Effective microorganisms input efficiently improves the vegetation and microbial community of degraded alpine grassland.

Soil beneficial microorganism deficiency in the degraded grasslands have emerged as the major factors negatively impacting soil quality and vegetation productivity. EM (effective microorganisms) has been regarded as a good ameliorant in improving microbial communities and restoring degraded soil of agricultural systems. However, knowledge was inadequate regarding the effects of adding EM on the degraded alpine grassland. Four levels of EM addition (0, 150, 200, 250 mL m-2) were conducted to investigate the effects of EM addition on soil properties and microorganisms of degraded alpine grassland. The addition of EM increased aboveground biomass, soil organic carbon, total nitrogen, available phosphorus, and microbial biomass, but decreased soil electric conductivity. Meanwhile, the relative biomasses of gram-negative bacteria decreased, while the ectomycorrhizal fungi and arbuscular mycorrhizal fungi increased after EM addition. The relationship between microbial communities and environmental factors has been changed. The restore effect of EM increased with the increase of addition time. These results indicated that EM addition could be a good practice to restore the health of the degraded alpine grassland ecosystem.

The histone demethylase Kdm6b regulates the maturation and cytotoxicity of TCRαß+CD8αα+ intestinal intraepithelial lymphocytes.

Intestinal intraepithelial lymphocytes (IELs) are distributed along the length of the intestine and are considered the frontline of immune surveillance. The precise molecular mechanisms, especially epigenetic regulation, of their development and function are poorly understood. The trimethylation of histone 3 at lysine 27 (H3K27Me3) is a kind of histone modifications and associated with gene repression. Kdm6b is an epigenetic enzyme responsible for the demethylation of H3K27Me3 and thus promotes gene expression. Here we identified Kdm6b as an important intracellular regulator of small intestinal IELs. Mice genetically deficient for Kdm6b showed greatly reduced numbers of TCRαß+CD8αα+ IELs. In the absence of Kdm6b, TCRαß+CD8αα+ IELs exhibited increased apoptosis, disturbed maturation and a compromised capability to lyse target cells. Both IL-15 and Kdm6b-mediated demethylation of histone 3 at lysine 27 are responsible for the maturation of TCRαß+CD8αα+ IELs through upregulating the expression of Gzmb and Fasl. In addition, Kdm6b also regulates the expression of the gut-homing molecule CCR9 by controlling H3K27Me3 level at its promoter. However, Kdm6b is dispensable for the reactivity of thymic precursors of TCRαß+CD8αα+ IELs (IELPs) to IL-15 and TGF-ß. In conclusion, we showed that Kdm6b plays critical roles in the maturation and cytotoxic function of small intestinal TCRαß+CD8αα+ IELs.

High-quality genome assembly of 'Cuiguan' pear (Pyrus pyrifolia) as a reference genome for identifying regulatory genes and epigenetic modifications responsible for bud dormancy.

Dormancy-associated MADS-box (DAM) genes serve as crucial regulators of the endodormancy cycle in rosaceous plants. Although pear DAM genes have been identified previously, the lack of a high-quality reference genome and techniques to study gene function have prevented accurate genome-wide analysis and functional verification of such genes. Additionally, the contribution of other genes to the regulation of endodormancy release remains poorly understood. In this study, a high-quality genome assembly for 'Cuiguan' pear (Pyrus pyrifolia), which is a leading cultivar with a low chilling requirement cultivated in China, was constructed using PacBio and Hi-C technologies. Using this genome sequence, we revealed that pear DAM genes were tandemly clustered on Chr8 and Chr15 and were differentially expressed in the buds between 'Cuiguan' and the high-chilling-requirement cultivar 'Suli' during the dormancy cycle. Using a virus-induced gene silencing system, we determined the repressive effects of DAM genes on bud break. Several novel genes potentially involved in the regulation of endodormancy release were identified by RNA sequencing and H3K4me3 chromatin immunoprecipitation sequencing analyses of 'Suli' buds during artificial chilling using the new reference genome. Our findings enrich the knowledge of the regulatory mechanism underlying endodormancy release and chilling requirements and provide a foundation for the practical regulation of dormancy release in fruit trees as an adaptation to climate change.

Alternative splicing of the dormancy-associated MADS-box transcription factor gene PpDAM1 is associated with flower bud dormancy in 'Dangshansu' pear (Pyrus pyrifolia white pear group).

