Genome-wide identification and expression analysis of the PP2C gene family in Apocynum venetum and Apocynum hendersonii.

BACKGROUND: Protein phosphatase class 2 C (PP2C) is the largest protein phosphatase family in plants. Members of the PP2C gene family are involved in a variety of physiological pathways in plants, including the abscisic acid signalling pathway, the regulation of plant growth and development, etc., and are capable of responding to a wide range of biotic and abiotic stresses, and play an important role in plant growth, development, and response to stress. Apocynum is a perennial persistent herb, divided into Apocynum venetum and Apocynum hendersonii. It mainly grows in saline soil, deserts and other harsh environments, and is widely used in saline soil improvement, ecological restoration, textiles and medicine. A. hendersonii was found to be more tolerant to adverse conditions. The main purpose of this study was to investigate the PP2C gene family and its expression pattern under salt stress and to identify important candidate genes related to salt tolerance. RESULTS: In this study, 68 AvPP2C genes and 68 AhPP2C genes were identified from the genomes of A. venetum and A. hendersonii, respectively. They were classified into 13 subgroups based on their phylogenetic relationships and were further analyzed for their subcellular locations, gene structures, conserved structural domains, and cis-acting elements. The results of qRT-PCR analyses of seven AvPP2C genes and seven AhPP2C genes proved that they differed significantly in gene expression under salt stress. It has been observed that the PP2C genes in A. venetum and A. hendersonii exhibit different expression patterns. Specifically, AvPP2C2, 6, 24, 27, 41 and AhPP2C2, 6, 24, 27, 42 have shown significant differences in expression under salt stress. This indicates that these genes may play a crucial role in the salt tolerance mechanism of A. venetum and A. hendersonii. CONCLUSIONS: In this study, we conducted a genome-wide analysis of the AvPP2C and AhPP2C gene families in Apocynum, which provided a reference for further understanding the functional characteristics of these genes.

Study on remediation of cadmium contaminated paddy field by ramie (Boehmeria nivea L.) floating island and its supporting technology.

Ramie (Boehmeria nivea L.) is an ideal crop for cadmium (Cd) pollution remediation due to its advantages of both remediating and utilizing, however, it is mainly carried out in dry land, whose restoration effect is relatively slow. Previously, we found that the ramie plants cultivated by hydroponics has several tens of times higher Cd absorption capacity than that planted in soil. However, the issue of how to use hydroponic ramie to remediate Cd contaminated paddy fields needs to be addressed. In this study, we innovatively developed the ramie floating island technology and studied its remediation model on simulated Cd contaminated paddy fields. Different ramie varieties were used to compare the remediation effects, and the results showed that there were differences in adaptability among different varieties on floating islands and the remediation ability of the tested ramie varieties was Z2 > Z1 > Z3. Different harvested times were set to analyze the effects of harvested model on remediation, and it was suggested that multiple harvests can be carried out according to the plant growth status of ramie floating island after 30 days of remediation to achieve better remediation effects. Low water level height (5 cm) of paddy field was beneficial for the accumulation of Cd in the roots, but considering the adaptability of various ramie varieties and the effect of long-term restoration, it was recommended that the water level height of 20 cm for the cultivation of ramie floating island was more suitable. Moreover, we found that low concentration of citric acid (≤2 g L-1) or polyaspartic acid (≤3 g L-1) can improve the remediation effects for ramie floating island. Our study opens up a novel approach for ramie to remediate heavy metal pollution and provides a technical reference for water body Cd remediation by plants.

Palladium-Catalyzed Dearomatization of Indoles with Alkynes: Construction of Spirocyclohexaneindolenines.

A palladium-catalyzed dearomatization of indoles with alkynes has been developed, providing an efficient route to access a variety of synthetically useful spirocyclohexaneindolenines in moderate to good yields. The current method features a simple catalytic system, operational simplicity, and good functional group compatibility, which will contribute substantially to the development of dearomatization to access spiro compounds. Besides, the ubiquitous existence of spiro molecules, including spirocyclohexaneindolenines, in drugs and biological active molecules suggests the potential application of this methodology in medicinal chemistry.

