Effects of conservation method on fatty acid composition of silage

This experiment was conducted to investigate effects of wilting and additives on the fatty acid (FA) composition of grass silage. The crop used was timothy (Phelum pratense L., cv. Grindstad), and the additives were Proens? (formic acid and propionic acid, 60–66 g/100 g and 25–30 g/100 g, respectively), the bacterial inoculant Siloferm® Plus (Pediococcus acidilactici and Lactobacillus plantarum) and water (control). The wilted material reached a dry matter (DM) content of 336 g/kg at the first cut and 350 g/kg at the second cut. Neither wilting nor the additives had any major effect on the FA proportions, with the only differences in the concentrations of C16:0 and C18:3. Silage treated with bacterial inoculant contained a higher proportion of C16:0 (P<0.05) than silage treated with acid, and a lower (P<0.05) concentration of C18:3 than silage treated with either of the other two additives. In the silages, there were lower (P<0.05) proportions of C16:0, C18:0, C18:1 and C18:3, and higher (P<0.05) proportions of C16:1, C18:2 and other identified FAs, than in the fresh material. A wilting process shorter than 24 h, to a DM content of 330–350 g/kg, did not have any effect on the proportions of FAs in P. pratense L., cv. Grindstad. Since the different additives and wilting strategies tested in this study did not affect the proportions of FAs in silage to a major extent, the results indicate that such a process offers a robust means to avoid losses of FAs that can occur during wilting, while retaining the positive effects of wilting, such as reduced losses of nutrients through effluents.     

Milk fatty acid composition of dairy cows grazing at two pasture allowances and supplemented with different levels and sources of concentrate

Milk fatty acid (FA) composition of dairy cows from two grazing studies was examined. In the first study, effects of concentrate supplementation and pasture allowance were evaluated using 20 multiparous Holstein cows in five 4 × 4 Latin squares. The four treatments resulted from the combination of two pasture allowances (i.e., low, 25 versus high, 40 kg dry matter/cow/day) and two concentrate supplementation levels (i.e., 0 versus 1 kg concentrate/4 kg milk). No interactions occurred between concentrate supplementation and pasture allowance for milk FA composition. Concentrate supplementation increased short-chain FA content, and reduced the content of long-chain FA, trans11 C18:1, and cis9, trans11 conjugated linoleic acid (CLA) (1.36 versus 1.18 g/100 g). Concentrate supplementation increased saturated FA (58.6 versus 54.0 g/100 g) and reduced unsaturated FA content (39.9 versus 43.8 g/100 g). Grazing at high pasture allowance increased short-, medium-, and long-chain FA content, without affecting cis9, trans11 CLA content. Saturated FA content was higher (57.1 versus 55.6 g/100 g), and unsaturated FA content was lower (41.3 versus 42.5 g/100 g), when cows grazed at high pasture allowance. Concentrate supplementation reduced unsaturated FA and cis9, trans11 CLA in milk of dairy cows grazing at two pasture allowances. In the second study, two experiments evaluated effects of different energy supplements in grazing dairy cows. In Experiment 1, 25 multiparous Holstein cows were assigned to a cracked corn (CC) or a steam flaked corn (SFC) supplement containing 667 g/kg of corn grain plus a pelleted protein/mineral supplement. In Experiment 2, 22 multiparous Holstein cows were assigned to a ground corn grain (GC) or a non-forage fiber (NFF) supplement. The GC supplement contained 850 g/kg of corn grain, and the NFF supplement contained 440 g/kg of non-forage fiber sources (i.e., beet pulp, soyhulls, wheat middlings) that partially replaced corn grain. Milk FA composition was not affected by corn processing or carbohydrate source. The content of short-, medium-, long-chain FA, and cis9, trans11 CLA (2.5 g/100 g in Experiment 1; 2.1 g/100 g in Experiment 2) was similar between supplements in both experiments. The type of supplement did not affect the content of saturated (62.6 g/100 g in Experiment 1; 65.4 g/100 g in Experiment 2) and unsaturated FA (37.5 g/100 g in Experiment 1; 34.6 g/100 g in Experiment 2). Supplementation with supplements differing in the rate and extent of ruminal carbohydrate digestion did not affect the milk FA composition of grazing dairy cows.     

