With the aim to challenge this hypothesis, we applied a primed, constant infusion of 1-13C-leucine in nine healthy male subjects at sea level and subsequently at high-altitude (4559 m) after 7–9 days of acclimatization. Physical activity levels and food and energy intake were controlled prior to the two experimental conditions with the aim to standardize these confounding factors. Blood samples and expired breath samples were collected hourly during the 4 hour trial and vastus lateralis muscle biopsies obtained at 1 and 4 hours after tracer priming in the overnight fasted state. Myofibrillar protein synthesis rate was doubled; 0.041±0.018 at sea-level to 0.080±0.018%⋅hr−1 (p<0.05) when acclimatized to high altitude. The sarcoplasmic protein synthesis rate was in contrast unaffected by altitude exposure; 0.052±0.019 at sea-level to 0.059±0.010%⋅hr−1 (p>0.05). Trends to increments in whole body protein kinetics were seen: Degradation rate elevated from 2.51±0.21 at sea level to 2.73±0.13 µmol⋅kg−1⋅min−1 (p = 0.05) at high altitude and synthesis rate similar; 2.24±0.20 at sea level and 2.43±0.13 µmol⋅kg−1⋅min−1 (p>0.05) at altitude. We conclude that whole body amino acid flux is increased due to an elevated protein turnover rate. Resting skeletal muscle myocontractile protein synthesis rate was concomitantly elevated by high-altitude induced hypoxia, whereas the sarcoplasmic protein synthesis rate was unaffected by hypoxia. These changed responses may lead to divergent adaptation over the course of prolonged exposure.As a consequence to hypobaric hypoxic exposure skeletal muscle atrophy is often reported. The underlying mechanism has been suggested to involve a decrease in protein synthesis in order to conserve O Long-term (75 days) exposure of healthy lowlanders to high-altitude (5250 m at Mount Everest base camp) induces loss of skeletal muscle mass and single fiber atrophy in m. vastus lateralis and m. biceps brachii in vitro models provide results that suggests that attenuated energy turnover and loss of muscle mass may be developed at hypoxia exposure due to a decrease in overall protein turnover rate In studies, applying indirect measures of protein turnover, A direct measurement of the protein kinetics processes is though more valid to decide whether altitude affects protein turnover. Using the stable isotope methodology, Imoberdorf and co-workers assessed muscle protein synthesis rate after exposure to high altitude We therefore hypothesized that skeletal muscle protein synthesis could be affected by high-altitude induced hypoxia if longer exposure time would have been allowed. Therefore, to study whole body and skeletal muscle protein kinetics, we completed trials with stable isotopically-labeled amino acid infusions at sea level and 7–9 days after ascent to high altitude (4559 m).Nine healthy, physically active male subjects participated in the study. They ranged in age from 24 to 30 years, had an average height of 180±1 cm (mean ± SEM), and a mean weight of 78±2 kg. Not all data points were obtained in all subjects during the altitude trials, and the exact n is given in tables/figures where needed. Results describing altitude induced changes in skeletal muscle proteins involved in iron transport, tricarboxylic acid (TCA) cycle, oxidative phosphorylation, and oxidative stress responses from the same study have been published elsewhere The study conformed with guidelines laid down in the Declaration of Helsinki II and was approved by the Copenhagen and Frederiksberg Ethics Committee, Denmark. After being given both written and oral information on the experimental protocol and procedures, the subjects gave their informed, written consent to participate.Experimental protocol.In April all subjects reported to the laboratory at sea level in Copenhagen three days prior to the experiments and were not allowed to leave the building until experiments had finished. Most of the time was passed watching TV, studying, working on computers, or conversation. During their indoor confinement the subjects had access to a broad variety of food and the subjects choose themselves what to eat at all times. All food intakes were registered. The day before the experiment no caffeine containing substances was allowed from 6 pm, and the subjects fasted from 8 pm until after completion of the experiments the following day. The next morning at 8 am the experiments were initiated and the protocol is described in the paragraph In late August all subjects were flown to Italy and brought to 3200 m altitude by bus and lift. After 2–3 hours of mountain walking all subjects arrived at the Gnifetti hut at 3647 m and spend two nights in order to minimize the risk of acute mountain sickness (assessed by Lake Louise scores). The experimental facilities of the Margeritha hut  were reached after 5–7 hours of mountain/glacier walking on day 3 and subjects resided here until the experiments were started 7–9 days after arrival to this altitude. The Margeritha hut is a modern building where parts of the second floor are allocated specifically for scientific projects. The building is well insulated, has electricity, and comfortable living and kitchen facilities, and the experimental settings cannot be compared to the previous studies conducted in for example the base camp of Mount Everest where low temperatures etc may influence the results.As at sea level, all subjects were confined to indoor activities three days prior to the experiments and were not allowed outside in an attempt to standardize energy expenditure. We had brought an exactly matched amount of food as the subjects ate during the sea level experiments and the subjects consumed a similar diet as they did at sea level during this period. The restrictions to food prior to the experiments were the same as they were initiated at sea level.Experimental protocolApproximately 10 days after the completion of the experiments at sea level an arterial blood sample was taken for other purposes 13C-leucine (7.0 µmol·kg−1) added H13CO3 (2.5 µmol·kg−1) to label the carbondioxide pool with 13C as well, the constant infusion of 1-13C-leucine (0.15 µmol·kg−1·min−1) was initiated and continued for 4 hours. The stable isotope tracers were sterility and pyrogenity tested and 99% enriched and purchased from Cambridge Isotopes Laboratories . They were dissolved in sterile saline prior infusion and sterilized through a 0.2 µm disposable filter . Muscle biopsies were obtained prior to any infusions and after 1 and 4 hours of infusions. Biopsies were obtained from vastus lateralis muscle through a new incision after local anesthesia (1% lidocaine) using a 5 mm Bergstrom needle with suction and were always obtained from the same, non-dominant leg.Subjects were studied overnight fasting and in the supine position. A 20 G venflon  was inserted in an antecubital vein in both arms, and one was used for infusions and the other for blood sampling. After a priming dose of 1-Before and one day after return to sea level all subjects were scanned for body composition in a dual-energy X-ray absorptiometer . For more detailed subject characteristic see reference 2 uptake (VO2), CO2 production (VCO2), and expired minute ventilation (VE) were measured for 15 minutes pre infusion and at every hour during the infusion . Based on ambient conditions on the experimental days the gas analyser and the flowmeter were calibrated with high precision gases. Expired air was also collected in a Douglas bag and transferred to vacuum glass containers  for later analysis of 13CO2 enrichments.Blood samples were obtained pre infusion and at every hour during the infusion in EDTA containing tubes and was centrifuged to obtain plasma, which was stored at –80°C for later analysis of hormone concentrations and stable isotope abundances. Whole body OPlasma concentrations for insulin (kit: DYC1544-2), human growth hormone (kit: DGH00), cortisol (kit: KGE008), and testosterone (kit: KGE010) were assessed by available ELISA kits  and insulin-like growth factor-1 (ELH-IGFBP1-001) with ELISA kits  applying the prescribed procedures.13C-leucine tracer concentration in the infusate was determined using the internal tracer standard method: adding a known amount of L--leucine to the sample and relating the