Nutrition is vital to health and the availability of resources has long been acknowledged as a key factor in the ability to fight off parasites, as investing in the immune system is costly. Resources have typically been considered as something of a “black box”, with the quantity of available food being used as a proxy for resource limitation. However, food is a complex mixture of macro- and micronutrients, the precise balance of which determines an animal's fitness. Here we use a state-space modelling approach, the Geometric Framework for Nutrition (GFN), to assess for the first time, how the balance and amount of nutrients affects an animal's ability to mount an immune response to a pathogenic infection.Spodoptera littoralis caterpillars were assigned to one of 20 diets that varied in the ratio of macronutrients (protein and carbohydrate) and their calorie content to cover a large region of nutrient space. Caterpillars were then handled or injected with either live or dead Xenorhabdus nematophila bacterial cells. The expression of nine genes  was measured 20 h post immune challenge. For two of the immune genes (PPO and Lysozyme) we also measured the relevant functional immune response in the hemolymph. Gene expression and functional immune responses were then mapped against nutritional intake.The expression of all immune genes was up-regulated by injection with dead bacteria, but only those in the IMD pathway (Moricin and Relish) were substantially up-regulated by both dead and live bacterial challenge. Functional immune responses increased with the protein content of the diet but the expression of immune genes was much less predictable.Our results indicate that diet does play an important role in the ability of an animal to mount an adequate immune response, with the availability of protein being the most important predictor of the functional  immune response. Importantly, however, immune gene expression responds quite differently to functional immunity and we would caution against using gene expression as a proxy for immune investment, as it is unlikely to be reliable indicator of the immune response, except under specific dietary conditions.  •High protein diets improved survival after live bacterial infection.•Injection with dead bacteria increased expression of Toll and IMD immune genes.•Injection with live bacteria inhibited immune gene expression (GE).•The ratio and concentration of macronutrients in the diet affected GE.•GE only predicted functional immune activity at high levels of dietary protein. Severalnfection  and bactnfection  when thenfection , 2014.per se. However, neither food restriction, nor the manipulation of macronutrient balance alone can determine the relative importance of either on host-parasite interactions. To address properly the role of nutrient availability on immunity, both the balance of nutrients in the diet and their quantity need to be simultaneously manipulated. The Geometric Framework for Nutrition (GFN) is a state-space model that allows the association of particular nutrient intakes with outcomes of interest . Similasophila: . In a resophila: . HoweverToll (fungi and gram-positive bacteria) or IMD pathways (Gram-negative bacteria), via host pattern recognition receptors (PRRs) that result in the bespoke production of antimicrobial peptides and the upregulation of constitutive lysozymes, which form the humoral component of the response , a generalist herbivore. We take a GFN approach, restricting caterpillars to diets that vary in their P:C ratio and energy content, thereby covering a large region of nutrient space. We then challenge the immune system by injecting caterpillars with live or dead bacteria, and measure the expression of 9 genes , and 3 functional immune responses, which are transcribed by two of the immune genes (PPO and lysozyme) in the hemolymph, thus allowing us to associate gene expression and functional immune responses to nutrient intake, and importantly, to assess how well gene expression predicts the immune response specifically associated with those genes under different dietary conditions.Here we address this gap using a model insect, 22.1Spodoptera littoralis culture was established from eggs collected near Alexandria in Egypt in 2011. The colony was reared using single pair matings with around 150 pairs established each generation. Following mating of unrelated adult moths; eggs were laid within 2 days with larvae hatching after a further 3 days. Spodoptera littoralis spend approximately 2 weeks in the larval stage, about 7 days of which are spent in the 5th and 6th instars. Larvae were reared individually from the 2nd instar on a semi-artificial wheat germ-based diet . Vortexing ensured that all X. nematophila F1D3 cells were coated in glycerol. To revive the stocks for use, 100 μl was added to 10 ml nutrient broth, and incubated at 28°C for up to 48 h . On the day of experimental bacterial challenge, a sub-culture of the stock was carried out, with 1 ml of the original stock added to 10 ml of nutrient broth and placed in a shaker-incubator for approximately 4 h. This ensured that the bacteria were in log phase prior to challenge. Following the sub-culture, a 1 ml sample was checked for purity under the microscope by looking for non-fluorescent cells, which would indicate contamination. The clean sample was then used to produce a serial dilution in nutrient broth from which the total cell count was determined with fluorescence microscopy, using a haemocytometer with improved Neubauer ruling. The remaining culture was diluted with nutrient broth to the appropriate concentration required for the bacterial challenge. The heat-killed treatment group was established by autoclaving the bacteria for 30 min at 121°C.Bacteria were originally supplied by the laboratory of Givaudan and colleagues . Pure X2.2AICc values and other model metrics  ratio and calorie density based on  Table 1Table 1.  metrics .Table 1N2.3Xenorhabdus nematophila is a highly pathogenic Gram-negative bacterium. In the wild, this species relies on the entomopathogenic nematode Steinernema carpocapsae, which vectors X. nematophila, to gain access to an insect host, where it rapidly multiplies, generally causing death within 24–48 h , placed individually into 90 mm diameter Petri dishes and provided with ∼1.5 g of one of the 20 chemically-defined diets (X. nematophila (averaging 1272 X. nematophila cells per ml nutrient broth) using a microinjector (Pump 11 Elite Nanomite) fitted with a Hamilton syringe (gauge = 0.5 mm). The syringe was sterilised in ethanol prior to use and the challenge was applied to the left anterior proleg. Every 24 h up to 72 h (i.e. 48 h post infection), larvae were transferred individually to clean 90 mm Petri dishes containing 1.5–1.8 g of their assigned chemically-defined diet. 96 h after moulting into L6, the larvae had either pupated or were placed on semi-artificial diet until death or pupation. The amount of food eaten each day was determined by weighing the wet mass of the chemically-defined diet provided each day to the caterpillars, as well as weighing uneaten control diets each day (3 control diets per diet). The uneaten diet and control diet were then dried to a constant mass (for approx. 72 h), allowing the consumption per larva to be estimated.ed diets . Within Experiment 2: The set up for this experiment was identical to Experiment 1, except that each of the 400 larvae was injected with 5 μl of either a heat-killed (control) or live LD50 dose of X. nematophila (averaging 1272 X. nematophila cells per ml nutrient broth).2.4Following challenge, hemolymph samples were obtained from all caterpillars at 20 h post infection. Hemolymph samples were obtained by piercing the cuticle next to the first proleg near the head with a sterile needle and allowing released hemolymph to bleed directly into an Eppendorf tube. Immediately following hemolymph sampling, 30 μl of fresh hemolymph was added to a sterile ice-cooled Eppendorf containing 350 μl of lysis buffer (RLT + Beta mercaptoethanol – 100:1) for later RNA extraction and qPCR analysis (Expts 1 and 2). The remainder of the hemolymph extracted was stored in a separate Eppendorf for further immune assays (Expt 2 only). All hemolymph samples were stored at −80 °C prior to processing.2.5RNA was extracted from hemolymph samples using Qiagen RNeasy mini kit following the manufacturers instructions with a final elution volume of 40 μl. Extracts were quantified using the Nanodrop 2000 and diluted to 0.5 μg/μl for cDNA synthesis. Prior to cDNA synthesis a genomic DNA elimination step was carried out by combining 12 μl RNA  plus 2 μl DNA wipeout solution and incubating at 42 °C for 2 min. cDNA synthesis was carried out using Qiagen Quantitect Reverse Transcription kit in a final reaction volume of 20 μl following the manufacturer's instructions, cDNA synthesis was carried out for 30 min at 42 °C followed by 3 min incubation at 95 °C and stored at −20 °C. cDNA was diluted 1:5 for use as a qPCR template.Primers and probes were synthesised by Primer Design and qPCR was performed in a reaction volume of 10 μl with 1x Taqman FAST Universal PCR Master mix (Thermo Fisher), 0.25 μM of each primer, 0.3 μM probe and 2 μl of a 1:5 dilution of cDNA. qPCR was carried on the ABI 7500 FAST, cycling parameters included an initial denaturation at 95 °C for 20 s followed by 40 cycles of denaturation at 95 °C, 3 s and annealing at 60 °C for 30 s. All PCRs were run in duplicate see .Table 2PTrichoplusia ni caterpillars , which is the precursor of the phenoloxidase enzyme (PO), responsible for production of melanin during the encapsulation response, and lysozyme, which produces the antimicrobial lysozyme enzyme, active against Gram positive bacteria. We also selected two genes from the IMD immune pathway, Moricin, which produces the AMP Moricin, active against Gram positive and negative bacteria, and Relish, which activates transcription of AMP genes . We alsorpillars  and so w2.6Micrococcus luteus (Merck: M3770) in potassium phosphate buffer, in a 2:1 ratio. 