Monoallelic expression of nine imprinted genes in the sheep embryo occurs after the blastocyst stage

The preimplantation embryos of a range of mammals can be susceptible to disruptions in genomic imprinting mechanisms, resulting in loss of imprinting. Such disruptions can have developmental consequences involving foetal and placental growth such as Beckwith-Wiedemann syndrome in humans and large offspring syndrome in sheep. Our objective was to investigate the dynamics of establishing monoallelic expression of individual sheep imprinted genes post-fertilisation. Semi-quantitative RT-PCR was used to amplify cDNA from the sheep blastocyst, day 21 foetus and day 21 chorioallantois, to compare expression levels between biparental and parthenogenetic embryos in order to indicate allelic expression status. In common with other mammals, IGF2, PEG1 and PEG3 were paternally expressed in the day 21 conceptus, while H19, IGF2R, GRB10 and p57KIP were maternally expressed. Interestingly, GNAS was maternally expressed in the foetus, but paternally expressed in the chorioallantois at day 21. Overall, the imprinting of ovine GRB10 and IGF2R was comparable with mouse but not with human. Contrary to the trophoblast-restricted maternal expression in both mouse and human, SASH2 (sheep homologue of Mash2/HASH2) was expressed in the ovine foetus and was biallelically expressed in the chorioallantois. Differential methylation of the H19 CTCF III upstream region and IGF2R DMR2 in the chorioallantois revealed predominantly paternal and maternal methylation respectively, indicating conservation of these imprinting regulatory regions. In blastocysts, IGF2R, GRB10 and SASH2 were expressed biallelically, while the other genes were not detected. Thus, for the majority of ovine imprinted genes examined, monoallelic expression does not occur until after the blastocyst stage.     

Imprinted gene expression in the rat embryo-fetal axis is altered in response to periconceptional maternal low protein diet

In our previous study, we have shown that maternal low protein diet (LPD, 9% casein vs 18% casein control) fed exclusively during the rat preimplantation period (0-4.25 day postcoitum) induced low birth weight, altered postnatal growth and hypertension in a gender-specific manner. In this study, we investigated the effect of maternal LPD restricted only to the preimplantation period (switched diet) or provided throughout gestation on fetal growth and imprinted gene expression in blastocyst and fetal stages of development. Male, but not female, blastocysts collected from LPD dams displayed a significant reduction (30%) in H19 mRNA level. A significant reduction in H19 (9.4%) and Igf2 (10.9%) mRNA was also observed in male, but not in female, fetal liver at day 20 postcoitum in response to maternal LPD restricted to the preimplantation period. No effect on the blastocyst expression of Igf2R was observed in relation to maternal diet. The reduction in H19 mRNA expression did not correlate with an observed alteration in DNA methylation at the H19 differentially methylated region in fetal liver. In contrast, maternal LPD throughout 20 days of gestation did not affect male or female H19 and Igf2 imprinted gene expression in fetal liver. Neither LPD nor switched diet treatments affected H19 and Igf2 imprinted gene expression in day 20 placenta. Our findings demonstrate that one contributor to the alteration in postnatal growth induced by periconceptional maternal LPD may derive from a gender-specific programming of imprinted gene expression originating within the preimplantation embryo itself.     

Blood as a surrogate marker for tissue-specific DNA methylation and changes due to folate depletion in post-partum female mice

