Background: Parenteral nutrition is central to the care of very immature infants. Current international recommendations favor higher amino acid intakes and fish oil–containing lipid emulsions.Objective: The aim of this trial was to compare 1) the effects of high [immediate recommended daily intake (Imm-RDI)] and low [incremental introduction of amino acids (Inc-AAs)] parenteral amino acid delivery within 24 h of birth on body composition and 2) the effect of a multicomponent lipid emulsion containing 30% soybean oil, 30% medium-chain triglycerides, 25% olive oil, and 15% fish oil (SMOF) with that of soybean oil (SO)-based lipid emulsion on intrahepatocellular lipid (IHCL) content.Design: We conducted a 2-by-2 factorial, double-blind, multicenter randomized controlled trial.Results: We randomly assigned 168 infants born at <31 wk of gestation. We evaluated outcomes at term in 133 infants. There were no significant differences between Imm-RDI and Inc-AA groups for nonadipose mass [adjusted mean difference: 1.0 g ] or between SMOF and SO groups for IHCL . There were no significant differences in any prespecified secondary outcomes, including adiposity, liver function tests, incidence of conjugated hyperbilirubinemia, weight, length, mortality, and brain volumes.Conclusion: Imm-RDI of parenteral amino acids does not benefit body composition or growth to term and may be harmful. This trial was registered at www.isrctn.com as ISRCTN29665319 and at eudract.ema.europa.eu as EudraCT 2009-016731-34. Parenteral nutrition (PN)A diet with a low protein:energy ratio results in lower lean body mass and greater adiposity . Thus, iWe designed a clinical trial  to test the hypotheses that the immediate delivery of RDI (Imm-RDI) of parenteral amino acids compared with incremental provision is more efficacious in increasing lean (nonadipose) body mass at term, and that a mechanism of action of 20% SMOF compared with 20% soybean-based lipid emulsion (SO) is to reduce intrahepatocellular lipid (IHCL).We conducted NEON , a 2-by-2 factorial, double-blind, multicenter randomized controlled trial in 4 National Health Service neonatal units in London and southeast England. The trial was preregistered and approved by the National Research Ethics Service and Medicines and Healthcare Products Regulatory Agency. The trial sponsor was Imperial College London. Recruitment commenced in July 2010 and ended in July 2013, with a final follow-up in October 2013.Preterm infants born at <31 wk of gestation were eligible for inclusion. Infants with life-threatening abnormalities and those for whom we were unable to administer trial PN within 24 h of birth were ineligible. When possible, the trial was discussed with parents antenatally, and written informed consent was sought within 24 h of birth. The interventions were 20% SMOFLipid (Fresenius Kabi) and Imm-RDI of amino acids. The comparators were 20% Intralipid (Fresenius Kabi) and incremental delivery of amino acids (Inc-AA). The amino acid source was Vaminolact (Fresenius Kabi). Trial formulations were investigational medicinal products prepared by a licensed facility (Bath ASU). Other PN components were identical across randomized groups.We randomly assigned eligible infants with the use of an interactive voice recognition telephone system to 1 of 4 groups  and incorporated minimization with a random element and stratification by gestational age (23–26 or 27–31 completed weeks), birth weight , and center. Hospital pharmacy staff dispensed trial PN between 0900 and 1700; attending clinicians were blinded to trial allocation. For consistency of capturing data, we defined day 1 as the period from birth to 1700. Subsequent days of capturing data were recorded from 1700 to 1700. The duration of day 1 was therefore variable and dependent on time of birth.−1 · d−1 from day 3; Imm-RDI infants received 3.6 g · kg−1 · d−1 from day 1. PN was provided in an aqueous volume of 90 mL · kg−1 · d−1 on days 1 and 2 and 120 mL · kg−1 · d−1 from day 3. Carbohydrate intake was 8.6 g · kg−1 · d−1 from day 1. An intravenous lipid was provided at 2 g · kg−1 · d−1 on day 1 and was increased to 3 g · kg−1 · d−1 from day 2. Weaning of trial PN was commenced once an infant received milk volumes >60 mL · kg−1 · d−1.PN and milk intake by a nasogastric tube commenced within 24 h of birth and was guided by prespecified protocols. The investigator manual provided instructions on managing electrolyte, glucose, and lipid disturbances. Inc-AA infants received 1.7 g/kg amino acids on day 1, 2.1 g/kg on day 2, and a maximum of 2.7 g · kg−1 · d−1 for ≥24 h. Any subsequent PN requirement was in accordance with local practice. Nutritional intake was recorded prospectively on a daily basis from birth until discharge or trial endpoint.Trial PN ceased when an infant had received and tolerated 150 mL milk · kgPrimary outcomes were nonadipose mass for the amino acid intervention and IHCL for the lipid intervention. Secondary outcomes were total adiposity, adipose tissue depots, insulin sensitivity (quantitative insulin sensitivity check index) , total a1H MR spectroscopy at 1.5T during natural sleep without sedation. We obtained serial axial images (5-mm slice and interslice thickness) to quantify total adipose tissue volume as the sum of 6 discrete depots  as previously described (3) × 0.9]]. Image analysis with the use of Slice-O-Matic  was undertaken independently by the Vardis Group and was blinded to participant identity and group allocation. We acquired MR spectra from the right lobe of the liver with the use of a point-resolved sequence  without water saturation and with 128 signal averages. Spectra were analyzed with the use of the advanced method for accurate, robust, and efficient spectral fitting algorithm in the jMRUI software package  by a single investigator who was blinded to allocation  to detect a difference in a mean IHCL of 0.53 on the logarithmic scale. Back-transforming to the original scale of measurement, this is equivalent to a 40% decrease in IHCL in the intervention group. We assumed there would be no interaction between the interventions. Allowing for 10% mortality and ≤10% dropout , we aimed to recruit 160 infants or until 64 infants in each pairwise group had undergone MRI and MRS, a total of 128 scans.We based the sample size on our estimate that 64 infants in each pairwise group (Imm-RDI compared with Inc-AA) would provide 80% power  to detect a 200-g difference in nonadipose mass assuming an SD of 400 g. This represents half the difference in the nonadipose mass we identified between very preterm and term infants in a prior experimental cohort . We haveWe used a modified intention-to-treat analysis because we anticipated we would be unable to obtain primary outcome measures in all infants. For the amino acid and lipid interventions, we used multiple regression with nonadipose mass (grams) or IHCL  as the dependent variables and the amino acid group (Inc-AA or Imm-RDI), lipid group (SO or SMOF), stratifying variables , sex, and age at assessment as the independent variables. We added an interaction term to assess whether the effect of amino acid regimen is influenced by lipid type. In a planned secondary analysis, we incorporated measures of illness severity and nutritional intake in the regression models to investigate their role as potential effect modifiers. We performed all analyses with the use of Stata version 13 (StataCorp LP).Supplemental Table 1). For ease of comparison between enteral and parenteral intakes, we expressed parenteral amino acid intake as protein (1 g amino acids = 0.89 g protein). Trial PN protein intake was higher in the Imm-RDI arms in the first 2 wk  , demonstrating that target intakes were reached; carbohydrate, lipid, and energy intakes were similar across the 4 groups  . Macronutrient and energy intake from milk and PN from the end of the second week onward did not differ between groups (The median (IQR) number of days to achieve a milk intake of 150 mL · kgP = 0.98] or between the SMOF and SO groups in IHCL  (P = 0.05) between the 2 interventions for nonadipose mass . There were no significant interactions between group differences for either primary outcome measure after adjustment for additional confounders ; P = 0.02] . Additional nutritional information, sepsis incidence, and length of hospital stay are shown in Supplemental Tables 7 and 8.Head circumference at term was smaller in the Imm-RDI group than in the Inc-AA groups  . There w = 0.02] . Weight P < 0.01) and with concentrations >10 mmol/L  . There were no significant differences in the proportion of infants with any other abnormal biochemical indexes, including incidence of conjugated hyperbilirubinemia . Serious adverse events are summarized in Supplemental Table 11.There were significantly more infants in Imm-RDI groups with blood urea nitrogen concentrations >7 mmol/L  were broadly equal across groups, making the trial results applicable to the most immature infants. NEON was adequately powered because the CIs for the mean differences in nonadipose mass and the SMOF:SO IHCL ratio, respectively, excluded the prespecified difference of 200 g and decrease of 40%. Because we know of no biological reason for lipid type to influence the quantity of nonadipose tissue, we consider it likely that the between-intervention interaction we detected resulted from chance.We acknowledge limitations. Our study was carried out in 4 neonatal units, with primary outcome assessments at the lead university hospital. We considered it unethical to transfer an infant between hospitals for research purposes so we were unable to obtain primary outcome measures for infants recruited in nonlead centers who remained inpatients at term. However, primary outcome measurements were available for 133 infants because the trial design allowed for recruitment to continue until 128  measurements were available. Recruitment during the weekend was not feasible in all centers because of the lack of availability of pharmacy staff trained in clinical trial procedures; thus, a number of eligible infants were unable to participate. We were able to assess brain volumes in only one-third of participants because we carried out MRI investigations without sedation , obtainiPN is a high-cost, widely used neonatal intensive care intervention, yet to our knowledge there have been few previous randomized controlled trials and none that have evaluated the effects on body composition . We achi−1 · d−1  because this was considered a standard of care in the United Kingdom at the time. However, total protein intakes were higher because milk feeds were commenced within 24 h of birth. Early commencement of milk feeds together with PN, as delivered in NEON, counter the assumption that very preterm infants inevitably