Abstract  C6 and C9 volatiles, originated from fatty acids, are important volatiles for 'Cabernet Sauvignon' grapes and wines. This study evaluated the influence of different training systems including Modified Vertical Shoot Positioned, (M-VSP); Fan training system with two trunks (F-TT); Fan training system with multiple trunks (F-MT) on these volatiles and the long-chain fatty acids (>C12) of grape berries and wines in the northwest of China. The expression profiles of genes from associated metabolic pathway were also analyzed. F-MT training resulted in lower vine vigor, larger yield, higher content of unsaturated fatty acids in grapes and lower C6 esters in wines in comparison with M-VSP and F-TT. M-VSP and F-TT enhanced C6 volatiles in grape berries. The concentrations of C6 volatiles were positively correlated with the expression of VvLOXA and VvHPL1. The results expanded the knowledge of the influence of training systems on fatty acids and their derived volatiles of grapes and wines.

Abstract  Tea is the second most consumed beverage in the world. A crop loss of up to 43 % has been reported due to blister blight disease of tea caused by a fungus, Exobasidium vexans. Thus, it directly affects the tea industry qualitatively and quantitatively. Solanum tuberosum class I chitinase gene (AF153195) is a plant pathogenesis-related gene. It was introduced into tea genome via Agrobacterium-mediated transformation with hygromycin phosphotransferase (hpt) gene conferring hygromycin resistance as plant selectable marker. A total of 41 hygromycin resistant plantlets were obtained, and PCR analysis established 12 plantlets confirming about the stable integration of transgene in the plant genome. Real-time PCR detected transgene expression in four transgenic plantlets (T28, C57, C9, and T31). Resistance to biotrophic fungal pathogen, E. vexans, was tested by detached leaf infection assay of greenhouse acclimated plantlets. An inhibitory activity against the fungal pathogen was evident from the detached leaves from the transformants compared with the control. Fungal lesion formed on control plantlet whereas the transgenic plantlets showed resistance to inoculated fungal pathogen by the formation of hypersensitivity reaction area. This result suggests that constitutive expression of the potato class I chitinase gene can be exploited to improve resistance to fungal pathogen, E. vexans, in economical perennial plantation crop like tea.

Abstract  Chronic relapsing experimental autoimmune encephalomyelitis (crEAE) in mice recapitulates many of the clinical and histopathological features of human multiple sclerosis (MS), making it a preferred model for the disease. In both, adaptive immunity and anti-myelin T cells responses are thought to be important, while in MS a role for innate immunity and complement has emerged. Here we sought to test whether complement is activated in crEAE and important for disease. Disease was induced in Biozzi ABH mice that were terminated at different stages of the disease to assess complement activation and local complement expression in the central nervous system. Complement activation products were abundant in all spinal cord areas examined in acute disease during relapse and in the progressive phase, but were absent in early disease remission, despite significant residual clinical disease. Local expression of C1q and C3 was increased at all stages of disease, while C9 expression was increased only in acute disease; expression of the complement regulators CD55, complement receptor 1-related gene/protein y (Crry) and CD59a was reduced at all stages of the disease compared to naive controls. These data show that complement is activated in the central nervous system in the model and suggest that it is a suitable candidate for exploring whether anti-complement agents might be of benefit in MS.

Abstract  Dearomatized hydrocarbon solvents in the C9-C14 aliphatic carbon number range were developed as alternatives to traditional solvents such as mineral spirits, but with lower aromatic content. Previous subchronic toxicity studies (both published and unpublished) have shown minimal to no systemic effects with exposure to dearomatized solvents, with the exception of rat-specific renal effects that have no relevance to humans. In this study, Sprague-Dawley rats were exposed to 0, 500, 2500 and 5000mg/kg/day of a C10-C13 dearomatized solvent for 90days by oral gavage. Liver enlargement and centrilobular hypertrophy were observed in all treated groups but were considered adaptive consequences of hydrocarbon-induced microsomal enzyme induction. Clinical chemistry data showed elevations of alanine aminotransferase (ALT), gamma glutamyltransferase (GGT) and total bilirubin in mid (ALT alone) and high dose groups, suggesting potential hepatobiliary effects with high dose exposure. Increased absolute kidney weight changes were restricted to male rats and associated with renal lesions indicative of alpha-2u globulin-mediated nephropathy. One limitation of the NOAEL/LOAEL approach in selecting points of departure for exposure limits is its dependence on dose selection/study design. Hence, a more robust approach that incorporates all data points on the dose-response curve, such as bench mark modeling, is preferred. Overall, benchmark dose analysis estimated a BMDL of 1857mg/kg/day based on increased serum ALT. This value is consistent with studies of similar hydrocarbon substances showing a lack of systemic effects at doses up to 1000mg/kg/day in the same rat strain.