10 ml plates of the resulting solution were poured and 2 mm diameter holes punched in each plate. Each hole was filled with 1 ml of ethanol saturated with phenylthiourea (PTU) in order to prevent melanisation of the samples. The ethanol evaporates, leaving the PTU in the hole. Following defrosting and vortexing of the stored hemolymph, each well was the filled with 1 μl of hemolymph, with two technical replicates per sample. The plates were incubated at 30°C for 24 h, and the clear zones around the samples were measured using digital callipers. Lytic activity (mg/ml) was then calculated from a serial dilution of a hen egg white lysozyme .Bacterial agar plates were used to determine lytic activity. These were made by mixing 1.5% water agar and 1.5% freeze-dried 2.72 l−1 sterile distilled water). The solution was then split into two Eppendorfs (each containing 225 μl), in order to carry out assays for both proPO and PO activity. To one Eppendorf, 25 μl of NaCac buffer was added (PO assay), and to the other, 25 μl of 20 mg per ml chymotrypsin in NaCac buffer was added (proPO activated). The samples were vortexed and incubated at 25 °C for 1 h. 90 μl of each solution was placed in a well of a 96-well microplate with 90 μl of 10 mM dopamine as a substrate. Two technical replicates were carried out per sample. Readings were taken every 15 s for 10 min at 490 nm and 25 °C using a Tecan infinite m200pro plate reader with Magellan software (V7.2). This range accounted for the linear stage of the reaction. The maximum rate of reaction was then used as an approximation of PO and proPO level. While many researchers still use L-dopa as a substrate for PO reactions, for insect POs, dopamine is the preferred substrate over L-dopa, it is the natural substrate for insects, it is more soluble than L-dopa and unlike L-dopa, is not subject to spontaneous darkening . Gene expression data were normalised using NORMA-Gene  to establish the most parsimonious models including likely nutritional attributes driving the observed data. AICc values and Akaike weights were estimated using the MuMin package  – the lysozyme gene for lysozyme activity and the PPO gene for PPO and PO activity.3.3survival , but this was indistinguishable from the same model that included the additive effects of treatment .The total amount of food consumed varied across the diets. For experiment 1, comparing handled caterpillars versus those injected with heat-killed bacteria, the best model predicting consumption was model   20 (Co*R + Co2+R2), but as for the handled versus dead treatments in experiment 1, this model was indistinguishable from the same model that included the additive effects of treatment .For experiment 2, comparing dead and live bacterial injections, the best model predicting consumption was model 20 ). However, although the fit of these models was generally good (r2 > 0.26–0.86), with the exception of Moricin, the amount of variation explained by the fixed part of the model was very low , injection with dead bacteria resulted in up-regulation of gene expression relative to handled caterpillars . In contVariation in the expression of all of the genes was explained by main and interactive effects of the amount of protein and carbohydrate eaten, and in interaction with the bacterial treatment, suggesting that the response to diet for each gene differed across treatments. A visualisation of these response surfaces , Fig. 5 4.3For the Lysozyme and PPO genes, we simultaneously measured functional lytic and PPO (and PO) activity in the hemolymph, allowing us to determine how well gene expression predicts the functional immune response. We had lytic and PO data only for Experiment 2, where larvae were challenged with live or dead bacteria.2 < 0.12; For PPO activity, AICc could not discriminate between several of the diet models, with seven being equally well supported . Whilst Moricin expression had negligible effects on survival in the dead bacterial treatment, high levels of expression were indicative of an  species  and afte exempta , 2014. HX. nematophila is a Gram-negative bacterium, and is clearly triggering Moricin and Relish expression, but as Toll is only marginally up-regulated in response, it is probably the IMD pathway that is controlling this response. Another possible explanation for why live bacteria appear to trigger a down-regulation of gene expression is that our sampling protocol  did not allow us to catch peak expression levels . Expression of lysozyme and PPO in the Glanville fritillary butterfly was not up-regulated 24 h after injection with M. luteus cells  and Micrococcus luteus (G + ve), so it is not clear if both or just one of the bacteria caused the response. Another possibility is that Arylphorin levels are already expressed at maximal levels and cannot be further up-regulated. In T. ni caterpillars, Arylphorin is the most abundant protein in the hemolymph during the final instar  typically improved with the protein content of the diet, gene expression varied non-linearly with diet composition. However, the expression of PPO and Lysozyme genes predicted PPO/PO and Lysozyme activity, but only when the availability of dietary protein was not limiting, suggesting that using gene expression as an indicator of investment in a trait is unlikely to be reliable, unless its relationship with diet is known.


During exercise in supine posture or under microgravity in space, the gravity-dependent component of local blood pressure in leg muscles at upright posture can be simulated by lower body negative pressure (LBNP). We hypothesized that during resistive exercise LBNP favors oxygen availability in lower extremities, benefiting energy levels and performance of working muscles.In permutated crossover design, nine subjects performed a series of fifteen slow-paced concentric (4 s) and eccentric contractions (4 s) without or with 40 mmHg LBNP and 4 s pause between repetitions. The force at knee flexion was 6% of the one repetition maximum (1-RM) and gradually increased to 60% 1RM in the first half of the individual range of motion, subsequently remaining constant until full extension.p < 0.01) and oxyhaemoglobin by about 10 µmol/l (p < 0.01). During continuous exercise, LBNP induced a trend towards a lower EMG increment. This LBNP effect was not found when the periods of low forces at knee flexion were extended by 4 s pauses. Increased respiratory oxygen uptake  indicated overall enhanced muscle energy turn-over.During the low force periods of continuous exercise, LBNP enhanced the refill of capillary blood measured by near infrared spectroscopy, amplifying the increase of total haemoglobin by about 20 µmol/l (Our results suggest stimulation of oxidative metabolism through LBNP enables working muscles to meet the energy demands of intense exercise. Further research is needed on the consequences for energy metabolism and the molecular control of growth and differentiation. In upright body posture, perfusion of working leg muscles profits from the muscle pump, which increases the arterio-venous difference of blood pressure. The mechanism involves a gravity-dependent pressure component on the arterial side and a reduction of pressure on the venous side by expulsion of blood and prevention of backflow through venous valves  leading to a diminished cardiac output. This is almost compensated for by a directly ensuing increase in heart rate (HR) and accompanied by a slow rise in total peripheral resistance stabilizing blood pressure  with similar profiles of moderate recreational activity completed the study. Subjects were fully acquainted with the experimental approach and provided a written informed consent prior to their participation. Approval was issued by the North Rhine Medical Association’s Ethics Committee .The study followed a crossover design testing the acute effects of 15 slow, high load, concentric and eccentric contractions of knee extensor muscles on a robotically controlled leg press (RCL) under four different conditions in which the following variables were either introduced or omitted: lower body negative pressure (LBNP) as means of substituting orthostasis and 4 s pauses enabling prolonged periods of very low muscle force and muscle perfusion between the contraction intervals. These four conditions were varied in a balanced permutated sequence with a minimal resting period of 7 days between different exercise sessions to avoid any interaction effects  was recorded for each subject. The subjects were advised to maintain their usual pattern of activity and not engage in more strenuous or additional exercise. Extensive physical activity (e.g. intense sports) was not allowed for up to two days prior to each session, to ensure that the subjects were well rested when attending their study appointments. Furthermore, subjects were advised to abstain from alcohol for 24 h prior to their appointments, as well as to eat 3 h beforehand and thereafter only consume water, to ensure an overall comparable metabolic state of the subjects during the sessions. As caffeine withdrawal can negatively impact the ability to perform, we decided against caffeine restriction and rather advised our subjects to retain their usual coffee intake. To maintain sufficient energy levels, subjects were also asked to consume a protein energy drink  2 h prior to their exercise sessions. All the above-mentioned points were followed-up on with each subject throughout their participation in the study. Subjects were introduced to these guidelines through an orientation before the study started. They additionally received all the details as handouts and were timely reminded of specific due points by phone or e-mail.Due to a risk of syncope during the sessions with LBNP, blood pressure and heart rate were monitored for pre-syncopal signs by an independent physician using ECG .RCL was developed at the German Aerospace Center  in cooperation with the companies Sensodrive G.m.b.H  and S.E.A. Datentechnik GmbH . The linear drive of the leg press is controlled by an electromotor enabling safe exercise without storage of energy by weights or springs. The force–distance profile is freely programmable and allows customized training adapted to individual requirements. The leg press is located within an LBNP chamber that previously contained a treadmill , directly followed by a 5 min baseline data collection phase on the RCL Table . In the N) determined as the average force over 2 s around the turning point in extension, mean test force (N) averaged over the entire 8 s contraction interval, single direction range of motion (mm), mean velocity (mm/s) of concentric and eccentric contractions, as well as the concentric and eccentric mean power .Sum force and foot position were continuously recorded together with the electromyogram data at a sampling rate of 1500 Hz. Analysis was performed for the peak test force  were measured with near infrared spectroscopy (NIRS) using the telemetric Portamon device , placed on the right leg, over the belly of the vastus lateralis muscle. Portamon is a small wireless device weighing 75 g, carrying one light sensor and three light sources with emission at wavelengths of 760 nm and 850 nm. Their distance towards the light sensor is 30, 35, and 40 mm, respectively. The proportion of light absorption by the skin is reduced to a minimum by an almost 1.5 mm elevation of the emitters and the receiver over the surface of the device. This elevation reduces thickness and blood content of the light-transmitting portion of the skin. Motion artifacts were minimized by a tight fixation of the device with black elastic kinesiology tape. NIRS signal was recorded continuously at a rate of 10 Hz. During data collection, the device automatically calculated the total haemoglobin content  and the tissue oxygen saturation index . BL1 and BL2 mean values were calculated for a 30 s period, from 2 to 1.5 min preceding the directly following phases. O2Hb and tHb minima occurred during the high load phases of the contraction intervals in the main exercise set (E), with corresponding maxima emerging during the low load phases, i.e., the transitions from one contraction interval to the next. During intermittent exercise, the maxima occurred in the 4 s pauses between the contractions. This recurrent pattern of minima and maxima gave a more precise reflection of the blood filling and Hb oxygenation levels than the corresponding mean values. Therefore, minima and maxima of tHb, O2Hb, HHb and TSI were determined for each contraction interval (C1–C15). As in case of the baseline values, the recovery values were also calculated as means during the 30 s period from the onset of the last 2 min of each phase .The concentrations of oxygenated and deoxygenated haemoglobin  was used for electromyography (EMG), with electrode pairs placed on the vastus lateralis and the biceps femoris muscle, respectively. In case of the right leg, the electrodes were placed distally from the Portamon device. EMG was recorded at 1500 Hz sampling rate throughout the main exercise set. The root mean square values of amplitudes were calculated for the concentric phases of each of the 15 contraction intervals and normalized to the initial values of the first contraction. Corresponding median frequency values are also presented.