Whilst olfactory dysfunction has been reported in Korsakoff's Syndrome (KS) patients, the diagnostic implications of this have not been fully explored. KS can be difficult to diagnose because cognitive symptoms are similar to other diagnoses. For instance, patients with Frontal Lobe (FL) Syndrome may present with memory impairments that are similar to KS. Participants were given the Benton Visual Retention Test-Fifth Edition (BVRT-V), to identify working memory dysfunction, and a Brief Smell Identification Test (B-SIT), to evaluate olfactory function. B-SIT scores were found to be significantly lower in the KS group compared to the control and FL groups. In contrast, the error scores on the BVRT-V were significantly higher in both the KS and FL groups compared to the healthy control subjects. Therefore, we suggest that olfactory function may aid in the differential diagnosis of patients presenting with working memory dysfunction. Olfactory dysfunction has been reported in several neurodegenerative conditions such as Alzheimer's disease (AD) and Parkinson's disease (PD)  and may KS patients usually first present with Wernicke's encephalopathy (WE), characterized by opthalmoplegia, gait abnormalities, confusion, and some signs of short-term memory loss, before developing KS. WE patients typically respond well to thiamine (vitamin B1), but unfortunately, the signs of WE are often overlooked , and witAcute KS may also benefit from early thiamine treatment ; howeverIn this study, we examine the relationship between olfaction and memory in KS patients, FL patients, and healthy volunteers to explore the implications of olfactory testing in the diagnosis of KS. We predicted that both patient groups would perform poorly on the memory test and that KS patients would show a more severe olfactory deficit than FL patients.Twelve KS patients, four age-matched FL patients  and nine healthy age-matched controls were tested. Ethical approval was obtained from the School of Psychology at Bangor University and the North West Wales Research Ethics Committee before this research began. Twelve individuals with a clinical diagnosis of alcoholic Korsakoff's syndrome and a history of Wernicke's encephalopathy were recruited from 4 nursing and private care facilities in the UK. Written and verbal informed consent was obtained from individual patients and their primary caregiver or family member. Medical or nursing care facility admission notes were obtained to confirm diagnosis. The diagnostic guidelines used to verify Wernicke-Korsakoff syndrome were based on consensus guidelines used for inclusion in an ongoing biomedical study investigating the genetic susceptibility of alcohol-related brain damage. The specific guidelines used wereWernicke's encephalopathy—confirmed by medical history of hospitalization with a presentation of confusion and either ataxia or nystagmus; Korsakoff's syndrome—confirmed by a medical history of memory impairment and a past presentation of either confabulation or disorientation, as well as a present indication of short-term memory impairment. Further selection processes were used to create a homogenous sample of Korsakoff's patients. Only adults under the age of 65 were included. Only individuals with an onset date of 6 months prior to recruitment were included to ensure that the patient group represented those in a chronic stage of the illness. The onset date of Korsakoff's syndrome recorded in their medical or care facility admission notes varied from 6 months to 10 years prior to recruitment. Only individuals who were currently abstinent from alcohol, and had been for at least 6 months, were included. Individuals with coexisting brain injuries, psychological, or neurological diagnoses were not included. There was no indication of spontaneous confabulation in any individual included in the Korsakoff's group. All Korsakoff's patients lived in a care environment where proper nutrition and medical attention were offered, although there was variation in the administration of prescription medication and vitamin supplementation, including thiamine. The Benton Visual Retention Test-Fifth Edition (BVRT-V) and the Brief Smell Identification Test (B-SIT). The Brief Smell Identification Test (B-SIT) was administered. A low score indicates poor performance. The Benton Visual Retention Test-Fifth Edition (BVRT-V) was administered. Performance was quantified by the numerical error score calculated relative to the participants IQ and age. A high score indicates a deficit in working memory. Testing time took approximately 15 minutes in total for each participant. Performance was assessed by the number error score calculated relative to the participants' IQ and age. The 12-item B-SIT  was usedF = 72.20, P < .001). Post hoc multiple comparisons using Tukey's HSD revealed significant differences between the KS and healthy controls groups  and also between the KS and FL groups . The B-SIT scores were significantly lower in the KS group than in either the healthy control or FL groups . One-wayF = 6.00, P > .005). Post hoc multiple comparisons using Tukey's HSD revealed significant differences interaction between the healthy control and KS groups  and also between the FL and control groups .In contrast, the KS and FL patients had similar high scores on the BVRT-V, compared to the healthy age-matched control subjects, whereas only the KS patients performed poorly on the B-SIT; therefore the poor performance of KS patients on the B-SIT cannot be attributed to a working memory dysfunction, rather it reflects olfactory dysfunction . One-wayThe present study revealed significant impairment in odour identification in KS patients only. The observed deficits in olfactory function support previous studies , 7. In cChronic alcohol misuse typically results in deficits in memory, planning, organizing, judgment making, and social skills. Since these are difficulties usually related to frontal lobe damage, the presentation of these symptoms in KS or FL patients might be easily confused. The diencephalic pathology seen in KS may be responsible for the selective impairment in olfactory perception observed, which may explain why the loss of perception is characteristic to the disorder . Clinical diagnosis of KS is subject to clinical error. When comparing two surveys conducted on the detection rate of KS, it was revealed that standardised tests can often miss the disorder altogether . The literature documenting olfactory dysfunction in KS patients is relatively limited and sometimes contradictory , 6. In HWe acknowledge that the present study involved a relatively small number of patients, but the results suggest that assessment of olfactory function may be an effective tool in the differential diagnosis of KS patients from patients with memory dysfunction due only to frontal lobe damage. The nature of KS that often keeps patients in long-term nursing care and the potential for treatment if diagnosed early highlight the importance of an early diagnosis. The B-SIT is an uncomplicated, easily administered assessment tool that is simple for KS and FL patients to complete. The use of the B-SIT could aid in diagnosis and referral for appropriate thiamine treatment. Future studies that include a greater number of patients, as well as comparisons to olfactory performance in other neurological disorders, may be useful in further determining the potential value of the B-SIT.


Prok1 and Prok2) was quantified in murine uteroplacental tissues by QPCR in the days preceding labour (days 16–19). Prok1 mRNA expression increased significantly on D18 in fetal membranes (compared with D16) but not in uterus or placenta. Intrauterine injection of PROK1 on D17 induced fetal membrane mRNA expression of the pro-inflammatory mediators Il6, Il1b, Tnf, Cxcl2 and Cxcl5, which are not normally up-regulated until D19 of pregnancy. However, intrauterine injection of PROK1 did not result in PTD. As expected, injection of lipopolysaccharide (LPS) induced PTD, but this was not associated with changes in expression of Prok1 or its receptor (Prokr1) in fetal membranes. These results suggest that although Prok1 exhibits dynamic mRNA regulation in fetal membranes preceding labour and induces a pro-inflammatory response when injected into the uterus on D17, it is insufficient to induce PTD. Additionally, prokineticin up-regulation appears not to be part of the LPS-induced inflammatory response in mouse fetal membranes.The mechanisms that regulate the induction of term or preterm delivery (PTD) are not fully understood. Infection is known to play a role in the induction of pro-inflammatory cascades in uteroplacental tissues associated with preterm pathological parturition. Similar but not identical cascades are evident in term labour. In the current study, we used a mouse model to evaluate the role of prokineticins in term and preterm parturition. Prokineticins are multi-functioning secreted proteins that signal through G-protein-coupled receptors to induce gene expression, including genes important in inflammatory responses. Expression of prokineticins ( The factors that trigger the onset of parturition at term in humans (∼39 weeks) are not yet fully understood, although inflammation in uteroplacental tissues and increased priming of leukocyte activation in maternal peripheral blood are well documented . IncreasPreterm delivery (PTD) in humans is considered to occur before 37 weeks of gestation and is the largest cause of neonatal mortality and morbidity in the developed world , resultiFetal membranes in humans comprise amnion and chorion and an extracellular matrix. At term parturition the fetal membranes overlying the cervix rupture; evidence suggests that this is due to reduced collagen levels and increased apoptosis. However, a role for fetal membrane activation in PTD has also been described as clinical and histological chorioamnionitis (inflammation of the amnion and chorion) can lead to preterm premature rupture of membranes (pPROM) , allowing the effect of preterm labour  on neonatal morbidity and mortality to be investigated  and allowed at least 6 days of acclimatisation prior to surgery. Pregnant females or virgin female and proven males were purchased pregnant and timed-mated within the University of Edinburgh facility. Surgery was performed on day 17 of pregnancy . Dams were anaesthetised with isoflurane and both uterine horns exposed following laparotomy. The number of viable fetuses in each horn was recorded and the horn containing the most fetuses was selected for intrauterine injection. Using a 33-gauge Hamilton syringe, pregnant mice were injected between the two most anterior fetuses with either a 25 μl volume of sterile saline (Sigma); recombinant mouse PROK1  diluted to 350 nM in saline . Dams were culled by cervical dislocation and uterus, placenta and fetal membranes separated, and collected into RNAlater ) for RNA extraction or fixed in 4% neutral buffered formalin and wax embedded for immunohistochemistry (see Actb) primers designed to span an intron and JOE-labelled probe  and diluted to 100 ng/μl in RNase-free water (Invitrogen) before RT using the Superscript VILO cDNA synthesis kit (Invitrogen). PCRs were carried out using Applied Biosystems 7500 and 7900 Fast instruments. Primer and FAM (6-carboxyfluorescein)-labelled probe sequences were designed to span an intron (where possible) and purchased from Sigma  or TaqMae Sigma; . Relative Sigma;  and 4. TParaffin-embedded tissue was processed for immunohistochemistry as described in Amniotic fluid collected from saline-, LPS- or PROK1-treated dams was quantified by ELISA Quantikine Kits (R&D Systems) for mouse IL1B (MLB00B), TNF (MTA00) and CXCL2 (MM200) as per kit instructions.t-test. Nonparametric data were subjected to Kruskal–Wallis test with Dunn's post-test or a two-tailed Mann–Whitney U test. Percentages were arcsine transformed prior to analysis as they cannot be normally distributed . Where more than one fetus was collected per dam the data was averaged per dam. All analysis was carried out using GraphPad Prism and GraphPad Software, Inc. . Significance was defined as P<0.05.Data that passed the normality test (Kolmogorov–Smirnov) were subjected to statistical analysis using a one-way ANOVA with Tukey's multiple comparison post-test or a two-tailed unpaired Prok1 