accrue substantial postnatal deficits and hence require protein intakes >4.5 g · kg−1 · d−1 (More than a decade ago, we found lean body mass at term to be almost 500 g lower in very preterm than in healthy term infants , which i−1 · d−1 . We used−1 · d−1  and are −1 · d−1  and youn−1 · d−1 . We also−1 · d−1 , althoug−1 · d−1 compared with a group that was randomly assigned to receive a lower intake had a lower mean mental development index at 18 mo and lower mean z scores for weight, length, and head circumference. We reassuringly identified no between-group differences in brain volume in NEON.We noted a smaller head circumference at term in infants receiving the higher amino acid intake. This observation is at odds with the Standardised, Concentrated Additional Macronutrients, Parenteral Nutrition in very preterm infants study. in which very preterm neonates randomly assigned to receive higher PN from birth had a larger head circumference at 28 d . Of note−1 · d−1 together with introducing milk feeds within 24 h of birth does not seem to be detrimental to body composition and may also be safer compared with the immediate provision of an amino acid intake of 3.6 g · kg−1 · d−1. We also conclude that SMOF does not reduce intrahepatic lipid accumulation. We found that a standardized PN regimen was well accepted by clinicians and well tolerated by infants. We intend to evaluate growth and cognitive, developmental, and other functional outcomes in childhood. Optimal amino acid intakes and intravenous lipid formulations for extremely preterm infants remain to be established.We conclude that commencing an Inc-AA regimen that provides a maximum of 2.7 g · kg


The most striking finding is the transformation of the AFM phase into a new reentrant-type magnetic glassy phase below Tg ≅ 10 K. We show that the behaviour at this transformation contrasts the typical behaviour of canonical spin glasses and is similar to the behaviour of relaxor ferroelectrics. Magnetoelectric effect is also observed in the AFM phase in the temperature range of the transition into electric relaxor phase at Tf ≅ 200. The mechanism of magnetic relaxor behaviour is supposed to arise from the frustrated interactions among the spins located at the AFM domain walls. Our results should inspire further studies of multirelaxor behaviour in other multiferroic systems.Significant quenched disorder in crystal structure can break ferroic (magnetic or electric) long-range order, resulting in the development of ferroic glassy states at low temperatures such as magnetic spin glasses, electric dipolar glasses, relaxor ferroelectrics, etc. These states have been widely studied due to novel physical phenomena they reveal. Much less known are the effects of quenched disorder in multiferroics, i.e. the materials where magnetic and electric correlations coexist. Here we report an unusual behaviour in complex perovskite Pb(Fe Multiferroic materials which exhibit both magnetic and ferroelectric orders and effects of coupling between magnetization and electric polarization have attracted much interest in the last decade and are considered to be extremely promising for a number of advanced applications such as spintronics and high-density data storage124Relaxor ferroelectrics are the crystalline materials whose structure is characterised by the presence of quenched (static) chemical disorder in the arrangement of different atoms on the equivalent crystallographic positions. Similar to normal ferroelectrics, the polar order appears in relaxors upon cooling at a particular temperature, but contrary to normal ferroelectrics, this order is local or short-ranged  and the dipolar arrangement inside the polar nanoregions is still a subject of intense debates. The dipolar transformation in relaxors takes place over a wide temperature range  in which unusual properties  are observed.0.98Mn0.02TiO3, frustrated dipolar and magnetic superexchange interactions give rise to both dipolar glass and spin glass states3-BaTiO3, where magnetic nanodomains were thought to be induced by electric polar nanoregions and to overlap with them in space1/2Nb1/2)O3-Pb(Mg1/2W1/2)O3 ceramics0.5Nb0.5O3Magnetoelectric correlations in disordered glassy ferroics have been poorly understood so far, mainly because only in a few of them the ferroelectric and ferromagnetic (FM) properties have actually been observed simultaneously. In perovskite solid solutions of Sr2/3W1/3)O3 (PFW) crystals in which alongside the electric relaxor phase below Tf ≅ 200 K and the AFM phase with a Neel temperature, TN = 350 K, we have found a new reentrant-type magnetic relaxor phase below Tg ≅ 10 K. The structure of PFW determined by neutron and x-ray diffraction methods is cubic at all temperatures with the space group Pm-3m, implying a fully disordered distribution of the Fe3+ and W6+ cations on the perovskite B-sites103+-O-Fe3+ superexchange interactions was also foundTN = (340–360) K was estimated from the anomaly in the temperature dependence of magnetic susceptibility10In the present work we study the complex perovskite Pb peak is observed. The temperature of the maximum susceptibility (Tm) varies with frequency, which excludes the possibility of FM or AFM transition as the origin of the χm(T) peak: dispersion in conventional magnets can be observed only at much higher frequencies (mega to giga Hz). Therefore, a spin glass-type nature of the low-temperature state of PFW has to be considered. Since this glassy state is observed in the temperature range below the ordered AFM phase, it points to a reentrant behaviour. However, we cannot conclude if the AFM order is destroyed and thereby a pure glassy phase is formed, or the glassy and AFM orders coexist at low temperatures  shift of Tm is known to follow the Vogel-Fulcher (VF) law:In electric relaxors5χ(T) peak is described by a Lorentzian-type function:and the shape of the high-temperature slope of the χA and TA (<Tm) are the magnitude and the (maximum) temperature of the Lorentzian peak, respectively, δ is the half-width of the peak at 2/3 of the maximum, and Em and TVF are the parameters. If the high-temperature slope of the peak is frequency independent , the measured χ represents the static value of susceptibility. It was shown16TA, but also at and below TA, where it cannot be directly measured.In the above equations χm in spin glassesχm(T) peak which is observed also in low-field ac measurements19Tm20Tm and 2Tm192324The VF law (1) is known to be also valid for Tm( f ) dependence in PFW can be reasonably well fitted to the VF law  crystallographic direction was determined with a SQUID magnetometer (Quantum Design MPMS XL). Before each measurement the sample was heated up to 400 K without magnetic field. Magnetic moment was measured upon cooling with a field of H = 200 Oe (in FC experiments) or the sample was cooled down to 2 K without the field, and afterwards the moment was measured upon heating with H = 100 Oe (in ZFC experiments). The isothermal M(H) dependences were measured at different temperatures after cooling in zero field from 400 K. The ac magnetic susceptibility (χm) was measured under the ZFC mode with H = 5 Oe. Electric ac susceptibility (χe) was studied using a Novocontrol Alpha broadband dielectric spectrometer. The measured dependences were fitted to empirical formulae using nonlinear least squares method.The PFW crystals which we studied were grown from high temperature solutionHow to cite this article: Chen, L. et al. Magnetoelectric relaxor and reentrant behaviours in multiferroic Pb(Fe2/3W1/3)O3 crystal. Sci. Rep.6, 22327; doi: 10.1038/srep22327 (2016).


Single crystals of the small-pore MOF, Sc2(BDC-NH2)3, , with well-defined crystal form have been investigated during CO2 uptake at partial pressures of 0.025-0.2 bar at 298–373 K. The enthalpy and diffusivity of adsorption determined from individual single crystals are consistent with values obtained from measurements on bulk samples. The brilliant SR IR source permits rapid collection of polarized spectra. Strong variations in absorbance of the symmetric stretch of the NH2 groups of the MOF and the asymmetric stretch of the adsorbed CO2 at different orientations of the crystals relative to the polarized IR light show that CO2 molecules align along channels in the MOF.Synchrotron radiation (SR) IR microspectroscopy has enabled determination of the thermodynamics, kinetics, and molecular orientation of CO The net CPh-N vector is close to parallel to the channel axis in the MOF . The intensity of the asymmetric stretch of the linear O=C=O molecule is enhanced relative to the NH2 symmetric stretch using IR light polarized along the crystal length when viewed down [001] (by ca. 90 %) and [011] (by 23 %) but is suppressed when viewed down [010] (by 17 %). The projection of the CO2 molecule is therefore more closely aligned to the channel axis than 17° when viewed down [001] and makes a larger angle than 11° when viewed down [010], in effect enabling triangulation of the relative orientation of the CO2 molecules’ orientation in the channel, which is on average closer than approximately 20° to the channel axis.A more quantitative estimation of the COd Figure . Intensi2 molecules within the pores of Sc2(BDC-NH2)3, even at rather low uptakes, a result which is experimentally difficult to attain by other means. Whereas previous diffraction measurements have located adsorbed CO2 on MOFs at room temperature at loadings down to approximately 1 mmol g−1,[2 at <0.1 mmol g−1 . Furthermore, the method has the potential to follow temporal and spatial variation over a single crystal and so to determine adsorbate diffusivities. Although the diffusion of CO2 in Sc2(BDC-NH2)3 is too fast to permit this, there are many examples of more slowly diffusing, IR-active molecules  in other porous solids than can be studied in this way, even when part of adsorbing mixtures, especially when developments in full field FTIR imaging using synchrotron radiation IR brilliance can offer faster acquisition and a larger field of view (see the Supporting Information).[A powerful new SR FTIR microspectroscopic technique has been developed which permits the rapid analysis of adsorption by porous materials under flowing gas conditions relevant to carbon capture. Investigations on an amino-functionalized MOF give a heat of adsorption and desorption rates on a single crystal and polarized IR light has been used to show a strong orientation dependency of adsorbed COmmol g−1, these IRrmation).The results reported here confirm the potential of amino-functionalized MOFs as carbon capture materials and indicate that in situ single-crystal gas adsorption and polarized IR microspectroscopy can rapidly provide unique insights into single- and eventually multi-component adsorption processes.


