Gag-Pol supplied in trans is efficiently packaged and supports viral function in human immunodeficiency virus type 1

The intracellular trafficking and subsequent incorporation of Gag-Pol into human immunodeficiency virus type 1 (HIV-1) remains poorly defined. Gag-Pol is encoded by the same mRNA as Gag and is generated by ribosomal frameshifting. The multimerization of Gag and Gag-Pol is an essential step in the formation of infectious viral particles. In this study, we examined whether the interaction between Gag and Gag-Pol is initiated during protein translation in order to facilitate the trafficking and subsequent packaging of Gag-Pol into the virion. A conditional cotransfection system was developed in which virion formation required the coexpression of two HIV-1-based plasmids, one that produces both Gag and Gag-Pol and one that only produces Gag-Pol. The Gag-Pol proteins were either immunotagged with a His epitope or functionally tagged with a mutation (K65R) in reverse transcriptase that is associated with drug resistance. Gag-Pol packaging was assessed to determine whether the Gag-Pol incorporated into the virion was preferentially packaged from the plasmid that expressed both Gag and Gag-Pol or whether it could be packaged from either plasmid. Our data show that translation of Gag and Gag-Pol from the same mRNA is not critical for virion packaging of the Gag-Pol polyprotein or for viral function.

Sesamin modulation of lipid class and fatty acid profile in early juvenile teleost, Lates calcarifer, fed different dietary oils

Sesamin, a major sesame seed lignan, has diverse biological functions including the modulation of molecular actions in lipid metabolic pathways and reducing cholesterol levels. Vertebrates have different capacities to biosynthesize long-chain PUFA from dietary precursors and sesamin can enhance the biosynthesis of ALA to EPA and DHA in marine teleost. Early juvenile barramundi, Lates calcarifer, were fed for two weeks on diets rich in ALA or SDA derived from linseed or Echium plantagineum, respectively. Both diets contained phytosterols and less cholesterol compared with a standard fish oil-based diet. The growth rates were reduced in the animals receiving sesamin regardless of the dietary oil. However, the relative levels of n-3 LC-PUFA in total lipid, but not the phospholipid, increased in the whole body by up to 25% in animals fed on sesamin with ALA or SDA. Sesamin reduced the relative levels of triacylglycerols and increased polar lipid, and did not affect the relative composition of phospholipid subclasses or sterols. Sesamin is a potent modulator for LC-PUFA biosynthesis in animals, but probably will have more effective impact at advanced ages. By modulating certain lipid metabolic pathways, sesamin has probably disrupted the body growth and development of organs and tissues in early juvenile barramundi.

Up-regulated Desaturase and Elongase Gene Expression Promoted Accumulation of Polyunsaturated Fatty Acid (PUFA) but Not Long-Chain PUFA in Lates calcarifer, a Tropical Euryhaline Fish, Fed a Stearidonic Acid- and γ-Linoleic Acid-Enriched Diet

The limited activity of Δ6 fatty acid desaturase (FAD6) on α-linolenic (ALA, 18:3n-3) and linoleic (LA, 18:2n-6) acids in marine fish alters the long-chain (≥C₂₀) polyunsaturated fatty acid (LC-PUFA) concentration in fish muscle and liver when vegetable oils replace fish oil (FO) in aquafeeds. Echium oil (EO), rich in stearidonic acid (SDA, 18:4n-3) and γ-linoleic acid (GLA, 18:3n-6), may enhance the biosynthesis of n-3 and n-6 LC-PUFA by bypassing the rate-limiting FAD6 step. Nutritional and environmental modulation of the mechanisms in LC-PUFA biosynthesis was examined in barramundi, Lates calcarifer, a tropical euryhaline fish. Juveniles were maintained in either freshwater or seawater and fed different dietary LC-PUFA precursors present in EO or rapeseed oil (RO) and compared with FO. After 8 weeks, growth of fish fed EO was slower compared to the FO and RO treatments. Irrespective of salinity, expression of the FAD6 and elongase was up-regulated in fish fed EO and RO diets, but did not lead to significant accumulation of LC-PUFA in the neutral lipid of fish tissues as occurred in the FO treatment. However, significant concentrations of eicosapentaenoic acid (EPA, 20:5n-3) and arachidonic acid (ARA, 20:4n-6), but not docosahexaenoic acid (DHA, 22:6n-3), appeared in liver and, to a lesser extent, in muscle of fish fed EO with marked increases in the phospholipid fraction. Fish in the EO treatment had higher EPA and ARA in their liver phospholipids than fish fed FO. Endogenous conversion of dietary precursors into neutral lipid LC-PUFA appears to be limited by factors other than the initial rate-limiting step. In contrast, phospholipid LC-PUFA had higher biosynthesis, or selective retention, in barramundi fed EO rather than RO.