Abstract  Chemoresistance prevents the curative cancer chemotherapy and presents a formidable challenge for both cancer researchers and clinicians. We have previously shown that miR-193a-3p promotes the multi-chemoresistance of bladder cancer cells via repressing its three target genes: SRSF2, PLAU and HIC2. Here, we showed that as a new direct target, the homeobox C9 (HOXC9) gene also executes the promoting effect of miR-193a-3p on the bladder cancer chemoresistance from a systematic study of multi-chemosensitive (5637) and resistant (H-bc) bladder cancer cell lines in both cell culture and tumor-xenograft/nude mice system. Paralleled with the changes in the drug-triggered cell death, the activities of both DNA damage response and oxidative stress pathways were drastically altered by a forced reversal of miR-193a-3p or HOXC9 levels in bladder cancer cells. In addition to a new mechanistic insight, our results provide a set of the essential genes in the miR-193a-3p/HOXC9/DNA damage response/oxidative stress pathway axis as the diagnostic targets for the guided anti-bladder cancer chemotherapy.

Abstract  For well over a decade, RNA interference (RNAi) has provided a powerful tool for investigators to query specific gene targets in an easily modulated loss-of-function setting, both in vitro and in vivo. Hundreds of publications have demonstrated the utility of RNAi in arrayed and pooled-based formats, in a wide variety of cell-based systems, including clonal, stem, transformed, and primary cells. Over the years, there have been significant improvements in the design of target-specific small-interfering RNA (siRNA) and short-hairpin RNA (shRNA), expression vectors, methods for mitigating off-target effects, and accurately interpreting screening results. Recent developments in RNAi technology include the Sensor assay, high-efficiency miR-E shRNAs, improved shRNA virus production with Pasha (DRGC8) knockdown, and assessment of RNAi off-target effects by using the C9-11 method. An exciting addition to the arsenal of RNA-mediated gene modulation is the clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas) system for genomic editing, allowing for gene functional knockout rather than knockdown.

Abstract  Systemic acquired resistance (SAR) is a form of inducible disease resistance that depends on salicylic acid and its upstream regulator ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1). Although local Arabidopsis thaliana defence responses activated by the Pseudomonas syringae effector protein AvrRpm1 are intact in eds1 mutant plants, SAR signal generation is abolished. Here, the SAR-specific phenotype of the eds1 mutant is utilized to identify metabolites that contribute to SAR. To this end, SAR bioassay-assisted fractionation of extracts from the wild type compared with eds1 mutant plants that conditionally express AvrRpm1 was performed. Using high-performance liquid chromatography followed by mass spectrometry, systemic immunity was associated with the accumulation of 60 metabolites, including the putative SAR signal azelaic acid (AzA) and its precursors 9-hydroperoxy octadecadienoic acid (9-HPOD) and 9-oxo nonanoic acid (ONA). Exogenous ONA induced SAR in systemic untreated leaves when applied at a 4-fold lower concentration than AzA. The data suggest that in planta oxidation of ONA to AzA might be partially responsible for this response and provide further evidence that AzA mobilizes Arabidopsis immunity in a concentration-dependent manner. The AzA fragmentation product pimelic acid did not induce SAR. The results link the C9 lipid peroxidation products ONA and AzA with systemic rather than local resistance and suggest that EDS1 directly or indirectly promotes the accumulation of ONA, AzA, or one or more of their common precursors possibly by activating one or more pathways that either result in the release of these compounds from galactolipids or promote lipid peroxidation.