5-Aminolevulinic acid production in engineered Corynebacterium glutamicum via C5 biosynthesis pathway

ALA (5-aminolevulinic acid) is an important intermediate in the synthesis of tetrapyrroles and the use of ALA has been gradually increasing in many fields, including medicine and agriculture. In this study, improved biological production of ALA in Corynebacterium glutamicum was achieved by overexpressing glutamate-initiated C₅ pathway. For this purpose, copies of the glutamyl t-RNA reductase HemA from several bacteria were mutated by site-directed mutagenesis of which a HemA version from Salmonella typhimurium exhibited the highest ALA production. Cultivation of the HemA-expressing strain produced approximately 204 mg/L of ALA, while co-expression with HemL (glutamate-1-semialdehyde aminotransferase) increased ALA concentration to 457 mg/L, representing 11.6- and 25.9-fold increases over the control strain (17 mg/L of ALA). Further effects of metabolic perturbation were investigated, leading to penicillin addition that further improves ALA production to 584 mg/L. In an optimized flask fermentation, engineered C. glutamicum strains expressing the HemA and hemAL operon produced up to 1.1 and 2.2 g/L ALA, respectively, under glutamate-producing conditions. The final yields represent 10.7- and 22.0-fold increases over the control strain (0.1 g/L of ALA). From these findings, ALA biosynthesis from glucose was successfully demonstrated and this study is the first to report ALA overproduction in C. glutamicum via metabolic engineering.     

Release of ferulic acid from corn cobs by alkaline hydrolysis

The process of corn cobs alkaline hydrolysis to produce solutions with high hydroxy-cinnamic acids content was investigated. In particular the attention was focused on the solubilisation of ferulic acid (FA) and related compounds, mainly p-coumaric acid (p-CA). Although these compounds have applications as antioxidants, the purpose of this work was to obtain FA solutions that can be used as feedstock for the biotechnological production of vanillin in future studies. The effects of different concentrations of NaOH (0.2 ≤ C_a ≤ 2.0N) and solid/liquid ratios (0.028 ≤ S/L ≤ 0.168 g/g) on the solubilisation of FA versus time have been investigated at room temperature. Optimal hydrolysis conditions (C_a = 0.5N, S/L = 0.084 g/g after 6 h) ensured the production of hydrolysates with relatively high contents of both FA (1171 ± 34 mg/L) and p-coumaric acid (2156 ± 64 mg/L), which can be used in future studies for the microbial transformation into vanillin.     

Revealing the three dimensional architecture of focal adhesion components to explain Ca2 +-mediated turnover of focal adhesions

BackgroundFocal adhesions (FAs) are large, dynamic protein complexes located close to the plasma membrane, which serve as the mechanical linkages and a biochemical signaling hub of cells. The coordinated and dynamic regulation of focal adhesion is required for cell migration. Degradation, or turnover, of FAs is a major event at the trailing edge of a migratory cell, and is mediated by Ca^2 +/calpain-dependent proteolysis and disassembly. Here, we investigated how Ca^2 + influx induces cascades of FA turnover in living cells.MethodsImages obtained with a total internal reflection fluorescence microscope (TIRFM) showed that Ca^2 + ions induce different processes in the FA molecules focal adhesion kinase (FAK), paxillin, vinculin, and talin. Three mutated calpain-resistant FA molecules, FAK-V744G, paxillin-S95G, and talin-L432G, were used to clarify the role of each FA molecule in FA turnover.ResultsVinculin was resistant to degradation and was not significantly affected by the presence of mutated calpain-resistant FA molecules. In contrast, talin was more sensitive to calpain-mediated turnover than the other molecules. Three-dimensional (3D) fluorescence imaging and immunoblotting demonstrated that outer FA molecules were more sensitive to calpain-mediated proteolysis than internal FA molecules. Furthermore, cell contraction is not involved in degradation of FA.ConclusionsThese results suggest that Ca^2 +-mediated degradation of FAs was mediated by both proteolysis and disassembly. The 3D architecture of FAs is related to the different dynamics of FA molecule degradation during Ca^2 +-mediated FA turnover.General significanceThis study will help us to clearly understand the underlying mechanism of focal adhesion turnover by Ca^2 +.     

Effects of total nonstructural carbohydrates and nitrogen balance on voluntary intake of goats and digestibility of gamagrass hay harvested at sunrise and sunset

