Molecular characterization of the cytochrome P450 enzyme CYP18A1 in Henosepilachna vigintioctopunctata.

In insects, the expression of 20E response genes that initiate metamorphosis is triggered by a pulse of 20-hydroxyecdysone (20E). The 20E pulse is generated through two processes: synthesis, which increases its level, and inactivation, which decreases its titer. CYP18A1 functions as an ecdysteroid 26-hydroxylase and plays a role in 20E removal in several representative insects. However, applying 20E degradation activity of CYP18A1 to other insects remains a significant challenge. In this study, we discovered high levels of Hvcyp18a1 during the larval and late pupal stages, particularly in the larval epidermis and fat body of Henosepilachna vigintioctopunctata, a damaging Coleopteran pest of potatoes. RNA interference (RNAi) targeting Hvcyp18a1 disrupted the pupation. Approximately 75% of the Hvcyp18a1 RNAi larvae experienced developmental arrest and remained as stunted prepupae. Subsequently, they gradually turned black and eventually died. Among the Hvcyp18a1-depleted animals that successfully pupated, around half became malformed pupae with swollen elytra and hindwings. The emerged adults from these deformed pupae appeared misshapen, with shriveled elytra and hindwings, and were wrapped in the pupal exuviae. Furthermore, RNAi of Hvcyp18a1 increased the expression of a 20E receptor gene (HvEcR) and four 20E response transcripts (HvE75, HvHR3, HvBrC, and HvαFTZ-F1), while decreased the transcription of HvßFTZ-F1. Our findings confirm the vital role of CYP18A1 in the pupation, potentially involved in the degradation of 20E in H. vigintioctopunctata.

Effects of overexpression of histone H3K9me3 demethylase on development of porcine cloned embryos.

The abnormal modification of histone is an important factor restricting development of porcine cloned embryos. Overexpression of histone H3K9me3 demethylase KDM4 family can effectively improve the developmental efficiency of cloned embryos. In order to explore the effects of overexpression of H3K9me3 demethylase on the development of porcine cloned embryos, KDM4A mRNA and KDM4D mRNA were injected respectively into porcine cloned embryos at the 1-cell stage and 2-cell stage to detect the blastocyst rate; 2-cell stage cloned embryos injected with KDM4A mRNA and embryo injection water (the control group) at the 1-cell stage were collected to detect the expression level of H3K9me3, and 4-cell stage cloned embryos were collected for single cell transcriptome sequencing, then the sequencing data was analyzed with KEGG and GO. The results showed that the blastocyst rate of porcine cloned embryos injected with KDM4A mRNA at 1-cell stage was significantly higher than that of the control group (25.32 ± 0.74% vs 14.78 ± 0.87%), while cloned embryos injected with KDM4D mRNA had a similar blastocyst rate with cloned embryos in control group (16.27 ± 0.77% vs 14.78 ± 0.87%). Porcine cloned embryos injected with KDM4A mRNA and KDM4D mRNA at 2-cell stage had a similar blastocyst rate with cloned embryos in control group (32.18 ± 1.67%, 30.04 ± 0.91% vs 31.22 ± 1.40%). The expression level of H3K9me3 in cloned embryos injected with KDM4A mRNA at 1-cell stage was lower than that in control group. There were 133 differentially expressed genes detected by transcriptome sequencing, including 52 up-regulated genes and 81 down-regulated genes. Pathways enriched by GO analyses were mainly related to protein localization. Pathways enriched by KEGG analyses were related to cellular senescence and acute myeloid leukemia. These results suggest that overexpression of histone H3K9me3 demethylase KDM4A can significantly improve the developmental efficiency of porcine cloned embryos.

[Regulation of ischemic stroke by circadian rhythm and intervention by traditional Chinese medicine].