− 5). For the baseline and recovery phases , mean values were calculated as previously described for the NIRS parameters. Mean values were also determined for each contraction interval of the main exercise set (C1–C15).Blood pressure was continuously measured using a plethysmographic device  with the finger cuff placed on the fourth finger of the right hand. Based on the blood pressure values, the BeatScope software  calculated beat-by-beat the following parameters: heart rate , stroke volume , cardiac output  and total peripheral resistance , carbon dioxide release , and respiratory exchange ratio (RER = V′CO2/V′O2) were determined in a breath-by-breath mode. Mean values were calculated for the last 2 min of baseline without LBNP, the entire main exercise set (E), as well as for the first 5 min and the final 15 min of recovery . Excess values of V′O2 and V′CO2 during E, R1 and R2 were determined by subtracting the corresponding baseline values. In addition, a representative time curve of V′O2, V′CO2 and ventilation (l/min) is presented for the initial recovery (R1 and first 5 min of R2).Spirometry was performed using the Innocor device . Oxygen uptake (V′OLactate concentration (mmol/l) in capillary blood obtained from the ear lobe was measured with a portable device  directly after the main exercise set, as well as post 1, 2, 4, 6, 10, 15 and 20 min.Rating of perceived exertion (RPE) based on Borg‘s Scale with a range of 6–20 Borg  was dete2Hb, HHb, TSI, HR, SV, CO, TPR) and time point (lactate). For exercise parameters , only the effect of having four different conditions (without/with LBNP or pause) was tested. Statistical analysis was performed with SPSS (IBM SPSS Statistics Version 21). Data are presented as means ± standard error (SE) or standard deviation (SD), as indicated in the accompanying legends. Statistical significance was rated as p < 0.05.Linear mixed effects models were used to test for significant effects of LBNP and pause, as well as their interactions. Additional tested fixed effects were contraction and side (EMG-amplitude), phase  and a decrease in stroke volume (SV) from approximately 110 to 80 ml . The resulting cardiac output (CO) decreased from approximately 8 to 6 l/min . Total peripheral resistance (TPR) was not significantly influenced by LBNP at rest . During the initial recovery with LBNP (R1), directly following the main exercise set, HR decreased in all conditions, but still remained higher with LBNP than under corresponding control conditions and was accompanied by a reduction in SV with LBNP  from around 70 to 80/min . This increase can be attributed to elevated deoxyhaemoglobin , since oxyhaemoglobin (O2Hb) levels were not significantly impacted. Consequently, a slight (under 5%) but significant decrease of the tissue oxygen saturation index (TSI) was observed (p < 0.01). The values of tHb, O2Hb, HHb and TSI relative to the initial baseline (BL1) did not significantly differ between the exercise sessions.Total haemoglobin (tHb) content was significantly elevated during baseline with LBNP (BL2) by approximately 20 µmol/l (p < 0.01), more evidently during continuous than during intermittent exercise . On the other hand, tHb minima were only significantly elevated during continuous exercise with LBNP, by around 10 µmol/l (p < 0.01). Likewise, TSI maxima were elevated in both LBNP exercise sessions by a margin of under 5% (p < 0.01 for continuous and p < 0.05 for intermittent exercise), whereas the TSI minima were only enhanced during continuous exercise, by a similar margin (p < 0.05). Maxima of O2Hb were elevated with LBNP, both during continuous exercise (p < 0.05), as well as during intermittent exercise (p < 0.001) by approximately 10–15 µmol/l, with no significant changes in O2Hb minima.During the subsequent exercise set (E), a pattern of minimal values occurring in the periods of high force and maximum values arising in the low force periods of the muscle contraction intervals (C1–C15) was observed for all NIRS-parameters, reflecting the stop and permission of blood flow and the activity of the muscle pump. Maxima of tHb were significantly elevated with LBNP , indicating a restoration of the pre-exercise tHb and O2Hb concentration. During R1 following intermittent exercise, TSI was slightly decreased with LBNP , accompanied by a small increase in HHb (approximately 5 µmol/l). This initial post-exercise response also receded before R2.Relative tHb and O2 and V′CO2 were significantly elevated with LBNP during the main exercise set  and the late recovery phase  compared to the corresponding control interventions, both without and with 4 s pause during flexion. No significant effect of LBNP on excess V′O2 and V′CO2 or RER was found in the initial recovery phase immediately prior to switching off LBNP. Intermittent exercise led to significantly diminished excess V′O2 and V′CO2 during the recovery phases under both control and LBNP conditions relative to sessions with continuous exercise, accompanied by an overall reduction in the respiratory exchange ratio . Lactate was only significantly elevated through LBNP after continuous exercise (p < 0.05).Lactate levels increased during the initial 4 min after continuous exercise and 1 min following intermittent exercise. They then gradually returned to baseline values during the overall 20 min recovery period. The observed increase was higher after continuous compared to intermittent exercise , with no significant influence of LBNP.After the main exercise set, subjects provided following ratings of perceived exertion (RPE) based on Borg’s scale Borg : for exeThrough our present work, we introduce a novel combination of a robotically controlled leg press within an LBNP chamber as an experimental model with promising results for simulating gravity like blood shifts in exercising leg muscles. Combined with an appropriate experimental design, this unique device enables studying the effects of simulated orthostasis on leg muscle perfusion without additional mechanical and neuromuscular effects linked with an actual change of body posture. In accordance with our introduced hypotheses, we were able to demonstrate the existence of a gravity-dependent muscle pump during slow paced high intensity resistive exercise. Our results demonstrate that LBNP superimposed on a slow concentric–eccentric resistive exercise can enhance blood volume and oxygenation of the knee extensor muscles, suggesting enhanced muscle perfusion.LBNP at 40 mmHg represents a reductive experimental model that simulates one component of upright posture, namely the fluid shift towards the lower body  in the forearm with a concomitant THI elevation in the calf muscle measured with NIRS  indicate that LBNP mediated a pooling of CO2-rich and oxygen-poor blood in the lower body that was released after LBNP shut-down and subsequently transported headwards, passing the lung. This finding is supported by the initial brief increase in V′O2 and in V′CO2 directly upon LBNP shut-down followed by a recovery of the values, indicating an initial transport of O2-diminished blood from the lower extremities into the lung rather than a sudden boost of the local energy metabolism in the recovering muscle. This distinct difference in the transport of blood gasses caused by LBNP decreases the comparability of respiratory values during exercise with and without LBNP at least outside long-term steady state periods.During exercise with pauses during flexion, V′OLower increase of oxygen uptake and a lower rise in lactate levels were previously observed during incremental load dynamic leg exercise with LBNP Eiken , in contPostural effects on the development of fatigue in lower limb muscles manifest at moderate to very high forces with regard to intermittent contractions . The RPE values did not significantly differ between the LBNP and their corresponding control sessions, suggesting a comparable intensity of exercise within individual training protocols. Furthermore, the average test force at the turning point between extension and flexion (± 1 s), mean force, as well as average work distance on the leg press and the concentric and eccentric velocity and power were all comparable between the four tested conditions (without/with LBNP and 4 s pause during flexion), eliminating the hypothetical influence of an uneven mechanical performance on the reported findings on motor unit activation. This is further reinforced by equivalent EMG signal patterns obtained through bilateral measurements, which suggested that the exercise was performed with equal contribution from both limbs.2 release indicate enhanced oxygen utilization in the working muscle. Furthermore, an ensuing tendency towards reduced motor unit recruitment suggests a possible benefit of LBNP in delaying muscle fatigue. Our findings indicate that LBNP is effective when ischemic periods by high muscle forces are preceded by equal periods of lower forces allowing blood flow through the muscle. Four seconds longer recovery periods between contractions allow full reflow of blood in all cases without an additional benefit of LBNP.In summary, we report an exercise protocol design involving a novel, robotically controlled leg press that enables even performance during intense resistive exercise, with added benefit of enhanced muscle perfusion through superimposed LBNP. This combination could ultimately facilitate muscle performance through increased oxygenation and energy provision, delaying the development of muscle fatigue. This is reflected in the enhanced total and oxyhaemoglobin content, and enhanced tissue oxygen saturation index of the knee extensor muscles. Concomitant increases in the overall oxygen uptake and COCombining LBNP and resistive exercise has shown promising results and potential benefits for diverse applications, spanning from advancing training techniques on Earth to countermeasure development for long-duration space flight. Due to the advantages of resistive exercise for muscle mass enhancement (Tesch et al.


