KS) are not well understood. This study was conducted to assess the impact of general intelligence, personality, and social engagement on cognitive performance among patients with KS and a group of controls matched for age and years of education.The determinants of cognitive deficits among individuals with Klinefelter syndrome  scale, and measures of cognitive performance reflecting working memory and executive function.Sixty‐nine patients with KS performed more poorly on memory and executive‐function tasks. Patients with KS also exhibited greater neuroticism and less extraversion, openness, and conscientiousness than controls. Memory deficits among patients with KS were associated with lower intelligence, while diminished executive functioning was mediated by both lower intelligence and less social engagement.Patients with KS, memory deficits are principally a function of lower general intelligence, while executive‐function deficits are associated with both lower intelligence and poorer social skills. This suggests a potential influence of social engagement on executive cognitive functioning (and/or vice‐versa) among individuals with KS, and perhaps those with other genetic disorders. Future longitudinal research would be important to further clarify this and other issues discussed in this research.Our results suggest that among patients with  The controls ranged in age from 19.4 to 59.1 years old, with an average of 36.4 years. Although these controls were not karyotyped, and none of them exhibited any characteristics associated with Klinefelter syndrome, and all of them had normal testosterone levels. All controls were of Danish heritage. All participants provided informed consent, and the study was reviewed and approved by The Danish Data Protection Agency and the local ethics committee . This research has also been registered at ClinicalTrials.gov . Certain genetic, anatomical, and neuropsychological data from this research has been presented previously to address separate research questions ; extraversion is characterized by pronounced engagement with the external world ; agreeableness refers to an individual's desire for social harmony, and to get along with others ; conscientiousness is a tendency to show self‐discipline, act dutifully, and strive for achievement ; and openness to experience reflects one's interest in, and appreciation for art, emotion, curiosity, novelty, adventure, and a variety of experiences . For all items, participants indicated how well each statement described them—relative to other people of the same sex and comparable age—on a scale from 1 (“very inaccurate”) to 5 (“very accurate”). Items within each trait were then combined to create an overall score for each personality type , short form ; attention switching reflects an individual's ability to transfer attentional focus from one target to another (e.g. “I frequently get so strongly absorbed in one thing that I lose sight of other things”); imagination refers to an individual's capacity for abstraction and envisioning alternative realities (e.g. “I find it very easy to play games with children that involve pretending”); communication reflects one's ability to relate his or her thoughts, feelings, and ideas to others (e.g. “I find making up stories easy”); and social skills include interests and abilities that facilitate positive social interactions and relationships . For all items, participants indicated how well each statement described them on a scale from 1 (“definitely disagree”) to 4 (“definitely agree”). Negatively worded items were reverse coded, so that higher scores for each item reflected more of each social characteristic. Items within each measure were then combined to create an overall score for each aspect of social engagement. Cronbach's alphas in the current study for these social engagement measures range from 0.61 to 0.74.Participants’ level of social engagement was assessed using the Autism Spectrum Quotient (AQ), which includes measures of attention to detail (10 items), attention switching (10 items), imagination (10 items), communication (10 items), and general social skills (10 items) , which combined two subscales of verbal IQ—vocabulary (V) and similarities (S)—and two subscales of performance IQ—matrix reasoning (MR) and block design (BD)—from the Wechsler Adult Intelligence Scale, Third Edition (WAIS‐III)  and letter‐number sequencing (LN)—with the Rey Auditory Verbal Learning Test (RAVL) , which is designed to measure an individual's cognitive flexibility in the face of changing schedules of reinforcement  in which each of these two dependent variables – after entering KS status – was regressed on following independent variables (1) personality traits, (2) social engagement measures, (3) intelligence, and (4) testosterone status, respectively. Finally, two path analyses—one for working memory and one for executive function—were conducted to identify the unique associations between each of the bivariate predictors of cognitive performance.t‐tests, with p‐values lower than .05 considered significant. All analyses were conducted using SPSS version 19.0 .Continuous measures were tested for normality using the Kolmogorov‐Smirnov test, and those with distributions of Dα > 0.23 were considered non‐normal, and were normalized using a log‐linear (ln+1) transformation. Differences between patients with KS and controls measures were assessed using two‐sample Student's 33.13.1.1ps ≤ .01), but the two groups did not differ significantly in terms of agreeableness (p > .33) , patients with KS scored significantly lower than controls on attention switching, imagination, communication, and social skills (ps < .01)(see Table ps ≤ .001).General intelligence scores of patients with KS were significantly lower than those of the controls (3.23.2.1p < .05). Testosterone levels among patients with KS receiving testosterone therapy (20.7 nmol/L ± 11.4) were significantly higher than those not receiving this therapy (9.61 nmol/L ± 5.38) (p < .001).As shown in Table 3.2.2ps ≥ .33), and because neither conscientiousness, attention switching nor testosterone levels were associated with cognitive performance, these variables were not included in subsequent analyses. As shown in Table ps < .01). Better memory and executive function were also associated with lower neuroticism, and more extraversion and openness to experience (ps < .05). Greater imagination, communication, and social skills were all related to better working memory (ps < .05), but only imagination and social skills predicted executive‐function performance (ps < .05).Because patients with KS and controls did not differ significantly in terms of age, agreeableness, or attention to detail ps ≥ .3, and be3.2.3R2 = .19, p < .001), but KS status was the only significant predictor (p < .001) , with both KS status and imagination emerging as significant predictors (ps < 0.01). Combining KS status and intelligence also predicted working memory , but this was attributable to the impact of intelligence (p < .001). Combining KS status and testosterone status, the full model was again significant , but this was attributable to the impact of KS status.When KS status and personality measures  were combined to predict working memory (dependent variable), the overall model was significant , with KS status again emerging as the lone significant predictor (p < 0.01). Executive function was also predicted by KS status and social engagement  , with KS status and social skills as the significant predictors , which was principally attributable to the impact of intelligence (p ≤ .01), although KS status was a marginal predictor (p < .06). When combining KS status and testosterone status, the overall model was significant , but this was attributable specifically to KS status.As shown in Table 3.2.4ps < .001). In the second path model . In addition, KS also led to poorer social engagement, which was in turn related to executive‐function deficits (ps ≤ .002). Finally, intelligence also mediated the impact of KS status on imagination (p = .002).In the first path model Figure a, intelll Figure b, intell4In this research, working memory deficits among patients with KS were attributable directly to lower intelligence (as measured by IQ), while their executive‐function deficits were associated with both lower intelligence and poorer social skills. Consistent with previous findings, linking KS status and intelligence with cognitive performance  path model are consistent with previous literature linking social skills and executive function in the general population  predict cognitive function. This is consistent with prior research finding. No cognitive differences observed between KS patients treated with testosterone and those who were not , health‐related behaviors other than substance abuse, and other personal experiences. Further research examining the impact of these and other factors on the cognitive function of patients with KS—and others—would help refine our understanding of these outcomes.Although this research was cross‐sectional, the directionality of genetic factors such as KS is clear. Similarly, personality traits are established early in life, social‐engagement skills are relatively stable over time, and both are thought to precede situational assessments of cognitive performance. On the other hand, while intelligence may influence imagination, the reverse is also possible. The directionality of these and other relationships can be assessed more directly by future longitudinal research, including tracking of developmental trajectories  of these factors among patients with KS and other populations associated with cognitive impairment, including Down′s syndrome (Roizen & Patterson, Given that patients with KS vary widely in their neuropsychological phenotype, the fact, that the patients with KS participating in this study were recruited through fertility clinics, endocrinology clinics and genetic departments may introduce ascertainment bias. Thus, these patients may differ systematically from others with KS who are either not diagnosed or followed by hospital departments and fertility clinics. It is estimated that, only 25% of males with KS are actually diagnosed, which may limit the external validity of KS research to date. However, the patients with KS in the current study exhibited a broad range of intelligence and neuropsychological phenotype, as well as age and testosterone treatment. In addition, the neuropsychological profiles of these patients correspond well to the profiles of patients with KS in the existing literature, supporting the generalizability of the current results to others with KS who have been identified.This research involved a comprehensive, biopsychosocial study of individuals with a sex chromosomal disorder, and is the first to examine the combined effects of intelligence, personality, and social engagement on cognitive performance among patients with KS and controls. In addition to clarifying the respective roles these factors play in working memory and executive function, these findings also have important implications for strategies to improve cognitive performance among patients with KS, and perhaps in the general population. This interdisciplinary approach can also serve as a model for future research, the results of which can help us better understand and address genetic disorders, as well as the profound personal, social, and economic problems they can cause.Authors do not have any potential sources of conflict of Interest.


Dopaminergic neurotransmission mediates a majority of the vital central nervous system functions. Disruption of these synaptic events provokes a multitude of neurological pathologies, including Parkinson's, schizophrenia, depression, and addiction. Growing evidence supports a key role for noncoding RNA (ncRNA) regulation in the synapse. This review will discuss the role of both short and long ncRNAs in dopamine signaling, including bioinformatic examination of the pathways they target. Specifically, we focus on the contribution of ncRNAs to dopaminergic dysfunction in neurodegenerative as well as psychiatric disease. Dopamine is a potent neurotransmitter in the central nervous system that governs a diverse panel of neuronal functions. Accumulating studies indicate that noncoding RNAs (ncRNAs) modulate almost all aspects of dopamine signaling, including its receptors, and transporter, as well as differentiation and viability of dopaminergic neurons. The processing and transcription of those implicated ncRNAs are also subject to control. Moreover, there are emerging roles for both short and long ncRNAs (lncRNAs) in synaptic transmission, including miRNAs, and antisense transcripts. This review discusses ncRNA-mediated mechanisms which influence dopaminergic signaling under control and pathogenic conditions.The diverse classes of ncRNAs target all stages in the lineage from stem cells to mature dopaminoceptive neurons (DN) which express dopamine receptors. One of the first ncRNAs reported to modulate midbrain DN (mDN) differentiation and viability was miR-133b , however, D1R, and D2R are the most abundant , these patients classically display upregulated expression of D1R and D2R with hypersensitive response to dopamine  allele, which could alter the allelic representation of DR1.Disease-associated polymorphic regions in the dopamine receptor genes can impact miRNA binding to their target sites. One such reported example is the D1R rs686 polymorphism linked to nicotine dependence  is critical for removal of dopamine in the synapse to halt neurotransmission , eliciting phenotypes which recapitulate PD   is significantly repressed in dopaminergic neurons of PD models , which are primary responders to drugs of abuse, revealed lncRNA modulators of addiction phenotypes (Bannon et al., A recent report now also suggests Gomafu, a schizophrenia associated lncRNA, modulates dopaminergic transmission (Ip et al., Epigenetic regulation of dopamine signaling by ncRNAs is a vital component to neuronal homeostasis. Correspondingly, impaired dopaminergic transmission provokes a range of cognitive, motor, and behavioral phenotypes in CNS disorders. The untapped pharmacological potential of ncRNA modulators to normalize aberrant synaptic transmission provides a wide-open opportunity for drug discovery efforts. Furthermore, as the dopamine pathway is extensive and partially driven by undefined mechanisms, the pleiotropic capacity of many implicated ncRNAs may enhance their efficacy.WC, BB, MC, and JK all wrote this manuscript.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.