fragment peak of [M+3] to the infused isotope peak [M+1] with M equal to m/z = 302 based on the obtained GC/MS chromatograms  using a capillary column  to isolate leucine and the EI-mode to create the fragment-ions. Based on the pump recorded infusion speed and the measured leucine tracer concentration, the delivery of leucine tracer to the subjects was calculated.The 2 was analyzed for 13C abundance originating from oxidation of the 13C-leucine tracer on the IRMS system  with CO2 separated by a CP-PoraPLOT Q column, 27.5 m×0.32 mm×10 µm .Breath CO13C-ketoisocaproic acid (KIC) abundance was used 2 at 50°C. With 200 µL acidified 2% w/v o-phenylenediamine in 200 µL Millipore water the substrates were prepared for the derivatization using pyridine and BSTFA +1% TMCS .The surrogate measure of the precursor pool for protein synthesis with leucine as the traced amino acid, plasma N-acetyl n-propyl (NAP) esters.The protein-bound leucine abundance was measured by GC-C-IRMS system , where the leucine was isolated by a capillary column . The myofibrillar and sarcoplasmic protein-fractions were separated from a muscle homogenate using a common solubility/centrifugation procedure 13C-KIC more accurately reflects the intracellular abundance of 13C-leucine than the venous 13C-leucine All whole body ventilation and leucine-based results are reported as the mean of data obtained at 3 and 4 hours whereas leucine incorporation into myofibrillar and sarcoplasmic protein fractions is determined from 1–4 hours of infusion. Intracellular leucine is in equilibrium with its transaminated product, the alpha-ketoisocaproic acid (KIC). Since the leucine metabolism occurs at the intracellular site, the venous enrichment of a, leu), which is an estimate of whole body protein breakdown, was calculated using: Infusion ratetracer ⋅ EKIC, estimate of intracellular tracer−1.Whole body leucine rate of appearance . Since leucine can be oxidized, not all of the ‘disappearing’ leucine goes for synthesis. Therefore, non-oxidative whole body leucine rate of disappearance (NOLD) rate, which is an estimate of whole body protein synthesis, is calculated: NOLDleu = Rd, leu – Oxleu. Leucine oxidation rate (Oxleu) was calculated using the following equation: ECO2 ⋅ CO2 production rate ⋅ bicarbonate correction factor 0.81 ⋅ EKIC, estimate of intracellular tracer−1, where the bicarbonate correction factor of 0.81 is a standard value for the detainment of CO2 in the metabolism 2 production rate was calculated using the equation: VCO2 ⋅ (22.4 ⋅ Body weight)−1 ⋅ 1000, where the CO2 production was found by analyzing the expired gas collected in the Douglas bags .The underlying assumption for the applied tracer calculations is that steady state is present for the tracer kinetics, i.e. leucine rate of appearance, a measure of whole body leucine release Rproduct⋅Eprecursor−1⋅Δtime−1, where ΔEproduct is the change in 13C-leucine tracer enrichment in two tissue samples taken with a time interval of Δtime hours. Eprecursor is the average precursor pool enrichment during Δtime hours determined by the valid surrogate measure of venous 13C-KIC enrichment The protein fractional synthesis rate (FSR) was calculated using the precursor-product equation: FSR = ΔE13C-KIC-enrichments, used as a valid estimate of the protein synthesis precursor pool 13C-leucine-enrichment 13C-leucine as a tracer and venous 13C-KIC as a derived estimate of the intracellular tracer abundance, those are thought to be equilibrated during such minor drifts in tracer abundance and thus are ascribed to reflect the actual precursor enrichment. Thus, by using a weighted mean of the precursor pool enrichment the minor drift towards higher enrichments is not supposed to violate the underlying assumptions for the FSR calculations.The group-mean Values obtained at sea level and at high altitude were compared by a parametric Students paired t-tests. The precursor enrichments at the single time points were compared by a two-way ANOVA with repeated measures. One subject could not complete the infusion trial at high altitude, thus total number of skeletal muscle protein synthesis rates is eight. Due to technical problems with the gas collections the whole body protein kinetics calculated from the leucine tracer data only exist for six subjects. Values are reported as mean ± SEM. Statistical significance was obtained when the p-value was <0.05. Analyses were performed in Prism 4.0 .−1) or VO2max measured before high altitude exposure and one day after return to sea level . DEXA scans revealed that also the body composition was not altered, i.e. at sea level and high-altitude fat mass was 13.4±4.2 and 13.2±4.5 kg, respectively and lean body mass was 63.7±6.1 and 63.6±6.1 kg, respectively.High altitude exposure did not influence body weight (78.3±2.3 to 77.9±2.9 kg), resting metabolic rate , an estimate of protein degradation, tended to be increased at high altitude compared to sea level  remained unchanged with altitude exposure, whereas insulin concentrations increased from 4.9±0.4 to 7.7±1.1 µlU·mL p<0.05, .Loss of skeletal muscle is a common observation when healthy native lowlanders are exposed to high altitude for prolonged periods The synthesis rate of human skeletal muscle protein has only sparsely been assessed during hypoxic conditions and data are somehow contrasting. Imoberdorf and co-workers assessed the fractional synthesis rate of mixed skeletal muscle protein at sea-level and divided the subjects into two groups subsequently; one group that was flown to high altitude and another that walked from 3220 m up to 3611 m (1.5 hr) on one day and then to 4559 m (4–5 hrs) the next day 2 at sea level and with acute normoxia at altitude). Whether, the turnover rates of the specific proteins leading to the metabolic changes and decreased oxidative capacity were affected by hypoxia or not or the changes yet were unable to exert functional changes are unknown. As tempted to outline, several adaptations have to take place along the path from changes in gene and protein expression till detectable physiological manifestations.In contrast, we found an unaffected synthesis rate of the sarcoplasmic proteins in skeletal muscle . Using aVenous plasma KIC is not solely originating from skeletal muscle, but is released from all cells making the venous plasma KIC a weighted mean of whole body intracellular leucine enrichments. It is generally agreed that since skeletal muscle make up such an abundant tissue, the venous plasma KIC enrichments is highly reflective of the status in the myocytes It is not believed that the observed protein turnover is driven by changes in systemic hormones as we  and othein vitro conditions, which lead to the formulation of the “Hypoxia Defence Strategies” by Hochachka and co-workers Defence and Rescue strategy may be expected. During an exposure to a mild hypoxic milieu as in the present setting, we propose a remodeling adaptive response in the skeletal muscle, which initiates an increase in protein degradation. Subsequently, it is left to the synthetic processes to reorganize and rebuild the tissue. Whether the net outcome over time then ends up with a steady or slightly decreased protein mass is depended on how well the protein synthetic processes are maintained. The stimulation of protein synthesis may primarily be through external factors, which at altitude may be compromised by decreasing food intake, decrease in physical activity level It has previously been demonstrated that the hypoxic response may not be as detrimental for the cell The applied tracer approach validly reveals that the adaptation of synthesis rates of various skeletal muscle protein fractions is different and future hypoxia-research should focus on describing the molecular signaling responsible for these divergent adaptations. With regard to the measurement of protein breakdown rate, no similar protein specific approach exists. However, the whole body amino acid flux rates assessed here indicate an elevation at altitude when compared to sea level but these results are underpowered and not protein specific. Therefore, the adaptation and regulation of the protein breakdown after acclimatization to high altitude demands thorough research and improvements in methodology.