Scope : DNA methylation patterns are tissue specific and may influence tissue‐specific gene regulation. Human studies investigating DNA methylation in relation to environmental factors primarily use blood‐derived DNA as a surrogate for DNA from target tissues. It is therefore important to know if DNA methylation changes in blood in response to environmental changes reflect those in target tissues. Folate intake can influence DNA methylation, via altered methyl donor supply. Previously, manipulations of maternal folate intake during pregnancy altered the patterns of DNA methylation in offspring but, to our knowledge, the consequences for maternal DNA methylation are unknown. Given the increased requirement for folate during pregnancy, mothers may be susceptible to aberrant DNA methylation due to folate depletion. Methods and results : Female mice were fed folate‐adequate (2 mg folic acid/kg diet) or folate‐deplete (0.4 mg folic acid/kg diet) diets prior to mating and during pregnancy and lactation. Following weaning, dams were killed and DNA methylation was assessed by pyrosequencing^® in blood, liver, and kidney at the Esr1, Igf2 differentially methylated region (DMR)1, Igf2 DMR2, Slc39a4CGI1, and Slc39a4CGI2 loci. We observed tissue‐specific differences in methylation at all loci. Folate depletion reduced Igf2 DMR1 and Slc39a4CGI1 methylation across all tissues and altered Igf2 DMR2 methylation in a tissue‐specific manner (p<0.05). Conclusion : Blood‐derived DNA methylation measurements may not always reflect methylation within other tissues. Further measurements of blood‐derived and tissue‐specific methylation patterns are warranted to understand the complexity of tissue‐specific responses to altered nutritional exposure.     

Saving energy and reducing emissions of both polycyclic aromatic hydrocarbons and particulate matter by adding bio-solution to emulsified diesel

Development of emulsified diesel has been driven by the need to reduce emissions from diesel engines and to save energy. Emulsification technology and bio-solution (NOE-7F) were used to produce emulsified diesel in this study. The experimental results indicated that there were no significant separation layers in W13 (13 wt % water + 87 wt % PDF), W16 (16 wt % water + 84 wt % PDF), W19 (19 wt % water + 81 wt % PDF), E13 (13 wt % NOE-7F water + 87 wt % PDF), E16 (16 wt % NOE-7F water + 83 wt % PDF), and E19 (19 wt % NOE-7F water + 81 wt % PDF) after premium diesel fuel (PDF) was emulsified for more than 30 days. In addition, there was no significant increase in damage from using these six emulsified fuels after the operation of the diesel generator for more than one year. The energy saving and reduction of particulate matter (PM) and total polycyclic aromatic hydrocarbons (PAHs) for W13, W16, W19, E13, E16 and E19, respectively, were 3.90%, 30.9%, 27.6%; 3.38%, 37.0%, 34.9%; 2.17%, 22.2%, 15.4%; 5.87%, 38.6%, 49.3%; 5.88%, 57.8%, 58.0%; and 4.75%, 31.1%, 47.3%, compared with PDF. The above results revealed that the bio-solution (NOE-7F) had a catalytic effect which elevated the combustion efficiency and decreased pollutant emissions during the combustion process. Furthermore, bio-solution (NOE-7F) can stabilize the emulsified fuels and enhance energy saving. Thus, emulsified fuels are highly suitable for use as alternative fuels. Due to the increasing price of diesel, emulsified diesel containing NOE-7F has potential for commercial application.     

Modelling the effect of ethanol on growth rate of food spoilage moulds

The effect of ethanol (E) on the radial growth rate (mu) of food spoilage moulds (Aspergillus candidus, Aspergillus flavus, Aspergillus niger, Cladosporium cladosporioides, Eurotium herbariorum, Mucor circinelloides, Mucor racemosus, Paecilomyces variotii, Penicillium chrysogenum, Penicillium digitatum, Rhizopus oryzae and Trichoderma harzianum) was assessed in Potato Dextrose Agar (PDA) medium at a(w) 0.99, 25 degrees C. In order to model this effect, the Monod type equation described previously by Houtsma et al. (Houtsma, P.C., Kusters, B.J.M., de Wit, J.C., Rombouts, F.M., Zwietering, M.H., 1994. Modelling growth rates of Listeria monocytogenes as a function of lactate concentration. Int. J. Food. Microbiol. 24, 113-123.) was re-parameterised: mu = mu(opt)[K(E(max)-E)/K E(max)-2KE+E(max)E]; E(max) (%, wt/wt): ethanol concentration at which no growth occurs, K (%, wt/wt): ethanol concentration at which mu = mu(opt)/2, mu(opt) (mm day(-1)): growth rate at 0% ethanol. The model was capable of describing curves, mu vs. E, with either a concave shape (KE(max)/2) with a good accuracy (root mean square error (RMSE) < or = 0.136) with the notable exception of R. oryzae and T. harzianum. After growth rate data were square-root transformed to stabilise the variance, E(max) was estimated in the range 3% to 5% for all moulds with the exception of T. harzianum (E(max) 2.14%) and P. variotii (E(max) 6.43%). Ethanol would appear an effective additional barrier to inhibit fungal growth in food products and would represent an interesting alternative to the use of preservatives.     