Alternative splicing (AS) plays a crucial role in plant growth, development and response to various environmental changes. However, whether alternative splicing of MADS-box transcription factors contributes to the flower bud dormancy process in fruit trees still remains unknown. In this work, the AS profile of genes in the dormant flower buds of 'Dangshansu' pear tree were examined. A total number of 3661 alternatively spliced genes were identified, and three mRNA isoforms of the dormancy associated MADS box (DAM) gene, PpDAM1, derived by alternative splicing, designated as PpDAM1.1, PpDAM1.2 and PpDAM1.3, were characterized. Bimolecular fluorescence complementation (BiFC) analysis indicated that AS of PpDAM1 didn't affect the nucleus localization and homo-/heterodimerization of PpDAM1.1, PpDAM1.2 and PpDAM1.3 proteins, but disturbed the translocation of PpDAM1.1/PpDAM1.1, PpDAM1.3/PpDAM1.3, PpDAM1.1/PpDAM1.3, and PpDAM1.2/PpDAM1.3 dimers to the nucleus. Constitutive expression of PpDAM1.2, but not PpDAM1.1 and PpDAM1.3, in Arabidopsis retarded the growth and development of transgenic plants. Further comparative expression analyses of PpDAM1.1, PpDAM1.2 and PpDAM1.3 in the flower buds of 'Dangshansu' and a less dormant pear cultivar, 'Cuiguan', exhibited that the expression of all the three isoforms in 'Dangshansu' were significantly higher than in 'Cuiguan', especially PpDAM1.2, which showed a predominantly higher expression than PpDAM1.1 and PpDAM1.3 in both cultivars. Our results suggest that alternative splicing of PpDAM1 could play a crucial role in pear flower bud dormancy process.

Strong Correlation between the Expression of CHEK1 and Clinicopathological Features of Patients with Multiple Myeloma.

Multiple myeloma (MM) is one of the most common malignancies, and the clinical outcome of patients with MM remains poor. Our objective is to screen biomarkers correlated with clinicopathological features and survival of patients with MM. A gene co-expression network was constructed to screen hub genes related to the three stages in the International Staging System (ISS) of MM. Functional analysis and protein-protein interaction analysis of the hub genes was performed. CHEK1, a gene most related to the ISS stages of MM, was selected for further clinical validation. A total of 780 hub genes correlated with ISS stages of MM were identified. Functional enrichment analysis of hub genes suggested that these genes were mostly enriched in several gene ontology (GO) terms and pathways from the Kyoto Encyclopedia of Genes and Genomes (KEGG) that were involved in cell proliferation and immune response. Expression of the gene for the protein checkpoint kinase I (CHEK1) was increased in MM cells from newly diagnosed patients (P = 0.0304) and relapsed patients (P = 0.0002) as compared to normal plasma cells. Meanwhile, CHEK1 was increased more in MM patients with stage II disease (P = 0.0321) and stage III disease (P = 0.0076) than in those with stage I disease. Survival analysis indicated that MM patients in the group characterized by low CHEK1 expression were associated with better clinical outcomes in terms of time to progression, event-free survival, and overall survival. High expression of CHEK1 predicted poor clinical characteristics of MM patient, and our results indicate that it can be considered a biomarker for the diagnosis of MM.

ABA-responsive ABRE-BINDING FACTOR3 activates DAM3 expression to promote bud dormancy in Asian pear.

Bud dormancy is indispensable for the survival of perennial plants in cold winters. Abscisic acid (ABA) has essential functions influencing the endo-dormancy status. Dormancy-associated MADS-box/SHORT VEGETATIVE PHASE-like genes function downstream of the ABA signalling pathway to regulate bud dormancy. However, the regulation of DAM/SVP expression remains largely uncharacterized. In this study, we confirmed that endo-dormancy maintenance and PpyDAM3 expression are controlled by the ABA content in pear (Pyrus pyrifolia) buds. The expression of pear ABRE-BINDING FACTOR3 (PpyABF3) was positively correlated with PpyDAM3 expression. Furthermore, PpyABF3 directly bound to the second ABRE in the PpyDAM3 promoter to activate its expression. Interestingly, both PpyABF3 and PpyDAM3 repressed the cell division and growth of transgenic pear calli. Another ABA-induced ABF protein, PpyABF2, physically interacted with PpyABF3 and disrupted the activation of the PpyDAM3 promoter by PpyABF3, indicating DAM expression was precisely controlled. Additionally, our results suggested that the differences in the PpyDAM3 promoter in two pear cultivars might be responsible for the diversity in the chilling requirements. In summary, our data clarify the finely tuned regulatory mechanism underlying the effect of ABA on DAM gene expression and provide new insights into ABA-related bud dormancy regulation.

