P<0.001). Furthermore, the present study found that DANCR expression level was correlated to T grade (P<0.01) and TNM stage (P=0.017). The present study demonstrated that DANCR was associated with PTC aggressive clinical features and may serve as a diagnostic biomarker for detecting PTC patients.Long non-coding RNAs (lncRNAs) have been reported to be dysregulated and play a crucial role in the progression of cancer. LncRNA DANCR has recently been revealed to be involved in tumorigenesis of numerous types of cancer, including osteosarcoma, gastric cancer, breast cancer, hepatocellular carcinoma, and colorectal cancer. However, the expression profiles and biological relevance of DANCR in papillary thyroid cancer (PTC) have not yet been reported. In the present study, the expression level of DANCR in PTC tissues and adjacent normal tissues was detected by reverse transcription-quantitative PCR in PTC patients, and then we analyzed the association with clinical pathological characteristics of patients and DANCR expressions. These results demonstrated that the expression of DANCR was notably decreased in tumor tissues in comparison with adjacent normal tissues ( Although the incidence of thyroid cancer is rapidly increased, the mortality rate remains flat ,2. But tLong non-coding RNAs (lncRNAs) constitute a newly identified class of RNAs, with more than 200 nucleotides in length and regulating gene expression through controlling transcription or post-transcription, epigenetic modification, and mRNA splicing ,10. AlthBy analyzing the GEO datasets, we found DANCR is significantly decreased in PTC tissues compared with normal adjacent tissues. In the present study, we detected the expression of DANCR in PTC tissues and adjacent normal tissues, and then investigated the correlation between DANCR expression and clinicopathologic characteristics. Finally, we investigated the feasibility of using DANCR to detect PTC patients.http://www.ncbi.nlm.nih.gov/geo/). GSE33630 was based on Affymetrix Human Genome U133 Plus 2.0 Array , GSE50901 was based on Agilent-028004 SurePrint G3 Human GE 8×60K Microarray , and GSE66783 was based on Agilent-060228 Human LncRNA v4 4×180K . In GSE33630, 49 PTC and 45 normal thyroid tissues were analyzed. A total of 57 PTC and nine adjacent normal thyroid tissues were employed in GSE50901; five PTC specimens and their paired adjacent noncancerous thyroid tissue samples were obtained in GSE66783. In order to obtain the expression of DANCR in GEO datasets, GEO2R was used to analyze the DANCR expression in PTC and normal tissues and the profile graph was employed to visualize DANCR expression in each sample.Three expression profiles  was signed by all patients involved in the study. Tissue samples were stored in liquid nitrogen immediately after surgery until total RNA was extracted. Clinical characteristics, such as age, gender, and TNM stage, were all collected from the case management system in Hunan Cancer Hospital.−ΔΔCt = ([Ct DANCR-Ct β actin] sample A − [Ct DANCR-Ct β actin] sample B)   and low DANCR expression group [n=38]) based on the median value (0.668).The relationship between DANCR and clinical characteristics of PTC was analyzed by χ2 test and summarized in 2 test, we found that the expression of DANCR was significantly associated with TNM stage (P<0.05) and T stage (P<0.01). However, there was no significant correlation of DANCR expression with other clinical features such as gender, age, tumor size, and local invasion (P>0.05). DANCR expression was significantly decreased in III/IV stage thyroid cancer compared with I/II stage thyroid cancer and normal tissues in 76-pair samples , these findings provided evidences for detecting PTC by using DANCR as a novel diagnostic marker.In the present research, we identified that lncRNA DANCR expression was significantly decreased in 76 PTC patients’ tissues compared with adjacent normal tissues. Moreover, we found that the expression of DANCR was negatively correlated with T staging and TNM staging in 76 PTC patients. Furthermore, multivariable logistic regression analysis indicated that DANCR was an independent protective factor for TNM staging . Finally, DANCR could discriminate PTC from normal adjacent tissues with AUC of 0.8233 (95% CI: 0.7545–0.8921, Previous functional studies on lncRNA provide evidence that DANCR is a tumor-associated lncRNA and up-regulated in numerous human cancers, including breast cancer, prostate cancer, gastric cancer, and osteosarcoma . DANCR iin vitro and in vivo experiments needed to be conducted to further validate the biological function of DANCR in thyroid cancer. Understanding the underlying mechanism of DANCR in PTC will be helpful to establish the novel diagnostic biomarker.However, our study still contains several limitations. For one thing, the diagnostic efficiency of DANCR should be based on large clinical samples. Therefore, in future experiments, we need to expand the sample size and continue to verify its value. For another thing, Overall, our results suggested that decreased DANCR can be used as a potent tumor biomarker for PTC detection, and DANCR is an independent factor for TNM stage.


Overcoming aversive emotional memories requires neural systems that detect when fear responses are no longer appropriate so that they can be extinguished. The midbrain ventral tegmental area (VTA) dopamine system has been implicated in reward and more broadly in signaling when a better-than-expected outcome has occurred. This suggests that it may be important in guiding fear to safety transitions. We report that when an expected aversive outcome does not occur, activity in midbrain dopamine neurons is necessary to extinguish behavioral fear responses and engage molecular signaling events in extinction learning circuits. Furthermore, a specific dopamine projection to the nucleus accumbens medial shell is partially responsible for this effect. In contrast, a separate dopamine projection to the medial prefrontal cortex opposes extinction learning. This demonstrates a novel function for the canonical VTA-dopamine reward system and reveals opposing behavioral roles for different dopamine neuron projections in fear extinction learning. Fear memories are overcome only when it is ascertained that fearful responses are not appropriate. Here the authors demonstrate that activity in dopamine neurons is necessary to extinguish fear responses and two distinct dopamine neuron projections exert opposing effects on extinction learning. Extinction of classically conditioned fear responses is a model of exposure therapy. In the laboratory, animals learn that a sensory stimulus predicts the occurrence of an aversive outcome through fear conditioning. During extinction, the omission of an expected aversive event signals a transition from fear responding to safety. To switch from fear responding to extinction learning, a brain system that recognizes when an expected aversive event does not occur is required. While molecular changes occurring in the ventromedial prefrontal cortex (vmPFC) and amygdala are known to be important for storing and consolidating extinction memories6. The omission of an expected aversive outcome, as occurs during extinction is also known to activate dopamine neurons and increase dopamine levels in the nucleus accumbens (NAc)11, particularly in the NAc medial shell (mShell)10. Dopamine neurons are also activated by the cessation of ongoing aversive stimuli and activity in VTA-dopamine neurons and their projections to the NAc are important in relief learning, a process which may be similar to extinction13. Dopamine neurons are heterogeneous in their response to rewarding and aversive outcomes14, with some cells responding specifically to reward and others more generally to salient stimuli. A recent study in Drosophila also reported that a distinct population of dopamine neurons are involved specifically in extinction of reward learning based on their connectivity with a specific area of the mushroom body15. In mammalian systems, separable populations of dopamine neurons project to the NAc, amygdala and mPFC16 and distinct calcium changes have been reported in different VTA-dopamine terminal fields18. Importantly, dopamine receptor activation in the vmPFC, amygdala and NAc modulates extinction learning and memory23. This suggests that based on their projection targets, distinct subpopulations of dopamine neurons may differentially regulate fear extinction. What is unknown is whether activation of VTA-dopamine neurons during the omission of an expected aversive outcome is necessary for fear extinction learning and, if so, whether this effect is mediated by specific populations and projections of dopamine neurons.One candidate circuit for this function is the midbrain ventral tegmental area (VTA)-dopamine system. VTA-dopamine neurons signal reward through phasic responses to better-than-expected outcomes. They are more strongly activated by unexpected, compared with expected, rewards and inhibited when an expected reward does not occur, thereby encoding reward prediction errorsUsing a circuit specific optogenetic-behavioral approach, we show that optogenetic inhibition of VTA-dopamine neurons during expected shock omission is necessary for extinction learning. Furthermore, we report that specific populations of VTA-dopamine neurons project to the NAc mShell or core and inhibition of the mShell projecting cells reduces the long-term retention of extinction. In contrast, a separate projection of the VTA-dopamine system to the vmPFC opposes extinction learning. Together, these results demonstrate that the VTA-dopamine system is necessary for detecting when aversive responses should be reduced and reveal that distinct populations of dopamine neurons make unique and specific contributions to this process.24 by injecting a Cre-dependent adeno-associated virus (AAV) into the VTA  was paired with an aversive footshock unconditioned stimulus (US). This was followed 24 h later by presentation of the auditory CS without the aversive US during an “extinction” learning session . To determine whether extinction was completely blocked we used a more specific analysis comparing freezing during the first CS of extinction to the last CS of extinction and to the first CS of extinction “retrieval”. This showed that compared with freezing to the first CS of extinction training, there was a significant reduction in freezing by the final CS of the extinction training session and this reduction persisted to the first CS of extinction retrieval  alone or the inhibitory Halorhodopsin fused to eYFP (eNpHR3.0-eYFP) specifically in VTA-dopamine cells of tyrosine hydroxylase-cre recombinase (TH-Cre) ratsVTA Fig. . Opticalion Fig.  in whichion Fig.  or durinion Fig.  of extinion Fig. . There w29. We next investigated whether inactivation of VTA-dopamine neurons during the shock omission period reduced extinction learning induced increases in pMAPK.29. We expressed eNpHR3.0 or eYFP in VTA-dopamine cells, optogenetically inhibited these cells during the shock omission period of fear extinction and used immunohistochemistry to quantify MAPK phosphorylation (pMAPK). We compared this to chamber exposed control animals who were fear conditioned but did not undergo extinction training  occurs in the amygdala and vmPFC and is necessary in these regions for normal extinction learninging Fig. . In the ing Fig. . In cont30. While activation of VTA-dopamine neurons is necessary during rewards for learning to occur and VTA-dopamine signaling has been implicated in aversive learning33, the role of shock-evoked activity in dopamine neurons in fear learning is not clear. We tested this question using the same approach described above to inhibit the activity of VTA-dopamine neurons during the shock US period of fear conditioning 36. To verify that optogenetic manipulation of dopamine nerve terminals inhibited dopamine release, we used fast-scan cyclic voltammetry (FSCV) to measure dopamine release in NAc 51. Interestingly, a recent study demonstrated that GABAergic D1 receptor expressing medium spiny neurons in NAc mShell and lShell have distinct connectivity with reciprocally connected and distinct mShell and lShell projecting VTA-dopamine neurons51, suggesting a possible mechanism for individualized regulation of these different populations of dopamine neurons. While another microdialysis study52 reported increases in dopamine in mShell in response to aversive predictive cues, because of the limited temporal specificity of the microdialysis technique this increase could reflect CS offset responses similar to what has been seen in previous work10. This may be related to the role of NAc dopamine in pain relief learning13, which may share similar neural substrates with fear extinction. Together with our results, these previous studies support a role for dopamine release in the NAc mShell in modulating fear extinction memory consolidation. The fact that inhibition of this dopamine projection only affected long-term retention of extinction, while the VTA-dopamine cell body manipulations affected both extinction learning and retention suggests that there may be other populations of dopamine neurons which project to different brain regions that participate in extinction learning. These populations may work together with substantia nigra dopamine projections to the dorsal striatum which have been implicated in fear extinction53.A notable aspect of our findings is the discovery of a specific dopamine projection to the NAc mShell which is distinct from projections to the NAc core