It was shown that the stabilizers affected exenatide stability in aqueous solutions, at water/dichloromethane interfaces, on PLGA surfaces, or during freeze-thawing and freeze-drying procedures. Sucrose predominantly reduces instability generated during freeze-thawing and freeze-drying. Phenylalanine prevents the destabilization at the water–dichloromethane (DCM) interface through decreased adsorption. Poloxamer 188 enhances stability in aqueous solutions and prevents adsorption to PLGA. Proline and lysine decrease adsorption on PLGA surfaces. Fourier transform infra-red spectroscopy (FT-IR) was used to find the molecular interaction of additives with exenatide or PLGA. Additives used in stability assessments were then added stepwise into the inner or outer water phase of the W/O/W double emulsion, and exenatide-loaded microspheres were prepared using the solvent evaporation method. The effect of each stabilizer on the encapsulation efficiency and release behavior of microspheres correlated well with the stability assessment results, except for the negative effect of poloxamer 188. Particle size analysis using laser diffractometry, scanning electron microscopy (SEM), water vapor sorption analysis, differential scanning calorimetry (DSC), and circular dichroism (CD) spectroscopy were also employed to characterize the prepared exenatide-loaded PLGA microsphere. This study demonstrated that an adequate formulation can be obtained by the study about the effect of stabilizers on peptide stability at the preformulation step. In addition, it can help to overcome various problems that can cause the destabilization of a peptide during the microsphere-manufacturing process and sustained drug release.The aim of this study was to investigate the effects of various stabilizers on the encapsulation efficiency and release of exenatide-loaded PLGA (poly(lactic- For such development, stability improvements to avoid peptide degradation and to ensure optimized sustained drug release are of particular interest [Many peptide drugs have been developed into commercialized medicines using PLGA  technique . In addiThe manipulation and optimization of various formulation factors is required to successfully develop new formulations of peptide drugs with desirable pharmaceutical properties. Such refinement should be carried out at very early stages of development using PLGA microsphere formulations to ensure sustained delivery of new peptide drugs as stated above. The properties of PLGA microspheres for different drugs may be highly variable, especially for mid-length peptides, like exenatide. To obtain an optimized formulation for the preparation of peptide-loaded PLGA microspheres, multiple factors affecting the formulation should be considered, including the pH of the inner/outer aqueous phases and the type and amount of stabilizers . FurtherExenatide instability and related problems in its commercial manufacture and clinical usage have been widely reported . For exaThe aim of this study was to show how useful the evaluation of stabilizer effects in the preformulation stage is for the optimized design of peptide-loaded PLGA microsphere formulation and improvement of the final product quality. Exenatide was used as a model peptide . We exam®F68, Ludwigshafen, Germany) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) was obtained from Corden Pharma . Acetonitrile, trifluoroacetic acid (TFA), and dichloromethane (DCM) were purchased from Merck . All other chemicals were of reagent grade and used without further purification.Exenatide was purchased from American Peptide Company, Inc. . PLGA 50/50 was obtained from Boehringer-Ingelheim . Sucrose, lysine, phenylalanine, proline, and poly  were purchased from Sigma Aldrich . Poloxamer 188 was kindly supplied by BASF (LutrolReverse phase high performance liquid chromatography (RP–HPLC) was used to analyze exenatide content. HPLC analysis was performed using an Agilent 1290 Infinity HPLC system  consisting of a pump (Model 1260 Quat Pump VL), an auto sampler (Model 1260 ALS), and UV detector (Model 1260 VWD DL) as previously described with slight modifications . The autThe conditions of the stability evaluations were set in consideration of the physicochemical properties of exenatide  and the The test solutions of exenatide (0.1 mg/mL) containing hydrophilic additives were prepared in pH 4.5 acetate buffer. They were transferred to a certified low adsorption amber glass vial , and then sealed with a cap. A pH condition of 4.5 was selected because this pH was used favorably for the marketed injectable liquid formulation of exenatide considering the stability and compatibility with PLGA ,39,40. T®, IKA®-WERKE GMBH & Co. KG, Staufen, Germany) at 21,500 rpm for 15 min. After homogenization, 4 mL of pH 4.5 acetate buffer was added then samples were centrifuged at 3000× g for 20 min to complete phase separation. After collecting as much water phase as possible, residual DCM was further removed by using a rotary vacuum evaporator  at ambient condition for 1 h  then frozen at −70 °C for 12 h. The frozen samples were then thawed at 10 °C for 1 h. This freeze-thawing process was repeated 7 times for each sample  was transferred to a CryoTubeh sample .TM  were lyophilized using a freeze dryer  at 50 mTorr of vacuum for 3 days. Lyophilized samples were then dissolved in 1 mL of DW  without additive were prepared using various buffered solutions with pH conditions of 3.5 (30 mM citrate buffer), 4.5 (30 mM acetate buffer), and 7.4 . A 100-µL volume of prepared test solution was added to each PLGA-coated well, and then the 24-well plate was sealed with sealing film. After shaking at 37 °C for 12 h, the amount of free exenatide was quantified using RP-HPLC . The perTo study the effect of hydrophilic additives on exenatide adsorption to PLGA film, 100 µL of test solution  containing hydrophilic additives were added to a PLGA-coated well. The concentration of hydrophilic additives was same as above stability test. For the amino acids, tests were also conducted in DW and pH 7.4 PBS. The pH 4.5 acetate buffer and water were chosen as aqueous phases taking into account the environment of the internal aqueous phase and outer aqueous phase during microsphere preparation, respectively. A pH of 7.4 was selected considering the pH condition of the in vitro release tests. Subsequent procedures were performed in the same manner as the above adsorption experiments with varying pH. The selected amphipathic additives  were dissolved in DCM solution with a PLGA concentration of 80 mg/mL. PLGA films containing hydrophobic additives were prepared using the same method as with blank PLGA film. A total of 100 µL of test solution (pH 4.2) without additive were transferred to a PLGA film. Subsequent procedures were performed in the same manner as the above experiments with hydrophilic additives .®, IKA®-WERKE GMBH & Co. KG, Germany) at 21,500 rpm for 5 min in an ice bath. This primary emulsion was then added to 12 mL of an outer aqueous phase containing 1% PVA ) with or without 0.1 M lysine. The emulsification continued at 6500 rpm for 1 min. An additional 12.5 mL of outer aqueous phase was poured slowly into the secondary (W/O/W) emulsion. The DCM in the double emulsion was removed using a rotary vacuum evaporator  at 35 °C. The resulting microspheres were washed three times with a 5% dextrose and 0.9% NaCl solution by centrifugation at 600× g , and then freeze-dried by the same lyophilization method as used above for the freeze-drying stability study. Exenatide-loaded microspheres were prepared by the double emulsion (W/O/W)–solvent evaporation method. Various formulations of exenatide-loaded microspheres are presented in Scanning electron microscopy  was used to observe the morphology of the PLGA microsphere after freeze drying. Microspheres were placed on a metal sample stub using conductive carbon double-sided adhesive tape. Gold-palladium coating was applied using an automatic sputter coater. The morphology and appearance of samples were examined using a SEM at an accelerating voltage of 5 kV.The distribution of particle sizes was determined using laser diffractometry with a Mastersizer 2000 containing a Hydro 2000S module  for wet sample dispersion. Particle sizes are expressed as the volume mean diameter (VMD) in micrometers (µm). The size distribution was determined using the apan value, which is calculated as the ratio of D90%–D10% to D50%, where DN% indicates the volume particle diameter at each cumulative volume percentage. Smaller span values represent narrow particle size distributions.A total of 10 mg of lyophilized microspheres were dissolved in 1 mL of acetonitrile, to which 1 mL of 30 mM acetate buffer solution (pH 4.5) were added. The mixture was centrifuged  at 12,000 rpm for 5 min. The supernatant was then collected and injected into an RP-HPLC system and the amount of exenatide in dried microsphere was determined. The loading capacity (LC) and encapsulation efficiency (EC) for the direct analysis method were calculated using the following equations:w/v) Tween 20 and 0.01% sodium azide was used for the in vitro release test. A total of 20 mg of microspheres were weighed then added into 2 mL of dissolution medium and incubated in a shaking water bath at 37 °C under continuous agitation (100 rpm) for 24 days. At predetermined time intervals, samples were centrifuged at 4000 rpm for 5 min. An aliquot of the resulting supernatant was then collected, and an equal volume of fresh release medium was added to the release tube. The supernatant was then centrifuged at 12,000 rpm (5 °C) for 10 min and the amount of exenatide in the supernatant was determined by RP-HPLC. The percentage of exenatide measured on the first day was considered the initial burst release (IBR) and used for comparison between samples.The phosphate buffer saline  containing 0.02%  to evaluate the moisture sorption/desorption behavior. Each sample was placed on the sample pan, which was equilibrated to 0% relative humidity (RH). The experimental cycle consisted of a sorption cycle followed by a desorption cycle. The RH was increased from 0% to 95% RH in steps of 5% RH for the adsorption isotherm and decreased from 95% to 0% RH in steps of 5% for the desorption isotherm. At each relative humidity step, the system controlled the RH and monitored the sample weight until it reached equilibrium conditions. The equilibrium weight and temperature at the RH step were then recorded.DSC measurements were carried out using a DSC S-650 . Calibration was performed first with indium (melting point = 156.6 °C) and zinc (melting point = 419.5 °C) standards at a heating speed of 10 °C/min. Accurately weighed 2-mg samples were placed in aluminum pans, and then sealed with an aluminum lid. A sealed empty pan was used as a reference. Measurements were conducted using a heating rate of 10 °C/min under a nitrogen purge with a flow rate of 20 mL/min.