Genome wide characterization of R2R3 MYB transcription factor from Apocynum venetum revealed potential stress tolerance and flavonoid biosynthesis genes.

MYB transcription factors are crucial in regulating stress tolerance and expression of major genes involved in flavonoid biosynthesis. The functions of MYBs is well explored in a number of plants, yet no study is reported in Apocynum venetum. We identified a total of 163 MYB candidates, that comprised of 101 (61.96%) R2R3, 6 3R, 1 4R and 55 1R. Syntenic analysis of A. venetum R2R3 (AvMYBs) showed highest orthologous pairs with Vitis vinifera MYBs followed by Arabidopsis thaliana among the four species evaluated. Thirty segmental duplications and 6 tandem duplications were obtained among AvMYB gene pairs signifying their role in the MYB gene family expansion. Nucleotide substitution analysis (Ka/Ks) showed the AvMYBs to be under the influence of strong purifying selection. Expression analysis of selected AvMYBs under low temperature and cadmium stresses resulted in the identification of AvMYB48, AvMYB97, AvMYB8, AvMYB4 as potential stress responsive genes and AvMYB10 and AvMYB11 in addition, proanthocyanidin biosynthesis regulatory genes which is consistent with their annotated homologues in Arabidopsis. Tissue specific expression profile analysis of the AvMYBs further supported the qPCR analysis result. MYBs with higher transcript levels in root, stem and leaf like AvMYB4 for example, was downregulated under the stresses and such with low transcript level such as AvMYB48 which had low transcript in the leaf was upregulated under both stresses. Transcriptome and phylogenetic analyses suggested AvMYB42 as a potential regulator of anthocyanin biosynthesis. Thus, this study provided valuable information on AvR2R3-MYB gene family with respect to stress tolerance and flavonoid biosynthesis.

Genome-Wide Identification and Expression Analysis of BnPP2C Gene Family in Response to Multiple Stresses in Ramie (Boehmeria nivea L.).

The protein phosphatase 2C (PP2C), a key regulator of the ABA signaling pathway, plays important roles in plant growth and development, hormone signaling, and abiotic stress response. Although the PP2C gene family has been identified in many species, systematic analysis was still relatively lacking in ramie (Boehmeria nivea L.). In the present study, we identified 63 BnPP2C genes from the ramie genome, using bioinformatics analysis, and classified them into 12 subfamilies, and this classification was consistently supported by their gene structures and conserved motifs. In addition, we observed that the functional differentiation of the BnPP2C family of genes was restricted and that fragment replication played a major role in the amplification of the BnPP2C gene family. The promoter cis-regulatory elements of BnPP2C genes were mainly involved in light response regulation, phytohormone synthesis, transport and signaling, environmental stress response and plant growth and development regulation. We identified BnPP2C genes with tissue specificity, using ramie transcriptome data from different tissues, in rhizome leaves and bast fibers. The qRT-PCR results showed that the BnPP2C1, BnPP2C26 and BnPP2C27 genes had a strong response to drought, high salt and ABA, and there were a large number of stress-responsive elements in the promoter region of BnPP2C1 and BnPP2C26. The results suggested that BnPP2C1 and BnPP2C26 could be used as the candidate genes for drought and salt tolerance in ramie. These results provide a reference for further studies on the function of the PP2C gene and advance the development of the mechanism of ramie stress response, with a view to providing candidate genes for the molecular breeding of ramie for drought and salt tolerance.

Genome-Wide Investigation of the NAC Transcription Factor Family in Apocynum venetum Revealed Their Synergistic Roles in Abiotic Stress Response and Trehalose Metabolism.