One potential strategy for the prevention of HIV infection is to induce virus specific mucosal antibody that can act as an immune barrier to prevent transmission. The mucosal application of chemokines after immunisation, termed “prime-pull”, has been shown to recruit T cells to mucosal sites. We wished to determine whether this strategy could be used to increase B cells and antibody in the vaginal mucosa following immunisation with an HIV antigen. BALB/c mice were immunised intranasally with trimeric gp140 prior to vaginal application of the chemokine CCL28 or the synthetic TLR4 ligand MPLA, without antigen six days later. There was no increase in vaginal IgA, IgG or B cells following the application of CCL28, however vaginal application of MPLA led to a significant boost in antigen specific vaginal IgA. Follow up studies to investigate the effect of the timing of the “pull” stimulation demonstrated that when given 14 days after the initial immunisation MPLA significantly increased systemic antibody responses. We speculate that this may be due to residual inflammation prior to re-immunisation. Overall we conclude that in contrast to the previously observed effect on T cells, the use of “prime-pull” has only a modest effect on B cells and antibody. One strategy for HIV vaccine development is to generate a local immune barrier at the site of infection We have previously observed that mucosal immunisation can induce local antibody responses to trimeric HIV envelope protein gp140 4β1, and α4β7 and the cytokines BAFF, APRIL and TSLP B cells are attracted to a range of factors, including the chemokines CCL19, CCL21, CCL28, CCL25, the integrins αIn this study we investigated the use of the chemokine CCL28 and TLR ligand MPLA as boost agents (without antigen) in a “prime-pull” regime following either mucosal or systemic immunisation with the HIV envelope protein gp140. We observed that the vaginal administration of MPLA alone after immunisation but not CCL28 led to an increase in vaginal IgA, systemic IgA and IgG and antigen specific B cells in the female genital tract. The timing of boost was important, with a greater response seen when “pull” stimulation was given 7 or 14 days after immunisation compared to when it is given on the day of immunisation. Interestingly mucosal administration of MPLA alone significantly increased systemic antibody responses to subsequent immunisations. Here we show that it is possible to increase the vaginal IgA using a “prime-pull” strategy, but the increase in antibody titre was modest and unsustained.Female BALB/c mice, 6–8 week old, were obtained from Harlan Olac Ltd . All procedures were performed in accordance with the United Kingdom's Home Office standards under the Animals Scientific Procedures Act, 1986, and approved by the Ethical Review Boards at Imperial College London and at St George's University of London. In the timecourse and comparison of TLR ligands studies, the same group of control animals were used to reduce animal usage. A clade C HIV-1 envelope clone p97CN54 was originally isolated from a Chinese patient Mice were immunized 3 times with 3 weeks interval, with 10 µg gp140 and 10µg MPLA. The gp140/MPLA formulation was either administered intranasally in a total volume of 20µl or subcutaneously in a volume of 50µl. At 0, 7, or 14 days after immunisation, animals received an intravaginal “pull” stimulation of 10µg of MPLA, CCL28, FSL-1 or CpG in a volume of 20µl.Serum and mucosal samples were obtained at various intervals before or after immunisation as described previously The mouse genital tract, including vagina, uterus, oviducts, and ovaries were dissected from the animal, and placed in cold complete medium , 2 mM glutamine, 10 mM HEPES, 100 IU/ml penicillin, 100µg/ml streptomycin, and 10µg/ml gentamycin). The tissue was finely cut with a scalpel, washed with complete medium and digested at 37°C for 1 h on a shaker with 5 ml of serum-free RPMI-1640 medium that contained 2 mg Collagenase Dispase and 0.1 mg/ml DNaseI (Roche Diagnostics). The digested tissue was spun and the cell pellet washed twice in CM. The lymphoid cell population was separated from the stromal cells by density gradient centrifugation .2SO4) and read at 450 nm. Reciprocal endpoint titres were calculated by using GraphPad Prism 4 using a cut-off value at OD450 of 0.1 for all samples.MaxiSorp 96-well plates were coated overnight with 1.0 µg/ml HIV-1 gp140 in PBS. Plates were blocked for 1 h at 37°C with 1% BSA in PBS. Serially diluted samples were incubated for 1 h at 37°C. Bound IgG was detected by incubation for 1 h at 37°C with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG  and IgA was detected with biotin-conjugated goat anti-mouse IgA . For IgA detection, plates were incubated with Streptavidin-HRP (R&D Systems) for 1 h at 37°C. Plates were developed using tetramethylbenzidine (TMB) substrate. The reaction was stopped with stop solution . Cells from genital tract were assessed immediately after isolation. ELISPOT assays were performed using a commercial kit from MABTECH  following the manufacturer's recommendations. The spots were counted using the AID ELISPOT reader ELR03 (Autoimmune Diagnostika).Analyses were performed using GraphPad Prism, version 4.00 (GraphPad Software). Statistical differences between groups were calculated using one-way analysis of variance (ANOVA) with appropriate post tests to measure significance between pairs of groups.nd intravaginal “pull” dose, 42 – before the 3rd intravaginal “pull” dose and 56 after the 3rd intravaginal dose. “Pull” stimulation with CCL28 had no significant effect on antibody specific IgG or IgA in serum or vaginal lavage compared to PBS treated animals. However, mice that received intravaginal MPLA had significantly greater levels of vaginal IgA levels after “pull” stimulation on day 34  express higher levels of CCL20, CCL28 and APRIL in intestinal epithelium There are a number of caveats that may influence translation of this approach to human vaccines. The first limitation is the modest effect we observed in this study, but there may be differences going into humans, especially due to the differences in TLR responses between mice and humans. The second limitation is of a practical nature – particularly as HIV vaccines are most relevant in resource poor settings. Requiring individuals to return for a second visit seven days after each immunisation would be extremely restrictive, this could potentially be circumvented with home administration of the boost – if formulated in a user friendly form. But, there may be issues with the cultural acceptability of the vagina for drug delivery. There is also a chance that inducing immune activation in the vagina could increase the risk of transmission


Exciton-Controlled Hybridization-sensitive fluorescent Oligonucleotide (ECHO) called Eprobe. Eprobes contain two dye moieties attached to the same nucleotide and their fluorescent signal is strongly suppressed as single-stranded oligonucleotides by an excitonic interaction between the dyes. Upon hybridization to a complementary DNA strand, the dyes are separated and intercalate into the double-strand leading to strong fluorescence signals. Intercalation of dyes can further stabilize the DNA/DNA hybrid and increase the melting temperature compared to standard DNA oligonucleotides. Eprobes allow for specific real-time monitoring of amplification reactions by hybridizing to the amplicon in a sequence-dependent manner. Similarly, Eprobes allow for analysis of reaction products by melting curve analysis. The function of different Eprobes was studied using the L858R mutation in the human epidermal growth factor receptor (EGFR) gene, and multiplex detection was demonstrated for the human EGFR and KRAS genes using Eprobes with two different dyes. Combining amplification and melting curve analysis in a single-tube reaction provides powerful means for new mutation detection assays. Functioning as “sequence-specific dyes”, Eprobes hold great promises for future applications not only in PCR but also as hybridization probes in other applications.Real-time monitoring of PCR is one of the most important methods for DNA and RNA detection widely used in research and medical diagnostics. Here we describe a new approach for combined real-time PCR monitoring and melting curve analysis using a 3′ end-blocked  Taq DNA polymerase will start to digest the probe, which leads to the release of the fluorescent dye at the 5′ end of the probe. During the PCR cycles, the released fluorescent dye will accumulate and thus indicate the progress of the PCR at the