Circadian rhythm is an internal regulatory mechanism formed in organisms in response to the circadian periodicity in the environment, which modulates the pathophysiological events, occurrence and development of diseases, and the response to treatment in mammals. It significantly influences the susceptibility, injury, and recovery of ischemic stroke, and the response to therapy. Accumulating evidence indicates that circadian rhythms not only regulate the important physiological factors of ischemic stroke events, such as blood pressure and coagulation-fibrinolysis system, but also participate in the immuno-inflammatory reaction mediated by glial cells and peripheral immune cells after ischemic injury and the regulation of neurovascular unit(NVU). This article aims to link molecular, cellular, and physiological pathways in circadian biology to the clinical consequences of ischemic stroke and to illustrate the impact of circadian rhythms on ischemic stroke pathogenesis, the regulation of NVU, and the immuno-inflammatory responses. The regulation of circadian rhythm by traditional Chinese medicine is reviewed, and the research progress of traditional Chinese medicine intervention in circadian rhythm is summarized to provide a reasonable and valuable reference for the follow-up traditional Chinese medicine research and molecular mechanism research of circadian rhythm.

Dissecting the molecular mechanism of russeting in sand pear (Pyrus pyrifolia Nakai) by metabolomics, transcriptomics, and proteomics.

Brown coloration and a rough appearance as russet and semi-russet (partial russet) are features unique to the popular Asian sand pear (Pyrus pyrifolia Nakai). The degree of russeting is different between different genotypes. Russeting is sensitive to water fluctuations, where excessive rainwater can trigger/stimulate its development. However, the molecular mechanism of russeting is currently unclear. Here, we employed multi-omics, i.e., metabolomics, transcriptomics, and proteomics, and analyzed the effect of different sand pear genotypes and artificial rainfall on russeting of pear fruits. This led to the identification of 79, 64, and 29 differentially produced/expressed metabolites, transcripts, and proteins that are involved in the biosynthesis of suberin, phenylpropane, cutin, and waxes. Further analysis of these differentially expressed genes and their encoded proteins revealed that four of them exhibited high expression at both transcript and protein levels. Transient expression of one such gene, PbHHT1 (accession number 103966555), which encodes ω-hydroxypalmitate-O-feruloyl transferase, in young green non-russet fruits triggered premature suberization in the russeting pear genotypes. This coincided with increased production of 16-feruloyloxypalmitic acid, a conjugated compound between phenols and esters during the polymerization for suberin formation. Collectively, our data from the combined three omics demonstrate that russeting in sand pear is a complex process involving the biosynthesis and transport of suberin and many other secondary metabolites.

Effect of ALA-PDT on inhibition of oral precancerous cell growth and its related mechanisms.

BACKROUND: Early treatment of oral precancerous lesions is considered as a key strategy for in oral carcinogenesis prevention. Increasing evidence has suggested that the transforming growth factor beta (TGF-ß) signaling pathway is tightly involved in the process of oral-carcinogenesis. In this study, we investigated the inhibition effect and potential mechanism of 5-aminolaevulinic acid photodynamic therapy (ALA-PDT) in human oral precancerous cells via TGF-ß pathway. MATERIALS AND METHODS: Here, the dysplastic oral keratinocyte (DOK) cells were incubated with ALA concentration of 1 mM/mL for 4 h and then irradiated with a Helium-Neon (He-Ne) ion laser at 633 nm (200 mW/cm2). The control cells were cultured in Dulbecco's modified Eagle's medium (DMEM) medium. We analyzed the differentially expressed genes and correlated pathways in oral precancerous cells following ALA-PDT using Affymetrix microarrays. TGF-ß pathway was analyzed by quantitative real-time polymerase chain reaction (RT-qPCR) and western blotting. Bioinformatics analysis was performed to evaluate the expression of TGF-ß1 in human oral cancer samples and adjacent normal samples. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), flow cytometry, 2'-7'-dichlorodihydrofluorescein diacetate (DCFH-DA), and wound healing assay were used to assess the effects of ALA-PDT plus TGF-ß receptor inhibitor (LY2109761) in DOK cells. RESULTS: The TGF-ß signaling could exert in suppressive effects on DOK cells after ALA-PDT. The cell proliferation and migration rate of DOK cells was significantly reduced and apoptosis and ROS generation induced more effectively by ALA-PDT combined with LY2109761. Furthermore, cell cycle analysis revealed that the combined treatment resulted in G0/G1 phase arrest. CONCLUSIONS: ALA-PDT suppresses the growth of oral precancerous cells by regulating the TGF-ß signaling pathway, and its suppressive effect was enhanced using LY2109761. These results indicate that it could be a promising alternative treatment against oral precancerous lesions.