NAC (NAM, ATAF1/2, and CUC2) transcription factors (TFs) are one of the most prominent plant-specific TF families and play essential roles in plant growth, development and adaptation to abiotic stress. Although the NAC gene family has been extensively characterized in many species, systematic analysis is still relatively lacking in Apocynum venetum (A. venetum). In this study, 74 AvNAC proteins were identified from the A. venetum genome and were classified into 16 subgroups. This classification was consistently supported by their gene structures, conserved motifs and subcellular localizations. Nucleotide substitution analysis (Ka/Ks) showed the AvNACs to be under the influence of strong purifying selection, and segmental duplication events were found to play the dominant roles in the AvNAC TF family expansion. Cis-elements analysis demonstrated that the light-, stress-, and phytohormone-responsive elements being dominant in the AvNAC promoters, and potential TFs including Dof, BBR-BPC, ERF and MIKC_MADS were visualized in the TF regulatory network. Among these AvNACs, AvNAC58 and AvNAC69 exhibited significant differential expression in response to drought and salt stresses. The protein interaction prediction further confirmed their potential roles in the trehalose metabolism pathway with respect to drought and salt resistance. This study provides a reference for further understanding the functional characteristics of NAC genes in the stress-response mechanism and development of A. venetum.

A comparative study of different methods for the determination of cadmium in various tissues of ramie (Boehmeria nivea L.).

Remediation of cadmium (Cd) pollution is one of the priorities of global environmental governance and accurate detection of Cd content is a key link in remediation of Cd pollution. This study aimed to compare three methods (inductively coupled plasma optical emission spectrometry (ICP-OES), inductively coupled plasma mass spectrometry (ICP-MS), and graphite furnace-atomic absorption spectrometry (GF-AAS)) for the determination of Cd with different tissues of various ramie varieties, and distinguish the advantage and disadvantage of each method. In total, 162 samples of ramie (Boehmeria nivea L.), which is an ideal plant for heavy metal remediation, were detected and the results showed that the three methods were all suitable for the de-termination of Cd content in ramie. ICP-OES and ICP-MS were simpler, faster, and more sensitive than GF-AAS. ICP-MS could be recommended for the determination of samples with various concentrations of Cd. ICP-OES could be used for measurement of samples with > 100 mg/kg Cd content, while GF-AAS was suitable for the detection of samples with very high (> 550 mg/kg) or very low (< 10 mg/kg) Cd content. Overall, considering the accuracy, stability, and the cost of measurement, ICP-MS was the most suitable method for determination of Cd content. This study provides significant reference information for the research in the field of Cd pollution remediation.

Total Flavonoids Extracts of Apocynum L. from the Ili River Valley Region at Different Harvesting Periods and Bioactivity Analysis.

In the current study, the total content from two Apocynum species leaves (Apocynum venetum and Apocynum hendersonii) collected from the Ili River Valley Region were extracted, and their bioactivities were investigated. The results showed a significant variation in the total flavonoid contents in the leaf samples collected at different periods (June, July, August, and September), with the highest content in August (60.11 ± 0.38 mg RE/g DW for A. venetum and 56.56 ± 0.24 mg RE/g DW for A. hendersonii), and the lowest in June (22.36 ± 0.05 mg RE/g DW for A. venetum and 20.79 ± 0.02 mg RE/g DW for A. hendersonii). The total flavonoid content was comparably higher in A. venetum than in A. hendersonii. Leaves extracts from the two species demonstrated strong bioactivity, which positively correlated with the total flavonoid contents. The anti-oxidative activity of A. venetum was higher than that of A. hendersonii in tandem with its higher flavonoid contents; the antibacterial activity, however, was conversely opposite. Furthermore, a total of 83 flavonoid metabolites were identified in the two species based on UPLC-ESI-MS/MS, out of which 24 metabolites were differentially accumulated. The variability in these metabolites might be the reason for the different bioactivities displayed by the two species. The present study provides insight into the optimal harvest time for Apocynum species planted in the major distribution area of the Ili River Valley and the specific utilization of A. venetum and A. hendersonii.

Transcriptome and metabolome analysis reveals anthocyanin biosynthesis pathway associated with ramie (Boehmeria nivea (L.) Gaud.) leaf color formation.