Crigler-Najjar syndrome (CNs) presents as unconjugated hyperbilirubinemia, as a result of UGT1A1 deficiency, and can be categorized in a severe (type I) and mild (type II) phenotype. CNs type II patients usually benefit from phenobarbital treatment that induces residual UGT1A1 activity.UGT1A1 gene. A 3 nucleotide insertion in the HNF-1α binding site in the proximal promoter previously reported in a Crigler-Najjar patient on one allele and a novel two nucleotide deletion in exon 1, resulting in a frameshift and a premature stop codon.Here we present a CNs type II patient that is not responsive to phenobarbital treatment, which can be explained by two heterozygous mutations in the In newly diagnosed CNs patients with unconjugated bilirubin levels consistent with CNs type II but that are unresponsive to phenobarbital treatment, disruption of the HNF-1α binding site in the proximal promoter should be considered as a probable cause. Upon confirming a mutation in the HNF-1α site, phenobarbital treatment should be stopped or at least be reconsidered because of its sedative effects and its teratogenic properties.The online version of this article (10.1186/s12887-019-1555-y) contains supplementary material, which is available to authorized users. Crigler-Najjar syndrome (CNs) is a rare inherited liver disorder with a severely impaired metabolism of bilirubin, resulting in the accumulation of neurotoxic unconjugated bilirubin.This deficiency of bilirubin glucuronidation is caused by mutations in the UGT1A1 gene encoding uridine diphosphate glucuronosyl transferase, resulting in impaired enzyme activity [A 14-year old female patient from Bangladesh presented with serum total bilirubin levels around 250 μmol/L and conjugated bilirubin (measured as direct bilirubin using the Diazo method) of around 10 μmol/L, indicating a predominantly unconjugated hyperbilirubinemia. According to her parents’ description, her weight at birth was around 2000 g and 4 days after birth, her skin turned yellow. Clinical assessment revealed an unconjugated hyperbilirubinemia of 220 μmol/L without signs of erythrocyte hemolysis (major cause: ABO or Rh incompatibility). After undergoing phototherapy for 4 h a day for 4 consecutive days the serum total bilirubin levels were reduced to 153 μmol/L. The parents were advised to keep their daughter in the sunlight, but after a few months her serum total bilirubin increased again to over 300 μmol/L. From this point onward, the patient did not receive treatment and no clinical data is available because the family lives in the country side and has limited access to medical care. Between the age of 14 and 17 years her serum total bilirubin levels have been monitored and where stable around 200–250 μmol/L. Liver damage markers in serum were low (ALT) and a hemolytic cause of the hyperbilirubinemia was excluded with normal hemoglobin and reticulocyte levels  and made the promoter non-responsive to CAR activation and to a potential alternative treatment via PXR activation with rifampicin which were confirmed by the functional promoter studies in that report. Due to this mutation, a minute amount of mRNA will be transcribed from this allele, which encodes for a minimal amount of normally active protein, and renders this allele unresponsive to UGT1A1 gene, inherited from her father, is a novel mutation. This mutation has not been described before and is predicted to result in a premature stop codon by frameshift leading to the formation of a truncated and inactive enzyme that will most likely be degraded.The two nucleotide deletion in exon 1 of the UGT1A1 gene by phenobarbital, phototherapy and liver transplantation are currently the only therapeutic options.The combination of these two mutated alleles results in a severely impaired UGT1A1 function, which is in line with the high levels of unconjugated hyperbilirubin seen in this patient. Unconjugated bilirubin levels in serum of this patient were comparable to that seen in the previous patient with an identical HNF-1a mutation, indicating the phenotype results from this mutation only. Since, the HNF-1α mutation renders the gene unresponsive to transcriptional activation of the We report a second patient with a CNs type II phenotype that is unresponsive to phenobarbital treatment due to a mutated HNF-1α binding site, in combination with a novel nonsense mutation. In newly diagnosed CNs patients with a similar phenotype, looking for mutations in the HNF1a binding site seems a good strategy. Upon confirming presence of a mutated HNF-1a binding site the use of phenobarbital should be reconsidered in view of its sedative effect and specifically in women, because of its reported teratogenic properties .Additional file 1:Table S1. Primer used to amplify UGT1A1 gene. (DOCX 18 kb)


Hordeum vulgare L.) accessions was uncovered. A genome wide association study (GWAS) allowed the identification of two genes located on chromosome 7H, homogentisate phytyltransferase (HPT-7H) and homogentisate geranylgeranyltransferase (HGGT) that code for key enzymes controlling the accumulation of tocopherols in leaves and tocotrienols in grains, respectively. Transcript profiling showed a correlation between HPT-7H expression and vitamin E content in leaves. Allele sequencing allowed to decipher the allelic variation of HPT-7H and HGGT genes corresponding to high and low vitamin E contents in the respective tissues. Using the obtained sequence information molecular markers have been developed which can be used to assist smart breeding of high vitamin E barley varieties. This will facilitate the selection of genotypes more tolerant to oxidative stress and producing high-quality grains.Tocopherols and tocotrienols, commonly referred to as vitamin E, are essential compounds in food and feed. Due to their lipophilic nature they protect biomembranes by preventing the propagation of lipid-peroxidation especially during oxidative stress. Since their synthesis is restricted to photosynthetic organisms, plant-derived products are the major source of natural vitamin E. In the present study the genetic basis for high vitamin E accumulation in leaves and grains of different barley ( The classification vitamin E nowadays refers to a group of eight structurally related lipophilic compounds. Four are classified as tocopherols and four as tocotrienols collectively known as tocochromanols2. The term tocopherol is an allusion regarding its necessity in the pregnancy of mice  and was first proposed after the successful isolation of an alcohol (ending: -ol) from wheat germ oil with the properties of vitamin E3.Nearly 100 years ago the report of the existence of a hitherto unrecognized dietary factor essential for reproduction of mice led to the discovery of vitamin E4. Common features of all tocochromanols are a hydrophobic polyprenyl side chain and a chromanol ring derived from homogentisate (HGA). The hydrophobic polyprenyl side chain anchors these compounds in biological membranes while a hydroxyl group at the chromanol ring confers vitamin E antioxidant activity: It can donate its phenolic hydrogen atom to scavenge radicals mainly to prevent propagation of lipid peroxidation in biological membranes (e.g. reviewed in5). While tocopherols comprise a saturated prenyl side chain the trivial name tocotrienol was chosen for tocochromanols with a tri-unsaturated aliphatic tail with double bonds at the 3′, 7′ and 11′ position6. Tocochromanols are exclusively synthesized by photosynthetic microorganisms like cyanobacteria and algae as well as plants7. While it is generally assumed that tocopherols are ubiquitously present in the plant kingdom tocotrienols are only present in certain plant groups8.The structure of this alcohol was solved when the reaction product (later: α-tocopherol) of phytyl-bromide and trimethyl-hydroquinone displayed identical chemical properties as vitamin E and gave excellent activity in biological tests9. The first committed step in tocotrienol biosynthesis depends on an enzyme designated as HGA geranylgeranyltransferase (HGGT). It displays enhanced affinity towards the tri-unsaturated polyprenyl compound geranylgeranyl pyrophosphate (GG-PP) over the saturated P-PP10. The enzymatic reactions downstream of HGA prenylation were also shown to take place at the inner chloroplast envelope membrane11. Furthermore, they do not discriminate between compounds with saturated or unsaturated side chains as proven by the introduction of the barley HGGT enzyme into Arabidopsis thaliana which is usually free of tocotrienols10. The HPT product MPBQ  as well as the HGGT product MGGBQ  can serve as the substrate for a MPBQ methyltransferase (MPBQ-MT - VTE3) which creates 2,3-dimethyl-6-phytyl-1,4-benzoquinol (DMPBQ) and 2,3-dimethyl-6-geranylgeranyl-1,4-benzoquinol (DMGGBQ), respectively. All four benzoquinones can act as substrates for tocopherol cyclase (TC - VTE1) which adds a second oxygen-containing ring at the junction between the aromatic head group and the prenyl tail to create the two-ring structure known as a chromanol ring12. TC activity generates δ-tocopherol (δ-tocotrienol) from MPBQ (MGGBQ) and γ-tocopherol (γ-tocotrienol) from DMPBQ (DMGGPQ). These isoforms can eventually be methylated at the aromatic ring by a γ-tocopherol methyltransferase (γTMT - VTE4) to generate β-tocopherol (β-tocotrienol) or α-tocopherol (α-tocotrienol), respectively. The methylation status of the chromanol ring therefore distinguishes the four tocopherol and tocotrienol species13.The first committed step in tocopherol biosynthesis the prenylation of HGA with the polyprenyl compound phytyl pyrophosphate (P-PP) is catalysed by a HGA phytyltransferase (HPT - VTE2) and was shown to take place in the chloroplast envelope membrane14 the substrate flow towards β- and α-tocopherol is not saturated in planta so that the progenitor isoforms δ- and γ-tocopherol can be observed in green parts15 and in plant-derived oils16 in a species- and tissue-specific manner. The ratio of the four tocopherol isoforms is influenced by the enzymatic activities and is especially well studied in Arabidopsis thaliana mutant and overexpression lines (reviewed in17). For the biosynthesis of tocochromanols the building blocks have to be provided by independent pathways. The aromatic compound HGA is derived from L-tyrosine. To this end the conversion from L-tyrosine to 4-hydroxyphenylpyruvate (HPP) is catalysed by a tyrosine aminotransferase (TAT) and from HPP to HGA by a HPP dioxygenase (HPD) which can reportedly limit tocopherol biosynthesis18. P-PP or GG-PP are possible products of the non-mevalonate pathway (DOXP pathway) and are in direct relation to each other via geranylgeranyl diphosphate reductase (GGDR) activity19. Desoxy-D-xylulose-5-phosphat-synthase (DXP) catalyses the first step of the DOXP pathway and a recent report speculates about a quantitative influence of DXP on the tocochromanol pool in maize20. Besides, it was long speculated that