Identification of putative cytochrome P450 monooxygenase genes from the small white butterfly, Pieris rapae (Lepidoptera: Pieridae), and their response to insecticides.

The small white butterfly, Pieris rapae (Lepidoptera: Pieridae), is an important pest on Brassicaceae plants, causing heavy crop loss each year. Cytochrome P450 monooxygenase (CYP) is a superfamily of enzymes involved in the detoxification of various xenobiotic compounds, including insecticides. However, little is known about the role of CYP genes in P. rapae. In this study, we identified 63 CYP genes in P. rapae, and analyzed their phylogenetic relationships, exon-intron structures and genomic locations. Moreover, our insecticide-response transcription profiling showed that LD5 doses of lambda-cyhalothrin, chlorantraniliprole, and abamectin significantly increased expression of five (CYP4M59, CYP6AE119, CYP6AE120, CYP6AE121, and CYP6BD18), three (CYP4AU1, CYP6AE120, and CYP6AW1), and five (CYP4L40, CYP4AU1, CYP6AE119, CYP6AW1, and CYP6BD19) CYP genes, respectively; and LD20 doses of the three pesticides significantly upregulated six (CYP4M59, CYP6AE119, CYP6AE120, CYP6AE121, CYP4AU1, and CYP6BD18), six (CYP4G168, CYP4L40, CYP4AU1, CYP6AE120, CYP6AW1, and CYP6BD19), and five (CYP4L40, CYP4AU1, CYP6AB108, CYP6AE119, and CYP6BD19) genes, respectively. When we used LD50 doses of the three insecticides, we reported significantly elevated expression levels of five (CYP4M59, CYP6AE119, CYP6AE120, CYP6BD17, and CYP6BD18), eight (CYP4G168, CYP4L40, CYP4AU1, CYP6AE120, CYP6AE121, CYP6AW1, CYP6BD18, and CYP6BD19), and six (CYP4L40, CYP4S34, CYP6AB108, CYP6AE119, CYP6AE120, and CYP6BD19) genes, respectively. Our expression analysis also revealed that five (CYP4G168, CYP4G169, CYP4S34, CYP6AW1, and CYP6CT3) and three (CYP4L40, CYP6AN33, and CYP6BD17) CYP genes were mainly expressed in the midgut and fat body, respectively, and one CYP gene (CYP6AE119) in the Malpighian tubules. This is the first large-scale report into the characterization of CYP genes in P. rapae.

Characterization of candidate odorant-binding proteins and chemosensory proteins in the tea geometrid Ectropis obliqua Prout (Lepidoptera: Geometridae).

Insects rely heavily on their sophisticated chemosensory systems to locate host plants and find conspecific mates. Although the molecular mechanisms of odorant recognition in many Lepidoptera species have been well explored, limited information has been reported on the geometrid moth Ectropis obliqua Prout, an economically important pest of tea plants. In the current study, we first attempted to identify and characterize the putative olfactory carrier proteins, including odorant-binding proteins (OBPs) and chemosensory proteins (CSPs). By analyzing previously obtained transcriptomic data of third-instar larvae, five OBPs and 14 CSPs in E. obliqua were identified. Sequence alignment, conserved motif identification, and phylogenetic analysis suggested that candidate proteins have typical characteristics of the insect OBP or CSP family. The expression patterns regarding life stages and different tissues were determined by quantitative real-time PCR. The results revealed that four transcripts (OBP2, OBP4 and CSP8, CSP10) had larvae preferential expression profiles and nine candidate genes (PBP1, OBP1 and CSP2, CSP4, CSP5, CSP6, CSP7, CSP11, and CSP13) were adult-biased expressed. Further specific tissue expression profile evaluation showed that OBP1, OBP2, OBP4, and PBP1 were highly expressed at olfactory organs, implying their potential involvement in chemical cue detection, whereas CSPs were ubiquitously detected among all of the tested tissues and could be associated with multiple physiological functions. This study provided a foundation for understanding the physiological functions of OBPs and CSPs in E. obliqua and will help pave the way for the development of a new environmental friendly pest management strategy against the tea geometrid moth.