Antiferromagnetic spintronics is an emerging field; antiferromagnets can improve the functionalities of ferromagnets with higher response times, and having the information shielded against external magnetic field. Moreover, a large list of aniferromagnetic semiconductors and metals with Néel temperatures above room temperature exists. In the present manuscript, we persevere in the quest for the limits of how large can anisotropic magnetoresistance be in antiferromagnetic materials with very large spin-orbit coupling. We selected IrMn as a prime example of first-class moment (Mn) and spin-orbit (Ir) combination. Isothermal magnetotransport measurements in an antiferromagnetic-metal(IrMn)/ferromagnetic-insulator thin film bilayer have been performed. The metal/insulator structure with magnetic coupling between both layers allows the measurement of the modulation of the transport properties exclusively in the antiferromagnetic layer. Anisotropic magnetoresistance as large as 0.15% has been found, which is much larger than that for a bare IrMn layer. Interestingly, it has been observed that anisotropic magnetoresistance is strongly influenced by the field cooling conditions, signaling the dependence of the found response on the formation of domains at the magnetic ordering temperature. During the recent years, antiferromagnetic (AF) materials1j). Therefore, AMR is useful to infer perpendicular magnetic states in AFs. Indeed AMR has been used to obtain different resistive states corresponding to different magnetic states in AF semiconductor (Sr2IrO4)1191114Ways to read/write on AF are currently under development72IrO4As mentioned, reports regarding AMR measurements on AF intrinsically modifying their microscopic magnetic moment vector are limited to Sr2CoMnO6) and magnetically coupled to it. Therefore, the unique metallic part in the bilayer is the AF IrMn layer. This distinct measurement configuration allows us to perform in-plane transport measurements and infer a sizeable AMR response (∼0.15%) in a metallic AF (IrMn) using exchange coupling. This value is one order of magnitude larger than that obtained measuring a bare IrMn film thus, confirming the intrinsic nature of the effect and the coupling between FM and AF layers.In the present work, we focus on the AMR effect in an IrMn layer grown on top of an insulating FM  bilayer is sketched in C) of LCMO at 250 K. M(H) magnetization loop recorded at 5 K along the [100]LCMO (in-plane) is also shown in C = 1 T is estimated. Note also that M(H) curve is still hysteretic up to about 3 T.The characterized AF-metal (IrMn-2 nm)/FM-insulator . Archetypical anisotropic magnetoresistance (AMR = (R(Φ) − R(Φ = 0))/R(Φ = 0)) measurements have been performed by rotating the applied magnetic field by an angle Φ in the plane of the sample as sketched in vanishes , reveali0·tanh[(H − HC)/δH] + χPM·H0 accounts for the switchable magnetization, HC for the magnetic coercive field, δH for the width of the magnetic susceptibility, and χPM for the paramagnetic contribution. Lines through data point correspond to the fitted data in EB = HC+ − HC−)/2] after the fitting is plotted in EB magnitude (HEB ≈ 1.5 mT) lead to an interface energy (ΔE = HEtFMMFM) of ≈0.01 erg/cm2, which is considered low, but in agreement with previous estimations for IrMn films grown on other ferromagnetic materialsThe existence of magnetic coupling between both layers is also signaled by the shift in the magnetic hysteresis loops recorded at 5 K, performed after cooling the LCMO/IrMn bilayer with a magnetic field applied along opposite directions. These loops are shown in c is about 1 T as shown by M(H) loops of The dependence of the AMR response on the intensity of the applied magnetic field is shown in C and TN18Finally, the temperature dependence of the AMR (at 3 T) is shown in We report sizeable AMR response of 0.15% in LCMO/IrMn bilayer system that is uniquely ascribed to IrMn, since the resistivity of LCMO layer is about 9 orders of magnitude above that of IrMn layer. This is observed in contrast with the negligible effect measured in a stand-alone IrMn layer, being former result the first isothermal measurement of AMR in a metallic antiferromagnetic material. The effect is observed owing to the existence of a magnetic exchange coupling between the LCMO and IrMn adjacent layers via exchange spring effect, also revealed by direct magnetometric data. The dependence of AMR response on the field cooling conditions and temperature together with the asymmetric behavior of the AMR response depending on the field cooling conditions, allows safely discarding uncompensated spins as the source of the observed behavior.2CoMnO6 (LCMO) layer, both prepared in-situ by RF magnetron sputtering. The LCMO layer was c-oriented, epitaxial and fully strained (see ref. 3 substrate. The stacking sequence is shown in An IrMn layer (2 nm) was grown on top of a ∼15.8 nm thick La(S)TEM images have been acquired by using FEI-TITAN 80–300 operated at 300 kV.The resistance of the bilayer has been measured using a 4-points contact method. The contacts are placed on top of the structure as shown by How to cite this article: Galceran, R. et al. Isothermal anisotropic magnetoresistance in antiferromagnetic metallic IrMn. Sci. Rep.6, 35471; doi: 10.1038/srep35471 (2016).


Compared to controls, t-AUCB–treated mice had increased CRH, including repayment volume (RV), repayment duration, and repayment/debt ratio (p < 0.05). Treatment with t-AUCB significantly changed oxylipin profiles, including an increase in EET/DHET ratio, increase in EpOME/DiHOME ratio, increase in the levels of HODEs, decrease in the levels of mid-chain HETEs, and decrease in prostanoids (p < 0.05). Treatment with MS-PPOH (CYP epoxygenase inhibitor) reduced CRH, including RV (p < 0.05). Involvement of PPARγ in the modulation of CRH was demonstrated using a PPARγ-antagonist (T0070907) and a PPARγ-agonist (rosiglitazone). T0070907 reduced CRH (p < 0.05), whereas rosiglitazone enhanced CRH (p < 0.05) in isolated mouse hearts compared to the non-treated. These data demonstrate that sEH inhibition enhances, whereas CYP epoxygenases-inhibition attenuates CRH, PPARγ mediate CRH downstream of the CYP epoxygenases-EET pathway, and the changes in oxylipin profiles associated with sEH-inhibition collectively contributed to the enhanced CRH.Coronary reactive hyperemia (CRH) is a physiological response to ischemic insult that prevents the potential harm associated with an interruption of blood supply. The relationship between the pharmacologic inhibition of soluble epoxide hydrolase (sEH) and CRH response to a brief ischemia is not known. sEH is involved in the main catabolic pathway of epoxyeicosatrienoic acids (EETs), which are converted into dihydroxyeicosatrienoic acids (DHETs). EETs protect against ischemia/reperfusion injury and have numerous beneficial physiological effects. We hypothesized that inhibition of sEH by  The left atrium was excised, and a water-filled balloon made of plastic wrap was inserted into the left ventricle through the mitral valve. The balloon was connected to a pressure transducer for continuous measurement of left ventricular developed pressure (LVDP) and heart rate (HR). The heart was then immersed in a water-jacketed perfusate bath (37°C) and left to beat spontaneously. Left ventricular diastolic pressure was adjusted to 2–5 mmHg. A flow transducer was installed above the cannulated aorta for continuous measurement of CF with an ultrasonic flow probe . A Power–Lab Chart data acquisition system  was used for data acquisition. Heart function was allowed to stabilize for 30–40 min before initiation of CRH. Only hearts whose CF increased by more than two fold after a 15-second total occlusion were included in the analysis. This demonstrated that the isolated heart preparation was intact to be included in the experiment. Hearts with persistent arrhythmias or LVDP <80 mmHg were excluded.Soluble epoxide hydrolase null (sEHt-AUCB (trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid , and 1 μM for MS-PPOH . These concentrations are the same, or less, as used in previous studies: rosiglitazone, 10 μM; , PG-F2α, thromboxane B2 [TxB2], PGD2, and PGE2) were determined in pre- and post-CRH heart perfusates of WT and t-AUCB-treated WT mice through liquid chromatography, tandem mass spectroscopy (LC-MS/MS) as described previously [t-AUCB to block further EET breakdown by sEH. Heart perfusates were collected two times before ischemia (baseline) and pooled together as one sample and two times after ischemia and pooled together as another sample for LC-MS/MS analysis. Samples were stored at –80°C until processing. Samples were spiked with 30 ng PGE2-d4, 10,11- DiHN, and 10,11-EpHep (Cayman) as internal standards, mixed with 0.1 vol of 1% acetic acid in 50% methanol, and extracted by serial passage through Oasis HLB C18 3mL columns . Columns were washed twice with 0.1% acetic acid in 5% methanol and eluted with methanol into glass tubes containing 6 μL of 30% glycerol in methanol. The methanol was then evaporated under a stream of nitrogen gas, and the dried tubes were frozen and stored at –80°C until analysis. Online liquid chromatography of extracted samples was performed with an Agilent 1200 Series capillary HPLC . Separations were achieved using a Halo C18 column , which was held at 50°C. Mobile phase A was 85:15:0.1 water: acetonitrile: acetic acid. Mobile phase B was 70:30:0.1 acetonitrile: methanol: acetic acid. Flow rate was 400 μL/min; Gradient elution was used. Mobile phase percentage B and flow rate were varied as follows: 20% B at 0 min, ramp from 0 to 5 min to 40% B, ramp from 5 to 7 min to 55% B, ramp from 7 to 13 min to 64% B. From 13 to 19 min the column was flushed with 100% B at a flow rate of 550 μL/min. Samples were solvated in 50 μl of 30% ethanol. The injection volume was 10 μL. Samples were analyzed in triplicate. Analyses were performed on an MDS Sciex API 3000 equipped with a TurboIonSpray source (Applied Biosystems). Turbo desolvation gas was heated to 425°C at a flow rate of 6 L/min. Negative ion electrospray ionization tandem mass spectrometry with multiple reaction monitoring was used for detection.Levels of oxylipins  were analyzed for each heart and averaged. MS-PPOH was infused at a final concentration of 1.0 μM for 15 minutes, after which the second CRH was induced. CRHs before and after MS-PPOH infusion were analyzed and compared.Isolated WT mouse hearts were stabilized for 30–40 min followed by 15 sec of total occlusion. Recordings of the first CRH  were analyzed for each heart and averaged for each group, as mentioned previously. T0070907 was infused at a final concentration of 10 μM and 1% of CF rate for 15 min, after which another CRH was induced and the same parameters  were recorded again and analyzed.t-AUCB. We evaluated the effects of selective sEH-inhibition on CRH followed by PPARγ-antagonist. Baseline CRH was induced in WT mice. t-AUCB was infused at a final concentration of 10 μM for 15 minutes, after which a second CRH was induced. T0070907 was infused while t-AUCB was still being infused, at a final concentration of 10 μM for another 15 min, and a third CRH was induced. The three CRH responses were analyzed and compared.In this experiment, we investigated a possible link between the role of PPARγ in CRH and pharmacological inhibition of sEH by After stabilization, WT mouse hearts were subjected to 15 sec of total occlusion. As described above, baseline CRH was induced in each mouse heart. Rosiglitazone was infused at a final concentration of 10 μM for 15 min, followed by another CRH. CRHs before and after rosiglitazone infusion were analyzed and compared.