MiR-9-5p inhibits mitochondrial damage and oxidative stress in AD cell models by targeting GSK-3ß.

This study aims to investigate the effects and underlying mechanisms of overexpression microRNA-9-5p (miR-9-5p) on the Aß-induced mouse hippocampal neuron cell line HT22. Different concentrations of Aß25-35 (10, 20, 40, 80, and 160 µM) treatment were used to establish AD model in HT22 cells. The CCK-8 assay was used to measure the cell viability. The mRNA expression levels of miR-9-5p and glycogen synthase kinase-3ß (GSK-3ß) were determined by RT-qPCR. HT22 cell apoptosis was analyzed flow cytometry. MiR-9-5p was down-regulated in Aß25-35-induced HT22 cells. GSK-3ß is a functional target for miR-9-5p. MiR-9-5p overexpression inhibited Aß25-35-induced mitochondrial dysfunction, cell apoptosis, and oxidative stress by regulating GSK-3ß expression in HT22 cells. Furthermore, through targeting GSK-3ß, overexpression of miR-9-5p partly activated nuclear factor Nrf2/Keap1 signaling, including part increases of Nrf2, HO-1, SOD-1, GCLC expression and slight decrease of Keap1 expression. Our results showed miR-9-5p may play a powerful role in the pathogenesis of AD.

[Laparoscopic cystogastrostomy with posterior approach for pancreatic pseudocyst drainage].

OBJECTIVE: To determine the clinical value of laparoscopic cystogastrostomy in the treatment of pancreatic pseudocyst. METHODS: Twenty-one patients with pancreatic pseudocyst received total laparoscopic cystogastrostomy. The data on intra-operative bleeding, operative time, post-operative time to get out of bed, time of first flatus/bowel motion, complication and duration of hospital stay were observed and analyzed retrospective1y. RESULTS: Twenty-one patients were successfully carried out the laparoscopic surgery. The average operation time was 90(62-120) min. The blood loss was less than 100 mL in all patients. The average time of hospital stay was 8 d. After 12-18 month follow-up, all patients recovered smoothly without any complication. CONCLUSION: Total laparoscopic cystogastrostomy with the posterior approach is a feasible, safe and minimal invasive procedure for pancreatic pseudocyst, which can be recommended to the clinical application.

Metabolic engineering of Saccharomyces cerevisiae for caffeine and theobromine production.

Caffeine (1, 3, 7-trimethylxanthine) and theobromine (3, 7-dimethylxanthine) are the major purine alkaloids in plants, e.g., tea (Camellia sinensis) and coffee (Coffea arabica). Caffeine is a major component of coffee and is used widely in food and beverage industries. Most of the enzymes involved in the caffeine biosynthetic pathway have been reported previously. Here, we demonstrated the biosynthesis of caffeine (0.38 mg/L) by co-expression of Coffea arabica xanthosine methyltransferase (CaXMT) and Camellia sinensis caffeine synthase (TCS) in Saccharomyces cerevisiae. Furthermore, we endeavored to develop this production platform for making other purine-based alkaloids. To increase the catalytic activity of TCS in an effort to increase theobromine production, we identified four amino acid residues based on structural analyses of 3D-model of TCS. Two TCS1 mutants (Val317Met and Phe217Trp) slightly increased in theobromine accumulation and simultaneously decreased in caffeine production. The application and further optimization of this biosynthetic platform are discussed.

Clinicopathological features of hepatitis B virus recurrence after liver transplantation: eleven-year experience.

OBJECTIVE: We sought to investigate new changes in the clinical pathology of hepatitis B virus (HBV) recurrence after orthotopic liver transplantation (OLT) in era of new nucleoside or nucleotide analogues. METHODS: One hundred and eighty-four adult patients who underwent OLT for HBV-related end-stage liver disease between 1999 and 2010 were enrolled in this study. Of these patients, 156 received lamivudine (LAM) plus hepatitis B immune globulin (HBIG) and 28 were treated with LAM. The liver function, serologic parameters and HBV-DNA of the 184 recipients were followed up, and clinical pathological characteristics of grafts with HBV recurrence were examined in this study. RESULTS: One hundred and seventy-nine (97%) were alive at their last follow-up and eleven (6%) had developed HBV recurrence at a median of 22 (range 6 to 46) months post-OLT. Two of the 11 recipients were detected with HBV-S gene mutation, and 5 were tested with YMDD mutation. Four recipients who died of irreversible graft dysfunction secondary to HBV recurrence, developed fibrosing cholestatic hepatitis (FCH) because of no effective antiviral agents available in the early stages of HBV recurrence after OLT. Six recipients who received adefovir (ADV) (and Entecavir, ETV) in the early stages of HBV recurrence after OLT achieved improvement in hepatic histology. CONCLUSIONS: HBV recurrence post-OLT could be controlled at an acceptable level for a long time and the recipients could achieve long-term survival by using new antiviral agents, instead of advancing into FCH in the short term after HBV recurrence.