Abstract  This study examined the effects of 6 weeks of conjugated linoleic acid (CLA) supplementation and moderate aerobic exercise on peak oxygen uptake (VO2 peak), the gas exchange threshold (GET), the respiratory compensation point (RCP), and serum concentrations of cholesterol, triacylglycerol, and glucose in humans. Thirty-four untrained to moderately trained men (mean ± SD; age = 21.5 ± 2.8 years; mass = 77.2 ± 9.5 kg) completed this double-blind, placebo controlled study and were randomly assigned to either a CLA (Clarinol A-80; n = 18) or placebo (PLA; sunflower oil; n = 16) group. Prior to and following 6 weeks of aerobic training (50% VO2 peak for 30 min, twice per week) and supplementation (5.63 g of total CLA isomers [of which 2.67 g was c9, t11 and 2.67 g was t10, c12] or 7.35 g high oleic sunflower oil per day), each participant completed an incremental cycle ergometer test to exhaustion to determine their [Formula: see text] peak, GET, and RCP and fasted blood draws were performed to measure serum concentrations of cholesterol, triacylglycerol, and glucose. Serum triacylglycerol concentrations were lower (p < 0.05) in the CLA than the PLA group. For VO2 peak and glucose, there were group × time interactions (p < 0.05), however, post hoc statistical tests did not reveal any differences (p > 0.05) between the CLA and PLA groups. GET and RCP increased (p < 0.05) from pre- to post-training for both the CLA and PLA groups. Overall, these data suggested that CLA and aerobic exercise may have synergistic, blood triacylglycerol lowering effects, although CLA may be ineffective for enhancing aerobic exercise performance in conjunction with a 6-week aerobic exercise training program in college-age men.

Abstract  The objective of the present study is to model the analogues of monosialoganglioside (GM3) by making modifications in its sialic acid residue with different substitutions in aqueous environment and to determine their structural stability based upon computational molecular dynamics. Molecular mechanics and molecular dynamics investigation was carried out to study the conformational preferences of the analogues of GM3. Dynamic simulations were carried out on the analogues of GM3 varying in the substituents at C-1, C-4, C-5, C-8 and C-9 positions of their sialic acid or Neuraminic acid (NeuAc) residue. The analogues are soaked in a periodic box of TIP3P water as solvent and subjected to a 10 ns molecular dynamics (MD) simulation using AMBER ff03 and gaff force fields with 30 ps equilibration. The analogue of GM3 with 9-N-succNeuAc (analogue5, C9 substitution) was observed to have the lowest energy of -6112.5 kcal/mol. Graphical analysis made on the MD trajectory reveals the direct and water mediated hydrogen bonds existing in these sialic acid analogues. The preferable conformations for glycosidic linkages of GM3 analogues found in different minimum energy regions in the conformational maps were identified. This study sheds light on the conformational preferences of GM3 analogues which may be essential for the design of GM3 analogues as inhibitors for different ganglioside specific pathogenic proteins such as bacterial toxins, influenza toxins and neuraminidases.

Abstract  The in vitro antifungal activity of nine dirutheniumpentadithiocarbamate complexes C1-C9 was investigated and assessed for its activity against four different fungal species with clinical interest and related to invasive fungal infections (IFIs), such as Candida spp. [C. albicans (two clinical isolates), C. glabrata, C. krusei, C. parapsolisis, C. tropicalis, C.dubliniensis (six clinical isolates)], Paracoccidioides brasiliensis (seven clinical isolates), Cryptococcus neoformans and Sporothrix schenckii. All synthesized complexes C1-C9 and also the free ligands L1-L9 were submitted to in vitro tests against those fungi and the results are very promising, since some of the obtained MIC (minimal inhibitory concentration) values were very low (from 10-6 mol mL-1 to 10-8 mol mL-1) against all investigated clinically relevant fungal pathogens, except for C. glabrata, that the MIC values are close to the ones obtained for fluconazole, the standard antifungal agent tested. Preliminary structure-activity relations (SAR) might be suggested and a strong influence from steric and lipophilic parameters in the antifungal activity can be noticed. Cytotoxicity assays (IC50) showed that the complexes are not as toxic (IC50 values are much higher-30 to 200 fold-than MIC values). These ruthenium complexes are very promising lead compounds for novel antifungal drug development, especially in IFIs, one of most harmful emerging infection diseases (EIDs).