Expression of the long (OB-RB) and short (OB-RA) forms of the leptin receptor throughout the oestrous cycle in the mature rat ovary

Leptin is secreted by adipocytes and exerts its effects by interacting with the long form of the leptin receptor, OB-RB. The leptin protein and leptin receptors have been localized in the ovary, and acute leptin treatment directly inhibits ovulation in the rat ovary. It was hypothesized that expression of the leptin receptor gene varies throughout the oestrous cycle to modulate the sensitivity of the ovary to leptin. In this study, expression of genes for the long and short isoforms of the leptin receptor in the adult ovary was investigated at different stages of the rat oestrous cycle. Vaginal cytology was used to determine the stage of the oestrous cycle. Ovaries were collected and RNA was extracted for real-time RT-PCR analysis of leptin receptor gene expression. OB-RB gene expression was low in pro-oestrus (3.13 +/- 0.18 fg RNA per microg total DNA) and dioestrus II (2.52 +/- 0.19 fg RNA per microg total DNA) of the oestrous cycle, whereas expression was high in oestrus (5.9 +/- 0.27 fg RNA per microg total DNA) and dioestrus I (4.6 +/- 0.24 fg RNA per microg total DNA) (P < 0.001). Expression of the gene for the short form of the leptin receptor (OB-RA) was at a maximum in dioestrus I (65.5 +/- 0.8 fg RNA per ng total DNA), high in oestrus (39.0 +/- 0.8 fg RNA per ng total DNA) and low at pro-oestrus (5.0 +/- 0.2 fg RNA per ng total DNA) and dioestrus II (1.1 +/- 0.09 fg RNA per ng total DNA) (P < 0.001). Plasma oestradiol concentrations (pg ml-1) were highest at pro-oestrus (19.38 +/- 1.3), and similar at the remaining three stages studied (oestrus: 13.7 +/- 1.9; dioestrus I: 12.4 +/- 1.0; dioestrus II: 10.3 +/- 0.9) (P < 0.05). Plasma progesterone concentrations (ng ml-1) were higher in the luteal phases of the oestrous cycle (dioestrus I: 18.6 +/- 2.3; dioestrus II: 14.7 +/- 2.5) than during pro-oestrus (5.12 +/- 0.6) and oestrus (5.9 +/- 0.8) (P < 0.05). Plasma leptin concentrations were detectable only in pro-oestrus (0.35 +/- 0.05 ng ml(-1)) and were below the detection limit of the assay at other stages of the oestrous cycle. In summary, mRNA content for the long and short isoforms of the leptin receptor is lower in pro-oestrus and dioestrus II than in oestrus and dioestrus I of the rat oestrous cycle. The fluctuations in leptin receptor mRNA content may be a response to the concentrations of circulating steroid hormones and leptin. This research supports the initial hypothesis and shows that ovarian leptin receptor concentrations vary throughout the oestrous cycle in response to the changing environment of the ovary.     