expense of the probe. The structure of Molecular Beacons is very similar to TaqMan probes, but unbound Molecular Beacons form an internal stem loop structure that links the fluorescent label and quencher for strong background suppression. The signal of Molecular Beacons depends on a conformation change, where the fluorescent label and quencher at the ends of the probe are separated upon hybridization of the probe to the PCR template. Molecular Beacons are not digested during PCR, and therefore allow for melting curve analysis after the PCR amplification has been completed. Many other approaches to real-time PCR and fluorescent probes have been described in the literature Many PCR applications directly monitor the amplification reaction in real-time such as research applications, commercial tests, and medical diagnostics. This can be achieved by fluorescent dyes like SYBR Green I The use of hybridization probes is preferable for the design of highly specific PCR reactions as these probes can distinguish between different reaction products obtained using the same primer set in situ hybridization (ECHO-FISH) assays M) than standard DNA oligonucleotides of same sequence. This has been studied in detail for thiazole orange, the most commonly used dye in ECHOs Exciton-Controlled Hybridization-sensitive Oligonucleotides (ECHO) are fluorescence labeled oligonucleotides that commonly possess a modified thymine carrying two dye moieties To use ECHOs as hybridization probes in real-time PCR and melting curve analysis, we prepared a modified version of ECHOs, so-called Eprobes, that are blocked at the 3′ end to prevent primer extension. Eprobes do not interfere with amplification reactions, but act as independent hybridization probes in signal detection and amplicon analysis.DNA oligonucleotides were purchased from Sigma-Aldrich Japan  and stored as 100 µM stock solutions in 10 mM Tris-HCl, 1 mM EDTA (pH 8.0). Wild-type human genomic DNA was purchased from Promega Japan ; heterozygote human genomic DNA with the mutation L858R in exon 21 of human EGFR gene was obtained from Horizon Diagnostics . Plasmids holding wild-type and mutated regions of the human EGFR gene were taken from Eprobe chemistry is derived from protocols for ECHO/Eprimer synthesis. Eprimers with paired thiazole orange moieties  are synthesized according to a modified version  of the previously published protocol M values for all Eprobes labeled by D514 were calculated using an online tool described by Kimura et al. +, 0.002 M Mg2+, 0.2 µM Eprobe, 60°C Temperature; TM values for Eprobe 215-21 wt TP are actual values as the tool cannot predict TM values for Eprobes labeled by D570. Predicted TM values for all Eprobes and DNA oligonucleotides of the same sequence are given in M values may vary by up to some 3°C. These differences cannot be predicted, because the makers do not disclose the components of their proprietary reagent mixes. The tool can be freely accessed at: http://genome.gsc.riken.jp/echo/thermodynamics/.The TEprobes (1 µM per reaction) with paired thiazole orange  or thiazole pink moieties  were fully digested with calf-intestine alkaline phosphatase , snake-venom phosphodiesterase , and P1 nuclease  at 25°C for 3 h in 100 µl reaction volume. The reaction mixtures with and without digestion were measured on a RF5300PC fluorescence spectrophotometer  with excitation at 483 nm for D514 when recording a spectrum from 495 nm to 800 nm, and an excitation at 540 nm for D570 when recording a spectrum from 550 nm to 800 nm.5′- CCTCACAGCAGGGTCTTCTC-3′, reverse primer: 5′-CCTGGTGTCAGGAAAATGCT-3′) were taken from 5′-TTATAAGGCCTGCTGAAAATGACTGAA-3′, reverse primer: 5′-TGAATTAGCTGTATCGTCAAGGCACT-3′) were taken from Specific primers for exon 21 in the human EGFR gene (forward primer: Taq DNA polymerase with an exonuclease activity (exo+) and LightCycler 480 Genotyping Master  as a Taq DNA polymerase having an N-terminal deletion lacking an exonuclease activity (exo−). Suitable Eprobes concentrations were tested in the range of 25 nM to 500 nM, where 200 nM was the optimal concentration for standard reactions. The template concentration per reaction varied from 1 ng to 1000 ng genomic DNA or from 150 to 150,000,000 copies for plasmid DNA.Different PCR reagents and enzyme master mixes were tested for use with Eprobes during PCR. We selected AmpliTaq Gold PCR Master Mix  as a Amplification reactions using AmpliTaq Gold were setup in 96-well plates using 5 µl template DNA, 12.5 µl of AmpliTaq Gold PCR Master Mix (with or without 1.0 µl of GC enhancer), 0.2 µM of each primer, and 0.2 µM Eprobe in a total volume of 25 µl. Real-time PCR experiments were run on a LightCycler 480  after activation of the hotstart enzyme for 10 min at 95°C, followed by 50 cycles of 15 s at 95°C, 30 s at 56°C, and 30 s at 72°C. Amplification signals were detected during the annealing step of each cycle at 56°C, using a SYBR Green I (483 nm–533 nm) filter for D514 and Red 610 (558–610 nm) filter for D570. For melting curve analysis, the PCR was followed by heating the reaction mixture to 95°C for 15 s, cooling to 37°C, holding at 37°C for 7 min, and then slowly heating again to 95°C at a ramp rate 2.2°C/s and continuous fluorescence acquisition at the indicated wave lengths. All PCR reactions and melting curve experiments were always performed in triplicate, and each experiment included a negative control where 1×TE Buffer  was added instead of template DNA.Alternatively, PCR reactions were setup using 4 µl of 5×LightCycler 480 Genotyping Master (with or without 5% Formamide), 5 µl template DNA, 0.2 µM of each primer, and 0.2 µM Eprobe in a total volume of 25 µl. Amplification reactions and melting curve analysis were performed as described above for the AmpliTaq Gold.PCR control experiments used SYBR Green I  or the EGFR specific TaqMan probe (50 nM) instead of Eprobes, and were run under the same reaction conditions as described above for use of AmpliTaq Gold.To achieve a higher sensitivity in the mutation detection assays, asymmetric amplification reactions were setup using 5 µl template DNA, 12.5 µl of AmpliTaq Gold PCR Master Mix (without GC enhancer), forward to reverse primers at a 1∶3 ratio and a final concentration of 1 µM, and 0.2 µM Eprobe in a total volume of 25 µl. Real-time PCR and melting curve experiments were run as described above for use of AmpliTaq Gold.Asymmetric amplification reactions were also performed for multiplexing detection of the human EGFR and KRAS genes using commercial genomic DNA. We mixed 500 nM of forward EGFR primer, 1500 nM of reverse EGFR primer, 250 nM of forward KRAS primer, 750 nM reverse KRAS primer, and 200 nM each of sequence specific Eprobe 215-21 wt TP for EGFR and Eprobe KWT14m3 TO for KRAS. Real-time PCR and melting curve analysis were performed as described above using AmpliTaq Gold.PCR products were analyzed on a 3% agarose gel in 1×TBE buffer. We mixed 10 µl of each PCR reaction with 2 µl of 6×loading dye  and loaded the gels together with a GeneRuler Low Range DNA Ladder, Ready-to-Use 25 to 700 bp . The electrophoresis was carried out for 1 h at 100 V, and gel images were taken by a Gel Doc™ XR+ System  after staining with Ethidium bromide. Images were analyzed using Image Lab 3.0 software .10(x) values which were transformed into ln(x) by multiplying the slope value by 2.303 [ln(x)  = 2.303 × log10(x)]. The slope values were used to calculate PCR efficiencies by a Thermo Fisher online web tool at: http://www.thermoscientificbio.com/webtools/qpcrefficiency/. PCR efficiencies are based on seven different DNA concentrations and one negative control using TE buffer instead of template DNA. Amplification curves, melting curves, and derivative melting curves were obtained from the LightCycler 480 instrument as indicated above, and the data points were transferred to Microsoft Excel for plotting.PCR standard curves were generated by analyzing Ct values acquired from the LightCycler 480 instrument with LightCycler 480 software release 1.2.0.0625, version 1.2.0.169. The data were transferred to Microsoft Excel, and Ct values were plotted against the template DNA concentrations. Logarithmic trendlines were added, and equations and R-squared values given by Microsoft Excel are shown in the graphs. Microsoft Excel provides slopes as logECHOs can easily be synthesized on a standard oligonucleotide synthesizer, where presently one deoxythymidine within the sequence is modified by an adapter to allow for the labeling with two dye moieties . For EprDespite the strong signal suppression by the excitonic interaction between the dye