We tested the effects of projected future CO2 levels (626 and 956 µatm) on the behaviour of male two-toned pygmy squid, Idiosepius pygmaeus. Exposure to elevated CO2 increased the number of active individuals by 19–25% and increased movement (number of line-crosses) by nearly 3 times compared to squid at present-day CO2. Squid vigilance and defensive behaviours were also altered by elevated CO2 with >80% of individuals choosing jet escape responses over defensive arm postures in response to a visual startle stimulus, compared with 50% choosing jet escape responses at control CO2. In addition, more escape responses were chosen over threat behaviours in body pattern displays at elevated CO2 and individuals were more than twice as likely to use ink as a defence strategy at 956 µatm CO2, compared with controls. Increased activity could lead to adverse effects on energy budgets as well as increasing visibility to predators. A tendency to respond to a stimulus with escape behaviours could increase survival, but may also be energetically costly and could potentially lead to more chases by predators compared with individuals that use defensive postures. These results demonstrate that projected future ocean acidification affects the behaviours of a tropical squid species.Carbon dioxide (CO Althoughannels) , evidenc species  suggestsd sharks . ConsequCephalopod defence can be categorized into primary defence (avoidance by using cryptic behaviours or camouflage), secondary defence  and escape responses . Seconda2 levels (626 and 956 µatm) on activity levels and behavioural responses to a visual startle stimulus in the two-toned pygmy squid, Idiosepius pygmaeus, in an aquarium setting. The CO2 treatments were selected to match projected CO2 levels in the atmosphere and ocean by the end of this century on a moderate (RCP6) or high (RCP8.5) emission trajectory ). Although I. pygmaeus is a less studied species compared to other cephalopods, its small size and adjustability to captivity make it highly suitable for laboratory studies. Average body size of this species is 8 mm in length and individuals have a life span of about 90 days . Animals were captured using a 500 µm-mesh triangular frame dip net and transported immediately to James Cook University, Townsville. Here, squid were housed in 53-litre rectangular tanks (56×38×25 cm) at a maximum of five individuals per tank, with six tanks at each of the three different CO2 levels. Each tank included rocks and PVC pipe structures that could be used as hiding places. I. pygmaeus readily adjust to captivity  a current-day control (447 µatm), 2) a lower end of the century projection (moderate CO2 626 µatm) and, 3) an upper end-of-century projection (high CO2 956 µatm)  and a temperature probe . Total alkalinity was estimated weekly by Gran Titration  using certified reference material from Dr. A.G. Dickson (Scripps Institution of Oceanography). Salinity was measured weekly using a conductivity sensor . Carbonate chemistry parameters  . A pH corameters  were calrameters  using thrameters  refit byrameters  for KHSO2 treatment. Individuals were carefully transferred from their holding tank to a clear plastic aquarium (34×20×18 cm) containing 11 litres of seawater from their CO2 treatment. The size of the observational tank is not expected to have a limiting effect on squid activity. During activity observations, movement was generally observed as a single short distance jet followed by a brief rest, then another short jet in a seemingly random direction. Individuals did not swim/move into the walls of the aquarium and, therefore, did not appear to be restricted in their movements. The front and left side of the aquarium contained gridded lines spaced 2 cm apart . A computer monitor was set up against one side of the tank displaying an all-white screen while the remaining three sides of the tank were covered with white paper to both minimize external distractions and match the white screen of the computer monitor. Each squid was observed from behind a black plastic sheet for 90 seconds as an acclimatisation period. A startle response trigger was then remotely activated when the computer monitor appeared to be in the squid's field of vision and the squid was a maximum distance of 8.5 cm (or one fourth of the tank length) from the side containing the monitor with the startle stimulus. The startle response was a black oval shape, which expanded in size from zero to 15×18.7 cm in 1.15 seconds. The non-natural stimulus used in this experiment was used as a means to elicit a startle response from the squid, not necessarily to resemble a predator. A very similar visual startle stimulus from a computer monitor elicits a natural defensive response in damselfish  count data (variances > mean), rather than Poisson distribution data (variance  =  mean). Coefficients from the negative binomial correlation of coefficients table  were converted into p-values. A Wilcoxon signed rank-sum test was used to test for differences in movement (number of line crosses) among the three CO2 treatments for all animals in the experiments , as the data were not normally distributed. Analysis of variance (ANOVA) on log-transformed data was then used to test for differences in movement among CO2 treatments for active animals only.Statistical analyses were performed using S-PLUS statistical software . Preliminary analyses showed that there were no significant differences among replicate treatment tanks for any of the traits measured; therefore data were pooled by CO2 treatments. The activity level of each individual from the observation study was included in the logistic regression as a continuous variable to determine if this correlated with their inking and reaction type in the startle response experiment. Logistic regression could not be used to compare body pattern choices among CO2 treatments, because there were more than 2 classes of behaviour in this analysis; therefore, a Fisher's Exact test was used for this comparison. These analyses are well suited to the relatively small sample sizes and zero-inflated data for these behavioural traits. One-way ANOVA with Tukey's HSD post-hoc test, with CO2 levels as a fixed factor, were used to determine the effect of CO2 treatments on squid reaction time and escape distance. Prior to the analysis of distance moved, we performed a Pearson's correlation to determine that the initial distance from the startle stimulus did not affect the subsequent escape distance moved (p = 0.356). Data for reaction time and escape distance met assumptions of normality and homogeneity of variances (Levene's test) confirming that the data were appropriate for ANOVA.Logistic regression  and Fisher's Exact tests were used to test for variation in squid behaviours among the three CO2 treatments  (2 treatment and 62% from the high CO2 treatment remained at rest (n = 27–29). Additionally, some squid from the control and moderate CO2 treatment, but not the high CO2 treatment, had a low level of activity (1–25 line crosses). Further analysis of movement levels showed control squid had a mean of 10 (SD = 28) line crosses compared to 61 (SD = 117) and 51 (SD = 100) line crosses in the moderate and high CO2 treatments, respectively, when all individuals (including those at rest) were included , but not between the control and high CO2 treatments . Among active individuals only (n = 6–12), there was a significant difference in the mean number of line crosses among the control and elevated CO2 treatments . A post hoc test showed that while there was no significant difference between control and moderate CO2 treatments , there was a significant difference between the control and high CO2 treatment levels . Activity levels were not affected by individual body size .There was a significant difference in the frequency of squid at rest and active among control and elevated CO= 0.044) . During included . For all2,37 = 1.442, p = 0.249). The mean reaction times among CO2 treatments were between 40 and 60 ms. Similarly, there was no significant difference among treatments in the distance moved in response to the stimulus (n = 19–23) , which ranged from 8.7 to 9.9 cm.In the startle response trials, there was no significant difference among treatments in the mean time to react to the looming stimulus (n = 10–16) . At control levels, only 26% of squid discharged ink when startled, whereas 50% at moderate and 60% at high CO2 treatments discharged ink (n = 20–23). When ink discharge was further compared by pairwise Fisher's Exact tests, there was a significant difference in the frequency of squid discharging ink between the control and high CO2 group (p = 0.033), but not between the control and moderate CO2 group (p = 0.129). There was no correlation between the observed activity level and the tendency to discharge ink in response to the visual startle stimulus .There was a non-significant trend towards an increased frequency of squid discharging ink in response to the looming stimulus increased at higher CO2 = 9.863, df = 2, p = 0.007). Controls had a 50% split between these two reaction types, whereas at higher CO2 levels, a greater proportion of animals jetted away from the stimulus . There was also no correlation between the observed activity level and the behavioural response to the visual startle stimulus .Behavioural responses to the looming stimulus were separated into two reaction type categories: jet and defensive anchor posture. There was a significant difference in reaction type among the three treatments (n = 17–20) (2 levels (n = 22–23) . The difference was most notable between the control and high CO2 treatments  at elevated CO2 (1000 µatm)  . This ma2 caused changes in the secondary defensive behaviours. While time to react and distance moved in response to the visual startle stimulus was not affected by elevated CO2, squid exhibited very different response types and defensive behaviours. In the elevated CO2 treatments, a much higher proportion of squid jetted instead of displaying the defensive anchor posture compared to controls. This demonstrates a shift from secondary defence behaviours towards escape responses at elevated CO2. Longfin squid  alter their defensive tactics depending on the type of predator encountered  in I. pygmaeus as a result of elevated CO2 is not yet known and further research is required. In fishes, behavioural alteration occurs due to interference with the function of GABAA neurotransmitter receptors  shows a stabilization of extracellular pH accompanied by an increase in blood HCO3− levels after prolonged exposure to elevated CO2  that the levels of ocean acidification tested here will occur (This study is the first to demonstrate the potential for near-future COal rates . Therefofication  and acidfication . The extfication . Therefoll occur . Due to 2 can alter activity and defensive behaviours in a tropical squid. Squid are important in ecosystems as both predator and prey, and any impacts of ocean acidification on squid may affect marine ecosystems. As more studies are conducted, it is becoming apparent that rising CO2 levels in the ocean could affect the behaviours of a wide range of marine species. Determining the extent of these effects as well as assessing the potential for adaptation will be necessary to properly predict the effects of ocean acidification on marine ecosystems.Our results suggest that elevated CO