and Prokr1 mRNA was examined daily in fetal membranes from D16 to 19 of pregnancy . On D18, expression of Prok1 mRNA was significantly greater when compared with expression on D16 and 17  and placenta .Expression analysis of Prok2 and Prokr2 was also examined in the same uteroplacental tissue samples from D16 to 19. Fetal membrane Prok2 expression increased by a small but significant amount on D18 when compared with expression on D16 . No significant changes were detected for either Prok2 or Prokr2 in the uterus and placenta .Expression of in vivo using a model of infection-induced PTD. For this, fetal membranes were collected 6 h after intrauterine injection with saline , LPS  or PROK1 . Compared with the saline treatment, relative expression of prostaglandin synthase 2 , Il6, Il1b, Tnf, chemokine (CXC motif) ligand 2 (Cxcl2) and Cxcl5 mRNAs were found to be significantly increased in response to LPS treatment (Ptgs1 (a gene chosen as a negative control) did not change in response to treatment with LPS. Similarly, Prok1 and Prokr1 expression levels were not regulated by LPS treatment at the 6-h time point  or lower than seen endogenously on D19 of pregnancy. Intrauterine injection of 20 μg LPS resulted in a greater fold increase in expression of pro-inflammatory genes compared with PROK1 and those measured endogenously on D19 (apart from Cxcl5). Interestingly, it was the chemokines Cxcl2 and Cxcl5 that showed the most striking differential expression in response to either term parturition or LPS treatment.We subsequently investigated the temporal expression of P<0.05; P<0.01).To compare the pro-inflammatory stimuli (LPS and PROK1), levels of secreted IL1B, TNF and CXCL2 were measured by ELISA in amniotic fluid collected 6 h after intrauterine injection (amniotic fluid was pooled from the amniotic sacs of four to six fetuses). Unexpectedly, a different pattern of results was obtained for each protein. IL1B was not regulated by either treatment, although PROK1 did demonstrate a trend for higher levels than LPS . The time to delivery in dams treated with PROK1  was not significantly different from the time to delivery in dams treated with saline. However, treatment with LPS significantly reduced the time to delivery  compared with treatment with saline (P<0.001) or PROK1 (P<0.05). To assess whether or not surgery itself affected time to delivery, the time taken for five dams to naturally deliver was recorded .In order to investigate the effect of PROK1 on PTD, three groups of mice received intrauterine injection of saline, LPS or PROK1 on D17 of pregnancy A. The tiP<0.001).Another measure of the effect of infection-induced PTD is to record pup survival B. The avPROK1 mRNA expression is increased in human placenta and myometrium at term, and ex vivo treatment of these tissues with PROK1 induces a pro-inflammatory response  and expression of both was significantly down-regulated after 10.5dpc, indicating a role in early placentation . We also demonstrated that these pro-inflammatory genes are significantly up-regulated in fetal membranes on D19 of pregnancy (the day before parturition). Indeed, in vivo PROK1-mediated induction of Ptgs2, Il6 and Il1b cytokines by 6 h after treatment was comparable to that seen endogenously on D19 of pregnancy. These pro-inflammatory mediators are well documented to have important roles in term labour and PTD; for example, PTGS2 converts arachidonic acid into prostaglandins, which stimulate uterine smooth muscle contractions necessary for parturition  in order to overcome any endogenous ‘brake’ on PTD.In this study, we demonstrated that intrauterine treatment with PROK1 or LPS induces significant up-regulation of pro-inflammatory mediators in fetal membranes  exhibit a significant increase in post hoc power calculation shows that we would need 21 mice in each of the natural delivery groups and the PROK1 group to have 90% power to show a difference (at the 5% significance level) in the interval to delivery in the two groups. This assumes equal numbers in each groups and that the mean and s.d. of the time to delivery remain the same in each group. Hence, our study is underpowered to determine whether the relatively subtle effect of PROK1 on shortening time to delivery by 28% is statistically significant. The effect of LPS was profound, shortening the interval to delivery by 74%. Our study had 89% power to detect a shortening effect of PROK1 of time to delivery by 37%. We can be confident that any effect of PROK1 on inducing PTD (if it exists) is less than half as powerful as LPS in shortening the interval to delivery (in the doses given). In contrast to the possible type II error for a subtle effect of PROK1 on inducing PTD, there was no difference in pup survival between the PROK1 and the saline groups; thus, even if there is a modest effect of PROK1 on PTD, it does not compromise offspring survival.Although the interval to birth was shorter after PROK1, this was not statistically significant. A in utero environment  may promote fetal maturation and potentially the induction of term labour. This hypothesis is consistent with our finding that PROK1 treatment, although pro-inflammatory, did not reduce pup survival. We can also speculate that as time to delivery was unaffected in PROK1-treated dams, the endogenous pro-inflammatory role of PROK1 is not to induce parturition through induction of a strong inflammatory response but to promote fetal maturation. This hypothesis may be supported by our finding that Prok1 expression peaks on D18, and not on D19 like the other pro-inflammatory mediators analysed, suggesting that in the mouse, transient endogenous expression occurs rather than a prolonged amplification of pro-inflammatory mediator expression driven by PROK1 as hypothesised to occur in humans (It has been demonstrated in fetal sheep lung that inflammation and immune cell recruitment in response to LPS treatment promotes lung maturation  and may Prok1 prior to the up-regulation of other pro-inflammatory mediators before parturition. This suggests an alternate as yet unidentified role similar to that in fetal maturation processes in term parturition in the mouse. We have also demonstrated differences between human and mouse prokineticin expression and regulation in response to LPS in uteroplacental tissues, demonstrating that caution should be applied when using mouse models of PTD. Lastly, we have also contributed to the building body of evidence that PTD is not simply the premature induction of the same pro-inflammatory cascades seen at term by showing the difference in regulation of chemokines at term and in response to LPS, highlighting potentially new targets for the treatment of PTD.In conclusion, we have shown that PROK1 has a pro-inflammatory effect on fetal membranes but intrauterine injection is insufficient to induce PTD. However, we have shown that fetal membranes exhibit dynamic regulation of http://dx.doi.org/10.1530/REP-13-0295.This is linked to the online version of the paper at


TiO2-PANI/PA6 composite nanofibers and PANI/PA6 composite nanofibers were fabricated for ammonia gas sensing. The ammonia sensing behaviors of the sensors were examined at room temperature. All the results indicated that the ammonia sensing property of TiO2-PANI/PA6 composite nanofibers was superior to that of PANI/PA6 composite nanofibers. TiO2-PANI/PA6 composite nanofibers had good selectivity to ammonia. It was also found that the content of TiO2 had a great influence on both the morphology and the sensing property of TiO2-PANI/PA6 composite nanofibers.Titanium dioxide-polyaniline/polyamide 6 (TiO High concentrations of ammonia constitutea threat to human health. Exposure to high ammonia concentrations of 1,000 ppm or more can cause pulmonaryoedema and accumulation of fluid in the lungs, leading to difficulty with breathing and tightness in the chest. Today, most of the ammonia in our atmosphere is emitted directly or indirectly by human activity. The majority of all man-made ammonia is used for the production of fertilizers and for use in refrigeration systems [2[2[2O3[2[2.Conducting polymers such as polypyrrole, polyaniline, polythiophene and their derivatives are being explored as promising materials for microsensors, because of their good ability to form chemical sensors either as a sensing element or as matrices to immobilize specific reagents. Among these conducting polymers, polyaniline nanomaterials are the most extensively studied because of their greater surface area that allows fast diffusion of gas molecules into the structure , and the2, and esp2–18. Thre2,20, PANI2,22 and P2[2, PANI-In2[2[2O3, PANI-Zn2[2[2O3 and PANI2[2[2O3[2,26). Recin situ polymerization. Then, TiO2-PANI/PA6 composite nanofibers were prepared by depositing TiO2 onto the PANI/PA6 substrate via RF magnetron sputtering. The TiO2-PANI/PA6 composite nanofibers were finally fabricated into sensing devices for sensing application.In this work, PA6 nanofibers obtained by an electrospinning technique were first used as template to fabricate PANI/PA6 composite nanofibers by 2.2.1.2SO4) were purchased from Sinopharm Chemical Reagent Co. Ltd. . Polyamide 6  was obtained from ZIG ZHENG Industrial Co. Ltd . All chemicals and reagents were used as received, except for aniline, which was distilled before use.Aniline, formic acid (FA), ammonium persulfate (APS), ammonia and sulfuric acid  of 50 mm diameter was used as sputtering targets. High purity argon (99.999%) and oxygen (99.999%) were used as the sputtering and reactive gases, respectively. A diffusion pump was used to get the desired 9.8 × 10−4 Pa base pressure. The argon and oxygen flow rates were controlled separately by mass flow meters. The distance between target and substrates was kept at 60 mm. Before each sputtering-deposition step, the target was pre-sputtered in argon for 10 min to clean the target surface. The sputtering conditions are listed in TiO2.4.2-PANI/PA6 composite nanofibers were observed with a field emission scanning electron microscope  with a golden coating. Fourier transform infrared (FTIR) spectrum of all the samples was obtained with a resolution of 4 cm−1 in the range of 400–4,000 cm−1 by using a NEXUS 470 spectrometer .The structure and surface morphology of PANI/PA6 and TiO2.5.2-PANI/PA6 composite nanofibers were pasted onto the open area between the two electrodes as shown in A sensing system for ammonia sensing test was fabricated by the following processes. A home-made Au electrode with a gap of 0.5 mm between two Au stripes was firstly prepared by depositing Au on phenolic resin, and then PANI/PA6 and TiO2-PANI/PA6 composite nanofibers sensors were monitored and recorded automatically by an Agilent electrometer and a computer. A constant voltage of 5.0 V was used as the DC power supply. All the tests were conducted at room temperature (25 ± 1 °C) with a relative humidity of 65 ± 1%.To test the ammonia sensitivity, the sensing electrode was placed in a lab-made sensing system as shown in i – R0)/R0, where Ri and R0 are the resistance of sensors in ammonia and in air, respectively. The response and recovery time are defined as the time of 90% total resistance change. Each result was the average value of fivetimed tests.During the measurements, the actual ammonia volumes injected were 0.67, 1.35, 2.02, 2.69 and 3.37 μL, corresponding to the ammonia vapor with the concentration of 50, 100, 150, 200 and 250 ppm, respectively. After the ammonia was introduced to the chamber, the resistance of the sensors was recorded for 250 s, then the test chamber was flushed with dry air consecutively for another 250 s to make sure that a relatively steady state had achieved before next cyclic test. The sensitivity is defined as (R3.3.1.2-PANI/PA6 composite nanofibers sputtered for different times are shown in 2-PANI/PA6 composite nanofibers had very rough surface with porous structures, as presented in 2. It is obvious that such porous structure exhibited higher specific surface area than PANI/PA6, which facilitated the diffusion of ammonia vapor in sensing materials. However, TiO2-PANI/PA6 with 90 min deposition time showed a distorted surface structure as the excessive sputtering time could damage the integrity of the PANI coating.It is well known that structure and morphology can have a significant effect on the