We evaluated differences in composition of Iuka gamagrass (Tripsacum dactyloides L.) hay harvested at 06:00 (AM harvest) or 18:00 h (PM harvest), and measured how protein supplementation and time of harvest interact to affect the voluntary intake, digestibility, and N balance of goats. Boer cross wethers (n = 28; 24 kg) were randomly assigned to be fed supplement (310 g/kg of crude protein (CP), fed at 110 g/kg of dry matter (DM) intake, 14 goats) or no supplement (14 goats). Within supplemented or not supplemented groups, goats were randomly assigned to a crossover design of AM harvest (seven goats) or PM harvest (seven goats), and housed individually in metabolism crates with free access to water and mineral blocks. They were fed twice daily, with supplement offered 30 min prior to the morning feedings. After a 7-d adaptation, voluntary intake (goats were offered 1100 g/kg of previous day's intake) was measured for 14 d, followed by a 4-d adjustment phase to equalize DM offered between periods, and finally a 5-d digestion and balance phase. After Period 1, goats were switched to their new hay harvest times, and the protocol was repeated. Compared to the AM harvest, the PM harvest had higher (P<0.03) proportions of total nonstructural carbohydrates (TNC, 70.8 g/kg DM versus 59.0 g/kg DM), monosaccharides (37.0 g/kg DM versus 28.6 g/kg DM), di- and polysaccharides (18.5 g/kg DM versus 15.4 g/kg DM) and less neutral detergent fiber (NDF, 700 g/kg versus 710 g/kg). Crude protein (79 g/kg DM) and starch (15.2 g/kg DM) were similar for the PM and AM harvest. Dry matter digestibility was higher (P<0.03) for the PM versus AM harvests (555 g/kg DM versus 531 g/kg DM) and for supplemented versus not supplemented (563 g/kg DM versus 522 g/kg DM). Voluntary gamagrass DM intake (550 g/d versus 548 g/d) and calculated total digestible DM intake (327 g/d versus 313 g/d) were similar for the PM and AM harvest. However, total digestible DM intake during the digestion and balance phase was higher (P<0.01) for the PM versus AM harvest (317 g/d versus 299 g/d). Time of harvest did not affect N intake, digestion, or calculated retention. Compared to no supplementation, the supplement improved (P<0.01) N digestion (6.1 g/d versus 3.7 g/d) and retention (2.2 g/d versus 1.1 g/d). The PM harvest increased DM digested, largely TNC and digestible DM intake by goats due to increased TNC and not because of a 2% increase in DM intake. Providing a protein supplement had very limited effects on intake and digestibility of gamagrass.     

Allometric scaling of fatty acyl chains in fowl liver,lung and kidney,but not in brain phospholipids

The phospholipid (PL) fatty acyl chain (FA) composition (mol%) was determined in the kidney, liver, lung and brain of 8 avian species ranging in body mass from 150 g (Japanese quail, Coturnix coturnix japonica) to 19 kg (turkey, Meleagris gallopavo). In all organs except the brain, docosahexaenoic acid (C22:6 n3, DHA) was found to show a negative allometric scaling (allometric exponent: B = ? 0.18; ? 0.20 and ? 0.24, for kidney, liver and lung, respectively). With minor inter-organ differences, smaller birds had more n3 FAs and longer FA chains in the renal, hepatic and pulmonary PLs. Comparing our results with literature data on avian skeletal muscle, liver mitochondria and kidney microsomes and divergent mammalian tissues, the present findings in the kidney, liver and lung PLs seem to be a part of a general relationship termed “membranes as metabolic pacemakers”. Marked negative allometric scaling was found furthermore for the tissue malondialdehyde concentrations in all organs except the brain (B = ? 0.17; ? 0.13 and ? 0.05, respectively). In the liver and kidney a strong correlation was found between the tissue MDA and DHA levels, expressing the role of DHA in shaping the allometric properties of membrane lipids.     

Influences of short-term pre-slaughter dietary manipulation in sheep and goats on pH and microbial loads of gastrointestinal tract