Changes in oil content, lipid class and fatty acid composition of the microalga Chaetoceros calcitrans over different phases of batch culture

 Abstract
The diatom Chaetoceros calcitrans is a microalgal species used as food for larva in aquaculture for many species worldwide. Chaetoceros calcitrans is an important source of omega 3 long chain (C ≥ 20) polyunsaturated fatty acids (n-3 LC PUFA), chiefly eicosapentaenoic acid (EPA, 20:5n-3). This article reports lipid content, lipid class composition and fatty acid profiles of each lipid class during the growth cycle of batch cultures of C. calcitrans. Total lipid content and the concentration of neutral lipid were highest in the late stationary growth phase (day 12). However, the amount of EPA was highest during the logarithmic growth phase (1.24 pg/cell on day 4). EPA was initially concentrated in the glycolipid fraction, but this fraction decreased during logarithmic growth, coinciding with the increase in neutral lipid. Docosahexaenoic acid (22:6n-3, DHA) (0.91 pg/cell) was reported as a major fatty acid (>10 mg/100 g) in all lipid classes on day 1. DHA was depleted quickly from the neutral lipid and glycolipid classes, but at a slower rate from the polar lipid fraction. This work confirms that C. calcitrans is a good source of lipid, in particular EPA, for larval and adult filter feeders in aquaculture.

Current status of biotechnological production of the cholesterol lowering drug lovastatin and its biomedical applications

Lovastatin is a potent hypercholesterolemic drug used for lowering blood cholesterol. It acts by competitively inhibiting the enzyme, 3-hydroxy-3-methylglutaryl coenzyme A reductase involved in the biosynthesis of cholesterol. It is produced by a variety of filamentous fungi including Penicillium species, Monascus ruber and Aspergillus terreus as a secondary metabolite. Production of lovastatin by biotechnology decreases the production cost compared to costs of chemical synthesis. In recent years, lovastatin has also been reported as a potential therapeutic agent for the treatment of various types of tumors and also play a tremendous role in the regulation of the inflammatory and immune response, coagulation process, bone turnover, neovascularization, vascular tone, and arterial pressure. This review focus on the structure, biosynthesis, biotechnological production and biomedical applications of lovastatin.

Response to long-distance relocation in Asian elephants (Elephas maximus) : monitoring adrenocortical activity via serum, urine, and feces

Understanding how elephants respond to potentially stressful events, such as relocation, is important for making informed management decisions. This study followed the relocation of eight Asian elephants from the Cocos (Keeling) Islands to mainland Australia. The first goal of this study was to examine patterns of adrenocortical activity as reflected in three different substrates: serum, urine, and feces. We found that the three substrates yielded very different signals of adrenocortical activity. Fecal glucocorticoid metabolites (FGM) increased as predicted post-transport, but urinary glucocorticoid metabolites (UGM) were actually lower following transport. Serum cortisol levels did not change significantly. We suggest that the differences in FGM and UGM may reflect changes in steroid biosynthesis, resulting in different primary glucocorticoids being produced at different stages of the stress response. Additional studies are needed to more thoroughly understand the signals of adrenocortical activity yielded by different substrates. The second goal was to examine individual variation in patterns of adrenal response. There was a positive correlation between baseline FGM value and duration of post-transfer increase in FGM concentration. Furthermore, an individual's adrenocortical response to relocation was correlated with behavioral traits of elephants. Elephants that were described by keepers as being “curious” exhibited a more prolonged increase in FGM post-transfer, and “reclusive” elephants had a greater increase in FGM values. Future research should investigate the importance of these personality types for the management and welfare of elephants.