Inhibition of Escherichia coli O157:H7 in ripening dry fermented sausage by ground yellow mustard

Compounds generated by the enzymatic hydrolysis of glucosinolates naturally present in mustard powder are potently bactericidal against Escherichia coli O157:H7. Because E. coli O157:H7 can survive the dry fermented sausage manufacturing process, 2, 4, and 6% (wt/wt) nondeheated (hot) mustard powder or 6% (wt/wt) deheated (cold) mustard powder were added to dry sausage batter inoculated with E. coli O157:H7 at about 7 log CFU/g to evaluate the antimicrobial effectiveness of the powders. Reductions in E. coli O157:H7 populations, changes in pH and water activity (aw), effects on starter culture (Pediococcus pentosaceus and Staphylococcus carnosus) populations, and effects of mustard powder on sausage texture (shear) were monitored during ripening. Nondeheated mustard powder at 2, 4, and 6% in dry sausage (0.90 aw) resulted in significant reductions in E. coli O157:H7 (P < 0.05) of 3.4, 4.4, and 6.9 log CFU/g, respectively, within 30 days of drying. During fermentation and drying, mustard powder did not affect P. pentosaceus and S. carnosus activity in any of the treatments. Extension of drying to 36 and 48 days reduced E. coli O157:H7 by >5 log CFU/g in the 4 and 2% mustard powder treatments, respectively. The 6% deheated mustard powder treatment provided the most rapid reductions of E. coli O157:H7 (yielding <0.20 log CFU/g after 24 days) by an unknown mechanism and was the least detrimental (P < 0.05) to sausage texture.     

Role of transforming growth factor-beta1 in gene expression and activity of estradiol and progesterone-generating enzymes in FSH-stimulated bovine granulosa cells

Survival and inhibitory factors regulate steroidogenesis and determine the fate of developing follicles. The objective of this study was to determine the role of transforming growth factor-beta1 (TGFB1) in the regulation of estradiol-17beta (E(2)) and progesterone (P(4)) secretion in FSH-stimulated bovine granulosa cells. Granulosa cells were obtained from 2 to 5 mm follicles and cultured in serum-free medium. FSH dose (1 and 10 ng/ml for 6 days) and time in culture (2, 4, and 6 days with 1 ng/ml FSH) increased E(2) secretion and mRNA expression of E(2)-related enzymes cytochrome P450 aromatase (CYP19A1) and 17beta-hydroxysteroid dehydrogenase type 1 (HSD17B1), but not HSD17B7. TGFB1 in the presence of FSH (1 ng/ml) inhibited E(2) secretion, and decreased mRNA expression of FSH receptor (FSHR), CYP19A1, and HSD17B1, but not HSD17B7. FSH dose did not affect P(4) secretion and mRNA expression of 3beta-hydroxysteroid dehydrogenase (HSD3B) and alpha-glutathione S-transferase (GSTA), but inhibited the amount of steroidogenic acute regulatory protein (STAR) mRNA. Conversely, P(4) and mRNA expression of STAR, cytochrome P450 side-chain cleavage (CYP11A1), HSD3B, and GSTA increased with time in culture. TGFB1 inhibited P(4) secretion and decreased mRNA expression of STAR, CYP11A1, HSD3B, and GSTA. TGFB1 modified the formation of granulosa cell clumps and reduced total cell protein. Finally, TGFB1 decreased conversion of androgens to E(2), but did not decrease the conversion of estrone (E(1)) to E(2) and pregnenolone to P(4). Overall, these results indicate that TGFB1 counteracts stimulation of E(2) and P(4) synthesis in granulosa cells by inhibiting key enzymes involved in the conversion of androgens to E(2) and cholesterol to P(4) without shutting down HSD17B reducing activity and HSD3B activity.     