Two B-box proteins, PpBBX18 and PpBBX21, antagonistically regulate anthocyanin biosynthesis via competitive association with Pyrus pyrifolia ELONGATED HYPOCOTYL 5 in the peel of pear fruit.

Light is indispensable for the accumulation of anthocyanin in the peel of red pear fruit (Pyrus pyrifolia Nakai). ELONGATED HYPOCOTYL 5 (HY5) is considered to be a critical regulator for induction of anthocyanin biosynthesis, but detailed characterization of its regulatory mechanism is needed. In this study, multiple genetic and biochemical approaches were applied to identify the roles of P. pyrifolia HY5 (PpHY5) and two B-box (BBX) proteins, PpBBX18 and PpBBX21, in the transcriptional regulation of PpMYB10. The functions of the two BBX proteins were analyzed in overexpression lines using pear calli-based approaches. On its own PpHY5 was unable to activate downstream genes. The two BBX proteins, PpBBX18 and PpBBX21, physically interacted with PpHY5 and antagonistically regulated anthocyanin biosynthesis in Arabidopsis and pear. PpBBX18 formed a heterodimer with PpHY5 via two B-box domains, in which PpHY5 bound to the G-box motif of PpMYB10 and PpBBX18 provided the trans-acting activity, thus inducing transcription of PpMYB10. PpBBX21 interacted with PpHY5 and PpBBX18 and hampered formation of the PpHY5-PpBBX18 active transcription activator complex, and subsequently repressed anthocyanin biosynthesis. The present results demonstrate the fine-tuned regulation of anthocyanin biosynthesis via transcriptional regulation of PpMYB10 by PpHY5-associated proteins and provide insights into light-induced anthocyanin biosynthesis.

BBX16, a B-box protein, positively regulates light-induced anthocyanin accumulation by activating MYB10 in red pear.

The red coloration of pear (Pyrus pyrifolia) results from anthocyanin accumulation in the fruit peel. Light is required for anthocyanin biosynthesis in pear. A pear homolog of Arabidopsis thaliana BBX22, PpBBX16, was differentially expressed after fruits were removed from bags and may be involved in anthocyanin biosynthesis. Here, the expression and function of PpBBX16 were analysed. PpBBX16's expression was highly induced by white-light irradiation, as was anthocyanin accumulation. PpBBX16's ectopic expression in Arabidopsis increased anthocyanin biosynthesis in the hypocotyls and tops of flower stalks. PpBBX16 was localized in the nucleus and showed trans-activity in yeast cells. Although PpBBX16 could not directly bind to the promoter of PpMYB10 or PpCHS in yeast one-hybrid assays, the complex of PpBBX16/PpHY5 strongly trans-activated anthocyanin pathway genes in tobacco. PpBBX16's overexpression in pear calli enhanced the red coloration during light treatments. Additionally, PpBBX16's transient overexpression in pear peel increased anthocyanin accumulation, while virus-induced gene silencing of PpBBX16 decreased anthocyanin accumulation. The expression patterns of pear BBX family members were analysed, and six additional BBX genes, which were differentially expressed during light-induced anthocyanin biosynthesis, were identified. Thus, PpBBX16 is a positive regulator of light-induced anthocyanin accumulation, but it could not directly induce the expression of the anthocyanin biosynthesis-related genes by itself but needed PpHY5 to gain full function. Our work uncovered regulatory modes for PpBBX16 and suggested the potential functions of other pear BBX genes in the regulation of anthocyanin accumulation, thereby providing target genes for further studies on anthocyanin biosynthesis.

PpCBFs selectively regulate PpDAMs and contribute to the pear bud endodormancy process.