Sheep (BW = 39.9 kg, n = 16) and goats (BW = 32.8 kg, n = 16) were used in a completely randomized design to determine the effect of short-term pre-slaughter diet and feed deprivation (FD) time on pH and microbial loads in the gastrointestinal tract (GIT) contents. In a 2 × 2 × 2 factorial treatment arrangement, the main effects of species, diet, and FD time prior to slaughter and their interactions were studied. Animals were fed either a hay or concentrate diet for 4 d and then feed deprived for either 12 or 24-h prior to slaughter. The pH of rumen and colon contents as well as weight of GIT was measured. The contents of rumen and rectum were also sampled for microbial analysis. The GIT of sheep (1.82 kg) was heavier (P < 0.05) than that of goats (1.46 kg). The 12-h FD group (1.74 kg) had a higher (P < 0.05) GIT weight than the 24-h FD group (1.53 kg). Hay-fed animals had higher (P < 0.05) rumen (7.08 vs. 6.43) and colon pH values (7.02 vs. 6.56) than those of the concentrate-fed animals. The 24-h FD group (3.39 ± 0.272 log₁₀CFU/g) contained more (P < 0.05) Escherichia coli in the rumen than did the 12-h FD (2.17 ± 0.272 log₁₀CFU/g) group. The concentrate-fed animals (3.49 ± 0.289 log₁₀CFU/g) had higher (P < 0.05) coliform counts in the rumen than the hay-fed animals (2.43 ± 0.289 log₁₀CFU/g). The 24-h FD group (3.42 ± 0.289 log₁₀CFU/g) had a higher (P < 0.05) concentration of coliform than did the 12-h FD group (2.50 ± 0.289 log₁₀CFU/g). The 24-h FD group (3.31 ± 0.259 log₁₀CFU/g) also had higher (P < 0.05) Enterobacteriaceae counts in the rumen than did in the 12-h FD group (2.47 ± 0.259 log₁₀CFU/g). Goats (5.71 ± 0.158 log₁₀CFU/g) had lower (P < 0.05) total plate counts in the rumen compared to sheep (6.27 ± 0.158 log₁₀CFU/g). The concentrate-fed animals had higher (P < 0.05) E. coli (6.44 vs. 4.01 ± 0.468 log₁₀CFU/g), total coliform (6.74 vs. 4.16 ± 0.469 log₁₀CFU/g), Enterobacteriaceae (6.93 vs. 3.83 ± 0.651 log₁₀CFU/g), and total plate counts (7.79 vs. 7.28 ± 0.170 log₁₀CFU/g) in the rectum than the hay-fed animals. The results indicate that microbial loads in the GIT of small ruminants may be reduced by either feeding hay for 4 d or depriving feed for 12-h prior to slaughter.     

The effect of benzoic acid concentration on nitrogen metabolism,manure ammonia and odour emissions in finishing pigs

A complete randomised block design experiment was conducted to investigate the effects of benzoic acid inclusion level on nitrogen (N) metabolism, and manure ammonia (NH₃) and odour emissions in finishing pigs. Sixteen boars (64 kg live weight ± 1.5 kg) were assigned to one of four dietary treatments (T) varying in benzoic acid concentration: (T1) 0 g benzoic acid/kg (as fed); (T2) 10 g benzoic acid/kg; (T3) 20 g benzoic acid/kg; (T4) 30 g benzoic acid/kg. Animals were housed in individual metabolism crates and feed was provided ad libitum. All diets were formulated to have similar concentration of digestible energy and ileal digestible lysine with benzoic acid replacing wheat in the diet. There was a linear decrease in NH₃ emission (P<0.001), as the dietary benzoic acid concentration increased (141.4 mg/g versus 40.5 mg/g N intake (S.E.M. 12.1) over the 240-h storage period). However, there was no effect (P>0.05) of benzoic acid on odour concentration. Urinary nitrogen (N) excretion, total N excretion and the urinary:faecal N ratio were linearly reduced (P<0.05) with increasing benzoic acid inclusion. Furthermore, N retention increased linearly (P<0.05) as benzoic acid concentration increased from 0 g/kg to 30 g/kg in the diet. In conclusion, the inclusion of benzoic acid in the diet of finishing pigs has the potential to reduce total and urinary N excretion and the urinary to faecal N ratio. This was mirrored by reductions in manure NH₃ emissions in the benzoic acid supplemented treatments.     

The effect of inclusion of a high lactose supplement in finishing diets on nutrient digestibility,nitrogen excretion,volatile fatty acid concentrations and ammonia emission from boars

An experiment (complete randomised design) was conducted to investigate the use of lactose in finishing pig diets on nitrogen excretion patterns and ammonia emissions. Diets were formulated to have similar digestible energy (13.8 MJ/kg) and total lysine (11.0 g/kg) contents. Thirty boars (58 kg) were assigned to one of the five dietary treatments (six per treatment) as follows: (T1) 0 g Lactofeed/kg (860 g whey permeate/kg, 140 g soya bean meal/kg, Volac International, UK) (LF70); (T2) 40 g LF70/kg; (T3) 80 g LF70/kg; (T4) 120 g LF70/kg; and (T5) 160 g LF70/kg. After a 14-day adaptation period, pigs were housed in metabolism crates and faeces and urine were collected for nitrogen balance and ammonia emission. NH₃–N emission was measured over 10 days using a laboratory scale procedure. Coefficient of total tract apparent digestibility (CTTAD) for dry matter and gross energy digestibility were affected by dietary LF70 inclusion (quadratic, P<0.05). T1 had a higher urinary N and total N excretion (P<0.1), a lower N retention (as a proportion of intake) (P<0.05) than the LF70 supplemented diets. T1 had a higher NH₃–N per gram of N intake from days 0 to 4 (P<0.05) than the LF70 supplemented diets. In the faeces isobutyric acid and isovaleric acid were affected by dietary LF70 level (quadratic, P<0.01). There was a linear decrease in the ratio of acetic acid: propionic acid with increasing LF70 levels (P<0.05). In the caecum and colon lactobacilli concentrations were affected by dietary LF70 inclusion (quadratic, P<0.05). In conclusion, the current study demonstrates that the incorporation of lactofeed in finisher pig diets increases CTTAD for gross energy and the concentration of lactobacilli in the hind-gut and decreases total nitrogen excretion. The inclusion of lactofeed also reduces the quantity of ammonia emitted from fresh manure in the first 4 days of storage.     