Aberrant DNA methylation at imprinted genes in testicular sperm retrieved from men with obstructive azoospermia and undergoing vasectomy reversal

Male factor infertility has been associated with abnormal DNA methylation at imprinted genes. Little information is available on the status of imprinting in the sperm of men with azoospermia, including the association between aberrant imprinting and obstructive azoospermia (OA) or non-OA (NOA). Analysis of DNA methylation at imprinted genes in the sperm of men undergoing vasectomy reversal would aid determination of whether aberrant imprinting is associated with obstruction. Testicular sperm was retrieved from testicular biopsies obtained from men with azoospermia (N=18), including OA (N=10), NOA (N=5), and unknown pathology (N=3), and from men undergoing vasectomy reversal (N=17). Sperm was also obtained from proven fertile men (N=9). DNA methylation was investigated at multiple CpG sites within the differentially methylated regions (DMRs) of three imprinted genes, H19, IG-GTL2 and MEST, using bisulphite sequencing. Unique clones representative of single cells were analyzed. We found a significant decrease in DNA methylation at the H19 DMR in testicular sperm of azoospermic men compared with proven fertile men. The decrease was also significant between OA and proven fertile men, and between men undergoing vasectomy reversal and proven fertile men, suggesting that aberrant DNA methylation may be associated with obstruction. Changes in DNA methylation at IG-GTL2 and MEST DMRs among groups were not significant. Our data suggest that imprinting abnormalities may be associated with obstruction and may occur in response to changes in testicular environment and not only spermatogenesis failure, as previously reported. Methylation at the H19 DMR was particularly prone to modification in testicular sperm.     

Hormonal regulation of expression of growth differentiation factor-9 receptor type I and II genes in the bovine ovarian follicle

Growth differentiation factor-9 (GDF-9) and bone morphogenetic proteins (BMPs) are crucial factors in follicular growth and development. GDF-9 and BMPs initiate signaling by assembling type I (ALK-3, ALK-5 and ALK-6) and type II (BMPRII) receptors. However, the mechanism regulating the expression of these receptors in the process of bovine follicle development is still unknown. The aim of the present study was to clarify the involvement of receptor systems for GDF-9 and BMPs in follicular selection by examining the effects of FSH and estradiol-17beta (E2) on the regulation of BMPRII, ALK-3, ALK-5 and ALK-6 mRNA expression in bovine granulosa cells (GCs). To observe mRNA expression during follicular development, follicles were obtained from heifers and classified into two groups: pre-selection follicles (PRFs) (an average of 7.7 mm follicles with low E2) and post-selection follicles (POFs) (an average of 15 mm follicles with high E2). Theca layer cells (TCs) and GCs were harvested from aspirated follicles. For in vitro studies, GCs were obtained from bovine follicles of 4-7 mm diameter and cultured in Dulbecco's modified Eagle's/F12 (DMEM/F-12) medium with 10% fetal calf serum for 24 h. The medium was then replaced with serum-free DMEM/F-12 supplemented with different doses of E2 (1, 10, 100 ng/ml) or FSH (1, 5, 10 ng/ml) or combinations of 1 ng/ml of E2 with different FSH doses. Total RNA was extracted and the mRNA expression of BMPRII, ALK-3, ALK-5 and ALK-6 was estimated by the quantitative real-time PCR method using a LightCycler. BMPRII and ALK-5 expression was significantly higher in the GCs of POFs than in those of PRFs, whereas ALK-3 expression was significantly lower in the GCs of POFs than in those of PRFs. There was no difference in ALK-6 expression in GCs between PRFs and POFs. The expression of BMPRII, ALK-5, ALK-3 and ALK-6 genes in the TCs was not significantly different between PRFs and POFs. Treatment of GCs with E2 alone increased BMPRII mRNA expression at a concentration of 100 ng/ml and ALK-5 mRNA expression at 10 ng/ml. BMPRII and ALK-5 mRNA levels were up-regulated by the combination of E2 (1 ng/ml) and FSH (5 ng/ml). On the other hand, FSH alone down-regulated the expression of BMPRII and ALK-5 in GCs. The results of the present study provide the first evidence that FSH and E2 regulate the expression of BMPRII and ALK-5 genes in bovine GCs. Thus, our data suggest that the GDF-9/BMPRII/ALK-5 system may be critically involved in the process of selection of bovine follicles.     

