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  1. Improving ethanol productivity through self-cycling fermentation of yeast: a proof of concept [Download]

    Title: Improving ethanol productivity through self-cycling fermentation of yeast: a proof of concept
    Creator: Wang, Jie
    Description: Background The cellulosic ethanol industry has developed efficient strategies for converting sugars obtained from various cellulosic feedstocks to bioethanol. However, any further major improvements in ethanol productivity will require development of novel and innovative fermentation strategies that enhance incumbent technologies in a cost-effective manner. The present study investigates the feasibility of applying self-cycling fermentation (SCF) to cellulosic ethanol production to elevate productivity. SCF is a semi-continuous cycling process that employs the following strategy: once the onset of stationary phase is detected, half of the broth volume is automatically harvested and replaced with fresh medium to initiate the next cycle. SCF has been shown to increase product yield and/or productivity in many types of microbial cultivation. To test whether this cycling process could increase productivity during ethanol fermentations, we mimicked the process by manually cycling the fermentation for five cycles in shake flasks, and then compared the results to batch operation. Results Mimicking SCF for five cycles resulted in regular patterns with regards to glucose consumption, ethanol titer, pH, and biomass production. Compared to batch fermentation, our cycling strategy displayed improved ethanol volumetric productivity (the titer of ethanol produced in a given cycle per corresponding cycle time) and specific productivity (the amount of ethanol produced per cellular biomass) by 43.1 ± 11.6 and 42.7 ± 9.8%, respectively. Five successive cycles contributed to an improvement of overall productivity (the aggregate amount of ethanol produced at the end of a given cycle per total processing time) and the estimated annual ethanol productivity (the amount of ethanol produced per year) by 64.4 ± 3.3 and 33.1 ± 7.2%, respectively. Conclusions This study provides proof of concept that applying SCF to ethanol production could significantly increase productivities, which will help strengthen the cellulosic ethanol industry.
    Subjects: Cellulosic ethanol, Batch, Self-cycling fermentation, Manual cycling fermentation, Ethanol volumetric productivity, Specific productivity, Overall productivity, Annual ethanol productivity, Production cost, Capital cost
    Date Created: 2017/11
  2. Multiple mechanisms contribute to increased neutral lipid accumulation in yeast producing recombinant variants of plant diacylglycerol acyltransferase 1 [Download]

    Title: Multiple mechanisms contribute to increased neutral lipid accumulation in yeast producing recombinant variants of plant diacylglycerol acyltransferase 1
    Creator: Xu, Yang
    Description: The apparent bottleneck in the accumulation of oil during seed development in some oleaginous plant species is the formation of triacylglycerol (TAG) by the acyl-CoA-dependent acylation of sn-1,2- diacylglycerol catalyzed by diacylglycerol acyltransferase (DGAT, EC 2.3.1.20). Improving DGAT activity using protein engineering could lead to improvements in seed oil yield, for example in canola-type Brassica napus. Directed evolution of B. napus DGAT1 (BnaDGAT1) previously revealed that one of the regions where amino acid residue substitutions leading to higher performance in BnaDGAT1 was in the ninth predicted transmembrane domain (PTMD9). In this study, several BnaDGAT1 variants with amino acid residue substitutions in PTMD9 were characterized. Among these enzyme variants, the extent of yeast TAG production was affected by different mechanisms including increased enzyme activity, increased polypeptide accumulation and possibly reduced substrate inhibition. The kinetic properties of the BnaDGAT1 variants were affected by the amino acid residue substitutions and a new kinetic model based on substrate inhibition and sigmoidicity was generated. Based on sequence alignment and further biochemical analysis, the amino acid residue substitutions that conferred increased TAG accumulation were shown to be present in the DGAT1-PTMD9 region of other higher plant species. When amino acid residue substitutions that increased BnaDGAT1 enzyme activity were introduced into recombinant Camelina sativa DGAT1, they also improved enzyme performance. Thus, the knowledge generated from directed evolution of DGAT1 in one plant species can be transferred to other plant species and has potentially broad applications in genetic engineering of oleaginous crops and microorganisms.
    Subjects: Acyltransferase, Enzyme kinetics, Plant biochemistry, Saccharomyces cerevisiae, Triacylglycerol, Brassica napus, Camelina sativa, DGAT, Oilseed rape, Substrate inhibition
    Date Created: 2017/09
  3. High-performance variants of plant diacylglycerol acyltransferase 1 generated by directed evolution provide insights into structure function [Download]