–1.g wet heart weight–1). Values are means ± standard error; n represents the number of animals. For data analysis, two-tailed paired t-tests were used for paired data analysis, repeated measures ANOVAs were used for populations measured 3 times, and two-way ANOVAs were used to compare data between groups. Differences were considered statistically significant when p < 0.05.Flow debt (baseline flow rate multiplied by occlusion duration) and RV  were calculated using “the integral relative to baseline” function in the data pad of Lab-Chart 7.0 software. Since absolute coronary flow rates change proportionally with heart mass, the RV and flow debt are presented as ml/g wet heart weight, and baseline and peak flow rate data are presented as , and 11,12-DHET were detected. An increasing trend in the level of 14,15-EET in t-AUCB-treated WT versus WT mice was observed at baseline and post-ischemia, but was not significant  and post-ischemia  compared to baseline, but this was only significant for 5-, 11-, and 15-HETE  in WT and t-AUCB-treated WT versus WT mice, but was not significant  epoxides  levels had an increasing trend at baseline and post-ischemia in t-AUCB-treated WT versus WT mice at baseline and post-ischemia  only for TxB2 .Pharmacologic inhibition of sEH by  earlier . This saInfusion of rosiglitazone enhanced CRH in WT mice. In rosiglitazone-treated WT versus WT mice, RV increased by 33%  Inhibition of sEH by t-AUCB enhanced CRH; 2) Treatment with t-AUCB significantly changed oxylipin profiles, including an increased EET/DHET ratio, increased EpOME/DiHOME ratio, increased HODEs, decreased mid-chain HETEs, and decreased prostanoids; 3) Inhibition of CYP epoxygenases (by MS-PPOH) attenuated CRH; 4) The PPARγ antagonist T0070907 decreased CRH, and 5) the PPARγ agonist rosiglitazone increased CRH.Pharmacologic inhibition of sEH in WT mice enhanced CRH in an isolated heart model after brief ischemia. sEH inhibition was also associated with changes in various oxylipin profiles. The relationship among inhibition of CYP epoxygenases, inhibition of sEH, oxylipins, and PPARγ in the modulation of CRH in isolated mouse hearts is not known. Therefore, this study was designed to investigate the role of sEH, oxylipins, and PPARγ in the modulation of CRH using isolated WT mouse hearts. Our data demonstrated that: t-AUCB significantly enhanced CRH after brief ischemia compared to non-treated WT mice. The association between compromised CRH and some cardiovascular pathologies [t-AUCB-enhanced CRH was accompanied by a 1.0- (at baseline) to 1.7- (post-ischemia) fold increase in the 14,15-EET/DHET ratio, primarily driven by a decrease in 14,15-DHET. Although, a trend of increase in 14,15-EET level was observed in t-AUCB–treated vs. non-treated WT mice, the difference was not statistically significant. This could be attributed to several factors including the acute use of t-AUCB for a short period, the short half-life of EETs compared to DHETs, and the short duration (15 seconds) of ischemic insult. Another dihydroxy-EET metabolite  decreased in t-AUCB–treated mouse hearts, further confirming the efficacy of the sEH-inhibitor, t-AUCB. The specificity of t-AUCB as a sEH inhibitor was tested using sEH knockout (sEH–/–) mice, where no difference in CRH between the t-AUCB-treated sEH–/–versus non-treated sEH–/–mice was observed. The rapid hydration of EETs [t-AUCB–treated or non–treated groups (data not presented). Based on our data, the enhancement of CRH associated with sEH inhibition in WT mice may be partially due to an increase in the EET/DHET ratio.Inhibition of sEH by hologies –28 confi of EETs  may expl of EETs –41, incl of EETs , kidney  of EETs , and bra of EETs . Inhibittabolite ,12-DHET  of EETs , whereas of EETs . Besides of EETs . In the  of EETs . Anothert-AUCB treatment at baseline and post-ischemia. Mid-chain HETEs are produced through allylic oxidation of AA by lipoxygenase (LOX) [t-AUCB) has also contributed to blocking the enhanced conversion of EETs to DHETS by HETEs. This in turn would contribute to the enhanced CRH through an increase in EET/DHET ratio. Based on these results, the decrease in mid-chain HETEs in both t-AUCB-treated and non-treated mice in response to ischemia suggests that these metabolites are down regulated in response to ischemic episodes. This is an interesting finding because HETEs were the only oxylipins analyzed in this paper whose levels were affected by the brief ischemia. Also, the further decrease of these HETEs by t-AUCB–treatment may have played a role in the enhanced CRH observed in t-AUCB–treated hearts.Mid-chain  HETE levels decreased in response to ischemia and to se (LOX) . They wese (LOX) –19. Alsose (LOX) –52. Unlise (LOX) , 12-HETEse (LOX) . In humase (LOX) , whereasse (LOX) . Also, tse (LOX)  suggestise (LOX) . Moreovese (LOX) . Therefot-AUCB was further confirmed by an increased EpOME/DiHOME ratio. This resulted from increased 9,10-,12,13-EpOMEs , and decreased 9,10-, 12,13-DiHOMEs in t-AUCB–treated versus non-treated mice. The physiological significance of EpOMEs and DiHOMEs remains poorly understood, and the evidence is somewhat contradictory, with a few studies suggesting toxic effects, while others indicating beneficial effects of these bioactive metabolites. EpOMEs and DiHOMEs were reported to increase oxidative stress in vascular endothelial cells [t-AUCB–treated mouse heart perfusates may have contributed to enhancing CRH.Linoleic acid (LA) epoxidation metabolites, such as 9,10-, and 12,13-EpOMEs, and their corresponding sEH-metabolized DiHOMEs were detected in the heart perfusate samples. As expected, inhibition of sEH by al cells ; DiHOMEsal cells ; and intal cells . The maial cells . Contraral cells . Mitchelal cells . Pretreaal cells . Also, tal cells . Based ot-AUCB-treated WT. Like EpOMEs, the physiologic functions of HODEs are still being investigated [2) biosynthesis, was involved in splenic and coronary artery relaxation in smooth muscle cells in Mongrel dogs [–/–compared to sEH+/+ [–/–(genetic deletion of sEH) and pharmacologic inhibition of sEH are not necessarily the same [t-AUCB–treated mouse heart perfusates might have played a role in enhancing CRH, whereas the role of 9-HODE is not yet known based on its, thus far, described biologic functions.Linoleic acid (LA) is also metabolized through hydroxylation by CYP epoxygenases to form hydroxyl-LA metabolites known as hydroxyoctadecadienoic acids (HODEs) . The detstigated . 13-HODEstigated –67. Alsorel dogs . 9-HODE,rel dogs , 69. In rel dogs . In conto sEH+/+ . This dio sEH+/+  used urithe same . Therefot-AUCB significantly decreased prostanoid levels, including PGs and TxB2. PG-G2 and PG-H2, AA metabolites formed by COX isoforms (1 and 2), are converted to the 4 main bioactive PGs  and thromboxanes (TxA2 and TxB2) [2 augments arterial dilation and increases microvascular permeability [2 was found to have an anti-inflammatory role as well by up-regulating cAMP and inducing secretion of the anti-inflammatory IL-10 [2 attenuated inflammation in experimental models of pleuritis and colitis [1α, the non-active hydrolysis product of prostacyclin (PG-I2), and TxB2, the inactive degradation product of TxA2 [t-AUCB. PG-F2α is elevated in patients with chronic inflammatory diseases [2, PG-E2, 6-keto-PG-F1α, and TxB2 [2 and PG-E2 [t-AUCB. However, the impact of these changes on the t-AUCB–enhanced CRH is not yet clear.Treatment of WT mice hearts with nd TxB2) , 71. Mosry IL-10 . Similarand TxB2 . Also, sand TxB2 . Combinand PG-E2 . TherefoAs mentioned earlier, EETs are generated from AA by cytochrome P450 (CYP) epoxygenase enzymes, primarily CYP2C and CYP2J. We targeted the CYP epoxygenase-EET pathway using pharmacologic inhibition of CYP-epoxygenases to further support our findings of the involvement of CYP-epoxygenases in modulating CRH. sEH-inhibition increases the level of EETs by blocking their breakdown; we investigated the effect of blocking the synthesis of EETs on CRH using a CYP epoxygenases-inhibitor. Evidently, inhibiting CYP epoxygenases would negatively impact EET synthesis and, therefore, their effects. P450 enzymes are highest in coronary arteries and in small arterioles . Our datt-AUCB–enhanced CRH in WT mouse hearts. Studies indicated that EET effects could be mediated by PPARγ receptors [t-AUCB–enhanced CRH in WT mice (by T0070907) suggests that PPARγ may mediate CRH downstream of CYP epoxygenases-EET pathway.This study also showed that CRH was decreased by T0070907, a PPARγ-antagonist, whereas rosiglitazone, a PPARγ-agonist, increased CRH. Moreover, T0070907 attenuated eceptors –23. Liu eceptors . Our resIn summary, the findings of this study suggest that the CYP epoxygenases-EET pathway may have a role in mediating CRH possibly through PPARγ activation, as noted by sEH-inhibition, which enhanced CRH, and CYP epoxygenases-inhibition, which decreased CRH. Also, sEH-inhibition is accompanied by changes in AA oxylipins  and LA oxylipins , which might have collectively accounted for the observed enhancement of CRH. Therefore, we conclude that sEH-inhibition enhances, whereas CYP epoxygenases-inhibition attenuates CRH, PPARγ activation mediates CRH downstream of the CYP epoxygenases-EET pathway, and the changes in oxylipin profiles associated with sEH-inhibition collectively account for the enhanced CRH.