Metabolic engineering study on the direct fermentation of 2-keto-L-gulonic acid, a key intermediate of L-ascorbic acid in Pseudomonas putida IFO3738

We have achieved production of 2-keto-L-gulonic acid (2-KLGA) in recombinant Pseudomonas putida IFO3738. Firstly, the genes for sorbose dehydrogenase (SDH)/sorbosone dehydrogenase (SNDH) were introduced into P. putida. The recombinant P. putida/pBBR-SDH produced 0.7 mg/ml of 2-KLGA in a culture broth containing 5% L-sorbose. Replacement of the native SNDH promoter by the Escherichia coli tufB promoter (pBBR-SDH-tufB) improved the productivity of 2-KLGA up to 11.4 mg/ml. Secondly, the sorbitol dehydrogenase (SLDH) gene was also introduced into P. putida. The recombinant P. putida/pUCP19-3DH carrying the genes for SDH, SNDH and SLDH had the ability to produce 2-KLGA (7.5 mg/ml) in a 5% d-sorbitol broth. The productivity of 2-KLGA was improved up to 9.8 mg/ml by changing to an expression system with two plasmids, pBBR-SDH-tufB (for SDH/SNDH) and pUCP19-SLDH (for SLDH), respectively. Moreover, the replacement of the native SLDH promoter by the E. coli tufB promoter (pUCP19-SLDH-tufB) improved the 2-KLGA productivity up to 11.6 mg/ml. Optimization of cultivation conditions increased the conversion yield of 2-KLGA to 32% and that of l-idonate, a metabolite of 2-KLGA, to 40%. These results indicate P. putida IFO3738 is one of the candidate strains for direct fermentation of 2-KLGA.     

Influence of autoinducer-2 (AI-2) and beef sample extracts on E. coli O157:H7 survival and gene expression of virulence genes yadK and hhA

ABSTRACT:  Bacterial cell-to-cell communication is mediated by autoinducer (AI) molecules such as AI-2 and has been reported to regulate gene expression in Escherichia coli O157:H7. We have previously shown that ground beef contains compounds that can inhibit sensing of AI-2 like activity. The hypothesis of this study was that AI-2 activity observed in conditioned medium (CM) will enhance E. coli O157:H7 survival and expression of virulence genes, whereas compounds inhibitory (such as those present in ground beef extracts) to AI-2 activity will negate these effects. E. coli O157:H7 luxS mutant strain VS 94 (incapable of synthesizing AI-2) was employed in these studies. The survival of this enteric bacterial pathogen as a function of AI-2 activity and the presence of AI-2 inhibitory compounds was studied at 4 °C. The number of survivors in the presence of AI-2 was significantly higher compared to the absence of AI-2, and the addition of ground beef extracts to conditioned medium negated the influence of AI-2 activity. Autoinducer AI-2 upregulated selected genes virulence genes ( yadK , and hha ), whereas the ground beef extract reversed the effect of AI-2 on the expression of the selected genes.     

Inhibition and inactivation of Listeria monocytogenes and Escherichia coli O157:H7 colony biofilms by micellar-encapsulated eugenol and carvacrol