Here we have applied hydrogen/deuterium exchange (HDX) mass spectrometry to characterize the effects of Bexarotene on the conformational plasticity of the intact RXRα:PPARγ heterodimer. Interestingly, addition of Bexarotene to PPARγ in the absence of RXRα induced protection from solvent exchange, suggesting direct receptor binding. This observation was confirmed using a competitive binding assay. Furthermore, Bexarotene functioned as a PPARγ antagonist able to alter rosiglitazone induced transactivation in a cell based promoter:reporter transactivation assay. Together these results highlight the complex polypharmacology of lipophilic NR targeted small molecules and the utility of HDX for identifying and characterizing these interactions.The retinoid x receptors (RXRs) are the pharmacological target of Bexarotene, an antineoplastic agent indicated for the treatment of cutaneous T cell lymphoma (CTCL). The RXRs form heterodimers with several nuclear receptors (NRs), including peroxisome proliferator-activated receptor gamma (PPAR No. Noγ wer Figures , mirroritagonist . To conf0 ~ 3 μM . A cotra0 ~ 3 μM . Howevertivation , confirm in vitro pharmacomic interactions with the advent of automated platforms and data processing software compatible with requisite screening throughputs [The strategy of repurposing pharmaceuticals has emerged in response to the challenges and expense of obtaining regulatory approval for new drugs , 23. Druoughputs .γ, also a target that has emerged for the treatment of Alzheimer's disease [γ may contribute to the reported insulin-sensitizing efficacy of retinoids through modulation of receptor posttranslational modifications [Bexarotene is approved for the treatment of CTCL and, like most chemotherapies, has been investigated for efficacy in other cancer types . Bexarot disease . A systeications .γ and confirmed this with a competitive binding assay. Bexarotene acts as a PPARγ antagonist in a cell based promoter:reporter transactivation assay, competing with rosiglitazone, and has a HDX profile consistent with other known PPARγ antagonists. The ability of Bexarotene to modulate PPARγ may contribute to the beneficial effects observed in animal models of insulin resistance and Alzheimer's disease. Together these results highlight the complex polypharmacology of NR ligands, the utility of HDX in characterizing these interactions, and the importance of characterizing ligands across the NR superfamily.Here we have applied HDX to identify the off-target binding of Bexarotene to PPAR


During viral infection, proper regulation of immune responses is necessary to ensure successful viral clearance with minimal host tissue damage. Proteasomes play a crucial role in the generation of antigenic peptides for presentation on MHC class I molecules, and thus activation of CD8 T cells, as well as activation of the NF-κB pathway. A specialized type of proteasome called the immunoproteasome is constitutively expressed in hematopoietic cells and induced in non-immune cells during viral infection by interferon signaling. The immunoproteasome regulates CD8 T cell responses to many viral epitopes during infection. Accumulating evidence suggests that the immunoproteasome may also contribute to regulation of proinflammatory cytokine production, activation of the NF-κB pathway, and management of oxidative stress. Many viruses have mechanisms of interfering with immunoproteasome function, including prevention of transcriptional upregulation of immunoproteasome components as well as direct interaction of viral proteins with immunoproteasome subunits. A better understanding of the role of the immunoproteasome in different cell types, tissues, and hosts has the potential to improve vaccine design and facilitate the development of effective treatment strategies for viral infections. The host outcome of viral infection depends on the successful balance of immune responses that contribute to control of viral replication, but those responses may also mediate aspects of host tissue damage that accompany infection. Antibodies, cytokines, CD4 T cells, and CD8 T cells can all play important roles in controlling viral replication and viral clearance from the host. CD8 T cells kill infected target cells by two major pathways: perforin/granzyme-mediated pathways and Fas–Fas ligand (FasL)-mediated pathways . CD8 T cFigure 1). Peptides that bind tightly to MHC class I are 8–11 amino acids in length and have anchor residues, which are generally in the C-terminus, but can be present elsewhere in the peptide sequence . The immunoproteasome is a specialized type of proteasome with altered peptide cleavage properties that is constitutively expressed in hematopoietic cells and induced in non-immune cells under conditions of inflammation. Evidence suggests that the immunoproteasome may play an important role during viral infection through regulation of CD8 T cell responses and proinflammatory cytokine production, activation of the NF-κB pathway, and management of oxidative stress .Figure 2A; Proteasomes are large complexes responsible for the regulated degradation of almost all cellular proteins, and as such proteasome activity is required for cell viability . ProteasFigure 2A). A third IFN-γ-inducible proteasome subunit, this one outside of the MHC region, was subsequently identified: proteasome subunit β2i , which is homologous to β2  and proteasome subunit β5i . Expression of β5t is essential for positive selection of T cells, and expression of the homologous subunits β5 or β5i cannot compensate for deficiency in this specialized subunit . This proteasome contains the immunosubunits β1i and β2i as well as a cTEC-specific proteasome subunit β5t , or could be due to complete absence of immunoproteasome expression in some cell types and not others .Constitutive expression of immunoproteasome subunits by immune cells appears to be independent of external signaling requirements, such as persistent stimulation by cytokines eceptors , but a seceptors . In sple reduced , suggest reduced . There iWhile non-immune cells express standard proteasomes almost exclusively, immunoproteasome expression can be induced in such cells following exposure to IFN-γ. As mentioned above, type I IFNs can also upregulate the immunoproteasome, although less efficiently than IFN-γ ,b. An inFigure 3).Proteasomes exist in many forms in cells, with different regulatory or activator cap complexes that associate with the 20S core to control access to the proteolytic inner chamber . The α rIt was originally thought that cells have little to no free 20S proteasome, and that the 20S complex is incapable of acting independently of its regulators or activators . WithoutThe changes in proteasome subunit composition from standard to immunosubunits in response to IFN-γ stimulation alter the proteolytic activity of the complex. Purified 20S and 26S immunoproteasomes from IFN-γ-treated cells substantially increase the rate at which they cleave after hydrophobic and basic residues  and decrIt was uncertain whether proteasomes cleave proteins to the exact length (8–10 residues) that would be directly loaded onto MHC class I molecules, or whether they produce larger precursors that are further cleaved by other peptidases. Some initial experiments indicated that isolated 20S proteasomes could cleave larger peptides to antigenic epitopes . HoweverAlthough immunoproteasomes degrade proteins at the same rate as standard proteasomes, immunoproteasomes generate more antigenic peptides than standard proteasomes . DegradaFigure 3). In mammals, PA28 is made of two homologous subunits, PA28α (REGα or PSME1) and PA28β , a large regulatory complex that binds the ends of the 20S proteasome in an ATP-independent manner (in vitro degradation of full-length proteins (insulin-like growth factor-1 and casein) by 20S, 26S, and PA28α/β-20S immunoproteasomes and analyzed the range of peptides released. Rather than increase the fraction of 8–10 residue peptides that is generated, association of PA28 with 20S immunoproteasomes reduces it from 10% to approximately 6% of the total, with the majority of peptides being <6 amino acids in length. This may occur through allosteric modification of proteasome active sites by PA28α/β. Alternatively, PA28α/β may control the eﬄux of longer peptides out of the proteolytic chamber and contribute to their ongoing hydrolysis  is important for positive selection of T cells . Most stSmall molecule peptide screens have led to the identification of inhibitors specific to immunoproteasome activity. The use of small molecule inhibitors offers several advantages over traditional gene deletion approaches, the most obvious of which is their potential for use as therapeutics. Because small molecule inhibitors are unlikely to affect the assembly or structure of the immunoproteasome, they allow for the study of how the catalytic activity of a specific subunit affects immune responses. Furthermore, these inhibitors are unlikely to affect the positive selection of T cells in the thymus, since most studies are undertaken in adult mice after maturation of the immune system.50 value of approximately 10 nM , inhibits β5i with an ICly 10 nM . ONX 091ly 10 nM . UK-101 ly 10 nM , with twly 10 nM . Leupeptly 10 nM . There aly 10 nM . While tly 10 nM .The nuclear factor-κB (NF-κB/Rel) family of transcription factors plays a central role in regulation of immunity and inflammation. NF-κB transcription factors interact as homodimers or heterodimers with other NF-κB family members, including p65 (RelA), RelB, c-Rel, p50 (NF-κB1), and p52 (NF-κB2). Under normal conditions, these factors exist in the cytoplasm in an inactive state because of interaction with inhibitory IκB proteins  or the unprocessed forms of NF-κB1 and NF-κB2 . The NF-κB pathway is activated in response to many different stimuli, including exposure to inflammatory cytokines such as TNF-α or IL-1 family members . In the -/- mice. The T2 human lymphocyte cell line, which lacks both β1i and β5i, has substantial defects in NF-κB activation compared to the parental T1 cell line and is sensitive to TNF-α-induced apoptosis . If that is the case, then the deficiencies in these cell types in β1i-/- mice are not a true reflection of deficient immunoproteasome function.In support of a role for the immunoproteasome in NF-κB activation, another study reported delayed termination of the classical NF-κB activation pathway and reduced activation of transcription factors associated with the alternative NF-κB pathway in β1iout mice . B cells and β2i . It is i-/- mice may instead be an artifact of mixed proteasomes. One study demonstrated reduced NF-κB activation in cardiomyocytes and B-cell-depleted splenocytes in β5i-/- mice following exposure to IFN-γ . Because β1i and β5i are encoded on the MHC locus, it was originally thought that the major function of the immunoproteasome is to regulate the immune response via optimization of MHC class I peptide processing. Although proteasome activity in general is required for MHC class I antigen presentation, the immunoproteasome does not appear to be essential for that function. In fact, some epitopes are processed more efficiently by the 20S proteasome than the immunoproteasome [-derived epitopes . InteresThe immunoproteasome (subunits β1i and β5i) moderately influences the magnitude and specificity of CD8 T cell responses to hepatitis B virus (HBV) polymerase and envelope proteins . AlthougThe majority of studies examining the effect of immunoproteasome deficiency on the generation of antigenic epitopes during viral infection have been performed with influenza virus or LCMV, two well-studied viruses for which the immunodominant CD8 T cell epitopes are known. The WE strain has been the most commonly used strain for LCMV-immunoproteasome studies, while the Armstrong and clone 13 strains have been used far less frequently. It is important to consider potential differences associated with the use of different LCMV strains, which vary greatly in terms of their interactions with host immune function and their ability to persist in an infected host.