix metalloproteinases (MMPs)/tissue inhibitors of metalloproteinase-1 (TIMP-1) and a loss of fibronectin are associated with postmyocardial infarction remodeling in rats. The present study was designed to examine this issue in patients with congestive heart failure (CHF). METHODS: We measured plasma levels and the cardiac protein expression of MMPs/TIMP-1 and fibronectin in 39 patients with CHF and 38 controls. RESULTS: Plasma levels of MMP-2, MMP-3, and MMP-9 tended to be higher in patients with CHF (NYHA II: 276 +/- 18, 613 +/- 118, and 245 +/- 43 microg/l, respectively; NYHA III: 302 +/- 20, 850 +/- 132, and 310 +/- 39 microg/l, respectively; NYHA IV: 367 +/- 15, 998 +/- 99, and 392 +/- 27 microg/l, respectively) than in controls (213 +/- 23, 485 +/- 102, and 158 +/- 31 microg/l, respectively), while the plasma TIMP-1 level tended to be lower in patients with CHF (NYHA II: 126 +/- 12 microg/l, NYHA III: 83 +/- 11 microg/l, and NYHA IV: 61 +/- 12 microg/l) than in controls (208 +/- 15 microg/l). Interestingly, the changes in protein expression of MMPs/TIMP-1 were consistent with their plasma concentration. Furthermore, the fibronectin level in the patients with CHF was significantly lower than in the controls. CONCLUSIONS: These data suggest that human CHF is associated with an imbalance of MMPs/TIMP-1 and a concurrent loss of fibronectin.

Establishment and biological characteristics of Ujumqin sheep fibroblast line.

A Ujumqin sheep ear marginal tissue (USEM) fibroblast line, frozen in 147 cryovials with 4 x 10(6) cell each, was successfully established from 33 Ujumqin sheep ear marginal tissues using explant culture and cryopreservation techniques. The cells were morphologically consistent with fibroblasts. The growth curve was typical S-shape and the cell population passed through a lag phase, a logarithmic phase and a plateau phase. The population doubling time (PDT) was approximately 72 h. Tests for bacteria, fungi, viruses and mycoplasma were all negative. Isoenzyme polymorphism indicated that the genetic characteristics of the cell line were stable in vitro. Karyotyping analysis indicated that the chromosome number of a normal cell was of 2n = 54 and 95.4% of the entire population was diploid. The transfection efficiencies of six fluorescent proteins (pEGFP-N3, pEGFP-C1, pDsRed-N1, pEYFP-N1, pECFP-N1 and pECFP-mito) optimal at 48 h were from 18.5% to 30.1%. The cell line met all criteria from the American Type Culture Collection (ATCC). Not only has the germline of this important sheep breed been preserved at the cell level, but also valuable material had been provided for genome, postgenome and somatic cloning research. Moreover, the establishment of this technical platform may provide both technical and theoretical support for storing the genetic resources of other animals and poultry at the cell level.

Mitotic deregulation by survivin in ErbB2-overexpressing breast cancer cells contributes to Taxol resistance.

PURPOSE: Taxol resistance remains a major obstacle to improve the benefit of breast cancer patients. Here, we studied whether overexpression of ErbB2 may lead to mitotic deregulation in breast cancer cells via up-regulation of survivin that confers Taxol resistance. EXPERIMENTAL DESIGN: ErbB2-overexpressing and ErbB2-low-expressing breast cancer cell lines were used to compare their mitotic exit rate, survivin expression level, and apoptosis level in response to Taxol. Survivin was then down-regulated by antisense oligonucleotides to evaluate its contribution to mitotic exit and Taxol resistance in ErbB2-overexpressing breast cancer cells. At last, specific PI3K/Akt and Src inhibitors were used to investigate the involvement of these two pathways in ErbB2-mediated survivin up-regulation and Taxol resistance. RESULTS: We found that ErbB2-overexpressing cells expressed higher levels of survivin in multiple breast cancer cell lines and patient samples. ErbB2-overexpressing cells exited M phase faster than ErbB2-low-expressing cells, which correlated with the increased resistance to Taxol-induced apoptosis. Down-regulation of survivin by antisense oligonucleotide delayed mitotic exit of ErbB2-overexpressing cells and also sensitized ErbB2-overexpressing cells to Taxol-induced apoptosis. Moreover, ErbB2 up-regulated survivin at translational level and PI3K/Akt and Src activation are involved. In addition, combination treatment of Taxol with PI3K/Akt and Src inhibitor led to increased apoptosis in ErbB2-overexpressing breast cancer cells than single treatment. CONCLUSIONS: Survivin up-regulation by ErbB2 is a critical event in ErbB2-mediated faster mitotic exit and contributes to Taxol resistance.