    Title: High-performance variants of plant diacylglycerol acyltransferase 1 generated by directed evolution provide insights into structure function
    Creator: Chen, Guanqun
    Description: Diacylglycerol acyltransferase 1 (DGAT1) catalyzes the acyl-CoA-dependent biosynthesis of triacylglycerol, the predominant component of seed oil. In some oil crops, including Brassica napus, the level of DGAT1 activity can have a substantial effect on triacylglycerol production. Structure–function insights into DGAT1, however, remain limited because of the lack of a three-dimensional detailed structure for this membrane-bound enzyme. In this study, the amino acid residues governing B. napus DGAT1 (BnaDGAT1) activity were investigated via directed evolution, targeted mutagenesis, in vitro enzymatic assay, topological analysis, and transient expression of cDNA encoding selected enzyme variants in Nicotiana benthamiana. Directed evolution revealed that numerous amino acid residues were associated with increased BnaDGAT1 activity, and 67% of these residues were conserved among plant DGAT1s. The identified amino acid residue substitution sites occur throughout the BnaDGAT1 polypeptide, with 89% of the substitutions located outside the putative substrate binding or active sites. In addition, cDNAs encoding variants I447F or L441P were transiently overexpressed in N. benthamiana leaves, resulting in 33.2 or 70.5% higher triacylglycerol content, respectively, compared with native BnaDGAT1. Overall, the results provide novel insights into amino acid residues underlying plant DGAT1 function and performance-enhanced BnaDGAT1 variants for increasing vegetable oil production.
    Subjects: triacylglycerol biosynthesis, diacylglycerol acyltransferase, directed evolution, topology, leaf oil production, Brassica napus, Nicotiana benthamiana
    Date Created: 2017/09/10
  4. Effects of dietary enrichment with a marine oil-based n-3 LCPUFA supplement in sows with predicted birth weight phenotypes on growth performance and carcass quality of offspring [Download]

    Title: Effects of dietary enrichment with a marine oil-based n-3 LCPUFA supplement in sows with predicted birth weight phenotypes on growth performance and carcass quality of offspring
    Creator: Smit, M. N.
    Description: Effects of a marine oil-based n-3 LCPUFA supplement (mLCPUFA) fed from weaning until the end of the next lactation to sows with a predicted low litter birth weight (LBW) phenotype on growth performance and carcass quality of litters born to these sows were studied, based on the hypothesis that LBW litters would benefit most from mLCPUFA supplementation. Sows were allocated to be fed either standard corn/soybean meal-based gestation and lactation diets (CON), or the same diets enriched with 0.5% of the mLCPUFA supplement at the expense of corn. The growth performance from birth until slaughter of the litters with the lowest average birth weight in each treatment (n=24 per treatment) is reported in this paper. At weaning, each litter was split between two nursery pens with three to six pigs per pen. At the end of the 5-week nursery period, two barrows and two gilts from each litter that had individual birth weights closest to their litter average birth weight, were moved to experimental grow–finish pens (barn A), where they were housed as two pigs per pen, sorted by sex within litter. Remaining pigs in each litter were moved to another grow–finish barn (barn B) and kept in mixed-sex pens of up to 10 littermates. After 8 weeks, one of the two pigs in each pen in barn A was relocated to the pens holding their respective littermates in barn B. The remaining barrows and gilts were individually housed in the pens in barn A until slaughter. Maternal mLCPUFA supplementation increased docosahexaenoic acid (DHA) concentration in the brain, liver and Semitendinosus muscle of stillborn pigs (P<0.01), did not affect eicosapentaenoic acid and DHA concentrations in sow serum at the end of lactation, and did not affect average daily gain, average daily feed intake or feed utilization efficiency of the offspring. BW was higher (P<0.01) in the second half of the grow–finish phase in pigs from mLCPUFA sows compared with controls in barn A, where space and competition for feed was minimal, but not barn B. Carcass quality was not affected by treatment for pigs from barn A, but maternal mLCPUFA supplementation negatively affected carcass quality in pigs from barn B. Collectively, these results suggest that nutritional supplementation of sows can have lasting effects on litter development, but that feeding mLCPUFA to sows during gestation and lactation was not effective in improving growth rates or carcass quality of LBW litters.
    Subjects: Litter, Growth, Birth Weight, n-3 LCPUFA, Swine
    Date Created: 2015
  5. Effects of dietary enrichment with a marine oil-based n-3 LCPUFA supplement in sows with predicted birth weight phenotypes on birth litter quality and growth performance to weaning [Download]