-/- mice show a reduced capacity to generate an influenza virus nucleoprotein-specific epitope, while presentation of OVA-derived antigens was unaffected  from wild-type mice. Because this defect was observed for epitopes produced equally by standard proteasomes and immunoproteasomes, it was suggested that immunoproteasomes might play a role in T cell activation and proliferation.APCs from β1iaffected . Two lataffected . Respons-/- mice. While B cells from influenza virus-infected β1i-/- mice proliferate as well as those from wild-type mice, they display a survival defect and impaired Ig isotype switching. DCs from the same mice show reduced innate cytokine production in response to influenza virus infection . This may explain the seemingly contradictory presence of mixed β2i/β5i proteasomes in β1i-/- mice. It is doubtful that mixed proteasomes containing β2i exist in wild-type mice, although this has not been formally analyzed in all tissues or cell types. Mixed proteasomes  are highly expressed in human immature and mature DCs. Human monocytes also contain a particularly high abundance of mixed proteasomes, up to 50% of the total proteasome content. The mixed proteasome content of B and T cells is unknown. However, the finding that mixed proteasomes are expressed at high levels in some cell types, particularly APCs, suggests that they may play an important role in shaping CD8 T cell responses. Indeed, work by Care must be taken in the interpretation of results obtained using β1iassembly . The aut-/-β2i-/- ONX 0914-treated mice and stimulated with LPS or α-CD3/CD28 had reduced production of IL-6, TNF-α, and IFN-γ. However, this defect was observed in stimulated splenocytes from wild-type mice treated with ONX 0914 alone, suggesting a specialized function of β5i in promoting cytokine production that is not shared by the other immunosubunits. Because the cytokine studies in mice lacking immunoproteasome activity were performed in splenocytes stimulated ex vivo or in other models, it is unknown whether these mice display defects in cytokine production in response to LCMV or other viruses in vivo. The relatively modest effect of impaired immunoproteasome activity on the generation of LCMV-specific IFN-γ+ CD8 T cells suggests that overall IFN-γ production may be unaffected. However, β1i-/-β2i-/- ONX 0914-treated mice may still have defects in production of other cytokines, such as IL-6 or TNF-α, in response to LCMV or other viruses.Mice deficient in β2i have ~20% fewer CD8 T cells in the spleen and reduced response to some LCMV-derived epitopes (WE strain). This is not due to impaired generation or presentation of these epitopes, but rather to either decreased precursor frequency or reduced expansion of the epitope-specific T cells, further supporting a role for the immunoproteasome in T cell survival or expansion rather than just antigen presentation . One str-/- mice, suggesting a role for microglial immunoproteasomes in exacerbating immunopathology. The lack of mature immunoproteasome assembly in astrocytes may be due to a posttranslational mechanism that prevents excess immunoproteasome assembly in the brain. Since cells in the CNS regenerate poorly or not at all, inhibition of immunoproteasome assembly might be a strategy to protect these cells from immunopathological destruction.Immunoproteasome subunits are transcriptionally induced in the brain following LCMV (WE strain) infection . Mature LMP2 and LMP7 genes  are closely linked on the same chromosome and flank the TAP1 transporter gene, so that breeding β1i-/- and β5i-/- mice with each other would not likely result in a double knockout but leave TAP1 unaffected. However, -/- mice to generate the triply deficient mice. APCs from these mice display profound defects in MHC class I antigen presentation, defects that are much more severe than those previously described in β1i, β2i, or β5i single knockout mice. These findings suggest that there may be functional overlap between the immunosubunits, and that the crucial role of immunoproteasomes in MHC class I antigen presentation has been obscured or underestimated by the use of mice deficient in only one immunosubunit. Triply deficient mice have an approximately 50% reduction in surface levels of MHC class I , triply deficient mice display substantially weaker CD8 T cell responses than wild-type mice. This is due to defects in antigen presentation (and not to pleiotropic effects on T cells), because weaker T cell responses are also observed in wild-type T cells transferred into triply deficient mice. MHC class II epitope presentation and CD4+ T cell responses to LCMV are similar in wild-type and triply deficient mice, suggesting that the immunoproteasome does not affect processing of MHC class II antigens. It remains to be seen whether complete immunoproteasome deficiency  affects LCMV replication or other virus-induced inflammatory responses, such as cytokine production.The above studies demonstrate subtle and possible organ- or virus-specific roles for the immunoproteasome during viral infection using mice deficient in only one or two immunosubunits. To assess the role of complete immunoproteasome deficiency, a recent study generated mice deficient in all three immunoproteasome subunits , express immunoproteasomes. A number of studies have suggested major roles for the immunoproteasome in T cell proliferation and survival, and there are hints from β1i-/- mice developed more severe myocardial tissue damage compared to wild-type mice  or during long-term persistence for ongoing evasion of the immune system.HIV-1 inhibits immunoproteasome function, likely by a number of mechanisms . ExpressHuman adenovirus E1A interacts with the immunoproteasome subunit β2i, but not its constitutive counterpart β2. E1A  also prevents IFN-γ-induced upregulation of immunoproteasome subunit expression by interfering with STAT1 phosphorylation . Of notein vitro  and MCMV inhibit IFN-γ-induced immunoproteasome formation in fibroblasts in vitro . InhibitCD8 T cells often play significant roles during viral infection. In endogenous antigen presentation, the proteasome is crucial for the generation of antigenic peptides for binding to MHC class I and promoting CD8 T cell responses. The immunoproteasome is a specialized type of proteasome with altered peptide cleavage properties that is constitutively expressed in hematopoietic cells and induced in non-immune cells under conditions of inflammation. Evidence suggests that the immunoproteasome may play an important role during viral infection through regulation of CD8 T cell responses, activation of the NF-κB pathway, and management of oxidative stress. Many viruses have mechanisms of interfering with MHC class I processing, including direct interaction of viral proteins with immunoproteasome subunits. It is essential to better understand the role of the immunoproteasome in different cell types, tissues, and hosts in the context of diverse inflammatory states. An improved understanding of the mechanisms of immunoproteasome function could aid in the development of vaccines and treatment strategies for viral infections.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The goal of this study is to use wireless sensor technologies to develop a smart clothes service platform for health monitoring. Our platform consists of smart clothes, a sensor node, a gateway server, and a health cloud. The smart clothes have fabric electrodes to detect electrocardiography (ECG) signals. The sensor node improves the accuracy of QRS complexes detection by morphology analysis and reduces power consumption by the power-saving transmission functionality. The gateway server provides a reconfigurable finite state machine (RFSM) software architecture for abnormal ECG detection to support online updating. Most normal ECG can be filtered out, and the abnormal ECG is further analyzed in the health cloud. Three experiments are conducted to evaluate the platform's performance. The results demonstrate that the signal-to-noise ratio (SNR) of the smart clothes exceeds 37 dB, which is within the “very good signal” interval. The average of the QRS sensitivity and positive prediction is above 99.5%. Power-saving transmission is reduced by nearly 1980 times the power consumption in the best-case analysis. As the population is becoming progressively older, higher quality of health and medical care is now expected in countries with significant aging problems. This care is particularly important for countries with 75% of the elders suffering from chronic diseases . Accordi2), and knee activity. The sensor data are sent via Bluetooth to mobile devices and are routed to nursing homes or workstations via the Internet. MIThril , P2[(x2∣x3∣x4∣x5∣x6)(y5∣y6)(z2)], P3[(x2∣x3∣x4∣x5∣x6)(y5∣y6)(z1∣z2∣z3)], P4[(x3∣x4∣x5∣x6)z1], P5[x4], P6[x6(y3∣y4∣y5∣y6)], and P7[(y4∣y5)z1]. Finally, all regular expressions are transmitted to the Deterministic Finite Automaton (DFA) to construct a look-up table [i, String Xi) → (State j). The matching process continues until it jumps to the final state to match a certain pattern and execute an action.For ease of explanation, we assume that there are seven patterns, named P1 to P7. These are defined by three attributes 2) is introduced to assess the performance of the smart clothes sensor. Signal quality is a vital factor that guarantees the accuracy of the platform. A criterion named SNR  in the MIT database is chosen as the standard comparison. Sensitivity and positive prediction are calculated using the “Bxb” programs from the PhysioToolkit [The performance of our algorithm is evaluated using the MIT database  by compaoToolkit . The expTo verify the performance of the algorithm for normal and abnormal subjects, we analyzed 18 normal subjects  and 48 abnormal subjects (MIT-BIH Arrhythmia Database). We calculated the HRV features, including AVNN , SDNN, RMSSD , and pNN50  . The fea∗ 128/sec ∗ 300 s + 20 bytes of package field + 32 bytes of HRV result). A 24-hour data set is emulated using 288 5-minute data sets. The normal ratio is defined as the percentage of normal HRV results analyzed by the multipattern abnormal disease matching in the gateway server. Once the HRV parameters are transmitted to the gateway server, they are analyzed to decide whether to transmit the raw data. For instance, if the normal ratio is 80%, the 24-hour power-saving transmission transmits 288∗32  bytes + 57652  bytes∗288∗(1 − 80%) = 3.176 Mb, and the efficiency is 15.84 Mb/3.176 Mb = 4.99. Suppose that 100% normal patterns can be detected from the subjects; the transmission efficacy is then 1980 times. This is very useful for the analysis of a huge amount of personalized ECG data in the health cloud (We compared the transmission efficacy every 5 minutes according to different abnormal subject ratios with “full transmission” and “power-saving transmission.” In the power-saving transmission mode, data is transmitted to the gateway server in a 32-byte package at a five-minute interval. In the full transmission mode, data is transmitted continuously at a one-second interval. In total, the size of continuous data transmitted over five minutes is 57,652 bytes (12 bits th cloud .In this paper, we present a smart-clothing platform for ECG acquisition, analysis, and transmission. This platform is dedicated to the detection of abnormal ECG. A washable, low-power consumption, and comfortable smart cloth is designed using fabric electrodes. The SNR values are more than 37 dB, which demonstrates that the smart clothes have very good signal acquisition capability. The reconfigurable firmware architecture enables online updating. In addition, the normal ECG detection process comprises a filtering phase and a pattern matching phase. Both the MWqrs detection algorithm and parameters of the whole platform have more than 99.5% accuracy performance in QRS detection and BPM calculation. The power-saving transmission strategy can smartly compress raw data depending on short-term analysis of HRV, which helps to achieve more than 500 times the transmission efficacy and power reduction.Our platform can be used for the long-term monitoring of elders' cardiovascular status with high accuracy, simplicity, and future expandability. Our future efforts will focus primarily on abnormal ECG pattern recognition with multichannels. Therefore, the platform can provide clinically meaningful information to doctors to reduce the harm caused by cardiovascular disease.