Abstract  Stearoyl-CoA desaturase is an enzyme from the class of oxidoreductase, which catalyzes the formation of a fatty acid double bond between C9 and C10. It plays a key role in composition of the fatty acid profile in adipose tissue and animal products such as meat and milk. Additionally, it is an important regulator of metabolic processes in the body, and it determines the maintenance of energy homeostasis. This enzyme is encoded by an SCD gene, which, depending on the species, may exist as different isoforms. mRNA expression of stearoyl-CoA desaturase is dependent on many factors, including diet, hormones, and the activity of other genes. In previous studies, several mutations were characterized within the sequence of Δ9-desaturase, which may affect the activity of the protein in the tissues, as well as the value of breeding animals. Effects of particular mutations of the gene encoding the enzyme appears to be particularly important for diseases associated with obesity, diabetes, hypertension, heart diseases or cancer in humans. Also, it seems that using sheep as a potential animal model could be helpful in uncovering and understanding the mechanisms regulated by stearoyl-CoA desaturase.

Abstract  Sphingolipids are involved in cell differentiation and morphogenesis in eukaryotic cells. In this study, YlLac1p, a ceramide synthase required for glucosylceramide (GlcCer) synthesis, was found to be essential for hyphal growth in Yarrowia lipolytica. Y. lipolytica GlcCer was shown to be composed of a C16:0 fatty acid, which is hydroxylated at C2, and a C18:2 long chain base, which is unsaturated at both C4 and C8 and methylated at C9. Domain swapping analysis revealed that the entire TRAM/Lag1/CLN8 (TLC) domain, not the Lag1 motif, is crucial for the function of YlLac1p. YlDes1p, the C4 desaturase of the ceramide synthesized by YlLac1p, was also required for Y. lipolytica morphogenesis. Both Yllac1Δ and Yldes1Δ mutants neither polarize lipid rafts nor form normal vacuoles. Interestingly, mutation in YlPEX5, which encode a peroxisomal targeting signal receptor, partially suppressed the defective hyphal growth of Yllac1Δ. The Yllac1ΔYlpex5Δ mutant restored the ability to polarize lipid rafts and to form normal vacuoles, although it could not synthesize GlcCer. Taken together, our results suggest that GlcCer or GlcCer derivatives may be involved in hyphal morphogenesis in Y. lipolytica, at least in part, by affecting polarization of lipid rafts and vacuole morphogenesis.

Abstract  The complement system is an intricate network of serum proteins that mediates humoral innate immunity through an amplification cascade that ultimately leads to recruitment of inflammatory cells or opsonisation or killing of pathogens. One effector arm of this network is the terminal pathway of complement, which leads to the formation of the membrane attack complex (MAC) composed of complement components C5b, C6, C7, C8 and C9. Upon formation of C5 convertases via the classical or alternative pathways of complement activation, C5b is generated from C5 by proteolytic cleavage, nucleating a series of association and polymerisation reactions of the MAC-constituting complement components that culminate in pore formation of pathogenic membranes. Recent structures of MAC components and homologous proteins significantly increased our understanding of oligomerisation, membrane association and integration, shedding light onto the molecular mechanism of this important branch of the innate immune system.

Abstract  BACKGROUND: Investigations concerned the mechanism of HT-29 cells radiosensitization by cis-9,trans-11-conjugated linoleic acid (c9,t11-CLA), a natural component of human diet with proven antitumor activity.

METHODS: The cells were incubated for 24h with 70μM c9,t11-CLA and then X-irradiated. The following methods were used: gas chromatography (incorporation of the CLA isomer), flow cytometry (cell cycle), cloning (survival), Western blotting (protein distribution in membrane fractions), and pulse-field gel electrophoresis (rejoining of DNA double-strand breaks). In parallel, DNA-PK activity, γ-H2AX foci numbers and chromatid fragmentation were estimated. Gene expression was analysed by RT-PCR and chromosomal aberrations by the mFISH method. Nuclear accumulation of the EGF receptor (EGFR) was monitored by ELISA.