Identification and characterisation of seventeen glutathione S-transferase genes from the cabbage white butterfly Pieris rapae.

Insect glutathione S-transferases (GSTs) play essential roles in the detoxification of insecticides and other xenobiotic compounds. The cabbage white butterfly, Pieris rapae, is an economically important agricultural pest. In this study, 17 cDNA sequences encoding putative GSTs were identified in P. rapae. All cDNAs include a complete open reading frame and were designated PrGSTd1-PrGSTz2. Based on phylogenetic analysis, PrGSTs were divided into six classes (delta, epsilon, omega, sigma, theta and zeta). The exon-intron organizations of these PrGSTs were also analysed. Recombinant proteins of eight PrGSTs (PrGSTD1, PrGSTD2, PrGSTE1, PrGSTE2, PrGSTO1, PrGSTS1, PrGSTT1 and PrGSTZ1) were heterologously expressed in Escherichia coli, and all of these proteins displayed glutathione-conjugating activity towards 1-chloro-2,4-dinitrobenzene (CDNB). Expression patterns in various larval tissues, at different life stages, and following exposure to sublethal doses of abamectin, chlorantraniliprole or lambda-cyhalothrin were determined by reverse transcription-quantitative PCR. The results showed that PrGSTe3, PrGSTs1, PrGSTs2, and PrGSTs4 were mainly transcribed in the fat body, while PrGSTe2 was expressed predominantly in the Malpighian tubules. Four genes (PrGSTe2, PrGSTo4, PrGSTs4 and PrGSTt1) were mainly expressed in fourth-instar larvae, while others were ubiquitously expressed in egg, larval, pupa and/or adult stages. Abamectin treatment significantly upregulated ten genes (PrGSTd1, PrGSTd3, PrGSTe1, PrGSTe2, PrGSTo1, PrGSTo3, PrGSTs1, PrGSTs3, PrGSTs4 and PrGSTt1). Chlorantraniliprole and lambda-cyhalothrin treatment significantly upregulated nine genes (PrGSTd1, PrGSTd2, PrGSTe1, PrGSTe2, PrGSTe3, PrGSTs1, PrGSTs3, PrGSTs4 and PrGSTz1) and ten genes (PrGSTd1, PrGSTd3, PrGSTe1, PrGSTe2, PrGSTo1, PrGSTo2, PrGSTs1, PrGSTs2, PrGSTs3 and PrGSTz2), respectively. These GSTs are potentially involved in the detoxification of insecticides.

Expression and regulation of pear 1-aminocyclopropane-1-carboxylic acid synthase gene (PpACS1a) during fruit ripening, under salicylic acid and indole-3-acetic acid treatment, and in diseased fruit.

In plants, the level of ethylene is determined by the activity of the key enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS). A gene encoding an ACC synthase protein was isolated from pear (Pyrus pyrifolia). This gene designated PpACS1a (GenBank accession no. KC632526) was 1488 bp in length with an open reading frame (ORF) encoding a protein of 495 amino acids that shared high similarity with other pear ACC synthase proteins. The PpACS1a was grouped into type-1 subfamily of plant ACS based on its conserved domain and phylogenetic status. Real-time quantitative PCR indicated that PpACS1a was differentially expressed in pear tissues and predominantly expressed in anthers. The expression signal of PpACS1a was also detected in fruit and leaves, but no signal was detected in shoots and petals. Furthermore, the PpACS1a expression was regulated during fruit ripening. In addition, the PpACS1a gene expression was regulated by salicylic acid (SA) and indole-3-acetic acid (IAA) in fruit. Moreover, the expression of the PpACS1a was up-regulated in diseased pear fruit. These results indicated that PpACS1a might be involved in fruit ripening and response to SA, IAA and disease.

Catalyst-Free Visible Light-Driven Hydrosulfonylation of Alkenes and Alkynes with Sulfonyl Chlorides in Water.