[Meta-analysis of total thyroidectomy for multinodular goiter].

OBJECTIVE: To systematically evaluate the efficiency and safety of total thyroidetomy (including near-total tyhroidectomy) versus subtotal thyroidectomy for multinodular goiter. METHODS: The literatures were searched from Cochrane Library, PubMed, Embase, Chinese Biological Medical Datebase, Chinese National Knowledge Infrastructure, and Chinese Science and Technology Journal Full-text Database as of November 2013. We included all randomizad controlled trials on total (including near-total) versus subtotal thyroidectomy in the treatment of multinodular goiter. The collecting of data and quality assessment were respectively completed by 2 researchers. RevMan5.1 software was used for Meta-analysis. RESULTS: We collected 7 literatures conforming to the standard, incuding 2 192 patients. The Metaanalysis outcomes showed that total thyroidectomy was associated with lower nodule recurrence rate (OR=0.13, 95% CI: 0.07-0.22, P<0.001) and higher in transient hypoparathyroidism rate (OR=2.33, 95% CI: 1.72-3.17, P<0.001). However, no statistical difference was seen comparing total and subtotal thyroidectomy in permanent recurrent laryngeal nerve paralysis rate (OR= 0.81, 95% CI: 0.24-2.74, P=0.74) and permanent hypoparathyroidism rate (OR=2.94, 95% CI: 0.48- 18.11, P=0.24). CONCLUSION: Nodule recurrence rate of total thyroidectomy for multinodular goiter is lower than subtotal thyroidectomy and does not increase permanent complications.

Genetic and biochemical basis for alternative routes of tocotrienol biosynthesis for enhanced vitamin E antioxidant production.

Vitamin E tocotrienol synthesis in monocots requires homogentisate geranylgeranyl transferase (HGGT), which catalyzes the condensation of homogentisate and the unsaturated C20 isoprenoid geranylgeranyl diphosphate (GGDP). By contrast, vitamin E tocopherol synthesis is mediated by homogentisate phytyltransferase (HPT), which condenses homogentisate and the saturated C20 isoprenoid phytyl diphosphate (PDP). An HGGT-independent pathway for tocotrienol synthesis has also been shown to occur by de-regulation of homogentisate synthesis. In this paper, the basis for this pathway and its impact on vitamin E production when combined with HGGT are explored. An Arabidopsis line was initially developed that accumulates tocotrienols and homogentisate by co-expression of Arabidopsis hydroxyphenylpyruvate dioxygenase (HPPD) and Escherichia coli bi-functional chorismate mutase/prephenate dehydrogenase (TyrA). When crossed into the vte2-1 HPT null mutant, tocotrienol production was lost, indicating that HPT catalyzes tocotrienol synthesis in HPPD/TyrA-expressing plants by atypical use of GGDP as a substrate. Consistent with this, recombinant Arabidopsis HPT preferentially catalyzed in vitro production of the tocotrienol precursor geranylgeranyl benzoquinol only when presented with high molar ratios of GGDP:PDP. In addition, tocotrienol levels were highest in early growth stages in HPPD/TyrA lines, but decreased strongly relative to tocopherols during later growth stages when PDP is known to accumulate. Collectively, these results indicate that HPPD/TyrA-induced tocotrienol production requires HPT and occurs upon enrichment of GGDP relative to PDP in prenyl diphosphate pools. Finally, combined expression of HPPD/TyrA and HGGT in Arabidopsis leaves and seeds resulted in large additive increases in vitamin E production, indicating that homogentisate concentrations limit HGGT-catalyzed tocotrienol synthesis.

Vitamin E biosynthesis: functional characterization of the monocot homogentisate geranylgeranyl transferase.