RESULTS AND CONCLUSIONS: C9,t11-CLA sensitized HT-29 cells to X-radiation. This effect was not due to changes in cell cycle progression or DNA-repair-related gene expression. Post-irradiation DSB rejoining was delayed, corresponding with the insufficient DNA-PK activation, although chromosomal aberration frequencies did not increase. Distributions of cholesterol and caveolin-1 in cellular membrane fractions changed. The nuclear EGFR translocation, necessary to increase the DNA-PK activity in response to oxidative stress, was blocked. We suppose that c9,t11-CLA modified the membrane structure, thus disturbing the intracellular EGFR transport and the EGFR-dependent pro-survival signalling, both functionally associated with lipid raft properties.

GENERAL SIGNIFICANCE: The results point to the importance of the cell membrane interactions with the nucleus after injury inflicted by X -rays. Compounds like c9,t11-CLA, that specifically alter membrane properties, could be used to develop new anticancer strategies.

Abstract  Vascular permeability, thrombocytopenia, liver pathology, complement activation, and altered hemostasis accompanying a febrile disease are the hallmarks of the dengue hemorrhagic fever/dengue shock syndrome, a major arthropod-borne viral disease that causes significant morbidity and mortality throughout tropical countries. We studied tissues from 13 children who died of acute dengue hemorrhagic fever/dengue shock syndrome at the Childrens' Hospital, Yangon, Myanmar. Dengue viral RNA from each of the 4 dengue viruses (DENVs) was detected by reverse transcriptase polymerase chain reaction in 11 cases, and dengue viral proteins (envelope, NS1, or NS3) were detected in 1 or more tissues from all 13 cases. Formalin-fixed and frozen tissues were studied for evidence of virus infection using monoclonal antibodies against DENV structural and nonstructural antigens (E, NS1, and nonsecreting NS3). In the liver, DENV infection occurred in hepatocytes and Kupffer cells but not in endothelial cells. Liver damage was associated with deposition on hepatocytes of complement components of both classical and alternative pathways. Evidence of dengue viral replication was observed in macrophage-like cells in spleens and lymph nodes. No dengue antigens were detected in endothelial cells in any organ. Germinal centers of the spleen and lymph nodes showed a marked reduction in the number of lymphocytes that were replaced by eosinophilic deposits, which contained dengue antigens as well as immunoglobulins, and complement components (C3, C1q, and C9). The latter findings had previously been reported but overlooked as a diagnostic feature.

Abstract  [Purpose] To investigate the correlation between the effect treadmill exercise and change in serum proteins in rats with osteoarthritis, a study of proteins was carried out using a mass spectrometer. [Subjects and Methods] Rats were randomly divided into five groups. After 4 weeks of treadmill training, serum from each rat was analyzed by Liquid chromatography-electrospray ionization tandem mass spectrometry. Complementary component 9 (C9) was discovered to be downregulated in the serum of the exercise groups, and this was validated by Western blot. [Results] Seventeen proteins were discovered to be elevated in the monosodium iodoacetate injection osteoarthritis group samples by more than 1.5 fold compared with the control group. One of the proteins upregulated, C9 protein, was validated, and it was found to decrease in the middle-intensity exercise group. [Conclusion] We showed that the serum level of C9, an inflammatory-related protein, decreased after treadmill exercise. Therefore, treadmill exercise with an appropriate intensity might be recommended for OA patients.

Abstract  AIMS: To assess strains of lactobacilli for their capacity to produce functional fatty acid-conjugated linoleic acid. To assess the linoleate isomerase for CLA production in the most efficient CLA producer.