P-PP and GG-PP can derive from chlorophyll degradation in vivo which releases phytyl21. This could be proven when a phytyl kinase (PK - VTE5)22 and a phytyl phosphate kinase (PPK - VTE6)23 were identified and tested to have relevant influence on tocochromanol biosynthesis.While the benzoquinol intermediates do not accumulate in notable amountsTC14 (vte1) and HPT24 (vte2) in Arabidopsis thaliana. Since then, overexpression of structural pathway genes, whether endogenous or heterologous, was used to successfully and significantly increase the vitamin E content of many plant species and tissues (e.g. reviewed in25). To influence the vitamin E content by classical breeding one relies on the existence of natural genetic diversity in the germplasm of a species and the ability to validate the heredity of the desired trait. An elegant way to dissect this variability is the identification of quantitative trait loci (QTLs) by genome wide association studies (GWAS). QTL mapping uses statistical techniques to localize chromosomal regions that most likely contain genes contributing to phenotypic variation in a complex trait of interest and thus make it accessible for marker-assisted breeding efforts26. QTLs associated with tocochromanol content have been detected for several crop species including tomato27, rapeseed30, maize33, soybean35, rice37 and barley39.Historically, the genetic dissection of tocochromanol biosynthesis started with the identification of the 39. Overall 57 metabolites have been analysed including those of primary carbon and nitrogen as well as antioxidant metabolism. Parallel genotyping of the investigated accessions allowed the identification of QTLs for tocopherol and other metabolites for plants grown under various environmental conditions. In the present study we set out to scrutinize whether the previously identified chromosomal regions determine the vitamin E composition and accumulation under field conditions. To achieve this goal two new but partially overlapping sets of genetically diverse spring barley genotypes were compiled and consecutively grown and probed under simulated drought conditions in a rain out shelter (RS) in the field. The study allowed us to confirm the previously determined QTLs for vitamin E accumulation in barley leaves39 and grains38 and enabled us to identify the underlying structural genes. Comparing genomic sequences of the different genotypes resulted in the determination of allelic variation which could further be associated with different expression levels of one structural gene of vitamin E biosynthesis, HPT-7H, correlating to high or low vitamin E accumulation. The uncovered allelic variation can be used to select for high quality barley genotypes in the future.Recently, we reported on a large-scale metabolic profiling approach with leaves from drought and heat stressed barley varieties grown under controlled conditions in phytochambers39. The first goal of the present study was to assess the stability of this association in field-grown plants. Therefore, two partially overlapping sets of spring barley genotypes were selected and analysed under drought and well-watered/irrigated conditions in the field. The new sets consisted of elite lines from North America and Germany as well as latest German breeding material and Mediterranean land races (please see Supplementary Table A QTL located on the long arm of the barley chromosome 7H at 118–122 cM (centimorgan) was identified to be associated with γ-tocopherol content of flag leaves of barley plants grown in different controlled environmental conditions including single drought and combined drought and heat stress39. In agreement with our previous work we confirmed significantly elevated contents of γ-tocopherol and additionally of α-tocopherol in genotypes carrying one of the two detected SNP haplotypes. This correlation was stable across growing seasons (2016 and 2017), genotype sets (2016 and 2017) and growth conditions (well-watered and drought) . To assess genetic diversity of the investigated barley varieties at the chromosomal region of interest we utilized the KASP-method (Competitive Allele Specific PCR) to specifically determine the single nucleotide polymorphism (SNP) patterns of three SNP-markers originally found in the QTLhttp://webblast.ipk-gatersleben.de/barley_ibsc/)42 we mapped the structural genes of the tocochromanol biosynthesis pathway in the barley genome as well as the position of several genes involved in provision of precursors  qualified for further investigation  and the Morex reference assembly pseudomolecules (MRA) in the IBSC database. By overlapping multiple reads, we were able to create approximately 6,500 bp long ungapped contigs of all three alleles. They surpassed the one Barke-, the one Bowman- and the two Morex-WGS_contigs in the IBSC database and in addition the newly generated contigs confirmed the orientation and distance of the five contigs of the MRA scaffold thereby closing all existing gaps  based identification. The transcribed region of HPT-7H harbours three SNPs which display a unique combination in each of the four identified alleles and give rise to the presence or absence of specific restriction enzyme recognition sites  is located at a distance of approx. 113,000 bp to the HPT-7H locus while the others are approx. 1,184,000 bp (i_SCRI_RS_225155) and 5,111,000 bp (i_SCRI_RS_237466) further apart and hence display a weaker genetic linkage to the HPT-7H  prevailed in the four days preceding sampling in 2017 that affected the plants, to which they apparently reacted by accumulating more tocopherol. The enhanced accumulation of tocopherols in response to drought and elevated temperatures had already been observed in pot-grown barley plants39 and is a well-known response of plants (e.g. reviewed in44).After grouping tocopherol contents of all cultivars based on ded Fig. . In geneded Fig. . In the HPT-7H haplotype we investigated the population structure. Based on 4320 SNP markers spread throughout the genome we inferred the population structure using a model-based approach implemented in the software package STRUCTURE. The calculated model proportionally assigns all genotypes to inferred population clusters based on allele frequencies. After estimating a proper population size , six-rowed genotypes displayed high inferred ancestry from one inferred population cluster (here: cluster 1) that is mostly absent in the two-rowed genotypes. In contrast the two-rowed genotypes display high inferred ancestry from the remaining inferred clusters. Despite the strong linkage of certain clusters to row types, linkage of HPT-7H haplotypes to any cluster seems to be absent in both years  but one allele appears to promote high and the other low foliar tocopherol contents. Therefore, we concluded that differences in transcript levels rather than gene-specific changes in enzyme kinetics are most likely responsible for the observed differences in vitamin E accumulation. We measured the relative transcript quantity of the HPT-7H by RT-qPCR in leaves of those 12 genotypes that we had previously used to identify further HPT-7H alleles. Since we had sequenced the genomic HPT-7H locus in these 12 genotypes completely we can unambiguously distinguish these genotypes by true alleles and not just by CAPS marker-based haplotypes. After grouping the results by allelic composition, we saw that HPT-7H transcript levels were highest in genotypes carrying the Morex allele followed by those bearing the Barke and the Umbrella allele . Additionally, more MYB and MYB-related binding sites can be found in the Morex allele which might explain the even higher responsiveness to drought stress compared to the other alleles (e.g. as reviewed in47). As our sequence of the Umbrella allele lacks large parts of the promotor region and since the Bowman allele contains a large deletion, it is virtually impossible to identify allele specific transcription factor binding sites that affect basal transcription level or HPT-7H inducibility based on two alleles (Morex and Barke) only. Therefore, we refrained from drawing any further conclusions from the detected promoter elements and focused on the analysis of the quantitative data. This revealed a close linkage between HPT-7H transcript amount and flag leaf minus one tocopherol content. Regarding all genotypes, the content of total-, α- as well as γ-tocopherol was directly proportional to the HPT-7H transcript quantity . No apparent linkage to a certain population cluster was found for the HGGT haplotypes . In line with literature48 characteristic amounts of tocopherols and tocotrienols could be detected . Since growing grains are transcriptionally and biosynthetically active, we expected a correlation between transcript levels and tocochromanol content. Parallel to leaf sampling in 2017 we harvested developing grains from plants of ten genotypes grown under well-watered and drought conditions in the RS. Each grain was handled individually. Since natural pollination from ear to ear and between genotypes is not synchronized the developmental stages varied between individual plants and genotypes. We tried to obtain a proxy for developmental age by determining the fresh weight (FW) of each grain. As a single grain could only be used for either RNA or tocochromanol extraction pairs of grains with similar FW and growth position in the ear were used to compare the transcript amount of one grain to the metabolite level of the other  increased exponentially over the growth stages covered Fig. . Puttingend Fig.  but a stend Fig. . Togetheage Fig. . In contage Fig.  whereas age Fig. . Togethe39 and grains38. By locating structural genes encoding pathway enzymes involved in vitamin E biosynthesis in the barley genome assembly . Similarly, an increase of tocochromanol levels in abiotic stress-conditions has also been reported several times for various plant species (e.g. reviewed in50). In contrast, the influence of environmental- or developmental-changes on transcript levels of structural genes of the vitamin E pathway has been studied less frequently. In context of senescence, jasmonate was shown to elevate mRNA levels of an Arabidopsis TAT causing an accumulation of γ-tocopherol51. After applying methyl jasmonate and ethylene to barley leaves or after inducing oxidative damage Falk and colleagues52 observed an increase of barley HPD mRNA levels and Singh and colleagues53 could directly show that the ethylene inducibility of the mango HPD resulted in increased foliar tocopherol levels. GWAS and RNAseq studies were combined in maize grown devoid of stress conditions by Diepenbrock and colleagues20 who concluded that genotype-specific differences in mRNA levels of vitamin E affiliated genes that map to some QTLs determine the tocochromanol composition. Further genotype-specific differences of transcript levels for structural genes of the tocopherol pathway were discovered in tomato introgression