The biosynthesis of the tocotrienol and tocopherol forms of vitamin E is initiated by prenylation of homogentisate. Geranylgeranyl diphosphate (GGDP) is the prenyl donor for tocotrienol synthesis, whereas phytyl diphosphate (PDP) is the prenyl donor for tocopherol synthesis. We have previously shown that tocotrienol synthesis is initiated in monocot seeds by homogentisate geranylgeranyl transferase (HGGT). This enzyme is related to homogentisate phytyltransferase (HPT), which catalyzes the prenylation step in tocopherol synthesis. Here we show that monocot HGGT is localized in the plastid and expressed primarily in seed endosperm. Despite the close structural relationship of monocot HGGT and HPT, these enzymes were found to have distinct substrate specificities. Barley (Hordeum vulgare cv. Morex) HGGT expressed in insect cells was six times more active with GGDP than with PDP, whereas the Arabidopsis HPT was nine times more active with PDP than with GGDP. However, only small differences were detected in the apparent Km values of barley HGGT for GGDP and PDP. Consistent with its in vitro substrate properties, barley HGGT generated a mixture of tocotrienols and tocopherols when expressed in the vitamin E-null vte2-1 mutant lacking a functional HPT. Relative levels of tocotrienols and tocopherols produced in vte2-1 differed between organs and growth stages, reflective of the composition of plastidic pools of GGDP and PDP. In addition, HGGT was able to functionally substitute for HPT to rescue vte2-1-associated phenotypes, including reduced seed viability and increased fatty acid oxidation of seed lipids. Overall, we show that monocot HGGT is biochemically distinct from HPT, but can replace HPT in important vitamin E-related physiological processes.

The cytochrome P450 CYP86A22 is a fatty acyl-CoA omega-hydroxylase essential for Estolide synthesis in the stigma of Petunia hybrida.

The stigmatic estolide is a lipid-based polyester constituting the major component of exudate in solanaceous plants. Although the exudate is believed to play important roles in the pollination process, the biosynthetic pathway of stigmatic estolide, including genes encoding the key enzymes, remains unknown. Here we report the cloning and characterization of the cytochrome P450 gene CYP86A22, which encodes a fatty acyl-CoA omega-hydroxylase involved in estolide biosynthesis in the stigma of Petunia hybrida. A CYP86A22 cDNA was isolated from a developing stigma cDNA library, and the corresponding gene was shown to express predominantly in the developing stigma. Among six P450 genes isolated from this library, only CYP86A22 was implicated in omega-hydroxylation following RNA interference (RNAi)-mediated suppression. Unlike wild-type plants in which omega-hydroxy fatty acids (mainly in the form of 18-hydroxy oleic acid and 18-hydroxy linoleic acid) compose 96% of total stigma fatty acids, the omega-hydroxy fatty acids were essentially absent in the stigmas from 18 of 46 CYP86A22-RNAi transgenic plants and had varying levels of suppression in the remaining 28 plants. Furthermore, lipids in the 18 CYP86A22-RNAi stigmas were predominantly triacylglycerols and diacylglycerols instead of the estolides, which characterize the wild-type stigma. Analyses of recombinant CYP86A22 conclusively demonstrated that this P450 is a omega-hydroxylase with a substrate preference for both saturated and unsaturated acyl-CoAs rather than free fatty acids. We conclude that the cytochrome P450 enzyme CYP86A22 is the key fatty acyl-CoA omega-hydroxylase essential for the production of omega-hydroxy fatty acids and the biosynthesis of triacylglycerol-/diacylglycerol-based estolide polyesters in the petunia stigma.

Cuticular wax biosynthesis in petunia petals: cloning and characterization of an alcohol-acyltransferase that synthesizes wax-esters.

The surface of plants is covered by cuticular wax, which contains a mixture of very long-chain fatty acid (VLCFA) derivatives. This wax surface provides a hydrophobic barrier which reduces non-stomatal water loss. One component of the cuticular wax is the alkyl esters, which typically contain a VLCFA esterified to an alcohol of a similar length. As part of an EST project, we recently identified an acyltransferase with 19% sequence identity (amino acid) to a bacterial 'bifunctional' wax-ester synthase/diacylglycerol acyltransferase (WS/DGAT). Northern analysis revealed that this petunia homologue was expressed predominantly within the petals. The cDNA encoding the WS/DGAT homologue was introduced into a yeast strain deficient in triacylglycerol biosynthesis. The expressed protein failed to restore triacylglycerol biosynthesis, indicating that it lacked DGAT activity. However, isoamyl esters of fatty acids were detected, which suggested that the petunia cDNA encoded a wax-synthase. Waxes were extracted from petunia petals and leaves. The petal wax extract was rich in VLCFA esters of methyl, isoamyl, and short-to-medium straight chain alcohols (C4-C12). These low molecular weight wax-esters were not present in leaf wax. In-vitro enzymes assays were performed using the heterologously expressed protein and 14C-labelled substrates. The expressed protein was membrane bound, and displayed a preference for medium chain alcohols and saturated very long-chain acyl-CoAs. In fact, the activity would be sufficient to produce most of the low molecular wax-esters present in petals, with methyl-esters being the exception. This work is the first characterization of a eukaryotic protein from the WS/DGAT family.