METHODS AND RESULTS: In this study, strains of food-derived lactobacilli were cultured in media with linoleic acid and CLA production was assessed. Most of the selected strains produced CLA at different levels, with Lactobacillus plantarum ZS2058 being the most efficient CLA producer converting over 50% of linoleic acid to c9, t11-CLA and t9, t11-CLA. Some intermediates 10-hydroxy-cis-12-octadecenoic acid, 10-oxo-cis-12-octadecenoic acid and 10-oxo-trans-11-octadecenoic acid were determined via GC-MS. The genes coding the multicomponent linoleate isomerase containing myosin-cross-reactive antigen, short-chain dehydrogenase/oxidoreductase and acetoacetate decarboxylase for CLA production in Lact. plantarum ZS2058 were cloned and expressed in Escherichia coli. With the mixture of recombinant E. coli, c9, t11-CLA and three kinds of intermediates were produced from linoleic acid, which were in line with those in the lactobacilli.

CONCLUSIONS: The ability for CLA production by lactobacilli exhibited variation. Lactobacillus plantarum and Lact. bulgaricus were the most efficient producers in the selected strains. Lact. plantarum ZS2058 converted linoleic acid to CLAs with 10-hydroxy-cis-12-octadecenoic acid, 10-oxo-cis-12-octadecenoic acid and 10-oxo-trans-11-octadecenoic acid as intermediates. The multiple-step reactions for CLA production catalysed by multicomponent linoleate isomerase in Lact. plantarum ZS2058 were confirmed successfully.

SIGNIFICANCE AND IMPACT OF THE STUDY: Multicomponent linoleate isomerase provides important results for the illustration of the mechanism for CLA production in lactic acid bacteria. Food-derived lactobacilli with CLA production ability offers novel opportunities for functional foods development.

Abstract  Our immunization strategy introduced recombinant mouse butyrylcholinesterase (BChE) to naïve BChE knockout mice. An extraordinarily strong immune reaction gave rise to a whole spectrum of antibodies with different properties. Two selective and highly efficient monoclonal anti-mouse BChE antibodies 4H1 (IgG1) and 4 C9 (IgG2a), with Kd values in the nanomolar range were generated. ELISA detected BChE in as little as 20-50 nl of mouse plasma using 2 μg (4H1) or 4 μg (4C9). Both antibodies cross-reacted with BChE in dog plasma but only 4 H1 reacted with rat BChE, suggesting that the antibodies are targeted towards different epitopes. Surprisingly, neither recognized human BChE. The anti-mouse BChE antibodies were used in immunohistochemistry analysis of mouse muscle where they specifically stained the neuromuscular junction. The antibodies enable visualization of the BChE protein in the mouse tissue, thus complementing activity assays. They can be used to study a long-lasting question about the existence of mixed acetylcholinesterase/BChE oligomers in mouse tissues. Moreover, monoclonal anti-mouse BChE antibodies can provide a simple, fast and efficient way to purify mouse BChE from small amounts of starting material by using a single-step immunomagnetic bead-based protocol.

Abstract  Polyunsaturated fatty acids (PUFA) can modulate the immune response, however the mechanism by which they exert this effect remains unclear. Previous studies have clearly demonstrated that the cis-9, trans-11 isomer of conjugated linoleic acid (c9,t11-CLA), found predominantly in beef and dairy products, can modulate the response of immune cells to the toll-like receptor (TLR) 4 ligand, lipopolysaccharide (LPS). This study aimed to investigate further the mechanism by which these effects are mediated. Treatment of macrophages with c9,t11-CLA significantly decreased CD14 expression and partially blocked its association with lipid rafts following stimulation with LPS. Furthermore the c9,t11-CLA isomer inhibited both nuclear factor-κB (NF-κB) and IRF3 activation following TLR4 ligation while eicosapentaenoic acid (EPA) only suppressed NF-κB activation. Given that the ability of LPS to activate IRF3 downstream of TLR4 depends on internalisation of the TLR4 complex and involves CD14, we examined TLR4 endocytosis. Indeed the internalisation of TLR4 to early endosomes following activation with LPS was markedly inhibited in c9,t11-CLA treated cells. These effects were not seen with the n-3 fatty acid, EPA, which was used as a comparison. Our data demonstrates that c9,t11-CLA inhibits IRF3 activation via its effects on CD14 expression and localisation. This results in a decrease in the endocytosis of TLR4 which is necessary for IRF3 activation, revealing a novel mechanism by which this PUFA exerts its anti-inflammatory effects.