sensing properties of materials. The SEM images of PA6 nanofibers, PANI/PA6 composite nanofibers and TiO3.2.2-PANI/PA6 composite nanofibers are shown in −1 and 1,559.92 cm−1 were assigned to C=C stretching vibrations of the benzenoid and quinoid rings, respectively. The peaks at 1,298.85 cm−1 and 798.54 cm−1 resulted from the C–N stretching vibration of the secondary aromatic amine and the C–H bending vibration, respectively. The characteristic peak of Q=NH+–B was also observed at around 1,118.85 cm−1. All these peaks were identical to those of PANI. On the other hand, the C=O stretching vibration peak of amide in PA6 was also observed at 1,636.84 cm−1, while the amide N–H stretching vibration peak overlapped with the C=C stretching peak of the PANI quinoid rings, thus the peak at 1,559.92 cm−1 was shown to be broader. All the characteristic peaks of PANI/PA6 could be observed in the spectrum of TiO2-PANI/PA6 composite nanofibers, and the characteristic bands around 616.44 cm−1 and 573.27 cm−1 was attributed to Ti–O bending vibration of TiO2. It also can be observed that incorporation of TiO2 nanoparticles leads peaks of PANI/PA6 to shift slightly to lower wave number, indicating that some interaction existed between PANI and TiO2.The FTIR spectra of PANI/PA6 and TiO3.3.3.3.1.2nanopaticles on the ammonia sensing properties, ammonia sensing comparison tests were carried out with PANI/PA6 and TiO2-PANI/PA6 sputtered for 30, 60 and 90 min. 2-PANI/PA6 composite nanofibers showed better ammonia sensitivity, as revealed in 2 deposition. It is also found that TiO2-PANI/PA6 sputtered for 60 min performed best among the samples prepared. Taking 250 ppm ammonia vapor for instance, the sensitivity of PANI/PA6 composite nanofibers was only 1.4, but the sensitivity of TiO2-PANI/PA6composite nanofibers sputtered for 30 min increased to 5.2, and the sensitivity of TiO2-PANI/PA6 sputtered for 60 min was as high as 18.3. However, when the sputtering time was extended to 90 min, the sensitivity of TiO2-PANI/PA6 composite nanofibers was 15.1 with a small decline.To investigate the effect of TiO2 nanoparticles were deposited randomly on the PANI/PA6 substrate, which contributed to the good contact between TiO2 and PANI. Sensing properties of PANI/PA6 were due to the reversible chemisorptions of PANI [2-PANI/PA6 composite nanofibers was the joint function of PANI and P-N junction formed between p-type PANI and n-type TiO2. When exposed to the ammonia vapor, PANI was deprotonated by ammonia, which would increase the resistance of PANI and broaden the depletion layer thickness of P–N junction, as shown in 2 content in the nanofibers could result in an increase in the sensitivity of the nanofiber sensors at low ranges of TiO2 content. However, too high a content of TiO2 resulting from overlong sputtering times would damage the continuous phase of PANI layer, as revealed in TiO of PANI ,28, whil3.3.2.2-PANI/PA6 composite nanofibers sputtered for 60 min to successive exposures to 250 ppm ammonia vapor. The resistance remained constant after repeat uses, indicating the good reproducibility of the material.The reliability of sensing materials also depends on their repeated use. 3.3.3.2-PANI/PA6 composite nanofibers to methanol, ethanol and acetone vapor in the range of 50–250 ppm were also conducted under the same conditions as the ammonia sensing experiments explained before. 2-PANI/PA6 composite nanofibers sputtered for 60 min to ammonia, methanol, and ethanol and acetone vapors. It is very obvious that the sensitivities of the sensor to methanol, ethanol and acetone vapors were much lower than those to ammonia, which indicates the selective sensing behavior of the TiO2-PANI/PA6 composite nanofibers. The TiO2-PANI/PA6 composite nanofibers thus showed excellent selectivity to ammonia vapor.Methanol, ethanol and acetone are common volatile liquids, whose vapor could show cross-sensitivity in the detection system. Therefore, in this work, the response and recovery experiments of TiO4.2-PANI/PA6 composite nanofibers were successfully fabricated via the combination of in situ chemical polymerization and sputtering, which was a new, easy-to-handle and inexpensive technique. P-N junctions formed between PANI and TiO2 played a key role in the sensing behavior of TiO2-PANI/PA6 composite nanofibers to ammonia, which lead to a better sensitivity to ammonia such as higher response sensitivity, better response and recovery, compared to PANI/PA6 composite nanofibers. It clearly appeared that the content of TiO2 component controlled by sputtering-deposition time influenced the morphology and sensing property of TiO2-PANI/PA6 composite nanofibers. The gas-sensing properties of TiO2-PANI/PA6 composite nanofibers to ammonia, methanol, and ethanol and acetone vapor indicated that TiO2-PANI/PA6 composite nanofibers had excellent selectivity for ammonia detection, but would not applicable for the fabrication of methanol, ethanol and acetone vapor sensors. Further work will be devoted to improving the stability of the TiO2-PANI/PA6 composite nanofibers sensor.From the above mentioned studies, it has been concluded that TiO


Environmental Health Perspectives (EHP) will need a new editor-in-chief. It has been a real privilege to lead this important scientific journal since 2008. I believe in EHP, the science it supports, and the communities it serves, and I am proud to have had a part in the journal’s continued success and growing impact.The first day of August 2014 will be a big day for me. I have decided to retire after nearly 40 years of federal service, and EHP must adhere to the principles of independence, transparency, and balance and be prepared to recognize emerging themes in order to attract a wider audience and have an impact on the field. Adherence to these standards has served the journal well. In 6 years, the journal’s impact factor increased from 5.86 to 7.26. EHP is one of the top-ranked journals in Public, Environmental, and Occupational Health and in Environmental Sciences. The journal receives nearly 1,500 research manuscripts each year and has an acceptance rate of 15%. We have more than 40 internationally recognized members of the Board of Associate Editors and more than 100 active members of the Editorial Review Board. The journal routinely publishes highly recognized news articles on a variety of emerging topics in environmental health research. In collaboration with the Shanghai Center for Disease Control and Prevention, we also publish news and other articles in a bimonthly issue of EHP Chinese Edition. The journal established and cultivated a thriving social media presence on Facebook and Twitter, and updated its format to provide easy access on personal communication devices. In 2013, EHP was one of the first environmental health journals to publish exclusively online, with the entire journal available electronically and free of charge.In my inaugural editorial as editor-in-chief , I wroteEHP staff members also deserve my gratitude and thanks. They are of some of the most dedicated and passionate individuals I have ever known. I believe that the articles and news we published have made a significant contribution to improving human health and have provided the best available information for decision makers and policy makers worldwide. I am confident that my successor will find the NIEHS and the EHP staff eager to carry on this tradition of excellence.I thank the leadership of the National Institute of Environmental Health Sciences (NIEHS) for providing continuous and enthusiastic support and for making good on its commitment to editorial independence and unfettered scientific peer review. The EHP will continue to receive manuscripts and publish research and news stories each month. Keep sending us your science, and by all means, keep reading EHP !The NIEHS is organizing a search committee to recruit my replacement. I have agreed to assist with the transition and, if necessary, will happily work as a volunteer until a new editor-in-chief is named. In the meantime,


The modified coaxial electrospinning is a useful tool to generate nanostructures with a tailored components and compositions in their different parts, and thus to realize the desired functional performances.This study reports a new type of drug-loaded core-shell nanofibers capable of providing dual controlled release with tunable dose in the second phase. The core-shell nanofibers were fabricated through a modified coaxial electrospinning using a Teflon-coated concentric spinneret. Poly(vinyl pyrrolidone) and ethyl cellulose were used as the shell and core polymer matrices respectively, and the content of active ingredient acetaminophen (APAP) in the core was programmed. The Teflon-coated concentric spinneret may facilitate the efficacious and stable preparation of core-shell nanofibers through the modified coaxial electrospinning, where the core fluids were electrospinnable and the shell fluid had no electrospinnability. The resultant nanofibers had linear morphologies and clear core-shell structures, as observed by the scanning and transmission electron microscopic images. APAP was amorphously distributed in the shell and core polymer matrices due to the favorite second-order interactions, as indicated by the X-ray diffraction and FTIR spectroscopic tests. The results from the  CoaxiHowever, a modified coaxial electrospinning, which is characterized by the usage of un-electrospinnable liquids as shell fluids, has been successfully developed for fabricating nanofibers ,16. BecaIn clinical applications, the desired drug release profiles should obey the biological rhythm for effective and safe drug delivery and convenient administration. For some active pharmaceutical ingredients (API) such as non-steroidal anti-inflammatory drugs (NSAIDs), as well as antihypertensive, antihistaminic and anti-allergic agents, an initial fast release of a fraction of the dose in the shortest time after administration is in favor of relieving the symptoms of the disease . MeanwhiThe dual release profiles often involve drug controlled release at two different rates or in two time periods. A typical dual release system can provide an immediate drug release followed by a constant release ,21. BecaElectrospinning can achieve this objective through strategies such as preparing multi-layered nanofiber mats using a sequential single fluid process or producing fibers containing drug-loaded nanoparticles through the electrospinning of suspensions . Core-shBased on the above-mentioned knowledge, herein for the first time, we report a new type of drug-loaded core-shell nanofibers capable of providing dual controlled release with tunable dose in the second phase. Using poly(vinyl pyrrolidone) (PVP) and ethyl cellulose (EC) as the shell and core polymer matrices, and acetaminophen  as the model API, a modified coaxial electrospinning with a Teflon-coated concentric spinneret was conducted for the preparation of core-shell drug-loaded nanofibers, in which the shell fluid was un-spinnable.2.2.1.A schematic diagram of the modified coaxial electrospinning process is shown in v/v), could not be electrospun into nanofibers or electrosprayed into microspheres.Two syringe pumps were separately used to drive the shell and core fluids, and an alligator clip was used to connect the inner stainless-steel capillary of the spinneret to the high-voltage power supply . Under tIn this study, four types of drug-loaded solid nanofibers were successfully produced and some key parameters are listed in 2.2.