EGFP reportergene in transgenic lines of Chinese hamster ovary (CHO) cells. Hence,transcription terminators can be used to create stable mammalian cells with ahigh and stable level of recombinant protein production.Mammalian cell lines are widely used to produce recombinant proteins. Stabletransgenic cell lines usually contain many insertions of the expression vectorin one genomic region. Transcription through transgene can be one of thereasons for target gene repression after prolonged cultivation of cell lines.In the present work, we used the known transcription terminators from the SV40virus, as well as the human β- and γ-globin genes, to preventtranscription through transgene. The transcription terminators were shown toincrease and stabilize the expression of the  An increasing number of drugs are currently produced in cell culturebioreactors cells) , 2. HoweTransfection of linearized plasmid DNA has become the most common method usedin bioengineering to generate cell lines for producing target proteins , 4.Thisin vitro experiments demonstrated that thesesequences interact with the matrix attachment region (MAR) and regulatory elements capable of blocking heterochromatin propagation  are also used in bioengineering.In general, it is fair to say that no universal regulatory elements with acomprehensible mechanism of action that could be efficiently used across alltypes of vector constructs intended for generating high-yield cell linesproducing various proteins have been found yet. Even the strongest promotersare obviously expected to have mechanisms for suppressing excessivetranscription. RNA interference is one of these mechanisms of transcriptionsuppression , 16. TheGγ- (γt) globin genes were used in this work to testthe role of transcription termination in the protection of transgene expression[Sequences of the well-studied transcription terminators from β- (βt)and pression, 21. Twopression. The traCreation of constructsEGFP and upstream of cytomegalovirus promoter 640 bp away fromthe transcriptional start point: two tandem copies of the HS4 core insulatorsequence from chicken β-globin locus (2×Ins) (476 bp), SV40transcription terminator (SV40pA) (868 bp), transcription terminator from thehuman β-globin locus (βt) (1130 bp), and a combined elementconsisting of both βt and 2×Ins. A 1336-bp-long terminator from thehuman Gγ-globin gene (γt) was inserted into the constructβt_EGFP at the restriction site AflII to obtain the construct βt_EGFP_γt.In order to create a series of constructs, different sequences were insertedinto the pEGFPN1 vector at the PciI restriction site located downstream ofCreation of transfected cell linesReporter gene expression was analyzed using Chinese hamster ovary (CHO-K1)cells, the cell culture most widely used in bioengineering to produce targetproteins.Lglutamine, 35 mg/LL-proline, and commercially available antibiotic Mycokill-AB(PAA Laboratories) at a working concentration. The cells were re-inoculatedevery 5 days at a concentration of 105 cells/cm2 and a1:20 dilution ratio. The cells were cultured at 37°C in an atmosphere of5% CO2 and high moisture content. The cell culture was transfectedwith recombinant plasmids. Plasmids were linearized with the ApaLI restrictionenzyme to ensure more efficient integration of the transgenic construct. Thetransfection protocol was as follows: The cells that reached 70–80% ofthe monolayer (8 × 104 cells/cm2) were washed with aserumfree cell culture medium. The transfection mixture was prepared: 3–4μg of linearized plasmid DNA was mixed with 375 μL of the serum-freecell culture medium. The commercially available transfection reagentLipofectamine 2000  was mixed with the same amount of theserum-free cell culture medium in a separate test tube. The solutions werecombined and incubated at room temperature for 30–40 min. The washedcells were coated with the transfection mixture. The transfection mixture wasreplaced with a serum-containing DMEM culture medium 4–6 h after thetransfection had been initiated.CHO-K1 cells were cultured on DMEM medium containing 10% of inactivated fetalbovine serum, 2mM The level of reporter gene expression was assessed according to thefluorescence intensity on day 2 after transfection (36–48 h) by flowcytometry on a MACSQuant Analyzer VYB (MiltenyiBiotec). Nontransfected CHO-K1cells were used as negative control.Cytofluorometric analysisPrior to the cytofluorometric analysis, the cells were washed with phosphatebuffered saline (PBS), treated with trypsin, and removed from the Petri dishes.They were subsequently washed twice to remove trypsin and thoroughlyre-suspended in phosphate buffered saline. The resulting suspension (106 cellsper mL of PBS) was transferred into 5-mL round-bottom test tubes.The voltage in the flow cytofluorometer channels was selected so as not to takeinto account the autofluorescence of nontransfected cells. After calibration,all the samples were measured at a constant voltage. Thus, if EGFP fluorescencewas detected, we counted the cells that emerged in the range of values above 10on the logarithmic scale for a proper channel and measured all the quantitativevalues of the gene expression level.Maintenance of the transfected cell poolsAfter the transfection, cell pools were cultured according to the aboveprotocol. Transgene-free cells were removed by selecting the transfected cellpools using the commercially available antibiotic Geneticin (Invitrogen) at aconcentration of 800 μg/mL. Since suppression of the transcriptionalactivity of the transgene reduces production of the antibiotic-resistance genewith time, the antibiotic concentration needs to be gradually reduced to 200μg/mL after the cells have been cultured for 77 days.Creation of individual clonesPools of cultured cells were removed from the plates and diluted in 10 mL ofthe culture medium. Cell concentration was then determined using a Scepterautomated cell counter (Millipore); the cells were diluted so that 1 mL of themedium contained 2–3 cells. The resulting suspension was transferred into24-well plates (1 mL per well).After cultivation in a DMEM medium containing antibiotic Geneticin at aconcentration of 800 μg/μL for two weeks, we performed acytofluorometric analysis of the clones that survived. The resulting cell lineswere further maintained, and the expression level of EGFP was determinedaccording to the procedure for transfected cell pools. EGFP fluorescenceintensity was measured every 15 days.EGFP gene underthe control of the CMV promoter (CMVpr) and SV40 transcriptional terminator(SV40pA) . Inaddition to the reporter gene, this plasmid contained the Neomycin resistance gene(NeoR) under the control of the SV40 promoter (SV40pr) and transcription terminatorof the herpes simplex virus thymidine kinase gene (HSVpA). In this case, the Neomycinresistance gene is needed for selecting transfected cells.The enhanced green fluorescent protein (EGFP) gene under control of the CMVpromoter was used as a reporter gene when studying the potential role oftranscription terminators in stabilizing the transgene expression level in CHOcells. All the experimental constructs were compared to the control, theplasmid pEGFPN1 (Clontech) that contained the Fig. 1).In order to completely isolate the reporter gene fromtranscription initiated in the surrounding chromatin, we created a derivativeconstruct βt_EGFP,which contained, in additionto the β-globin terminator, the transcription terminator from theGγ-globin gene (βt_EGFP_γt) inserted in reverse orientation atthe 3’- end of the reporter gene. In this construct, the reporter gene isprotected on both sides against transcription initiated from the surroundingchromatin.The well-studied transcription terminators of the β-globin gene (βt)and SV40pA were used to assess the effect of termination of transcriptioninitiated downstream of the inserted transgene on reporter gene expression. Inthe constructs (SV40_EGFP and βt_EGFP), these terminators were inserted inforward orientation immediately upstream of the CMV promoter.Finally, a βt_2×Ins_EGFP construct with theglobin terminator inserted downstream of the two copies of the insulator wasgenerated to study the cooperative effect of two regulatory elements withdifferent functions: the insulator and transcription terminator.In order to compare the efficiency of transcription terminators with thecurrently known regulatory elements stabilizing transgene expression in CHOcells, we used an element consisting of two copies of the HS4 core insulatorsequence inserted immediately downstream of the CMV promoter . Themean expression level of the EGFP gene in the cell pool at acertain moment was determined according to the percentage of cells with afluorescence intensity higher than 10 on the logarithmic scale . The ratio between the percentageof EGFP-expressing cells in the pool of cells carrying theconstruct with this element and the percentage ofEGFP-expressing cells with the control plasmid (EGFP) was anindicator of the efficiency of a particular regulatory element in achieving ahigh and stable level of protein production.The levels of reporter gene expression were estimated every 15 days using flowcytofluorometry to determine the degree of transgene suppression in the cellpools transfected with the control (pEGFP_N1 and 2×Ins_EGFP) andexperimental  constructs indicate that the use of a transcriptionterminator downstream of the target gene promoter significantly enhances theefficiency of protein production during prolonged cultivation: over threefoldfor terminator SV40 and fivefold for terminator βt by the end of theexperiments.The regulatory element consisting of twocopies of the insulator also had a similar effect.The results of determining the Fig. 2).Thus, protection of the reporter gene (on both sides) against transcription initiatedoutside the transgene enhances stability and expression efficiency. Finally,the best results (a 12-fold increase) were achieved when the transcriptionterminator and two copies of the insulator were combined in the plasmidβt_2×Ins_EGFP.A conclusion can be drawn that two regulatory elements with differentmechanisms of action exhibit an additive effect on the stability andefficiency of regulatory gene expression.The increase in the level of EGFP expression was higher in the pool of cellstransfected with βt_EGFP_γt plasmid, where the reporter gene issurrounded by transcription terminators.It should be mentioned that clones containing the control construct initiallyshowed a much lower level of EGFP expression in stable cell clones containingthe EGFP and 2×Ins_EGFP constructs decreased approximately tenfold aftercultivation for 2.5 months. The average expression activity of the clonescontaining the SV40_pEGFP and βt_pEGFP constructs was ~ 75% of the initialvalue.The level of The results of measuring stable cell pools demonstrated that the most efficientcells characterized by a stable expression of the reporter gene were obtainedafter culturing for ~ 90 days. Hence, in order to study the clones generatedfrom a stabilized cell population in more detail, we repeated the procedure ofgenerating individual cell clones from total populations using the limitingdilution procedure after culturing stable pools for 90 days.Fig. 3).The measurements demonstrate that the resulting EGFPclones are characterized by a stable, but extremely low, level of fluorescence. The levels of fluorescence areslightly higher for 2×Ins_EGFP clones: only one clone has low activity,while the activities of the remaining ones lie in a range between 10 and 100.βt_EGFP clones are characterized by even higher average fluorescencevalues compared to 2×Ins_EGFP clones. Furthermore, one of the βt_EGFPclones exhibited ultra-high levels of fluorescence (between 10 and 1000). Theβt_2×Ins_EGFP clones unexpectedly divide into two groups: one group(two clones) was characterized by low levels of fluorescence comparable tothose of the clones containing the EGFP construct, while the second group wascharacterized by much higher values (about 100), which were on average higherthan those for the 2×Ins_EGFP and βt_EGFP clones. This discrepancy inresults probably arises from the fact that we used a more complex combinationof regulatory elements (instable in certain genomic regions). Similar activitywas observed for βt_EGFP_γt clones. Among them, one clone wascharacterized by an extremely low level of fluorescence, while the other onesexhibited a high level of fluorescence (about 100). The results of thisexperimental series can be used to draw a conclusion that transcriptionterminators are very efficient in establishment and maintenance of a high levelof target protein production. The combined element (terminator attached to theinsulator) and the variant containing the reporter gene surrounded byterminators are characterized by a higher efficiency of target proteinproduction. However, these constructs require a more careful selection ofclones, since some clones turned out to be ineffective due to some unknownreasons.Identically to the previous experiment, the median cell distribution over thefluorescence intensity was the main quantitative indicator of the activity ofreporter gene expression. Twelve individual clones with the EGFP,2×Ins_EGFP, βt_EGFP, βt_2×Ins_EGFP, andβt_EGFP_γt constructs were initially obtained. Some clones died bythe end of the experiment (culturing the cell clone for 300 days); hence, wereport the data for five clones containing the EGFP construct, 12 clonescontaining the 2×Ins_EGFP construct, nine clones containing theβt_EGFP construct, nine clones containing the βt_2×Ins_EGFPconstruct, and five clones containing the βt_EGFP_γt construct(It has been demonstrated that transcription terminators, which can potentiallyisolate the transgene from transcriptional signals, are capable of maintainingthe transgene transcription level stably high for an appreciably long period oftime when culturing CHO-K1 cell lines. The terminator was found to maintain astable level of transgene expression more effectively compared to theinsulator. Furthermore, a combination of the transcription terminator and theinsulator exhibits an additive effect, which enhances and stabilizes transgeneexpression.