A convenient and sustainable method for synthesizing sulfonyl-containing compounds through a catalyst-free aqueous-phase hydrosulfonylation of alkenes and alkynes with sulfonyl chlorides under visible light irradiation is presented. Unactivated alkenes, electron-deficient alkenes, alkyl and aryl alkynes can be hydrosulfonylated with various sulfonyl chlorides at room temperature with excellent yields and geometric selectivities by using tris(trimethylsilyl)silane as a hydrogen atom donor and silyl radical precursor to activate sulfonyl chlorides. Mechanistic studies revealed that the photolysis of tris(trimethylsilyl)silane in aqueous solution to produce silyl radical is crucial for the success of this reaction.

Novel Flavonol Alkaloids in Green Tea: Synthesis, Detection, and Anti-Alzheimer's Disease Effect in a Transgenic Caenorhabditis elegans CL4176 Model.

Novel N-ethy-2-pyrrolidinone-substituted flavonols, myricetin alkaloids A-C (1-3), quercetin alkaloids A-C (4a, 4b, and 5), and kaempferol alkaloids A and B (6 and 7), were prepared from thermal reaction products of myricetin, quercetin, kaempferol─l-theanine, respectively. We used HPLC-ESI-HRMS/MS to detect 1-7 in 14 cultivars of green tea and found that they were all present in "Shuchazao," "Longjing 43", "Fudingdabai", and "Zhongcha 108" green teas. The structures of 1-4 and 6 were determined by extensive 1D and 2D NMR spectroscopies. These flavonol alkaloids along with their skeletal flavonols were assessed for anti-Alzheimer's disease effect based on molecular docking, acetylcholinesterase inhibition, and the transgenic Caenorhabditis elegans CL4176 model. Compound 7 strongly binds to the protein amyloid ß (Aß1-42) through hydrogen bonds (BE: -9.5 kcal/mol, Ki: 114.3 nM). Compound 3 (100 µM) is the strongest one in significantly extending the mean lifespan (13.4 ± 0.5 d, 43.0% promotion), delaying the Aß1-42-induced paralysis (PT50: 40.7 ± 1.9 h, 17.1% promotion), enhancing the locomotion (140.0% promotion at 48 h), and alleviating glutamic acid (Glu)-induced neurotoxicity (153.5% promotion at 48 h) of CL4176 worms (p < 0.0001).

Pear ACO genes encoding putative 1-aminocyclopropane-1-carboxylate oxidase homologs are functionally expressed during fruit ripening and involved in response to salicylic acid.

1-Aminocyclopropane-1-carboxylate (ACC) oxidase catalyzes the final reaction of the ethylene biosynthetic pathway, converting ACC into ethylene. Past studies have shown a possible link between ACC oxidase and salicylic acid during fruit ripening in pear, but the relationship has received no more than modest study at the gene expression level. In this study, two cDNA clones encoding putative ACC oxidase, PpACO1 and PpACO2, were isolated from a cDNA library constructed by our own laboratory and produced using mRNA from mesocarp of pear (Pyrus pyrifolia Nakai. cv.Whangkeumbae). One cDNA clone, designated PpACO1 (GenBank accession No. JN807390), comprised an open reading frame of 945 bp encoding a protein of 314 amino acids. The other cDNA, designated PpACO2 (GenBank accession No. JN807392), encodes a protein with 322 amino acids that shares high similarity with the known plant ACOs. Using PCR amplification techniques, two genomic clones corresponding to PpACO1 and PpACO2 were isolated and shown to contain independently three introns with typical GT/AG boundaries defining the splice junctions. The PpACO1 gene product shared 99 % identity with an ACC oxidase from pear (Pyrus × bretschneideri Rehd.cv.Yali), and phylogenetic analyses clearly placed the gene product in the ACC oxidase cluster of the pear 2-oxoglutarate-dependent dioxygenase superfamily tree. Quantitative RT-PCR analysis indicated that the two PpACO genes are differentially expressed in pear tissues. PpACO1 and PpACO2 were predominantly expressed in fruit. The transcripts of PpACO1 were accumulated at relatively low levels in early fruit, but strongly high levels in fruit ripening and senescence stages, while the transcripts of PpACO2 were accumulated at higher levels in early fruit and much lower levels with further fruit cell development than the transcripts of PpACO1. In addition, PpACO1 gene was down-regulated in fruit by salicylic acid (SA). Nevertheless, PpACO2 gene was dramatically up-regulated in fruit by SA. These results suggested that the PpACOs may participate in regulation of fruit ripening and in response to SA in pear.