In this study, a precursor for carbon nanofibers (CNF) was fabricated via electrospinning and carbonized through a thermal process. Before carbonization, oxidative stabilization should be applied, and the oxidation mechanism also plays an important role during carbonization. Thus, the understanding of the oxidation mechanism is an essential part of the production of CNF. The oxidation process of polyacrylonitrile was studied and nanofiber webs containing graphene oxide (GO) are obtained to improve the electrochemical properties of CNF. Structural and morphological characterizations of the webs are carried out by using attenuated total reflectance Fourier transform infrared spectroscopy and Raman spectroscopy, scanning electron microscopy, atomic force microscopy and transmission electron microscopy. Mechanical tests are performed with a dynamic mechanical analyzer, and thermal studies are conducted by using thermogravimetric analysis. Electrochemical impedance spectroscopy, and cyclic voltammetry are used to investigate capacitive behavior of the products. The proposed equivalent circuit model was consistent with charge-transfer processes taking place at interior pores filled with electrolyte. Carbon nanofibers are of great interest because of their chemical similarity to fullerenes and carbon nanotubes. Carbon nanofibers (CNF) have promising electrochemical and mechanical properties and a potential for a variety of applications; such as supercapacitor applications, battery applications, and catalyst support materials. Polyacrylonitrile (PAN) is one of the well-known precursor for obtaining carbon nanofibers that have a diameter ranging between nanometers and micrometers and exhibit a high surface area and a high electrical conductivity.Also, nanofibers can be used with polymeric structures to generate composite materials to improve the electrochemical properties of polymeric structures –3. Nanof2, O2, H2) and structural impurities is accelerated and the carbon concentration inside the structure is simultaneously increased.Oxidative stabilization is a crucial heat-treatment process to produce carbon fibers from PAN fibers. PAN chains start to cross-link during this process and the newly composed polymeric structure can endure the rigors of high-temperature processing –11. OxidTg) of PAN homopolymer is reduced by the addition of a co-monomer to form P(AN-co-AA), enhancing cyclization reactions and the formation of thermally stable aromatic ladder polymer chains [The most common co-monomers of acrylonitrile in the acrylonitrile copoymers are: vinyl acetate, itaconic acid, methyl metacrylate and acrylic acid –20. Co-mr chains . Acidic r chains ,18.Graphene has several desirable features, such as high surface area, high aspect ratio and other properties comparable to those of carbon nanotubes. Thus, graphene attracts attention in science as a new class of material for polymer-based composites . GraphenIn this paper, graphene oxide was used as an additive to increase the capacitance of oxidized PAN-based nanofibers. Further, GO addition was studied to improve electrochemical properties of CNF webs.Mw 150,000 g/mol) was purchased from Sigma-Aldrich and was used as received. Dimethylformamide , sulfuric acid , acetonitrile  were chosen as solvents and were used without any further purification. Graphene oxide  was purchased from Grafen Chemical Industries and used as received. The properties of the few-layered GO are: GO consists of a few layers (1–10 layers) and the average thickness of the layer is smaller than 4 nm. The specific surface area of GO is larger than 550 m2/g. GO consists of 68.44 atom % C, 30.92 atom % O and 0.63 atom % S.Polyacrylonitrile  DMF evaporated and nanofiber formation was achieved on the collector. Those nanofibers stacked and formed a web. Different collectors were used to fabricate PAN-based nanofiber webs via electrospinning. Before the electrospinning process, GO was also added to the PAN/DMF solution to obtain PAN/GO nanofibers. After electrospinning of the PAN/GO nanofibers, the PAN/GO samples underwent the same heat treatment (oxidation and carbonization) as the PAN nanofibers. Rotating and fixed collectors were used to vary the samples and investigate physical and chemical changes.Electrospinning solutions were prepared at different PAN/DMF ratios. Electrospinning parameters  effect the nanofiber diameter and homogeneity. Lower viscosity helps to produce finer nanofibers, and an increased polymer weight percentage results in higher viscosities. Thus it is one of the significant parameters for electrospinning .In this study, graphene oxide was used as an additive to increase the capacitance of oxidized PAN-based nanofibers. Thus, the nanofibers were produced via electrospinning using a mixture of PAN (10% w/v) and a given amount of GO (at different weight-to-volume percentages) in DMF. The solutions were poured into a 2 mL syringe and delivered at a constant flow rate of 1.0 mL/h  to a needle with a blunt tip connected to a high-voltage power supply (Gamma high voltage research) producing a voltage of 15 kV. Aligned nanofibers were deposited on the rotating drum collector at 21.50 Hz rotating frequency at a distance of 15 cm. After producing the nanofibers, oxidative stabilization was performed at 250 °C for 3 h in air atmosphere and carbonization was performed at 900 °C for 1 h under nitrogen atmosphere.Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and Raman spectroscopy were used to record the characteristic peaks of the oxidized and carbonized nanofibers. Mechanical properties of nanofiber webs were characterized by using a dynamic mechanical analyzer (DMA) .Thermal behavior of nanofiber webs was examined with thermogravimetric analysis . The structure of the nanofiber webs was characterized by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) . The microstructure of the carbonized nanofiber webs was investigated by Raman spectroscopy . The sample morphologies were characterized by scanning electron microscopy (Gemini Leo Supra 35 VP) and samples were coated with thin gold film using a sputter coater to prevent the accumulation of charge on their surface.TM software. AFM analyses were performed with a non-contact mode by using NCLR-10 model Al-coating silicon tips with 7 μm thickness, 225 μm length, 38 μm width, 190 kHz resonance frequency and 48 N/m force constant. Surface morphology of the nanofibers was observed with scanning electron microscopy (SEM) at Namık Kemal University and transmission electron microscopy (TEM). Fiber diameters were measured within electron micrographs from a population between forty and fifty nanofibers taken from each sample and then the average values were calculated by ImageJ software.Electrochemical performances of nanofibers were analyzed by using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Electrochemical measurements were performed by using potentiostat 2263 Electrochemical Analyser . EIS data were simulated with the electrical equivalent circuit by ZSimpWin V.3.10 analysis program . The surface topography of the fibers was observed by atomic force microscopy (AFM) with Nanosurf Easy-Scan2Oxidative stabilization is a complex process and should be applied to the webs before carbonization. The mechanism plays an important role in the carbonization. Therefore, a detailed understanding of the mechanism of oxidation has an important part in the success of the production of CNF.Nanofiber webs are produced with different collectors to achieve fiber alignment. The results for webs of aligned and non-aligned nanofibers are compared. A rotating collector that produces aligned nanofiber webs reduces the nanofiber diameter as shown in SEM images in −1 represent the absorption of C≡N triple bond [−1 can be assigned to a combined effect of C=N, C=C, N–H groups [−1 is connected to C–H bonds [−1 because of the formation of =C−H bonds [2O [The oxidation process including conversions of C≡N bonds to C=N and also dehydrogenation leading to aromatic and supramolecular structures was studied . Structuple bond ,22,35. TH groups ,17,35–36–H bonds –38. ATR-–H bonds ,36,39. A–H bonds . During −H bonds –41. In tThe oxidation ratio can be calculated from the absorbance ratio obtained from ATR-FTIR results ,39–40. T2 and in O2 atmosphere) exhibit the same trend. However, the region with no weight loss is shifted in N2 atmosphere because of N2 suppresses the reactions compared to O2, according to TGA measurements, the PAN polymer stays stable up to ca. 300 °C. This stable phase can be explained by cyclization reactions [2 atmosphere, weight loss starts above 100 °C, after a stable cyclization phase, dehydrogenation in O2 atmosphere is observed between 100 and 140 °C. In N2 atmosphere this temperature shifts to 300–400 °C. The reaction propagation is faster under O2 atmosphere compared to N2.During the stabilization process, the cyclization of the nitrile groups and cross-linking of the chain molecules is followed by dehydrogenation . This reeactions . Above teactions –38. In O2O, which was released from the structure [The same conditions as in the oxidation procedure were applied during TGA. A 5 °C/min ramp was applied till the samples reached the desired oxidation temperature . After the samples reached the oxidation temperature TGA was carried out for further 300 minutes. It can be seen from the TGA curves that the 300 °C/300 min oxidation process shows the highest weight loss. For the TGA measurement of nanofiber webs in oxygen environment the curves are similar to those of the PAN polymer. However, the temperature ranges are shifted because of the presence of oxygen. In the presence of reactive atmospheres, such as air or oxygen, the oxidation process is faster at lower temperatures . At temptructure –38. Figutructure ,18. A drtructure .2SO4 electrolyte in the frequency range of 100 mHz to 100 kHz at open circuit potential with an AC perturbation of 10 mV. A standard three-electrode cell was used to study the electrochemical performances of PAN nanofibers which were stabilized at 250 °C for 1 h in air. Oxidized PAN nanofiber mats were used as free standing working electrodes, a platinum wire was used as counter electrode, and a silver wire was used as pseudo-reference electrode. EIS data were simulated with electrical equivalent circuit by using the ZSimpWin V.3.10 analysis program.Electrochemical properties of oxidized PAN nanofibers were analyzed by using electrochemical impedance spectroscopy (EIS). EIS measurements were performed in 0.5 M H2 ≈ 5·10−4 . Rs is the ohmic resistance of the solution, Rct represents the charge-transfer resistance between nanofiber electrodes and electrolyte interface and Qdl (constant phase element (CPE)) is the double-layer CPE, a frequency-dependent element.Experimental and calculated measurements were fitted by equivalent circuit modelling. EIS plots with measured and calculated data are shown in Rct) can be calculated from measuring the diameter of the semicircle. According to the Bode phase plot in The Nyquist plot in Rs, Rct and Qdl were determined as 552 Ω, 340 Ω and 2·10−2 µS·sn according to the Randles circuit model for non-ideal electrodes described as Rs(QdlRct) in short hand. The CPE (Qdl) can also be attributed to the double-layer capacitance (Cdl) in the non-homogeneous systems [The values of  systems . Double- systems .The impedance of the non-ideal electrode is defined byj is the imaginary unit √−1, ω is the angular frequency, and TCPE and n are frequency-independent experimental constants; TCPE relates to the size, thickness, and materials properties, while n relates to the degree of energy dissipation and measures the arc depression, which is