lines. By dissecting a previously identified QTL for the tocopherol content of ripe tomato fruits27 Quadrana and colleagues54 identified two epialleles of a VTE3 gene derived from a domesticated (Solanum lycopersicum) and a wild tomato variety (Solanum pennellii) which confered different tocopherol levels by different VTE3 transcript levels. Similar, ten haplotypes of a yTMT where identified in 137 rice accessions37. After defining two phylogenetic groups based on the promotor sequence Wang and colleagues37 showed that enhanced yTMT transcript levels in one group are linked to elevated α-tocopherol contents37. In summary, Quadrana and colleagues54 and Wang and colleagues37 were able to decipher allelic variation of tocopherol biosynthetic genes and link these variations to gene expression. They also established that elevated expression of these genes resulted in elevated tocopherol levels in tomato and rice, respectively. Here we show allelic variation of tocopherol biosynthetic genes in barley and identified alleles resulting in different levels of expression and tocopherol content. Unlike previous studies we were able to demonstrate that increased stress-inducibility of individual HPT-7H alleles is the basis for improved tocopherol accumulation in barley leaves during drought stress under field conditions . In case of barley, Templer and colleagues39 observed a positive correlation between leaf tocopherol content and drought tolerance of pot-grown barley plants. Based on these previous studies we speculated that tocopherol accumulation should enhance stress tolerance of field grown barley plants. To validate this assumption, we analysed a broad range of barley accessions with different basal tocopherol levels in leaves and grains. Unfortunately, we were not able to resolve a clear positive correlation between tocopherol content and plant performance under drought stress. This obvious discrepancy might be explained by short-term and long-term responses of plants to adverse environmental conditions. Vitamin E accumulation might be more beneficial during short-term adaption to rapidly emerging stress as seen in pot-grown plants upon abrupt water withdrawal of previously non-stressed plants39. Munné-Bosch50 already reported that tocopherol accumulation follows a biphasic pattern in which an initial increase can be followed by a drastic decline in tocopherol pool size. The gradual decline of water availability in the silty clay soil in the RS . In our field trails, we reproducibly observed a stress-mediated increase of tocopherol content in barley leaves in two consecutive years and the accumulation of tocopherol always correlated with the severeness of the stress applied  were sampled. From the harvested leaves 1–2 cm from the base and 3–5 cm from the tip were removed prior to deep-freezing in liquid nitrogen and long-term storage at −80 °C. Four sample pools comprising four to six leaves were generated for each genotype and condition. Due to the large sample number (>450) a longer sampling period from late morning to late afternoon could not be avoided. To distribute daytime effects as good as possible replicate one for each genotype grown under well-watered conditions was taken before replicate one for each genotype under drought conditions, followed by replicate two for well-watered conditions for each genotype and replicate two for drought conditions and so on.At the same day leaf samples were taken in 2017 immature grains of ten selected genotypes were harvested. Per genotype and condition one representative ear was chosen that still had grains in the early to late milk stage (BBCH-stage 73–77). Starting from the bottom of the ear each grain was removed and frozen separately leaving the husks in the ear. The fresh weight (FW) of all grains was determined afterwards to find pairs of grains grown at similar position and with similar FW Fig. . Mature 76. Tocochromanols of mature barley grains were extracted as adapted from the methods of76: ten grains were ground using a household MC 23200 coffee grinder  and aliquots (80–100 mgFW) of this fine powder were used for extraction. Successively four times the powder was extracted with 1 ml 100% methanol for 1 h at 4 °C at constant horizontal shaking at 1,500 rpm. To prevent sedimentation of the powder during shaking stainless steel beads were added to the reaction tubes. The supernatants were pooled accordingly, concentrated using a vacuum concentrator and suspended in 250 µl methanol and stored on ice. We combined both methods to extract tocochromanols from immature grains: entire grains were ground individually in liquid nitrogen and extracted three times with the horizontal shaking method with stainless steel beads. The tocochromanols in the final extracts were detected and quantified with a Dionex Ultimate 3000 HPLC system  system as described in76.The tocochromanols of leaf samples (40–80 mgFW) were extracted as described in77 scaled down by a factor of 100. Additionally, the DNA was treated with 0.4 µl RNAse (10 µg/µl) in 500 µl 10:1 TE (Tris-HCl/EDTA) puffer (pH 7.5) for 1 h at 60 °C followed by a precipitation step with 500 µl Chloroform:Isoamyl alcohol  at 8,000 g for 10 min. DNA was then precipitated from the aqueous phase by addition of 50 µl 3 M Sodium acetate (pH 5.2) and 2 volumes 100% ethanol, incubation for 1 h at −20 °C and centrifugation for 20 min at 17,000 g. It was washed twice with 1 ml 70% ethanol, dried and suspended in 50 µl H2O bidest. and stored at −20 °C until further use.For genotyping genomic DNA was extracted from barley leaf tissue using an adapted protocolHPT-7H and HGGT was performed with Phusion® High-Fidelity DNA Polymerase  according to the manufacturer’s guidelines. Primers  were designed by LGC  and supplied as a primer mix which could directly be used in the KASP reaction. 3 µl 2 × KASP PCR mix , 3 µl genomic DNA template (10 ng/µl) and 0.084 µl primer mix were incorporated into the KASP reaction. Amplification was performed in a Light Cycler® 480 instrument II , starting with 15 min at 94 °C, a touchdown phase of 10 cycles at 94 °C for 20 s and at 61 °C for 60 s with a 0.6 °C decrease in temperature per cycle, followed by 26 cycles of 94 °C for 20 s and 55 °C for 60 s. After the initial PCR reactions a recycling step was included with 94 °C for 20 s followed by an annealing/elongation step at 57 °C for 60 s. The fluorescence signals were acquired at 520 nm and 556 nm for 2 min at 25 °C.Allele-specific primers for the SNP markers i_SCRI_RS_225155, i_SCRI_RS_237466, and i_SCRI_RS_18448880 that already had been used in our previous study39. Population structure was investigated independently for each genotype set . To this end 4320 SNPs were chosen that had a minor allele frequency of at least 10% and maximum missing data frequency of 10% in both sets simultaneously. We choose a model based approach implemented in the software package STRUCTURE v2.3.484 to infer population structure. We choose ploidy of data = 2 to take heterozygosity into account, used the admixture model, no prior population information and correlated allele frequencies. As suggested in the documentation of the program when using sets of SNP-data it can be favourable to estimate λ  first by running the program at K = 1 (number of populations) and then fix it for subsequent runs. This was done five times for both sets and λ was fixed at the mean of these runs as following (2016: 3.13606 (±0.01373) and 2017: 2.86704 (±0.02380)). All remaining parameters were kept at the default settings. Then for each K from 1 to 15 the population structure was inferred 20 times. All runs were performed using a burn-in length of 100,000 iterations followed by 500,000 MCMC (Markov Chain Monte Carlo) iterations. Based on the posterior probability of K (ln Pr(X|K)) provided by the program after each run as a parameter called estimated ln prob of data, we choose K = 5 (2016) and K = 6 (2017) as they plateau at these values stable. Additionally, we looked at the ΔK values as suggested by Evanno and colleagues85. Those values display maxima at the chosen K values and certify the choice  was compiled  with an Illumina iSelect 9 K SNP array containing 7864 genome-wide single nucleotide polymorphism (SNP) markers86. Quickly, leaf material or grains were ground in liquid nitrogen cooled mortars and homogenised in 800 µl GITC-containing solution  N-Lauroylsarcosine sodium salt, pH 7.0 (NaOH)). The homogenate was transferred to a 2 ml reaction tube, 1/10 volume (80 µl) 0.2 M sodium acetate (pH 4.0) was added and vortexed briefly to extract DNA. One volume phenol was added, vortexed vigorously and incubated on ice for 30 min. ¼ volume CI was added, vortexed vigorously and incubated on ice for 15 min. Phases were separated by spinning in a benchtop centrifuge at 6,000 g for at least 10 min. The aqueous phase was transferred to a fresh 2 ml reaction tube and organic compounds were extracted with 800 µl PCI ) after a 10 min incubation on ice and subsequent centrifugation at 6,000 g for at least 10 min. This PCI step was repeated once. In the end total RNA was precipitated from the aqueous phase be adding 1/10 volume 1 M acetic acid and 1 volume 100% ethanol. After incubation for at least 15 min at −20 °C RNA was precipitated by full speed centrifugation in a benchtop centrifuge for at least 20 min. The RNA pellet was washed once with 800 µl 3 M NaOAc (pH 6.0) and twice with 800 µl 80% ethanol (v/v) at full speed for at least 20 min each. Residual ethanol was let evaporate and the RNA dissolved in 50 µl DEPC-treated water. DEPC-treated water and the 3 M NaOAc solution were prepared as follows: 0.1% (v/v) Diethyl pyrocarbonate (DEPC) was added to H2O bidest. or 3 M NaOAc, mixed well and kept at room temperature overnight. Afterwards the solutions were autoclaved twice to heat denature the DEPC. DEPC-treated water was also used to dilute 100% to 80% (v/v) ethanol. RNA purity and integrity were verified with a NanoDrop ND-1000  and an Agilent 2100 Bioanalyzer .For expression profiling total RNA was extracted from approximately 50 mg fresh barley leaf tissue or single immature grains using a guanidinium thiocyanate (GITC)-based protocol adapted from the method of Chomczynski and SacchiHPT-7H and HGGT were determined in technical triplicates using an Agilent Technologies G8830A AriaMx Real-Time PCR machine  in combination with Agilent Technologies Brilliant III Ultra-Fast SYBR® Green QPCR Master Mix  according to the manufacturer’s guidelines using the HvActin3 transcript for normalization. Please see Supplementary Table A total of 1 µg leaf or 100 ng grain total RNA were used for cDNA synthesis using RevertAid H Minus Reverse Transcriptase  according to the manufacturer’s guidelines. Relative transcript quantities of Fisher’s exact test was performed using the statistical language R . All remaining statistical analyses were performed with GraphPad Prism 5 software as stated in each figure legend.Supplementary FiguresSupplementary Table S1Supplementary Table S2Supplementary Table S3Supplementary Table S4Supplementary Table S5Supplementary Table S6