−1 at a 4 cm−1 resolution and 256 scan numbers. Prior to the collection of spectra for the samples, background spectra were scanned first by purging the detector with nitrogen gas to minimize interference by water vapor and CO2. Second derivative data were obtained by OriginPro 8 software,  and then processed with 7-point smoothing.FT-IR analysis was conducted using a Nicolet 380 FT-IR spectrometer  equipped with an attenuated total reflectance (ATR) accessory. Spectra were collected from 400 to 4000 cmCD spectroscopy  was exerted to investigate the secondary structure of exenatide in PLGA microsphere. The exenatide was extracted from microspheres by dissolving about 10 mg of exenatide microspheres in 3 mL acetonitrile followed by the addition of 3 mL of distilled water. The presence of acetonitrile from the solution was removed by rotary evaporation under vacuum. After that, samples were centrifuged  at 12,000 rpm for 5 min. Supernatant was collected as sample solution. Unprocessed raw exenatide was used for a comparison of the secondary structural integrity. The detection was performed using a 0.1-cm path cuvette with a scan rate of 50 nm/min at 30 °C, with 24 scans averaged to generate each spectrum. For comparison, unprocessed exenatide dissolved in distilled water was analyzed by CD spectroscopy in the same way. Peptides can be degraded through hydrolysis and temperature-dependent destabilization. The recovery of exenatide for samples without additives was only 34.2%. The test temperature of 50 °C probably induced acceleration of exenatide destabilization in aqueous solution. Significant improvements in aqueous exenatide stability were observed from all additives . Many reAmong the water-soluble additives used in this study, poloxamer 188 had the largest effect. This may be due to synergic effects by other complex mechanisms as well as preferential interaction. Peptides can be adsorbed to air/water interfaces followed by subsequent reorientation and rearrangement of the adsorbed molecule. Nonionic surfactant, like poloxamer 188, can decrease the surface tension and induce a decrease in peptide adsorption at the air/water interfaces, thereby reducing changes in the secondary structure and destabilization of the peptide . In addiThe stabilization effect of amino acids, including proline , lysine Exenatide recovery after the W/O interface stability tests was 59.4%, confirming that exenatide instability can be caused by W/O emulsification during PLGA microsphere preparation. The hydrophobic surface of DCM makes the circumjacent water molecule become highly ordered. The surrounding water molecules that stabilize the peptide can be dehydrated at the W/O interface, thus the peptide molecule can contact the DCM directly. This peptide adsorption to the W/O interface spontaneously displaces ordered water and induces chemical and physical destabilization of peptides because this dehydration increases entropy . Except In particular, phenylalanine, with a hydrophobic phenyl group, showed dramatic stabilizing effects, which may be due to the replacement of water molecules by its good affinity for the DCM/water interface. p > 0.05). This may be due to the lack of its surface activity and affinity for the DCM/water interface. Several studies also presented similar results [Lysine and proline showed a small effect. This may be due to their low surface activity considering their chemical structure without a hydrophobic moiety. The W/O interface-induced instability was not significantly reduced by the addition of sucrose  (PEO) and poly(propylene oxide) (PPO). In the water phase, it is expected that the hydrophobic PPO and hydrophilic PEO will face the DCM and water phase, respectively, and be arranged with high affinity at the W/O interface. Therefore, exenatide in the water phase may be prevented sterically from contacting with DCM. In addition, there may be a stabilizing effect by micelle formation, but no further experiment for this micelle formation was performed in this study . In cont−1, which contains bands assigned to amide I and II of exenatide  of PLGA carboxylate, and the incubation time may not be enough to proceed much of the ionization of the carboxyl end group of PLGA . For theThe amount of adsorbed exenatide was decreased by an increase in pH to 7.4 over pI, but it was higher than that of pH 3.5. At pH values higher than pI, peptides are negatively charged, hence the repulsion between peptide molecules increases. At pH 7.4, even though the incubation was short, ionization of carboxylic acid groups could occur by hydration because used PLGA503H is uncapped PLGA. A negative or neutral net charge of the peptide could mask the fact that positively charged residues can still exist and may interact electrostatically with negatively charged carboxylic acid end-groups of PLGA . NeverthThe interaction of peptide drugs with PLGA can improve the encapsulation efficiency of those drugs, although such interactions can also cause problems during drug release from microspheres . An initThe prevention of adsorption of exenatide to PLGA by various additives was evaluated in aqueous solution at pH 4.5. Proline, lysine , and polProline and lysine were effective in preventing exenatide from adsorbing to PLGA, whereas phenylalanine was ineffective. In the FT-IR spectra of exenatide mixtures with amino acids obtained by lyophilization, proline and lysine showed shifts in the amide peak due to interaction with exenatide, but phenylalanine showed no significant change . ThroughAs mentioned above, this adsorption is a very important factor that greatly affects the stability of the peptide during the manufacturing process and drug release ,58. DuriAll PLGA-blended amphipathic additives tested had no effect of inhibiting the adsorption of exenatide to PLGA . There wBased on the results of the above various stability evaluations of exenatide in aqueous solution, we selected the additives to be added to each phase of the double emulsion and observed how they affected the final microsphere. Poloxamer 188, sucrose, proline, and phenylalanine were added separately or in combination to the inner water phase of pH 4.5 in consideration of water-induced instability, lyophilization stability, desirable release behaviors, and W/O interface stability, respectively . Lysine In this study, the volume mean diameter (VMD) and span value of exenatide-loaded PLGA microsphere (ELPM) were used to evaluate the particle size and its distribution. The VMD and span value of ELPM5 with lysine in the outer aqueous phase were smaller than that of ELPM1, which means the addition of lysine into the outer aqueous phase of the double emulsion resulted in a reduced particle size with a narrow distribution . As discParticle size and surface morphology are important properties of microspheres and can affect degradation rates, drug loading, and the initial burst release of a drug. In the following section, we will address these correlations in detail for a discussion of the consequences of encapsulation efficiency (EE) and in vitro release.Comparing ELPM1 with ELPM2 and ELPM3, it was revealed that the addition of sucrose and proline to the inner aqueous phase resulted in a significant increase in EE. It is estimated that sucrose and proline can contribute to stabilization of exenatide in the internal water phase during the preparation process, as predicted by the stability evaluation results. In contrast, the initial burst release (IBR) of ELPM2 and ELPM3 increased slightly compared to ELPM1. This might be due to the higher osmotic pressure in the inner aqueous phase, as induced by the addition of sucorose or proline, enhancing the leakage of exenatide to the outer phase during the preparation process . The addELPM5 with lysine in the outer water phase showed a desirable effect of increasing EE and decreasing IBR despite decreasing the particle size. In particular, moisture sorption was reduced compared to ELPM1, as shown by the result of the water vapor sorption analysis . This reInterestingly, ELPM3, ELPM5, and ELPM6 showed a decrease in lag time during drug release compare to ELPM 1, ELPM 2, and ELPM4. In order to clarify this difference, a representative comparison of ELPM2 and ELPM6 will be given. A typical biphasic release pattern was observed for ELPM 2, with an initial burst release followed by a more controlled secondary phase. However, the amount of exenatide release for ELPM2 was less than 10% from 3 to 17 days in the secondary phase. The final accumulated release percentage after 24 days was 72%, which indicates the incomplete release or destabilization of exenatide. Generally, ionic interaction may occur between peptide and carboxylic acid end-groups in PLGA, then non-specific adsorption on the degrading PLGA surface, covalent/non-covalent aggregation, and denaturation of peptide can be induced when the PLGA starts to degrade. These factors can cause the incomplete release of the drug from the PLGA microsphere. In addition, chemical degradation, such as acylation and deamination, could also occur during release. It is assumed that these factors are not controlled by the addition of sucrose alone, resulting in an undesirable release pattern. On the other hand, a typical triphasic release pattern was observed for ELPM6. After the initial burst release, the drug was released constantly for 24 days and the final accumulated release percentage after 24 days was 91%. The accelerated drug release in the third phase can be caused by a dramatic change of the conditions for the mass transport processes when the drug is still present as the original state without aggregation and/or destabilization by interaction with PLGA and the PLGA polymeric structure of the system becomes unstable and the macromolecular network breaks down. This in vitro release result is in good agreement with the results of the adsorption experiments and shows that proline and lysine can actually reduce the adsorption to PLGA and ionic interaction with PLGA during drug release at the inside and outside surfaces of the microsphere, respectively, hence improving the release profile through the stabilization of exnatide in the environment where release occurs. The decrease in EE of ELPM6 was expected compared to ELPM5 because sucrose and proline were added in the inner aqueous phase, and they can decrease EE due to the internal osmotic pressure. Interestingly, EE of ELPM6 did not decrease but was rather higher than that of ELPM5. The higher EE of ELPM6 than ELPM5 may be due to more destabilization during the microsphere preparation process because no additives with a protective effect were added to the inner aqueous phase of ELPM5. Moreover, IBR of ELPM6 was not only reduced compared to ELPM5, but exenatide was preferably released by more than 90% in 4 weeks due to the significantly reduced lag time. This result for ELPM6 shows that the positive role of lysine to the surface of PLGA microspheres, as shown in SEM images, can improve effectively the encapsulation of exenatide into the microsphere during the preparation process and the release behavior during the release period. As described in the adsorption test section, this positive effect of lysine may be due to the two mechanisms of not only molecular interaction with exenatide but also the role as a barrier on the surface of PLGA. Therefore, it was revealed obviously that the addition of proline to the internal water and the addition of lysine to the external water had a synergistic effect on the modification to a more desirable sustained release profile.gT) decreased to 38.4 °C from 41.6 °C of ELPM6  spectroscopy to examine the changes in secondary structure in aqueous solution compared to unprocessed exenatide ,18. The −1 in the amide I region is assigned to α-helix, and two peaks at 1692 cm−1 and 1629 cm−1 are identified as an intermolecular β-sheet [To confirm the CD results and observe differences in the secondary structure in a solid state, second derivative FT-IR spectra were also obtained for unprocessed exenatide powder and exenatide extracted and lyophilized from ELPM1 and ELPM6 microspheres b. The pe β-sheet ,20,31. TIn this study, we determined the effects of various additives, such as carbohydrates, amino acids, and surfactants, on the stability of exenatide in aqueous solutions, at water/DCM interfaces, and during freeze-thawing and freeze-drying procedures. Furthermore, we also examined the effects of additives on the adsorption of exenatide to the PLGA polymer. Stability data were well correlated to the EE of microspheres and the release behavior of exenatide from microspheres. Our selected formulation used a combination of additives based on the stability data and showed synergic improvement of EE and IBR. Consequently, this study demonstrates that the evaluation of various stability at the preformulation step is important in the development of PLGA microsphere injections containing peptides and proteins. This type of study can help to develop a formulation with a desirable quality of injectable PLGA microspheres for sustained release by predicting the stability and performance of the final formulation as well as stability issues in the manufacturing process.