frequency-independent. Moreover, n is a parameter describing the deviation from an ideal capacitor and arises from the slope of the log Z versus log f plot. The values for n vary from 0 to 1, and n = 1 describes an ideal capacitor, while n = 0 describes the behavior of a resistor. The n value of oxidized PAN was equal to 0.83.where −1 and 1580–1600 cm−1, respectively [2-hybridized C–C bonds [−1) represents ordered graphitic crystallites [−1 is related to disordered turbostratic structures [R = ID/IG) indicates structurally ordered graphite crystallites [R value of CNF is around 0.9. A lower R value means a more crystalline material with higher conductivity [The Raman spectroscopic measurements show characteristic peaks of carbon materials, namely D band and G band at around 1360 cmectively –52  and Ox.PAN/GO(2) indicating concentration of 0, 1.25 and 2.5% graphene oxide relative to PAN, respectively.A standard three-electrode cell was used to study electrochemical performances of nanofibers by using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Carbonized nanofibers were used as free standing electrodes whereas oxidized nanofibers were deposited on fluorine-doped tin oxide (FTO) glass to use as working electrodes. EIS analysis were investigated in 0.1 M NaClORct) were evaluated by using equivalent circuit modelling. Rct is attributed to the pore size of the electrodes. The values of Rct of Ox.PAN, Ox.PAN/GO(1) and Ox.PAN/GO(2) were equal to 1180 kΩ, 119700 kΩ and 182800 kΩ, respectively, Rct increases with GO content.Nyquist plots in According to the Bode phase plots, the sample of Ox.PAN/GO(1) and Ox.PAN/GO(2) show similar properties while Ox.PAN behaves differently . After aRs) of the electrochemical system changes very slightly, which can be seen in The Bode magnitude plots exhibit two different shapes for high and low frequencies . At high2 values around 10−3. The result shows two different models. Rs(QelRct) circuit modeling was compatible with Ox.PAN, while a Rs(Qel(RctCdl)) circuit modelling was chosen for Ox.PAN/GO(1) and Ox.PAN/GO(2).The parameters of the simulated equivalent circuit models obtained from the Nyquist and Bode phase plots are given in Rs corresponds to the solution resistance, Rct corrresponds to the charge-transfer resistance of electrode surface and solution interface, and Qel corrresponds to the combined capacitance of nanofibers and FTO glass electrode. Rct and Cdl change linearly with the amount of GO and the values of n of Ox.PAN and Ox.PAN/GO(1) are is very similar . The Cdl value of Ox.PAN/GO(2) is 3.16 times higher than that of Ox.PAN/GO(1). A CPE is generally used in heterogeneous systems associated with non-ideal capacitive behavior resulting from electrode roughness, inhomogeneous conductivity, or even diffusion [ similar . After iiffusion . CPE is −1 between −0.5 V and 1.2 V in 0.1 M NaClO4/ACN electrolyte. It can be seen that CNF/GO electrode exhibits a larger CV area than the CNF electrode, indicating a higher specific capacitance compared to CNF. Adding GO increases the O/C ratio, which could result in an enhanced capacitive behavior of the carbon nanofibers.Heat treatment was applied to Ox.PAN and Ox.PAN/GO nanofibers to produce carbon nanofibers (CNF) and GO-containing carbon nanofibers (CNF/GO). CNF and CNF/GO, which include very small amount of graphene oxide (1.25% relative to PAN) were used as free standing working electrodes during CV. Cdl value of GO-containing oxidized nanofibers. The Cdl value of Ox.PAN/GO(2) is the highest as being 0.600 µF. Individual layered sheets of GO with high surface area are supposedly exposed to the electrolyte, which can result in the increase of the double layer capacitance. GO functional groups enhance the capacitance performance of CNF webs. As a result, CNF/GO can be a potential candidate for capacitive applications.In this paper, CNF webs and GO-containing CNF webs were successfully fabricated. Nanofiber webs were fabricated via electrospinning. Nanofiber alignment was achieved with a rotating collector, which also had the definite and expected effect of increasing modulus and reducing the strain to break of the webs. Different oxidation temperatures were studied and 250 °C was selected as optimum temperature for this study. Increased the oxidation temperature increases the oxidation level of the sample. However, thermal oxidation between 200 and 300 °C was not enough to eliminate all C≡N triple bonds. GO-containing oxidized nanofibers have a rough surface. Nanopores of around 38.5 ± 11 nm pore size on the nanofiber surface can help to store large amounts of charge. GO addition into PAN makes a significant change on the EIS results, i.e., the capacitive behavior increases with the increase in the


The Global Burden of Diseases, Injuries, and Risk Factors (GBD) Study 2015 provides an up-to-date analysis of the burden of lower respiratory tract infections (LRIs) in 195 countries. This study assesses cases, deaths, and aetiologies spanning the past 25 years and shows how the burden of LRI has changed in people of all ages.We estimated LRI mortality by age, sex, geography, and year using a modelling platform shared across most causes of death in the GBD 2015 study called the Cause of Death Ensemble model. We modelled LRI morbidity, including incidence and prevalence, using a meta-regression platform called DisMod-MR. We estimated aetiologies for LRI using two different counterfactual approaches, the first for viral pathogens, which incorporates the aetiology-specific risk of LRI and the prevalence of the aetiology in LRI episodes, and the second for bacterial pathogens, which uses a vaccine-probe approach. We used the Socio-demographic Index, which is a summary indicator derived from measures of income per capita, educational attainment, and fertility, to assess trends in LRI-related mortality. The two leading risk factors for LRI disability-adjusted life-years (DALYs), childhood undernutrition and air pollution, were used in a decomposition analysis to establish the relative contribution of changes in LRI DALYs.In 2015, we estimated that LRIs caused 2·74 million deaths  and 103·0 million DALYs (95% UI 96·1 million to 109·1 million). LRIs have a disproportionate effect on children younger than 5 years, responsible for 704 000 deaths (95% UI 651 000–763 000) and 60.6 million DALYs (95ÙI 56·0–65·6). Between 2005 and 2015, the number of deaths due to LRI decreased by 36·9% (95% UI 31·6 to 42·0) in children younger than 5 years, and by 3·2% (95% UI −0·4 to 6·9) in all ages. Pneumococcal pneumonia caused 55·4% of LRI deaths in all ages, totalling 1 517 388 deaths (95% UI 857 940–2 183 791). Between 2005 and 2015, improvements in air pollution exposure were responsible for a 4·3% reduction in LRI DALYs and improvements in childhood undernutrition were responsible for an 8·9% reduction.LRIs are the leading infectious cause of death and the fifth-leading cause of death overall; they are the second-leading cause of DALYs. At the global level, the burden of LRIs has decreased dramatically in the last 10 years in children younger than 5 years, although the burden in people older than 70 years has increased in many regions. LRI remains a largely preventable disease and cause of death, and continued efforts to decrease indoor and ambient air pollution, improve childhood nutrition, and scale up the use of the pneumococcal conjugate vaccine in children and adults will be essential in reducing the global burden of LRI.Bill & Melinda Gates Foundation. Lower respiratory tract infections (LRIs) are a substantial public health problem and a leading cause of illness and death in people of all ages. Previous estimates found that in 2013, LRIs caused more than 2·6 million deaths worldwide, making them the fifth leading cause of death overall and the leading infectious cause of death in children younger than 5 years.Estimates of the burden of LRIs and their aetiologies are being produced annually as part of the Global Burden of Diseases, Injuries, and Risk Factors (GBD) Study, which provides a unique source for tracking trends in LRI-related morbidity and mortality. Results from the GBD study that quantify the burden of LRI will help to measure progress towards the Sustainable Development Goals, including Goal 3, which is to ensure healthy lives and wellbeing for people of all ages.Evidence before this studyLower respiratory tract infections are a leading cause of morbidity and mortality, particularly in children younger than 5 years, and the global burden has been estimated by several groups, including the Global Burden of Disease study (GBD). We build on previous GBD studies with updated data and methods. Updated cause-of-death data came from additional years of vital registration systems  and by searching PubMed and Google Scholar for “verbal autopsy” on March 3, 2015. Updated non-fatal and aetiology data came from a PubMed search on June 1, 2015, for “lower respiratory infections”, “bronchiolitis”, and “pneumonia”. Articles published between Jan 1, 2012, and Dec 31, 2015, were selected without language restrictions.Added value of this studyThis manuscript provides a comprehensive assessment of LRI burden based on GBD 2015, including newer and more robust evidence on the mortality, morbidity, and risk factors associated with LRIs, including four aetiologies, and is the first cause-specific description of LRI in a GBD study. In addition to descriptions of trends in morbidity and mortality, we use the Socio-demographic Index to relate changes in LRI burden to demographic transitions and assess the effect of changing population characteristics and risk factor exposure to decompose the trends in LRI burden.Implications of all the available evidenceWe show a decreasing burden of LRI in children younger than 5 years, but an increase in the burden in adults. Furthermore, we show the change in risk factor exposure globally, providing health professionals with valuable information needed to design and implement effective programmes and policies to reduce the burden of LRI. We also identify high-burden LRI regions that need more attention. Expanded use of the pneumococcal conjugate vaccine, interventions to improve under-5 nutrition, and a focus on appropriate case management could reduce the burden of LRI. Comprehensive and reliable data on LRI morbidity and mortality globally are still needed.Haemophilus influenzae type B [Hib], Streptococcus pneumoniae [pneumococcal pneumonia], influenza, and respiratory syncytial virus [RSV]), covering deaths, episodes, disability-adjusted life-years (DALYs), risk factors contributing to the burden of LRIs, and the relationship between LRIs and social development for 195 countries from 1990 to 2015 for both sexes and by age.Here, we present results from the GBD study 2015, describing the burden of LRIs and four aetiologies  showing the 2·5 and 97·5 percentiles of the distribution.The GBD Cause of Death database contains all available data from vital registration systems, surveillance systems, and verbal autopsies  framework.The sum of all cause-specific mortality models must be equal to the all-cause mortality estimate.LRIs were defined as clinician-confirmed or radiologically confirmed pneumonia or bronchiolitis and were divided into moderate and severe or very severe episodes on the basis of WHO case definitions for pneumonia.LRI morbidity  was modelled using DisMod-MR version 2.1 (DisMod), a Bayesian, hierarchical, mixed-effects meta-regression platform.DALYs are the sum of YLLs and YLDs and represent the cumulative burden of disease due to LRI.We estimated LRI aetiologies separately from overall LRI mortality and morbidity using two distinct counterfactual modelling