and regulates fear extinction. This is consistent with a previous in vivo microdialysis study which found that dopamine in mShell increased at the cessation of an aversive predictive cue58. Furthermore, previous work has demonstrated that the NAc core and mShell are functionally distinct59 and maintain differential connectivity with the rest of the basal ganglia54. For example, anatomical studies have found that vmPFC projects preferentially to NAc mShell while dorsomedial PFC preferentially innervates NAc core, though these distinctions are not absolute56. NAc projects to ventral pallidum, which projects in turn to distinct thalamic subregions which innervate either vm- or dorsal mPFC (dmPFC) as well as amygdala58. From the perspective of fear and reward learning these distinctions could be important as vm- and dmPFC have been implicated in distinct reward and aversive functions60, with vmPFC participating in extinction and dmPFC being important in producing fear responses61. Providing functional support for the idea that NAc regulates extinction related networks, deep brain electrical stimulation of NAc facilitates fear extinction and MAPK phosphorylation in vmPFC and amygdala and this effect is dependent on neural activity in the NAc62. Furthermore, stimulation of amygdala inputs to NAc also enhanced extinction learning and activated vmPFC and, to a lesser extent, dmPFC neurons45. Together, this suggests that the NAc mShell modulates vmPFC and amygdala function during fear extinction learning, possibly through distributed, but preferential connectivity with these regions.These results also suggest a differential involvement of NAc core vs. mShell in extinction learning, but how dopamine in mShell relates to MAPK phosphorylation in vmPFC and amygdala is not clear. One possibility is that the mShell regulates vmPFC and amygdala through basal ganglia connectivity loops. In fact, the NAc projects indirectly to both the amygdala and vmPFC63. Similarly, pharmacologically enhancing dopamine neurotransmission in humans facilitates fear extinction learning64. Exposure therapies such as extinction are first line treatments for anxiety disorders such as PTSD. Our results combined with the human work implicating the dopamine system in extinction suggest that enhancing dopamine signaling, and particularly dopamine release in the NAc during extinction could be an important therapeutic adjuvant when combined with exposure therapy. This could be accomplished through pharmacological means64 and/or through counterconditioning66 (but see ref. 67), a treatment technique which combines exposure therapy with rewarding experiences and may preferentially engage the NAc projecting dopamine circuit.These findings could also have relevance for the treatment of human anxiety disorders. The importance of dopamine in fear extinction learning in humans was highlighted by one study reporting that a polymorphism in the dopamine transporter 1 (DAT1) gene, which likely increases dopamine levels, enhanced fear extinction24 9–20-week old were housed individually in temperature and humidity controlled environments on 12-h light-dark cycles (lights on from 8AM to 8PM) and provided with food and water ad libitum. TH-Cre breeding males were mated with 9 weeks old or older Long-Evans females  in 2 week breeding periods to generate the TH-Cre colony. Littermates were assigned to experiment and control groups whenever possible. Experiments were all conducted during the animal’s light cycle. All procedures conformed to protocols approved by the Animal Care and Use Committees of the RIKEN Brain Science Institute.Male Long-Evans TH-Cre ratsAAV5-EF1α-DIO-eNpHR3.0-eYFP, AAV5-EF1α-DIO-eYFP, AAV9-CAG-FLEX-ArchT-GFP, AAV9-CAG-FLEX-GFP were produced by the University of North Carolina Vector Core . CAV2-FLEX-flp was produced by the Montpellier vector core. AAV9-CAG-fDIO-eArchT3.0-eYFP and AAV9-CAG-fDIO-eYFP were produced and packaged in our lab.Animals were induced and maintained under isoflurane anesthesia (Pfizer) at 1.5–3%. Animals were then placed in a stereotaxic device (Kopf Instruments) for virus injection and optical fiber insertion. For VTA manipulation experiments, AAV5 constructs were injected bilaterally into the VTA  using a Hamilton syringe  attached to a Harvard Apparatus pump (PHD2000) at a rate of 0.1 µl/min (1 µl per side). 20 min post-injection, injection cannulae were withdrawn, and a dual fiber optic cannula  was inserted bilaterally dorsal to the injection sites . Fiber optic cannulae were secured using head screws, a layer of Super Bond cement  followed by application of acrylic dental cement. Animals were trained 3 weeks post-surgery. For terminal manipulation and voltammetry experiments, AAV9 constructs were injected bilaterally at 0.1 µl/min into four sites (0.5 µl per site) in VTA . For terminal manipulations, 6–9 weeks after the virus injection animals were anesthetized again and implanted with two optical fibers  on each side of the NAc , LA , or IL . For manipulation of mShell or core projecting VTA-dopamine neuron experiments, CAV2-FLEX-flp was injected bilaterally at 0.1 µl/min (0.3 µl/site) in either NAc shell  or core  and flp-dependent AAV constructs (1.0 µl/side) were injected into VTA . A dual fiber optical cannula was implanted above the VTA using the same procedures described above. Animals were then allowed to recover for one week before behavioral experiments began at which time they were handled for 5 min each, 4 days before fear conditioning. For retrograde tracer experiment, 0.3 μl of Alexa Fluor 555 or 647 conjugated cholera toxin subunitB was ipsilaterally injected into Nac shell  and core .For voltammetry experiments, rats injected with AAV9 contructs in VTA 6–12 weeks previously were anesthetized with isoflurane and placed in a stereotaxic frame. Anesthesia was maintained with 0.5–1% isoflurane inhalation concentration. Craniotomies were made over the nucleus accumbens  and medial forebrain bundle  in one hemisphere (left) for stimulating and recording electrode insertion. A third craniotomy was made over the opposite hemisphere for reference and auxiliary electrode insertion. A carbon fiber (cf)-optrode was lowered into the NAc (~7.2 mm V) with the cf-containing silica tube placed at the coordinates above. One silver wire (auxiliary electrode) and one silver/silver chloride wire (reference electrode) was inserted into the cortex of the contralateral hemisphere. A pair of stainless steel bipolar stimulating electrodes were lowered to the MFB. The dorsal–ventral placement of the stimulating electrode was adjusted to obtain maximal dopamine overflow during initial stimulation . After stable and maximal striatal dopamine release was confirmed, trials with stimulation preceded by light application (Laser) alternated with trials without light application (No Laser).10 showed that dopamine levels increased in the NAc mShell after an expected shock was omitted and that the dopamine peaked after the offset of where the shock would have occurred. Intertrial intervals were variable averaging 150.9 s. Before each experiment laser power was adjusted to 12–15 mW of power at the tip of optical fibers. During extinction retrieval trials 24 h later, animals were returned to the extinction context with the same lighting and scent conditions as extinction training, and given five presentations of the same CS with variable ITIs averaging 150 s. Freezing levels were averaged in two trial bins during extinction training, and in a five trial average during testing. In the pMAPK study, control animals were placed in the same environment but without tones for the same amount of time. In the LA/B terminal inhibition studies, six of the ArchT and seven of the GFP injected animals were trained using a 1 kHz-pitch tone CS after previously being trained with 5 kHz-pitch tones. Twenty-four hours later, the animals were given extinction trials with light inhibition as in other animals, but with 1 kHz tones. The rest of the LA/B animals received the same training as the other experiments described above. No significant differences in freezing rates were found between groups experiencing 1 kHz vs. 5 kHz extinction and extinction retrieval trials, so their data were grouped. Freezing behavior during the 20 s CS period of fear training and during the 5 CS presentations of extinction retrieval were scored automatically using the Video Freeze program v2.7.1  or a customized optical flow-based freezing program designed in Matlab. To avoid conflating freezing with sleeping during the longer extinction trials, all extinction freezing was scored manually by a rater blind to the identity of the treatment groups using a stopwatch. Freezing was defined as the cessation of all bodily movements except for breathing or sleep. The methods used to quantify freezing were identical across all experimental runs and were the same across all experimental groups.Animals were randomly assigned to groups prior to the start of each experiment. Animal numbers/groups were decided based on standard practices in fear conditioning and extinction experiments. During fear conditioning, animals were placed in a sound-isolating chamber  and presented with auditory conditioned stimuli (CS) paired with an aversive footshock unconditioned stimulus (US) 3X. The CS was a series of 20  kHz-pitch tone pips presented at 1 Hz . The shock US  began at the onset of the last pip and lasted for 1 s . Variable intertrial intervals (ITIs) averaged at 130 s were used. During fear extinction training 24 h later, animals were attached to a laser through fiber optic cabling and placed in a novel chamber with fluorescent lights off and a different scent in the box and the CS was presented repeatedly (22 × ) without the shock US. During each CS presentation laser illumination occurred starting either at the onset of the final pip and lasting for 3 s (the shock omission period) or throughout the auditory CS period . For the inhibition during shock omission experiments, the 3 s laser stimulation period was selected because a previous studyFor fear conditioning studies Fig.  stimuli Within 3 days of the final experiment, animals were overdosed with 25% chloral hydrate and perfused with paraformaldehyde (4% in PBS). Brains were then post-fixed in a 30% sucrose PBS solution and then embedded in optimal cutting temperature (OCT) compound (Sakura Finetek) and sliced into 40 µm coronal sections using cryostat. Slices were then mounted on subbed glass slides , coverslipped using ProLong Gold antifade reagent (Molecular Probes) for microscopic examination. Additional sections were stored in 0.05% Sodium Azide PBS. In the pMAPK study, animals are sacrificed and perfused one hour after extinction training or one hour after being placed in the box for the same amount of time. Animals were excluded from analyses if virus expression was minimal/off-target or if fiber optic placements were incorrect (assessed blindly).For verification of TH specificity of expression and visualization of terminals, immunohistochemistry was performed prior to mounting/coverslipping. Sections were washed 3 × in PBST (0.3% Triton-X in PBS) and blocked for 30 min in 2% bovine serum albumin (BSA) in PBST. Slices were then incubated in primary antibodies diluted in BSA-PBST overnight at 4°C in the dark. Next day, sections were washed 3× in PBS and incubated for 1 h in secondary antibodies (if necessary) diluted in BSA-PBST. After rinsing with PBS, slices were mounted as stated above. Primary antibodies used were goat anti-GFP Alexa Fluor 488 conjugate , rabbit or mouse anti-TH , rabbit or mouse anti-GFP . Secondary antibodies used were goat anti-rabbit or goat anti-mouse Alexa Fluor 488 or 594 . In the pMAPK study, sections were incubated in 1% hydrogen peroxide in PBS for 30 min before wash. The primary antibody used was rabbit anti-phospho-p44/42 MAPK Erk1/2 Thr202/204 . Sections were incubated for 72 h at 4°C. After incubation and 3 × PBS wash, sections were placed in biotinylated goat anti-rabbit IgG for 30 min . Following another 3 × PBS wash, sections were incubated in avidin-biotinylated HRP for 30 min (ABC HRP Kit) and washed again 3 × PBS. For visualization, slices were placed in a solution containing diaminobenzidine (DAB), hydrogen peroxide, and nickel  for 5–10 min until staining of cell bodies was apparent. We used the same staining time for sections from all groups once the sufficient amount of time was determined. Sections were washed in distilled water, mounted on gelatin-coated slides, dehydrated using a series of 70%–95%–100% EtOH and finally xylene solutions, and coverslipped in Permount (Fisher).Electrochemical recordings using FSCV were made in anesthetized rats with carbon fiber (cf) microelectrodes. The cf electrodes consisted of 7-μm-diameter carbon fibers  threaded through silica tubing  backfilled with epoxy resin. To make a cf optrode, the cf-threaded silica tubing was appended with silver paint  to the metal holder for a 200 µm-diameter optic fiber such that the tubing extended parallel with the optic fiber at a distance of approximately 0.5 mm. The cf tip was cut to extend approximately 300 µm from the tip of the silica tubing. A commercial counter electrode-grounded type potentiostat  was used for electrochemical recordings. Data acquisition was performed by a commercial control/recording system  implemented on a Windows PC (MODEL). The gain of the amplifier was 500 nA/V and the low-pass filter time-constant was 0.2 ms. Voltage scans from -0.4 V to 1.3 V vs. Ag/AgCl reference, and back from 1.3 V to -0.4 V, were applied to