By proteomics based mass spectrometry we observed a high abundance of agalactosylated IgG1 Fc-glycans in ASS/IIM patients (n = 44) compared to healthy age matched controls (n = 24). Using intra-individual normalization of the main agalactosylated glycan (FA2) of IgG1 vs FA2-IgG2, ASS/IIM and controls were distinguished with an area under the curve (AUC) of 79 ± 6%. For Jo1+ patients (n = 19) the AUCs went up to 88 ± 6%. Bisected and afucosylated Fc-glycans were significantly lower in Jo1+ compared to Jo1− patients. Anti-Jo1-IgG enriched from eleven patients contained even significantly lower abundances of bisected, afucosylated and galactosylated forms compared to matched total-IgG. ASS and ILD diagnosis, as well as lysozyme and thrombospondin correlated with Jo1+ characteristic Fc-glycan features. These results suggest that the anti-Jo1+ patient Fc-glycan profile contains phenotype specific features which may underlie the pathogenic role of Jo1 autoantibodies.IgG Fc-glycans affect IgG function and are altered in autoimmune diseases and autoantibodies. Anti-histidyl tRNA synthetase autoantibodies (anti-Jo1) are frequent in patients with idiopathic inflammatory myopathies (IIM) and anti-synthetase syndrome (ASS) with associated interstitial lung disease (ILD). Thus, we hypothesized that the total-IgG Fc-glycans from Jo1 Extra-muscular organs are commonly affected such as skin, joints and lung, the latter often as interstitial lung disease (ILD), being a major cause of morbidity and mortality3. Collectively, this clinical entity is termed anti-synthetase syndrome (ASS)3. Not all clinical settings coexist in the same patient and at the same time. However, up to 90% of patients with IIM and ILD present anti-Jo1 autoantibodies4.Autoantibodies are frequently found in patients with IIM, the most common being anti-Jo1 antibodies targeting histidyl transfer RNA synthetase (HisRS aka Jo1), which is an enzyme belonging to the family of aminoacyl-tRNA synthetases (aaRS). To date, HisRS and seven additional aaRS are recognized IIM-specific autoantigens. Autoantibodies targeting this family of enzymes  are, mainly monospecific, and associated with specific clinical manifestations: myositis, ILD, arthritis, Raynaud’s phenomenon, skin rash in the form of mechanic´s hands and fever6; and anti-Jo1 positive serum induces interferon (IFN) production from plasmacytoid dendritic cells7. However, direct evidence that anti-Jo1 autoantibodies cause or contribute to ASS/IIM pathogenesis is still lacking. One possible mechanism to explore is the effect of IgG Fc-glycans  response in early rheumatoid arthritis (RA) patients10. Notably, Fc-glycans from anti-citrullinated protein autoantibodies 12, are more agalactosylated and lack sialic acid residues, compared to non-ACPA IgG15. A significant decline in Fc-galactosylation and afucosylation of ACPAs has been reported prior to RA onset, which could suggest that Fc-glycosylation is a disease-driving mechanism16. In patients with IIM, more agalactosylated forms of IgG have likewise been found compared to healthy siblings and age/sex matched controls17. Together, these findings indicate the importance of antibody Fc-glycosylation for mediating immune regulating response and thus a potential role in pathogenicity of autoimmune disorders.Accumulated evidence suggests that anti-Jo1 autoantibodies may be involved in the pathogenesis of ASS and/or IIM (ASS/IIM): serum levels of anti-Jo1 autoantibodies vary with disease activity+ and anti-Jo1− ASS/IIM patients. The Fc-glycan profiles were correlated with clinical manifestations and protein information (which was obtained by simultaneous proteomics screening of the IgG enriched samples).To better understand the potential role of anti-Jo1 autoantibodies in IIM pathogenesis we investigated the Fc-glycans of these autoantibodies as well as total IgG from anti-Jo1+) and 25 Jo1 negative (Jo1−). For seven Jo1− patients we had access to enriched sera IgG from two time points, , Supplemental Table + patients, anti-Jo1 auto-immune IgG was further enriched from the total serum IgG from respective patient using either pools from several sampling time points per patient (n = 8), or from one sampling time point (n = 1). Furthermore, we had access to anti-Jo1 specific IgG from two additional patients both sampled at one occasion. For statistical comparisons, the patients were sub-grouped according to ASS and/or IIM (ASS/IIM), ILD and/or ASS (ILD/ASS), Jo1− and Jo1+, respectively  and 44 patients participated in the study. Of these patients, 15 were diagnosed with IIM but not ASS, 12 were diagnosed with ASS and IIM, one patient was diagnosed with ILD and IIM and 16 patients were diagnosed with ILD, ASS and IIM, (Table + patients (84%) was significantly higher than in Jo1− . A larger  percentage of Jo1+ patients (58%) were diagnosed with arthritis compared to Jo1− patients (28%).Clinical characteristics of ASS/IIM patients are summarized in Table Efficient purification of total IgG and anti-Jo1-IgG was confirmed by well-defined protein gel bands localized in the antibody heavy and light chain regions of total IgG and anti-Jo1-IgG  in serum and IgG percentage among total serum proteins (median < 16.6 mg/mL) at first available sample was similar in patients and controls  and twelve from IgG3/4 were quantified. The glycoform abundances were normalized to total content (100%) of Fc-glycosylated IgG1, total content (100%) of Fc-glycosylated IgG2/(3) and total content (100%) of Fc-glycosylated IgG4/(3) peptides, respectively. In Supplementary Table 1 was altered with half of the glycans being significantly different (p < 0.05). Of these significant glycans seven (out of nine) indicated a decrease in galactosylation. In contrast, the galactosylation status of IgG2/(3) and of IgG3/4 remained relatively stable. For example, compared to the main agalactosylated form FA2 , FA2 of IgG2/(3) and FA2 of IgG3/4 were not significantly different between the controls and the ASS/IIM patients , Supplementary Table 18, we tested if the unchanged FA2 of IgG2/(3) (FA2_2) could be used as an intra-individual control of the change in FA2 of IgG1 (FA2_1). Thus, we used log(FA2_1/FA2_2), and tested how well this factor could distinguish patients with ASS/IIM compared to controls. In terms of significance, log(FA2_1/FA2_2) was significantly different  as well as FA2G1 and FA2G2 (decreasing), the most prominent differences in the Fc-glycan profiles between Jo1+ and Jo1− patients were observed in significantly lower abundances of bisected (6 out of 16) and afucosylated (2 out of 7) glycopeptides, Supplementary Table 1 specific with particularly the bisected and galactosylated forms of IgG1 and IgG2  and afucosylated (p = 4.8E-2) forms (Table 2/(3) glycans (p = 9.7E-4) and afucosylated IgG1 glycans (p = 3.3E-2)  in one corner of the 3D plot . The Jo1+ patients total-IgG (black circles) are closest in space to the specific anti-Jo1-IgG (red circles). Inversely, the Jo1− patients total-IgG (yellow circles) and control total-IgG (blue circles) are furthest away from the anti-Jo1-IgG cluster.In a next step the Fc-glycans from the disease specific anti-Jo1 autoimmune reactive IgG (enriched from the IgG from the Jo1 positive patients) were interrogated and compared intra-individually to the IgG prior to anti-Jo1 enrichment, (“PE” in Table ls Table  and 4. I+ patients (compared to Jo1− patients). For details of the ASS/IIM vs control analysis, see Supplementary material.Multivariate statistical modelling (MVA) was done to interrogate how well features within the Fc-glycan profile would correlate with clinical and diagnostic patient information and protein data obtained from the IgG enriched samples. Two MVA models were created and assessed to distinguish (1) ASS/IIM (compared to controls) and (2) Jo12 = 0.53, 8 components) of the ASS/IIM patient data is presented. Noteworthy, a majority of the Jo1+ patients (dark circles) cluster in one part of the plot. This clustering indicates that there are features within the model that are similar for these patients and different from the Jo1− patient subgroup (white circles). However, there are also some overlap between the two subgroups. Noteworthy, these “outliers” are Jo1− patients with ILD symptoms. To distinguish which factors most prominently separated the Jo1+ and Jo1− patient, an OPLS-DA model of the data was created  and in Fig. + patients) ranked as high contributing factors in distinguishing Jo1+ patients from Jo1− patients. Other prominent (Jo1+ positively correlating) factors were clinical information such as ASS, ILD, MSA and higher MMT8 as well as the agalactosylation status factor (log[FA2_1/FA2_2]). Noteworthy, of the top ten positively Jo1+ correlating factors several were proteins . Additionally, the conserved lambda chain 2, one kappa variable chain, complement factor H related protein and platelet factor 4 correlated with Jo1+ status. For more details, see Supplementary Table + and Jo1− patient groups (p = 4.2E-6). To validate the model further, we used the remaining patient data from other measured patient time points (0.5–14 years from time point 1), and treated these patient profiles as unknowns. The validation cohort  were even more pronounced in anti-Jo1 specific enriched IgG.This study confirms that the Fc-glycan profile of IgG20. Furthermore, a recent study has connected Fc-glycosylation regulation with the axis of IL-23 and TH17-cell activity and suggest that this is a determining factor for autoimmune disease onset21. The Fc-galactosylation status of IgG can regulate binding to complement 1q factor and complement-dependent toxicity (CDC), thereby influencing the inflammatory status. Furthermore, sialylated Fc-glycans , have been directly linked to anti-inflammatory activation via SIGN receptor interactions, and reduce CDC efficacy23.It is well known that Fc-glycans contain less galactosylated epitopes in other autoimmune disorder and that these correlate with disease activity and severity1 specific we tested if the Fc-galactosylation status of IgG2 could serve as an internal control used to intra-individually correct for other factors that are also know to affect the galactosylation status of the N-glycan profile such as age, gender and heritability24. Hence, by using the factor log(FA2_1/FA2_2), ASS/IIM and controls were distinguished with an AUC of 79 ± 6%. This is an improvement to other Fc-galactosylation status measuring factors such as log(FA2/FA2G2) of IgG1, which on this data generated an AUC of 71 ± 6% and log(FA2/(FA2G1 + FA2G2) of IgG1 which on this data gave an AUC of 73 ± 6%, respectively25. From investigating the patients with most sampling time points, it was also evident that the log(FA2_1/FA2_2) factor is more stable than log(FA2/FA2G2), Supplemental Fig. 1 and of IgG2 have been shown to be skewed in RA10. Noteworthy, both the subgroup of Jo1+ patients and the subgroup of patients diagnosed