KEY MESSAGE: PpCBF2 directly binds to the promoters of PpCBF3 and PpCBF4 to activate their expressions and selectively regulates PpDAMs during the leaf bud endodormancy process of 'Wonhwang' pear (Pyrus pyrifolia). Endodormancy is critical for temperate plant survival under freezing winter conditions, and low temperature is a vital environmental factor in endodormancy regulation. A C-repeat binding factor (CBF) has been found to regulate important DAM transcription factors during endodormancy in pear (Pyrus pyrifolia). In this study, we analyzed the regulation of pear DAM genes by CBFs in further detail. Four CBF and three DAM genes were identified in the pear cultivar 'Wonhwang'. Under natural conditions, PpDAM1 expression decreased from the start of chilling accumulation, while the other two DAM and three CBF genes peaked during endodormancy release. Under chilling treatment, the expressions of PpDAM1, PpDAM2 and PpCBF1 genes were similar to those under natural conditions. Different biochemical methods revealed that PpCBF2/4 can bind to the promoter of PpDAM1 and activate its expression and that PpCBF1/4 can activate PpDAM3. Interestingly, we found that PpCBF2 can activate PpCBF3/4 transcription by directly binding to their promoters. The ICE-CBF regulon is conserved in some plants; three ICE genes were identified in pear, but their expressions did not obviously change under natural and artificial chilling conditions. On the contrary, the selective transcriptional induction of PpCBFs by PpICE1s was observed in a dual-luciferase assay. Considering all these results, we propose that the PpCBF1-PpDAM2 regulon mainly responds to low temperature during endodormancy regulation, with further post-translational regulation by PpICE3. Our results provide basic information on CBF genes functional redundancy and differentiation and demonstrate that the CBF-DAM signaling pathway is involved in the pear bud endodormancy process.

Genome wide identification and predicted functional analyses of NAC transcription factors in Asian pears.

BACKGROUND: NAC proteins contribute to diverse plant developmental processes as well as tolerances to biotic and abiotic stresses. The pear genome had been decoded and provided the basis for the genome-wide analysis to find the evolution, duplication, gene structures and predicted functions of PpNAC transcription factors. RESULTS: A total of 185 PpNAC genes were found in pear, of which 148 were mapped on chromosomes while 37 were on unanchored scaffolds. Phylogeny split the NAC genes into 6 clades (Group1- Group6) with their sub clades (~ subgroup A to subgroup H) and each group displayed common motifs with no/minor change. The numbers of exons in each group varied from 1 to 12 with an average of 3 while 44 pairs from all groups showed their duplication events. qPCR and RNA-Seq data analyses in different pear cultivars/species revealed some predicted functions of PpNAC genes i.e. PpNACs 37, 61, 70 (2A), 53, 151(2D), 10, 92, 130 and 154 (3D) were potentially involved in bud endodormancy, PpNACs 61, 70 (2A), 172, 176 and 23 (4E) were associated with fruit pigmentations in blue light, PpNACs 127 (1E), 46 (1G) and 56 (5A) might be related to early, middle and late fruit developments respectively. Besides, all genes from subgroups 2D and 3D were found to be related with abiotic stress (cold, salt and drought) tolerances by targeting the stress responsive genes in pear. CONCLUSIONS: The present genome-wide analysis provided valuable information for understanding the classification, motif and gene structure, evolution and predicted functions of NAC gene family in pear as well as in higher plants. NAC TFs play diverse and multifunctional roles in biotic and abiotic stresses, growth and development and fruit ripening and pigmentation through multiple pathways in pear.

DHCR24 predicts poor clinicopathological features of patients with bladder cancer: A STROBE-compliant study.

To investigate the prognostic value of DHCR24 for patients with bladder cancer (BC). We used public bladder cancer microarray studies to evaluate the expression of DHCR24 between normal bladder tissues and BC cells, to investigate the relationship between the expression of DHCR24 and the clinical features of BC patients. Survival analysis was performed to investigate the correlation between DHCR24 expression and the survivals of BC patients. Gene set enrichment analysis was conducted to identify relevant mechanisms. The results showed that DHCR24 was up-regulated in BC cells compared with that in normal bladder tissues (P = .0389). Results of chi-square test suggested that BC patients in DHCR24 low expression group were proved to have better clinical characteristics (including tumor grade, disease progression, T staging, and N staging) as compared with those in DHCR24 low expression group (P < .0001, P = .002, P = .005, and P = .002, respectively). BC patients in DHCR24 low expression group were associated with better cancer-specific survival and overall survival (P < .0001 and P = .0008, respectively). DHCR24 might promote the proliferation of BC cells through several oncogenesis-associated biological processes (estrogen response, heme metabolism, P53 pathway, cholesterol homeostasis, mTORC1 signaling, peroxisome, xenobiotic metabolism, glycolysis, and protein secretion). Thus, DHCR24 might be a therapeutic target for patients with BC.