moieties, unbound Eprimers and Eprobes show a weak signal in fluorescence excitation spectra as shown in M value below the elongation temperature. The Eprobe should bind to the template DNA during the annealing step, when the fluorescent signal is detected, and would dissociate from the template again before the elongation step not to infer with the DNA polymerase. To test those considerations, we synthesized a set of specific Eprobes for detection of exon 21 in the human EGFR gene having a temperature range below and above the elongation temperature of the Taq DNA polymerase . M values for all Eprobes used in this study, where the TM values of Eprobes labeled by dye D514 are higher than the TM values estimated for DNA oligonucleotides of the same sequence. All PCR reactions in the presence of an Eprobe yielded DNA fragments of the correct 229 bp length  provided the weakest signals of all tested Eprobes suggesting an incomplete binding of the Eprobe at the higher annealing temperature  as a template lymerase . The thep length , indicatp length . Moreovep length  and provp length ; all prop length . While aperature . Since operature . HoweverTaq DNA polymerase having a 5′–3′ exonuclease activity. The consecutive reduction in the Eprobe concentration would lead to reduced signals although the amount of PCR product did no longer change at the late phase of the PCR. To test this hypothesis, we compared Eprobe 215-21 wt TO along with the shorter Eprobe 205-13 wt TO using a Taq DNA polymerase with a 5′–3′ exonuclease activity (Applied Biosystems AmpliTaq Gold) and a Taq DNA polymerase lacking the 5′–3′ exonuclease activity (Roche Genotyping Master). As shown in Taq DNA polymerase with a 5′–3′ exonuclease activity (Taq DNA polymerase lacking the 5′–3′ exonuclease activity (M below the elongation temperature are stable during PCR even in the presence of a DNA polymerase having a 5′–3′ exonuclease activity.For the longest Eprobe (215-21 wt TO) we noticed in the forgoing PCR experiments a strong hook-like shape of the amplification curves . This coactivity . Eprobe activity . The difM values for the wild-type and mutated allele is in line with our estimations, although the observed TM values did not match exactly with our predictions (see above). We confirmed the results of these experiments by analyzing wild-type and mutated genomic DNA with Eprobe 215-21 wt TP. Again the wild-type and mutated allele could be clearly distinguished by their different TM values .Next we tested the ability of our Eprobes to distinguish between the wild-type and mutated allele of exon 21 in the human EGFR gene. In the first experiment, we mixed plasmid DNA comprising the wild-type and mutated allele at three different ratios and performed asymmetric PCR to enrich for the reverse strand in the presence of Eprobe 205-13 wt TO. The melting profiles indicated two distinct melting temperatures that are more clearly shown in the differential melting curve analysis . The obsM values , where tM value (In a last set of experiments, we confirmed that Eprobes having two different dyes can be used in multiplex PCR in the same way as previously shown for isothermal amplification reactions using Eprimers M value  as compaM values can be used to distinguish single nucleotide changes in a sequence dependent manner as shown by our data for the mutation L858R in the human EGFR gene. Hence Eprobes can be applied for both quantification and qualitative analysis of amplicons on real-time PCR instruments in a single consecutive procedure combining PCR amplification and melting steps. This allows for the design of sensitive and highly specific PCR assays, for example, detection of genetically modified organisms or infectious agents Our data demonstrate that Eprobes allow for sensitive real-time PCR monitoring. Moreover, since Eprobes under optimal conditions neither interfere nor get destroyed during the amplification reactions, they can directly be used for melting curve analysis of amplification products. Melting curve analysis is one of the most specific methods to look for mutations in DNA M range, their design is more flexible than for example Molecular Beacons that are also used in melting curve analysis. Molecular Beacons often have a narrow temperature range for detecting mutations. At higher temperatures, their stem loop structure melts and the high background fluorescence does not allow target detection. In contrast, the background signal of Eprobes is less affected by temperature changes. For different Eprobes we observed characteristic baseline changes with increasing temperatures (compare Since Eprobes can be designed over a wide T compare  and 7. AM range allowing for variable lengths and lower sequence requirements than other hybridization probes used in PCR. Shorter Eprobes could avoid G/C or A/T rich regions, whereas longer Eprobes could more easily distinguish between problematic target sequences, e.g. containing repeat elements. Additional considerations should be given to the use of Taq DNA polymerases with or without a 5′–3′ exonuclease activity M value of about 5°C below elongation temperature or lower when using a DNA polymerase having a 5′–3′ exonuclease activity. Switching to DNA polymerases lacking any 5′–3′ exonuclease activity offers a wider temperature range for designing Eprobes, which may prove very useful for reactions using multiple Eprobes having distinct TM values. Eprobes with TM values below the annealing temperature are not suitable for real-time monitoring of PCR, but are still functional in melting curve analysis using a wider temperature range. Combining multiple Eprobes with different dyes and melting temperatures could become an attractive approach for the design of highly complex “superplexed” PCR assays. Here we demonstrated the use of Eprobes having two different dyes in multiplex PCR. Besides dyes D514 and D570, additional dyes have been described in the literature for use in ECHOs ECHOs labeled by dye D514 have been studied in detail for their thermodynamic properties M values in For genotyping experiments, we performed asymmetric PCR reactions followed by melting curve analysis in situ hybridization assays for ex vivo and in vivo detection in FISH assays making them universal hybridization probes.We recommend Eprobes rather than previously published ECHOs or Eprimers for all PCR applications involving real-time monitoring and melting curve analysis for extended template characterization. Only Eprobes can provide the additional specificity offered by working with a primer-independent hybridization probe during PCR. Moreover, Eprobes hold great potential for the development of


It has been proposed that F420 has an important role in the oxidoreductive reactions of Mycobacterium tuberculosis, possibly associated with anaerobic survival and persistence. The protein encoded by Rv0132c has a predicted N–terminal signal sequence and is annotated as an F420–dependent glucose-6-phosphate dehydrogenase. Here we show that Rv0132c protein does not have the annotated activity. It does, however, co–purify with F420 during expression experiments in M. smegmatis. We also show that the Rv0132c–F420 complex is a substrate for the Tat pathway, which mediates translocation of the complex across the cytoplasmic membrane, where Rv0132c is anchored to the cell envelope. This is the first report of any F420–binding protein being a substrate for the Tat pathway and of the presence of F420 outside of the cytosol in any F420–producing microorganism. The Rv0132c protein and its Tat export sequence are essentially invariant in the Mycobacterium tuberculosis complex. Taken together, these results show that current understanding of F420 biology in mycobacteria should be expanded to include activities occurring in the extra-cytoplasmic cell envelope.F Mycobacterium tuberculosis (Mtb) bacilli, the causative agent of this disease, infect one–third of the world's population, while TB claims nearly two million lives a year Mtb make TB a worldwide concern Tuberculosis (TB) is a devastating and contagious infectious disease. It is estimated that 420 is a 5-deazaflavin derivative that has been recently proposed to play a substantial role in the redox reactions of Mtb420 contains an isoalloxazine chromophore with a side chain composed of ribitol and phospholactate moieties and a poly–glutamate tail of variable length (420 is very similar to that of the flavins (FMN and FAD), with the major difference being the atoms involved in the oxidoreductive reactions. Oxidoreduction of F420 is achieved by hydride transfer between a substrate molecule