Fish oil replacement in current aquaculture feed : is cholesterol a hidden treasure for fish nutrition?

Teleost fish, as with all vertebrates, are capable of synthesizing cholesterol and as such have no dietary requirement for it. Thus, limited research has addressed the potential effects of dietary cholesterol in fish, even if fish meal and fish oil are increasingly replaced by vegetable alternatives in modern aquafeeds, resulting in progressively reduced dietary cholesterol content. The objective of this study was to determine if dietary cholesterol fortification in a vegetable oil-based diet can manifest any effects on growth and feed utilization performance in the salmonid fish, the rainbow trout. In addition, given a series of studies in mammals have shown that dietary cholesterol can directly affect the fatty acid metabolism, the apparent in vivo fatty acid metabolism of fish fed the experimental diets was assessed. Triplicate groups of juvenile fish were fed one of two identical vegetable oil-based diets, with additional cholesterol fortification (high cholesterol, H-Chol) or without (low cholesterol, L-Chol), for 12 weeks. No effects were observed on growth and feed efficiency, however, in fish fed H-Col no biosynthesis of cholesterol, and a remarkably decreased apparent in vivo fatty acid b-oxidation were recorded, whilst in LChol fed fish, cholesterol was abundantly biosynthesised and an increased apparent in vivo fatty acid b-oxidation was observed. Only minor effects were observed on the activity of stearyl-CoA desaturase, but a significant increase was observed for both the transcription rate in liver and the apparent in vivo activity of the fatty acid D-6 desaturase and elongase, with increasing dietary cholesterol. This study showed that the possible effects of reduced dietary cholesterol in current aquafeeds can be significant and warrant future investigations.

Bioconversion of α-Linolenic Acid into n-3 Long-Chain polyunsaturated fatty acid in hepatocytes and ad hoc cell culture optimisation

This study aimed to establish optimal conditions for a cell culture system that would allow the measurement of 18:3n-3 (ALA) bioconversion into n-3 long-chain polyunsaturated fatty acid (n-3 LC-PUFA), and to determine the overall pathway kinetics. Using rat hepatocytes (FaO) as model cells, it was established that a maximum 20:5n-3 (EPA) production from 50 mM ALA initial concentration was achieved after 3 days of incubation. Next, it was established that a gradual increase in the ALA concentration from 0 up to 125mM lead to a proportional increase in EPA, without concomitant increase in further elongated or desaturated products, such as 22:5n-3 (DPA) and 22:6n-3 (DHA) in 3 day incubations. Of interest, ALA bioconversion products were observed in the culture medium. Therefore, in vitro experiments disregarding the medium fatty acid content are underestimating the metabolism efficiency. The novel application of the fatty acid mass balance (FAMB) method on cell culture system (cells with medium) enabled quantifying the apparent enzymatic activities for the biosynthesis of n-3 LC-PUFA. The activity of the key enzymes was estimated and showed that, under these conditions, 50% (Km) of the theoretical maximal (Vmax = 3654 mmol.g21 of cell protein.hour21) Fads2 activity on ALA can be achieved with 81 mM initial ALA. Interestingly, the apparent activity of Elovl2 (20:5n-3 elongation) was the slowest amongst other biosynthesis steps. Therefore, the possible improvement of Elovl2 activity is suggested toward a more efficient DHA production from ALA. The present study proposed and described an ad hoc optimised cell culture conditions and methodology towards achieving a reliable experimental platform, using FAMB, to assist in studying the efficiency of ALA bioconversion into n-3 LC-PUFA in vitro. The FAMB proved to be a powerful and inexpensive method to generate a detailed description of the kinetics of n-3 LC-PUFA biosynthesis enzymes activities in vitro.