The Clinical Relevance of Fragile Histidine Triad Protein (FHIT) in Patients with Bladder Cancer.

BACKGROUND The present study aimed to investigate the clinical relevance of fragile histidine triad protein (FHIT) in patients with bladder cancer (BC). MATERIAL AND METHODS Three independent BC microarray studies were collected and reanalyzed. The expression of FHIT was evaluated between BC samples and normal bladder tissues. The correlation between the expression of FHIT and clinicopathological features was analyzed using the chi-square test. Log-rank based survival analysis was conducted to detect the survival significance of FHIT in patients with BC. Gene set enrichment analysis (GSEA) was performed to identify the mechanisms. RESULTS FHIT was significantly downregulated in BC cells (p=0.0044). BC patients in the FHIT high expression group had better clinical characteristics (including invasiveness, tumor grade, disease progression, and T staging) than those in the FHIT low expression group (p<0.0001, p<0.0001, p=0.031, p<0.0001, and p=0.056, respectively). Patients in the FHIT high expression group had better cancer-specific survival (p<0.0001) and overall survival (p=0.0008) than those in the FHIT low expression. GSEA results indicated that BC samples in the FHIT low expression group were enriched in interferon alpha response, apoptosis, androgen response, interferon gamma response, heme metabolism, and transforming growth facto r(TGF) beta signaling. CONCLUSIONS FHIT predicts better clinical relevance for patients with BC, which may be a promising therapeutic target.

Development and Validation of a 9-Gene Prognostic Signature in Patients With Multiple Myeloma.

Background: Multiple myeloma (MM) is one of the most common types of hematological malignance, and the prognosis of MM patients remains poor. Objective: To identify and validate a genetic prognostic signature in patients with MM. Methods: Co-expression network was constructed to identify hub genes related with International Staging System (ISS) stage of MM. Functional analysis of hub genes was conducted. Univariate Cox proportional hazard regression analysis was conducted to identify genes correlated with the overall survival (OS) of MM patients. Least absolute shrinkage and selection operator (LASSO) penalized Cox proportional hazards regression model was used to minimize overfitting and construct a prognostic signature. The prognostic value of the signature was validated in the test set and an independent validation cohort. Results: A total of 758 hub genes correlated with ISS stage of MM patients were identified, and these hub genes were mainly enriched in several GO terms and KEGG pathways involved in cell proliferation and immune response. Nine hub genes (HLA-DPB1, TOP2A, FABP5, CYP1B1, IGHM, FANCI, LYZ, HMGN5, and BEND6) with non-zero coefficients in the LASSO Cox regression model were used to build a 9-gene prognostic signature. Relapsed MM and ISS stage III MM was associated with high risk score calculated based on the signature. Patients in the 9-gene signature low risk group was significantly associated with better clinical outcome than those in the 9-gene signature high risk group in the training set, test, and validation set. Conclusions: We developed a 9-gene prognostic signature that might be an independent prognostic factor in patients with MM.

Endoplasmic reticulum stress protects human thyroid carcinoma cell lines against ionizing radiation-induced apoptosis.

Radiotherapy is one of the most effective forms of cancer treatment, used in the treatment of a number of malignant tumors. However, the resistance of tumor cells to ionizing radiation remains a major therapeutic problem and the critical mechanisms determining radiation resistance are poorly defined. In the present study, a cellular endoplasmic reticulum (ER) stress microenvironment was established through the pretreatment of cultured thyroid cancer cells with tunicamycin (TM) and thapsigargin (TG), in order to mimic the ER stress response in a tumor microenvironment. This microenviroment was confirmed through the X­box binding protein 1 splice process, glucose­regulated protein 78 kD and ER degradation­enhancing α­mannosidase­like mRNA expression. A clonogenic assay was used to measure cancer cell resistance to 60Co­Î³ following TM pretreatment; in addition, human C/EBP homologous protein (CHOP) mRNA expression was determined and apoptosis assays were performed. The results showed that TM or TG pretreatment inhibited CHOP expression and reduced the apoptotic rate of cells. Furthermore, the results demonstrated that the induced ER stress response rendered cancer cells more resistant to ionizing radiation­induced apoptosis. Therefore, the ER stress pathway may be a potential therapeutic target in order to improve the clinical efficiency of radiotherapy.