BACKGROUND: The bast fiber crop ramie can be used as high-quality forage resources, especially in tropical or subtropical region where there is lack of high-quality protein feed. Hongxuan No.1 (HX_1) is a unique ramie variety with a light reddish brown leaf color, which is obviously different from elite cultivar, Zhongzhu No.1 (ZZ_1, green leaf). While, the regulatory mechanism of color difference or secondary metaboliates synthesis between these two varieties have not been studied. RESULTS: In this study, phenotypic, transcriptomic and metabolomic analysis of HX_1 and ZZ_1 were conducted to elucidate the mechanism of leaf color formation. Chromaticity value and pigment content measuring showed that anthocyanin was the main metabolites imparting the different leaf color phenotype between the two varieties. Based on LC/MS, at least 14 anthocyanins were identified in leaves of HX_1 and ZZ_1, and the HX_1 showed the higher relative content of malvidin-, pelargonidin-,and cyanidin-based anthocyanins. Transcriptome and metabolome co-analysis revealed that the up-regulated expression of flavonoids synthesis gene was positively correlated with total anthocyanins accumulation in ramie leaf, and the differentfially expression of "blue gene" (F3'5'H) and the "red gene" (F3'H) in leaves bring out HX_1 metabolic flow more input into the cyanidin branch. Furthermore, the enrichment of glycosylated modification pathway (UGT and AT) and the expression of flavonoid 3-O-glucosyl transferase (UFGT), anthocyanidin reductase (ANR), in leaves were significantly influenced the diversity of anthocyanins between HX_1 and ZZ_1. CONCLUSIONS: Phenotypic, transcriptomic and metabolomic analysis of HX_1 and ZZ_1 indicated that the expression levels of genes related to anthocyanin metabolism contribute to the color formation of ramie variety. Anthocyanins are important plant secandary metabilates with many physiological functions, the results of this study will deepened our understanding of ramie leaf color formation, and provided basis for molecular breeding of functional forage ramie.

Discovery of ß-carboline-(phenylsulfonyl)furoxan hybrids as potential anti-breast cancer agents.

The cytotoxicity properties of the ß-carboline alkaloids have been broadly investigated. However, the potential application of ß-carbolines was hindered due to the moderate activity in cancer. In the present study, thirty ß-carboline-(phenylsulfonyl)furoxan hybrids (11a-j, 12a-j and 13a-j) were designed and synthesized through esterification and amidation reaction strategy, and their inhibitory activities against the human breast cancer cell lines MCF-7 and MDA-MB-231 were evaluated by CCK-8 assay. Biological evaluation presented that the most promising amide derivative 13h, substituted with p-methoxyphenyl group at position 1, generated high concentration of NO and evidently depressed the MCF-7 (IC50 = 0.89 µM) and MDA-MB-231 (IC50 = 0.62 µM) cells proliferation. Particularly, the wound healing and transwell assays demonstrated that 13h significantly inhibited the migration and invasion of MDA-MB-231cells. Furthermore, the preliminary mechanisms studies indicated that 13h induced G2/M phase arrest and apoptosis possibly causing by ROS accumulation and ROS-mediated DNA damage. Based on these considerations, 13h may be a promising antimetastatic agent for breast cancer, which is noteworthy for further exploration.

Marine Antitumor Peptide Dolastatin 10: Biological Activity, Structural Modification and Synthetic Chemistry.

Dolastatin 10 (Dol-10), a leading marine pentapeptide isolated from the Indian Ocean mollusk Dolabella auricularia, contains three unique amino acid residues. Dol-10 can effectively induce apoptosis of lung cancer cells and other tumor cells at nanomolar concentration, and it has been developed into commercial drugs for treating some specific lymphomas, so it has received wide attention in recent years. In vitro experiments showed that Dol-10 and its derivatives were highly lethal to common tumor cells, such as L1210 leukemia cells (IC50 = 0.03 nM), small cell lung cancer NCI-H69 cells (IC50 = 0.059 nM), and human prostate cancer DU-145 cells (IC50 = 0.5 nM), etc. With the rise of antibody-drug conjugates (ADCs), milestone progress was made in clinical research based on Dol-10. A variety of ADCs constructed by combining MMAE or MMAF (Dol-10 derivatives) with a specific antibody not only ensured the antitumor activity of the drugs themself but also improved their tumor targeting and reduced the systemic toxicity. They are currently undergoing clinical trials or have been approved for marketing, such as Adcetris®, which had been approved for the treatment of anaplastic large T-cell systemic malignant lymphoma and Hodgkin lymphoma. Dol-10, as one of the most medically valuable natural compounds discovered up to now, has brought unprecedented hope for tumor treatment. It is particularly noteworthy that, by modifying the chemical structure of Dol-10 and combining with the application of ADCs technology, Dol-10 as a new drug candidate still has great potential for development. In this review, the biological activity and chemical work of Dol-10 in the advance of antitumor drugs in the last 35 years will be summarized, which will provide the support for pharmaceutical researchers interested in leading exploration of antitumor marine peptides.