−1) [The morphologies of the four electrospun products are shown in −1) . The forThe nanofibers F3 and F4 had obvious core-shell structures, with an estimated core diameter and sheath thickness of 490 and 220 nm for F3  and a va2.3.XRD analyses were performed to determine the physical status of APAP in the core-shell nanofibers. As shown in −1 is assigned to the N–H stretching vibration, and the peaks around 3164 cm−1 might be due to the OH stretching vibration plus other combination bands. The peaks at 1655 and 1260 cm−1 are assigned to the C=O stretching vibration of amide I band and/or C–N stretching of amides II and III vibrational bands, respectively. The peaks at 1611 cm−1 is due to the C=C bond stretching of aromatic benzene ring [−1 (C–H stretch) and 1664 cm−1 (C=O).Compatibility between components is essential for producing high-quality and stable composite nanofibers. Second-order interactions such as hydrogen bonding, electrostatic interactions, and hydrophobic interactions can often improve compatibility . The molene ring ,28. The −1 and peaks around 3164 cm−1 in the pure APAP spectrum have changed to broad but weak bands in the nanofibers F1, F3 and F4. The characteristic peaks of APAP corresponding to the C=C stretching of the aromatic benzene ring attenuated, shifted, or even disappeared in the nanofibers’ spectra. In the fingerprint regions, almost all of the peaks of APAP were shifted, decreased in intensity, or even disappeared from the spectra of the three nanofibers. All these were attributed to the hydrogen bonding between the hydroxyl group of APAP and the carboxyl group of PVP in the outer shell of the nanofibers, or the hydrogen bonding between the hydroxyl group of EC and the carboxyl group of APAP in the inner core of the nanofibers. These favorite second-order interactions were not only useful for the formation of amorphous drug-loaded nanocomposites during the electrospinning processes, but also desired for preservations of these nanoproducts for aftertreatment and further development of DDS.The sharp peak of 3326 cm2.4.max = 257 nm. Thus, the amount of APAP released from the fibers was determined by UV spectroscopy using a predetermined calibration curve C = 13.37A – 0.2115 (R = 0.9996) where C is the concentration of APAP (μg/mL) and A is the solution absorbance at 257 nm (linear range: 2 to 20 μg/mL).APAP has a UV absorbance peak at λin vitro drug release profiles of the three types of nanofibers are shown in The actual content of APAP in all the nanofibers was equivalent to the theoretical calculation value, suggesting little drug loss during the electrospinning process. The Later nanofibers F3 and F4 released 51.1% and 62.7% of the remaining APAP sustainably for 24 h . The cori.e. a mixed solvent with the addition of DMAc would effectively prevent the clogging of spinneret due to its high boiling point; and (4) The usage of PVP k25 would promote the fast exhaustion of the shell drug than its analogues such as PVPK30, PVP K60 and PVPK90. The amorphous APAP dissolved in the dissolution medium through the erosion of polymer matrix PVP. Often the polymer dissolution involve two sequential processes, i.e., absorbing solvent to form a swollen gel, and then followed by chain disentangles and disperses into the solution of the swollen polymer. Thus, a higher molecular weight meaning a longer time period of dissolutions and the drug dissolution processes. Here, the PVP K25, although could not be electrospun into nanofibers or electrosprayed into microparticles, but could be solidified as shell parts based on the electrospinnable core fluids and it could enhance the fast dissolution of APAP. In a word, for the first time, the drug concentrations in the core fluids were exploited as a useful tool to tailor the dose of drug released in the second phases.Thus, the dual release profiles could be achieved through a core-shell structure, wherein the shell hydrophilic polymer provides a pulsatile release initially and the core matrix furnishes the later sustained release. Previous reports have also demonstrated the usefulness of core-shell structures in providing this type of controlled release profiles ,29. Howe3.3.1.Mw = 30, 000) was purchased from Shanghai Yunhong Pharmaceutical Aids and Technology Co., Ltd. . EC (6 to 9 mPa·s) was obtained from Aladdin Chemistry Co., Ltd. . Methylene blue, N,N-dimethylacetamide (DMAc), and anhydrous ethanol were purchased from Sinopharm Chemical Reagent Co. Ltd. . All other chemicals used were analytical grade. Water was immediately double distilled before use.APAP (purity > 99%) was obtained from the 4th Pharmaceutical Factory of Weifang . PVP K25 (3.2.w/v) PVP K25 and a fixed APAP concentration of 5% (w/v) in a mixture of ethanol and DMAc ; The core fluid consists of 24% (w/v) EC and a varied concentration of APAP in ethanol, as described in −1, Shanghai Sute Corp., Shanghai, China) was used for electrospinning, which was performed under ambient conditions . The modified coaxial electrospinning processes were recorded using a digital video recorder . After optimization, the applied voltage was fixed at 15 kV and the fibers were collected on an aluminum foil 20 cm from the spinneret. All other parameters are listed in The shell fluid consists of 15% . Prior to examination, the samples were subject to platinum sputter-coating in nitrogen atmosphere for electrical conductivity. The average fiber diameter was determined by measuring their sizes in FESEM images at more than 100 different places using NIH Image J software . TEM images of the samples were recorded on a JEM 2100F field-emission instrument . TEM images of the core-shell nanofibers were obtained by fixing a lacey carbon-coated copper grid on the collector. The topographies of the raw particles of APAP, EC and PVP, and also the nanofibers F4 was observed under cross-polarized light using an XP-700 polarized optical microscope .3.3.2.−1 at a resolution of 2 cm−1.XRD patterns were obtained over the 2θ range of 5° to 60° on a D/Max-BR diffractometer  with Cu Kα radiation at 40 mV and 30 mA. FTIR analysis was conducted on a Nicolet-Nexus 670 FTIR spectrometer  from 500 to 4000 cm3.3.3.In vitro dissolution tests were carried out according to the Chinese Pharmacopoeia, 2005 ed. Method II, a paddle method, was performed using a RCZ-8A dissolution apparatus . An equal amount APAP of 20 mg  were placed in 600 mL of physiological saline  at 37 ± 1 °C. The instrument was set to stir at 50 rpm, providing sink conditions with C < 0.2Cs. At predetermined time points, 5.0 mL aliquots were withdrawn from the dissolution medium and replaced with fresh medium to maintain a constant volume. After filtration through a 0.22 μm membrane  and appropriate dilution with PS, the samples were analyzed at λmax = 257 nm using a UV/Vis spectrophotometer . The accumulative APAP released was back-calculated from the data obtained against a predetermined calibration curve. The experiments were carried out six times, and the accumulative release amount reported as mean values was plotted as a function of time .4.In vitro dissolution tests exhibited that the core-shell nanofibers could provide dual release profiles consisting of an immediate and a sustained release. The amount of drug released in the second phase was tunable. The present study showed a simple and useful approach for the systematic design and fabrication of novel biomaterials with structural characteristics for providing complicated and programmed drug release profiles using the modified coaxial electrospinning.A modified coaxial electrospinning was carried out to prepare core-shell nanofibers that provided dual drug release profiles of APAP, and the drug contents in the core fluids were successfully exploited as a tool to tailor the dose of drug released in the second phase. A Teflon-coated concentric spinneret was used to carry out the modified coaxial electrospinning. Although the shell fluid had no electrospinnability, the addition of DMAc in the shell solvent system may facilitate the coaxial electrospinning process through prevent the clogging of spinneret, and the usage of PVP K25 with a relatively small molecules could promote the fast dissolution of APAP in the first immediate release phase. XRD results verified that APAP was present in an amorphous status in the outer shell with PVP and in the inner core with EC in the core-shell nanofibers. FTIR spectra demonstrated that the shell polymer and core EC matrix were compatible with APAP owing to hydrogen bonding.


Mice are commonly used in biomedical research, and euthanasia is an important part of mouse husbandry. Approved, humane methods of euthanasia are designed to minimize the potential for pain or discomfort, but may also influence the measurement of experimental variables.2)-induced asphyxiation or by cervical dislocation.We compared the effects of two approved methods of mouse euthanasia on the levels of retinal dopamine. We examined the level of retinal dopamine, a commonly studied neuromodulator, following euthanasia by carbon dioxide (CO2 overdose substantially differed from that in mice euthanized through cervical dislocation.We found that the level of retinal dopamine in mice euthanized through CO2 as a method of euthanasia could result in an experimental artifact that could compromise results when studying labile biologic processes.The use of CO CO2 overdose has been favored for euthanasia, because the lowering of pH in the cerebrospinal fluid (CSF) that occurs secondary to CO2 overdose is associated with anesthetic depth and insensibility to pain in humans [2 overdose alters the retinal level of dopamine, an important regulator of visual function [Mice are commonly used in biomedical research today as genetic manipulation in mice is becoming more feasible and affordable. An important part of mouse husbandry is euthanasia, which should be performed as painlessly and efficiently as possible. There are several ways in which laboratory mice are euthanized, two of which include carbon dioxide  and conformed to the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Five-month-old adult male 129/Sv mice  were used for this study. Animals were kept in a 12 h:12 h light-dark cycle. Lights were on from 7 a.m. to 7 p.m., and food and water were provided ad libitum. At approximately 1 p.m., all animals were euthanized by either COt et al.  for adul4 solution containing 0.01% of sodium meta-bisulfite and 25 ng/ml 3,4-dihydroxybenzylamine hydrobromide . Samples were subsequently centrifuged at 15,000 ×g for 10 min at 4 °C, and 50 µl of the supernatant was used for HPLC analysis using an Ultrasphere ODS 5 µm 250×4.6 mm column  with a mobile phase containing 0.1 M sodium phosphate, 0.1 mM EDTA, 0.35 mM sodium octyl-sulfate, and 5.5% acetonitrile, pH 2.7. Standards of dopamine and DOPAC ranging from 2 to 20 ng/ml were analyzed with the samples. The precipitate obtained by centrifuging the homogenized retinas was resuspended in 100 µl of 1 N NaOH by sonication. An aliquot of 5 µl was used to estimate the amount of protein in each retina sample [Levels of retinal dopamine and its major metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), were determined with ion-pair reverse-phase high-performance liquid chromatography (HPLC) with coulometric detection  using a modification of the method as described by Pozdeyev et al. [t test using SigmaPlot 12 . Error bars represent the standard error of the mean (SEM), and p<0.05 was considered significant.Comparisons of two groups were made with the Student 2 overdose compared to those in mice euthanized with cervical dislocation (2 overdose method was 0.78 ng per mg of retinal protein. However, the level of retinal DOPAC, dopamine’s major metabolite, was not statistically different between the two groups of mice (2 overdose method was 0.50 ng per mg of retinal protein.The level of retinal dopamine was significantly different in mice euthanized with COlocation ; p=0.003 of mice ; p>0.05.2 overdose was a major change in experimental condition that significantly altered the level of retinal dopamine.The current study compared two methods of euthanasia approved by the American Veterinary Association on the level of retinal dopamine. We found that changing the euthanasia method from cervical dislocation to CO2 overdose, we can conjecture that the mechanism involves factors of time, hypoxia, and respiratory acidosis that result from having excess CO2 in the blood. Dopamine turnover in the mouse retina exposed to light is rapid [2-induced asphyxiation requires several minutes, and changes in oxygenation and pH could affect dopamine synthesis and metabolism during that period. In contrast, cervical dislocation is rapid, and retinas are typically dissected and frozen within 1–2 min. The rate-limiting step in dopamine biosynthesis is catalyzed by tyrosine hydroxylase, which requires molecular O2 for activity [2 could inhibit tyrosine hydroxylation and dopamine biosynthesis. Normally blood has a buffering capacity for CO2 in which water combines with CO2 to produce hydrogen and bicarbonate ions to keep the CO2 levels tightly regulated. However, when the capacity of the buffering system in the blood is exceeded, the excess CO2 leads to acidosis. Acidosis alters dopamine reuptake by affecting the activity of dopamine transporters [O-methyl transferase rather than monoamine oxidase, which requires oxygen to metabolize dopamine. These factors could explain why the level of retinal dopamine was affected while the level of retinal DOPAC was preserved.Although it is not clear why dopamine levels are diminished in mice euthanized through COis rapid . CO2-indactivity . Thus, hsporters ,9. As a 2 overdose have significantly different retinal dopamine levels compared to mice euthanized with cervical dislocation. Thus, the use of CO2 as a method of euthanasia could result in an experimental artifact that could compromise results when studying labile biologic processes.In conclusion, we showed that mice euthanized with a CO