the cf electrode. This triangle-positive waveform was repeated at 10 Hz. Using this waveform, in vitro calibration of the cf electrodes was performed where a flow of phosphate-buffered saline (PBS) was switched to PBS containing known concentrations of dopamine, pH shifts or adenosine. The pH shifts were set using a pH-meter and adjusted with HCl or NaOH solution. Further calibration of light-generated artifact signals was made in vivo .For quantification of the specificity of TH expression . Significant interactions or main effects in the ANOVAs were analyzed for multiple comparisons (α = 0.05) using Holm–Sidak test, which gives greater power than the Bonferroni and Tukey methods68. In the pMAPK study, immunoreactivity was quantified using four slices for each animal imaged with 10 × tiles on a microscope in bright field . An experimenter blind to the identity of the groups cut out the IL and LA areas in the images and thresholded the brightness levels to obtain a count of the number of cells in the predefined areas using ImageJ. Results were calibrated by dividing by the area where counts were made to get counts of number of positive cells per mm2. All behavioral experiments were run at least 2 × with groups consisting of experimental and control animals.Two-way ANOVA, two-way repeated measures ANOVA, and unpaired Student’s 69 . The differential currents across the voltage scan were analyzed with principal component regression analysis using modified TH-1 CV-Analysis software (Paul Phillips Laboratory). Calibration templates were constructed using in vivo dopamine and pH templates that corresponded to known values obtained in vitro. In vivo adenosine templates were additionally used but were assigned nA current values for regression instead of concentration. Although optic fibers were not directly oriented toward the cf electrodes, 600 nm wavelength light application generated a voltammetric artifact similar to that observed previously with 460 nmThe code that support the findings of this study are available from the corresponding author upon reasonable request.The data that support the findings of this study are available from the corresponding author upon reasonable request.Supplementary InformationPeer Review File


Improving robustness of farm animals is one of the goals in breeding programmes. However, robustness is a complex trait and not measurable directly. The objective of this study was to quantify and characterize (elements of) robustness in growing pigs. Robustness can be analysed by examining the animal’s response to perturbations. Although the origin of perturbations may not be known, their effect on animal performance can be observed, for example, through changes in voluntary feed intake. A generic model and data analysis procedure was developed (1) to estimate the target trajectory of feed intake, which is the amount of feed that a pig desires to eat when it is not facing any perturbations; (2) to detect potential perturbations, which are deviations of feed intake from the estimated target trajectory; and (3) to characterize and quantify the response of the growing pigs to the perturbations using voluntary feed intake as response criterion. The response of a pig to a perturbation is characterized by four parameters. The start and end times of the perturbation are ‘imposed’ by the perturbing factor, while two other parameters describe the resistance and resilience potential of the pig. One of these describes the immediate reduction in daily feed intake at the start of the perturbation  while another parameter describes the capacity of the pig to adapt to the perturbation through compensatory feed intake to rejoin the target trajectory of feed intake . The procedure has been employed successfully to identify the target trajectory of feed intake in growing pigs and to quantify the pig’s response to a perturbation. The study provides a data analysis procedure to detect the impact of perturbations on feed intake in growing pigs, and a mathematical model to quantify traits related to resistance and resilience. When pigs are kept in the same environment and are facing a common perturbing factor, the model can be used to identify differences in resistance and resilience among pigs, which can be used in selection programmes. Although this procedure uses feed intake as a response criterion and is applied to growing pigs, it is generic and can be applied to other species and with other response criteria.et al., et al., Growing pigs, like other animals, are confronted with variation in their environment to which they may have to respond. This includes the effects of climate change , infectious diseases, but also management practices and interactions with other animals. Robustness deals with the way animals respond to changes in their environment. Knap  defined et al., et al., et al., et al., Because of the dynamic nature of the response of animals to a perturbation, it is difficult to use single time-point measurements to quantify resistance and resilience . Moreover, feed intake is among the first measurable and non-invasive traits affected by perturbations, and was therefore considered as a suitable trait to quantify the response of a pig to a perturbation.Only perturbations that have a negative impact on feed intake are considered in this study. Perturbations that result in an increase in feed intake  are not considered here, but the proposed method is generic and can be adapted to account for these types of perturbations.CFI) of a pig is the combination of a target trajectory curve  and a change in feed intake due to perturbations. During a perturbation, the feed intake of the pig will deviate from the target trajectory but, once the perturbing factor is over, the pig will strive to increase its feed intake through compensatory feed intake to rejoin the target trajectory of CFI (target CFI).It is hypothesized that the observed cumulative feed intake  is often used as a production trait, fluctuations in DFI data make detection of perturbations difficult. Moreover, after a perturbed period, the overall reduction in feed intake needs to be compensated for by an equal increase in feed intake during the recovery period, which should surpass the target trajectory of DFI (target DFI). The cumulative feed intake  has the advantage over DFI of being less variable and, more importantly, allows for an easier representation of a trajectory including deviations and recovery. In the absence of a perturbation, it is hypothesized that the observed CFI is identical to the target CFI. During a perturbation, the observed CFI deviates from the target CFI  and, once the perturbing factor is over, the animal seeks to rejoin the target CFI through compensatory feed intake, without surpassing it in a systematic way.Although daily feed intake  and ‘Target_CFI(t)’is the target CFI at day ‘t’. The parameters a, b and c are the classical parameters of a polynomial function, and ts is the day when the quadratic segment of the curve changes to the linear segment.where ‘a, b and c by t0 (the estimated age at which CFI = 0), CFImid-point  and CFIlast (the estimated CFI at the last observation). Details of this re-parametrization are described in Supplementary Material S1.To facilitate the biological interpretation of the parameters, equation  was repat0 and CFIlast). Secondly, to avoid the estimation of ts being too close to either the first or the last observation, a linear function was then used to describe DFI, resulting in a quadratic function for CFI .Using the reparametrized equation  to estimTo estimate the target CFI, the reparametrized equation  has to bDeviations from the target CFI correspond to potential perturbations. As indicated earlier, small oscillations in feed intake patterns exist. The aim here is to detect the most important deviations that are the result of perturbations. These perturbations can then be characterized by the duration and magnitude of the deviation from the target CFI. A deviation was considered as a perturbation if it lasted at least 5 days, to ensure that a reasonable number of data were used to estimate the model parameters. The magnitude was determined by calculating the maximum reduction of a deviation from the target trajectory. Because CFI is increasing continuously, deviations from the target CFI were expressed as a percentage and an arbitrary value of 5% was set as a threshold value to identify a perturbation. To identify perturbations among all the deviations from the target CFI, a B-spline function of order 6 was fitted to the difference between the observed CFI and the target CFI. Any period during which the observed data deviated from the target CFI for more than 5 days and for more than 5% was considered to be the result of a perturbation. The interest of using a B-spline function is its high flexibility and its smoothing properties that allow to capture small deviations from the target CFI  and deviations from the target CFI of more than 5% occur frequently due to the small value of CFI during the first week. However, deviations that started during the first week and for which the selection criteria of duration and magnitude continue to hold during the second week were considered the result of a perturbation.The model to characterize the animal’s response to a perturbation is based on an ordinary differential equation and includes two components: the immediate impact of the perturbation and the response of the pig to the perturbation Figure . A pertuk1) describes the constant negative impact of the perturbation on DFI. The fourth parameter (k2) is the marginal response in DFI due to a change in the ratio between the CFI and the target CFI, and describes the capacity of the pig to adapt to the perturbation through resilience and compensatory feed intake. The perturbation model is therefore the result of resistance and resilience mechanisms and can be written as:t)’ takes the value of k1 between the t_start and t_stop time, and is zero otherwise, while ‘Resilience(t)’is described by:The perturbation model was conceptualized in a way that the impact of a perturbation on feed intake can be characterized by four parameters. Two parameters indicate the start (t_start) and end times (t_stop) of the perturbing factor, while the third parameter  as described by Labroue et al. (RFID) ear tag or malfunctioning of the feeder), a procedure to deal with missing data was developed . There were no missing feed intake records for the five pigs used here to illustrate the procedure. Because pigs were fasted one day before leaving to slaughterhouse, the last observation of feed intake of each pig was ignored.Data were collected from an experimental farm of INRA in Le Magneraud . Five pigs from the same batch  were chosen to demonstrate the procedure. The pigs entered the same growing facility at 68 days of age and stayed there until reaching their slaughter weights (124 kg on average). Feed was provided e et al. . Becausehttp://cran.r-project.org/). To account for scale differences in the target CFI , a weigequation  and equaequation  and 2b)http://cr et al., . All equThe test for auto-correlation was performed by a Wald-Wolfowitz runs test. To fit the B-spline function to the difference between the observed CFI and target CFI, the package ‘fda’ was used. To characterize the pig’s response to a perturbation, equations  and 2b)2b) were The procedure is illustrated step by step for one of the pigs, and the results for all five pigs are presented in Table The target trajectory of CFI of pig 01 was estimated using the quadratic-linear function of the reparametrized equation 2b) were k1 and k2) varied between these pigs .Table Characterization and quantification of the response of animals to perturbations are important in animal management and breeding. The recent and rapid development of monitoring devices in combination with data analysis and modelling techniques offer a great potential to progress in this area. This study illustrated how DFI records can be used to characterize and quantify elements of resistance and resilience in growing pigs.et al., et al. , the power function and a Gamma-function describing feed intake relative to maintenance energy requirements  four parameters. Although the choice of 20 observations was arbitrary, it is a compromise between the number of remaining data and the presence of auto-correlation in the target CFI. If the filtration procedure was allowed to go further, there would be no data with auto-correlated residuals, but the estimation of the target CFI (which is described by maximum four parameters) would be based on a small number of observations. In contrast, if the procedure was stopped earlier (with more remaining observations), the target CFI would include more data with auto-correlated residuals, some of which could be due to a perturbation.et al.  may be given to animals that deviate from their target CFI to limit the impact of the (known or unknown) perturbing factors and to facilitate the recovery of the animals so that they can regain their target CFI.The recent development of monitoring technologies offers new opportunities for livestock management. Recording of individual feed intake in group-housed pigs is becoming more accessible, and feed intake can be very informative about the health and welfare status of the animal. The model and data analysis procedure proposed in this study showed to have the potential to detect the impact of a perturbation on the feed intake and to quantify the response of the animal in terms of traits related to resistance and resilience.