SMA1, a homolog of the splicing factor Prp28, has a multifaceted role in miRNA biogenesis in Arabidopsis.

MicroRNAs (miRNAs) are a class of small non-coding RNAs that repress gene expression. In plants, the RNase III enzyme Dicer-like (DCL1) processes primary miRNAs (pri-miRNAs) into miRNAs. Here, we show that SMALL1 (SMA1), a homolog of the DEAD-box pre-mRNA splicing factor Prp28, plays essential roles in miRNA biogenesis in Arabidopsis. A hypomorphic sma1-1 mutation causes growth defects and reduces miRNA accumulation correlated with increased target transcript levels. SMA1 interacts with the DCL1 complex and positively influences pri-miRNA processing. Moreover, SMA1 binds the promoter region of genes encoding pri-miRNAs (MIRs) and is required for MIR transcription. Furthermore, SMA1 also enhances the abundance of the DCL1 protein levels through promoting the splicing of the DCL1 pre-mRNAs. Collectively, our data provide new insights into the function of SMA1/Prp28 in regulating miRNA abundance in plants.

Naringin Protects Against Interleukin 1ß (IL-1ß)-Induced Human Nucleus Pulposus Cells Degeneration via Downregulation Nuclear Factor kappa B (NF-κB) Pathway and p53 Expression.

BACKGROUND Low back pain (LBP) is regarded as a frequent disease that causes disability. We aimed to explore the effect of naringin on intervertebral disc degeneration (IDD) in IL-1ß-induced human nucleus pulposus (NP) cells and its corresponding molecular mechanisms. MATERIAL AND METHODS Human NP cells were identified by toluidine blue and Safranin O staining. Cell viability was determined by MTT assay. The expression levels of matrix metalloproteinases (MMP-3, MMP-13, ADAMTS-4, ADAMTS-5, collagen II, aggrecan), inflammatory genes (tumor necrosis factor [TNF]-alpha, interleukin [IL]-6), kappa B kinase alpha (IkappaBalpha), p65 and p53 were determined by quantitative real-time polymerase chain reaction (qPCR) and western blotting. Immunofluorescence study was performed to detect the position and expression of p65 protein in IL-1ß-induced human NP cells. RESULTS Human NP cells were successfully separated from intervertebral disc tissue. We found that naringin could significantly reduce the expressions of matrix metalloproteinases (MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5) and inflammatory genes in IL-1ß-stimulated human NP cells, while collagen II and aggrecan were increased at mRNA and protein level. Immunofluorescence showed that naringin pretreatment decreased the p65 protein expression in the nucleus and suppressed the phosphorylation of IkappaBalpha and p65. CONCLUSIONS These results demonstrated that naringin could attenuate matrix metalloproteinase catabolism and inflammation in IL-1ß-treated human nucleus pulposus cells via downregulating NF-kappaB pathway and p53 expression, suggesting that naringin has the potential to prevent and treat IDD.

The miRNAome of ramie (Boehmeria nivea L.): identification, expression, and potential roles of novel microRNAs in regulation of cadmium stress response.