Abstract  Conjugated linoleic acid (CLA), a dietary lipid, has been proposed as an antidiabetic agent. However, studies specifically addressing the molecular dynamics of CLA on skeletal muscle glucose transport and differences between the key isomers are limited. We demonstrate that acute exposure of L6 myotubes to cis-9, trans-11 (c9,t11) and trans-10, cis-12 (t10,c12) CLA isomers mimics insulin action by stimulating glucose uptake and glucose transporter-4 (GLUT4) trafficking. Both c9,t10-CLA and t10,c12-CLA stimulate the phosphorylation of phosphatidylinositol 3-kinase (PI3-kinase) p85 subunit and Akt substrate-160 kDa (AS160), while showing isomer-specific effects on AMP-activated protein kinase (AMPK). CLA isomers showed synergistic effects with the AMPK activator, 5-aminoimidazole-4-carboxamide-1-β-d-ribonucleoside (AICAR). Blocking PI3-kinase and AMPK prevented the stimulatory effects of t10,c12-CLA on AS160 phosphorylation and glucose uptake, indicating that this isomer acts via a PI3-kinase and AMPK-dependent mechanism, whereas the mechanism of c9,t11-CLA remains unclear. Intriguingly, CLA isomers sensitized insulin-Akt-responsive glucose uptake and prevented high insulin-induced Akt desensitisation. Together, these results establish that CLA exhibits isomer-specific effects on GLUT4 trafficking and the increase in glucose uptake induced by CLA treatment of L6 myotubes occurs via pathways that are distinctive from those utilised by insulin.

Abstract  The aim of the present study was to perform a short-term safety evaluation of dietary mono-conjugated α-linolenic acid isomers (CLNA; c9-t11-c15-18:3+c9-t13-c15-18:3) using a neonatal pig model. CLNA diet was compared with three other dietary fats: (1) conjugated linoleic acid (CLA; c9-t11-18:2+t10-c12-18:2), (2) non-conjugated n-3 PUFA and (3) n-6 PUFA. Thirty-two piglets weaned at 3 weeks of age were distributed into four dietary groups. Diets were isoenergetic and food intake was controlled by a gastric tube. Mono-CLNA diet did not significantly change body or organ weight, carcass composition and most biochemical parameters including; glucose, cholesterol, triglycerides, creatinine, blood urea nitrogen, hepatic enzymes and electrolytes levels in blood (P⩾0.09). Conversely, the n-3 PUFA composition of the brain, liver and heart decreased by 6-21% in the CLNA-fed group compared to animals fed nonconjugated n-3 PUFA (P<0.01). Responses to dietary treatments were tissue-specific, with the liver and the brain being the most deprived in n-3 PUFA. Our results support that short-term intake of mono-CLNA is safe in neonatal pigs but n-3 PUFA reduction in tissues deserves to be further investigated before using long-term nutritional supplementation in pigs and other animal models and before moving to clinical trials.

Abstract  The finding that a GGGGCC (G4C2) hexanucleotide repeat expansion in the chromosome 9 ORF 72 (C9ORF72) gene is a common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) links ALS/FTD to a large group of unstable microsatellite diseases. Previously, we showed that microsatellite expansion mutations can be bidirectionally transcribed and that these mutations express unexpected proteins by a unique mechanism, repeat-associated non-ATG (RAN) translation. In this study, we show that C9ORF72 antisense transcripts are elevated in the brains of C9ORF72 expansion-positive [C9(+)] patients, and antisense GGCCCC (G2C4) repeat-expansion RNAs accumulate in nuclear foci in brain. Additionally, sense and antisense foci accumulate in blood and are potential biomarkers of the disease. Furthermore, we show that RAN translation occurs from both sense and antisense expansion transcripts, resulting in the expression of six RAN proteins (antisense: Pro-Arg, Pro-Ala, Gly-Pro; and sense: Gly-Ala, Gly-Arg, Gly-Pro). These proteins accumulate in cytoplasmic aggregates in affected brain regions, including the frontal and motor cortex, hippocampus, and spinal cord neurons, with some brain regions showing dramatic RAN protein accumulation and clustering. The finding that unique antisense G2C4 RNA foci and three unique antisense RAN proteins accumulate in patient tissues indicates that bidirectional transcription of expanded alleles is a fundamental pathologic feature of C9ORF72 ALS/FTD. Additionally, these findings suggest the need to test therapeutic strategies that target both sense and antisense RNAs and RAN proteins in C9ORF72 ALS/FTD, and to more broadly consider the role of antisense expression and RAN translation across microsatellite expansion diseases.