Electrospun nanofibers have been widely used as substrata for mammalian cell culture owing to their structural similarity to natural extracellular matrices. Structurally consistent electrospun nanofibers can be produced with synthetic polymers but require chemical modification to graft cell-adhesive molecules to make the nanofibers functional. Development of a facile method of grafting functional molecules on the nanofibers will contribute to the production of diverse cell type-specific nanofiber substrata.Small molecules, peptides, and functionalized gold nanoparticles were successfully incorporated with polymethylglutarimide (PMGI) nanofibers through electrospinning. The PMGI nanofibers functionalized by the grafted AuNPs, which were labeled with cell-adhesive peptides, enhanced HeLa cell attachment and potentiated cardiomyocyte differentiation of human pluripotent stem cells.PMGI nanofibers can be functionalized simply by co-electrospinning with the grafting materials. In addition, grafting functionalized AuNPs enable high-density localization of the cell-adhesive peptides on the nanofiber. The results of the present study suggest that more cell type-specific synthetic substrata can be fabricated with molecule-doped nanofibers, in which diverse functional molecules are grafted alone or in combination with other molecules at different concentrations. Although the invention of electrospinning was disclosed in the 1930s, electrospinning-related studies have exponentially increased in the last few decades owing to the demands of nanofibrous structures for diverse applications ,3, whichPSCs attract a lot of attention for their potential to supply any kind of somatic cells in the body. In addition to embryonic stem cells (ESCs), which are mainly produced from a preimplantation embryo -26, the Herein, we examined the potentiating activity of a functionalized nanofiber substratum for cardiomyocyte differentiation in comparison with that of laminin-211. The PMGI nanofiber was selected to be functionalized through electrospinning owing to its proven rigidity that enables incorporation of small fluorescent molecules through co-electrospinning , similarw/v) PMGI polymer solution  as described previously [v/v) and mixed completely: concentrated stock solutions were 10 mM fluorescent molecules such as fluorescein and porphine dissolved in ethanol, 1 mM fluorescent peptides dissolved in dimethylsulfoxide (DMSO), and 10.7 nM peptide-labeled AuNPs dissolved in water. The mixed solution was loaded into a syringe equipped with a 21 one-fourth-gauge blunt-ended steel needle . To produce nanofibers, 8 kV was applied between the needle and a grounded collector, which was a silicon wafer covered by aluminum foil, separated 10 cm apart, while the PMGI solution was released continuously out of the syringe at a speed of 0.8 μL/min via a syringe pump. The diameters of the collected nanofibers ranged from 300 to 500 nm. Synthetic nanofiber substrata were fabricated by repeated pipeting of the collected nanofibers, which were fully soaked in autoclaved Milli-Q water , against the surface of the polystyrene dishes. Approximately, nanofibers produced with 4 μL of PMGI solution were used to coat a 35 mm dish. The unbound nanofibers were washed out with autoclaved Milli-Q water. The substratum was sterilized under UV light for 4 h before use.Electrospinning was processed with 13 % , was dissolved in DMSO at a 10-mM concentration and added to the 13 % PMGI solution at a 10 % (v/v) ratio. The fluorescent molecule-doped nanofibers were used to construct nanofiber substrata using 10-cm polystyrene dishes. Each substratum was incubated at 37°C in 10-mL phosphate-buffered saline (PBS). Every 5 d, the residual fluorescence intensities of the nanofiber substrata were measured using a fluorescent microscope  and the accompanying MetaMorph image analysis software . The size of the measured area, exposure time, and threshold were preset for the equal measurement of the fluorescence intensities of 5 different areas in each substratum. For the peptide-releasing test, 3 peptides were custom synthesized : a hydrophobic peptide composed of 6 leucines, a negatively charged peptide composed of 6 glutamic acids, and a positively charged peptide composed of 6 lysines. To monitor their release, a lysine labeled with fluorescein isothiocyanate (FITC) was added to each of the peptides. The FITC intensities in the nanofiber substrata were measured as described for the small fluorescent molecules, with which the amounts of the residual peptides in the nanofiber substrata were quantified.Each fluorescent molecule, the sodium salt of fluorescein (fluorescein) and the 5,10,15,20-Tetra(4-pyridyl)-21 Solutions of 20-nM AuNP were used to dope the nanofibers through electrospinning. The AuNP-doped nanofibers were fully soaked in Milli-Q water and fragmented by pipeting. For transmission electron microscopy (TEM) observation, microdroplets of the fragmented nanofibers or the AuNPs were deposited and dried on a plastic holey film covering a copper grid. TEM was done using a JEM-2200FS , operating at 200 kV. The fast Fourier transform (FFT) analysis was done with the DigitalMicrograph software package .t-butyl alcohol for 1 h. After washing with PBS, the cells were dried at 4 °C for 30 min. Then, the cells were covered with a 5-nm-thick gold layer through a sputtering at 200A for 15 sec. Samples were observed with a microscope . Using the same method, plain PMGI nanofibers were prepared for structural analysis with the SEM microscope.Mouse R1 ESCs were spread over unfixed PMGI nanofiber mesh and cultured for 1 week under growth medium, which was composed of DMEM-F12 supplemented with 15 % fetal bovine serum, 0.1 mM 2-mercaptoethanol, non-essential amino acids, and 1,000 U/ml mouse leukemia inhibitory factor  from Millipore . The cells were fixed using 1 % glutaraldehyde solution in PBS for 1 h, and then soaked in 100 % N-hydroxysuccinimide . To the AuNP solution being stirred at room temperature, we added 0.3-mM EDC and 0.75-mM NHS, stirred the solution for 45 min to activate the carboxyl group, and then filtered the solution 3 times with 25-mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer. A solution containing a 10-M excess of a mixture of cRGD peptide  and/or heparin-binding peptide I  was added dropwise to the AuNP solution, which was preadjusted to pH 7.5 with 100-mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, and stirred for 2 h. The AuNP solution was filtered 3 times using a 10,000-MWCO filter to remove the unreacted molecules and finally reconstituted with Milli-Q water. Each step of the labeling was confirmed by measuring the size of the AuNPs using a Zetasizer .The unconjugated AuNPs  were labeled with functional peptides using a 2-step method. First, a 1-mM mixture of 16-mercapto-hexadecanoic acid  and the polyethylene glycol (PEG)-based molecule, which was dissolved in ethanol at a 1:3 ratio (MHDA/PEG), was added to a basic AuNP solution  and stirred for 24 h. In most cases, the mole fraction of the thiols in the solution was similar to the mole fraction of the thiols bound to the nanoparticles. The solution was then filtered 3 times with a 10,000-MWCO filter  by adding Milli-Q water at each step. The second step involved linking the peptides to the AuNPs via 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide  and Nanofiber meshes (1-mm thickness) were laid on a solid surface on which a water drop was placed. Static contact angles of the water drops were measured using the sessile drop method on a homemade contact angle instrument and the Low Bond Axisymmetric Drop Shape Analysis plugin  for Imag2) were seeded and incubated at 37°C in the presence of 5 % CO2 for 6 h. HeLa cell attachment was monitored using a microscope, and the bound cells were quantified by counting the detached cells after trypsinization from the 3 dishes of each nanofiber substratum.The nanofiber substrata were constructed with the PMGI nanofibers containing plain AuNPs, FLAG and PEG-labeled AuNPs, or cRGD and PEG-labeled AuNPs. Over the nanofiber substrata, the same number of HeLa cells (500 cells/cmA human ESC line (KhES-3) and a human iPSC line (IMR 90-1) were maintained on mitomycin C-treated mouse embryonic fibroblasts (MEFs) with primate ESC culture medium  containing 5 ng/mL basic fibroblast growth factor . The human ESC line was used in conformity with the guidelines for derivation and use of human embryonic stem cells of the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan. Cardiac differentiation was carried out as described in a previous study , with miGenerally, molecule-doped nanofibers have been developed for controlled release of the doped molecules -41. In cSynthetic substrata for human PSCs is required to have a large amount of cell-adhesive molecules because of the low adhesion of human PSCs. To achieve this, we used a gold nanoparticle (AuNP) because its large surface area enables high-density labeling of functional molecules with low cytotoxicity ,43. In aNext, we investigated whether a peptide can be localized on the nanofiber surface using the PEG-labeled AuNP and electrospinning. The surface localization of the peptide was confirmed via molecular recognition by an antibody. To this end, AuNPs were labeled with the 3X FLAG peptide, which is widely used as a tagging sequence, through a MHDA linker, with or without PEG as depicted in the diagram  were spread over each substratum and incubated for 6 h. The cells grown on the nanofiber substratum containing cRGD revealed enhanced attachment and spreading, as determined by the formation of its unique shape indicated by the red arrows, whereas there was no cell or only a limited number of cells with such attachment and spreading on the substrata containing FLAG-AuNPs or PEG-AuNPs, respectively  on cell adhesion, respectively. HeLa cells (500 cells/cmP < 0.01). Unexpectedly, the mean colony number from the nanofiber substrata containing PEG-AuNPs was comparable with that from the laminin-coated substrata  and iPSCs (201B7), which were maintained over MEFs in the growth medium, became embryoid bodies (EBs) via suspension culture. The same number of the EBs were spread over 3 dishes of each substratum and then induced to differentiate into cardiomyocytes using the differentiation medium Figure A. After a Figure C. Thus, 2 nanofibers through electrospinning for the wavelength-dependent photoelectric response and for enhancing lithium-ion diffusion and charge transfer, respectively [Nanofibers are proven substrata for culturing somatic cells, but their application to PSCs has recently been investigated . Herein,ectively ,45. Unliectively . Therefoectively , and we ectively .The method described here has attractive points in terms of cost, convenience, and applicability: the PMGI nanofiber is cheap, and functionalization is achieved through simple co-electrospinning and can be applied to examine concentration-dependent effects and combinatorial effects of different functional molecules to the cells. The produced functionalized nanofibers can be applied to lithographic patterning of the nanofibers as described  for moreOur findings indicate that PMGI nanofibers can integrate small molecules, peptides, and functionalized AuNPs through electrospinning. Using this technique, we were able to fabricate synthetic nanofiber substrata that enhanced HeLa cell adhesion or potentiated cardiomyocyte