Modulation of blood oxylipin levels by long-chain omega-3 fatty acid supplementation in hyper- and normolipidemic men

IntroductionLong chain omega-3 polyunsaturated fatty acids (LC n-3 PUFA) such as EPA and DHA have been shown to possess beneficial health effects, and it is believed that many of their effects are mediated by their oxygenated products (oxylipins). Recently, we have shown that serum levels of several hydroxy, epoxy, and dihydroxy FAs are dependent on the individual status of the parent FAs in a cohort of normo- and hyperlipidemic subjects. So far, the effect of an increased dietary LC n-3 PUFA intake on hydroxy, epoxy, and dihydroxy FA levels has not been investigated in subjects with mild combined hyperlipidemia.Subjects and methodsIn the present study, we compared oxylipin patterns of 10 hyperlipidemic (cholesterol >200 mg/dl; triglyceride >150 mg/ml) and 10 normolipidemic men in response to twelve weeks of LC n-3 PUFA intake (1.14 g DHA and 1.56 g EPA). Levels of 44 free hydroxy, epoxy and dihydroxy FAs were analyzed in serum by LC-MS. Additionally, oxylipin levels were compared with their parent PUFA levels in erythrocyte membranes; a biomarker for the individual PUFA status.ResultsDifferences in the oxylipin pattern between normo- and hyperlipidemic subjects were minor before and after treatment. In all subjects, levels of EPA-derived oxylipins (170–4800 pM) were considerably elevated after LC n-3 PUFA intake (150–1400%), the increase of DHA-derived oxylipins (360–3900 pM) was less pronounced (30–130%). The relative change of EPA in erythrocyte membranes is strongly correlated (r≥0.5; p<0.05) with the relative change of corresponding epoxy and dihydroxy FA serum levels. The effect on arachidonic acid (AA)-derived oxylipin levels (140–27,100 pM) was inconsistent.Discussion and conclusionsThe dietary LC PUFA composition has a direct influence on the endogenous oxylipin profile, including several highly biological active EPA- and DHA-derived lipid mediators. The shift in oxylipin pattern appears to be dependent on the initial LC PUFA status particularly for EPA. The finding that also levels of other oxylipins derived from ALA, LA or AA are modified by LC n-3 PUFA intake might suggest that at least some of the effects of EPA and DHA could be mediated by a shift in the entire oxylipin profile.     

Safety of Prunus africana and Warburgia ugandensis in asthma treatment

The aim of the study was to determine the possible cytotoxicity of the aqueous stem bark extracts of Prunus africana and Warburgia ugandensis to Vero E6 cells and acute toxicity in BALB/c mice. Despite being some of the most popular medicinal plants used in Africa, little is known about the safety. In-vitro cytotoxicity tests on Vero E6 cells were investigated using MTT assay to assess the safety of the two plant extracts. Vero E6 cells on growing to confluence were incubated with different drug concentrations for 48 h for the drug to take effect. Viability of the cells was measured by a scanning multiwell spectrophotometer, color intensity being equivalent to viable cells which reduce MTT to soluble formazan crystals. This was done by determining the CC₅₀ of the extracts, CC₅₀ being the concentration of the dose of the compound/extract that kills 50% of the cells. In acute toxicity a total of 55 mice were used. Mice were divided into eleven groups of 5 mice, one group served as negative control and ten groups received oral gavage doses at 500, 889.56, 1581.6, 2812.15 or 5000 mg/kg body weight once. Mortality and other signs of toxicity were recorded within 24 h and the weights of the surviving mice taken for 14 days thereafter. P. africana had CC₅₀ of 104.08 μg/ml while W. ugandensis had CC₅₀ > 250 μg/ml and both were classified as not cytotoxic. There was no mortality observed in groups of mice that received P. africana extracts at 500 and 889.56 mg/kg body weight. There was 20%, 60% and 100% mortality observed within 24 h for mice that received P. africana extracts at 1581.64, 2812.15 and 5000 mg/kg body weight respectively. Lethal dose (LD₅₀) for P. africana was 2201.207 mg/kg body weight. W. ugandensis extracts had no mortality recorded in all dose levels and the LD₅₀ was > 5000 mg/kg body weight. The weights of mice that survived the entire 14 days in all groups increased and were not significantly different from that of controls p > 0.05. From the in vitro and in vivo studies, the two extracts were safe to use. Though with their customary value among many Kenyan communities in management of asthma among other ailments there is a need for further validation of any anti-asthmatic properties and responsible chemical compounds to augment the findings.     