Abstract  PURPOSE: The aim of the current study was to determine the incorporation of cis (c) 9, trans (t) 11-conjugated linoleic acid (CLA) and t10, c12-CLA into porcine erythrocytes-both isomers were supplemented in equal proportions.

METHODS: The study group consisted of 16 piglets randomly assigned into experimental and control group. For the period of 5 weeks, the piglets from the experimental group were receiving a 1.2% CLA supplement while the controls were supplemented with the same amount of sunflower oil. For the remaining 7 weeks, the piglets were fed without a supplement. Blood samples to evaluate incorporation of CLA into erythrocyte membranes were taken from all animals on weekly basis.

RESULTS: Compared to t10, c12-CLA isomer, proportion of c9, t11-CLA isomer in the membrane of erythrocytes was higher for the whole time of the study period. After 4 weeks of feeding, it approaches the plateau. The peak value for both isomers was measured at the end of week 5, with a value of 3.24 g c9, t11-CLA/100 g of fatty acids and a 1.09 g t10, c12-CLA/100 g of fatty acids (p < 0.0001). After cessation of supplementation, the proportion of both isomers gradually decreased to be almost completely washed out-in 7 weeks.

CONCLUSIONS: During supplementation with equivalent amounts of CLA isomers, their proportion in membranes of porcine erythrocytes increases with time, with higher proportion of c9, t11-CLA. CLA isomers probably differently incorporate into different cell membranes at different species which could explain its various biological functions.

Abstract  Complement is a humoral factor of innate immunity and plays an essential role in altering the host of the presence of potential pathogens and clearing of invading microorganisms. The third complement component (C3) not only is regarded as the crossing of the three pathways of complement activation, but also serves one of the bridges linking innate and acquired immunity. The nine complement component (C9) can combine with C5b, C6, C7 and C8 to form MAC which bounds to the surface of microorganisms to kill them. The evidence of evolution on C3 genes which have multiple functions and plays central role in innate immunity was documented in our previous study. Now we were interested in the evolution of C9 genes which were the terminal complement components. For these reasons, we want to explore the evolutionary patterns of C9 and whether C3 and C9 experience different evolutionary patterns. In our study, we used the sliding window method to separately calculate the values of ω among fishes and mammals of C3 and C9 codons. In order to detect the positive selection sites, we used the maximum likelihood (ML) method to study the evolutionary pattern on C3 and C9 genes. Positive selection sites were detected in mammalian C9 genes and no positive selection sites were detected in fishes C9 genes. However, no positive selection sites were detected in mammalian C3 genes and positive selection sites were detected in fishes C3 genes. The result indicated that C3 and C9 had different evolutionary patterns on mammals and fishes. In conclusion, different living environments lead to different evolutionary patterns on C3 and C9 in mammals and fishes. Besides, different complement components may have different evolutionary patterns on mammals and fishes.

Abstract  Carotenoid-derived aroma compounds play an important role in the composition of aroma and grapes, and consequently of wine. The volatile composition of forty-five grape varieties harvested in 2011 and 2012 was investigated by solid phase extraction of samples and gas chromatography-mass spectrometry (GC-MS) analysis. In grape extracts of cultivars Barbera, Refosco dal Peducolo Rosso, Ribolla gialla and Rossese, the C9-norisoprenoid compound β-isophorone (3,5,5-trimethyl-3-cyclohexen-1-one) was found and quantified. This carotenoid-derived compound is characterised by a saffron aroma and is here reported in grape for the first time.