The MN12SX density functional, in connection with the Def2TZVP basis set, was assessed, together with the SMD solvation model (Solvation Model based on the Density), for calculation of the molecular properties and structure of a group of peptides of marine origin named Mirabamides A–H. All the chemical reactivity descriptors for the systems were calculated via Conceptual Density Functional Theory (CDFT). The active sites suitable for nucleophilic, electrophilic, and radical attacks were chosen by linking them with the Fukui function indices, nucleophilic and electrophilic Parr functions, and condensed Dual Descriptor  The sea is an inexhaustible source of natural resources that give rise to molecules that can serve as a guide for the development of new medicines. For this reason, numerous investigations have been carried out in recent years dedicated to the search for new natural products that can be obtained from the knowledge of marine species .Among the chemical species that can be obtained from natural products of marine origin, the peptides that are molecules of intermediate size between amino acids and proteins stand out. The therapeutic application of these peptides, called for this reason therapeutic peptides, is currently one of the most active fields of research due to the great possibilities they represent as aids for the treatment of numerous diseases .For the consideration of therapeutic peptides from the point of view of medicine, it is necessary to know their molecular properties and their bioactivity. It is our belief that the bioactivity of these peptides is intimately related to their chemical reactivity from a molecular perspective ,4. For tConsidering that the knowledge of the chemical reactivity is essential for the development of new medicines from natural products, we have decided in this work to study a series of peptides obtained from marine sponges that have been recently isolated and characterized, and that could be the foundation of new therapeutic peptides ,11.The objective of this work is to study the chemical reactivity of the Mirabamides A–D  and the ed by us , the abied by us , and theed by us ,15.rN) in case of the exact Kohn–Sham (KS) theory as well as generalized KS theory for an N electron system, should be −IP(N). Here, IP represents the vertical ionization potential, which is calculated as the energy difference, E(N− 1) − E (N), by considering a particular functional. If approximate functionals are used, it could possibly lead to considerable differences between N) and −IP(N). Optimal tuning involves determining the system-specific range-separation parameter, N) = −IP(N), are optimally satisfied [N + 1) = −EA(N), which facilitates obtaining the optimized value of N) = −IP(N) satisfaction and, at the same time, a comparison between the N) of the neutral species (the LUMO) and the N − 1) for the anionic system .The descriptor related to this comparison is called As this work is a part of an ongoing study related to our project on Computational Medicinal Nanochemistry, the theoretical background will be similar to that presented in previous works ,20,21,22atisfied ,36,37,38ferences ,20,21,22the LUMO ,20,21,22https://pubchem.ncbi.nlm.nih.gov), a website that serves as the public repository for information pertaining to chemical substances, along with their associated biological activities. The pre-optimization of the resultant system involved selecting the most stable conformers. The selection was done using random sampling that involved molecular mechanics techniques and the inclusion of the various torsional angles via the general MMFF94 force field [https://www.chemaxon.com). After that, the chemistry of the structures was checked and the 3D structures of the stereoisomers were generated using the same MarvinView 17.15 program. The chirality at the stereogenic centers was verified in accordance to the Cahn-Ingold-Prelog priority rules. The resulting geometries were further refined, as was previously explained, and the lowest energy conformation for each molecule was chosen to calculate the electronic energy and the HOMO and LUMO orbitals at the DFT functional level, as mentioned in the next paragraph.This study obtained the molecular structure of the Mirabamides A–H peptides from PubChem  for the calculation of the molecular properties  and for the prediction of the bioactivity score for different drug targets . The bioactivity scores were compared with those obtained through the use of other software, such as MolSoft from Molsoft L.L.C.  (http://molsoft.com/mprop/) and ChemDoodle Version 9.02 from iChemLabs L.L.C.  (www.chemdoodle.com).The SMILES (Simplified Molecular Input Line Entry Specification) notations of the studied compounds were fed in the online Molinspiration software from Molinspiration Cheminformatics   ligands, kinase inhibitors, ion channel modulators, enzymes and nuclear receptors). The results are named Bioactivity Scores and the values for the Mirabamides A–H are presented in These bioactivity scores for organic molecules can be interpreted as active (when the bioactivity score is >0), moderately active (when the bioactivity score lies between −5.0 and 0.0) and inactive (when the bioactivity score As the ICferences ,11, we cIn this paper we have presented the results of a study of the chemical reactivity of a group of peptides with therapeutic potential, Mirabamides A–H, based on the Conceptual DFT as a tool to explain the molecular interactions.It must be remarked that the tools used did not generate a huge error despite the relative low computational they have, which is itself a good advantage.The knowledge of the values of the global and local descriptors of the molecular reactivity of the Mirabamides A–H peptides studied could be useful in the development of new drugs based on these compounds.In a similar manner, the pA point of special interest has been the quantification of the ability of each peptide to act as an inhibitor in the formation of AGEs, and this could be of importance for the design of medicines for fighting diseases like Diabetes, Alzheimer’s or Parkinson’s.Finally, the molecular properties related to bioavailability have been predicted using different methodologies already described in the literature, and the descriptors used for the quantification of the bioactivity allowed the characterization of the studied peptides as moderately bioactive towards several ligands and enzymes.


Körner’s septum (KS) is a developmental remnant formed at the junction of mastoid and temporal squama, representing the persistence of the petrosquamosal suture. During mastoid surgery, it could be taken as a false medial wall of the antrum so that the deeper cells might not be explored. The aim of the study was to assess a Körner’s septum prevalence and to analyze its topography.The study was performed on 80 sets of cone-beam computed tomography (CBCT) images of temporal bone . Körner’s septum was identified and its thickness was measured on axial sections at three points: at the level of superior semicircular canal (SCC), at the level of head of malleus (HM) and at the level of tympanic sinus (TS).KS was encountered at least in one point of measurements in 50 out of 80 sets of CBCT images (62.5%). The average thickness at the level of SCC was 0.87 ± 0.34 mm, at the level of HM was 0.99 ± 0.37 mm and at the level of TS was 0.52 ± 0.17 mm.Körner’s septum is a common structure in the temporal bone–air cell complex. It is more often encountered in men. In half of the patients, it occurs bilaterally. However, in most of the cases it is incomplete with anterior and superior portions being the most constant. Körner’s septum (KS) also known as petrosquamous lamina is a bony plate formed at the junction of the mastoid and temporal squama and it represents the persistence of the petrosquamous suture , 21. It Despite the fact that some authors point to A. H. Cheatle as the first to describe KS , 17 usinSince Körner highlighted the clinical relevance of the septum , the impAlthough some authors mentioned the KS in their papers, no papers describing its structure have been published to our knowledge. Therefore, we aim to assess in our study the Körner’s septum prevalence and analyze its topography.The study was performed on anonymized sets of cone-beam computed tomography (CBCT) images of temporal bone gathered from the Department of Dental and Maxillofacial Radiology, Medical University of Warsaw, between February 2013 and June 2013. All the scans were obtained due to clinical  indications with the scanner Planmeca Promax 3D Mid  with parameters: voxel dimensions 400 × 400 × 400 µm; the exposition was performed with source voltage of 90 kV and current of 12 mA. From the initial group (141 patients), the images with any temporal bone pathology were excluded (61 patients). Thus, a final group of 80 sets of images  was enrolled to the study. The age of the patients involved in the analysis ranged from 12 to 77 (mean age 37 ± 16 years).In the next phase, the scans were analyzed in RadiAnt DICOM Viewer 4.0.3 (64-bit), obtained data were analyzed statistically with the use of StatSoft Statistica 13.1 software.The subsequent step was to identify the Körner’s septum and to measure its thickness on horizontal sections at three points (landmarks): at the level of superior semicircular canal (SSC), at the level of head of malleus (HM) and at the level of tympanic sinus (TS) Fig. . The KS p > 0.05) and it was present bilaterally in half of the cases (25 people out of 50). A complete KS, present at all three points, was encountered in 19 out of 75 temporal bones (25%) but it was found bilaterally only in 3 subjects—2 women and 1 man, which made 5% and 2% of our population, respectively. An incomplete KS was encountered in 56/75 temporal bones (75%). The name for each portion of KS was adopted from anatomical directions, taking into consideration the position of KS in the space of the antrum. Therefore, the level of SCC corresponded to superior portion of KS, the level of HM to anterior portion and the level of TS to its posterior portion.We were able to find all anatomical landmarks  for every part of Körner’s septum on all evaluated CBCT scans. KS was recognized at least in one point of measurements in 50 out of 80 sets of CBCT images (62.5%), which constituted 75/160 temporal bones. The KS was more frequent in males than females —32 in males (39%) and 22 in females (28%). KS was recognized at the level of superior semicircular canal in 53/160 cases (33%)—29 males (35%) and 24 females (31%). At the last point (sinus tympani), KS was found in 31 bones (19%)—16 males and 15 females (19% each).p < 0.05). It was the greatest at the level of HM and the lowest at the level of TS. The average thickness with standard deviation, median, minimal and maximal values of thickness are presented in Table The average thickness of KS was significantly different in every portion  starting from the anterior edge of temporomandibular joint and forming its roof, (2) through the superior wall of external acoustic meatus to the (3) mastoid or posterior part overlaying a great part of the petrous bone. They also described the internal petrosquamous suture positioned anterosuperiorly to the tympanic sulcus  and the posterior external petrosquamous suture which represented the contact of the deep aspect of the retromeatal portion of the squama with the pars petrosa. Interestingly, they claimed that structure can be called a Körner’s septum or petrosquamous lamina when the junction of the pars petrosa and squama persists only inside the mastoid process . The samIn our study, we have chosen three areas in which we expected to find the Körner’s septum. At the level of tympanic sinus, in close relationship with a mastoid part of facial canal, we have searched for posterior portion of KS. Superior semicircular canal was a landmark for the superior portion of the KS. This one can also occur as the only persistent part of KS which can lead an otosurgeon straightforward to the posterior crus of the incus . In certAccording to the literature, the presence of Körner’s septum may vary in different groups from approximately 6.5%  in tempop < 0.05). The thickness of KS at the level of SSC is the same in males and females. The thickness at the level of HM is greatest than in other portions.None of the published papers comments on the portions of KS and their dimensions. In the present study, we found that KS is remarkably thinner at the level of TS than in other portions , superior  and posterior .The thickness of the Körner’s septum is the greatest in anterior and the lowest in its posterior portion.