Intravenous injection of urocortin 1 induces a CRF2 mediated increase in circulating ghrelin and glucose levels through distinct mechanisms in rats

Urocortins (Ucns) injected peripherally decrease food intake and gastric emptying through peripheral CRF₂ receptors in rodents. However, whether Ucns influence circulating levels of the orexigenic and prokinetic hormone, ghrelin has been little investigated. We examined plasma levels of ghrelin and blood glucose after intravenous (iv) injection of Ucn 1, the CRF receptor subtype involved and underlying mechanisms in ad libitum fed rats equipped with a chronic iv cannula. Ucn 1 (10 μg/kg, iv) induced a rapid onset and long lasting increase in ghrelin levels reaching 68% and 219% at 0.5 and 3 h post injection respectively and a 5-h hyperglycemic response. The selective CRF₂ agonist, Ucn 2 (3 μg/kg, iv) increased fasting acyl (3 h: 49%) and des-acyl ghrelin levels (3 h: 30%) compared to vehicle while the preferential CRF₁ agonist, CRF (3 μg/kg, iv) had no effect. <!-- no-mfc -->Ucn 1's<!-- /no-mfc --> stimulatory actions were blocked by the selective CRF₂ antagonist, astressin₂-B (100 μg/kg, iv). Hexamethonium (10 mg/kg, sc) prevented Ucn 1-induced rise in total ghrelin levels while not altering the hyperglycemic response. These data indicate that systemic injection of Ucns induces a CRF₂-mediated increase in circulating ghrelin levels likely via indirect actions on gastric ghrelin cells that involves a nicotinic pathway independently from the hyperglycemic response.     

Engineering Escherichia coli to convert acetic acid to free fatty acids

Fatty acids (FAs) are promising precursors of advanced biofuels. This study investigated conversion of acetic acid (HAc) to FAs by an engineered Escherichia coli strain. We combined established genetic engineering strategies including overexpression of acs and tesA genes, and knockout of fadE in E. coli BL21, resulting in the production of ~1 g/L FAs from acetic acid. The microbial conversion of HAc to FAs was achieved with ~20% of the theoretical yield. We cultured the engineered strain with HAc-rich liquid wastes, which yielded ~0.43 g/L FAs using waste streams from dilute acid hydrolysis of lignocellulosic biomass and ~0.17 g/L FAs using effluent from anaerobic-digested sewage sludge. ¹³C-isotopic experiments showed that the metabolism in our engineered strain had high carbon fluxes toward FAs synthesis and TCA cycle in a complex HAc medium. This proof-of-concept work demonstrates the possibility for coupling the waste treatment with the biosynthesis of advanced biofuel via genetically engineered microbial species.     

Interaction kinetics of liposome-incorporated unsaturated fatty acids with fatty acid-binding protein 3 by surface plasmon resonance

The role of heart-type fatty acid-binding protein (FABP3) in human physiology as an intracellular carrier of fatty acids (FAs) has been well-documented. In this study, we aimed to develop an analytical method to study real-time interaction kinetics between FABP3 immobilized on the sensor surface and unsaturated C18 FAs using surface plasmon resonance (SPR). To establish the conditions for SPR experiments, we used an FABP3-selective inhibitor 4-(2-(1-(4-bromophenyl)-5-phenyl-1H-pyrazol-3-yl)-phenoxy)-butyric acid. The affinity index thus obtained was comparable to that reported previously, further supporting the usefulness of the SPR-based approach for evaluating interactions between FABPs and hydrophobic ligands. A pseudo-first-order affinity of FABP3 to K⁺ petroselinate (C18:1 Δ6 cis), K⁺ elaidate (C18:1 Δ9 trans), and K⁺ oleate (C18:1 Δ9 cis) was characterized by the dissociation constant (K_d) near micromolar ranges, whereas K⁺ linoleate (C18:2 Δ9,12 cis/cis) and K⁺ α-linolenate (C18:3 Δ9,12,15 cis/cis/cis) showed a higher affinity to FABP3 with K_d around 1 × 10⁻⁶ M. Interactions between FAPB3 and C18 FAs incorporated in large unilamellar vesicles consisting of 1,2-dimyristoyl-sn-glycero-3-phosphocholine and FAs (5:1 molar ratio) were also analysed. Control DMPC liposomes without FA showed only marginal binding to FABP3 immobilized on a sensor chip while liposome-incorporated FA revealed significant responses in sensorgrams, demonstrating that the affinity of FAs to FABP3 could be evaluated by using the liposome-incorporated analytes. Significant affinity to FABP3 was observed for monounsaturated fatty acids (K_d in the range of 1 × 10⁻⁷ M). These experiments demonstrated that highly hydrophobic compounds in a liposome-incorporated form could be subjected to SPR experiments for kinetic analysis.     