with ILD/ASS further improved the sensitivity of the analysis  in ASS/IIM patients is IgG26 and lung cancer patients display a high degree of Fc-agalactosylation which is inversely correlated with good diagnostic performance27. Furthermore, note that RA often is affecting the lungs28.Our hypothesis is that the agalactosylated features of ASS/IIM IgG (and anti-Jo1-IgG) are enhanced due to underlying lung disease related mechanisms in these patients. This hypothesis is in line with a growing body of research that indicate a possible immunological role of IgG Fc-glycans in the respiratory tract. For example, the Fc-galactosylation in chronic lung disease (sarcoidosis and severe asthma) is skewed+ and in the ASS/ILD subgroup, the lower abundance of bisected- and core afucosylated Fc-glycans is more Jo1+ phenotype explicit. This hypothesis was further strengthened by analysis of Jo1 specific IgG which contained even lower abundance of bisected- and core afucosylated Fc-glycans than total IgG from the same patients. The profiling of IgG Fc-glycans of specific disease autoantibody reactivities is well characterized in ACPA from RA patients15. Noteworthy, anti-Jo1-IgG and ACPA Fc-glycan profiles have several similarities. In addition to higher content of agalactosylated epitopes ACPA-IgG1 contains a lower abundance of afucosylated and bisected forms. This might indicate that IgG-Fc-regulatory properties of ACPA and anti-Jo1 are similar and potentially consistent with other autoantibodies. Both bisected- and core afucosylated Fc-glycans increase IgG affinity to FcγRII and III receptors, potentially resulting in more potent antibody dependent cell-mediated cytotoxicity (ADCC)30. Thus, lower abundance of these epitopes might indicate an inverse effect.In contrast to the galactosylation status that was more prominently skewed both in Jo1+ Fc-glycan profile correlate with clinical manifestations and proteomic information of IgG chain distributions and trace proteins (measured in the IgG enriched samples), MVA-analyses were performed. As expected (and in line with the hypothesis that the anti-Jo1 Fc-glycan characteristic features are inflammatory regulating), several of the trace proteins found to positively correlate with the Jo1+ patients are involved in inflammatory processes  and the log Σ[B] data correlated weakly but significantly  with accessible C reactive Protein (CRP) data linked to 70 of the measuring time points, (from 23 of the Jo1− and of the 17 Jo1+ patients). However, in contrast to the Fc-glycan profile, the CRP data was not significant between the two ASS/IIM phenotypes (p = 0.8), thus indicating that the Fc-glycan profile give more precise information on the underlying immunological patient status.To further evaluate and test how the anti-Jo1In conclusion, our study shows that both total IgG and specifically anti-Jo1-IgG from ASS/IIM patients display IgG Fc-glycans that comprise features associated with pro-inflammation , which were also overrepresented in patients diagnosed with ILD/ASS. Furthermore, the lower abundance in bisected and afucosylated forms appears to be directly linked to anti-Jo1 autoimmune IgG. Functional studies with glycan manipulation recurring to anti-Jo1-IgG (and other autoantibodies) will shed insights into the role of these Fc-glycan aberrations in anti-Jo1 autoantibodies.36 criteria for definite, probable or possible polymyositis (PM) or dermatomyositis (DM). Inclusion Body Myositis (IBM) was diagnosed according to Griggs criteria37. Because samples were collected between 1996 and 2016, the new EULAR/ACR classification criteria for adult and juvenile IIM had not been approved and therefore the old classification criteria was applied38. The diagnosis of ASS was based on the presence of aaRS antibodies, plus one of the following features: ILD, myositis, arthritis, Raynaud’s phenomenon, fever, or mechanic hands39. Clinical characteristics of ASS/IIM patients are summarized in Table IIM patients (n = 44) fulfilled the Bohan and Peter+ n = 19; Jo1− n = 25, and 24 age/sex-matched healthy controls (HC) was isolated as previously described40. Longitudinal sera samples were available from eleven Jo1+ (i.e. T1–T10) and seven Jo1− (i.e. T1–T2) patients,  was available at one time point for isolation of anti-Jo1-IgG. For details, see Supplemental Fig. Total IgG from serum 0.35–1.5 mL) of 44 ASS/IIM myositis patients, Jo1.5 mL of 25. Briefly, ten µg of IgG/sample were dissolved to a final volume of 70 µL with a final concentration of 50 mM ammonium bicarbonate (pH 8). The protein was reduced  and alkylated . Trypsin was added in a ratio of 1:50 (enzyme:protein) for overnight digestion at 37 °C. Peptides were then desalted using C18 HyperSep Filer Plates . For peptide elution, 60% acetonitrile in 0.1% formic acid was used. Samples were dried using SpeedVac and stored at −20 °C until LC-MS/MS analysis. Samples were randomized both during the sample work up and during the LC-MS/MS analyses. One microgram of digest/sample was analysed using an UltiMate 3000 system connected to an Elite Orbitrap mass spectrometer (both Thermo Fisher Scientific). Reversed phase nano-LC-separation of the peptides was performed on a 15 cm long EASY spray column . LC-gradient and MS conditions are described in the supplementary information. Information on run-to-run variation  and from variation between IgG digests are given in Supplemental Table Samples were digested with trypsin in duplicates according to previously described protocols41, and as previously described25, glycopeptide ion abundances were integrated and quantified in a label free manner. The program was set to detect and quantify doubly and triply charged ions from Fc-glycopeptides  as well as triply and quadruply charged ions from  using their accurate monoisotopic masses  and within a ± 2 min interval around expected retention times. Note that IgG3 occurs as both EEQFNSTFR and EEQYNSTFR, with EEQYNSTFR being less frequent in Caucasians (which were predominant in this study)43. In total 63 glycopeptide variants were screened for  are shown in Supplementary Fig. 1, total content (100%) of Fc-glycosylated IgG2/(3) and total content (100%) of Fc-glycosylated IgG4/(3) peptides, respectively. Glycoforms detected at levels constituting less than 0.05% of the total IgG Fc-glycan profiles were discarded. The sub-grouping of respective glycan feature  was also performed on the relative distribution values (%) obtained by IgG1 and IgG2, respectively.Using in-house developed software (Quanti)25. For details, see supplemental material.In addition to Fc-glycan profiling, samples were simultaneously subjected to regular peptide sequencing of MS/MS spectraUnivariate analyses on glycopeptides were performed using two tailed Student’s t-test  or paired t-test. P-values were FDR corrected according total number of comparisons (n = 754). Prism, GraphPad Software was used for ROC-curve and linear regression analysis as well as for analysis of other factors via 1) Wilcoxon matched-pairs signed rank test, 2) Fisher’s exact test or Chi-square test and 3) Kruskal-Wallis or Mann-Whitney tests. Multivariate modelling using Principal Component Analysis (PCA) and Orthogonal projections to latent structures discrimination analysis (OPLS-DA) was performed using SIMCA 15.0. For details, see Supplementary Material.Supplementary information


Piperazine-based compounds bearing two phenylboronic acid or two benzoxaborole groups (PBPA and PBBB) were applied as dopamine receptors in polymeric membranes (PVC/DOS) of ion-selective electrodes. The potentiometric sensitivity and selectivity of the sensors towards dopamine were evaluated and compared with the results obtained for 2-phenylethylamine. Since the developed electrodes displayed strong interference from 2-phenylethylamine, single-molecule geometry optimizations were performed using the density functional theory (DFT) method in order to investigate the origin of dopamine/2-phenylethylamine selectivity. The results indicated that phenylboronic acid and benzoxaborole receptors bind dopamine mainly through the dative B–N bond (like 2-phenylethylamine) and the potentiometric selectivity is mainly governed by the higher lipophilicity of 2-phenylethylamine. Nowadays, boronic acids are important group of compounds widely used in synthetic and medicinal chemistry, i.e., Suzuki-Miyaura cross-coupling reactions , protectpKa depression. Consequently, benzoxaboroles are able to bind diols under neutral physiological conditions. Another important class of diol receptors are ortho-substituted aminomethyl arylboronic acids  3−/4− reporter couple was added  borate (KTFPB) were obtained from Fluka (Selectophore). Freshly distilled tetrahydrofuran from Fluka was used as a solvent for the membrane components. Dopamine, 2-phenylethylamine and acetylcholine were purchased from Sigma-Aldrich. Organoboron receptors 4-octyloxyphenylboronic acid (OPBA) ,54, 3,3′The methods of membranes and electrodes preparation were the same as for classical liquid-membrane ion-selective electrodes . The mem3COOLi 1 M/sample solution//membrane//internal filling solution; AgCl, Ag.All potentiometric measurements were carried out with cells of the following type: Ag, AgCl; KCl 1 M/CH−6–10−2 M). Potentiometric selectivity coefficients  of the polymeric membranes were determined by the separate solution method (SSM) using 0.01 M solutions. The activities of anions in aqueous solutions were calculated according to the Debye–Hückel approximation.A potentiometric multiplexer  was used for the EMF measurements. The calibration curves of the electrodes were examined by measuring the EMFs in buffered solution (pH 4.5), increasing the concentration of the neurotransmitter in steps of 0.5 log c  arene derivative-bearing boronic acid substituent . In bothNext, single-molecule theoretical calculations were performed in order to elucidate the origin of 2-phenylethylamine/dopamine selectivity. The results revealed that dopamine complexation through the dative B–N bond is energetically more favored than the formation of catechol ester in the case of bifunctional receptors (PBPA and PBBB), whereas the catechol binding is more preferred for OPBA. Therefore, similar interactions can be considered when discussing the selectivity of membranes, containing PBPA or PBBB, toward dopamine and 2-phenylethylamine. Despite the fact that the calculations showed higher affinities of bifunctional receptors to 2-phenylethylamine, the determined potentiometric selectivity can be rationalized in terms of the different lipophilic characters of guest molecules.To conclude, the results obtained clearly demonstrated that, taking into account the lipophilicity of the neurotransmitters, the synthesis of an organoboron derivative strongly favoring dopamine binding is required to develop potentiometric sensors that can exhibit the desired discrimination between dopamine and 2-phenylethylamine.