differentiation of human PSCs.We believe that these results would serve as a foundation to fabricate diverse cell type-specific substrata.The authors declare that they have no competing interests.DJ, SP, JL MI, JB, QY, LL, and SK performed the research; DJ, YC, KL, and NN analyzed the data and wrote the manuscript. All the authors read and approved the final manuscript.Figure S1. SEM images of PMGI nanofibers. Nanofibers maintained their structure. (A) Structure of newly produced PMGI nanofibers. (B) A mouse ESC colony cultured over PMGI nanofibers for 1 week was observed.Click here for fileFigure S2. The surface of a polystyrene dish can be coated with PMGI nanofibers by pipeting. Rhodamine 6B  was premixed in 13 % PMGI solution and then incorporated into the nanofibers via electrospinning. A polystyrene dish (10 cm) was coated with the Rhodamine-doped nanofibers by pipeting the nanofibers repeatedly against the surface. The fluorescence image indicates that Rhodamine remains in the nanofiber under PBS, which was monitored with a fluorescence confocal microscope. Scale bars: 50 μm.Click here for fileFigure S3. TEM images of PEG-labeled AuNPs. High resolution TEM images showed formation of PEG layer on the surface of AuNPs. Red arrows indicate the PEG layer. Scale bars: A = 10 nm and B = 5 nm.Click here for fileFigure S4. TEM images of PMGI nanofiber containing higher number of AuNPs. Plain AuNPs (≅ 50 nM) were integrated into PMGI nanofiber through co-electrospinning with which a TEM image was taken. Scale bars: A = 0.5 μm and B = 100 nm.Click here for fileFigure S5. Contact angle measurements. The high-density PMGI nanofibers were fixed on a solid polydimethylsiloxane (PDMS) gel using adhesive silicon on which a drop of Mili-Q water was placed on the nanofiber meshes to measure its contact angle. Surface localization of the PEG-labeled AuNP made the nanofiber substratum hydrophilic, resulting in a decreased contact angle of the water drop. Au and Au-PEG indicate plain gold nanoparticle and polyethylene glycol-labeled gold nanoparticle, respectively.Click here for fileFigure S6. Labeling process of a AuNP with functional peptides. The surface of a 15-nm AuNP was labeled with 75 % PEG and 25 % MHDA using equal amounts of cRGD and HBP covalently conjugated through EDC/NHS coupling. EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, NHS: N-hydroxysuccinimide, and MHDA: 16-mercaptohexadecanoic acid.Click here for fileFigure S7. Size measurement of a functionalized AuNP.The size of an AuNP labeled with the 3X FLAG peptide (NH2-MDYKDHDGDYKDHDIDYKDDDDK-OH) through MHDA was measured. The surface of a 15-nm AuNP (A) was covered with 75 % PEG and 25 % MHDA (B), and the FLAG peptide was conjugated at the MHDA (C). The growing size of the AuNP after each labeling process was measured using a Zetasizer. The actual size of the functionalized FLAG-AuNP almost exactly matched the predicted size, which was calculated based on the length of the linker and 3X FLAG peptide. The results represent 3 independent measurements.Click here for file


The once-weekly (QW) formulation of the glucagon-like peptide-1 receptor agonist exenatide has been demonstrated to improve A1C, fasting plasma glucose (FPG), body weight, serum lipid profiles, and blood pressure in patients with type 2 diabetes through 52 weeks of treatment. In this report, we describe the 2-year results of the open-label, open-ended extension to the DURATION-1 trial of exenatide QW for type 2 diabetes.A 2-stage protocol was used: patients received either exenatide QW (2 mg) or exenatide twice daily for 30 weeks (5 μg for the first 4 weeks and 10 μg thereafter), followed by 1.5 years of treatment with exenatide QW (2 mg), for a total of 2 years (104 weeks) of exenatide treatment. Of the 295 (intent-to-treat [ITT]) patients who entered the trial, 73% (n = 216) completed 2 years of treatment (completer population). Baseline characteristics (mean ± SE) for these patients were: A1C, 8.2 ± 0.1%; FPG, 168.4 ± 43.0 mg/dL; body weight, 101.1 ± 18.7 kg; and diabetes duration, 7 ± 5 years.In the completer population, significant improvements (LS mean ± SE [95% CI]) were maintained after 2 years of treatment in A1C (-1.71 ± 0.08% [-1.86 to -1.55%]), FPG (-40.1 ± 2.9 mg/dL [-45.7 to -34.5 mg/dL]), and body weight (-2.61 ± 0.52 kg [-3.64 to -1.58 kg]) compared with baseline. The percentages of patients who achieved an A1C of <7.0% and ≤6.5% at 2 years were 60% and 39%, respectively. A significant reduction in systolic blood pressure  was maintained through 2 years of treatment. Serum lipid profiles were also significantly improved, including triglycerides , total cholesterol (-8.6 ± 2.8 mg/dL [-14.0 to -3.1 mg/dL]), and low-density lipoproteins (-4.5 ± 2.2 mg/dL [-8.9 to -0.01 mg/dL]). Changes in A1C, body weight, FPG, SBP, and lipids in the ITT population were similar to those seen in the completer population. Nausea (predominantly mild in intensity) was the most common adverse event, although the frequency and intensity of nausea decreased over time. No severe hypoglycemia was observed.Exenatide QW was well tolerated during the 2-year treatment period. This study demonstrated sustained glucose control and weight loss throughout 2 years of treatment with exenatide QW.NCT00308139ClinicalTrials.gov  Type 2 diabetes is characterized by progressive insulin resistance and pancreatic beta-cell dysfunction resulting in key defects in insulin secretion and function . UltimatTreatment of type 2 diabetes often begins with lifestyle modifications, but as the disease progresses, most patients require pharmacotherapy. However, many patients with type 2 diabetes do not maintain the recommended glycemic targets over time with traditional therapies and must transition to combination therapy and/or increasing doses of insulin as their glycemic control deteriorates ,6,10,11.Exenatide is a potent GLP-1 receptor agonist with multiple glucoregulatory effects for the treatment of type 2 diabetes -18. TreaThe sustained-release formulation of exenatide, exenatide once-weekly (QW), allows for continuous drug release over time, resulting in sustained therapeutic levels of exenatide - and thus, 24-hour glycemic control - with a single weekly injection ,22. ProvThe DURATION-1 trial was designed to compare the safety and efficacy of exenatide BID with exenatide QW in patients with type 2 diabetes. This 2-stage protocol consisted of a randomized, open-label comparison of exenatide QW and exenatide BID for 30 weeks, followed by an open-ended assessment period in which all patients received exenatide QW. The results from the 30- and 52-week assessment periods have previously been reported ,26. The Patients were randomized to one of two open-label treatment groups: 1) weekly subcutaneous (SC) injections of 2 mg exenatide QW or 2) SC injections of exenatide 5 μg BID for the first 4 weeks followed by a dose increase to 10 μg BID for the remainder of the 30-week assessment period. The inclusion and exclusion criteria have previously been reported . At 30 wA common clinical protocol was approved for each study site by the appropriate Institutional Review Board. Patients provided written informed consent prior to participation. The study was conducted in accordance with the principles described in the Declaration of Helsinki, including all amendments through the South Africa revision of 1996 .The objectives of this study were to examine the effects of exenatide QW on glycemic control, body weight, fasting lipids, and blood pressure after 2 years of treatment. Efficacy, safety, and tolerability were assessed in all patients, including intent-to-treat (ITT) and completer populations. Plasma analytes and A1C were quantitated by Quintiles Laboratories  using standard methods .All randomized patients who received at least one injection of exenatide in the 30-week phase of this study were defined as the ITT population. The 2-year (104-week) completer population consisted of patients from the ITT population who completed the study procedures through week 100 in compliance with the protocol and who received at least 96 weeks of treatment. At week 52, there were no significant differences in change from baseline A1C or the percentage of patients who achieved target A1C between the group initially randomized to exenatide QW and the group initially randomized to exenatide BID (who switched to exenatide QW at week 30) . Thus, iTreatment-emergent adverse events (TEAEs) were defined as events that occurred during or after the first injection of randomized treatment. Hypoglycemic episodes were classified as major  if 1) the event resulted in loss of consciousness, seizure, coma, or other change in mental status consistent with neuroglycopenia (as judged by the investigator or physician), in which symptoms resolved after administration of intramuscular glucagon or intravenous glucose, or 2) the event required third-party assistance because of severe impairment in consciousness or behavior and was accompanied by a blood glucose concentration of <54 mg/dL (3.0 mmol/L). Minor hypoglycemia was defined as a report of symptoms consistent with hypoglycemia and a glucose value of <54 mg/dL prior to treatment of the episode. Safety analyses are provided for the ITT population.2; A1C, 8.3; FPG, 169 mg/dL; duration of diabetes, 7 y) were nearly identical to the 2-year completer population.Demographic characteristics of the completer population n = 216) are described in Figure 6 are desThe results reported here for the completer population and ITT population consist of all patients regardless of initial randomization. Overall, patients in the 2-year completer population maintained improvements in A1C Figure , with LSBody weight reduction was generally maintained during the study; at the end of the 2-year assessment period, body weight (mean ± SE [95% CI]) was reduced by 2.6 ± 0.5 kg (-3.64 to -1.58 kg) in the completer population and 2.4 ± 0.4 kg (-3.19 to -1.53 kg) in the ITT population Figure . The disSignificant improvements in systolic blood pressure (SBP) were observed in patients treated with exenatide QW Figure : the meaFrequent (≥5%) TEAEs that occurred for the first time or worsened after receiving exenatide QW from weeks 0 to 30 and weeks 30 to 104 (ITT population) are described in Table Guidelines for the treatment of type 2 diabetes are currently focused on the use of agents that provide effective lowering of A1C, have beneficial effects on extraglycemic factors associated with long-term complications, and have favorable safety and tolerability profiles ,28. ReceThe improvements in glycemic control that were previously reported with 52 weeks of exenatide QW treatment  were susPreviously reported results from this trial demonstrated that improvements in glycemic control achieved with exenatide QW were greater than those achieved with exenatide BID, with similar or greater improvements in other outcomes including body weight, blood pressure, and serum lipid profiles ,26. AfteOther GLP-1-related therapies currently available for the treatment of type 2 diabetes are the once-daily GLP-1 receptor agonist, liraglutide, and the once-daily DPP-4 inhibitors sitagliptin and saxagliptin -33. The Treatment with exenatide QW was safe and well tolerated during the 2-year assessment period, with a retention rate of 73%. Adverse events (AEs) were generally mild to moderate. Few patients who received exenatide QW withdrew due to an AE (1.4% in the uncontrolled period) and there were no withdrawals from the study due to serious AEs. Mild nausea was common, but the frequency and severity of nausea decreased or abated over time. To date, no safety signals specific to exenatide QW have been identified in clinical trials.There were several potential limitations of this study. The absence of a comparator arm during the 1.5-year uncontrolled period limits the conclusions that can be drawn from the results, and the open-label trial design inherently creates the potential for bias and can affect patient expectations. The trial design also complicates interpretation of the modest increases in A1C, FPG, and body weight seen from week 52 to week 104. Furthermore, the population was heterogeneous for background therapy with other antidiabetic drugs; however, the earlier results at 30 and 52 weeks in this study suggest that the