The electron conductivity of an extended metal atom chain (EMAC) that consisted of penta-nickel(II) ions bridged by oligo-α-pyridylamino ligands was examined by density functional theory (DFT) and elastic scattering Green’s functions (ESGF) calculations. The calculated results revealed that an intramolecular ferromagnetic (FM) coupling state showed a higher conductivity in comparison with an anti-ferromagnetic (AFM) coupling state. The present results suggest the potential of the complex as a molecular switch as well as a molecular wire. A technology to manipulate single molecules has been developing to realize molecule-based devices. At the same time, it is necessary to find more suitable molecules for such devices ,2,3. Fro5(tpda)4X2], [M7(teptra)4X2], [Ni11(tentra)4Cl2](PF6)4  [11(bnatpya)4Cl2]4+  strongly depends on its intra-molecular spin-coupling states  (1). First, we examined the electronic structures and electron conductivities of the AFM and FM states. On the other hand, as a development of computational procedures to calculate properties of the single-molecule, the electron conductivities of various magnetic molecules have been estimated by theoretical calculations ,24. Our hain see B 20]. W. WS = 1)g states . The spiThe conductivity of the FM state was obviously different from that of the AFM state reported in previous paper . FurtherThe conductivity of small systems is usually evaluated by a non-equilibrium Green’s function (NEGF) method using molecular orbitals obtained from, for example, extended-Hückel, DFT, and other quantum chemical calculations ,24. Beca1 is connected to gold electrodes by the sulfur atoms of NCS ligands, as illustrated in 1 was connected to the gold dimers i.e., an extended molecular system, as illustrated in AFM1 and FM1, respectively. When a voltage (VD) is applied to the right (R) and left (L) electrodes using the extended ESGF method, the static carrier conduction of the system  and the lowest unoccupied molecular orbital (LUMO) of the extended molecular system corresponded to the Fermi energy. In the extended ESGF method, a square of the transition probability for the spin σ at a given voltage E; m-th basis set of atom n for spin σ. In the case of complex 1, however, the LUMO and LUMO+1 were quasi-degenerate as explained below, so that we also included the effect of those quasi-degenerate orbitals by weighting with the Boltzmann factor. The effective strength of k represents the number of the degenerate orbitals around LUMO, and Bk is the Boltzmann factor, with the temperature assumed to be 300 K. In order to depict the I–V characteristics, voltage from 0.1 to 3 V was applied. Here, we noted that there might be a possibility that the applied maximum voltage (3V) was over the breakdown voltage. Let us consider that complex 1 for the extended molecular system are summarized in 1, we performed DFT calculations for both AFM and FM states . For the spin-polarized electronic structures, all calculations were performed using the spin-unrestricted DFT method with the Becke 3-parameter Lee–Yang–Parr hybrid functional set (UB3LYP). As a basis sets, Tatewaki–Huzinaga MIDI plus p-type (533(21)/53(21)/(41)), 6-31+G*, and 6-31G were used for Ni atoms, axial ligand atoms, and bridging ligand atoms, respectively. All DFT calculations were performed in the gas phase by using Gaussian09  and [Ni5(tpda)4(NCS)2] were 4.86 and 9.33 Å, respectively [AFM1 and FM1. The two spin states, therefore, coexisted because of a Boltzmann distribution at 300K, so that the real conductance of the extended molecular system was considered to be the averaged value of the two states. So, one must perform the experiment under the much lower temperature condition. By decreasing the temperature, the conductance is decreased because the population of the ground state, i.e., the AFM state that indicates lower conductance, becomes dominant. Then, a change in the conductance by changing the spin states into the FM state appears more significantly. In this paper, we examined electron conductivities of EMAC consisting of penta-nickel(II) ions bridged by oligo-α-pyridylamino ligands by the DFT and ESGF calculations. We especially analyzed the calculated results in terms of the difference in electron conductivities between the spin states. The calculated results indicated that the conductivity of  complex . The difectively , and theIt is, however, necessary to increase the total electron conductivity and differentiate the electron conductance between the AFM and FM states for a more reliable molecular switch, suggesting a necessity of a rational molecular design based on quantum chemistry. For example, our results indicate the importance of selection of the axial ligand species and of the metal ions for higher conductivity. By using the axial ligands with the same orbital energy level as that of the dσ orbitals, the conductivity of the system was considered to be raised because the site-overlap increased, as mentioned above. In addition, it is, of course, crucial to decrease the HOMO–LUMO energy gap. On the other hand, it was also reported that the orbital overlap between metal ions can be controlled by the functional groups of the bridging ligands , suggestIn this way, our studies about the relationship between electronic structures, spin structures, and electron conductivities of nickel EMACs have obviously indicated a difference in the conductivities between spin states. In conclusion, the open-shell EMACs are considered to be promising materials for molecular switches as well as for molecular wires, and a rational investigation based on quantum chemical calculations is a powerful approach for their molecular design.


This investigation aims to study the characteristics and release properties of lutein-loaded polyvinyl alcohol/sodium alginate (PVA/SA) nanofibers prepared by electrospinning. In order to increase PVA/SA nanofibers’ water-resistant ability for potential biomedical applications, the electrospun PVA/SA nanofibers were cross-linked with a mixture of glutaraldehyde and saturated boric acid solution at room temperature. The nanofibers were characterized using scanning electron microscopy (SEM) and X-ray diffractometer (XRD). Disintegration time and contact angle measurements testified the hydrophilicity change of the nanofibers before and after cross-linking. The lutein release from the nanofibers after cross-linking was measured by an ultraviolet absorption spectrophotometer, which showed sustained release up to 48 h and followed anomalous (non-Fickian) release mechanism as indicated by diffusion exponent value obtained from the Korsmeyer–Peppas equation. The results indicated that the prepared lutein-loaded PVA/SA nanofibers have great potential as a controlled release system. Lutein, also known as “plant lutein”, is a natural pigment and an excellent antioxidant  widely fElectrospinning has attracted more and more attention from the past decade due to its potential use in biomedical materials, filtration, catalysis, optoelectronics, food engineering, cosmetics, and drug delivery devices ,6,7. AmoElectrospun PVA/SA nanofibers as drug carriers are limited due to the burst release of drugs. For example, Li et al.  reportedIn this study, a controlled drug delivery system was developed from electrospun PVA/SA nanofibers. Lutein was utilized as a model drug. Lutein-loaded nanofibers were cross-linked by using glutaraldehyde (GA) and saturated boric acid solution as a cross-linking agent. The properties of nanofibers before and after cross-linking and the lutein release behavior were investigated.2HPO4, 10039-32-4, 99%), potassium phosphatemonobasic , potassium chloride , sodium chloride , and N,N-dimethylformamide  were purchased from Aladdin, Shanghai China. Lutein  was provided by Shandong Tian Yin Biotechnology co.ltd, Zibo, China.Polyvinyl alcohol , 87%–89%(purity), Mw = 72600–81400), sodium alginate , glutaraldehyde , boric acid , sodium phosphate dibasic  was dissolved in 5 mL DMF at room temperature. The blended above PVA/SA solution and lutein solution were mixed and stirred for 5 h at room temperature to ensure homogeneous distribution.Electrospinning was performed to fabricate lutein-loaded PVA/SA nanofibers at room temperature. The mixed solution was poured into a 10 mL plastic syringe with a needle having an inner diameter of 0.41 mm. Output voltage applied to the solution was 15 kV. Besides, the flow rate of the injection pump was set to 0.3 mL/h. The nanofibers collector was cylindrical and covered by aluminum foil. At the same time, the distance from the syringe needle to the receiver collector was 150 mm. After completing the electrospinning process, the nanofibers were placed in a vacuum oven for 12 h to remove residual traces of solvents.Cross-linking of the electrospun lutein-loaded nanofibers were carried out using a mixture of GA and saturated boric acid solution. Electrospun lutein-loaded nanofibers were carefully peeled from the aluminum foil and weighed exactly using a digital balance. A weight of 0.020 g of each sample was immersed in the cross-linking fluid at room temperature for various times  to carry out cross-linking. After completing the required cross-linking time for each sample, all the samples were dried with filter paper and then exposed to a vacuum oven for 12 h at room temperature to remove residual GA and water.The morphology of the lutein-loaded PVA/SA nanofibers before and after cross-linking was observed by quanta 250 field emission environment scanning electron microscope (SEM). The average diameter of the lutein-loaded PVA/SA nanofibers before cross-linking was calculated based on the SEM image. The distribution of lutein was observed by fluorescence microscope .The surface static contact angles of the lutein-loaded PVA/SA nanofibers before and after cross-linking were investigated using a contact angle meter analysis system .X-ray diffractometer  was used to observe the physical state of lutein in PVA/SA nanofibers in the range from 5° to 50°.The cross-linking effectiveness of lutein-loaded PVA/SA nanofiber mat was analyzed using Fourier transform infrared (FTIR) spectroscopy .To determine the encapsulation efficiency (EE) of lutein in PVA/SA nanofibers, the lutein-loaded PVA/SA nanofibers were completely dissolved in water and lutein content was measured using UV-Vis spectrophotometer. The encapsulation efficiency of lutein was determined using the following equation:The release profile of lutein from the cross-linked lutein-loaded PVA/SA nanofibers was studied in phosphate buffer saline  solution. Lutein-loaded PVA/SA nanofibers (20 mg) were placed in 50 mL of PBS solution at 37 °C with constant stirring. At defined time intervals, 1 mL of sample was taken from the release medium and replaced with fresh PBS to maintain the original volume. The amount of lutein released at different time intervals in PBS solution was measured by a UV-Vis spectrophotometer . The lutein release percentage was calculated and release profile was drawn. All the measurements were performed in triplicates.SEM images of un-crosslinked lutein-loaded PVA/SA nanofibers are shown in θ = 20.54° and 14.06°), which were attributed to the crystallinity of lutein and consistent with the literature [X-ray diffraction (XRD) measurements were used to study the physical state and distribution of the lutein-loaded PVA/SA nanofibers. The XRD patterns of free lutein, PVA/SA nanofibers, and lutein-loaded PVA/SA nanofibers are shown in terature ,41. As sHydrophilic assessment of biological materials is a very important parameter in the field of drug delivery. Therefore, in order to determine the hydrophilicity of the nanofibers, the water contact angle of the lutein-loaded PVA/SA nanofibers before and after cross-linking was measured using a contact angle meter analysis system . The un-−1. The FTIR spectra of the lutein-loaded PVA/SA nanofibers with different cross-linking time are presented in −1 is ascribed to a hydroxyl group, at 2939 cm−1 to C–H stretching (CH2), at 1728 cm−1 and 1268 cm−1 to acetate groups , and at 1427 cm−1 to C–H stretching (CH3) [−1 is due to the hydrogen bonding between hydroxyl groups of PVA and SA [−1 (C=O) remains constant [−1 could be an indicator of cross-linking degree [OH) and carbonyl functions (hCO) decreased from 2.02 to 0.60 as the nanofibers cross-linking time increased from 1 to 5 h. This reflects a higher cross-linking effectiveness in the lutein-loaded PVA/SA nanofibers.The