BACKGROUND: MicroRNAs (miRNAs) regulate numerous crucial abiotic stress processes in plants. However, information is limited on their involvement in cadmium (Cd) stress response and tolerance mechanisms in plants, including ramie (Boehmeria nivea L.) that produces a number of economic valuable as an important natural fibre crop and an ideal crop for Cd pollution remediation. RESULTS: Four small RNA libraries of Cd-stressed and non-stressed leaves and roots of ramie were constructed. Using small RNA-sequencing, 73 novel miRNAs were identified. Genome-wide expression analysis revealed that a set of miRNAs was differentially regulated in response to Cd stress. In silico target prediction identified 426 potential miRNA targets that include several uptake or transport factors for heavy metal ions. The reliability of small RNA sequencing and the relationship between the expression levels of miRNAs and their target genes were confirmed by quantitative PCR (q-PCR). We showed that the expression patterns of miRNAs obtained by q-PCR were consistent with those obtained from small RNA sequencing. Moreover, we demonstrated that the expression of six randomly selected target genes was inversely related to that of their corresponding miRNAs, indicating that the miRNAs regulate Cd stress response in ramie. CONCLUSIONS: This study enriches the number of Cd-responsive miRNAs and lays a foundation for the elucidation of the miRNA-mediated regulatory mechanism in ramie during Cd stress.

Genome-wide association study discovered favorable single nucleotide polymorphisms and candidate genes associated with ramet number in ramie (Boehmeria nivea L.).

BACKGROUND: Ramie (Boehmeria nivea L.) is one of the most important natural fiber crops and an important forage grass in south China. Ramet number, which is a quantitative trait controlled by multigenes, is one of the most important agronomic traits in plants because the ramet number per plant is a key component of grain yield and biomass. However, the genetic variation and genetic architecture of ramie ramet number are rarely known. RESULTS: A genome-wide association study was performed using a panel of 112 core germplasms and 108,888 single nucleotide polymorphisms (SNPs) detected using specific-locus amplified fragment sequencing technology. Trait-SNP association analysis detected 44 significant SNPs that were associated with ramet number at P < 0.01. The favorable SNP Marker20170-64 emerged at least twice in the three detected stages and was validated to be associated with the ramie ramet number using genomic DNA polymerase chain reaction with an F1 hybrid progeny population. Comparative genome analysis predicted nine candidate genes for ramet number based on Marker20170-64. Real-time quantitative polymerase chain reaction analysis indicated that six of the genes were specific to upregulation in the ramie variety with high ramet number. These results suggest that these genes could be considered as ramet number-associated candidates in ramie. CONCLUSIONS: The identified loci or genes may be promising targets for genetic engineering and selection for modulating the ramet number in ramie. Our work improves understanding of the genetics of ramet number in ramie core germplasms and provides tools for marker-assisted selection for improvement of agricultural traits.

Hydroponic method for ramie and removal of nitrogen and phosphorus from livestock wastewater.

By using a hydroponic culture system, the terrestrial fiber crop ramie can growth optimally in aquatic environment and enhance exponentially quantities of high quality seedlings for subsequent field cultivation. In this study, the survival rate of ramie seedling was more than 97% when cultured using the novel hydroponic method. Further physiological analysis of the hydroponic ramie to different concentration of livestock wastewater demonstrated that all of these ramies can survival in livestock wastewater, but the 4 times diluted livestock wastewater (total N: 100.9 mg L-1, total P: 2.69 mg L-1) was more appropriate for ramie growth. The nutrients N and P in livestock wastewater were significantly decreased by the growth of ramie, and the removal efficiency of total N and total P in the 4 times diluted livestock wastewater achieved 78.1% and 43.1% respectively within 5 weeks. In conclusion, our studies highlight that the combination of ramie and the hydroponic technology resulted to be effective in the phytoremediation of livestock wastewater.

Antioxidant Activity of Coconut (Cocos nucifera L.) Protein Fractions.

Coconut cake is an abundant and good potential edible protein source. However, until now it has not been extensively used in the food industry. To promote its usage, the characterization, nutrition value and antioxidant activity of coconut cake protein fractions (albumin, globulin, prolamine, glutelin-1 and glutelin-2) were studied. Results revealed that all the albumin, globulin, glutelin-1 and glutelin-2 fractions showed a high nutrition value. The prolamine, glutelin-1 and glutelin-2 all exhibited good radical scavenging activity and reducing power, and the globulin and prolamine showed high ion chelating ability (89.14-80.38%). Moreover, all the fractions except glutelin-2 could effectively protect DNA against oxidative damage. Several peptides containing five to eight amino acids with antioxidant activity were also identified by LC-MS/MS from the globulin and glutelin-2 fractions. The results demonstrated that the coconut cake protein fractions have potential usages in functional foods.