These opposing reactions may provide new and interesting applications for CNT wires. Yet, before attempting to move to any real-world harsh environment applications, for the CNT wires, it is essential that this area of their operation be thoroughly examined. To investigate this, CNT wires were treated with multiple combinations of the strongest acids and halogens. The wires were then subjected to conductivity measurements, current carrying capacity tests, as well as Raman, microscopy and thermogravimetric analysis to enable the identification of both the limits of oxidative conductivity boosting and the onset of physical damage to the wires. These experiments have led to two main conclusions. Firstly, that CNT wires may operate effectively in harsh oxidizing environments where metal wires would easily fail and secondly, that the highest conductivity increase of the CNT wires can be achieved through a process of annealing, acetone and HCl purification followed by either H Their yarn-like, tubular, highly anisotropic and nanostructured, carbon-based morphology makes them intrinsically different to any existing metallic conductors, this potentially paving the way for significant advancements in the performance of existing wiring systems, as well as completely new applications, such as super lightweight electrical machines3, smart textiles4 or high performance coaxial cables5. However one of the quite unusual aspects of CNT wires is their response to oxidants9.Carbon nanotube (CNT) fibre conductors are a very interesting new class of materials3 + 3HCl) or chlorine.It is well understood that both copper and aluminum conductors are very sensitive to oxidation. Low levels of oxidation cause the formation of copper/aluminum oxides that are resistive and this oxidization decreases the cross-sectional area of the wires that is available for current transport. Fortunately, passivation of the wires tends to hinder their further oxidation, unless strong oxidants are present in large amounts. For example, dipping both copper and aluminum wires in strong acids causes them to dissolve with a rate dependent on the type, concentration, strength and redox potential of the acid. In order to mitigate these effects more oxidation resistant metals such as gold are used, for example, as electronic bonding wires. But even this metal reacts with aqua regia  method described previously3H6O), toluene (C7H8), chloroform (CHCl3), n-hexane (C6H14), methanol (CH3OH), hydrogen peroxide (H2O2 (60%)), acetic acid (CH3COOH), formic acid (HCOOH), trichloroacetic acid (CCl3COOH), hydrochloric acid (HCl (35–38%)), sulfuric acid (H2SO4 (98%)), nitric acid (HNO3 (65%)), perchloric acid (HClO4 (70%)), chlorosulfonic acid (HSO3Cl (97%)), 20% oleum, I2, Cl2 (obtained via the reaction of KMnO4 with HClaq), ICl, and Br2.The following list of chemical compounds were used in the purifying and doping treatments: acetone  Sigma-Aldrich , Acros Organics (HSO3Cl), ichemia.pl (I2) and REKO PR-W (Br2). .All acid treatments were conducted by soaking the samples for 24 hours. ICl and BrThe samples were annealed in air using a laboratory muffle furnace Czylok FCF 7 SHM and the samples were sonicated in acetone using Utrasonix Proclean 2.0 M sonication bath.Measurements of the CNT wire diameter were made on a digital optical microscope Keyence VHX-900 F. Weighing was performed on Radwag Ultra-Microbalance UYA 2.4Y. The electrical resistance measurements were conducted using the “two-point method” using a True RMS multimeter UNI-T UT804. Connections between electrodes and CNT wires were facilitated by the use of a silver conductive paint.The current carrying capacity measurements were performed with a setup specifically designed for this activity comprising a DC power supply TTi QL564P, and a DC Keithley 2000 multimeter, all controlled via proprietary software written in LabVIEW.2 - 18 ml/min), from room temperature up to 800 °C.Thermal analysis  was performed on an STA 449 F1 Jupiter (NETZSCH) apparatus. A standard platinum crucible was loaded with 1.4–2 mg pieces of a carbon-nanotube fabric with these flattened to improve the heat transfer between the sample and the crucible. The measurement was conducted with a heating rate of 5 K/min, under a flow of “synthetic air”  with numerical aperture (NA) equal to 0.5, this leading to a laser beam diameter of approximately 2 μm. Motorized stages were used to perform Raman surface mapping measurements with an area of 40 × 40 µm. Renishaw WiRE 3.4 software was used for deconvolution of the obtained spectra, by the curve fitting method using a mix of Lorentz and Gaussian functions.The Raman scattering spectra of samples were investigated in the spectral range of 100 cmThe CNT specimens were subjected to Scanning Electron Microscope (SEM) investigations with low energy (less than 5 keV) electrons using the Auriga CrossBeam Workstation (Carl Zeiss) equipped with an In-lens SE (true SE1) detector and an Energy selective Backscattered electron  detector, both positioned on the optical axis of the Gemini (TM) column. In front of the entry system on the axis of the BSE detector there is an energy filtering grid integrated into the electron optical detection system that can be adjusted in its retarding potential from 0 to 1500 volts. This grid allows only BSEs with energies greater than the grid energy to be detected. The energy of the primary electrons was adjusted to effectively reveal all the features of the studied samples in terms of the impurities present within the CNT bundles.a = 1.76 × 10−5), formic acid (Ka = 1.77 × 10−4), trichloroacetic acid (Ka = 0.16), hydrochloric acid (Ka = 1.3 × 106) and perchloric acid (Ka > 1010)  Fig. .Figure 1After immersion in these acids, the samples – on a macroscopic scale – did not show any visual structural changes. However, as shown in Fig. 19. All of these factors can influence the doping effectiveness  and behavior of CNT wires. Hence, previous research on CNT wire doping often included purification steps, in various configurations26. The most popular treatments included annealing which was reported to cause the removal of amorphous carbon21. Also soaking and sonication in organic solvents, has been reported to remove organic contaminants27. While catalyst nanoparticles were supposed to be disposed of, by mineral acids treatment.Currently produced CNT fibres/films can contain CVD production derived side-products such as amorphous carbon, hydrocarbons and their derivatives or catalyst particles. Additionally, there is always the small influence of air dopants which may be correlated with the presence of the above impurities22. There are also reports involving soaking in concentrated hydrogen peroxide as a way to enable the cracking of amorphous carbon and the oxidative dissolution of catalysts particles from the material15.In the case of iron-based nanoparticles present in our material , hydrochloric acid could be indicated as a first choice chemical solventWith the aforementioned purification methods considered, a set of tests starting from annealing in air at a range of temperatures was performed.1, was 20%. At a temperature 250 °C, a decrease of conductivity was observed . And annealing at 500 °C led to a structural disintegration of the wires.For this purification step, the greatest increase in both absolute and specific conductivity was recorded for an annealing at 400 °C. Here, the percentage change in absolute conductivity (Δσ) was equal to 18%, while the percentage change in specific conductivity (Δσ*), a measurement which takes into account the weight of the materialThe results indicated that a 400 °C annealing may indeed quantitatively remove amorphous carbon, paving the way for better electron transport in the fibre, while a lower temperature treatment only removes some air dopants.Following the annealing, further tests involved organic solvent treatments.28  (48 hours at room temperature) and concentrated HCl(aq) (24 hours at room temperature). It should be noted that the HCl treatment was followed by deionized water rinsing (30 sec long immersion repeated 3 times).Following the purification procedure proposed by Zhao 2O2) resulted in an approximately 117% and 133% increase in absolute and specific conductivity, respectively. This indicates that the conductivity changes observed may be not only due to catalyst removal, but also their doping action.This procedure resulted in an approximately 87% and 95% increase in absolute and specific conductivity, respectively. Interestingly a change in the sequence of these two treatments  was increased by approximately 30% only. Both these results lead to the conclusion that the mechanism responsible for the conductivity increase is the increase in the amount of adsorbed acid due to opening/swelling of the structure.It can be seen in Fig. 2O2 for 48 hours.Based on this optimization of the absolute and specific conductivity, the purification procedure was recorded and defined as: 1) annealing at 400 °C for one hour, 2) 10 min sonication in acetone with a subsequent soaking for a minimum of 24 hours and 3) soaking in concentrated HCl for 24 hours. Samples doped with acids were also pre-treated by soaking in 60% H2SO4, HNO3, HClO4, HSO3Cl, SO3, aqua regia (HNO3 + 3HCl) and HCl, which is a selection of common, strong oxidizing acids and a strong non-oxidizing mineral acid for comparison. Figure The above pre-treatment procedure was used to condition all of the samples used for further acid doping tests. As mentioned above, only strong acids were used for these experiments and these included H30.However, there is also a physical limit to these doping procedures. Since sulfuric acid and its compositions (oleum) or derivatives (chlorosulfonic acid) are hygroscopic, , hence fibres treated with those reagents disintegrate 4, which are both strong and non-oxidizing acids in water (at room temperature). This result may be explained by the fact that these strong acids dope by a different chemical route, yet they do not damage the fibres physically. Indeed, in acidic aqueous solutions ClO4− oxidizes Cl− to chlorine (Cl2) since hydronium-doping is enhanced by doping with chlorine (HClO4 + 7HCl = 4Cl2 + 4H2O).With a view to finding the further limits of the physical endurance of the samples and checking the possibility of further conductivity boosting, the wires were treated with various combinations of two acids one after the other. Figure 2, Br2, I2 and ICl. The pre-treatment did not involve the H2O2 step as the pre-tests showed that soaking in hydrogen peroxide had no influence on the effectiveness of halogenation. As shown in Fig. 2 had the most pronounced effect on the conductivity. This effect could be ascribed to the most time-stable adsorption of bromine on the CNT outer walls and its lower viscosity