cross-linking effectiveness of lutein-loaded PVA/SA was analyzed using FTIR. Infrared scanning was performed in the range of 4000 to 600 cmA and SA . During g degree . The ratThe lutein-loaded PVA/SA nanofibers were cut into a size of 1 cm × 1 cm and dissolved in deionized water to verify its disintegration time. As shown in The EE and in vitro drug release assessment is necessary in order to determine the bioavailability and extent of drug assimilation, which subsequently determines the drug therapeutic efficiency . The EE Since both PVA and SA polymers are hydrophilic, the composite of these two elements  by electrospinning is readily soluble in water, as shown by the disintegration test. The slow release of lutein can be achieved by cross-linking the PVA/SA nanofibers with a cross-linking agent. In order to study the effect of different levels of cross-linking on the release behavior of lutein from PVA/SA nanofibers, an in vitro release study was performed for 48 h in PBS  respectively.t is the mass of the released drug at t time, M∞ is the mass of the released drug when the time approaches infinity, k is a constant, and n is the diffusion exponent. This expression depicts a proportional mass release out of the polymer matrix with time. The value of n is dependent on the type of drug delivery mechanism, geometry, and polydispersity. When n < 0.5, 0.5 < n < 1.0, and 0.5 < n < 1.0, the type of release follows Fickian diffusion, non-Fickian diffusion, and Case-II transport, respectively.The Korsmeyer–Peppas equation is as follows:k1 is the diffusion rate constant.Higuchi model is another widely used pattern for analyzing the mechanism of drug release:n, k, k1, and the correlation coefficient (R2) from fitting curves with two models. The R2 values of the Korsmeyer–Peppas model (matrix system) were closer to 1, compared to that of the Higuchi model for all the three nanofibers with different cross-linking time. From the Korsmeyer–Peppas model, n values were found to be 0.7398, 0.5840, and 0.6278 for lutein release from PVA/SA nanofibers cross-linked for 1 h, 3 h, and 5 h, respectively, indicating that lutein release from PVA/SA nanofibers occurred through non-Fickian  diffusion suggesting that more than one mechanism process was involved in lutein release. These mathematical models could be purely empirical through the insignificant changes of R2. Although the release mechanism requires further clarification, the phenomenon of the study proves that drugs can be released from the electrospun PVA/SA nanofibers matrices in a continuous manner.The release of the drug in the drug-loaded nanofibers is controlled by the diffusion of drug within the polymer matrix and/or matrix degradation, which involves bulk and surface-polymer erosion, depending on the polymer composition . When thIn this research, the lutein-loaded PVA/SA nanofibers with uniform and smooth morphology were obtained by electrospinning. The PVA/SA nanofibers were cross-linked for different time points and their hydrophilicity was measured with a contact angle measurement experiment. XRD analysis showed that lutein was present in the stable amorphous state in the PVA/SA nanofibers. The sustained release was achieved after the tuning the hydrophilicity of the lutein-loaded PVA/SA nanofibers as it released the lutein in a controlled manner extending up to 48 h. The drug release kinetics revealed that the release of the lutein was through non-Fickian diffusion mechanism. The results indicate that encapsulation of lutein utilizing polymer matrices by electrospinning is an effective method in drug delivery and cross-linking could further help to achieve the sustained lutein release by tuning the hydrophilicity. Therefore, the drug-loaded PVA/SA nanofibers developed in this study has great potential to be used as the delivery system in the near future.


GAS5) has been shown to be involved in several biologic processes, including inflammatory and autoimmune diseases, vascular endothelial cells apoptosis and atherosclerosis, as well as cellular growth and proliferation, cellular development and cell-to-cell signaling and interaction. The lncRNA GAS5 expression profile in KS patients has never been evaluated so far.Klinefelter syndrome (KS) is characterized by the presence of at least one supernumerary X chromosome. KS typical symptoms include tall stature, gynecomastia, hypogonadism and azoospermia. KS patients show a higher risk of developing metabolic and cardiovascular diseases, inflammatory and autoimmune disorders, osteoporosis and cancer. Long non-coding RNA (lncRNA) growth arrest-specific 5  technology and qRT-PCR assay in 10 patients with KS and 10 age-matched controls.NGS results showed a significantly lncRNAGAS5up-regulation by 5.171-fold in patients with KS. Theresults of qRT-PCR confirmed the NGS data.These findings showed the occurrence of lncRNA GAS5 over-expression in KS patients. Whether this lncRNA is involved in the pathogenesis of inflammation and autoimmune diseases, atherogenesis or germ cell depletion in KS patients is not known. Further studies are needed. Klinefelter syndrome (KS) is the most common sex-chromosome disorder in men, with an estimated prevalence of 1:660 newborns . The mosThe chromosomal abnormality leads to a progressive germ cell degeneration starting from mid-puberty, impaired Sertoli cell function , total tSeveral clinical manifestations associate with the syndrome. These include learning and language disability, reduction in intelligence quotient (IQ) scores of 10 to 15 points, but not into the intellectual disability range , increasGAS5) gene, mapping on the 1q25.1 chromosome, encodes for a long non-coding RNA (lncRNA) which is involved in the modulation of gene expression, targeting many different downstream miRNAs [Molecular mechanisms underlying the variability in KS phenotype have not been clearly understood yet. Epigenetic mechanisms have been suggested to play a role . Growth m miRNAs , 15.LncRNA GAS5 was initially identified as a tumor suppressor gene –21. HoweNo study evaluated the lncRNA GAS5 expression profile in KS patients so far. On this account, the present study was undertaken to investigate differently expressed genes and, specifically, lncRNA GAS5 expression in patients with KS and controls.The present study belongs to a broad project investigating transcriptome differences between patients with KS and normal control subjects (NC). To accomplish this, 10 patients with non-mosaic KS and 10 healthy controls of similar age were selected. All men with KS (mean age 32.5) had 47,XXY karyotype, as confirmed by cytogenetic investigation performed on at least 50 metaphases. NC (mean age 32) had negative history of genetic diseases, normal testis volume and normal reproductive hormones  levels. All men enrolled in this study (KS patients and controls) were Italians.They underwent to transcriptome and qRT-PCR analysis which were performed on peripheral blood mononuclear cells (PBMCs) isolated with ficoll gradient.RNA Sequencing, Next Generation Sequencing (NGS) and Data Analysis of NGS methods have been detailed in previous articles presenting NGS data from the same project , 25.https://www.bioinformatics.babraham.ac.uk/projects/fastqc/).Indexed libraries were prepared from 1 μg/ea. purified RNA with TruSeq Stranded Total RNA (Illumina) Library Prep Kit according to the manufacturer’s instructions. Libraries were quantified using the Agilent 2100 Bioanalyzer (Agilent Technologies) and pooled such that each index-tagged sample was present in equimolar amounts, with final concentration of the pooled samples of 2bnM. The pooled samples underwent cluster generation and sequencing using an IlluminaHiSeq 2500 System (Illumina) in a 2 × 100 paired-end (RNA-seq) format. The raw sequence files generated (.fastq files) underwent quality control analysis using FastQC , Italy). The quality checked reads were trimmed with cutadapt v.1.10 and then aligned to the human genome (hg19 assembly) using STAR v.2.5.2 , with stp-values (computed according to Benjamini-Hochberg). Differential expression data were further confirmed using Cuffdiff [Gene annotation, as provided by Ensembl (GRCh37), was obtained for all known genes in the human genome. We calculated the number of reads mapping to each transcript with HTSeq-count v.0.6.1 . These rCuffdiff . Functiohttps://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-6107/) with accession number E-MTAB-6107.Raw data are available in ArrayExpress database repository . Briefly, 600 ng of total RNA from each sample was then reconstituted in a final volume of 20 μl and the generated cDNA was used as a template for real-time quantitative PCR analysis using gene expression products. Quantification with qRT-PCR was performed using the 2t method  by matchGAS5 and the reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) TaqMan assays (Assay ID:Hs99999905_m1) were obtained from Applied Biosystems .The target GAS5 TaqMan assays (Assay ID: Hs03464472_m1) used amplifies an amplicon between exon 10 and exon 11 (it is possible to check the characteristics of the probe used at the following link: https://www.thermofisher.com/taqman-gene-expression/product/Hs03464472_m1?CID = &ICID = &subtype=). The mean was obtained with the Software Version 1.5 supplied with the LightCycler 480, as previously reported [The reported .t-test and bivariate linear regression analysis. Graph Pad Prism 5 software was used for statistical analysis. A p value < 0.05 was accepted as significant.Distribution analysis of measured gene transcript levels was performed using Shapiro – Wilk test and inferential statistical analysis of results was carried out using paired two tailed GAS5  was up-regulated in the patients with KS by 5.171-fold  . Therefore, we carried out a statistical inferential analysis using the parametric test paired two tailed t-test. Data acquired from this analysis revealed a significant increase of GAS5mRNA levels in KS patients compared to relative controls (p < 0.01), as shown in Fig. p < 0.05). Linear regression analysis reveals no significant correlation between GAS5 mRNA levels and age of subjects involved in our study (p > 0.05).Distribution of expression values was normal in both KS patients and NC  actions, inflammatory and autoimmune diseases, vascular endothelial cells apoptosis and atherosclerosis, FGSC proliferation.LncRNA GAS5 could directly interact with the DNA binding domain of the glucocorticoid receptor (GR) . This prLncRNA GAS5 expression is involved in atherogenesis. Its expression is significantly higher in plaques of atherosclerosis both from human and from animal models. Its over-expression enhances vascular endothelial cells apoptosis after lipoperoxidation. Therefore, lncRNA GAS5 has been suggested as a target for the therapy of atherosclerosis . KS patiDue to the involvement of lncGAS5 in several biological functions required for normal spermatogenesis, such as cellular growth and proliferation, cellular development and cell-to-cell signaling and interaction Fig. , we specIGF1R expression [IGF1R expression was down-regulated in KS patients compared to controls. Growing evidence addressed to IGF1R a role in human testicular function [Insr) in Sertoli cells from mice affected the final testicular size, Sertoli cell number and the sperm output [IGF1R in Sertoli cells might hypothetically explain germ cell loss in KS patients.Very recently, lncRNA GAS5 has been regarded as a promoter of proliferation and survival of FGSC . In greapression . Accordifunction  and in Sfunction . Indeed,m output . TherefoFinally, apoptosis is a mechanism responsible for the normal regulation of germ-cell death during differentiation and maturation of normal human germ cells. It is a prerequisite for a normal spermatogenesis , 42. TheIn conclusion, the results of this study showed a significant over-expression of lncRNA GAS5 in patients with non-mosaic KS compared to controls. The lncRNA seems to play a role in various biologic processes, such as GC actions, inflammatory and autoimmune diseases, vascular endothelial cells apoptosis and atherosclerosis and in FGSC proliferation. Furthermore, its involvement in cellular growth and proliferation, cellular development and cell-to-cell signaling and interaction suggests a possible role in germ cell loss. However, whether the lncRNA GAS5 over-expression is really involved in the pathogenesis of some of the typical features of KS patients  needs to be further investigated.