strategies to calculate population attributable fractions (PAFs) for influenza, RSV, Hib, and pneumococcal pneumonia. The PAF is the proportional reduction in LRI morbidity or mortality that would be observed if the exposure to the pathogen were zero. We did not attribute aetiologies to neonatal pneumonia cases or deaths because of an absence of reliable data in this age group, and we did not consider Hib in age groups older than 5 years for the same reason.We used a vaccine probe design to estimate the PAF for pneumococcal pneumonia and Hib by first calculating the ratio of vaccine effectiveness against non-specific pneumonia to pathogen-specific pneumonia at the study level.For Hib, we assumed that the vaccine efficacy against invasive Hib disease is the same as against Hib pneumonia. However, we did not make the same assumption for pneumococcal pneumonia because a study of pneumococcal conjugate vaccine (PCV) found that the vaccine efficacy against invasive pneumococcal disease might be significantly higher than against pneumococcal pneumonia.Influenza and RSV were estimated by calculating an attributable fraction that relates the odds ratio (OR) of LRI given pathogen detectionWe conducted a systematic literature review of the proportion of LRI cases that test positive for influenza and RSV and used the meta-regression tool DisMod to estimate the proportion of people with LRI who are positive for influenza and RSV, separately, by location, year, age, and sex.Different PAFs were measured for non-fatal and fatal LRI episodes. Fatal PAFs were adjusted using a scalar from the DisMod proportion models that represents the relative frequency of detection in inpatient versus non-inpatient sample populations. In the absence of aetiological data from fatal cases of LRI after death, we assumed that episodes of LRI requiring hospital admission were a reasonable proxy of severe and fatal episodes. Mortality is lower in patients with viral pneumonia than in those with pneumonia with bacterial causes. Therefore, we adjusted the fatal PAF estimates by establishing the ratio of case fatality in viral to bacterial causes of pneumonia from hospital data coded specifically to these causes, representing the relative fatality in people who were treated .On the basis of methods used to construct the Human Development Index, GBD 2015 used the Socio-demographic Index (SDI), a summary measure of a country's development based on lag-distributed income per capita, average educational attainment, and total fertility rate.Methods for risk factor attribution to LRI are described in detail elsewhere.27The sponsor of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.At the global level, under-5 LRI mortality occurred in 104·8 children per 100 000 (95% UI 97·0–113·6) and varied by region and country . AccordiWe calculated that, in 2015, LRIs caused 103·0 million DALYs (95% UI 96·1 million to 109·1 million) in all ages and 60·6 million DALYs (95% UI 56·0 million to 65·6 million) in children younger than 5 years , the USA (235·2 per 100 000 [224·0–247·0] vs 2·7 per 100 000 [2·4–3·0]), and Japan (613·7 per 100 000 [588·4–639·2] vs 2·8 per 100 000 [2·4–3·2]).Although nearly 60% of LRI DALYs were from children younger than 5 years, our findings suggest that LRI mortality was substantial across all ages, and in elderly people in particular. In adults aged 70 years or older, 1·27 million deaths (95% UI 1·15–1·34 million) were estimated to be caused by LRIs in 2015. In some countries, we estimated a much larger number of deaths due to LRIs in older adults (≥70 years) than in children younger than 5 years—eg, in China , and influenza was responsible for 10 151 (5 731–16 790) in children younger than 5 years, together accounting for 6·6% of LRI deaths in this age group . The burWe estimated that the leading risk factors for LRI DALYs in 2015 were childhood wasting (responsible for 44·6% [95% UI 31·7–52·8] of DALYs worldwide), household air pollution , and ambient particulate matter . Suboptimal breastfeeding was the third-leading risk factor for under-5 DALYs globally and the leading risk factor for under-5 DALYs in high SDI locations. Other risk factors for LRI, such as smoking, alcohol use, and zinc deficiency, were responsible for less than 10% of LRI DALYs globally.Health Metrics and Evaluation visualisations.A decomposition of the change in attributable DALYs between 2005 and 2015 by country is shown in The GBD 2015 study estimated that LRIs were the fifth-leading cause of death (of 249 causes) and the leading infectious cause, responsible for 2·74 million deaths (95% UI 2·50 million to 2·86 million). LRIs were the third-leading cause of under-5 mortality behind preterm birth and neonatal encephalopathy, accounting for 12·1% of deaths in this age group. Our analysis suggests that the number of deaths due to LRI in children younger than 5 years decreased by 37% between 2005 and 2015. We found that, although all-age LRI mortality rate decreased, growing and ageing populations have contributed to no significant decline in total LRI deaths between 2005 and 2015. Despite dramatic improvement in the under-5 LRI mortality rate, LRI remains a preventable cause of death in young children and elderly adults, particularly in south Asia and sub-Saharan Africa, and was the second-leading cause of DALYs in 2015. The findings call for renewed efforts to control and prevent LRIs across all age groups.Some solutions to prevent LRI deaths do not require major advances in technology. Measures to protect, prevent, and treat LRIs are highlighted in the Global Action Plan for Pneumonia and Diarrhoea.Although such interventions were not estimated in GBD 2015, improved access to health care and emphasis on appropriate treatment have probably played a crucial role in reducing LRI mortality, with proper treatment reducing mortality by 20–42%.In 2015, approximately 65% of children younger than 5 years received the Hib vaccine and 40% received the PCV.The expanded use of PCV might have several indirect effects on LRI burden. PCV might prevent influenza and RSV mortality, because up to half of severe viral infections are complicated by pneumococcal pneumonia.Our results suggest that LRIs were the second-leading cause of DALYs globally in 2015 after ischaemic heart disease.Household solid fuel use as a risk factor for LRI has decreased since 2005, particularly in Latin America and southeast Asia, which are undergoing rapid urbanisation and economic development. Economic development that shifts energy requirements away from household burning of biomass might reduce exposure to indoor air pollution at the expense of outdoor and ambient particulate matter from large-scale energy production facilities like coal-burning power plants.Our estimates of LRI mortality, morbidity, and aetiology attribution are limited by data availability and especially the sparsity of data in sub-Saharan Africa, the region with the greatest LRI burden and need for high-quality data . Only exStaphylococcus aureus, or intracellular pathogens like Mycoplasma pneumoniae, might be important aetiologies for LRIs and are not included in GBD 2015.Even with the application and expanded use of PCR diagnostic techniques, data on the aetiology of pneumonia remain sparse, particularly in areas with high disease burden. This scarcity of data is largely due to the difficulty of obtaining appropriate samples for testing, particularly in children, the relatively high cost of PCR, and challenges in culturing and diagnosing many pathogens that cause respiratory infections.The attributable fraction strategy for Hib and pneumococcal pneumonia assumes that the vaccine efficacy against invasive disease is the same as for pneumonia. A study by Bonten and colleaguesOnly four randomised controlled trials on Hib vaccine efficacy have been done in children younger than 5 years. Despite a plausible disease burden in older children and adults, we decided to apply the attributable fraction of LRI episodes and deaths due to Hib pneumonia to the under-5 age group only. The lower bound of the Hib PAF estimates is below zero  at the global level, reflecting in part the scarcity of reliable data on Hib vaccine efficacy.The attribution of the viral pathogens to LRI mortality was based on the relative case fatality of bacterial to viral aetiologies, and cases of LRI admitted to hospital might not be representative of cases not admitted to hospital. Efforts to improve surveillance, such as the African Network for Influenza Surveillance and Epidemiology,The GBD 2015 estimates of LRI mortality and burden are generally similar to the GBD 2013 estimates.Our estimates of pneumonia mortality in children younger than 5 years differ from those produced by the WHO Department of Evidence, Information and Research and the Maternal and Child Epidemiology Estimation (MCEE) group. The GBD 2015 estimates for under-5 mortality due to LRI in 2015  were much lower than those from the MCEE (920 000 deaths).Despite substantial reductions in under-5 LRI mortality in many countries, the burden remains high, particularly in areas of low sociodemographic development, and has increased in some populations, particularly elderly people. Estimates of the global burden of LRI will be improved by more high-quality data on mortality, morbidity, and aetiologies, especially in sub-Saharan Africa where the burden is highest and data are most scarce. The creation and expansion of civil registration systems in Africa and south Asia are gaining momentum, and such data will not only improve global comparative mortality assessments such as the GBD study, but also increase the evidence for guiding decision about local policy.mokdaa@uw.eduCorrespondence to: Dr Ali H Mokdad, Institute for Health Metrics and Evaluation, University of Washington, Seattle, WA 98121, USA


There is large variation in treatment responses in children with cerebral palsy. Experimental and clinical results suggest that dopamine neurotransmission and brain-derived neurotrophic factor (BDNF) signalling are involved in motor learning and plasticity, which are key factors in modern habilitation success. We examined whether naturally occurring variations in dopamine and BDNF genes influenced the treatment outcomes.Thirty-three children (18–60 months of age) with spastic unilateral cerebral palsy were enrolled in the study. Each child had participated in a training programme consisting of active training of the involved hand for 2 h every day during a 2-month training period. The training outcome was measured using Assisting Hand Assessment before and after the training period. Saliva was collected for genotyping of COMT, DAT, DRD1, DRD2, DRD3, and BDNF. Regression analyses were used to examine associations between genetic variation and training outcome.There was a statistically significant association between variation in dopamine genes and treatment outcome. Children with a high polygenic dopamine gene score including polymorphisms of five dopamine genes , and reflecting higher endogenous dopaminergic neurotransmission, had the greatest functional outcome gains after intervention.Naturally occurring genetic variation in the dopamine system can influence treatment outcomes in children with cerebral palsy. A polygenic dopamine score might be valid for treatment outcome prediction and for designing individually tailored interventions for children with cerebral palsy. •Naturally occurring variation of dopamine genes is associated with treatment outcomes in children with cerebral palsy.•Children with polymorphisms reflecting higher endogenous dopaminergic neurotransmission had the greatest functional gains.