Taking advantage of the high functionalization capacity of poly (PVA), bead-free homogeneous nanofibrous mats were produced. The addition of functional groups by means of grafting strategies such as the sulfonation and the addition of nanoparticles such as graphene oxide (GO) were considered to bring new features to PVA. Two series of sulfonated and nonsulfonated composite nanofibers, with different compositions of GO, were prepared by electrospinning. The use of sulfosuccinic acid (SSA) allowed crosslinked and functionalized mats with controlled size and morphology to be obtained. The functionalization of the main chain of the PVA and the determination of the optimum composition of GO were analyzed in terms of the nanofibrous morphology, the chemical structure, the thermal properties, and conductivity. The crosslinking and the sulfonation treatment decreased the average fiber diameter of the nanofibers, which were electrical insulators regardless of the composition. The addition of small amounts of GO contributed to the retention of humidity, which significantly increased the proton conductivity. Although the single sulfonation of the polymer matrix produced a decrease in the proton conductivity, the combination of the sulfonation, the crosslinking, and the addition of GO enhanced the proton conductivity. The proposed nanofibers can be considered as good candidates for being exploited as valuable components for ionic polyelectrolyte membranes. The development of nanofibrous polyelectrolyte structures is a topic of interest in different fields of application, such as the preparation of proton exchange membranes for fuel cells , electroElectrospinning is one of the main approaches to obtain functionalized nanofibrous mats, including homopolymers, blends, or composites ,6,7. GivMany materials have been considered for the production of nanofibrous polyelectrolytes, such as polyvinylidenefluoride (PVDF) , polystyRegarding its application, PVA requires functionalization to incorporate specific ionic domains to induce polyelectrolyte performance. Different strategies, such as copolymerization, polymer grafting, or combining with inorganic particles to create composite materials, have been proposed . The sulGraphene oxide (GO) has received considerable interest because of its unique two-dimensional structure with excellent dimensional, chemical, thermal and mechanical stability, low permeability, low cost, surface functionality, and minimum thickness . GO can In this context, raw and sulfonated PVA combined with graphene oxide (GO) and crosslinked with sulfosuccinic acid (SSA) have been proposed in this work for developing functionalized nanofibers using the electrospinning technique ,47,48,49wM 130,000 g·mol−1) was supplied by Sigma Aldrich . The sulfonation of poly (PVA) was performed with the use of 1,3-propanesultone 97%, purchased from Acros Organics . The sulfonation reaction was carried out in two stages that have been described elsewhere [The poly (PVA)  was added to the dissolution and homogenized under stirring for 24 h [A main aqueous solution of 8%wt of PVA in 50 mL of deionized water was prepared and stirred at 90 °C for 6 h in a reflux system. After that, a 0.25%wtsolution ,52. To afor 24 h ,37.PVA in 10 mL of deionized water to achieve a good dispersion. These GO solutions were sonicated for 30 min in a ATM40-2LCD ultrasonic bath . Subsequently, the GO solutions were added to the PVA dissolution and stirred for 30 min. Taking into account the addition of 10 mL of the GO solution to the mixture of PVA/Triton®/SSA, the concentration of the latter was adjusted to achieve the desired final concentrations. The same process was repeated for the sulfonated PVA (SPVA), with equal concentrations of Triton®, SSA, and GO, as defined for PVA.Simultaneously, the GO solutions were prepared with a concentration of 0.25, 0.50, 0.75, and 1.00%wt−1 and a voltage ranging from 16 to 18 kV. The time of electrospinning varied as a function of the flow rate, in order to obtain the same quantity of electrospun material in all the samples, and ranged between 2 and 3 h. A nanofibrous mat of 4 × 4 cm2 surface with a thickness of approximately 20 μm was obtained in all cases. The electrospinning process was carried out by means of a Fluidnatek LE-10 equipment . The solutions cited in the previous section were taken and each filled a plastic syringe (5 mL) with a diameter of 11.99 mm . The distance from the tip of the needle to the flat collector was set at 17 cm. The electrospinning was carried out with a variable feeding rate between 300 and 800 μL·h® reinforced with glass fiber and, in turn, introduced between two metallic discs to ensure the crosslinking reaction in a planar arrangement. The entire assembly was then subjected to 110 °C for 2 h in a UT-6060 convection oven . Finally, the crosslinked nanofibers were stored in zip bags and placed into a desiccator at room temperature for further analyses.The PVA/SSA and SPVA/SSA-based nanofibers were subjected to a crosslinking reaction to provide sulfonic functional groups and bring stability. For this purpose, the nanofibrous mats were placed between two sheets of TeflonThe field-emission scanning electron microscopy (FE-SEM) was performed by means of a Zeiss Ultra 55 microscope . Nanofibers were mounted onto metallic sample holders and sputter coated with a K950 Sputter Coater device  under an inert atmosphere and vacuum conditions. Samples were platinum coated for 15 s. The micrographs were taken with a working distance of 7 mm and a voltage of 2 kV at different degrees of magnification. The diameter of the nanofibers was obtained from the average of 100 measurements by means of the Image J software. Three different electron microscopy images representative of different regions of the nanofibrous mat were analyzed.−1 and a resolution of 4 cm−1 along 64 scans. Five assays were performed from different points in the same sample and the mean spectra were taken as representative.The Fourier-transformed infrared spectroscopy (FT-IR) was performed in a Nicolet 5700 spectrometer , equipped with a total attenuated reflectance (ATR) module. The FT-IR spectrum was taken between 4000 and 400 cm−1. The tests were carried out under an oxidative atmosphere with an O2 feeding rate of 50 mL·min−1. The samples were studied in triplicates and the averages and deviations were taken as representative values.The thermogravimetric assays (TGA) were performed by means of a TGA 851 analyzer . The samples (5–8 mg) were introduced into 70 μL alumina capsules and subjected to a dynamic assay, based on a heating segment from 25 °C to 800 °C with a heating rate of 10 °C·mine setup  equipped with a refrigeration system. The samples (3–5 mg) were placed into 40 μL aluminum crucibles. The method of analysis consisted of different consecutive heating/cooling/heating segments between 50 °C and 250 °C with a heating/cooling rate of 10 °C·min−1. The assays were carried out under an inert atmosphere of N2 at a flow rate of 50 mL·min−1. The samples were studied in triplicates and the averages and deviations were taken as representative values.The differential scanning calorimetry (DSC) was performed in a DSC 822protσ) was measured by means of a dielectric thermal analyzer (DETA) composed of an alpha mainframe frequency analyzer in conjunction with a Concept 40 active cell . The response was measured in the frequency range from 10−2 to 107 Hz at room temperature (25 °C). The sample electrode assembly (SEA) consisted of two stainless steel electrodes filled with the polymer. The diameter of the electrodes was 20 mm and the thickness was kept around 30 μm. Preliminarily, the polyelectrolytes were hydrated in ultra-pure water for 24 h. The protσ (S·cm−1) was calculated according to Equation 1:l is the thickness of the conducting nanofibrous membranes in cm, A is the area of the electrode in contact with the sample in cm2, and 0R is the proton impedance taken from the Bode plot at high frequencies in ohms (Ω) [The proton conductivity (ohms (Ω) .elecσ) was measured at room temperature using the same equipment, at low frequencies, where the measured real part of the conductivity (σ’) reaches a plateau that is strictly correlated to the direct current (DC) conductivity (0σ).The electrical conductivity , as previously described.The characterization of the stability of the nanofibers under simulated service conditions was assessed by means of the immersion in ultra-pure water, which can be correlated to the performance of these materials for application as polyelectrolytes in fuel cells . For thiIn this study, the effects of the addition of SSA to the PVA matrix, the crosslinking reaction, the sulfonation of the PVA (SPVA), and the combination with GO nanoparticles on the electrospun nanofibers—summarized in The surface morphology of the obtained PVA/SSA/GO and SPVA/SSA/GO nanofibers was characterized by means of field-emission scanning electron microscopy (FE-SEM). The acquired electronic micrographs, along with the fiber diameter histograms are gathered in In general, smooth bead-free electrospun nanofibers were obtained. The SPVA-based nanofibers showed histograms displaced towards lower values in comparison to those of PVA. The SPVA showed an average diameter around 200 nm while that of pure PVA nanofibers was around 350 nm. This effect can be correlated to the higher conductivity of the SPVA solution during electrospinning [For the crosslinked PVA/SSA and SPVA/SSA nanofibers, the average fiber diameter decreased by 10%. Crosslinking may have reduced the free volume inside the fibers and, subsequently, resulted in a more compact structure due to the intra- and inter-molecular chemical interactions between the PVA and the SSA ,56. Addi−1, associated with the stretching of the ‒OH groups of PVA. The peaks between 1660 and 1640 cm−1 are correlated to the ‒OH bending of the H2O molecules retained in the material, due to its high hydrophilicity. The peaks between 2800 and 3000 cm−1 and between 1300 and 1500 cm−1 corresponded to the stretching and deformation vibrations of the ‒CH2‒ groups of the PVA molecule, respectively [−1 can be associated with the vibration stretching of the C‒O bonds of the PVA [−1 related to the presence of sulfonic groups (‒SO3H) was observed, confirming the sulfonation strategy for this material [Both PVA and SPVA-based nanofibers, revealed a wide absorption band between 3000 and 3400 cm the PVA . For thematerial .−1, associated with the carboxyl (‒COOH) and acetate (‒COO‒) groups, was observed. The stretching vibration of the O‒H bond of the carboxyl groups appeared between 2500 and 3000 cm−1. In addition, an increase in the peak intensity of the 1040 cm−1 signal associated with the stretching of the sulfonic groups (‒SO3H) was perceived [When the SSA was added to the nanofibers, an increase of the bands between 1780 and 1710 cmerceived . After c−1, assigned to the –OH group stretching vibrations, stretching vibrations of C=O, skeletal vibrations of nonoxidized graphite domains and C=C bonds, and to deformation vibrations of the O‒H and C‒O bonds, respectively [The GO spectrum was characterized by peaks at 3450, 1720, 1600, 1400, and 1100 cmectively . Given tThe thermal properties of the nanofibers were studied by means of differential scanning calorimetry (DSC), which brings valuable information about the microstructure and morphology of the polymer-based materials . The theThe pure PVA nanofibers showed an endothermic peak between 50 and 100 °C, associated with the evaporation of free water, followed by an endothermic peak at 229 °C, ascribed to the melting of crystalline domains. In the cooling scan, a crystallization peak was shown with its maximum located at 191 °C. Finally, when the thermal story and water content were removed, a melting peak was observed with its maximum at 220 °C. These results confirmed the typical semicrystalline morphology of the pure PVA .In contrast, the SPVA nanofibers revealed an amorphous morphology due the absence of melting and crystallization peaks. The hydrogen bonding interactions between the PVA molecules and the sulfonic groups may have prevented the PVA from crystallization . Indeed,The crosslinked nanofibers also revealed an amorphous morphology, as reported for PVA with concentrations of SSA greater than 15% ,36,61. FThe PVA/SSA/GO and SPVA/SSA/GO nanofibers showed the absence of melting and crystallization peaks in the cooling and heating scans, and therefore an amorphous morphology is expected for these materials ,36,61. OThe thermo-oxidative stability was further assessed by means of thermogravimetry (TGA). The thermogravimetric curves (TG) along with the first derivative thermogravimetric curve (DTG) obtained under an oxidative atmosphere for all the developed nanofibers are plotted in 3H groups.The neat PVA nanofibers underwent a first mass loss stage between 100 and 200 °C and comprised a gradual loss of free and bounded water, respectively. The second degradation stage, between 250 °C and 300 °C, was dominated by the cleavage reactions of the ‒OH groups of the PVA molecules. The thermogram of the SPVA nanofibers revealed an additional peak around 300 °C related to the decomposition of the grafted ‒SOThe crosslinked nanofibers showed thermo-oxidation behavior in multiple stages. The first stage with a mass loss of around 10% was observed from 50 to 100 °C. In the second stage, between 100 and 230 °C, a mass loss of 20% took place, attributed to bounded water release. In this case, water molecules are assumed to be bonded to the polymer chains in the available hydroxyl, sulfonic, and carboxylic groups by hydrogen bonding . The thiRegarding the PVA/SSA/GO and SPVA/SSA/GO nanofibers, the mass loss of the water release stage remained almost constant. However, the bound water release temperature increased as a function of the GO content. The functional groups of the GO nanoparticles available to interact with water molecules may have formed stronger hydrogen bonds, which may have inhibited the release of water.elecσ), as obtained by means of dielectric thermal analysis (DETA), is plotted in elecσ was considered at low frequencies, where the measured real part of the conductivity (σ’) reached a plateau that was strictly correlated to the DC conductivity (0σ). All the developed nanofibers revealed an elecσ in the range between 10−7 and 10−8 S·cm−1. Therefore, they can be considered as good insulators that prevent the flow of electrons through them, especially relevant for some polyelectrolyte applications.The electrical conductivity (protσ). Thus, the protσ will strongly depend on the degree of hydration of the material. For this reason, the nanofibers were measured after being subjected to a hydration process through immersion for 24 h in ultra-pure water. Afterwards, the effects of the sulfonation of the PVA, the addition of the SSA, as well as the incorporation of the GO with respect to the proton conductivity were studied. protσ values, as calculated by means of Equation (1) are shown in There are two main proton conduction pathways in the PVA/SSA and SPVA/SSA-based polyelectrolytes . On the protσ of the proposed PVA-based nanofibers must still be enhanced if compared to Nafion®-based nanofibers [protσ of the PVA/SSA/GO nanofibers may be comparable to that found for sulfonated poly(ether sulfone) (SPES) (9.3 × 10−4 S·cm−1) [−4 S·cm−1) [−3 S·cm−1) [−4 S·cm−1) [The nofibers ,62. Howe S·cm−1) , sulfona S·cm−1) , and sul S·cm−1) , all of protσ of the PVA/SSA/GO nanofibers progressively increased as a function of the GO content. In contrast, for SPVA/SSA/GO nanofibers, the protσ depended on the proportion of GO. The protσ decreased by the presence of 0.25% of GO but increased from 1.87 to 4.28 ×10−4 S·cm−1 when the proportion of GO increased from 0.25% to 0.75%. Finally, for 1.00% of GO it slightly decreased again. Given these results, the PVA-based crosslinked nanofibers combined with GO allowed better control of the proton conductivity. In general, it can be considered that they showed relatively high proton conductivity values, regardless of the small thickness of the nanofibrous membranes [The embranes ,64.The evaluation of the behavior when exposed to service conditions is essential for the corroboration of the suitability of a polymeric material for a given application ,66,67,68Both PVA and SPVA non-crosslinked nanofibers completely lost the nanofibrous morphology after immersion. The nanofibers swelled until coalescence, which would result in the disappearance of the nanoscaled structure. In contrast, the crosslinked nanofibers retained their fibrous morphology after the hydrothermal treatment in all cases. A general increase in the average fiber diameter was observed, caused by swelling due to water absorption. The largest increase in the fiber diameter was perceived for the PVA/SSA and SPVA/SSA nanofibers. However, the use of SPVA promoted a lower increase after immersion, which may be correlated to the lower availability of hydrophilic sites, mainly hydroxyl groups, for the incorporation of water into the structure . The preFunctionalized nanofibrous membranes based in poly (PVA) or sulfonated poly (SPVA) combined with graphene oxide (GO) were developed by means of electrospinning. The subsequent crosslinking reaction with sulfosuccinic acid (SSA) brought thermal and hydrothermal stability, as well as the required electrical insulator performance and proton conductivity, to the nanofibers, especially relevant for polyelectrolyte applications. A good interaction of the GO nanoparticles with the PVA/SSA and SPVA/SSA matrices was found. The GO contributed strongly to retain humidity, both as free and bounded water, essential to promote proton conductivity. The crosslinked nanofibrous membranes were stable during simulated service conditions. Although fiber diameter increased after immersion, the presence of GO and the use of SPVA contributed to higher diameter stability. Finally, the developed nanofibrous composite membranes can be considered as good candidates for being exploited as components for the preparation of ionic multilayered polyelectrolyte membranes.