data are consistent across populations, regardless of background therapy.Results from both the ITT and completer populations were included in this study, and both populations present certain limitations to the interpretation of the results. For the ITT analysis, using a LOCF method is a conservative approach that accounts for early study withdrawal and provides a prediction of treatment effect for any subject who ever received treatment. The completer analysis mitigates the potentially large variations in treatment exposure by representing the actual efficacy for the full term of drug exposure. The results of the ITT analysis should, thus, be weighed against the results of the completer analysis, as efficacy data collected early in the trial  may not be representative of the true treatment effect, which is established with increased duration of treatment exposure. However, subject retention is often a challenge in long-term trials and the results of the completer analysis may be affected by the composition of the completer population, which often includes a high proportion of subjects who experienced favorable outcomes with treatment and underrepresents subjects who had issues with tolerability or lack of efficacy.The present analysis demonstrated durable efficacy with long-term exenatide QW therapy and showed that common AEs, especially nausea, subsided with time. The continuous release of exenatide in the once-weekly formulation produced continuous GLP-1 receptor activation leading to sustained control of both fasting and postprandial hyperglycemia, which resulted in enhanced improvements in A1C. Additionally, the convenience of the once-weekly injection of exenatide offers an attractive alternative to once- and twice-daily injections, and could improve treatment adherence in the clinical setting . This coQW: once-weekly; SBP: systolic blood pressure; SC: subcutaneous; SD: standard deviation; SE: standard error; SFU: sulfonylurea.AE: adverse event; BID: twice daily; BL: baseline; FPG: fasting plasma glucose; GLP-1: glucagon-like peptide-1; ITT: intent-to-treat; LOCF: last observation carried forward; LS: least squares; This study was funded by Amylin Pharmaceuticals, Inc. and Eli Lilly and Company. Dr. Gurney, Ms. Han, Dr. Pencek, and Dr. Walsh are employees of Amylin Pharmaceuticals, Inc. Dr. Taylor was previously an employee of Amylin Pharmaceuticals, Inc. Dr. Trautmann is an employee of Eli Lilly and Company. Amylin Pharmaceuticals, Inc. has a global agreement with Eli Lilly and Company to collaborate on the development and commercialization of exenatide. All authors own company stock and have stock options.KT, JH, and RP participated in the design of the analysis. JH and RP performed the statistical analysis. All authors were involved in the interpretation of the analysis. All authors were involved in drafting the manuscript or revising it critically for important intellectual content. All authors approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6823/11/9/prepub


Objective(s): The dopamine level of the nucleus accumbens changes during some stereotyped behaviors. To study dopamine level of the nucleus accumbens in intra infralimbic apomorphine-induced climbing, microdialysis probes were implanted into the nucleus accumbens shell of male Sprague Dawley rats weighting 275–400 g.Materials and Methods: The rats were divided into two groups (apomorphine and control) of least eleven rats in each group. Apomorphine at dose of 5 μg/0.5 μl or its vehicle was microinjected into the infralimbic in apomorphine and control groups respectively. Then, changes in dopamine levels in the nucleus accumbens shell were monitored. The concentration of dopamine was measured by High-Performance Liquid Chromatography-Electochemical (HPLC-ECD). Finally, the stereotyped behaviors were recorded. Results:  The mean of dopamine levels for all of after microinjection period in control and drug groups were 450% and 150% respectively compared to those of before microinjection period. However, there was no significant difference between groups of apomorphine and control. In addition, the return of dopamine level to the baseline was faster in apomorphine group than the control group. Conclusion: The intra infralimbic apomorphine -induced climbing at dose of 5 μg/0.5 μl was not modulated via the increase of dopamine level in the nucleus accumbens area.  The pathological changes in the prefrontal cortex are involved in schizophrenia -4. DysfuThe medial prefrontal cortex is one of the regions of the prefrontal cortex involved in higher cognitive functions , 8, 9. TThe dopamine transmission of nucleus accumbens integrates a wide range of limbic and motor information -18. BaseDue to the importance of the medial prefrontal cortex in the pathogenesis of schizophrenia, the pathophysiologic study models have focused on the cortical dysregulation on subcortical dopamine neurotransmission . In addiIt has been previously shown that the microinjection of apomorphine in the infralimbic could induce climbing significantly at dose of 5 μg/0.5 μl . TherefoIt is important to know whether induction of climbing via microinjection of apomorphine is a behaviors related to the medial prefrontal cortex or mediated by dopamine transmission of the nucleus accumbens via its relation with the medial prefrontal cortex. In the current study, apomorphine was therefore microinjected into the infralimbic subarea of the medial prefrontal cortex and released dopamine was measured by microdialysis method in the shell of nucleus accumbens to investigat if there was any possible involvement of the shell of nucleus accumbens in intra infralimbic apomorphine -induced climbing.Ethics and AnimalsThe protocol used in this study was approved by the Ethics Committee of Shiraz University of Medical Sciences. Eleven male Sprague Dawley rats (275–400 g) free access to food and water were used in each group for the experiments. Drugs and MaterialsApomorphine, 3, 4-dihydroxyphenylalanine (DOPA), Dopamine, Mandelic Acid and Tyrosine , Ascorbic acid, calcium chloride, glucose, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, sodium dihydrogen phosphate and sodium hydroxide , Dialysis membrane, cut-off =6000D, fused silica connecting tube with Inner diameter (ID) =75 μm andouter diameter (OD) =150 μm , Acetonitrile , ketamine , xylazine , perchloric Acid , polyethylene tubing 10 and 20 .Preparation of microdialysis probein vitro environment. Their recovery was about 25%. Microdialysis probes were generated based on the Sharp and Zetterstrom method under stereomicroscope  , 25. TheImplantation of probe and guide cannula The rats were anesthetized by Ketamine + Xylazine . The guide cannulas and probes were then implanted in the infralimbic and the nucleus accumbens shell respectively by stereotaxis . The coordinate of the infralimbic was  and for probes into the nucleus accumbens shell at angle 15° from vertical was  . Microinjection, microdialysis and behavioral recordingThe microdialysis experiments were performed 24 hr after the surgery. Apomorphine at dose of 5 µg/0.5 µl and 0.5 µl ascorbic acid 0.1% (as solvent of apomorphine and stabilizer) was delivered into the infralimbic as drug group and control or vehicle group respectively. The stereotyped behaviors were recorded as well  followed2 1, MgSO4 2, NaH2PO4 1.25, NaHCO3 26, NaOH 1 and Glucose 10, pH 7.4 (The compositions of ACSF include (in mmol) NaCl 114, KCl 3, CaCl, pH 7.4 .Dialysis procedure was 180 min divided into two parts, 60 and 120 min as before and after microinjections , 37. TheHPLC-ECD analysis2PO4 in HPLC grade water and 1% acetonitrile (pH3) was delivered into the reverse phase column  at a flow rate of 1 ml/min . The mobile phase consisting of 100 mmol NaH1 ml/min , 40 The 1 ml/min . However1 ml/min .The analysis of chromatograms was done by the Peak track software . The area under the curve (AUC) of standard solutions was used for the comparison surement of dopamine concentrations in the samples.Statistical analysisP<0.05.The analysis of data was performed by SPSS ver. 13. The data were analyzed by two and one way repeated-measures ANOVA using time as a repeated factor followed by LSD (Least Significance Difference) test as post hoc. The Significance level was set at Histological verificationAt the end of each test, serial sections were provided coronally by a microtome  and the positions of cannula and probe tracing were compared with the rat brain atlas  Figure .In this study, the mean of dopamine concentration of three samples prior to the microinjection period was considered as baseline level for each probe. The percentage of changes of dopamine concentration of samples during after microinjection period was estimated related to its own baseline level. The time-course graph presents the percentage of changes in extracellular dopamine from the baseline level . It was P<0.01). However, there was not any significant difference between two groups of apomorphine and control  = 0.259). There was not any significant interaction between time and treatment (control and apomorphine) factors . One-way-repeated measures ANOVA for each group of control or apomorphine using time as a repeated measure factor showed that factor of time was not significant for the control group, but it was significant for apomorphine group  = 2.857;post hoc revealed that there were significant differences in changes of percent of dopamine level among the fourth (20th min), fifth (40th min) and sixth (60th min) 20-min episodes with ninth 20-min episode (120th min), also between sixth (60th min) and seventh 20-min (80th  min) episode (P<0.05) (LSD test as (P<0.05) . In addiThe nucleus accumbens  is a heterogeneous area and has been subdivided into two different compartments, a medioventral shell and a laterodorsal core. The nucleus accumbens shell is involved in the control of motivation, emotional responses and most functionally reactive to the stress and abused drugs , 41. Furthermore, dorsal and ventral areas of the medial prefrontal cortex preferentially innervate the core and shell of nucleus accumbens respectively . TherefoIn the present study, it was observed that the microinjection of either apomorphine or ascorbic acid 0.1% as apomorphine vehicle into the infralimbic subarea significantly increases the dopamine level in the nucleus accumbens shell .Apomorphine as a nonselective dopamine agonist is a non-specific dopamine agonist for dopamine D1 and dopamine D2-like receptors , 49. More.g locomotor activity and changes of dopamine concentrations to stress and stimulants are dampened by the concurrent activation of meso-prefrontal cortex dopamine neurons (It has been reported that the increase of dopamine transmission in the ventral or deeper parts of medial prefrontal cortex including the prelimbic and infralimbic area has an inhibitory effect on turn-over of dopamine transmission in the mesolimbic system and its response to stimulants , 52-57.  neurons , 55, 58. neurons . Thus, t neurons -62. This neurons . The neu neurons . It is w neurons , 55, 58. neurons , 63, 64.The findings of this study with no significant change in the dopamine neurotransmission of the nucleus accumbens shell after microinjection of apomorphine into the infralimbic vs. control group can also confirm the results of previous reports. With respect to another study , it seemThe changes of dopamine release after microinjection were significant in the drug group. The microinjection-handling stress has not been excessively influential to induce any significant differences of dopamine release between two groups in the nucleus accumbens, but because of the inhibitory effect of dopamine in the medial prefrontal cortex on dopamine neurotransmission of the nucleus accumbens, the return of dopamine level to baseline near to its level before microinjection was faster in drug group than that of control group . The incMicrodialysis probes could not keep their dialysis ability more than 24h after implantation ; therefoThis study showed that intra infralimbic apomorphine -induced climbing at dose of 5 μg/0.5 μl is not modulated via the increase of dopamine level in the nucleus accumbens area. This behavior may be a direct effect related to the modulation of dopamine receptors of the infralimbic subarea and not via the nucleus accumbens.