Physiological parameters correlated with Tomato Mosaic Virus inducing defensive response in Datura metel

Programed cell death resembles a real nature active defense in Datura metel against TMV after three days of virus infection. This adaptive plant immune response was quantitatively assessed against Tomato Mosaic Virus infection by the following physiological markers; Chlorophyll-a (mg/g), Chlorophyll-b (mg/g), total protein (mg/g), hydrogen peroxide H₂O₂ (μmol/100 mg), DNA (μg/100 mg), RNA (μg/100 mg), Salicylic acid (μg/g), and Comet Assays. Parameters were assessed for asymptomatic healthy and symptomatic infected detached leaves. The results indicated H₂O₂ and Chlorophyll-a as the most potential parameters. Chlorophyll-a was considered the only significant predictor variant for the H₂O₂ dependent variant with a P value of 0.001 and R-square of 0.900. The plant immune response was measured within three days of virus infection using the cutoff value of H₂O₂ (⩽1.095 μmol/100 mg) and (⩽3.201 units) for the tail moment in the Comet Assay. Their percentage changes were 255.12% and 522.40% respectively which reflects the stress of virus infection in the plant. Moreover, H₂O₂ showed 100% specificity and sensitivity in the symptomatic infected group using the receiver-operating characteristic (ROC). All tested parameters in the symptomatic infected group had significant correlations with twenty-five positive and thirty-one negative correlations where the P value was <0.05 and 0.01. Chlorophyll-a parameter had a crucial role of highly significant correlation between total protein and salicylic acid. Contrarily, this correlation with tail moment unit was (r = −0.930, P < 0.01) where the P value was <0.01. The strongest significant negative correlation was between Chlorophyll-a and H₂O₂ at P < 0.01, while moderate negative significant correlation was seen for Chlorophyll-b where the P value < 0.05. The present study discloses the secret of the three days of rapid transient production of activated oxygen species (AOS) that was enough for having potential quantitative physiological parameters for defensive plant response toward the virus.     

Molt cycle related changes and effect of short term starvation on the biochemical constituents of the blue swimmer crab Portunus pelagicus

Synthesis and hardening of a new exoskeleton are essential to the arthropod molting process. The present study emphasizes the variations in the levels of hemolymph total free sugars, hepatopancreas glycogen and cuticular proteins during the molting stages of Portunus pelagicus. It also reports the effect of short-term starvation conditions on the biochemical constituents of the hemolymph. Intermolt crabs were subjected to 6 days of starvation and hemolymph samples were taken. Standard biochemical procedures were followed toward the quantification of total proteins, total free sugars and total lipids. The total free sugar level in the hemolymph of P. pelagicus was observed to increase during early premolt D₀ (3.108 ± 0.032 g/ml) and a gradual decrease till late postmolt B stage (0.552 ± 0.124 g/ml), suggesting the need for total free sugars to provide energy for the apolysis process. Increase in the levels of hepatopancreas glycogen was observed from 1225 ± 0.04 μg/mg in early premolt D₀ to 1700 ± 0.3 μg/mg in late premolt D_2–3. This is in correlation with the decreased levels of free sugars during premolt stages, suggesting an increase in the storage of glycogen reserves in the hepatopancreas. Cuticular proteins increased during stage B (2.702 ± 0.093 g/ml) and stage C (3.065 ± 0.012 g/ml), indicating exoskeleton hardening and mineralization. Results of the starvation studies clearly showed a steady decline in the level of total free sugars till day 6 (0.099 ± 0.00 g/ml) when compared to the control (8.646 ± 0.08 g/ml). Gradual decrease of total lipids was also observed from the first day of the experiment (6.088 ± 2.44 g/ml) to the last day of the study (0.401 ± 0.20 g/ml) which was 85% lesser than the control (8.450 ± 0.49 g/ml)suggesting the efficient usage of total sugars to consolidate the loss of energy reserves during starvation. The knowledge of Molt-cycle events can be used as a tool for the evaluation of the developmental state providing a morphological reference system for physiological and biochemical studies related to crab aquaculture. Starvation studies enlightens that increasing carbohydrate levels in crab feed together with good protein content could alleviate the natural effects of starvation, improve farm productivity and reduce the deleterious impact of nitrogen pollution generated by rich-protein feeds used in crab farming.     