and C5 of the 5-deazaflavin moiety, whereas transfer occurs to N5 in FMN and FAD. Despite its structural similarity to the flavins, F420 is functionally similar to NAD(P)+, being involved in hydride transfer reactions [for a review see The coenzyme Fe length . The iso420–dependent proteins in mycobacterial species is growing, and a few of such activities have been experimentally shown, including F420–dependent glucose-6-phosphate dehydrogenase (FGD) 420H2–dependent reductase (FDR) 420–dependent families in Mtb with at least 28 members; the luciferase–like monooxygenase (LLM), pyridoxamine 5′-phosphate oxidase (PNPOx), and the deazaflavin–dependent nitroreductase (DDN) families Mtb and showed that it has the annotated activity, providing reduced F420 for cell metabolism 420 produced by FGD1 to activate a promising anti–TB prodrug PA–824 420 can convert NO2 to NO in vitro, implying a possible protective role against nitrosative damage in mycobacteria in vivo420 has an important role in the physiology, and likely in the pathogenesis, of Mtb.The number of known FMtb genome in vitroΔsigF mutant of Mtb in late stationary phase 420 and whether it has the annotated F420–dependent glucose-6-phosphate dehydrogenase activity. We also noted that Rv0132c has a predicted N–terminal signal sequence that contains motifs suggestive of export via the twin-arginine translocation (Tat) pathway The 420 metabolism in Mtb. We report experimental evidence regarding the cellular location of Rv0132c, consistent with post–translational lipidation of the protein. In addition, we show that the Rv0132c protein does bind the cofactor F420, has a functional twin–arginine translocation (Tat) signal sequence, and is exported to the cell envelope by the Tat-dependent pathway. These results demonstrate that Rv0132c and the Mtb Tat pathway have a direct role in transferring the cofactor F420 across the cytoplasmic membrane, and suggest that the known roles of F420 should be expanded to include activities in the cell envelope.Here we investigate the possible significance of the Rv0132c protein in FMycobacterial strains and plasmids used in this study are in www.ncbi.nlm.nih.gov/gene) and unpublished (http://www.sanger.ac.uk/cgi-bin/blast/submitblast/mycobacterium) sequences and the alignments were carried out using CLUSTALW Sequence data for Rv0132c and its orthologues in various mycobacterial species were retrieved from the published  using the same buffer as for lysis (with no imidazole). Analytical SEC was used to determine the oligomeric state of the purified Rv0132c protein, using low molecular weight markers  to prepare a standard curve (The ORF encoding Rv0132c was PCR–amplified from omic DNA . When itomic DNA . The Rv0rd curve . The elu420 (25 µM) and glucose-6-phosphate (0.01–1 mM). The change in cofactor F420 absorbance at 420 nm was monitored over 5 min using UV–visible spectroscopy on a SpectraMAX microplate spectrophotometer (Molecular Devices). The reactions contained 100 µL of 20 mM HEPES pH 7.0, 150 mM NaCl, 1 mM β–ME and were performed in 96–well format .Assays for the annotated FGD activity of Rv0132c were performed using previously published protocols g. The resulting supernatant was adjusted to pH<7 with formic acid and applied to a 10 mg C-18 reversed phase solid phase extraction (SPE) cartridge. The SPE cartridge was then washed with 300 µL water and then 150 µL of 50 mM ammonium bicarbonate. The alkaline elution was collected and concentrated to 10 µL and was then diluted to 20 µL with 20% acetonitrile. The resulting solution was back–filled into a static nanoelectrospray needle (tip diameter 4 µm) which was then mounted into a nanoelectrospray interface of a Finnigan LTQ FT mass spectrometer. Ion trap and ion cyclotron resonance (ICR) cell data were obtained using a source voltage of 1.3 kV, capillary temperature of 225°C and capillary voltage of 26 V. MS/MS spectra were obtained by isolating key molecular ions and fragmenting using helium as the collision gas and 35% collision energy.The protein off the gel filtration column was boiled at 100°C for 15 min and centrifuged at 16000×420 coenzyme was purified from large scale preparations of M. smegmatis mc24517 cells expressing Mtb–FGD1 or FbiABC (three ORFs involved in the biosynthesis of F420) constructs 420 at 400 nm (ε400 = 25 mM−1 cm−1) was used to determine its concentration The FM. smegmatis ΔlysΔblaS (PM759) M. smegmatis ΔlysΔblaSΔtatA (JM578) M. tuberculosis ΔblaC (PM638) M. smegmatis and Mtb strains 500–1000 bacteria were plated on +/− carbenicillin containing agar. Carbenicillin resistance (+) was scored when >90% of colonies plated grew on carbenicillin, whereas carbenicillin sensitivity (−) was defined in strains showing 0% (no single colony growth) on carbenicillin containing media (The first 42 residues of the Rv0132c protein were used to generate a BlaC fusion construct (Rv0132cSS–'BlaC), which was then transformed into ng media .M. smegmatis mc2155 (wild type) ΔtatC mutant) As an independent method of proving Rv0132c is exported out of the cytoplasm by the Tat pathway, epitope-tagged full-length Rv0132c (Rv0132c-HA) was introduced into Mtb was determined by western blotting and immunoelectron microscopy , cytoplasmic membrane (CM), and soluble (SOL) fractions Mtb lipoproteins were prepared using Triton X–114 partitioning Mtb H37Ra cells were fixed and dehydrated in ethanol, pelleted and transferred to fresh resin in gelatin capsules for polymerization overnight at 60°C. Ultrathin sections (∼80 nm) were cut with a 45 degree diamond knife (Diatome) on an EM UC6 and collected onto 400–mesh nickel grids. For immunogold labelling, grids were incubated with either antiserum or preimmune serum, washed, blotted and transferred to drops of secondary antibody . Grids were washed, stained, air–dried and viewed in either a Philips CM12 or an FEI Tecnai 12 TEM, both operating at 120 kV.The subcellular localization of native Rv0132c in croscopy , using pM. smegmatis from an Rv0132c–Δ38 construct lacking the predicted N–terminal signal sequence, was purified by Ni–NTA and SEC. The protein eluted in the Ni–NTA step was resolved as a single band on an SDS–PAGE gel  and suggest that the suffix 1 should be removed from FGD1 to prevent further confusion with the incorrectly–annotated FGD2.Of the two  Rv0132c  show thaAnalysis of the Rv0132c sequence  using thMtb complex  have homologues of Rv0132c with full conservation (99–100% identity) of the mature protein sequence and the signal sequence, including the Tat motif. Other pathogenic microbacteria such as M. kansasii, M. avium and M. avium subsp. paratuberculosis have homologues with lower sequence identity (75–80%) that in most cases retain the Tat export sequence. In contrast, non-pathogenic mycobacteria such as M. smegmatis have homologues of Rv0132c that are of much lower sequence identity (30–40%) and all appear to lack a Tat signal sequence.Sequence searches show that all members of the Mtb (and BlaS for M. smegmatis) is a secreted β–lactamase that confers resistance to β–lactam antibiotics and can be used as a reporter for Tat–dependent export. This is because BlaC is normally exported via the Tat pathway The Tat pathway is responsible for transporting folded proteins across the cytoplasmic membrane M. smegmatis ΔblaS mutant is sensitive to carbenicillin, a β–lactam antibiotic, expression of Rv0132cSS–'BlaC conferred resistance to carbenicillin, indicating export , Rv0132css–'BlaC failed to confer resistance to carbenicillin, confirming Tat dependent export  as a control. The cell wall and cytoplasmic membrane fractions were free of cytoplasmic contamination, as shown by the lack of GroEL , cytoplasmic membrane (CM), and soluble (SOL) fractions. Equal cell equivalents of the fractions were subjected to western blot analyses, which showed that the native Rv0132c protein is present in the cell wall and membrane fractions of Mtb  and FGD2 (Rv0132c) 420–dependent nature; either Rv0132c does not bind F420, or it does not have the annotated activity, or is physically separated from Ddn where the activation process occurs.The presence of F420-binding protein, but that it does not have the annotated FGD activity. Differences in the substrate-binding region of the active site are consistent with preference for a different, as yet unknown, substrate. What sets Rv0132c apart from FGD1, apart from the differences in the substrate binding site, is its possession . In contrast, although homologues of Rv0132c can be found in non-pathogenic bacterial species they have much lower sequence identity with Rv0132c (<40%) and