Limnodynastes peronii. We tested the hypotheses that a) the embryonic environment affects mean trait values only; b) temperature at which performance of tadpoles is maximal shifts with egg incubation temperatures so that performance is maximised at the incubation temperatures, and c) incubation temperatures modulate the capacity for reversible acclimation in tadpoles. Growth rates were greater in warm (25°C) compared to cold (15°C) acclimated (6 weeks) tadpoles regardless of egg developmental temperatures . The breadth of the performance curve of burst locomotor performance  is greatest when egg developmental and acclimation temperatures coincide. The mode of the performance curves shifted with acclimation conditions and maximum performance was always at higher temperatures than acclimation conditions. Performance curves of glycolytic (lactate dehydrogenase activities) and mitochondrial (citrate synthase and cytochrome c oxidase) enzymes were modulated by interactions between egg incubation and acclimation temperatures. Lactate dehydrogenase activity paralleled patterns seen in burst locomotor performance, but oxygen consumption rates and mitochondrial enzyme activities did not mirror growth or locomotor performance. We show that embryonic developmental conditions can modulate performance curves of later life-history stages, thereby conferring flexibilty to respond to environmental conditions later in life.Performance curves of physiological rates are not fixed, and determining the extent to which thermal performance curves can change in response to environmental signals is essential to understand the effect of climate variability on populations. The aim of this study was to determine whether and how temperatures experienced during early embryonic development affect thermal performance curves of later life history stages in the frog  Temperature influences physiological rate processes, and the temperature breadth across which animals can perform will determine their distribution and abundance across environments Conditions experienced during embryonic development can influence physiological responses to the environment Limnodynastes peronii) affect thermal performance curves of later life history stages. Our study follows a three-factor design with egg incubation temperature, tadpole acclimation temperature, and acute test temperatures as factors. We tested the hypotheses The aim of this study was to determine whether and how temperatures experienced by during early embryonic development in the frog (Limnodynastes peronii) as study organism, because tadpoles of Lim. peronii are known to acclimate swimming performance and metabolism Limnodynastes in the egg is fairly rapid (3–5 days) but the tadpole stage is prolonged and variable between individuals (60–300 days) We chose the striped marsh frog  and glycolytic (lactate dehydrogenase) ATP-producing pathways Animals were collected under NSW National Parks and Wildlife Service Scientific License SL100518. All experiments were conducted under approval from the University of Sydney Animal Ethics Committee .Limnodynastes peronii) were collected from ponds (mean water temperatures at time of collection 19.5±0.5°C) in the Sydney, Australia, metropolitan area . Ponds were checked in the evening and eggs were collected early morning to ensure that nests were laid that night. Eggs were transported to the University of Sydney and each egg mass was divided between two developmental treatments: a “warm” temperature treatment (water temperature: 25.0±0.5°C) and a “cold” temperature treatment (water temperature: 15.0±0.5°C). These temperatures were chosen because they represent mean conditions at the warm midpoint and cool beginning of the breeding season at this location, respectively Freshly-laid egg masses (n = 6) of striped marsh frogs  throughout the acclimation period (6 weeks) and euthanised in a solution of buffered MS222 . Total body length was measured with callipers to the nearest 0.1 mm, and the developmental (Gosner) stage was determined.Tadpoles were gently dried, weighed (tadpoles did not have any yolk left at this stage) and placed into a plastic respirometer (43 ml). The respirometers were provided with an inflow and outflow port through which aged oxygenated water was supplied before measurements started, and which could be closed-off without disturbance to the animals. Inside each respirometer we placed an oxygen sensor spot  that was monitored with a fibre-optic cable  mounted on the outside of the respirometer and connected to an oxygen meter .−1g−1 wet mass After tadpoles were placed into the respirometers, the respirometers were left in a temperature controlled room set to the appropriate test temperature for at least one hour, which is sufficiently long for recovery from handling stress We used independent tadpoles  to measure rates of oxygen consumption at each of five test temperatures , and tadpoles within each treatment were tested in random order. The test temperatures were chosen because they represent the thermal extremes experienced by tadpoles at this location. We also measured oxygen consumption in respirometers not containing tadpoles as controls, and we subtracted oxygen consumption by the controls from the experimental values. In the event, oxygen consumption by controls was kept to a minimum by routinely cleaning equipment and regular water changes. It was not possible to keep tadpoles stationary during trials so that the measurements represent routine rates of oxygen consumption that represent a mixture of oxygen consumed during low levels of voluntary activity, and maintenance and growth.−1 (BL s−1).To measure burst swimming speed, individuals were placed into a plastic tray (40 cm×25 cm×5 cm) filled with water to a depth of 2 cm and were allowed to equilibrate for 15 minutes; trays were kept in constant temperature rooms set at the desired experimental temperature, and water temperature was allowed to equilibrate to room temperature before measurements. The tray dimensions were large relative to the distance moved during an escape response so that data were not confounded by edge effects. Individuals were within the range of developmental stages (Gosner 25–30) in which the relationship between total body length and burst swimming performance remains constant max) of lactate dehydrogenase (LDH), an indicator of glycolytic ATP production, and the two mitochondrial enzymes citrate synthase (CS) and cytochrome c oxidase (COX). Samples were homogenised in 1∶20 (CS and COX) or 1∶100 (LDH) w/v extraction buffer , 0.1% Triton X-100, and 1 mM glutathione, pH = 7.5). Enzyme assays were conducted at 10, 15, 20, 25, and 30°C according to published protocols After euthanasia in a solution of buffered MS222 , tadpoles  were weighed, measured, and their developmental stage was determined before we dissected axial tail muscle. Tail muscle was frozen at −80°C for later analysis. We measured actitivites  to analyse differences between treatments. Permutational analyses use the data per se rather than an assumed underlying distribution for statistical inference. This approach is superior for laboratory-based experiments with relatively small sample sizes relative to the total population Additionally, we analysed the performance curve of swimming speed explicitly to determine changes in performance breadth, maxima, and temperatures at which maxima occurred. We could conduct this analysis on swimming data only because it was the only response that was non-linear across the range of test temperatures (see below). Thermal reaction norms can be described by Gaussian (inverted “U”-shaped) distributions 2/c2), where a = amplitude (maximum), b = mode that aligns the curve on the temperature (x) axis, and c = breadth of the curve. To compare these parameters between treatments, we performed a bootstrap analysis on our data to determine mean parameter values and 95% confidence intervals Gaussian functions are in the form: Y = a*exp(−0.5((X−b)5,419 = 5.33, p<0.001; 1,419 = 0.25, p = 0.673) and there were no interactions between incubation temperature and time  or acclimation temperature . At the end of the 6 week acclimation period, tadpoles from the 15°C incubation temperature on average reached Gosner stage 25.3±0.1 (s.e.) at 15°C acclimation temperature and 25.8±0.2 (s.e.) at 25°C acclimation temperature; tadpoles from the 25°C incubation temperature reached Gosner stage 25.4±0.1 (s.e.) after 6 weeks of acclimation to 15°C, and 27.6±1.4 (s.e.) after acclimation to 25°C.There was an interaction between acclimation temperature and time , and tadpoles that developed from eggs incubated at 15°C had higher swimming performance (12.39 BL s−1 ±0.34 s.e.) than tadpoles from eggs incubated at 25°C (11.47 BL s−1 ±0.35 s.e.). Additionally, there was a significnat interaction between acclimation and test temperatures . None of the other interactions were significant .Incubation temperature had a significant main effect on swimming speed .Cold acclimated tadpoles had significantly higher routine oxygen consumption rates, particulalry at higher test temperatures .Citrate synthase activity was similar in all tadpoles from 15°C incubated eggs, but cold acclimated tadpoles had higher activity than warm acclimated animals from eggs incubated at 25°C .Similar to citrate synthase, cytochrome c oxidase activity of 15°C acclimated tadpoles that were incubated at 25°C had greater activity than 25°C acclimated animals, but there were no differences in tadpoles incubated at 15°C , but not those of any other traits. Additionally, acclimation of tadpoles changed the mode and breadth so that curves for swimming performance were determined by a combination of thermal conditions experienced by embryos and tadpoles. However, we found no evidence in any trait that incubation temperatures shift performance curves so that temperatures at which performance maxima of tadpoles occur coincide with egg incubation temperatures . Incubation temperatures did modulate acclimation of performance curves of enzyme activities . Lastly, growth was the only response that was independent from incubation conditions and tadpoles from warm acclimation treatments grew at a faster rate.Locomotor performance was the only trait that showed a non-linear response to acute temperature increases across our range of test temperatures. Hence, unlike locomotor performance, growth, oxygen consumption and enzyme activities were maximal at temperatures above 30°C. Mean environmental conditions at the study site are below 30°C Rana sylvatica) Rana, however, in Lim. peronii tadpoles the fastest growth rates occurred in animals that experienced high temperatures towards the later developmental stages. Under natural conditions, this would be tadpoles that hatched from eggs laid early in the season but subsequently experience warm temperatures, or from those laid during summer. Tadpoles hatching from eggs laid late in summer when temperatures are still high but then cool or those laid late in the season at low temperatures will grow slowly and overwinter. These latter animals also have the slowest locomotor performance. Hence, the interaction between incubation and acclimation temperatures produced locomotor phenotypes that are responsive to variation in environmental conditions and are matched to growth patterns.Egg incubation temperature did not affect growth rates of