Astrocyte activation is a common pathological feature in many brain diseases with neuroinflammation, and revealing the underlying mechanisms might shed light on the regulatory processes of the diseases. Recently, soluble epoxide hydrolase (sEH) has been proposed to affect neuroinflammation in brain injuries. However, the roles of astrocytic sEH in brains with neurodegeneration remain unclear.−/−) background were also applied.The expression of astrocytic sEH in the brains of APPswe/PSEN1dE9 (APP/PS1) mice developing Alzheimer’s disease (AD)-like pathology was evaluated by confocal imaging. LPS-activated primary astrocytes with mRNA silencing or overexpression of sEH were used to investigate its regulatory roles in astrocyte activation and the induction of pro-inflammatory markers. Primary astrocytes isolated from a sEH knockout  and pro-inflammatory markers. Moreover, our data show that the phosphorylation of the signal transducer and activator of transcription 3 (STAT3) was upregulated in activated astrocytes from sEH mouse brains, and the pharmacological blockade of STAT3 activity alleviated the pro-inflammatory effects of sEH deletion in LPS-activated primary astrocytes.The immunoreactivity of sEH was increased in activated astrocytes in parallel with the progression of AD in APP/PS1 mice. Our data from primary astrocyte cultures further demonstrate that the overexpression of sEH ameliorated, while the silencing of sEH mRNA enhanced, the lipopolysaccharides (LPS)-induced expression of pro-inflammatory markers, such as inducible nitric oxide, cyclooxygenase 2 (COX-2), and pro-inflammatory cytokines. These findings suggest that sEH negatively regulates astrocyte immune responses. Enhanced immune responses found in LPS-activated sEHOur results provide evidence, for the first time, showing that sEH negatively regulates astrocytic immune responses and GFAP expression, while the underlying mechanism at least partly involves the downregulation of STAT3 phosphorylation. The discovery of a novel function for sEH in the negative control of astrocytic immune responses involving STAT3 activation confers further insights into the regulatory machinery of astrocyte activation during the development of neurodegeneration.The online version of this article (10.1186/s12974-019-1508-2) contains supplementary material, which is available to authorized users. Astrocytes are major glial cells residing in the brain. Astrocytes become activated during the pathogenesis of many neurodegenerative diseases such as Alzheimer’s disease (AD). In AD, activated astrocytes are in proximity to β-amyloid (Aβ) plaques, one of the pathological hallmarks of AD, which have been implicated in the progression of AD , 2. A plGlia-mediated neuro-inflammation and subsequent neurodegeneration are common pathological features shared by many neurodegenerative diseases, and the inflammatory responses derived from activated astrocytes exacerbate the pathogenesis of these diseases . HoweverSoluble epoxide hydrolase (sEH) has recently gained increasing attention because the inhibition of hydrolase activity by sEH can result in sustained levels of epoxyeicosatrienoic acids (EETs) and provide neuroprotection in cardiovascular disease and brain injuries –28. EpoxEscherichia coli O55:B5) was purchased from Calbiochem. Antibodies for inducible nitric oxide synthase (iNOS) and β-actin were purchased from BD Transduction Lab and Novus Biologicals, respectively. Cyclooxygenase-2 (COX-2) and glial fibrillary acidic protein (GFAP) were purchased from Abcam. Total STAT3 and phospho-STAT3 (p-STAT3) were purchased from Cell Signaling Technology. ELISA kits for TNFα and IL-6 were purchased from Invitrogen. 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA) and N-acetyl-S-farnesyl-L-cysteine (AFC) were purchased from Sigma-Aldrich. Poly-ornithine was purchased from BD Biosciences. Papain and DNase I were purchased from Worthington Biochemical. Fetal bovine serum was purchased from HyClone and heat-inactivated. Culture media and penicillin/streptomycin were purchased from Gibco, and other common chemicals were from Sigma-Aldrich, unless stated otherwise.Lipopolysaccharide , and sEH−/− mice (C57BL/6/B6.129X-Ephx2tm1Gonz/J) were generously provided by Dr. Tzong-Shyuan Lee. Mice were maintained at the NHRI Laboratory Animal Center with free access to food and water. All experiments were performed as approved by the NHRI IACUC.Breeding pairs of wild-type (Wt) controls and APPswe/PSEN1dE9 (APP/PS1) transgenic mice, overexpressing both human APP695 Swedish mutations (HuAPP695swe) and a mutant human presenilin 1 (PS1-dE9), were originally obtained from the Jackson laboratory. The transgenic genotypes of both APP and PS1 were detected using PCR, according to the manufacturer’s instruction. APP/PS1 transgenic mice that were heterozygous for the transgenes and Wt littermates were used as controls. Given that LPS has been widely used to induce inflammatory responses , 22, a s5 cells per well, followed by GFAP staining, to determine the purity of the astrocytes. The purity of primary astrocyte cultures was approximately 95%. Primary cultures were treated with 10 ng LPS/ml of culture medium for 24 h. To inhibit STAT3 phosphorylation in LPS-activated astrocytes, 1-h pretreatment with 10 μM of the STAT3 inhibitor, stattic (Abcam), was applied.Primary astrocytic cultures were derived from the cortices of P1 to P3 neonates. Cells were dissociated using an enzyme solution, containing Dulbecco’s modified Eagle’s medium (DMEM), ethylenediamine tetraacetic acid (0.5 mmol/L), L-cysteine (0.2 mg/ml), papain (15 U/ml), and DNase I (200 μg/ml), followed by trituration. Culture medium  was changed after 24 hours of initial seeding. To obtain enriched primary astrocytes, the astrocytic monolayer was repeatedly trypsinized (at 90% confluence) and reseeded twice. On the 14th day in vitro (14 DIV), primary astrocytes were reseeded in poly-ornithine-coated 24-well culture plates for experiments, and a fraction of the cells was cultivated on chambered slides at 1 × 10Small interfering RNA duplexes targeting sEH (si-sEH), consisting of a pool of 3 target-specific siRNAs designed to knock down sEH gene expression, and a scramble control were purchased from Santa Cruz Biotechnology, and an Ephx2 mouse cDNA clone for the overexpression of sEH and a vehicle control were purchased from OriGene. Primary cell cultures were transfected with siRNAs, plasmid, or controls at a final concentration of 30 nM in serum-free Opti-MEM by using the TransIT®-siQUEST™ transfection reagents (Mirus) for 48 h, followed by 24-h treatments of LPS. Quantification of sEH gene expression was evaluated by quantitative polymerase chain reaction (qPCR), as described below.x = 2−ΔCt, in which ΔCt = Cttarget gene − CtGADH. For cell cultures, each experimental condition was collected from at least 3 independent cultures.Total RNA was extracted with an RNeasy Plus Mini Kit (Qiagen) according to the manufacturer’s instructions. RNA (1 μg) was reverse transcribed into cDNA using a random primer and a SuperScript III Reverse Transcriptase Kit (Invitrogen). Quantitative PCR was conducted using Luminaris Color HiGreen qPCR Master Mix (Thermo) and an ABI PRISM 7500 Real-Time PCR System. Forward and reverse primer sets for each cDNA were used as follows: 5′-TGGTGGTGACAAGCACATTT-3′ and 5′-AAGGCCAAACACAGCATACC-3′ ; 5′-GGCCATGGAGTGGACTTAAA-3′ and 5′-CACCTCTCCACCAATGACCT-3′ ; 5′-GAGGGACAACTTTGCACAGG-3′ and 5′-TCCTGTCTATACGCAGCCAG-3′ ; 5′-ATCTCATACCAGGAGAAAGTCAACCT-3′ and 5′-TGGGCTCATACCAGGGTTTG-3′ ; 5′-GACCAAGACCATCCAATTCATCTT-3′ and 5′-GGAATGTCCACAAACTGATATGCT-3′ ; 5′-TGGTGTGGAACATGGCTCTCT-3′ and 5′-ACTGGGATAGATCGGATAACTTTCA-3′ ; and 5′-TGTGTCCGTCGTGGATCTGA-3′ and 5′-GATGCCTGCTTCACCACCTT-3′ . The average cycle threshold (Ct) value was normalized using the GAPDH signal. Relative transcript levels were calculated as Confocal microscopy was used for semi-quantification and qualitative analysis. Brain samples were subjected to paraformaldehyde (4% in PBS) fixation, overnight, followed by cryoprotection with sucrose (30% in PBS). Cryosections, at 30 μm thick, were subjected to immunohistochemical analysis using antibodies against Aβ , GFAP , p-STAT3 , or sEH (1:100) for overnight incubation at 4 °C. The corresponding secondary antibodies conjugated with Alexa Fluor (Invitrogen) as indicated in the results were applied for 2 h. Tissues were coverslipped with mounting medium (Vector Lab) containing 4,6-diamiino-2-phenylindole (DAPI) for nuclei counterstaining. Images were acquired using a Leica confocal microscopy imaging system. GFAP immunoreactivity has been widely used for the evaluation of morphological alterations in activated astrocytes in vivo and thus the quantification of GFAP immunoreactivity was used as a measure of astrocyte activation in this study. Semi-quantification of the immunoreactivity from two sections per mouse was performed using MetaMorph imaging software.Homogenates of the hippocampus or cortex were subjected to measurements of sEH activity, using an epoxide hydrolase activity assay kit (Cayman Chemistry) according to the manufacturer’s instructions. Briefly, the assay utilized Expox Fluor 7, a sensitive fluorescent substrate for sEH that can be used to monitor the activity of sEH. Hydrolysis of the substrate epoxide yielded a highly fluorescent product, 6-methoxy-2-naphthaldehyde, that was monitored at excitation and emission wavelengths of 330 and 465 nm, respectively, using a microplate reader. sEH activity was presented as pmol/min per mg of protein.The expression levels of COX-2, iNOS, GFAP, total STAT3, p-STAT3, and β-actin were determined using cell lysates or brain homogenates. Samples were lysed in lysis buffer  and were subjected to electrophoresis, followed by protein transfer onto PVDF membranes and Western blot analyses using antibodies against COX-2 (1:750), iNOS (1:750), GFAP (1:1000), total STAT3 (1:1000), p-STAT3 (1:1000), and β-actin (1:5000). Targets were detected