•A polygenic dopamine score might be valid to predict treatment outcome.New evidence-based therapies including active motor learning and training for children with cerebral palsy improve motor function at a group level, but there are also large inter-individual variations. Naturally occurring variations in dopamine and BDNF genes affect motor learning and cortical plasticity.This study showed that naturally occurring genetic variation of five dopamine genes was associated with the outcome of a 2-month long active upper limb motor training intervention in children with unilateral cerebral palsy. The results suggest that a polygenic dopamine gene score can be used to predict the outcome of motor training programmes for children with cerebral palsy. Researchers and clinicians have only during the past few decades adopted an evidence-based approach to identify and use effective interventions . New theThese observed large inter-individual differences in treatment outcomes have negative consequences for non-responders because time and effort will be expended without any gains in functional improvement. Identification of the causes of the variability is an important step in the advancement of personalized rehabilitation medicine. Each child with cerebral palsy can then receive an individually tailored intervention. The concept of personalized medicine has evolved mainly from the variability observed in response to various drugs. It has developed into the field of pharmacogenetics, which examines how genetic differences  affect individual responses to drugs . The genSeveral factors may influence the outcome of interventions in cerebral palsy, which encompasses heterogeneous clinical phenotypes and aetiologies. Factors that can affect the outcome of an intervention can be associated with the white and/or grey matter brain injuries of varying location, size, and time of origin. These conditions are known to affect cognitive and motor functions. Many children have no detectable risk factors, and 30% of the cases of cerebral palsy may be of genetic origin . However66met polymorphism in the gene for brain derived neurotrophic factor (BDNF). BDNF is highly expressed throughout the brain and has important roles in development, plasticity, and repair. The presence of the BDNF val66met polymorphism is associated with poor short-term motor learning gains and altered short-term cortical plasticity  . Rodent The results of human and animal studies thus suggest that dopamine and BDNF are involved in motor learning, and that variation in dopamine and BDNF genes might contribute to the inter-individual differences in treatment response. The aim of this study was to examine the influences of functional genetic variation in the dopamine system and BDNF on the outcome of an intervention programme for children with cerebral palsy. The intervention used was modified Constraint Movement Therapy (CIMT) , which i22.1The participants were recruited from two previous intervention studies of children with spastic unilateral cerebral palsy. The first study was a controlled clinical trial that included 21 children (18–48 months of age) . The secEach participant provided written informed consent prior to the collection of the saliva sample. The study was approved by the Regional Ethical Review Board in Stockholm (Dnr: 2015/61–31/2).2.2The training programme for both studies comprised active training of the involved hand for 2 h each day during a 2-month training period. Toys and activities relevant for the age and ability of the child were used. During this modified Constraint-Induced Movement Therapy (CIMT), a comfortable fabric glove with a built in volar stiff plastic splint was worn on the less impaired hand to encourage each child to use the impaired hand . Parents2.3The change in the Assisting Hand Assessment (AHA)  performeIn this study, we used the change in AHA-units  as the primary outcome variable to examine the effect of genetic variation on intervention outcome.2.4Saliva samples were collected from 35 children with spastic unilateral cerebral palsy using the Oragene DNA Sample Collection Kit . DNA was extracted within 3 days of collection following the manufacturer's protocol. DNA quantity and quality were evaluated using a NanoDrop TM 2000 spectrophotometer . Data from 33 participants were included in the analysis because two poor-quality saliva DNA samples were excluded. All samples were stored at − 20 °C until use. Genotype was determined for COMT rs4680, DRD1 rs4532, DRD2/ANKK1 rs1800497, DRD3 rs6280, DAT1 VNTR, and BDNF rs6265 using polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis as previously described . All salA combined “polygenic” dopamine gene score was determined as previously described . Briefly2.5All statistical analyses were performed using R (version 3.2.3). In the main analysis, we used a general linear model to explain the changes in AHA-units in terms of the following variables used together: combined dopamine gene score , BDNF gene score (0 for VAL/MET and MET/MET or one for VAL/VAL), and sex and age (months at the start of the 2-month intervention period). In subsequent explorations of single-dopamine gene relationships, a four-gene score  was inserted into the same model used with the multi-gene score, and each single gene score, to evaluate each dopamine gene independently of the other genes. Finally, in a separate linear model, we evaluated the effect of AHA-units at baseline on the changes in AHA-units, while maintaining all other explanatory variables. Chi-Square and exact tests were used to assess the Hardy-Weinberg equilibrium. The significance level for effects was set at p < 0·05.3The results for the characteristics of the 33 children with spastic unilateral cerebral palsy are presented in The results for the distributions of alleles for the six genes in the 33 children with cerebral palsy are presented in The results for the distribution of dopamine score in relation to sex, age, and AHA-units at baseline are presented in The effect of genetic variation, sex, and age on training outcome was examined using a general linear model. We used the change in AHA-units as the outcome variable; age, sex, dopamine gene score, and BDNF were considered together as independent variables. Age and dopamine score had statistically significant effects on training outcome . The results for BDNF and sex were not statistically significant . In Next, we explored the individual contributions of the five dopamine genes involved in the combined dopamine gene score by removing one gene at a time or by calculating the effect of only one gene at a time. The results are presented in We then explored whether the AHA-unit value at baseline had an effect on outcome. We used the linear model described above and added the values for the AHA-units before training as an explanatory variable. The effect of the dopamine five-gene score remained statistically significant  even after controlling for AHA-units at baseline. The effect of AHA-units at baseline was also statistically significant . However, the result using this model indicated that the effect of age was no longer statistically significant, but there was a tendency towards significance . The effects of sex and BDNF remained statistically insignificant .4The results of this study are the first to suggest that naturally occurring genetic variation can predict the outcome of a rehabilitation intervention for patients with movement disorders. The results indicated that there was a statistically significant association between a polygenic dopamine gene score reflecting the collective effects of five dopamine gene polymorphisms , and CIMT outcome in 33 children with spastic unilateral cerebral palsy. Consistent with our hypothesis, children with a high gene score, and thus higher endogenous dopaminergic neurotransmission, had the greatest functional outcome gains after intervention. The gains were measured using changes in AHA units. We found that there was no statistically significant effect of the BDNF val66met polymorphism on intervention outcome. The results thus suggested that a polygenic dopamine gene score can be useful for predicting the outcome of motor interventions in individuals with motor disabilities.C57BL/6 and BALB/c mice) revealed that genetically driven variation in midbrain dopamine neurotransmission is a major contributor to individual differences in motor skill learning (The association between dopamine neurotransmission (as reflected by the polygenic dopamine gene score) and functional CIMT outcome in children with spastic unilateral cerebral palsy likely reflects a more general principle of dopamine-dependent brain plasticity. This finding may therefore be relevant for other ages and other types of movement disorders. The association suggests that the effectiveness of interventions based on active motor learning and training depends on dopamine-mediated regulation of cortico-striatal plasticity. Abundant experimental evidence indicates that dopamine has a key role in motor learning and associated cortical plasticity. Rodent studies have found that mesocortical dopaminergic transmission is required for learning new motor skills, but not for executing learned movements . Intracelearning  One majolearning  addresselearning . These rlearning .Our resuThe five proteins  involved in the polygenic dopamine gene score have different roles in dopamine neurotransmission and have different patterns of distribution in various brain regions. Therefore, these five gene polymorphisms may affect training-induced plasticity to differing degrees. We adapted the five-gene score model and examined the effects of single dopamine genes by calculating a four-gene score . Removal of the DRD3 gene increased the strength of the association; elimination of any of the other genes  weakened the association . EnterinOur results  also sugOur initial analysis also found that age had a statistically significant effect on the intervention outcome . YoungerPearson-Fuhrhop et al.  also fouThe strengths of this study include the use of a well-characterized cohort of children with cerebral palsy who were exposed to the same type of intervention program and assessment. The limitations of the study were the small size of the cohort and the retrospective study design. Saliva was collected for genotyping 6–15 years after the intervention. However, gene polymorphisms are stable over time and age-related changes likely did not affect the results.In conclusion, our findings indicate the importance of naturally occurring genetic variation in the dopamine system for treatment outcomes in children with spastic unilateral cerebral palsy. We propose that the polygenic dopamine score used in the current study might be useful for prediction of treatment outcome, while it needs to be replicated in other studies before it can be translated into clinical practise. The designs of individually tailored interventions for children with cerebral palsy could benefit from application of this scoring system.Swedish Research Council (5925), Foundation Olle Engkvist Byggmästare, Swedish Brain Foundation, Foundation Frimurarna Barnhuset, Promobilia Foundation, Strategic Research Programme of Neuroscience at Karolinska Institutet. The funding bodies had no role in planning the study, analysing the data or writing the manuscript.All authors declare that there are no conflicts of interest to report.RDH and HF conceived, designed and organized the study. ACE was responsible for the intervention studies and analysed the AHA data. RDH performed the genetic analysis. RA performed the statistical analysis. HF wrote the first manuscript draft and all authors contributed to the subsequent drafts and approved the final version.