Effect of rare earth elements on in vitro rumen microbial fermentation and feed digestion

The objectives of this study were to investigate the effects of rare earth elements (REEs) on in vitro rumen fermentation, gas production, microbial protein synthesis and nutrient digestion using in vitro batch culture and continuous culture technique. A mixture of REE containing (g/kg) 380 g of LaCI₃·6H₂O, 521 g of CeCI₃·6H₂O, 30 g of PrCI₃·6H₂O and 69 g chlorides of other light REEs. The experimental diet consisted of 885 g/kg barley grain, 84 g/kg barley silage and 31 g/kg supplement (dry matter (DM) basis). Diet supplemented with different dosages of REE (control, no additional REE; low, 400 mg/kg REE; and high, 800 mg/kg REE, DM basis) were incubated for 4, 8, 14 and 24 h in diluted rumen fluid. At the end of 24 h of incubation, gas production and concentration of volatile fatty acid (VFA) linearly increased with increasing REE supplementation; whereas, influence of REE supplementation on VFA profile was marginal. Dry matter disappearance was not affected (P>0.10). Six dual-flow continuous culture fermenters were used in a replicated 3 × 3 Latin square with same treatments and same diet used in the batch culture. Mean ruminal pH (5.71) and total VFA (93.6 mM) concentration were not affected by supplementation of REE. The molar proportion (mol/100 mol) of acetate (39.1) and propionate (50.5) was similar among the treatments. However, the proportion (mol/100 mol) of butyrate was higher with the high REE (6.6) than with low REE (5.3) or the control (5.8). Ruminal true digestibilities of organic matter (OM) (0.785, 0.811 and 0.828), acid detergent fibre (0.360, 0.431 and 0.432) and crude protein (0.496, 0.590 and 0.589) for control, low and high REE, respectively, linearly increased with increasing REE supplementation, whereas, the increase in ruminal digestibility from low to high dosage of REE was minimal. Microbial nitrogen (N) production (g/day) and microbial efficiency (g N/kg of truly fermented OM) were not affected by treatments. Improvement of ruminal digestibility of OM due to REE supplementation was attributed to the increase in digestibility of fibre and degradability of protein. The results suggest that REE supplementation improved ruminal fibrolytic and proteolytic activities.     

Antioxidative,antimicrobial and cytotoxic effects of the phenolics of Leea indica leaf extract

This study investigated the phytochemical, antioxidative, antimicrobial and cytotoxic effects of Leea indica leaf ethanol extract. Phytochemical values namely total phenolic and flavonoid contents, total antioxidant capacity, DPPH radical scavenging effect, FeCl₃ reducing power, DMSO superoxide scavenging effect and Iron chelating effects were studied by established methods. Antibacterial, antifungal and cytotoxic effects were screened by disk diffusion technique, food poison technique and brine shrimp bioassay, respectively. Results showed the total phenolic content 24.00 ± 0.81 g GAE/100 g, total flavonoid content 194.68 ± 2.43 g quercetin/100 g and total antioxidant capacity 106.61 ± 1.84 g AA/100 g dry extract. Significant (P < 0.05) IC₅₀ values compared to respective standards were recorded in DPPH radical scavenging (139.83 ± 1.40 μg/ml), FeCl₃ reduction (16.48 ± 0.64 μg/ml), DMSO superoxide scavenging (676.08 ± 5.80 μg/ml) and Iron chelating (519.33 ± 16.96 μg/ml) methods. In antibacterial screening, the extract showed significant (P < 0.05) zone of inhibitions compared to positive controls Ampicillin and Tetracycline against Gram positive Bacillus subtilis, Bacillus cereus, Bacillus megaterium, and Staphylococcus aureus and Gram negative Salmonella typhi, Salmonella paratyphi, Pseudomonas aeroginosa, Shigella dysenteriae, Vibrio cholerae, and Escherichia coli. Significant minimum inhibitory concentrations compared to tetracycline were obtained against the above organisms. In antifungal assay, the extract inhibited the growth of Aspergillus flavus, Candida albicans and Fusarium equisetii by 38.09 ± 0.59, 22.58 ± 2.22, and 22.58 ± 2.22%, respectively. The extract showed a significant LC₅₀ value compared to vincristine sulfate in cytotoxic assay. The results evidenced the potential antioxidative, antimicrobial and cytotoxic capacities of Leea inidica leaf extract to be processed for pharmaceutical use.