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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 TCRαβ+CD8αα+ IELs. In the absence of Kdm6b, TCRαβ+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 TCRαβ+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 TCRαβ+CD8αα+ IELs (IELPs) to IL-15 and TGF-β. In conclusion, we showed that Kdm6b plays critical roles in the maturation and cytotoxic function of small intestinal TCRαβ+CD8αα+ IELs.

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.

Speech network analysis and anomaly detection based on the FSS model is analyzed in this research. Aiming at the problem of parallel detection and processing of massive data in distributed intrusion detection, a data segmentation algorithm based on capability and load is proposed. The algorithm evaluates the capabilities and actual load of the nodes, weighs the actual state of each node and the data distribution relationship in the cluster, and allocates more data to be processed to nodes with strong data processing capabilities and light loads. High-order statistics are used to describe the intrusion characteristics of persistent attacks in the link layer of the speech sensor networks, and vector quantitative decomposition is used to analyze the fusion characteristics of advanced persistent intrusion symbols in mobile terminals. Different terminals are estimated based on the machine learning algorithms, and the FSS model is integrated to achieve the comprehensive analysis of the speech analytic models. The experiment compared with the state-of-the-art methods have proven the efficiency of the framework. The detection accuracy is higher than the latest methodologies.

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.

Aims Nitrogen (N) deposition is a global environmental problem that can alter community compositions and functions, and consequently, the ecosystem services. In this study, we assessed the responses of aboveground vegetation, surface soil properties and microbial communities to N addition, and explored the drivers of microbial community in a semiarid steppe ecosystem in northwest of China. Methods Thirty-six 6×10-m2 plots composed of six N addition levels and six replicates were distributed in six columns and six rows. Nine vegetation characteristics and seven soil properties were measured and calculated. Soil microbial characteristics were analyzed by 16S rRNA high-throughput sequencing. Results N addition positively affected aboveground vegetation traits such as the community weighted-mean of leaf nitrogen content (LNCWM). High N inputs significantly altered the microbial community assembly process from random to deterministic. The microbial community diversity and composition, however, were not sensitive to N addition. A piecewise structural equation model (SEM) further showed that the microbial community composition was affected by both aboveground vegetation and soil properties. The composition of bacterial communities was mainly regulated by the composition of plant communities and soil total N. In contrast, the composition of fungal communities was driven by soil pH and the community weighted-mean of specific leaf area (SLACWM). Microbial diversity and composition remained unchanged because their drivers were not affected by N addition. The results of this research improved our understanding of the response of grassland ecosystems to N deposition, and provided a theoretical basis for grassland utilization and management under N deposition.

Fertilization and grazing are two common anthropogenic disturbances that can lead to unprecedented changes in biodiversity and ecological stability of grassland ecosystems. A few studies, however, have explored the effects of fertilization and grazing on community stability and the underlying mechanisms. We conducted a six-year field experiment to assess the influence of nitrogen (N) fertilization and grazing on the community stability in a long-term enclosure and grazing grassland ecosystems on the Loess Plateau. A structural equation modeling method was used to evaluate how fertilization and grazing altered community stability. Our results indicated that the community stability decreased in the enclosure and grazing grassland ecosystems with the addition of N. The community stability began to decline significantly at 4.68 and 9.36 N g m&minus, 2 year&minus, 1 for the grazing and enclosure grassland ecosystems, respectively. We also found that the addition of N reduced the community stability through decreasing species richness, but a long-term enclosure can alleviate its negative effect. Overall, species diversity can be a useful predictor of the stability of ecosystems confronted with disturbances. Also, our results showed that long-term enclosure was an effective grassland management practice to ensure community stability on the Loess Plateau of China.

Dormancy-associated MADS-box(DAM) genes serve as crucial regulators of the endodormancy cycle in rosaceous plants. Although pearDAMgenes 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 pearDAMgenes 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 ofDAMgenes 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.