[Research progress of single nucleotide polymorphisms in forest trees.].

Single nucleotide polymorphisms (SNPs) are the most abundant form of genetic variation in many organisms. Forest is an important plant group and key component of the terrestrial plant ecosystem. As a new marker system, SNP has been applied in genetic and breeding studies in some tree species of genera Pinus, Populus, Pseudotsuga, Eucalyptus, and Picea, and the related genetic information, such as nucleotide diversity, linkage disequilibrium (LD) and population structure has been generated. These studies are mainly based on sequencing and analysis of candidate genes. SNP-based association genetics analysis or LD mapping has become a useful tool for dissection of complex traits in forest trees. Association studies in Eucalyptus spp. and Pinus taeda L. found that some SNP sites in various genes were associated with distinct wood property traits. Meanwhile, estimation of genetic parameters has revealed the evolutionary and ecological significance at different extents in several tree species. Intensive applications of SNP approach are expected to greatly accelerate the development of research on forest genetics and breeding.

[Progress in research on forest tree genomics].

This is a review on forest tree genomics. In structural genomics, genetic maps have been constructed for up to 40 forest tree species, more than 30 commercially important QTLs have been detected, comparative mapping has been done for a few of forest tree taxa, and whole genome sequencing was completed for Populus and is under way for Eucalyptus. For functional genomics, huge EST databases from multiple tissues of a number of tree species have been rapidly accumulated, and molecular analyses on secondary growth and wood formation, flowering, and cold hardiness have given some insights into the metabolic pathways of those tree-specific development processes. The prospects of development in tree genomics are discussed, which may be implicative for accelerating forest tree genomics studies in China.

[The role of angiotension II receptor and calpain in myocardium remodeling].

AIM: To investigate the relation among myocardial angiotension II receptor (AT1/AT2) expression, calpain and cardiac function in patients with congestive heart failure (CHF). METHODS: Message RNA (mRNA) expression of AT1/AT2 receptor in myocardial tissue of 39 patients with CHF due to valvular heart disease and 8 control subjects were analyzed using reverse transcriptase-polymerase chain reaction (RT-PCR). Immunoprecipitation was used to assay the protein expression of u-calpain and m-calpain. RESULTS: Pathological changes of myocardial tissue in CHF due to valvular heart disease showed typical myocardial remodeling. AT1 receptor mRNA expression was slightly increased in the patients with mild CHF than in the control subjects, but decreased in the moderate and severe CHF patients. No difference was observed in AT2 receptor mRNA expression among all the groups, but the ratio of AT1/AT2 decreased. The protein expression of u-calpain and m-calpain were positively correlated to the levels of cardiac function in patient with CHD. CONCLUSION: The expression of AT1 receptor is down-regulated in moderate and severe CHF, the dominant receptor subtype is transformed to AT2. Cardiomyocyte apoptosis or death may be induced via AT2 receptor to activate calpain system, leading the deterioration of cardiac function.

[SSR molecular markers related to wood density and fibre traits in poplar].

In this study, field experiment was carried out following the randomized complete block design with five replications in 1999. F2 population was derived from a cross between the female ZH3 and the male ZH1 (ZH3, ZH1 were selected from F1 individuals derived from the cross of Populus deltoides with P. cathayana, produced by HUANG Dong-Sen in 1973). Sixty-eight F2 populations, four F1 individuals (included ZH3, ZH1) and both parents (Populus deltoides and P. cathayana) were measured for wood densities, fibre lengths, fibre widths and fibre angles. The results indicated that the trait of fibre length has obviously heterosis, there may have positive effects among the genes controlling the trait of fibre length, but may have negative effects among the genes controlling the trait of wood density. Using the method of single factor variance, the SSR markers correlated with wood density, fibre length, fibre width and fibre angle were identified to be 5, 7, 4 and 2, respectively. There were some useful markers among the above traits in P. cathayana, such as PMGC2873-1 for wood density (its contribution was 4.88%), PMGC456-3, PMGC2702-2 for fibre length (their contribution were 22.96% and 9.17%, respectively), PMGC2408-1 for fibre width (its contribution was 7.18%), and PMGC2525-1 for fibre angle (its contribution was 16.59%), these markers might be useful for wood property improvement if using them correctly in breeding program.