Role of Runx2 polymorphisms in risk and prognosis of ossification of posterior longitudinal ligament.

BACKGROUND: Our study was aimed at finding out if Runx2 SNPs (single-nucleotide polymorphisms) are related to susceptibility to and prognosis of ossification of posterior longitudinal ligament (OPLL). METHODS: We selected 80 OPLL patients and another 80 independent patients without OPLL from September 2013 to November 2014. Serum was collected to detect the genotypes of rs1321075, rs12333172, and rs1406846 on Runx2 with direct sequencing analysis. RESULTS: Differences in clinical characteristics, including age, weight, height, sex ratio, as well as smoking and drinking history, between OPLL and control groups appeared to be insignificant (all P-value >.05). The allele of rs1406846 (A) emerged as a key element in raising OPLL risk with the biggest statistical significance (P<.001). Conversely, alleles of rs967588 (T) and rs16873379 (C) were associated with reduced predisposition to OPLL less remarkably (both P=.033). Regarding rs16873379, the case group exhibited a smaller frequency of homozygote CC in comparison with TT genotype than the control group (P=.016). Furthermore, the improvement rate based on calculation of JOA score suggested that genotype AA of rs6908650 was beneficial for OPLL patients' recovery from posterior laminoplasty surgery (P<.05), while genotypes of rs16873379 (CC), rs1406846 (AA), and rs2677108 (CC) significantly restrained this process (P<.05). Besides, rs16873379, rs1406846, and rs2677108 were significantly associated with number of ossification segments (P<.05). CONCLUSIONS: Runx2 SNPs (e.g., rs16873379, rs1406846, and rs2677108) were strongly correlated with onset and treatment efficacy of OPLL, and they might regulate severity of OPLL.

Efficient yeast cell-surface display of an endoglucanase of Aspergillus flavus and functional characterization of the whole-cell enzyme.

The endoglucanase gene endo753 from Aspergillus flavus NRRL3357 strains was cloned, and the recombinant Endo753 was displayed on the cell surface of Saccharomyces cerevisiae EBY100 strain by the C-terminal fusion using Aga2p protein as anchor attachment tag. The results of indirect immunofluorescence and Western blot confirmed the expression and localization of Endo753 on the yeast cell surface. The hydrolytic activity test of the whole-cell enzyme revealed that Endo753 immobilized on the yeast cell surface had high endoglucanase activity. The functional characterization of the whole-cell enzyme was investigated, and the whole-cell enzyme displayed the maximum activity at pH 8 and 50 °C. The enzyme was stable in a pH range of 7.0-10.0. Furthermore, the whole-cell enzyme displayed high thermostability below 50 °C and moderate stability between 50 and 70 °C. These properties make endo753 a good candidate in bioethanol production from lignocellulosic materials after displaying on the yeast cell surface.

IDENTIFICATION AND MOLECULAR CHARACTERIZATION OF TWO SERINE PROTEASES AND THEIR POTENTIAL INVOLVEMENT IN PROPHENOLOXIDASE ACTIVATION IN Plutella xylostella.

The proteolytic activation of prophenoloxidase (proPO) is a humoral defense mechanism in insects and crustaceans. Phenoloxidase (PO) is produced as an inactive precursor namely, proPO and is activated via specific proteolytic cleavage by proPO-activating proteinase. The current research reports two novel serine proteinase genes (PxSP1-768 bp and PxSP2-816 bp) from Plutella xylostella, encoding 255 and 271 amino acid residues, respectively. Tissue distribution analyses by semiquantitative reverse transcription-PCR (RT-PCR) revealed the resultant genes to be primarily expressed in the hemocytes, while quantitative-RT-PCR (qRT-PCR) assay showed that transcription level of PxSP1 and PxSP2 increased significantly after injection of the fungal pathogen Beauveria bassiana. Purified recombinant fusion proteins of PxSP2 and PxSP1 were injected to New Zealand white rabbits and polyclonal antibodies were generated with the titers of 1:12,800. After silencing the expression of PxSP2 by RNAi, the PO activity decreased significantly. The results show that PxSP2 is involved in prophenoloxidase activation in P. xylostella.