    Title: Effects of dietary enrichment with a marine oil-based n-3 LCPUFA supplement in sows with predicted birth weight phenotypes on birth litter quality and growth performance to weaning
    Creator: Smit, M. N.
    Description: The effects of a marine oil-based n-3 long-chain polyunsaturated fatty acid (mLCPUFA) supplement fed to the sow from weaning, through the rebreeding period, during gestation and until end of lactation on litter characteristics from birth until weaning were studied in sows with known litter birth weight phenotypes. It was hypothesized that low birth weight (LBW) litters would benefit more from mLCPUFA supplementation than high birth weight litters. A total of 163 sows (mean parity=4.9±0.9) were rebred after weaning. Sows were pair-matched by parity and litter average birth weight of the previous three litters. Within pairs, sows were allocated to be fed either standard corn/soyabean meal-based gestation and lactation diets (CON), or the same diets enriched with 0.5% of the mLCPUFA supplement at the expense of corn. Each litter between 9 and 16 total pigs born was classified as LBW or medium/high average birth weight (MHBW) litter and there was a significant correlation (P<0.001) between litter average birth weight of the current and previous litters within sows (r=0.49). Sow serum was harvested at day 113 of gestation for determination of immunoglobulin G (IgG) concentrations. The number of pigs born total and alive were lower (P=0.01) in mLCPUFA than CON sows, whereas the number of stillborn and mummified pigs were similar between treatments. Number of stillborns (trend) and mummies (P<0.01) were higher in LBW than MHBW litters. Tissue weights and brain : tissue weight ratios were similar between treatments, but LBW litters had decreased tissue weights and increased brain : tissue weight ratios compared with MHBW litters. Placental weight was lower (P=0.01) in LBW than MHBW litters, but was not different between treatments. Average and total litter weight at day 1 was similar between treatments. mLCPUFA increased weaning weight (P=0.08) and average daily gain (P<0.05) in MHBW litters, but not in LBW litters. Pre-weaning mortality was similar between treatments, but was higher (P<0.01) in LBW than MHBW litters. IgG concentration in sow serum was similar between treatments and litter birth weight categories. In conclusion, litter birth weight phenotype was repeatable within sows and LBW litters showed the benchmarks of intra-uterine growth retardation (lower placental weight and brain sparing effects). As maternal mLCPUFA supplementation decreased litter size overall, only improved litter growth rate until weaning in MHBW litters, and did not affect pre-weaning mortality, maternal mLCPUFA supplementation was not an effective strategy in our study for mitigating negative effects of a LBW litter phenotype.
    Subjects: Birth Weight, Growth, Litter, n-3 LCPUFA, Swine
    Date Created: 2015
  6. Infant gut microbiota and the hygiene hypothesis of allergic disease: Impact of household pets and siblings on microbiota composition and diversity [Download]