Escherichia coli from high cell density fermentation process: The recombinant inclusion body product is expressed by using a mixed feed fed-batch process which allows expression tuning via adjusting the specific uptake rate of the inducing substrate. Then, bacteriophage ΦX174-derived lysis protein E is expressed to induce cell lysis. Inclusion bodies in empty cell envelopes are harvested via centrifugation of the fermentation broth. A subsequent solubilization step reveals the recombinant protein. The process was investigated by analyzing the impact of fermentation conditions on protein E-mediated cell lysis as well as cell lysis kinetics. Optimal cell lysis efficiencies of 99% were obtained with inclusion body titers of >2.0 g/l at specific growth rates higher 0.12 h−1 and inducer uptake rates below 0.125 g/(g × h). Protein E-mediated cell disruption showed a first-order kinetics with a kinetic constant of −0.8 ± 0.3 h−1. This alternative inclusion body protein isolation technique was compared to the one via high-pressure homogenization. SDS gel analysis showed 10% less protein impurities when cells had been disrupted via high-pressure homogenization, than when empty cell envelopes including inclusion bodies were investigated. Within this contribution, an innovative technology, tuning recombinant protein production and substituting cost-intensive mechanical cell disruption, is presented. We anticipate that the presented method will simplify and reduce the production costs of inclusion body processes to produce technical enzymes and biopharmaceutical products.Production of recombinant proteins as inclusion bodies is an important strategy in the production of technical enzymes and biopharmaceutical products. So far, protein from inclusion bodies has been recovered from the cell factory through mechanical or chemical disruption methods, requiring additional cost-intensive unit operations. We describe a novel method that is using a bacteriophage-derived lysis protein to directly recover inclusion body protein from The online version of this article (doi:10.1007/s00253-017-8281-x) contains supplementary material, which is available to authorized users. Escherichia coli is the primary microbial cell factory ): F– ompT hsdSB  gal dcm (DE3) strain  was used. It contained two plasmids coding for the recombinant human bone morphogenetic protein 2  as well as the phage protein E.An pR, mut-cI857 P/O-system  of D-glucose and of L-arabinose. As qs D-glucose and qs L-arabinose differed between cultivations, so did the ratio of the sugars in the medium.Media were used as described in DeLisa et al. . The batMultiple fermentations were conducted in a Techfors-S bioreactor  of 10-l maximum working volume with a bioreactor setup and instrumentation as described elsewhere . Furthe−1 until an estimated biomass concentration of 15 g/l was reached. In this phase of the process, D-glucose functioned as the sole carbon source. After the uninduced fed-batch, an L-arabinose pulse was performed to a final concentration of 2.5 g/l to adapt the cells. During this period, no additional sugar was fed.Parallel to starting the fed-batch, a first-principle soft sensor based on elemental balancing was initialized. To do this, the biomass concentration was estimated via OD correlation as a starting value, a procedure more thoroughly described elsewhere  was used. The specific substrate uptake rates of D-glucose and L-arabinose varied between cultivations based on a design of experiments (DoE) to investigate the influence on product titer, on product purity, and on E-lysis. To achieve the desired set points of As soon as the estimated biomass concentration in the induction phase approached a value of 35 g/l, the feed was changed back to D-glucose feed, which had been previously used for the non-induced fed-batch. The reactor temperature was shifted to 42 °C to induce protein E formation and, consequently, protein E-mediated lysis of the cellular population. An annular type Permittivity probe  was used for biomass measurement during the E-lysis phase and, hence, was the basis for the control of the specific D-glucose uptake rate. Biomass estimation via Permittivity measurement was conducted as described in Meitz et al. , with thqs D-glucose and qs L-arabinose as described in the following section.The batch, the uninduced fed-batch, the L-arabinose pulse, and the E-lysis phase were the same for all cultivations. Only the induced fed-batch phase differed in Recently, we reported induced state metabolic capabilities in respect to catabolite repression (maximum uptake of L-arabinose in the presence of uptake of D-glucose) for the pBAD mixed feed system  and the high point qs D-glucose/low qs L-arabinose (fed-batch 1/2) were conducted in duplicate to evaluate the experimental replicate error. In fed-batch 2, the process was stopped after the induction phase. An overview of the cultivations and process conditions is given in Table Due to primary results of the DoE cultivations, the DoE was slightly changed. In total, ten fermentations were conducted, including different combinations of these three In this study, a previously reported soft-sensor control strategy designed for process development was applied , the biomass concentration (cx), the density of the feed (ρfeed), the volume of the cultivation broth (VR), and the concentration of substrate in the feed (cs).Equation More details about the first-principle soft-sensor applied in this study are found in Sagmeister et al.  and Wechg, 10 min, and 4 °C. The supernatant was put on −20 °C for later investigation while the pellet was washed, centrifuged again, and then dried for 48 h at 105 °C. The DCW was determined gravimetrically.The dry cell weight concentration (DCW) determination was done in duplicate. A 2-ml sample was centrifuged at 4332×g, 10 min, 4 °C) and washed once. For homogenization, the pellets were resuspended to 20 ml in a buffer . All samples were suspended in the buffer, but only samples taken before the E-lysis phase were homogenized  in a continuous mode at 1500 bar for 3 passages.A duplicate of 2-ml fermentation broth was centrifuged . For homogenized samples, the resulting pellet and the supernatant were separately reduced on a heating block  in a buffer described by Laemmli  Coomassie Brilliant Blue G 250, 5% (w/v) aluminum sulfate-(14–18)-hydrate, 10% (v/v) ethanol, 2% (v/v) orthophosphoretic acid, distilled water). Stained gels were scanned and analyzed using the software ImageLab . To investigate the purity of rhBMP-2, reference bands were marked and the intensity of the bands was then compared. The reference bands were selected in respect to their impact on downstream processing. Each sample was evaluated in triplicate on three different gels.After running the gels , they were stained overnight in a sensitive Coomassie solution , the supernatant was discarded, and the pellet was resuspended in the solubilization buffer  to a final protein concentration of 5 ± 1 g/l. For solubilization, samples were shaken overnight in 15-ml plastic tubes .For rhBMP-2 titer determination, homogenized samples and lysed samples were used. For better comparability, the samples taken after E-lysis were solved in the homogenization buffer. One milliliter of each of these samples was centrifuged .Then, samples were transferred to a 1.5-ml reaction tube  and centrifuged . The supernatant was analyzed via HPLC  with a reversed phase column  using a gradient method  was used. The machine had a 488-nm blue solid state laser, whereas, five optical parameters were available. These were three fluorescence channels as well as a forward scatter (FSC) and side scatters (SSC). The wavelengths detected by the fluorescence channels (FL) were 527 nm for FL1 , 590 nm for FL2 , and 630 nm for FL3 (long-pass filter).A combination of fluorescence stains RH 414  and DiBAC4(3)   as well as FSC were used to evaluate the E-lysis efficiency which was defined as the percentage of BGs in the total cell population. Sample preparation, flow cytometry measurement, and data treatment were conducted as described by Langemann et al. .To better understand the four different phases of the cultivations presented here as well as the mentioned process control strategies, one point of the DoE (FB5) is now going to be described in more detail. This cultivation is represented divided into process phases in Fig. 2 off-gas signal and in the Permittivity signal , a second feed (mixture of D-glucose and L-arabinose) was started to begin the mixed feed induction phase.During the batch phase, biomass accumulated to a concentration of approximately 10 g/l. This increase of biomass concentration is reflected in the COqs D-glucose, using pre-established calibration to estimate biomass concentration during induction phase. Subsequently, the reactor temperature was shifted from 35 to 42 °C to induce the expression of the recombinant gene E, thereby starting the E-lysis phase . The model for these linear effects had an F value of 14.4 . Both trends were significant with a p <0.05.E-lysis efficiency is defined as the percentage of lysed cells in respect to the total cell population. In this study, achieved maximal E-lysis efficiencies varied between 31% in FB10 and 99% in FB3. Evaluation using multilinear regression analysis showed that the specific growth rate and the concentration of L-arabinose in the feed significantly impact the E-lysis efficiency .Equation N) over time (t) described as Eq. k being the reaction constant. The function of E-lysis competent cells as a function of time was fitted for all process runs as exemplified in Fig. The number of E-lysis competent cells decreases with on-going E-lysis phase until it approaches 0 asymptotically at the end of this same phase. A negative exponential curve showed to appropriately fit the trend of E-lysis competent cells over time. Hence, the amount of E-lysis competent cells (N) over time of the E-lysis phase (tL) and the fitted reaction constant k.Equation k was found to vary between −0.97 and −0.47, and, additionally, there was one outlier with a k of −1.25. This outlier belonged to FB3, which had the highest E-lysis efficiency of all fermentations (99%).The fitted reaction constant k was determined as −0.76 ± 0.29 and can be used to estimate the time necessary to reach maximal protein E-mediated lysis.Multivariate investigation of interdependencies between the process and physiological parameters with E-lysis kinetics did not yield in any significant connections. Nevertheless, the mean reaction coefficient F value 58.9 . Both trends were significant with a p <0.05. Predictions of the product titer based on the model are shown in Fig. As for the E-lysis efficiency, the influence of process parameters on the product titer at the end of the induction phase was investigated. The total specific substrate uptake rate showed a negative linear correlation to the final product titer, while L-arabinose in the feed was positively correlated. The model for these linear effects was significant with an −1 (FB9). Maximal titers at E-lysis efficiencies higher than 90% were predicted to be 2.6 g/l at a growth rate of 0.09 h−1 and 19.8% L-arabinose in the feed.The conducted cultivations revealed a maximal product titer of 6.1 g/l at a growth rate of 0.05 ht test showed . These time-space-yields did not consider downstream steps like homogenization. Thus, the 3-h elongated upstream process, resulting from the addition of the E-lysis phase, led to this difference. This was underlined by the findings that the rhBMP-2 recovery in grams per liter after solubilization of the homogenized respective of the E-lysed sample measured via HPLC analysis were the same. A paired t test revealed missing significance and thus supported the hypothesis that there was no significant difference in rhBMP-2 titer .For the upstream process, time-space-yields (gram rhBMP-2 per liter fermentation broth and process hour) were calculated for homogenized samples from the end of the induction phase and E-lysed samples. Yields were significantly higher for homogenized samples from the end of induction phase (0.74 ± 0.25 g/(l × h)) than E-lysed samples (0.47 ± 0.17 g/(l × h)), as a paired In order to compare protein E-mediated lysis to “classical” cell rupture by means of high-pressure homogenization, the inclusion body purity was compared for the respective last samples of the induction phase in combination with homogenization to the sample after E-lysis (without homogenization). Inclusion body purity refers to the ratio of recombinant protein to other proteins found via SDS gel analysis in pellets of homogenized samples and BGs. Pellets of the latter contained the cell envelope with sealed periplasmatic space and inclusion bodies, while pellets of homogenized samples were expected to contain—besides inclusion bodies—only cell debris.SDS gel analysis was carried out for all homogenized samples at the end of the mixed feed process, as well as for non-homogenized samples taken after the protein E-mediated lysis, in triplicate each. Furthermore, the fermentation broth supernatant and the homogenization supernatant were checked for the presence of soluble product (rhBMP-2). A typical SDS gel is displayed in Fig. t test . However, protein E-mediated ruptured samples were also found to be 40% purer compared to non-lysed and non-homogenized samples.The impact of process parameters and cell rupture methods on the inclusion body purity was investigated. SDS gel analysis was carried out in triplicate for all homogenized end mixed feed phase samples and E-lysis phase samples for all processes. Densiometric evaluation showed that homogenized samples are significantly purer (61 ± 10%) than lysed non-homogenized samples (52 ± 7%), which was verified with a paired In high-pressure homogenization, all host cell proteins are expelled. Hence, only inclusion bodies and cell debris are found in the pellet used for SDS gel analysis. Opposed to this, solely cytoplasmic proteins are expelled during protein E-mediated lysis and the periplasmic space stays intact  on the inclusion body purity after homogenization  as well as E-lysis  while the feed concentration of the inducer L-arabinose was found to be insignificant. At lower specific growth rates, inclusion bodies were found to be purer. At a specific growth rate of 0.05 h−1 (FB9), an inclusion body purity of 71 ± 4% in reference to key contaminating proteins was achieved after homogenization.The evaluation of a regression study identified a significant impact of the specific growth rate during the induction phase . This highlights the power and versatility of this technology and suggests the application of the pBAD mixed feed system for other processes where the metabolic load on the cell factory is to be reduced by simple process technological means.As stated above, state-of-the-art rupture methods to release recombinant protein (soluble or in the form of inclusion body aggregates) from the cytoplasm are detergent treatment, cell rupture by lysozyme, sonication, and freeze-thaw , is to be considered. An improvement of the solubilization procedure of the inclusion body in the empty cell envelope may increase product purity by omitting to open the periplasmic space. Thereby, the decrease of inclusion body purity by periplasmatic protein extraction could be omitted. An option could be the usage of milder detergents for solubilization. Applying non-denaturating detergents for solubilization enables gaining active protein from inclusion bodies without further renaturation steps  and steam in place (SIP) of the homogenizer. However, a significant quality loss in inclusion body purity (10%), due to retaining periplasmic proteins, as well as lower product titer, due to physiological constraints