The antimicrobial efficacy of carvacrol and eugenol, two essential oil compounds, encapsulated in a micellar nonionic surfactant solution on four strains of Listeria monocytogenes (Scott A, 101, 108, and 310) and four strains of Escherichia coli O157:H7 (H1730, E0019, F4546, and 932) growing as colony biofilms was investigated. Carvacrol and eugenol were encapsulated in Surfynol 485W at concentrations ranging from 0.3 to 0.9% (wt/wt) at a surfactant concentration of 5% (wt/wt). Colony biofilms were grown on polycarbonate membranes resting on agar plates containing antimicrobial formulations. Cells were enumerated after 0, 3, 6, 9, 24, 48, and 72 h of incubation. Colony biofilms of all E. coli O157:H7 strains were more sensitive to both antimicrobial systems than L. monocytogenes strains. Surface-grown E. coli O157:H7 viable cell numbers decreased below detectable levels after exposure to encapsulated essential oil compounds for > 3 h at all tested concentrations, except for E. coli O157:H7 F4546, which grew slowly in the presence of < 0.5% (wt/wt) eugenol. L. monocytogenes Scott A and 101 were more resistant to eugenol than carvacrol at sublethal concentrations (< 0.5% [wt/wt]). Carvacrol was effective at any concentration against L. monocytogenes 108, whereas concentrations of > 0.5% (wt/wt) eugenol were required for inactivation. L. monocytogenes 310 was equally sensitive to both essential oil compounds. Results suggest that surfactant-encapsulated generally recognized as safe essential oil compounds may offer a new means to control the growth of food pathogens such as E. coli O157:H7 and L. monocytogenes on food contact surfaces.     

Effect of modified atmosphere packaging on the persistence and expression of virulence factors of Escherichia coli O157:H7 on shredded iceberg lettuce

Fresh-cut leafy greens contaminated with Escherichia coli O157:H7 have caused foodborne outbreaks. Packaging conditions, coupled with abusive storage temperatures of contaminated lettuce, were evaluated for their effect on the potential virulence of E. coli O157:H7. Shredded lettuce was inoculated with 5.58 and 3.98 log CFU E. coli O157:H7 per g and stored at 4 and 15°C, respectively, for up to 10 days. Lettuce was packaged under treatment A (modified atmosphere packaging conditions used for commercial fresh-cut produce, in gas-permeable film with N(2)), treatment B (near-ambient air atmospheric conditions in a gas-permeable film with microperforations), and treatment C (high-CO(2) and low-O(2) conditions in a gas-impermeable film). E. coli O157:H7 populations from each treatment were determined by enumeration of numbers on MacConkey agar containing nalidixic acid. RNA was extracted from packaged lettuce for analysis of expression of virulence factor genes stx(2), eae, ehxA, iha, and rfbE. E. coli O157:H7 populations on lettuce at 4°C under all treatments decreased, but most considerably so under treatment B over 10 days. At 15°C, E. coli O157:H7 populations increased by at least 2.76 log CFU/g under all treatments. At 15°C, expression of eae and iha was significantly greater under treatment B than it was under treatments A and C on day 3. Similarly, treatment B promoted significantly higher expression of stx(2), eae, ehxA, and rfbE genes on day 10, compared with treatments A and C at 15°C. Results indicate that storage under near-ambient air atmospheric conditions can promote higher expression levels of O157 virulence factors on lettuce, and could affect the severity of E. coli O157:H7 infections associated with leafy greens.     

Control of Escherichia coli O157:H7 with sodium metasilicate

Three intervention strategies-trisodium phosphate, lactic acid, and sodium metasilicate--were examined for their in vitro antimicrobial activities in water at room temperature against a three-strain cocktail of Escherichia coli O157:H7 and a three-strain cocktail of "generic" E. coli. Both initial inhibition and recovery of injured cells were monitored. When 3.0% (wt/wt) lactic acid, pH 2.4, was inoculated with E. coli O157:H7 (approximately 6 log CFU/ml), viable microorganisms were recovered after a 20-min exposure to the acid. After 20 min in 1.0% (wt/wt) trisodium phosphate, pH 12.0, no viable E. coli O157:H7 microorganisms were detected. Exposure of E. coli O157:H7 to sodium metasilicate (5 to 10 s) at concentrations as low as 0.6%, pH 12.1, resulted in 100% inhibition with no recoverable E. coli O157:H7. No difference in inhibition profiles was detected between the E. coli O157:H7 and generic strains, suggesting that nonpathogenic strains may be used for in-plant sodium metasilicate studies.     