O-glucoside and flavanols, suggests that non-Saccharomyces yeasts are able to provide increased levels of colour intensity and larger polymeric pigment ratios and polymerization indexes. The selection of non-Saccharomyces genera, in particular Lachancea thermotolerans and Schizosaccharomyces pombe in sequential fermentation, have provided experimental wines with increased fruity esters, as well as producing wines with potential pigment compositions, even though there is an important reduction of total anthocyanins.Anthocyanins in red grape musts may evolve during the winemaking process and wine aging for several different reasons; colour stability and evolution is a complex process that may depend on grape variety, winemaking technology, fermentative yeast selection, co-pigmentation phenomena and polymerization. The condensation of flavanols with anthocyanins may occur either with the flavylium ion or with the hemiacetal formation in order to produce oligomers and polymers. The kinetics of the reaction are enhanced by the presence of metabolic acetaldehyde, promoting the formation of pyranoanthocyanin-type dimers or flavanol-ethyl-anthocyanin structures. The experimental design carried out using white must corrected with the addition of malvidin-3- There are different factors that determine the colour evolution and colour stability of red wines, including grape variety as a sole source of anthocyanins ; practicSaccharomyces yeasts have been shown to produce a concentration of pyruvic acid and acetaldehyde that is different from Saccharomyces cerevisiae + (m/z) 639 and fragment ion(m/z)331 + (m/z) 517 and fragment ion with (m/z) 355 [O-glucoside-4-vinylphenol with molecular ion [M]+ (m/z) 609 was the only vinylphenolic pigment identified in the trials where Sc was used as pure culture and sequential fermentation. The concentration in the pure culture trials ranged between 0.25 and 0.35 mg/L, while the concentration in the trials with the fermentative strains Lt-Sc was between 0.18 and 0.22 mg/L  355  was idenm/z) 355 , the mal.22 mg/L . The ferO-glucoside-vinyl-catechin and malvidin-3-O-glucoside-vinyl-epicatechin with molecular ion [M]+ (m/z) 805 [O-(p-coumaroyl)-glucoside-8-ethyl-(epi) catechin with molecular ion [M]+ (m/z) 955 and fragment ions with (m/z) 803 and 647 [O-glucoside-epicatechin with molecular ion [M]+ (m/z) 781 and fragment ion (m/z) 619, malvidin-3-O-glucoside-4-ethyl-epicatechin with molecular ion [M]+ (m/z) 809, malvidin-3-O-glucoside-4-vinyl-epicatechin with molecular ion [M]+ (m/z) 805 and malvidin-3-O-glucoside-4-vinyl-diepicatechin with molecular ion [M]+ (m/z) 931 + (m/z) 805 [O-glucoside-(epi) catechin with molecular ion [M]+ (m/z) 781 and fragment ion (m/z) 619 [O-glucoside-4-vinyl-diepicatechin with molecular ion [M]+ (m/z) 931 + (m/z) 781 and fragment ion (m/z) 619, the flavanol pyranoanthocyanin dimer malvidin-3-O-glucoside-4-vinyl-epicatechin with molecular ion [M]+ (m/z) 805 and flavanol pyranoanthocyanin trimer malvidin-3-O-glucoside-4-vinyl-diepicatechin with molecular ion [M]+ (m/z) 931  × 100%; total phenolics (TPI) is the indeX obtained from multiplying A280 nm × 10 (dilution correction); chemical age (ChA) is eXpressed as the ratio of A520 nmSO2/A520 nmHCl; polymerization indeX (PI) obtained from ChA × 100; polymerized pigments (PP) as the absorption A520SO2, and; total pigments (TP) as the absorption A520HCl.The colour intensity (I) as well as colour shade (N), anthocyanin ionization degree (α), total phenolics (TPI), chemical age (ChA), polymerization indeX (PI), polymerized pigments (PP) and total pigments (TP) of red wine samples were determined through the use of a UV-visible spectrometer Unicamp™ UV4  using 1 mm and 1 cm path length cuvettes following the Somers and Evans  methodolP < 0.05.Means and standard deviations were calculated and differences eXamined using ANOVA and the least significant difference (LSD) test. All calculations were made using the PC Statgraphics v.XI software . Significance was set at Saccharomyces cerevisiae and Schizosaccharomyces pombe in sequential fermentation increased the production of acetaldehyde and pyruvic acid toward the end of the fermentation process, making possible the formation of stable pigments during this stage. Therefore, the colour of wines is a result of the concentration of monomeric pigments and the oligomeric fraction formed during fermentation. The different treatments, meaning the addition of flavanols to musts before fermentation, did not seem to have an influence on the metabolic pathways for fermentative volatile production for samples with the same fermentative conditions; most of the differences in fermentative volatiles found in wines were related to the yeasts used for fermentation. Less 2,3-butanediol and more fruity esters were formed when using non-Saccharomyces yeasts. Not only the aromatic profile, but also the colour observed in the wines changed as a result of the nature of the fermentative strains used, some of which may have had higher pigment adsorption on their cell wall structure.The use of (+)-catechin in musts produced a higher concentration of oligomeric pigments, most of which are linked to acetaldehyde. The use of both


Brucella strains. Individual and herd prevalence for BTB in cattle of 20.4% (95% CI 18%-23%) and 57.7% (95% CI 48%-66%), respectively, were observed in this study. The prevalence of brucellosis in cattle at individual and herd level was 1.9% (95% CI 1.2%-2.8%) and 9% (95% CI 4.5%-1.5%), respectively. Brucella pathogens were isolated from three cattle milk samples and were identified as B. abortus using Bruceladder® multiplex PCR and B. abortus biovar 1 by classical phenotyping. All small ruminants were seronegative to sRBT, two were positive to mRBT. A higher risk of BTB and brucellosis was observed in cattle in intensive livestock systems, in imported and crossed breeds and in animals from larger herds (>15). The three risk factors were usually present in the same herds, leading to higher transmission risk and persistence of both zoonoses. These results highlight the importance of implementing control strategies for both BTB and brucellosis to reduce productivity losses and the risk of transmission to humans. Prioritising control for BTB and brucellosis in intensive livestock production systems is essential for human and animal health.Bovine tuberculosis (BTB) and brucellosis are major endemic zoonoses in ruminants in Morocco that impact on both animal and human health. This study presents an assessment of the epidemiological and socioeconomic burden of bacterial zoonoses in Sidi Kacem Province in Northern Morocco from a cross-sectional survey of 125 cattle and/or small ruminant-owning households. In total, 1082 sheep and goats were examined from 81 households. The single intradermal comparative cervical test to screen for bovine tuberculosis was undertaken on 1194 cattle from 123 households and all cattle were blood sampled. Cattle and small ruminant sera were tested for brucellosis using the standard Rose Bengal Test (sRBT) and the modified Rose Bengal Test (mRBT). Bacteriology was performed on 21 milk samples obtained from cattle that were seropositive for brucellosis for isolation and phenotyping of circulating  Bovine tuberculosis (BTB) and brucellosis are bacterial zoonoses endemic in cattle, and brucellosis is endemic in small ruminants, in Morocco. These diseases are prioritized in Moroccan veterinary legislation ,2 but reMycobacterium bovis, a member of the Mycobacterium tuberculosis complex. Despite a host preference for cattle . Br. BrBrucesmission . BrucellB. abortus biovar 1 and 3 in the 1980s . Deampaigns , recent ampaigns . Thus, aBrucella strains isolated from the three Friesian/Holstein cows and belonging to the same herd in the irrigated zone were found to be B. abortus biovar 1, and phylogenomic studies (not described here) show that these strains have some homogeneity with Spanish strains. This is in line with a study conducted in the mid-1980s yielding 8 B. abortus biovar 1 and 28 B. abortus biovar 3 strains from 500 samples [B. abortus strains of African origin and found the 12 Moroccan strains to be identical to B. abortus biotypes isolated in Europe.Using these methods, individual apparent brucellosis prevalence found in this work in cattle (1.9%) is near the 2.1% reported for the national survey of 2010–2011. National statistics from 1982 to 1992 registered a mean herd prevalence ranging from 2.1% to 4.9%, which is lower than brucellosis herd prevalence (9%) found in the present study . A recen samples ,25. A suB. abortus infected cattle. These sheep could have developed antibodies but cleared the infection and hence be of negative infection status or alternatively be infected and a source of contagion to other ruminants or humans. A study performed in Northern Morocco and the Middle Atlas in 2013, investigated brucellosis among 23 sheep and goat herds using mRBT. The results of the study showed an individual and herd prevalence of 13.3% and 43% respectively. The high prevalence registered could be explained by the condition of animals sampled, which were exclusively aborting females, so abortion could be considered as a confounding factor for that study [B. abortus in sheep, which seems to be a very rare event even in areas free of B. melitensis and where cattle are infected by B. abortus [In this study only two sheep from two different herds in the irrigated area showed a positive mRBT result. The mRBT positive sheep were from a herd that had several cases of cattle brucellosis. Livestock from this village grazed on shared pasture and the sheep may have developed seropositivity due to contact with at study . Very li abortus . RegrettBrucella and Mycobacteria between animals. The higher stocking density, larger herd sizes, and propensity to buy in animals, together with higher calving frequency in intensive systems may also increase the risk of disease transmission. On the other hand, extensive systems, where animals are grazed in lower density on communal pasture, may have a lower risk of transmission because of the effect on the sun and heat on Brucella and Mycobacteria environmental contaminants [Consistent with a recent review , we obseaminants . The shiaminants ,66.B. abortus infection [As has been previously reported, Indigenous animals showed a lower risk of BTB,  comparednfection . In the nfection ,70, althnfection ,72. Low nfection , but in Based on the findings of this study we conclude that bovine tuberculosis and brucellosis are prevalent in Sidi Kacem. Considering the economic losses caused by BTB and brucellosis, in addition to their public health impact, additional efforts should be deployed to design an integrated control strategy. Test and slaughter has been shown to be the most efficient elimination strategy for BTB in several countries. Many factors contribute to the success of a control and elimination campaign. Trust between all stakeholders especially the farming industry, the government and the farmers are a very important component which contributed to the success of brucellosis and BTB control program in other contexts. Correct application of livestock biosecurity measures, early diagnosis of the disease, and application of movement restrictions also affect the success of a control campaign . HoweverThe best strategy for controlling brucellosis in Morocco would be conjunctival mass vaccination every two years just before the natural breeding season or immediately after calving/lambing/kidding. This is the best option as prevalence is high and the