Chinese Herbal Medicine Adjusting Brain Microenvironment via Mediating Central Nervous System Lymphatic Drainage in Alzheimer's Disease.

Due to its complex pathogenesis and lack of effective therapeutic methods, Alzheimer's disease (AD) has become a severe public health problem worldwide. Recent studies have discovered the function of central nervous system lymphatic drainage, which provides a new strategy for the treatment of AD. Chinese herbal medicine (CHM) has been considered as a cure for AD for hundreds of years in China, and its effect on scavenging ß-amyloid protein in the brain of AD patients has been confirmed. In this review, the mechanism of central nervous system lymphatic drainage and the regulatory functions of CHM on correlation factors were briefly summarized. The advances in our understanding regarding the treatment of AD via regulating the central lymphatic system with CHM will promote the clinical application of CHM in AD patients and the discovery of new therapeutic drugs.

Reference Genes for Expression Analyses by qRT-PCR in Phthorimaea operculella (Lepidoptera: Gelechiidae).

Due to a lack of effective internal references, studies on functional genes in Phthorimaea operculella, a serious Lepidopteran pest attacking potatoes worldwide, have been greatly limited. To select suitable endogenous controls, ten housekeeping genes of actin (ACT), α-tubulin (α-TUB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor 1α (EF1α), 18S and 28S ribosomal RNA (18S, 28S), ribosomal protein genes RPL4, RPL13 and RPL27 and superoxide dismutase (SOD) were tested. Their expression levels were determined under three different experimental conditions (developmental stages, tissues/organs and temperatures) using qRT-PCR technology. The stability was evaluated with five methods (Ct value, geNorm, NormFinder, BestKeeper and RefFinder). The results clarified that RPL13, EF1α and RPL27 are ranked as the best reference gene combination for measuring gene expression levels among different developing stages and under various temperatures; EF1α and RPL13 are recommended to normalize the gene expression levels among diverse tissues. EF1α and RPL13 are the best reference genes in all the experimental conditions. To validate the utility of the selected reference pair, EF1α and RPL13, we estimated the tissue-biased expression level of chitin synthase A gene (PoChSA). As expected, PoChSA was abundantly expressed in ectodermally derived epidermal cells, and lowly transcribed in the midgut. These findings will lay the foundation for future research on the molecular physiology and biochemistry of P. operculella.

Dynamics of Aboveground Natural Enemies of Grasshoppers, and Biodiversity after Application of Paranosema locustae in Rangeland.

Substantial harm to ecosystems from the use of chemical pesticides has led to an increasing interest in the use of biopesticides to control grasshoppers in rangelands, including China. One such potential biopesticide for control of grasshoppers is the fungus Paranosema locustae. In this study, the dynamics of aboveground natural enemies of grasshoppers and arthropod diversity 0-9 years after application of P. locustae were investigated in rangeland in Qinghai Plateau, China. We found that the number of species and of individuals of aboveground natural enemies increased by 17-250% and 40-126%, respectively, after spraying P. locustae, and that the main natural enemies showed three peaks after treatment. The conventional indices of species diversity (H') and evenness (J') increased by 11-267% and 13-171%, respectively, after treatment with P. locustae. The results showed the positive effects of P. locustae on aboveground natural enemies and biodiversity in an arthropod community in Chinese rangeland. Paranosema locustae is thought to be a safe biological control agent for grasshopper management in Northwestern China.

Fracture strength of custom-fabricated Celay all-ceramic post and core restored endodontically treated teeth.