lack a Tat motif. This suggests a role for Rv0132c and its Tat motif in mycobacterial pathogenesis.Our results show unequivocally that Rv0132c is indeed an Fssession  of a sig+M. smegmatis we show Rv0132c is exported in a Tat-dependent manner and we further show the native Rv0132c is exported to the cell envelope of pathogenic M. tuberculosis. This further implies translocation of F420 to the cell envelope. This is the first report of F420 translocation across the cytoplasmic membrane in any F420–producing microorganism and expands the number of Tat–dependent cofactors.The Tat pathway transports folded proteins across energy–transducing membranes (e.g. the cytoplasmic membrane) 420 in the cytosol, after which the Rv0132c–F420 complex is translocated across the cytoplasmic membrane via the Tat pathway, where the protein is anchored to the cell envelope. Although the specific biochemical function of Rv0132c is not yet known, its Tat–dependent translocation may have evolved in pathogenic mycobacteria to enable F420 utilization for metabolic or biosynthetic processes in the dense, lipid–rich cell wall. The conservation of Rv0132c and its specific Tat signal sequence in the Mtb complex, but not in non–pathogenic mycobacteria implies that the presence of F420 outside the cytosol is important to the pathogenesis of tuberculosis. There is growing evidence that F420 plays an important part in defense against the host immune system and in non-replicating persistence of Mtb. Transition to the persistent state involves major changes in energy metabolism 420, with its low redox potential, may have a role in reactions associated with anaerobic survival Putting our experimental results together, it is reasonable to conclude that the Rv0132c protein binds FFigure S1Calibration curve for estimation of Rv0132c–Δ38 molecular weight using analytical SEC. The molecular weight calibration curve was obtained by plotting Kav values against LogMW of protein standards. The Kav value determined from the elution volume of Rv0132c–Δ38, indicated with *, corresponded to a molecular weight of 75.4 kDa, which is indicative of a dimer in solution.(TIF)Click here for additional data file.Table S1Mycobacterial strains and plasmids used in this study.(DOCX)Click here for additional data file.Table S2Primers used in the amplification of different Rv0132c constructs.(DOCX)Click here for additional data file.Text S1Supplementary methods. Details of bacterial growth; PCR amplification, cloning and preparation of constructs; western blotting; immunoelectron microscopy and homology modeling.(DOC)Click here for additional data file.


Fish meal and fish oil are increasingly replaced by ingredients from terrestrial sources in the feeds for farmed salmonids due to expanding production and reduced availability of marine feed raw material. Fish oil that is rich in n-3 polyunsaturated fatty acids is considered beneficial to human health in general and to prevent intestinal inflammation and carcinogenesis in particular. In contrast, n-6 fatty acids that are present in many vegetable oils have been associated with increased risk of colitis and colon cancer in rodents and humans, as well as lowered transcription levels of certain stress and antioxidant-related genes in Atlantic salmon.The aim of the present study was to investigate the intestinal health in Atlantic salmon fed with different vegetable oils as partial substitutes of fish oil in the diet. A feed trial lasting for 28 weeks included one reference diet containing fish oil as the sole lipid source and three diets where 80% of the fish oil was replaced by a plant oil blend with either olive oil, rapeseed oil or soybean oil as the main lipid source. These plant oils have intermediate or low n-3/n-6-ratios compared to fish oil having a high n-3/n-6-ratio. The protein and carbohydrate fractions were identical in all the feeds.Morphometric measurements showed significantly shorter folds in the mid intestine in all groups fed vegetable oils compared to the group fed fish oil. In the distal intestine, the complex folds were significantly shorter in the fish fed soybean oil compared to the fish fed rapeseed oil. Histological and immunohistochemical examination did not show clear difference in the degree of inflammation or proliferation of epithelial cells related to dietary groups, which was further confirmed by real-time RT-PCR which revealed only moderate alterations in the mRNA transcript levels of selected immune-related genes.Shortened intestinal folds might be associated with reduced intestinal surface and impaired nutrient absorption and growth, but our results suggest that partial substitution of dietary fish oil with vegetable oils does not have any major negative impact on the intestinal health of Atlantic salmon. Salmo salar) and rainbow trout (Oncorhynchus mykiss). Due to limited sources of marine raw materials, a fast growing aquaculture industry and increased focus on sustainability, the salmon farming industry has searched for alternative feeds [Salmonids are indigenous carnivores, and fish meal and fish oil have traditionally been the main ingredients in feed for farmed Atlantic salmon  such as linoleic acid LA, 18:2n-6) that can be further metabolized to arachidonic acid . In contrast, fish oils are rich in n-3 PUFAs such as docosahexaenoic acid  and eicosapentaenoic acid  , cluster of differentiation 3ζ (CD3ζ), immunoglobulins, the intracellular receptor nucleotide-binding oligomerization domain-containing protein 2 (NOD2) and COX-2a in the intestine of Atlantic salmon when dietary fish oil was partially replaced by different vegetable oil blends with varying n-3/n-6-ratio.Salmo salar) with mean initial weight of 815 ± 28 g were equally distributed into 12 tanks, and triplicate groups of fish were fed a diet with either fish oil as the sole lipid source or a diet where 80% of the fish oil was replaced by one of three vegetable oil blends with olive oil, rapeseed oil or soybean oil as the main lipid source . Six hundred Atlantic salmon (-1 and euthanized according to regulations (Forskrift om drift av akvakulturanlegg §34. Avlivning av fisk). The design of the feed trial and feed composition is described in detail elsewhere [The fish were vaccinated intraperitonally  to give protection towards furunculosis, vibriosis, cold-water vibriosis, winter ulcer disease and infectious pancreas necrosis one month prior to seawater transfer. The trial lasted for 28 weeks, and the mean final weight was 3399 ± 76 g with no significant difference between dietary groups. The fish were anesthetized with MS222 at a concentration of 7 g LFor histological evaluation, tissues from the mid and distal intestine Figure , also caet al. [Formalin-fixed and paraffin-embedded tissues from the mid intestine and the distal intestine from nine fish in each dietary groups (three fish in each tank) were prepared for detection of antigen presenting cells expressing MHC class II and T lymphocytes expressing CD3ϵ by immunohistochemistry with salmon specific polyclonal rabbit antisera as previously described by Koppang et al. ,30 with et al. [Micrographs of intestinal sections from nine fish in each dietary group (three fish per tank) were captured and morphometric measurements were performed in the software NIS-Elements D version 3  using Nikon digital sight camera configured with a Nikon eclipse 80i microscope. The measurements were performed as previously described by Løkka et al. . The heiTissues from the pyloric caeca, mid intestine and distal intestine Figure  from ninThe samples from the pyloric caeca and the distal intestine were homogenized in Buffer RLT added mercaptoetanol using stainless steelbeads (5 mm) in a Retsch MM 300 homogenizer (Retsch GmbH). RNA was extracted with RNeasy Mini kit  using the protocol “RNeasyMini Animal Tissues and Cells Standard V3” followed by the protocol “Cleanup RNeasyMini RNA Standard V3” in a QIAcube (QIAGEN).The concentration of RNA was measured using a Biospec-Nano  or Nanodrop ND-1000 UV-Vis Spectrophotometer . To verify acceptable quality of the RNA, 24 random samples were selected and tested on an Agilent 2100 Bioanalyzer . Total RNA was stored at -80°C.The cDNA synthesis from 1 μg of total RNA was prepared with oligo(dT), random hexamer primers, M-MLV Reverse transcriptase  and RNase inhibitor (Promega) to prevent RNA degradation. Real-time PCR was carried out in 13 μl reactions using TaqMan Gene Expression Master Mix  with cDNA template corresponding to 15 ng of RNA in each reaction in a 7900HT fast real-time PCR system (Applied Biosystems) according to the producer’s