by the incubation of HRP-labeled secondary antibodies. The corresponding bands, revealed by electrochemiluminescence (ECL) reaction, were analyzed using ImageJ.The culture media from primary astrocytic cultures was subjected to measurements of pro-inflammatory cytokines, including IL-6 and TNFα, using ELISA kits. The assay was performed according to the manufacturers’ instructions. Detection of the results was performed using an ELISA plate reader  at a wavelength of 450 nm.t test was used to test significance. For ANOVA, significance for post hoc multiple comparisons between groups was determined with the Bonferroni test using GraphPad Prism software. Data are presented as the mean ± SEM. Statistical significance was set at p < 0.05.A two-tailed independent Student’s Because activated astrocytes are closely associated with the progression of AD, we first evaluated the expression levels of sEH in activated astrocytes in the vicinity of Aβ plaques. Brain tissues from APP/PS1 transgenic mice at different ages, ranging from 5 to 14 months, were used for fluorescent immunohistochemistry  compared to non-treated control . After genetic manipulation of sEH for 24 h, data show that the expression levels of sEH did not affect the immunity of astrocytes at the basal condition  and 183 ± 25% (p < 0.05) of the LPS-treated controls, respectively. In contrast, over-sEH astrocytes showed a reduction in LPS-induced expression levels of iNOS  and COX-2 . Although levels of iNOS and COX-2 were not affected in astrocytes derived from sEH knockout (sEH−/−) mice  and IL-6  in sEH−/− astrocytes as measured by ELISA was significantly increased compared to the LPS-treated controls . TNFα  and IL-6  secretion in over-sEH astrocytes was reduced compared to the LPS-treated controls   secretion was significantly increased compared to the LPS-treated controls (68 ± 4 pg/ml), while TNFα (740 ± 176 pg/ml) was slightly increased compared to the LPS-treated controls (332 ± 141 pg/ml).To reveal the possible role of astrocytic sEH observed in the activated astrocytes in vivo, we established in vitro models, using LPS to activated primary astrocytes, and then examined the effects of sEH expression on the regulation of astrocyte activation using genetic manipulations. The expression levels of sEH in primary astrocytes derived from wt mice were genetically manipulated by the RNA silencing of sEH (si-sEH) and the overexpression of sEH (over-sEH). The levels of sEH mRNA in si-sEH and over-sEH astrocytes were 28 ± 3% and 5.8 ± 1.2 × 10−/− primary astrocytes than those in the respective controls as shown in Fig. The effects of sEH expression on the mRNA levels of the pro-inflammatory markers were then evaluated by qPCR. Similar to that observed at protein levels, the mRNA levels of the pro-inflammatory markers, including iNOS, COX-2, IL-6, and TNFα, were higher in si-sEH or sEH−/− mice exacerbated astrocyte activation in the brain as evidenced by the enhanced reactive morphology of GFAP-positive astrocytes at 48 h after the treatments  and hippocampus  were increased in LPS-treated sEH−/− mice compared to those in the Wt counterparts  and sEH−/− mice . An increased hippocampal iNOS at protein levels was also found in sEH−/− mice that received LPS , whereas cortex iNOS was slightly increased.The effect of sEH in the negative regulation of astrocyte activation was further tested using animal models, with cerebral inflammation induced by the peripheral delivery of LPS. Our data show that intraperitoneal injections of LPS (5 mg/kg body weight) in sEH−/− mice. One explanation for the slight increase of these inflammatory markers induced by LPS might be due to the time-dependent effects of LPS on mRNA expression. As shown in Fig. −/− mice expressed higher mRNA levels of iNOS , IL-6 , and TNFα  in the hippocampus than the LPS-treated Wt mice. However, mRNA levels of GFAP and COX-2 in the hippocampus were slightly increased in the LPS-treated sEH−/− mice as compared to the LPS-treated wt mice. In the cortex, the LPS-treated sEH−/− mice expressed higher mRNA levels of COX-2  and IL-6  than the LPS-treated wt mice, whereas mRNA levels of GFAP, iNOS, and TNFα were slightly increased. These results support the findings of our in vitro study, suggesting that sEH may function as a suppressor in the regulation of the LPS-activated immune response in astrocytes, while the genetic deletion of sEH may exacerbate astrocyte activation and the associated immune response. However, the levels of GFAP and the pro-inflammatory markers at the basal conditions were not affected by the genetic deletion of sEH. These findings suggest that the functional effects of sEH on the regulation of the astrocytic immune response might be associated with the activation status, and it is conceivable to speculate that the LPS-activated signaling pathways may be involved in the regulatory function of sEH during astrocyte activation. In line with this speculation, our data indeed showed that sEH activity in the hippocampus and cortex was significantly suppressed in the brains of mice that received intraperitoneal injections of LPS  than in Wt counterparts (161 ± 13% of Wt control). Although a slight elevation was found in the hippocampus of sEH−/− mice (541 ± 42% of Wt control) compared to the control that received LPS (387 ± 19% of Wt control), phosphorylation of STAT3 in the hippocampus of LPS-treated sEH−/− mice was significantly increased (p < 0.05) in the hilus of the dentate gyrus as demonstrated by confocal imaging with quantification , P38, and janus kinase 2 (JAK2)/STAT3 in sEHins Fig. a, but nop < 0.001) and IL-6  mRNA were higher in si-sEH astrocytes than those in the scrambled control astrocytes   compared to the control with LPS (100 ± 5%)  Fig. c. These −/− or si-sEH, enhanced the production of LPS-induced pro-inflammatory markers, whereas the overexpression of sEH ameliorated the immune response. However, the involvement of microglial activation in the enhanced pro-inflammatory markers found in sEH−/− mice cannot be excluded due to the constant interactions between microglia and astrocytes in the brain. Furthermore, our findings identify sEH as a novel inhibitory effector in the regulation of STAT3 activity and demonstrate the critical role of STAT3 in astrocyte activation. Based on these findings, we propose that the immune-suppressing function of sEH involving STAT3 inhibition may play an important role in the regulation of astrocyte activation.Astrocyte activation is an important pathological feature in AD and many other neurodegenerative diseases , 35. UndThe number of GFAP-positive astrocytes near Aβ plaques increases as AD progresses. Similarly, our data show that astrocytic sEH immunoreactivity increased in parallel with the extent of astrogliosis and was limited to the activated astrocytes in proximity to Aβ plaques. However, astrocytes in the brain of AD mice appear to express different levels of sEH, and less activated astrocytes that were close to Aβ plaques expressed little or no sEH, as shown in the abovementioned results. One possible explanation for these findings is that the different levels of sEH expression in response to an inflammatory microenvironment may be due to the heterogeneity of astrocytes . Indeed,Nevertheless, the fact that levels of sEH modulate immune response in activated astrocytes may have significant impacts on AD pathogenesis. We speculate that high sEH-expressing astrocytes could be a subtype of astrocytes that are attempting to cope with inflammation because the expression of sEH could be anti-inflammatory in activated astrocytes. Therefore, it is conceivable to propose that activated astrocytes with high expression levels of sEH could be beneficial for counteracting the over-activation of astrocyte. However, sEH-overexpressing astrocytes, on the other hand, may reduce their beneficial functions of immune response against the disease progression. In fact, the beneficial functions of activated astrocytes have been recently reported, showing that Aβ plaques with little or no activated astrocytes were associated with dementia status in AD patients , 38. TheOur study support the notion that STAT3 activation can be pro-inflammatory and is involved in triggering the expression of pro-inflammatory markers in activated astrocytes . In addiIn conclusion, we demonstrated, for the first time, that the deletion of sEH in astrocytes was pro-inflammatory, whereas the overexpression of sEH suppressed the immune response. The underlying mechanism may be at least partly attributed to the regulation of STAT3 activation. The sEH-mediated STAT3 inhibition in a cellular context suggests that astrocytic sEH may play an important role in the regulatory machinery of pro-inflammatory response in activated astrocytes during the progression of the brain diseases. Revealing the underlying mechanisms through which sEH orchestrates STAT3 activity and developing means to manipulate astrocytic sEH expression could facilitate the future development of treatment for brain diseases.Additional file 1:Figure S1. The expression levels of sEH by genetic manipulations in astrocytes. The expression levels of sEH in primary astrocytes derived from Wt mice were genetically manipulated by using commercially available reagents for the RNA silencing of sEH (si-sEH) and the overexpression of sEH (over-sEH). The levels of sEH mRNA in si-sEH and over-sEH astrocytes were 28 ± 3% (n = 9) and 5.8 ± 1.2 × 106% (n = 6) of the control, respectively, as measured by qPCR  . ***p < 0.001. Figure S2. Pro-inflammatory markers in primary astrocytes from sEH−/−. Protein levels of iNOS and COX-2 were significantly increased by LPS treatments in primary astrocytes derived from either Wt or sEH−/− mice. However, the LPS-induced expressions of these pro-inflammatory markers were not affected by sEH−/−. Data are presented as the mean ± SEM. One-way ANOVA and Bonferroni multiple comparison test were performed. *p < 0.05, **p < 0.01, ***p < 0.001. (DOCX 99 kb)