veterinary services are not able to apply individual tagging allowing vaccination of young replacements only . HoweverA BTB transmission model for Morocco indicated that BTB could be controlled within 25 years, if 50% of cattle were tested annually, and infected animals were slaughtered at an estimated cost of 1.55 billion Euros . Taking M. bovis in humans is required, as well as a calculation of the direct and indirect cost of a human TB case. Cross-sectoral socio-economic analysis of the cost of both diseases is needed. In addition, support from private industry (e.g. the milk industry) could sustain BTB and brucellosis control campaigns, as they will also benefit from the control measures. Novel methods for innovative financing should be examined to mobilize investment for interventions that contribute towards elimination of neglected zoonosis [As for other neglected zoonosis, evidence and advocacy is necessary to convince policy-makers and communities of the benefits of disease control ,83. The zoonosis ,85 and bThe present study confirms that there is added value in investigating multiple zoonoses simultaneously, especially for zoonoses with a reservoir overlap. Undertaking brucellosis and BTB screening in parallel and in multiple hosts is logistically and technically feasible. The added value of an integrated approach to epidemiological investigations on zoonoses has been demonstrated in Chad . MoroccoThe methodology of the study including the household questionnaire was reviewed and validated by international and national research expert partners within the ICONZ project. In addition, the Moroccan veterinary services (ONSSA) approved and granted authorization for the study. The purpose of the study was explained to the local authorities and to the farmers. The tests used for the screening of BTB and brucellosis are routinely used by the veterinary services of Morocco.S1 Dataset(XLSX)Click here for additional data file.S2 Dataset(XLSX)Click here for additional data file.S3 Dataset(XLSX)Click here for additional data file.


By using quantum chemical calculations, we demonstrated that the rate-limiting step is the formation of a hydroxide ion from a water molecule, which attacks the amino group that enters intramolecular Michael addition, giving rise to a pharmacologically inert aminochrome. We have shown that for dopamine stability on a time scale of days, it is essential that the pH value of the synaptic vesicle interior is acidic. The pathophysiologic correlates of the results are discussed in the context of Parkinson's disease as well as the pathology caused by long-term amphetamine and cocaine administration.We studied the reaction mechanism of dopamine autoxidation using quantum chemical methods. Unlike other biogenic amines important in the central nervous system, dopamine and noradrenaline are capable of undergoing a non-enzymatic autoxidative reaction giving rise to a superoxide anion that further decomposes to reactive oxygen species. The reaction in question, which takes place in an aqueous solution, is as such not limited to the mitochondrial membrane where scavenging enzymes such as catalase and superoxide dismutase are located. With the experimental rate constant of 0.147 s Dopaminergic neurons, that project to many central nervous system (CNS) areas, have been implicated in a myriad of neurologic diseases including Parkinson's disease, dementia with Lewy bodies and Alzheimer's disease, which are all caused by neurodegeneration  or diamine oxidases (DAO). Dopamine first reacts with oxygen, giving rise to the quinone form and superoxide anion, the latter further decomposing to various ROS. The quinone form is capable of intramolecular Michael addition (cyclization), yielding aminochrome, which at physiological conditions represents the rate-determining step. The aminochrome can further polymerize into dark neuromelanin polymer Free energy differences, associated with proton transfer from the ionizable group with a certain pKWe considered a few possible reaction mechanisms and the results are described below.2), and the other in the weak hydrogen bond involving the scissile C-H hydrogen atom. To prevent rupture of the hydrogen bonds in the geometry optimization process, both C-O and N-O distances were constrained to 3 Å for the reactants complex. For the products complex, consisting of one aminochrome molecule and two hydronium ions, all six O-H distances in both hydronium ions were constrained to 0.963 Å, as suggested by Kollman et al. , and the other in the weak hydrogen bond involving scissile C-H hydrogen atom. The products complex involved one aminochrome molecule and two water molecules. A comparison of energies between the minima shows the reaction is strongly exergonic with the reaction free energy of −62.70 kcal mol−1. However, this reaction mechanism is not plausible since the formation of two hydroxide ions at pH = 7.4 requires a = 15.7 value for water molecule  is therefore the only constituent of the energy barrier for this second step, and consequently does not control the overall reaction rate. The rate-limiting reaction mechanism is depicted in Figure In order to calculate the complete activation free energy for this rate-limiting step, which would be comparable to the experimental value, it is necessary to add the following to the intrinsic activation-free energy: the free energy of hydroxide ion formation Suggested mechanism of dopamine reaction with one hydroxide ion allows us to analytically express the activation-free energy as a function of the pH value of the solution using the following equation (Equation 2):≠ with the reaction rate constant krate.Note that at acidic pH, the free energy cost for hydroxide ion and neutral amino group formation is much higher than at neutral pH. Inserting this expression into the transition state equation which links activation energy ΔGyields the pH-dependence:from which we can derive the following (Equation 5):The above derived functional equation yields results that are fully consistent with the dependence of the dopamine autoxidation rate constant upon pH-described by Lloyd .rate described by Tse et al. and Lloyd, who performed the experiments in the presence of magnesium ions that might change the kinetic data to certain extent, while the shape is practically identical to the function shown by Lloyd  formation of a hydroxide ion in water; (2) dopamine o-quinone charged amino group (-−1 and a half-life of 4.7 s, making the reaction mechanism almost impossible to investigate experimentally (Tse et al., Autoxidation of dopamine has been investigated since the past few decades (Young and Babbitt, −1. This is reasonably close to the experimental barrier of 18.5 kcal mol−1 (Tse et al., a values of reaction intermediates and ionizable protein groups is not easy (Mildvan et al., a values are extremely demanding, typically involve errors of at least one pKa unit, and thus represent a very strict test of the applied computational methodology (Warshel, −1 (Kelly et al., The calculated activation-free energy for the cyclization reaction equals 21.0 kcal molWarshel, . An impoGu et al. demonstrated that in dopaminergic neurons, dopamine is released by a transient reversible fusion mechanism known as “kiss and run” (Gu et al., +-ATPase into the presynaptic membrane (Sinning and Hübner, corpus striatum, where dopamine uptake by DAT is the most important determinant of dopamine signaling (Rice and Cragg, In the case of classic synaptic transmission from presynaptic to the postsynaptic neuron, the synaptic gap is only 20–30 nm wide, which makes it very sensitive to pH change resulting from the release of protons from synaptic vesicles and the transient incorporation of vesicular Hsubstantia nigra are especially susceptible to dopamine neurotoxicity compared to the more resistant dopaminergic neurons in the ventral tegmental area. They partially attributed this effect to higher cytosolic dopamine concentrations in neurons of substantia nigra (Mosharov et al., Parkinson's disease is a neurodegenerative disease localized to the nigrostriatal pathway, where the major pathophysiological process is a degeneration of dopaminergic neurons. Despite intensive research into Parkinson's disease pathophysiology, the initial trigger of neuron degeneration is still unknown. It is generally accepted that α-synuclein aggregation, mitochondrial dysfunction, neuroinflammation, dysfunction of protein degradation and oxidative stress are involved in neurodegeneration. In the last decades, it has been shown that aminochrome, the product of dopamine autoxidation, can induce most of these changes and that the rate of some of them is pH dependent (García-Moreno et al., The rate of dopamine autoxidation outside the synaptic vesicles is probably comparable to a MAO-B-catalyzed reaction (Pavlin et al., The amount of autoxidized dopamine and therewith associated ROS production is proportional to the level of cytosolic dopamine. Inhibition of DAT and VMAT-2 by amphetamines, leads to increased levels of dopamine in the synaptic gap, cytoplasm and extracellular space (Ciccarone, in vivo. Moreover, Pham et al. showed that the formation of insoluble α-synuclein oligomers is increased at acidic pH (Pham et al., Since pH is physiologically very strictly regulated, its manipulation probably cannot be used to modulate dopamine autoxidation rate Our study has a few limitations. First, the applied computational methods were limited to the quantum-chemical description of the reactive species, while solvent effects were included on the level of solvent reaction field. Explicit description of solvent, counterions and thermal fluctuations on the level of multiscale simulation is computationally demanding and would not significantly change the results (Warshel and Levitt, We have studied the reaction mechanism of dopamine autoxidation using quantum chemical methods, and shown that at the physiologic pH the rate-limiting step is the formation of a hydroxide ion. An analytical solution for describing the pH-dependence of the rate constant is derived. The proposed model is fully consistent with experimental data. A strongly pH-dependent rate constant supports the assumption that the rate-limiting step involves a heterolytic reaction and that a reaction mechanism involving free radicals is not plausible at physiological conditions. Therefore, for stability of dopamine stored in synaptic vesicles, an acidic interior is essential, since at neutral pH, dopamine would quickly almost completely disappear. We believe that dopamine autoxidation is one of the most important mechanisms in pathophysiology of Parkinson's disease, and the major factor for its localized nature on the nigrostriatal pathway.NU, JM, and BG performed the calculations; NU, JM, BG, NV, and MŠ collected the literature and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In order to maintain the perfect fibrous architecture of the Na3V2(PO4)3/C samples after calcining, a series of heat treatment parameters are studied in detail. It is found that the heat treatment process shows important influence on the morphology and electrochemical performance of Na3V2(PO4)3/C composite nanofibers. Under the calcining conditions of 800°C for 10 h with a heating rate of 2.5°C min−1, the well-crystallized uniform Na3V2(PO4)3/C nanofibers with excellent electrochemical performances are successfully obtained. The initial discharge specific capacities of the nanofibers at 0.05, 1, and 10C are 114.0, 106.0, and 77.9 mAh g−1, respectively. The capacity retention still remains 