instructions and running 40 cycles.B (EF1AB) as the reference gene [The following genes were analyzed by real-time RT-PCR: Cluster of differentiation 3ζ (CD3ζ), cyclooxygenase-2a (COX-2a), interleukin 1β (IL-1β), immunoglobulin M (IgM), immunoglobulin T (IgT), major histocompatibility class II (MHC class II), nucleotide-binding oligomerization domain-containing protein 2 (NOD2), transforming growth factor β (TGF-β), tumour necrosis factor α (TNF-α) and elongation factor 1Ance gene  . Morphometric data within each dietary group were pooled before calculating the group mean.Data are given as mean ± standard error of the mean (SEM). The data were analyzed for normality using a Shapiro Wilk’s test and for homogeneity of variance using a Brown-Forsythe’s test. For data that were non-normal and/or with non-homogeneous residuals, a log transformation was performed prior to one way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. The significance level was set to 0.05.Histological examination of the mid intestine did not show any obvious pathological changes in any fish, while there was shortening, widening and fusion of the simple folds with leucocyte infiltration in the lamina propria in the distal intestine in a few individuals without any obvious association with the different dietary groups. Examination of sections stained with antibodies against MHC class II and CD3ϵ showed positive cells both in the epithelium and in the lamina propria and with similar density and distribution of antigen presenting cells and T lymphocytes regardless of diet Figure . The proMid intestine: The folds of the mid intestine were tallest in the fish oil group (1393 ± 36.4 μm), intermediate in the olive oil and rapeseed oil groups (1134 ± 31.2 μm and 1131 ± 42.4 μm respectively) and lowest in the soybean oil group (1012 ± 24.8 μm). The differences were highly significant (P < 0.0001) between the fish oil group and all vegetable oils groups. The folds of the mid intestine were widest in the soybean oil group (142.0 ± 5.4 μm) and most slender in the olive oil group (122.6 ± 4.3 μm) (P = 0.0249). There was no significant difference between the dietary groups regarding the wall thickness. The results from the morphometric analyses of the mid intestine are shown in Table Distal intestine: The complex folds were tallest in the rapeseed oil group (3598 ± 100.2 μm) and lowest in the soybean oil group (3123 ± 79.7 μm) (P = 0.0113), while the wall was thickest in the fish oil group (705.2 ± 20.4 μm) and thinnest in the olive oil group (616.8 ± 22.8 μm) (P = 0.0080). There was no significant difference between the dietary groups regarding the height of the simple folds or the width of either simple or complex folds. The results from the morphometric analyses of the distal intestine are shown in Table Only small differences in the relative transcript levels of the various genes between the dietary groups were detected, and there was generally larger individual variation within a group than between the groups. Neither the transcript levels of the pro-inflammatory cytokines IL-1β and TNF-α, the intracellular receptor NOD2 nor the enzyme COX-2a that is involved in the synthesis of prostaglandins from fatty acids were significantly altered in any dietary group in any of the intestinal segments.Pyloric caeca: In the pyloric caeca, the olive oil group had significantly higher transcript levels of CD3ζ and MHC class II (P = 0.004 and 0.048) while the rapeseed oil group had a significantly higher transcript level of TGF-β (P = 0.022) and the soybean oil group had significantly higher transcript levels of CD3ζ and TGF-β (P = 0.033 and 0.017) compared to the fish oil group . The transcript levels of both IgM and IgT was significantly lower in the soybean oil group than in the fish oil group (P = 0.014 and 0.016) and the olive oil group (P = 0.015 and 0.038)  and pea protein concentrate ,24,34. SIn fish, the gastrointestinal microbiota is known to change with different feeding regimes ,37 and mSignificant reductions of mucosal folds in the mid intestine of all vegetable dietary groups were observed, in contrast to the mildly affected distal intestine. This finding might be related to the fact that long chain fatty acids (LCFAs) mainly are absorbed in the pyloric caeca and mid intestine and only to a limited extent in the distal parts of the intestine ,39. AlteShortening of mucosal folds has been linked to altered proliferation pattern of the intestinal epithelium in Atlantic salmon; decrease in cell proliferation and apoptosis in smolt exposed to sublethal levels of inorganic mercury  and incrShortening of the mid intestinal folds probably reduces the total surface of the intestine and hence the absorption of nutrients, which may in turn influence the growth of the fish. A substantial proportion of starch and lipids is absorbed in the mid intestine . Fish feIn the pyloric caeca, the transcript levels of TGF-β and CD3ζ in the soybean oil group, the transcript level of TGF-β in the rapeseed oil group and the transcript levels of MHC class II and CD3ζ in the olive oil group was significantly higher than in the fish oil group. TGF-β is produced by cells of the innate immune system and by regulatory T lymphocytes and is both a pro- and anti-inflammatory cytokine that is involved in cell growth, migration, differentiation and apoptosis including inhibition of lymphocyte proliferation . In SBM-In contrast to mammals harboring mesenteric lymph nodes and distinct lymphoid follicles in the intestinal mucosa, the immune competent cells including antigen presenting cells as well as T and B lymphocytes are more diffusely spread in the intestinal tissue of teleosts like Atlantic salmon . The modThe significantly lower transcript level of CD3ζ in the distal intestine of the soybean oil group compared to the olive oil group might in contrast to the pyloric caeca, indicate a lower number of T lymphocytes. Furthermore, the soybean oil group had significantly lower transcript levels of IgM and IgT than the fish fed both fish oil and olive oil. These immunoglobulins are expressed by different subpopulations of B lymphocytes in the teleost intestine  and are In mammals, replacement of n-3 fatty acids with n-6 polyunsaturated acids has been associated with intestinal inflammation and promotion of intestinal cancer ,11. FeedThe amount of fish oil still present in the feed might be of importance for the ability of the fish to cope with the increased amount of n-6 fatty acids. The regular feed composition used in the salmon industry has changed dramatically over the last decades as the lipid fraction has increased from 10% till about 35% . This meThe folds in the mid intestine were significantly shorter in all groups of Atlantic salmon fed vegetable oils compared to the group fed fish oil. In the distal intestine, the complex folds were significantly shorter in the fish fed soybean oil compared to the fish fed rapeseed oil. Histological examination did not reveal clear difference in degree of inflammation related to dietary groups, and this finding was confirmed by real-time RT-PCR that only revealed moderate alterations in the mRNA transcript levels of selected immune-related genes.The shortening of the intestinal folds was most pronounced in the fish fed soybean oil and might be associated with reduced intestinal surface and impaired nutrient absorption and growth. Kept together with significantly higher transcript levels of TGF-β and CD3ζ in the pyloric caeca and significantly lower transcript levels of IgM and IgT in the distal intestine in the fish fed soybean oil compared to the fish fed fish oil, it can be concluded that inclusion of high levels of soybean oil in the feed for Atlantic salmon should be done with caution.The authors declare that they have no competing interests.TM did the sampling, planned the experiments, performed the histological examination, morphometric measurements, immunohistochemical stainings, parts of the cDNA synthesis and PCR and statistical analyses and drafted the manuscript, GL planned the experiments, performed immunohistochemical stainings, examination of immunohistochemical stainings, parts of the cDNA synthesis and PCR and statistical analyses and drafted the manuscript, JWN performed parts of the RNA isolation, cDNA synthesis and PCR, LA planned the experiments, designed the probes and supervised the PCR work, BT and GR designed and supervised the dietary experiment and samplings, and OBD, EOK and MK planned the experiments and supervised the study. Everyone commented on the manuscript and approved the final version.