    Title: Infant gut microbiota and the hygiene hypothesis of allergic disease: Impact of household pets and siblings on microbiota composition and diversity
    Creator: Azad, Meghan B.
    Description: Background Multiple studies have demonstrated that early-life exposure to pets or siblings affords protection against allergic disease; these associations are commonly attributed to the “hygiene hypothesis”. Recently, low diversity of the infant gut microbiota has also been linked to allergic disease. In this study, we characterize the infant gut microbiota in relation to pets and siblings. Methods The study population comprised a small sub-sample of 24 healthy, full term infants from the Canadian Healthy Infant Longitudinal Development (CHILD) birth cohort. Mothers reported on household pets and siblings. Fecal samples were collected at 4 months of age, and microbiota composition was characterized by high-throughput signature gene sequencing. Results Microbiota richness and diversity tended to be increased in infants living with pets, whereas these measures were decreased in infants with older siblings. Infants living with pets exhibited under-representation of Bifidobacteriaceae and over-representation of Peptostreptococcaceae; infants with older siblings exhibited under-representation of Peptostreptococcaceae. Conclusions This study provides new evidence that exposure to pets and siblings may influence the early development of the gut microbiota, with potential implications for allergic disease. These two traditionally protective “hygiene hypothesis” factors appear to differentially impact gut microbiota composition and diversity, calling into question the clinical significance of these measures. Further research is required to confirm and expand these findings.
    Subjects: Siblings, Microflora Hypothesis, Gut Microbiota, Hygiene Hypothesis, Allergic Disease, Infants, Gut Microbiome, Atopy, Environmental Exposures, Pets
    Date Created: 2013
  7. Responses to n-3 fatty acid (LCPUFA) supplementation of gestating gilts, and lactating and weaned sows [Download]

    Title: Responses to n-3 fatty acid (LCPUFA) supplementation of gestating gilts, and lactating and weaned sows
    Creator: Smit, M. N.
    Description: Feeding n-3 long-chain polyunsaturated fatty acids (LCPUFA) to gilts or sows has shown different responses to litter growth, pre-weaning mortality and subsequent reproductive performance of the sow. Two hypotheses were tested: (1) that feeding a marine oil-based supplement rich in protected n-3 LCPUFAs to gilts in established gestation would improve the growth performance of their litters; and (2) that continued feeding of the supplement during lactation and after weaning would offset the negative effects of lactational catabolism induced, using an established experimental model involving feed restriction of lactating primiparous sows. A total of 117 primiparous sows were pair-matched at day 60 of gestation by weight, and when possible, litter of origin, and were allocated to be either control sows (CON) fed standard gestation and lactation diets, or treated sows (LCPUFA) fed the standard diets supplemented with 84 g/day of a n-3 LCPUFA rich supplement, from day 60 of first gestation, through a 21-day lactation, and until euthanasia at day 30 of their second gestation. All sows were feed restricted during the last 7 days of lactation to induce catabolism, providing a background challenge against which to determine beneficial effects of n-3 LCPUFA supplementation on subsequent reproduction. In the absence of an effect on litter size or birth weight, n-3 LCPUFA tended to improve piglet BW gain from birth until 34 days after weaning (P = 0.06), while increasing pre-weaning mortality (P = 0.05). It did not affect energy utilization by the sow during lactation, thus not improving the catabolic state of the sows. Supplementation from weaning until day 30 of second gestation did not have an effect on embryonic weight, ovulation rate or early embryonic survival, but did increase corpora lutea (CL) weight (P = 0.001). Eicosapentaenoic acid and docosahexaenoic acid (DHA) levels were increased in sow serum and CL (P < 0.001), whereas only DHA levels increased in embryos (P < 0.01). In conclusion, feeding n-3 LCPUFA to gilts tended to improve litter growth, but did not have an effect on overall subsequent reproductive performance.
    Subjects: Sow, Reproduction, Growth, Gilt, Fatty Acids
    Date Created: 2012
  8. A characterization of inflammatory and structural markers within the rumen epithelium during grain-induced ruminal acidosis in lactating dairy cattle [Download]