tadpoles, which is a similar pattern as in the woodfrog (Lim. peroni tadpoles are not straightforward to reconcile with growth patterns. The high oxygen consumption rates of cold-acclimated tadpoles (regardless of incubation temperatures) indicates that oxygen consumption, and hence mitochondrial flux, is not driven predominantly by growth as would have been expected for larval animals. Cold-acclimated tadpoles were therefore either more active than warm acclimated animals, or more oxygen was used by cold-acclimated tadpoles for a given level of activity. An alternative is that activity at low temperature requires more ATP per unit muscle power output than at high temperature. Isolated gastrocnemius muscle from the African clawed frog Xenopus laevis used more oxygen at low (15°C) than at high (25°C) acute temperatures per Joule of work performed regardless of acclimation conditions. The reason for this difference is at least partly that the stiffness of the muscle is greater at low temperature and therefore requires greater cross-bridge activity to achieve force production Lim. peronii tadpoles. Additionally, cold acclimation can lead to increased proportions of oxidative fibres in fish The metabolic responses of Gambusia holbrookiUnlike growth and routine metabolic rates, incubation temperatures did affect the capacities for glycolytic and mitochondrial ATP production, and tadpoles from cross-over temperatures (cold incubation and warm acclimation or vice versa) had the greatest activities. The high lactate dehydrogenase activities of tadpoles developed from cool eggs but acclimated to warm temperatures matches their high burst swimming performance. Lactate dehydrogenase provides ATP quickly for short bursts of activity and it is commonly associated with burst performance max of enzymes is not necessarily rate-limiting. Flux through metabolic pathways can be controlled by changes in substrate concentrations and allosteric regulators at numerous points in a pathway rather than by single rate limiting steps The high activities of the mitochondrial enzymes at low acclimation temperature are consistent with our possible explanations for increased metabolic rates above, that is increased ATP demand for a given muscle power output, and increased proportions of oxidative fibres. However, the pattern of high mitochondrial enzyme activities at cold acclimation temperatures was apparent only in tadpoles from eggs developed at 25°C. Mitochondrial enzyme activities do not predict swimming performance and growth rates. It would not be expected that burst swimming performance is constrained by mitochondrial activities Developmental modification of phenotypes is increasingly recognised as a crucial mechanisms that underlies animal function


Self-heating induced failure of graphene devices synthesized from both chemical vapor deposition (CVD) and epitaxial means is compared using a combination of infrared thermography and Raman imaging. Despite a larger thermal resistance, CVD devices dissipate >3x the amount of power before failure than their epitaxial counterparts. The discrepancy arises due to morphological irregularities implicit to the graphene synthesis method that induce localized heating. Morphology, rather than thermal resistance, therefore dictates power handling limits in graphene devices. Epitaxial (Epi) graphene realized via the sublimation of silicon (Si) from silicon carbide (SiC) is limited only by the size of the supporting substrateBeyond the curiosity of an atomic dimension, it is the transport properties of graphene that differentiates it from traditional materials. Exhibiting a mobility that can exceed 200,000 cmThe same syntheses processes that provide scale, however, also induce morphological imperfections that limit device performance. Both epitaxial and CVD syntheses result in small regions of non-uniform layer number71012Self-heating of exfoliated graphene devices has been examined previously using infrared-thermography (IR)14151714141517e.g., SiO2) separating the graphene from the more thermally conductive substrate. While transfer of CVD graphene to SiC can capitalize upon the high thermal conductivity of the substrate, weak van der Waals bonding will result in a comparatively large thermal resistance at the interface 232425Solely from the perspective of thermal resistance then, epitaxial graphene provides an avenue to minimize self-heating and maximize power dissipation. Silicon carbide has a thermal conductivity ~3x that of silicon at room temperature21Self-heating is not determined exclusively from the thermal resistance, however, but also the area over which heat is generated. Factors localizing heat generation will exacerbate its effect. Here, we demonstrate that morphological imperfections localizing heat generation in a graphene device ultimately determine its power handling capability. Specifically, the breakdown power of epitaxial graphene on SiC devices is shown to be <3x that of comparable devices made up of CVD graphene atop a similar substrate. Correlations between the temperature distribution obtained with infrared–(IR) thermography and the morphology acquired using Raman imaging indicate that the difference arises from heat localization stemming from morphological features characteristic of the synthesis process. Altogether, the results demonstrate that the morphology of the graphene, rather than the system’s thermal resistance, dictates the amount of power that it can sustain.2 deposited via plasma enhanced chemical vapor deposition. This is termed— epitaxial/covered (Epi/Cov). The final architecture is composed of commercially synthesized CVD graphene  that is transferred to a SiC substrate and then covered with 50 nm of HfO2 realized via atomic layer deposition (ALD). This is termed—CVD/covered (CVD/Cov). Device geometries ranged in size from from 5 × 15 μm to 20 × 60 μm for each architecture and were contacted utilizing a Ti/Au stack (10/100 nm). Resistivity varied by ≤2x between the differing architectures and had comparable Raman responses largely absent of any defect signature (I(D)/I(G) ≤ 0.15, see Three graphene device architectures were fabricated at chip scale using standard photo-lithographic processes as shown schematically in 28For each architecture, six different devices of varying size were subject to increasing levels of Joule-heating until breakdown occurred. Breakdown was defined when channel resistance suddenly increased by several orders of magnitude. Only devices in which failure occurred in the channel—as opposed to the contacts—were included in the analysis. Practically, heating was induced by ramping current across the outer two leads to a target value held constant for 30s to facilitate thermography. Powers were measured by monitoring voltage across the inner contacts see . UtiliziBreakdown power differs significantly between the architectures see . Quantit2 carbon bonds limits the susceptibility of the graphene to electromigration2030Heating is of particular salience as the breakdown of graphene devices is thermal in origin203031et al.T > 1400 °C) than during the self-heatingExposed to the environment, graphene reacts with oxygen upon reaching temperatures of ~450 °C202/Si stack of an exfoliated device exhibits an out of plane thermal resistance of at least Ri = Li/Ki assuming thicknesses of 300 nm and 400 μm for the SiO2 and Si, respectively. Thermal conductivities of the two layers are presumed to be: KSi = 148 W/mKμm of SiC (KSiC = 450 W/mK) induces a resistance of RSiC = 0.9 m2K/MW —3x less than the exfoliated condition. Second, the exfoliated device possesses two thermally resistive interfaces—graphene to SiO2 and SiO2 to Si—to the epitaxial devices one—graphene to SiC. Third, the exfoliated devices have an area that is at least 8x less than the epitaxial devices. Under the assumption of uniform heating, these effects together will cause the exfoliated devices to heat up much faster and, by extension, fail at significantly lower powers. This is contrary to observation as, in reality, the epitaxial devices do not heat uniformly.Bare epitaxial devices failed at similar powers compared to previous reports of exfoliated devices see 20. This Epitaxial graphene emerges from the sublimation of Si from SiC resulting in concentration gradients of carbon that drive layer formation. The morphology of the underlying SiC affects the carbon concentration and thus the resulting graphene layerFor example, graphene multilayers form near atomic steps of the SiC leading to monolayer multilayer boundaries9Similarly, electrical resistance, and by extension heating, will increase locally in regions of lower mobility. Lower mobility has been correlated with regions of high variation in the 2D-mode peak positionLocalized heating and failure was also observed in epitaxial devices covered in an oxide implying that hot spot formation observed in 2. SiO2 degradation initiates at temperatures of 900–1000 °C3839402 as shown by the inset of Relative to the Epi/Bare device, the Epi/Cov architecture is capable of withstanding much larger powers see . The hig2, ALD layers of hafnia seeded with alumina begin to degrade near 900 °C43442 and HfO2 is ~900 °C. These oxides have negligible influence on the heat dissipation in either case since they are thermally insulative and extremely thin. Taken together, the difference in oxide species cannot be the cause for the 3x larger power handling capability of the CVD devices as compared to those epitaxially derived  Detector. A 20X objective having a lateral resolution of ~2 μm in size using standard photolithographic processes. Analogous CVD devices were fabricated by transferring ACS “Trivial Transfer” graphene onto 6H-SiC substrates using a procedure specified by the vendor. Conventional photolithographic techniques were employed to define and contact the devices2 was deposited using an established plasma enhanced chemical vapor deposition process.Quasi-free standing epitaxial graphene monolayer was synthesized using established methodologies292 was deposited using atomic layer deposition. To prevent graphene decomposition during the ALD process, a thin alumina layer is first grown onto the graphene surface. This protective alumina layer is formed by the controlled oxidization of a 10 Å, 0.2 Å/s electron beam evaporated aluminum thin film in pure oxygen at a partial pressure of 10 Torr. After a twenty minute exposure of the aluminum to this oxygen enriched environment, the high quality protective alumina layer is completely formed. This alumina layer not only serves to protect the graphene from potential reactants, it also appears to enhance the ALD hafnia nucleation process. Hafnia ALD was grown using a Picosun Sunale R150 hot-wall reactor operating at 250 °C using tetrakis(dimethylamido)hafnium(IV) (TDMAH) and deionized H2O precursors. At a source temperature of 75 °C, the TDMAH is introduced into the reaction space for a period of 1.5 s followed by a 15 s, N2 purge. To complete the formation of a single monolayer of hafnia, deionized water is then introduced for a period of 0.1 s followed by a 40 s, N2 purge. Both the TDMAH and H2O reaction cycles are repeated 550 times to grow a 50 nm hafnia gate dielectric.For the CVD/Cov devices, HfOHow to cite this article: Beechem, T. E. et al. Self-Heating and Failure in Scalable Graphene Devices. Sci. Rep.6, 26457; doi: 10.1038/srep26457 (2016).