Vps35 p.D620N implicates retromer dysfunction in the pathogenesis of Parkinson’s disease (PD). We have characterized a knock-in mouse with a Vps35 p.D620N substitution (hereafter referred to as VKI) at 3 months of age. Standardized behavioral testing did not observe overt movement disorder. Tyrosine hydroxylase (TH)-positive nigral neuron counts and terminal expression in striata were comparable across genotypes. Fast scan cyclic voltammetry revealed increased dopamine release in VKI striatal slices. While extracellular dopamine collected via striatal microdialysis of freely moving animals was comparable across genotypes, the ratio of dopamine metabolites to dopamine suggests increased dopamine turnover in VKI homozygous mice. Western blot of striatal proteins revealed a genotype-dependent decrease in dopamine transporter (DAT) along with an increase in vesicular monoamine transporter 2 (VMAT2), albeit independent of changes in other synaptic markers. The reduction in DAT was further supported by immunohistochemical analysis. The data show that the dopaminergic system of VKI mice is profoundly altered relative to wild-type littermates. We conclude early synaptic dysfunction contributes to age-related pathophysiology in the nigrostriatal system that may lead to parkinsonism in man.Vacuolar protein sorting 35 (VPS35) is a core component of the retromer trimer required for endosomal membrane-associated protein trafficking. The discovery of a missense mutation,  Vps35), in young adult mice. Although no significant effects on motor function or neurodegeneration were found, an increase in dopamine release and turnover in the dorsolateral striatum were observed in the mutant mice. Deficits in the transport and recycling of transporters required for dopamine re-uptake and storage may underlie these effects. Further analyses of Vps35 mutant mice could improve our understanding of the synaptic dysfunction that precedes neurodegeneration in PD.A mutation linked to late-onset Parkinson’s disease (PD) disrupts dopaminergic neurotransmission in mice. A study led by Matthew J. Farrer at the Centre for Applied Neurogenetics, University of British Columbia, Canada, examined the effects of a missense mutation in the gene encoding a core subunit of a complex required for recycling membrane-associated proteins, Vacuolar Protein Sorting 35 (VPS35;  Non-motor dysfunction including autonomic, cognitive, psychiatric, sensory, and sleep disorders are also associated with motor symptoms and typically antedate them.3 While dopamine-replacement therapies alleviate motor symptoms, they fail to halt disease progression and often lead to dyskinesia. Thus, new therapeutic targets and strategies to combat neuronal cell loss are necessary.Parkinson’s disease (PD) is an age-related neurodegenerative disease characterized by the loss of midbrain dopaminergic neurons and Lewy pathology.5 Affected subjects in >61 unrelated families have been described with a clinical syndrome indistinguishable from idiopathic PD.6 VPS35, VPS26, and VPS29 are structural components of the retromer trimer8 that complex with a pair of sorting nexins to recycle transmembrane cargo to the cell surface or trans-Golgi network.9 Within the synapse, the retromer recycles neurotransmitter receptors including β-adrenergic, AMPA-type glutamate, and D1-type dopamine receptors.15Dominantly-inherited, late-onset parkinsonism has recently been linked to an aspartate to asparagine substitution in vacuolar protein sorting 35 (VPS35 p.D620N).Vps35 p.D620N knock-in mice was recently reported without a behavioral phenotype, although striatal microdialysis revealed a reduction in evoked striatal dopamine in homozygous animals.16 Here we report an independent Vps35 p.D620N knock-in mouse model (VKI) developed to preserve the physiological 1:1:1 stoichiometry of the retromer complex. Given the selective vulnerability and loss of dopamine neurons in PD, we hypothesized Vps35 p.D620N may impair retromer-dependent recycling of specific cargo with consequent effects on dopaminergic neurotransmission. Hence, at 3 months of age, our characterization has focused on motor behavior, the integrity of the nigrostriatal system, and dopamine release.A line of Vps35 p.D620N mutation at physiological levels, and avoid the confounds of random insertion and overexpression, we engineered C57Bl/6J mice to express a Vps35 exon 15 g.85,263,520G>A (p.D620N) nucleotide mutation . Similarly, total Vps35 protein levels were unchanged . VKI mice are viable, show no evidence of overt distress, breed well and produce heterozygous (Het) and homozygous (Homo) pups