The A622 gene in Nicotiana glauca (tree tobacco): evidence for a functional role in pyridine alkaloid synthesis

Nicotiana glauca (Argentinean tree tobacco) is atypical within the genus Nicotiana, accumulating predominantly anabasine rather than nicotine and/or nornicotine as the main component of its leaf pyridine alkaloid fraction. The current study examines the role of the A622 gene from N. glauca (NgA622) in alkaloid production and utilises an RNAi approach to down-regulate gene expression and diminish levels of A622 protein in transgenic tissues. Results indicate that RNAi-mediated reduction in A622 transcript levels markedly reduces the capacity of N. glauca to produce anabasine resulting in plants with scarcely any pyridine alkaloids in leaf tissues, even after damage to apical tissues. In addition, analysis of hairy roots containing the NgA622-RNAi construct shows a substantial reduction in both anabasine and nicotine levels within these tissues, even if stimulated with methyl jasmonate, indicating a role for the A622 enzyme in the synthesis of both alkaloids in roots of N. glauca. Feeding of Nicotinic Acid (NA) to hairy roots of N. glauca containing the NgA622-RNAi construct did not restore capacity for synthesis of anabasine or nicotine. Moreover, treatment of these hairy root lines with NA did not lead to an increase in anatabine levels, unlike controls. Together, these results strongly suggest that A622 is an integral component of the final enzyme complex responsible for biosynthesis of all three pyridine alkaloids in Nicotiana.

Transcriptomics-based analysis using RNA-Seq of the coconut (Cocos nucifera) leaf in response to yellow decline phytoplasma infection

Invasive phytoplasmas wreak havoc on coconut palms worldwide, leading to high loss of income, food insecurity and extreme poverty of farmers in producing countries. Phytoplasmas as strictly biotrophic insect-transmitted bacterial pathogens instigate distinct changes in developmental processes and defence responses of the infected plants and manipulate plants to their own advantage; however, little is known about the cellular and molecular mechanisms underlying host–phytoplasma interactions. Further, phytoplasma-mediated transcriptional alterations in coconut palm genes have not yet been identified. This study evaluated the whole transcriptome profiles of naturally infected leaves of Cocos nucifera ecotype Malayan Red Dwarf in response to yellow decline phytoplasma from group 16SrXIV, using RNA-Seq technique. Transcriptomics-based analysis reported here identified genes involved in coconut innate immunity. The number of down-regulated genes in response to phytoplasma infection exceeded the number of genes up-regulated. Of the 39,873 differentially expressed unigenes, 21,860 unigenes were suppressed and 18,013 were induced following infection. Comparative analysis revealed that genes associated with defence signalling against biotic stimuli were significantly overexpressed in phytoplasma-infected leaves versus healthy coconut leaves. Genes involving cell rescue and defence, cellular transport, oxidative stress, hormone stimulus and metabolism, photosynthesis reduction, transcription and biosynthesis of secondary metabolites were differentially represented. Our transcriptome analysis unveiled a core set of genes associated with defence of coconut in response to phytoplasma attack, although several novel defence response candidate genes with unknown function have also been identified. This study constitutes valuable sequence resource for uncovering the resistance genes and/or susceptibility genes which can be used as genetic tools in disease resistance breeding.

In situ synthesis of natural rubber latex-supported gold nanoparticles for flexible SERS substrates

Natural rubber latex (NRL) from Hevea brasiliensis was used as a matrix to synthesize gold nanoparticles (AuNPs), leading to an organic-inorganic hybrid latex of NRL-supported AuNPs (AuNPs@NRL). The in situ and environmentally friendly preparation of AuNPs in an NRL matrix was developed by thermal treatment without using any other reducing agents or stabilizers because natural rubber particles and non-rubber components present in serum can serve as supporters for the synthesized AuNPs. As a result, the nanosized and well-dispersed AuNPs not only are decorated on the surface of natural rubber particles, but also can be found in the serum of NRL. The size of the AuNPs presented in NRL matrix can be controlled by adjusting the concentration of NRL. Furthermore, the flexible surface-enhanced Raman scattering (SERS) substrates made from the AuNPs@NRL through vacuum filtration presented good enhancement of the Raman probe molecule of 4-mercaptopyridine and outstanding SERS reproducibility. The capability of synthesizing the bio-supported nanohybrid latex provides a novel green and simple approach for the fabrication of flexible and effective SERS substrates.