BACKGROUND: The increased use of all-ceramic crown provides a rationale for tooth-colored core. Due to superior mechanical properties, Vita Celay infiltration ceramic developed for crown and bridge works presents the potential for fabricating all-ceramic posts and cores in one piece. This study was conducted to compare the fracture strength of endodontically treated teeth which were thereafter given different types of posts and cores and crowns restoration, respectively. The evaluated post-and-core systems are: custom-fabricated Celay all-ceramic post-core, custom cast metal post-core, and prefabricated stainless steel post (Parapost) with and without 2.0 mm dentine ferrule. METHODS: Sixty freshly extracted human maxillary central incisors were endodontically treated and randomly divided into five groups with 12 samples each. Group A: Celay ceramic post-cores restored teeth with 2.0 mm dentine ferrule. Group B: Celay ceramic post-cores restored teeth with no dentine ferrule. Group C: cast metal post-cores restored teeth with 2.0 mm dentine ferrule. Group D: cast metal post-cores restored teeth with no dentine ferrule. Group E: prefabricated post and composite cores restored teeth with 2.0 mm dentine ferrule. All teeth were restored with Celay ceramic crowns. Each specimen was subjected to a load at a 45-degree angle to the long axis on MTS 810 material testing machine until failure, at crosshead speed of 0.02 cm/minute. Analysis of variance followed by the Newman-Keuls pairwise multiple comparison tests were used to compare the results of the groups tested. RESULTS: There was a statistically significant difference among the five groups (P < 0.01). Celay ceramic post-cores restored teeth with 2.0 mm dentine ferrule [(758.35+/-19.26) N] and cast metal post-cores restored teeth with 2.0 mm dentine ferrule [(756.63+/-66.22) N] had a significantly greater mean fracture strength than the other three groups in which no significant difference was observed. The 2.0 mm dentine ferrule could cause significant fracture resistance alteration of Celay post-core restored teeth. CONCLUSIONS: When covered with Celay ceramic crowns, Celay post-cores restored teeth with 2.0 mm dentine ferrule and cast metal post-cores restored teeth with 2.0 mm dentine ferrule have similar fracture strength. There was a statistically significant difference between the fracture resistance of Celay post-core restored teeth with and without 2.0 mm dentine ferrule.

Complementary DNA cloning of the pear 1-aminocyclopropane-1-carboxylic acid oxidase gene and agrobacterium-mediated anti-sense genetic transformation.

The aim of the present study was to genetically modify plantlets of the Chinese yali pear to reduce their expression of ripening-associated 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) and therefore increase the shelf-life of the fruit. Primers were designed with selectivity for the conserved regions of published ACO gene sequences, and yali complementary DNA (cDNA) cloning was performed by reverse transcription quantitative polymerase chain reaction (PCR). The obtained cDNA fragment contained 831 base pairs, encoding 276 amino acid residues, and shared no less than 94% nucleotide sequence identity with other published ACO genes. The cDNA fragment was inversely inserted into a pBI121 expression vector, between the cauliflower mosaic virus 35S promoter and the nopaline synthase terminator, in order to construct the anti­sense expression vector of the ACO gene; it was transfected into cultured yali plants using Agrobacterium LBA4404. Four independent transgenic lines of pear plantlets were obtained and validated by PCR analysis. A Southern blot assay revealed that there were three transgenic lines containing a single copy of exogenous gene and one line with double copies. The present study provided germplasm resources for the cultivation of novel storage varieties of pears, therefore providing a reference for further applications of anti­sense RNA technology in the genetic improvement of pears and other fruit.

Two pear glutathione S-transferases genes are regulated during fruit development and involved in response to salicylic acid, auxin, and glucose signaling.

Two genes encoding putative glutathione S-transferase proteins were isolated from pear (Pyrus pyrifolia) and designated PpGST1 and PpGST2. The deduced PpGST1 and PpGST2 proteins contain conserved Glutathione S-transferase N-terminal domain (GST_N) and Glutathione S-transferase, C-terminal domain (GST_C). Using PCR amplification technique, the genomic clones corresponding to PpGST1 and PpGST2 were isolated and shown to contain two introns and a singal intron respectively with typical GT/AG boundaries defining the splice junctions. Phylogenetic analysis clearly demonstrated that PpGST1 belonged to Phi class of GST superfamilies and had high homology with apple MdGST, while PpGST2 was classified into the Tau class of GST superfamilies. The expression of PpGST1 and PpGST2 genes was developmentally regulated in fruit. Further study demonstrated that PpGST1 and PpGST2 expression was remarkably induced by glucose, salicylic acid (SA) and indole-3-aceticacid (IAA) treatments in pear fruit, and in diseased fruit. These data suggested that PpGST1 and PpGST2 might be involved in response to sugar, SA, and IAA signaling during fruit development of pear.