at expected Mendelian ratios.To examine the effects of the p = 0.76). Basic motor function was normal in VKI as compared to WT littermates. There were no changes in open field exploration, as shown by similar total distance traveled, moving time  and center path ratio . Similarly, there were no differences in cylinder test rearing activity  or performance on an accelerating rotarod .At 3 months there were no differences in appearance or weight of VKI mice when compared to WT littermates  and substantia nigra pars compacta (SNpc)] by immunohistochemical staining of the dopamine synthesis enzyme tyrosine hydroxylase (TH). No significant differences were observed between VKI and WT littermates in the intensity of TH-signal produced by nigral terminals in the dorsolateral striatum , or in the number of TH-positive nigral neurons assessed by stereological counts . Consistent findings were observed by western blotting of striatal tissue probed for TH . Together, the data indicate that physiological expression of Vps35 p.D620N does not overtly impair motor function or induce dopaminergic neurodegeneration in young adult mice.We examined the integrity of the nigrostriatal system  and oxygenated  at room temperature for >1 h prior to experiments. Individual slices were then placed in a recording chamber at temperature 22–26 °C, and perfused at 1–2 mL/min with ACSF. Stimuli (150 μs duration) were delivered by nickel-chromium bipolar electrodes (made in house) placed in the dorsolateral striatum, optically isolated  and controlled/sequenced with ClampEx software. Voltammetric responses were recorded, standardized and analyzed with an Invilog Voltammetry system and software . Carbon fiber electrodes  were purchased prefabricated (Invilog) and placed within 100–200 μm of the stimulating electrode in the dorsolateral striatum  and a stainless steel screw. During surgery, animals were given lidocaine prior to incision , meloxicam  and warm 0.9% saline (s.q.) at the end of the procedure. Following surgery, mice were allowed to recover and experiments were run 24 h after probe implantation. Microdialysis probes were perfused at a flow rate of 1.5 µL/min with a modified Ringer’s solution . Samples were collected every 15 min, starting 2 h after the onset of probe perfusion, into vials containing 2 µL of 10 mM acetic acid.Under isoflurane anesthesia, a CMA 7 microdialysis probe  was lowered into the mouse striatum according to the following coordinates: AP +0.5, ML +2.1 from bregma, DV −2.8 below dura, as previous.Dopamine and its metabolites (DOPAC and HVA) were measured by HPLC coupled to electrochemical detection . Five microliters of sample were automatically injected  onto an Acquity UHPLC BEH C18 analytical column  perfused at a flow rate of 50 µL/min  with a mobile phase containing 100 mM phosphoric acid, 100 mM citric acid, 0.1 mM EDTA, 600 mg/l octan sulfonic acid sodium salt and 8% acetonitrile (pH 3). Dopamine, DOPAC and HVA were detected by means of an electrochemical detector  with cell potential set at 0.8 V vs salt bridge. Retention times were: 2.57 ± 0.2 min for DOPAC, 3.82 ± 0.2 min for dopamine and 5.18 ± 0.2 min for HVA. Mice were euthanized after the experiment and brains collected to confirm probe placement by immunohistochemistry.g for 10 min at 4 °C and supernatant separated from pellet, then dopamine and its metabolites were measured by HPLC as above. The pellet was lysed in HEPES buffer  containing protease and phosphatase inhibitors (Roche), homogenized, and incubated . Lysates were cleared by centrifugation at 4000×g for 12 min at 4 °C and supernatant was quantified by BCA assay (Pierce), to normalize monoamines levels to protein concentration.Striatal tissue was mechanically homogenized in a solution of 100 uM EDTA and 0.1 mM perchloric acid. Samples were cleared by centrifugation at 14,000×n” equals the number of animals, or else are shown as per slice “n” for the total number of animals (indicated in parentheses). Sample sizes estimates used the resource equation method41 but were refined using G power software42 with pilot data (n = 3) on effect size (mean/standard deviation within groups) for 1- beta = 0.8 and alpha = 0.05. Throughout the study, comparisons were conducted by 1- or 2-way ANOVA with appropriate post-hoc tests, as detailed in the text, using Prism 6.0 .Data are presented throughout as mean ± SEM where “Supplementary figures and legends