植物甾醇酯对非酒精性脂肪肝大鼠肝脏部分代谢物的影响

本实验通过考察植物甾醇酯（phytosterol esters,PSE）对高脂饮食诱导的非酒精性脂肪肝（nonalcoholic fatty liver disease,NAFLD）大鼠肝脏部分小分子代谢物的影响,深入研究PSE对NAFLD发挥预防作用的代谢分子机制。利用高脂饮食建立NAFLD大鼠模型的同时,PSE干预组分别灌胃低剂量（0.05 g/（100 g?d mb））、高剂量（0.10 g/（100 g?d mb））PSE强化牛奶,利用超高效液相色谱-四极杆串联飞行时间质谱仪（ultraperformance liquid chromatography tandem time-of-fight mass spectrometry,UPLC-Q-TOF-MS）分析大鼠肝脏组织代谢物,通过Progenesis QI v2.3软件进行无监督和有监督的模式判别及UNIFI数据分析平台初步筛选各组差异代谢物,利用Metabo Analyst（http://www.metaboanalyst.ca/faces/upload/PathUploadView.xhtml）等数据库寻找所参与的代谢通路。结果：筛选出磷脂酸（16∶0/20∶2（11Z,14Z）、20∶1（11Z）/0∶0）、磷脂酰胆碱（16∶0/18∶1（11E）、18∶1（6Z）/0∶0、18∶1（9Z）/18∶0、20∶3（8Z,11Z,14Z）/0∶0、15∶0/18∶1（11Z）、16∶0/16∶1（9Z）、16∶0/2∶0、17∶1（10Z）/0∶0、19∶3（10Z,13Z,16Z）/0∶0、20∶4（5Z,8Z,11Z,14Z）/14∶0）、磷脂酰乙醇胺（20∶3（8Z,11Z,14Z）/22∶6（4Z,7Z,10Z,13Z,16Z,19Z）、22∶6（4Z,7Z,10Z,13Z,16Z,19Z）/17∶1（9Z））、磷脂酰甘油（17∶2（9Z,12Z）/22∶6（4Z,7Z,10Z,13Z,16Z,19Z））、鞘磷脂（d18∶0/16∶0）、甘氨胆酸、溶血卵磷脂（18∶1（9Z）、20∶3（5Z,8Z,11Z））、20∶3（8Z,11Z,14Z））共20 种差异代谢物,高剂量PSE可更好地调节高脂饮食诱导的甘油磷脂代谢通路的紊乱。结论：PSE可通过调节磷脂类和胆汁酸类的小分子代谢物含量来改善NAFLD的发生发展。     

Biodegradation of Jatropha curcas phorbol esters in soil

BACKGROUND: Jatropha curcas seed cake is generated as a by‐product during biodiesel production. Seed cake containing toxic phorbol esters (PEs) is currently used as a fertiliser and thus it is of eco‐toxicological concern. In the present study the fate of PEs in soil was studied. RESULTS: Two approaches for the incorporation of PEs in soil were used. In the first, silica was bound to PEs, and in the second, seedcake was used. At day 0, the concentration of PEs in soil was 2.6 and 0.37 mg g^?1 for approach 1 and 2 respectively. PEs from silica bound PEs were completely degraded after 19, 12, 12 days (at 130 g kg^?1 moisture) and after 17, 9, 9 days (at 230 g kg^?1 moisture) at room temperature, 32 °C and 42 °C respectively. Similarly at these temperatures PEs from seed cake were degraded after 21, 17 and 17 days (at 130 g kg^?1 moisture) and after 23, 17, and 15 days (at 230 g kg^?1 moisture). Increase in temperature and moisture increased rate of PEs degradation. Using the snail (Physa fontinalis) bioassay, mortality by PE‐amended soil extracts decreased with the decrease in PE concentration in soil. CONCLUSION: Jatropha PEs are biodegradable. The degraded products are innocuous. Copyright © 2010 Society of Chemical Industry