than for ICl, enabling more efficient penetration of CNT fibres. The moisture-assisted reaction of bromine with water (Br2 + H2O = 2HBr + [O]) could also yield an additional mechanism of doping based on protonation by strong acid HBr and oxidation by in situ formed reactive oxygen. Importantly, none of the halogens caused any visible damage to the wires.Another set of strong p-dopants includes halogens and interhalogen compounds. To compare the effect of these oxidizers, the purified CNT fibres were treated with vapors or infiltrated with Cl4 treatment defined above and the results examined. Tests showed that although this combination is not harmful to the structural integrity of the fibres it is also not beneficial to the conductivity , σ’ = 8,76∙102 S∙m2/kg (Δσ’ = 295%); and for Br2 treatment: σ = 7,02∙105 S/m (Δσ = 631%), σ’ = 6,51∙102 S∙m2/kg (Δσ’ = 158%).The values obtained for the samples amounted to: for HClO33. The only paper currently reporting higher values, is that mentioned earlier by Zhao et al.15 in which the authors claim that their doped fibres have higher specific electrical conductivity than copper and aluminium. However, these results were questioned by Behabtu et al. 17 Our tests, based on the procedures of Zhou et al. are also not producing the results predicted in their work. Therefore, there is certainly room for further discussion/research in this area.These values are higher than for most CVD spun doped fibres2O2 treatment have visibly larger spaces between the CNT bundles at the microscale  or H2SO4-5b).More insight into the changes taking place in the material during the purification and doping procedures was obtained from the thermogravimetric analysis (TGA). This was performed on the samples as they underwent each step of purification and doping, including: 1) samples annealed at 400 °C, 2) samples annealed and treated with acetone, 3) samples annealed, treated with acetone and soaked in HCl, 4) samples annealed, treated with acetone, soaked in HCl and HExamples of the TGA curves are presented in Fig. 27), which is a less graphitized carbon phase than carbon nanotubes, thus having increased reactivity towards oxygen. The weight loss at temperatures between 350 °C and 500 °C is expected to be caused by the removal of hydroxyl functionalities34. (Oxidation of well-graphitized structures takes place at temperatures over 500 °C).It is clearly visible that the annealed sample, from the outset shows a small steady decrease in mass, which can be related to the air adsorbates (including molecular water) released from the structure. The further decrease in weight may be related to removal of amorphous carbon . Similar calculations for annealed and acetone and HCl purified sample, give 6.2% and 4.34% for Fe2O3 and Fe residue respectively.The temperature of the elimination of catalyst particles is not straightforward to determine specifically. Both the amounts and microstructures of metal particles could affect the position of the peaks. However, for the undoped structures, the dry matter residue can mostly be associated with the remaining iron oxide catalyst. By analyzing successive TGA graphs, it can be seen that after treating the CNT fibers with HCl the dry matter residue is reduced, which may suggest that the elimination of the catalyst nanoparticles occurred. Comparing the sample after annealing and after HCl treatment, it can be seen that for the annealed sample there is a mass change of 1.4% at temperature ~200 °C, then at 600 °C a mass change of 33.5%, and 55.6% above 600 °C. This gives a total mass change of 90.5%. Thus, the catalyst residue as Fe7. It is also worth noting that the weight drop recorded below ~500 °C was higher for H2SO4 than for the HClO4 treatment . For the sample doped with perchloric acid the peaks appeared at lower temperatures than for sulfuric acid, which suggest that the CNT wires are well loaded by both acids, and their boiling point (HClO4 = 203 °C and H2SO4 = 338 °C) influences the decomposition of the material.This comparison indicates that the samples treated with HClOAll the above presented results indicate that the strongly oxidized CNT wires are more sensitive to temperature than their pure/annealed counterparts.In order to obtain more information on the doped state of CNT wires, a set of Raman spectroscopy measurements were performed, these being a recognised technique for the investigation of the structure and doping state of CNTs.One of the most important features of CNTs in Raman spectra are: RBM  however this is only visible for single wall carbon nanotubes (SWNT) at a sufficiently high concentration. The CNT fibres, tested here, were composed of single-wall, double-wall and multi-wall CNTs in varying concentrations, therefore RBMs were not observable.−1) which is the result of the double-resonance Raman effect in sp2-carbon and corresponds to the presence of amorphous or disordered carbon in the CNT structure35. It has been reported that p-doped CNTs respond with an up shift of the D-band36. However, the acid treatment can also result in an increase of the amount of CNT structural defects and therefore the relative intensity of the D peak37.Another important feature is D-band (~1350 cm−1). The G-band signature comes from the stretching of carbon-carbon bonds within the graphene plane7. Analysis of the G-band may be used to distinguish metallic from semiconducting nanotubes or to examine the electron flow involved with the doping37. The G-band is composed of two main components. G+ (~1590 cm−1) and G− (~1570 cm−1), where the G+ and G− features are related to carbon atom vibrations along and across the nanotube axis respectively. The charge transfer induced by the presence of dopants in the CNT material results in the frequency offset of component G+. The up-shifted G+ frequency indicates the presence of acceptor dopant in the material38.Another feature, important for CNT material characterization is the G-band (~1580 cm−1), it is the overtone to the D-feature, not related to the disorder39.The last feature to be mentioned is the 2D ; (6) fibres purified, treated with H2O2 and doped with 6a- HClO4 or 6b- H2SO4 as well as 6c- purified fibre treated with Br2.A 488 nm laser wavelength was used for the Raman spectra acquisition and the following steps of doping procedure were measured: (1) CNT fibres as made; (2) CNT fibres annealed at 400 °C; (3) CNT fibres annealed and treated with acetone; (4) CNT fibres annealed, treated with acetone and soaked in HCl; (5) fibres annealed, treated with acetone, soaked in HCl and HConsidering the results shown in Fig. The next steps involved a medium increase of all 3 bands positions, which corresponds with the inferred earlier mild doping of CNT fibres with the chemical compounds used for purification.2SO4 treatment does not cause any shift in the 2D-band position  but for HClO4 there is clear up-shift in the 2D-position. Moreover, the H2SO4 treatment induces a visible disturbance of the typical Raman spectrum for CNT , which is a very important parameter when considering electrical wires. In this study, a number of tests had been conducted to verify how doping affects the CCC. The current was applied in steps, with a predetermined increment and duration. For comparison of different samples in the same plot, the dependence of the current on time is presented Fig. .Figure 62SO4 appeared to be visibly the worst. The best results were obtained for Br2 and HClO4 .4 must have only partly evaporated as the final CCC decreased by 25% as compared to small increment measurements. (For bromine doping there was no clear difference in the results compared to the short measurement suggesting its superior performance over the other dopants).These “longer time measurement” results showed that the HClOThis research and its associated results allow us to form a clearer picture of both the opportunities and limitations of CNTs fibres operating in harsh oxidizing environments.The research has facilitated the identification of the most effective purification and pre-treatment methods, which are often used before doping procedures. Interestingly these have been found to also lead to an increase in the effectiveness of further doping. Moreover, measurements have shown that in using these methods the wires can increase in both absolute and specific conductivities by up to 100%.Further, the tests included doping with a wide range of acids including HCl and oxidizing acids of varying strengths with particular attention paid to the strongest ones as well as with halogens. The acids and halogens were applied in various configurations including multi-compound treatments.The absolute and specific conductivity measurements were supported by CCC testing, TGA and SEM analyses as well as Raman spectroscopy. These tests showed that the most beneficial doping procedures involved annealing at 400 °C (which partly removes the amorphous carbon), short sonication and soaking in acetone , soaking in HCl  and using pre-doped fibres.2 doping or H2O2 soaking (which swelled the fibres to enable better acid penetration) and rarely reported HClO4 acid treatment.The above procedures were followed by either BrThese treatments resulted in a very significant doping of the CNT wires, this observed in Raman spectra, and also negligible damage to the structure of the CNT wires at room temperature. In all these produced an increase of approximately 700% in absolute conductivity, an increase of 150% in specific conductivity, and a 341% improvement in current carrying capacity.2SO4 with HClO4 doped fibres, showed a higher degree of damage to the fibres and a greater sensitivity to temperature related damage caused by H2SO4 treatment.However, it has been shown that the action of some of the strongest, hygroscopic and water-reactive acids resulted in the destruction of thinner fibres. Also, treatment by two acids resulted in an increased share of damaged fibres. Comparison of the Raman and TGA features as well as current carrying capacity of sulfur containing acid HThis research and the tests performed led to the formulation of the “operational window of the oxidation of CNT wires”. This identifies the factors limiting the performance of the wires in strongly oxidizing environments. These covering temperature, the redox potential of the doping agent and the value of current flowing through the wire Fig. .Figure 7Despite the fact that there are identified limits to the performance of CNT wires in harsh oxidation environments, these materials exhibit not only a much higher resistance to such conditions, compared to conventional metals used in electrical wiring, but, against intuition, they may also benefit greatly from their operation in such environments.This is both an intriguing and a practical fact that should not be neglected when considering the future design of industrial implementations requiring electrical wiring to function successfully, in harsh oxidizing environments.