The Mn2O3-Ag nanofibers were used as facilitator electron transfer between Hb and glassy-carbon-modified electrode. The Mn2O3-Ag nanofibers are studied by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The hemoglobin showed a quasireversible electrochemical redox behavior with a formal potential of −49 mV (versus Ag/AgCl) in 0.1 M potassium phosphate buffer solution at pH 7.0. The designed biosensor possesses good stability and reproducibility and achieves 95% of the steady-state current in less than five seconds.We investigated the electrochemical behavior of hemoglobin by glassy carbon electrode modified with Mn To accomplish these functions, red cells have hemoglobin (Hb). The Hb has iron in center of its structure [Hemoglobin is a remarkable molecular machine that uses motion and small structural changes to regulate its action. Oxygen binding at the four heme sites in hemoglobin does not happen simultaneously. Once the first heme binds oxygen, it introduces small changes in the structure of the corresponding protein chain. The main function of red blood cell is transfer of Otructure . The irotructure –5. Glasstructure , 7. Theytructure . Becausetructure . Small ftructure , 11. Pretructure . While structure  and in ptructure . Electrotructure , 16. By tructure –19. PVPsElectrospinning is a process of applying a high-voltage electric field  to generate electrically charged jets from polymer solutions or melts and further to produce polymer (nano) fibers. This technique is quite similar with the commercial process for drawing microscale fibers; however, it is more suitable for generating nanofibers, because the elongation can be accomplished by a contactless scheme through the application of an external electric field . General2HPO4 and NaH2PO4 and adjusting the pH values with NaOH and H3PO4 solution. All solutions used in the experiments were prepared with deionized water generated by a Barnstead water system.Hb, manganese(II) nitrate tetrahydrate, and poly(vinylpyrrolidone)  were purchased from Sigma. Silver nitrate and other materials were supplied by Merck. 0.1 M phosphate buffer solutions with various pH values were prepared by mixing stock standard solutions of Na3)2, 11 wt% AgNO3, and 44 wt% PVP were dissolved in DMF. The solution was kept under magnetic stirring for 2 h and then loaded into a plastic syringe equipped with a 23-gauge needle made of stainless steel. Electrospinning process was conducted at an applied voltage of 20 kV with a feeding rate of 0.3 mL/h and a collection distance of 15 cm. The nanofibers were collected on aluminum foil and then calcined under air atmosphere at 500°C for 3 h for the degradation of PVP and the decomposition of Mn(NO3)2 and AgNO3:44 wt% Mn to generate electrically charged jets from polymer solutions or melts and further to produce polymer (nano) fibers. So, in this study, we used from this method to produce Mnμm and 0.05 μm alumina slurries sequentially and then rinsed with DI water. After that, the electrode was sonicated in deionized water and finally dried under ambient conditions. To prepare the modified GCE, The Mn2O3-Ag nanofibers/glassy carbon electrode was placed into a fresh PBS including 5 mg mL−1 Hb  for 8 h. At the end, the modified electrode was washed in deionized water and placed in PBS (PH 7.0) at a refrigerator (3 to 5°C), before being employed in the electrochemical measurements as the working electrode.The most commonly used carbon-based electrode in the analytical laboratory is glassy carbon (GC). It is made by pyrolyzing a carbon polymer, under carefully controlled conditions, to a high temperature like 2000°C. An intertwining ribbon-like material results with retention of high conductivity, hardness, and inertness. Glassy carbon electrode  was polished with 1 2O3-Ag nanofibers were obtained with a Tecnai T12 transmission electron microscope (TEM) operated at 120 kV. XRD pattern was obtained with Oxford diffraction XcaliburTM PX Ultra with ONYX detector to study the crystal structure of Mn2O3-Ag nanofibers measurements which were carried out with a potentiostat/galvanostat  using a single compartment voltammetric cell, equipped with a platinum rod auxiliary electrode. All experiments were conducted using a three-electrode electrochemical cell (10 mL volume with a working volume of 5 mL), with a working electrode, an Ag/AgCl reference electrode, and a platinum wire counter electrode. Electrochemical measurements were carried out with a potentiostat/galvanostat  using a single compartment voltammetric cell, equipped with a platinum rod auxiliary electrode.A JEOL 6335F field-emission scanning electron microscope (SEM) was used to examine the morphology and the size of the as-prepared nanofibers. More detailed morphology and selected area electron diffraction (SAED) patterns of Mn2O3-Ag nanofibers. 3)2-AgNO3 nanofibers. PVP was used for characterization of Mn2O3-Ag nanofibers, because PVP binds to polar molecules exceptionally well, owing to its polarity. This has led to its application in coatings for increase of photo quality in microscopic studies. The molecular structure of PVP can be observed in 2O3-Ag nanofibers have polar groups too, a good bind was created between this nanofiber and PVP.SEM was first employed to investigate the morphology of the Mn2O3-Ag composite nanofibers (3)2) with PVP and its calcined product (Mn2O nanofibers) are presented in Figures  After calcination, the as-prepared Mnnofibers  exhibit 2O3-Ag nanofibers matches the combination of the standard spectrum of JCPDS 41-1442 (Mn2O3) and JCPDS 04-0783 (Ag). The formation of face-centered cubic crystalline Mn2O3 is revealed by the diffraction peaks at 2θ values of 32.951, 38.234, 45.178, 49.347, 55.189, and 65.806, corresponding to (111), (200), (220), (311), (222), and (400) crystal planes, respectively, while the diffraction peaks at 2θ values of 38.116, 44.277, and 64.426, which correspond to (111), (200), and (220) crystal planes, respectively, indicate the formation of cubic crystalline Ag.The composition and crystal structure were characterized by XRD . The XRD2O3-Ag-nanofibers-modified electrode is apparently larger, which indicates that the effective electrode surface area is significantly enhanced by use of Mn2O3-Ag nanofibers to modify the electrode. However, the CVs of Hb/Mn2O3-Ag nanofibers/GC electrode give a pair of well-defined redox peaks at −75 mV at scan rate of 100 mV/s, characteristic of heme Fe(III)/Fe(II) redox couples of Hb, suggesting that direct electron transfer has been achieved between Hb and modified electrode. The electron transfer of the proteins, at the bare electrodes is very slow so that the redox peak of proteins can usually be observed. 2O3-Ag nanofibers/glassy carbon electrode in 0.1 M phosphate buffer at pH 7.0. The Hb showed quasireversible electrochemical behavior with a formal potential of −49 Mv (versus Ag/AgCl); cathodic and anodic peaks were not observed using the bare graphite electrode. This shows that Mn2O3-Ag nanofibers act as a facilitator of electron transfer from the redox species of Hb to the electrode surface and vice versa. These results are in line with the previous work that explains the behavior of nanoparticles as the facilitators of electron transfer.Cyclic voltammetry (CV) was used to characterize the modification of electrodes. The cyclic voltammograms (CVs) of different modified electrodes were obtained in 0.1 M PBS with pH 7.0. No redox peak is observed for the CVs of bare GC electrode. Compared with bare GC electrode, the background current of Mn2O3-Ag nanofibers were successfully assembled on the glassy carbon electrode surface. The redox peak currents increase linearly with the square root of the scan rate. With an increasing scan rate, the anodic peak potential of adsorbed Hb shifted to a more positive value, while the cathodic peak current shifted in a negative direction. The redox peak currents were proportional to the scan rate  s−1. These statements discussed below indicate that Hb is strongly adsorbed onto the surface of modified electrode. As could be seen in −1, the catholic peak potential (Epc) changed linearly versus lnv with a linear regression equation of y = −0.2425x − 0.4254, R2 = 0.945, according to the following equation [In can rate ; thus, tequation –28,α is the cathodic electron transfer coefficient, n is the number of electrons, R, T, and F are gas, temperature, and Faraday constant, respectively , and αn is calculated to be 0.52. Given 0.3 < α < 0.7 in general [n = 1 and α = 0.52. From the width of the peak at midheight and low scan rate, we can also obtain n = 1 [ks), the following equation was used [was used –28:ks was calculated to be 1.90 s−1. Moreover, this ks indicates that transfer of electron is good and fast in Hb-modified glassy carbon electrode. The 2+ changes to Fe3+, the oxidation reaction occurred, and when Fe3+ changes to Fe2+, the reduction reaction occurred. The reduction state is an important matter for designing hydrogen peroxide biosensors [The designed biosensor is introduced as a hydrogen biosensor because it has oxidation and reduction reactions in Hb; when Feosensors .2O3-Ag nanofibers/GC electrode toward H2O2 was investigated by CV. 2O2 in the absence of oxygen. The catalytic reduction of hydrogen peroxide at the biosensor can be seen clearly in 2O2, the reduction peak current increases obviously, while the oxidation peak current decreases  is shown in In order to obtain an efficient biosensor for H2O2 was prepared based on Hb/Mn2O3-Ag nanofibers/glassy carbon electrode. Hb retained well in Mn2O3-Ag nanofibers/GCE, which combined the utilities of Mn2O3-Ag nanofibers facilitating the electron transfer. At Hb/Mn2O3-Ag nanofibers/GCE, the cyclic voltammogram exhibits a pair of redox peaks corresponding to a surface-controlled electrode process with a single-proton transfer. The designed biosensor displays a high affinity and sensitivity to H2O2. The sensor shows good reproducibility and stability.A new biosensor for H