    Title: A characterization of inflammatory and structural markers within the rumen epithelium during grain-induced ruminal acidosis in lactating dairy cattle
    Creator: Dionissopoulos, Louis
    Description: The objective of this study was to characterize the mRNA and protein expression of inflammatory and structural genes in the rumen epithelium during grain-induced ruminal acidosis in lactating dairy cattle. A total of 16 rumen-fistulated, lactating Holstein dairy cattle (618±35 kg of body weight, 221±32 days in milk) were used in a randomized complete block design study. All cattle were initially fed a high-forage diet (HF; 88.9% of dry matter) and after a baseline (wk 0) measurement, half of the cattle were randomly assigned and transitioned to a high-concentrate diet (HC; 62.2% of dry matter) which was fed for 3 weeks (weeks 1, 2 and 3). Continuous ruminal pH, ruminal LPS and plasma LPS-binding protein were measured each week followed by a rumen papillae biopsy used for mRNA and protein quantification. After the baseline period, ruminal LPS was higher in HC compared to HF cattle (28851±6905 vs. 5771±3042 EU mL-1). There was no difference in mRNA expression of inflammatory and structure genes in rumen papillae between HF and HC cattle during all weeks. With regard to protein expression, there was an up regulation (p = 0.02) of nuclear factor of activated T-cells cytoplasmic 2 expression during weeks 1, 2 and 3; however, all other inflammatory markers within the rumen epithelium were unchanged by treatment. These results suggest that although grain-induced ruminal acidosis leads to characteristic whole-animal inflammatory response, only marginal changes in inflammatory and structural gene and protein expression in the rumen epithelium were detected.
    Subjects: Ruminal Acidosis, Rumen Epithelium, Gene Expression, Inflammation
    Date Created: 2012
  9. Skeletal muscle anabolism is a side effect of therapy with the MEK inhibitor: Selumetinib in patients with cholangiocarcinoma [Download]

    Title: Skeletal muscle anabolism is a side effect of therapy with the MEK inhibitor: Selumetinib in patients with cholangiocarcinoma
    Creator: Prado, Carla M.
    Description: background: Cancer cachexia is characterised by skeletal muscle wasting; however, potential for muscle anabolism in patients with advanced cancer is unproven. methods: Quantitative analysis of computed tomography images for loss/gain of muscle in cholangiocarcinoma patients receiving selumetinib (AZD6244; ARRY-142886) in a Phase II study, compared with a separate standard therapy group. Selumetinib is an inhibitor of mitogen-activated protein/extracellular signal–regulated kinase and of interleukin-6 secretion, a putative mediator of muscle wasting. results: Overall, 84.2% of patients gained muscle after initiating selumetinib; mean overall gain of total lumbar muscle cross-sectional area was 13.6 cm2/100 days (~2.3 kg on a whole-body basis). Cholangiocarcinoma patients who began standard treatment were markedly catabolic, with overall muscle loss of −7.3 cm2/100 days (~1.2 kg) and by contrast only 16.7% of these patients gained muscle. conclusion: Our findings suggest that selumetinib promotes muscle gain in patients with cholangiocarcinoma. Specific mechanisms and relevance for cachexia therapy remain to be investigated.
    Subjects: Interleukin-6, Cholangiocarcinoma, Skeletal Muscle, Cachexia
    Date Created: 2012
  10. Initial differences in lipid processing leading to pig- and beef-derived mature adipocyte dedifferentiation [Download]

    Title: Initial differences in lipid processing leading to pig- and beef-derived mature adipocyte dedifferentiation
    Creator: Chen, Jie
    Description: Clonal cultures of pig-derived mature adipocytes are capable of dedifferentiating and forming proliferative-competent progeny cells in vitro. Initial lipid processing, is different to that observed in cultures of beef-derived adipocytes. Mature pig adipocytes extrude lipid before proliferation, whereas beef-derived adipocytes symmetrically, or asymmetrically, divide without expelling lipid. These observations suggest that dedifferentiation of mature adipocytes relies on species-specific mechanisms, or that different culture conditions are required for pigderived adipocytes to dedifferentiate in a manner similar to beef adipocytes. This in vitro system will aid in our understanding of lipid metabolism, regulation of single cells, processes involved in dedifferentiation of cells, and/or characteristics of putative stem cells residing in adipose tissue.
    Subjects: Dedifferentiation, Adipocyte, Beef, Lipid Extrusion, Pig
    Date Created: 2009