97.0% after 100 cycles at 0.05C. This smooth, uniform, and continuous Na3V2(PO4)3/C composite nanofibers prepared by simple electrospinning method, is expected to be a superior cathode material for sodium-ion batteries.Na The theoretical specific capacity of Na3V2(PO4)3 is 117 mAh g−1 , and its average operating voltage is as high as 3.4 V. Most importantly, the NASICON structure can provides faster channels for the insertion/extraction of Na-ions, making Na3V2(PO4)3 has relatively higher ionic conductivity than many other polyanionic compounds 3 further need to be improved 3 nanofibers prepared by electrospinning can weave a three-dimensional (3D) conductive network which is beneficial for the fast transmission of electrons and sodium-ions 3 fibers wrapped with amorphous carbon are expected to exhibit better electrochemical performances.As an alternative strategy to lithium-ion batteries (LIBs), sodium-ion batteries (SIBs) recently have been paid increasing attention due to the low cost and the abundant reserves of sodium resource in the earth 3/C composites have been synthesized by electrospinning method 3/C nanofibers obtained at 800°C tends to have a more uniform nanorod shape, but the rate performance of the nanofibers still needs to be enhanced. Li et al. (3V2(PO4)3/C with budding willow branches shape by electrospinning method as well, yielding a discharge capacity of 116.2 mAh g−1 at 0.05 C rate, but its rate performance was not investigated further in the paper.Recently years, one-dimensional Nai et al.  synthesi3V2(PO4)3/C composite fibers are successfully prepared by our group through a pre-reduction assisted electrospinning method 3/C nanofibers present significantly improved electrochemical performances. It is generally believed that the particle size, morphology, and structure can greatly affect the material electrochemical properties. While heat treatment conditions  have direct effects on the morphology, structure, and particle size of nanofiber materials 3/C wires after electrospinning has not been studied yet. Therefore, a series of electrospun Na3V2(PO4)3/C nanofibers under different heating conditions were studied in detail in this paper. As the heat treatment parameters are adjusted, the morphologies of the Na3V2(PO4)3/C nanofibers represent a series of significant and regular changes. The effect of morphology on its electrochemical performance is further discussed in particular below.Lately, the smooth and uniform nano-sized Na3V2(PO4)3/C nanofibers were synthesized by the following procedures. (1) 0.12 M solution (30 mL) was prepared using H2C2O4·2H2O and deionized water and (2) NH4VO3 and NaH2PO4  were added into the above solution, and a green solution (Solution I) was got after mixing at 70°C for 2 h. (3) Polyvinylpyrrolidone  was added into the deionized water. After stirring for 2 h at room temperature, 1.5 g mL−1 PVP solution  was obtained. (4) Solution I was mixed with solution II under stirring, and then the final spinning solution (Solution III) was obtained after 2 h. (5) Solution III was pumped into an injector with a stainless steel needle pipe (inside diameter: 0.6 mm). In the spinning process, the distance, and voltage between the collector (Al-foil) and needle tip is 25 cm and 12 kV, respectively. The injection speed of spinning is 0.05 mL min−1. Then the nanofibers of precursor were collected and dried at 120°C in oven for 12 h. (6) The nanofibers of precursor were calcined at 750–900°C in Ar atmosphere for 6–12 h with the different heating rate of 1–5°C min−1. And a series of Na3V2(PO4)3/C composite nanofibers were obtained after cooling to ambient temperature.Na3V2(PO4)3/C samples were characterized by X-ray diffraction . The morphology of Na3V2(PO4)3/C nanofibers was observed by scanning electron microscopy . The amount of residual carbon of samples was measured by a C-S analyzer .The phase and crystal structure of Na3V2(PO4)3/C samples, acetylene black and PVDF  by using N-methylpyrrolidone (NMP) solvent with an Al-foil as current collector. The electrode loading density is about 2.5 mg cm−2. The button batteries (CR2025) were assembled in an Ar-filled glove box. A glass fiber membrane  and a metallic Na-foil were used as the separator and negative electrode, respectively. The NaClO4 (1 M) solution in propylene carbonate (PC) and fluoroethylene carbonate (FEC) (1:0.05 in volume) was used as the electrolyte. The electrochemical performances of cells were tested on a LAND BT2013A battery tester at ambient temperature. The cells were charged/discharged at 0.05–10 C rates (1C = 118 mAh g−1) between the potentials (vs. Na/Na+) of 2.5 and 4.2 V. The electrochemical impedance spectroscopy (EIS) was measured by a CHI 660D workstation with the amplitude of 5 mV and the frequency range of 0.01–100 kHz.The positive electrode plate was prepared with the Na3V2(PO4)3/C nanofibers is the structure stability during the heat treatment process, which requires to prevent the damage to the carbon layer and the fiber morphology. 3/C nanofibers prepared at 750–900°C. As shown, the samples calcined at 750 and 800°C exhibit continuous fibrous morphology. Furthermore, the diameter of nanofibers synthesized at 800°C is finer and more mean. When the heating temperature rises to 850°C, the nanofibers are broken. The surface of the sample prepared at 900°C can be unable to withstand the thermal stress so that the nanowires are completely disconnected and tend to grow into larger particles. Thus, the synthesis temperature exhibits a significant effect on the morphology of Na3V2(PO4)3/C nanofibers. And if the calcining temperature is not higher than 800°C, the filamentous morphology of nanowires is more likely to be preserved. In addition, the TEM images in 3 phase (space group of R-3C) 3/C samples synthesized at different temperatures are shown in Figure −1 at 0.05 C rate, close to the theoretical capacity, and still maintains a capacity of 77.9 mAh g−1 at 10 C.The charge-discharge curves at 0.05–10 C rates of Na3V2(PO4)3/C nanofibers synthesized at 800°C exhibit continuous, smooth, and uniform filamentous architecture 3 fibers are wrapped with a carbon layer with a suitable thickness. In contrast, the nanofibers obtained at 750°C are non-uniform and the average diameter is thicker, and samples at the region of 850–900°C evenly cannot maintain fibrous structure. Thus, there are significant interconnections between the morphological characteristics of Na3V2(PO4)3/C nanowires and their electrochemical properties. In order to obtain better comprehensive electrochemical performances for the materials, the core-shell fibers should be continuous, smooth, and uniform and coated by proper carbon layer. Therefore, adjusting the electrospinning preparation conditions  can lead to excellent electrochemical properties of the material.Although all samples are well-crystallized according to the XRD results, there are large differences in electrochemical properties among these samples. The reasons can be ascribed to the various morphologies of samples obtained at different temperatures. In particular, the core-shell Na Figures –g. The N−1 at 800°C for 6–12 h, respectively. Figure 3V2(PO4)3/C materials obtained under the above conditions are shown in Figure 3V2(PO4)3 without obvious diffraction peak of carbon can be obtained. The presence of Na3V2(PO4)3 grains on the surface of the nanowires responds to a higher intensity diffraction peak in the XRD patterns. As the calcining time is prolonged, the intensity of the diffraction peak slightly decreases. When the calcining time reaches 12 h, the Na4P2O7 (JCPDS 10-1087) heterogeneous phase appears. The carbon contents of the samples calcined for 6, 8, 10, and 12 h are 12.7, 12.2, 11.9, and 11.5%, respectively.Further, the samples are synthesized at a heating rate of 2.5°C min−1 at 0.05 C, but only 13.0 mAh g−1 at 10 C. The capacity decay is more pronounced in the charge and discharge test of the sample synthesized for 6 h. These indicate that the electrochemical performances of Na3V2(PO4)3/C grains are much worse than the intact Na3V2(PO4)3/C nanofibers, which is consistent with the above results. Compared with the granular samples, the continuous, smooth, and uniform nanofibers evenly coated by a carbon layer can build up a perfect network with high electronic conductivity, which will greatly enhance the rate performances of Na3V2(PO4)3, especially at high current rates. An additional small platform around 3.9 V appears in the charging curve of the sample synthesized for 12 h, corresponding to the appearance of the heterogeneous phase in the XRD pattern. Thus, as the calcining time extended, the nanofibers gradually become more smooth, and continuous, and exhibiting a better rate performance. However, if the calcining time is too long, heterogeneous phase appears in the synthesized samples would lead to a decreased electrochemical property.The charge and discharge curves of the samples with heat treatment time of 6–12 h are shown in Figure −1, the filament structure of the precursor is completely destroyed due to the excessive warm-up time, which is similar to the sample morphology at 900°C in Figure −1. Obviously, the faster the heating rate is, the smaller the diameter of the wire gets.Based on the above results, another batch of samples are synthesized at 800°C for 10 h with different heating rates. The SEM images of the samples prepared at different heating rates are shown in Figure 3V2(PO4)3/C materials obtained under the above conditions are shown in Figure −1 sample, the intensity of diffraction peak is lower, and the half width is larger which may be the result of insufficient diffusion of elements during the process of heat treatment under a fast heating rate.The XRD patterns of the Na−1 is even worse than that of the 900°C sample because of the most loss of filamentous morphology. The initial discharge capacity of the sample heated at the rate of 5°C min−1 is only 108.8 mAh g−1 at 0.05 C, and its capacity decay is obvious at low current density. This may be due to the unsatisfied crystallization of the Na3V2(PO4)3.As shown in Figure −1. For the Na3V2(PO4)3/C sample synthesized at the optimal synthesis conditions, the rate-cycle curves and cycling perormance at 0.05 C rate are shown in Figure 3V2(PO4)3/C sample. And when 100 times cycle at 0.05 C 3/C nanofibers are synthesized through a pre-reduction assisted electrospinning method. Under the different synthesis conditions, the morphology, and electrochemical performance of the Na3V2(PO4)3/C nanofibers are obviously different. As the temperature increases, the nanofibers become thinner, but gradually lose the filamentous morphology. With the elongation of the calcining time, shoot-like crystalline particles on the fiber surface can be re-melted into nanowires. The heating rate also play a critical role in the structure and morphology of the Na3V2(PO4)3/C samples. As a result, the structure, morphology, and electrochemical performance of Na3V2(PO4)3/C composite nanofibers can be controlled by adjusting the heat treatment parameters. Under the optimum synthesis conditions of 800°C, 10 h and heating rate of 2.5°C min−1, the obtained Na3V2(PO4)3/C composite nanofibers present excellent electrochemical performances.A series of NaLW, YH, and SS did the main experiment and write the manuscript. XZ and HL envolved the discussion of the experiment and revised the manuscript. LY assisted the material synthesis. YS and SZ made the research plan. SZ and LW also provided the financial support.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.