Bud dormancy establishment is essential for perennial plants to survive cold winters, while dormancy release is important for the resumption of growth and flowering. Gibberellins (GAs) and abscisic acid (ABA) are involved in bud dormancy release, but the details of the regulatory pathway are still obscure. First, we measured the GAs contents in pear buds during dormancy transition. The content of GA1, one of the bioactive GAs, increased during dormancy release while GA4 was not detectable. Based on a ‘Suli’ pear (Pyrus pyrifolia White Pear Group) RNA sequencing analysis, we found that PpyGAST1, a GA-stimulated transcripts (GAST) gene family member, was rapidly up-regulated during dormancy release, with a concurrent up-regulation of PpyGA20OX2 and active GA level. Overexpression of PpyGAST1 in Arabidopsis resulted in earlier seed germination under mock-, ABA- and GA-treatment conditions, but there was no difference under paclobutrazol-treatment conditions, compared with the wild type. Additionally, the transgenic Arabidopsis lines showed higher AtGA20ox2, AtGA3ox1 and AtEXPA1 expression levels. We identified 17 GAST genes differentially expressed during dormancy transitions, with higher transcript abundancy levels during dormancy release. The expression levels of some GAST genes in the buds were regulated by both GA and ABA treatments during dormancy release. Together, these results indicate that GAST genes may be involved in the ABA- and GA-responsive pathway and their expressions are correlated with pear bud dormancy release. Our findings provide new insights into the possible molecular mechanisms integrating GA biosynthesis and ABA signaling during bud dormancy release.

Summary 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.

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.

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.

Heat map presentation of all PpNAC TFs according to group wise distributions during fruit color development in P. pyrifolia â Meirensuâ (DOCX 242 kb)

Heat map presentation of all PpNAC TFs according to group distributions during fruit developments in three pear cultivars viz. P. calleryana, P. pashia and P. pyrifolia. (DOCX 925 kb)

Frequency Distribution of PpNAC genes on each chromosome. (DOCX 117 kb)

Phylogenetic tree of apple, pear and Arabidopsis (DOCX 350 kb)

Heat map presentation of all PpNAC TFs according to group distributions during bud endodormancy in P. pyrifolia Chinese â Suliâ (DOCX 144 kb)

Fourier transform infrared spectroscopy (FTIR) was employed to study the human epidermis larynx carcinoma cell lines (Hep-2) which were irradiated by different doses of X-ray. The results show that (1) the irradiation of X-ray damages the structure of the CH3 groups of the thymine in DNA, which restrains the reproduction of Hep-2 cells effectively, (2) the 8 Gy dose of X-ray irradiation changes the framework and the relative contents of some proteins, lipids and the nucleic acid molecules intercellular in the greatest degree, and (3) the 8 Gy dose of X-ray irradiation is the best irradiation dose for lowering the degree of the cancerization of Hep-2 cells according to the criteria for the degree of the cancerization reported recently. Meanwhile, the apoptosis of these cells were detected by using flow cytometry (FCM) primarily. It shows that the apoptotic ratio of the Hep-2 cells depends on the irradiation dose to some extent, but is not linearly. And the apoptotic ratio of the 12 Gy dose group is the maximum (20.36%), but the apoptotic ratios of the 2 to 8 Gy dose groups change little.

A near-infrared surface-enhanced Raman spectroscopy (NIR-SERS) method was employed for oxyheamoglobin (OxyHb) detection to develop a simple blood test for liver cancer detection. Polyvinyl alcohol protected silver nanofilm (PVA-Ag nanofilm) used as the NIR-SERS active substrate to enhance the Raman scattering signals of OxyHb. High quality NIR-SERS spectrum from OxyHb adsorbed on PVA-Ag nanofilm can be obtained within 16 s using a portable Raman spectrometer. NIR-SERS measurements were performed on OxyHb samples of healthy volunteers (control subjects, n = 30), patients (n = 40) with confirmed liver cancer (stage I, II and III) and the liver cancer patients after surgery (n = 30). Meanwhile, the tentative assignments of the Raman bands in the measured NIR-SERS spectra were performed, and the results suggested cancer specific changes on molecule level, including a decrease in the relative concentrations and the percentage of aromatic amino acids of OxyHb, changes of the vibration modes of the CaHm group and pyrrole ring of OxyHb of liver cancer patients. In this paper, principal component analysis (PCA) combined with independent sample T test analysis of the measured NIR-SERS spectra separated the spectral features of the two groups into two distinct clusters with the sensitivity of 95.0% and the specificity of 85.7%. Meanwhile, the recovery situations of the liver cancer patients after surgery were also assessed using the method of discriminant analysis-predicting group membership based on PCA. The results show that 26.7% surgeried liver cancer patients were distinguished as the normal subjects and 63.3% were distinguished into the cancer. Our study demonstrated great potentials for developing NIR-SERS OxyHb analysis into a novel clinical tool for non-invasive detection of liver cancers. Copyright © 2013 John Wiley & Sons, Ltd.