Predictive growth budgets in terns and gulls

Energy budgets for nestling growth are presented for sandwich tern Sterna sandvicensis, common tern S. hirundo, Arctic tern S. paradisaea, and herring gull Larus argentatus. Energy used in the production of body tissue averaged 27% (of which 7% for biosynthesis) while BMR accounted for 45%, the remainder being cost of activity and thermoregulation (28%). Where quantified, cost of temperature regulation accounted for only 10% of the total expenditure under field conditions. A regression made of metabolic energy (ME) intake over the entire nestling period against body mass of the fledgling based on eight studies of gulls and terns resulted in ME=35.14×M1.0105. -from Authors

Synthetic biology based metabolic engineering of saccharomyces cerevisiae for omega 6 and 3 PUFA production

Metabolic engineering of PUFA biosynthesis pathway using codon optimized DGA1 (Diacylglycerol acyltransferase), FAA3 (Acyl-CoA synthetase), desaturase genes named D9D, D12D, D5D, D6D, D17D and D6E elongase gene was studied in S. cerevisiae. Engineered yeast strains successfully demonstrated increase in lipid accumulation, and heterologous biosynthesis of linoleic, γ-linolenic, dihomo γ-linolenic, arachidonic and eicosapentaenoic acid.

Dietary creatine supplementation during pregnancy: a study on the effects of creatine supplementation on creatine homeostasis and renal excretory function in spiny mice

Recent evidence obtained from a rodent model of birth asphyxia shows that supplementation of the maternal diet with creatine during pregnancy protects the neonate from multi-organ damage. However, the effect of increasing creatine intake on creatine homeostasis and biosynthesis in females, particularly during pregnancy, is unknown. This study assessed the impact of creatine supplementation on creatine homeostasis, body composition, capacity for de novo creatine synthesis and renal excretory function in non-pregnant and pregnant spiny mice. Mid-gestation pregnant and virgin spiny mice were fed normal chow or chow supplemented with 5 % w/w creatine for 18 days. Weight gain, urinary creatine and electrolyte excretion were assessed during supplementation. At post mortem, body composition was assessed by Dual-energy X-ray absorptiometry, or tissues were collected to assess creatine content and mRNA expression of the creatine synthesising enzymes arginine:glycine amidinotransferase (AGAT) and guanidinoacetate methyltransferase (GAMT) and the creatine transporter (CrT1). Protein expression of AGAT and GAMT was also assessed by Western blot. Key findings of this study include no changes in body weight or composition with creatine supplementation; increased urinary creatine excretion in supplemented spiny mice, with increased sodium (P < 0.001) and chloride (P < 0.05) excretion in pregnant dams after 3 days of supplementation; lowered renal AGAT mRNA (P < 0.001) and protein (P < 0.001) expressions, and lowered CrT1 mRNA expression in the kidney (P < 0.01) and brain (P < 0.001). Creatine supplementation had minimal impact on creatine homeostasis in either non-pregnant or pregnant spiny mice. Increasing maternal dietary creatine consumption could be a useful treatment for birth asphyxia.

Changes in lipid class content and composition of Isochrysis sp. (T-Iso) grown in batch culture

© 2015, Springer International Publishing Switzerland. For decades, the microalgae Isochrysis spp. have been widely utilised as a live feed in aquaculture practices. This species possesses a number of favourable characteristics, notably its long-chain omega-3 polyunsaturated fatty acid (LC n-3 PUFA) content; primarily docosahexaenoic acid (DHA, 22:6n-3). This article describes the lipid class content and composition of this microalga grown in batch culture, covering the entirety of lag, log and stationary growth phases. The total lipid was highest in the lag phase (27 pg/cell). Total lipid significantly decreased in the exponential growth (7 pg/cell), then steadily increasing for the remainder of growth. The increase in total lipid was due to the accumulation of neutral lipid in the form of triacylglycerides. The DHA content (pg/cell) of the neutral lipid remained relatively unchanged for the duration of growth, with the influx of fatty acids being primarily myristic and palmitic acids. DHA (pg/cell) was found at relatively uniform amounts across all lipid classes. However, the DHA content as a percentage differed greatly between classes. The polar lipid class had a significantly higher DHA content, which peaked at 38 % of all polar lipid in log growth. The primary PUFA species present in the glycolipid class was stearidonic acid (18:4n-3). This work gives an overview of the lipid content and composition of Isochrysis sp. (T-Iso) over the entirety of its growth under batch culture. The lipid profile for this species at different stages of culture provides a basal data set that is useful for comparative studies using this organism.