Colorectal cancer screening: comparison of transferrin and immuno fecal occult blood test.

AIM: To evaluate the sensitivity and specificity of transfesrrin dipstick test (Tf) in colorectal cancer (CRC) screening and precancerous lesions screening. METHODS: Eight hundreds and sixty-one individuals at high-risk for CRC were recruited. Six hundreds and eleven subsequently received the three fecal occult blood tests and colonoscopy with biopsy performed as needed. Fecal samples were obtained on the day before colonoscopy. Tf, immuno fecal occult blood test (IFOBT) and guaiac fecal occult blood test (g-FOBT) were performed simultaneously on the same stool. To minimize false-negative cases, all subjects with negative samples were asked to provide an additional stool specimen for a second test even a third test. If the results were all negative after testing three repeated samples, the subject was considered a true negative. The performance characteristics of Tf for detecting CRC and precancerous lesions were examined and compared to those of IFOBT and the combination of Tf, IFOBT and g-FOBT. RESULTS: A total of six hundreds and eleven subjects met the study criteria including 25 with CRC and 60 with precancerous lesions. Sensitivity for detecting CRC was 92% for Tf and 96% for IFOBT, specificities of Tf and IFOBT were both 72.0% (95% CI: 68.2%-75.5%; χ² = 0.4, P > 0.05); positive likelihood ratios of those were 3.3 (95% CI: 2.8-3.9) and 3.4 (95% CI: 2.9-4.0), respectively. In precancerous lesions, sensitivities for Tf and IFOBT were 50% and 58%, respectively (χ² = 0.8, P > 0.05); specificities of Tf and IFOBT were 71.5% (95% CI: 67.6%-75.1%) and 72.2% (95% CI: 68.4%-75.8%); positive likelihood ratios of those were 1.8 (95% CI: 1.3-2.3) and 2.1 (95% CI: 1.6-2.7), respectively; compared to IFOBT, g-FOBT+ Tf+ IFOBT had a significantly higher positive rate for precancerous lesions (83% vs 58%, respectively; χ² = 9.1, P < 0.05). In patients with CRC and precancerous lesions, the sensitivities of Tf and IFOBT were 62% and 69% (χ² = 0.9, P > 0.05); specificities of those were 74.5% (95% CI: 70.6%-78.1%) and 75.5% (95% CI: 71.6%-79.0%); positive likelihood ratios of those were 2.5 (95% CI: 2.0-3.1) and 2.8 (95% CI: 2.3-3.5). Compared to IFOBT alone, combining g-FOBT, IFOBT and Tf led to significantly increased sensitivity for detecting CRC and cancerous lesions (69% vs 88%, respectively; χ² = 9.0, P < 0.05). CONCLUSION: Tf dipstick test might be used as an additional tool for CRC and precancerous lesions screening in a high-risk cohort.

[Preliminary report about the screening program for colorectal cancer by sequential fecal occult blood in Wuhan area for 4 years].

OBJECTIVE: To evaluate the sequential fecal occult blood test (SFOBT) program for the screening of colorectal cancer and elucidate the prevalence of colorectal cancer in Wuhan area. METHODS: At 19 screening sites, 63,961 residents were recruited as target population according to random cluster and stratified sampling for four years (between 2005 and 2008). Residents aged over 40 years old received SFOBT. Those with positive SFOBT underwent colonoscopy. RESULTS: The target population was 63,961. There were 25,837 people whose age was over 40. Finally, 7784 participants received the SFOBT screening, with a medium age of 56 years old. The positive rate of SFOBT was 12.3% (956 persons). Of the 956 persons, 240 participants underwent colonoscopy. Colorectal cancer was found in 14 cases (6.5%), gastric cancer in 2 cases (0.9%), colorectal adenoma in 53 cases(24.8%), colorectal inflammation in 80 cases (37.3%) and hemorrhoids in 65 cases (30.4%). CONCLUSIONS: The prevalence of colorectal cancer is relatively high in Wuhan area. The SFOBT is available and feasible in screening early changes of colorectal cancer.