Hydrophobically modified acrylamide copolymers dewater oil sands tailings more effectively than anionic polyacrylamide, but the root causes for this enhanced performance have not been investigated systematically. Polyacrylamide‐poly(ethylene oxide methyl ether methacrylate) copolymers with different comonomer compositions, hydrophobic chain lengths, and molecular weights to map out these effects systematically are synthetized. Through a statistical design of experiments, it is found out that all three variables above significantly affected flocculation performance and that certain combinations achieve optimal results. The effect of centrifugation on the flocculation and dewatering performance of these polymers is also investigated. One of the main contributors to this stigma is the generation of large volumes of waste stored in tailings ponds. Tailings ponds are composed of water, residual bitumen, fine mineral particles, and organic compounds, covering an area more than 175 kmThe industry has practiced several technologies to dewater and consolidate tailings. Thin lift and freeze‐thaw drying, centrifugation, and composite tailing are examples of some of these practices.−1) anionic polyacrylamide (PAM), but the sediments from PAM‐induced flocculation are loosely packed and have a gel‐like structure because they trap water in their matrix via hydrogen bonding among polymer chains and water molecules.The industry standard flocculant is ultrahigh molecular weight (>10 million g molg‐PPO) copolymer, but only compared the data points in terms of flocculation performance, and did not inquire into the reason for differing results at different copolymer compositions.−1 or 1000 g mol−1 macromonomers, but with only two different values it is not possible to determine a correlation between graft size and flocculation/dewatering performance. Their results did show that the PPO graft with the lowest molecular weight (300 g mol−1) achieved better results, such as lower turbidity and faster settling rate, which was a unique outcome, since most tests ascertain the opposite: higher molecular weight polymers usually performs better than lower molecular weight ones.g‐PPO flocculant. Ren et al. flocculated kaolin suspensions with PAM, poly (PDADMAC), and 3‐acrylamido‐2‐hydroxypropyltrialkylammonium chloride copolymers, where the alkyl group was ethyl, butyl, or octyl.Though some research groups have tested acrylamide copolymers as flocculants for oil sands tailings, to the best of our knowledge, there are only few studies that have performed a systematic study of the causes for improved dewatering and consolidation of oil sands tailings in the open literature.−1, sought in gravity thickeners, may not be as important if dewatering is done by postflocculation centrifugation or by thin lift drying. The Alberta Energy Regulator, however, has approved Directive 085 for fluid tailings management for oil sands mining projects, where it states some minimum acceptable criteria for treated tailings before they are ready to be reclaimed , which is not helpful for flocculation performance characterization.Laboratory scale screening of polymer flocculants for oil sands tailings is still controversial, mainly because of the lack of clear procedures and accepted values to quantify flocculation and dewatering, as well as different performance metrics required by different dewatering technologies. For example, a high settling rate of 20 m hIn this study, we used a central composite rotatable design of experiment to analyze our results and evaluated the performance of our novel copolymers through CST, supernatant turbidity, initial settling rate (ISR), and solid content of sediments. To observe the effect of centrifugation on flocculation performance, we also tested the turbidity, solid content, and CST after centrifugation. By studying the cause of this increased flocculation performance, we will be able to understand the reason for improvement and tailor polymers for optimal performance during oil sand tailing flocculation and dewatering. It is worth noting that the intention of this study was not to optimize the proposed flocculant composition for what are arbitrarily selected conditions or a particular MFT concentration, but rather to study how certain polymer microstructure properties  affected their performance in small‐scale tests under controlled conditions.22.1TableA Dean Stark apparatus was used to determine the amount of solids, water, and bitumen in the tailings we used for testing. The Dean Stark apparatus comprises of a trap, round bottom flask, and reflux condenser.2.2FiguresAll normal and residual plots are displayed in the L) and quadratic (Q), and their two factor interaction effects, where the notation used is linear effect by linear effect  and PEOMA wt% (2).In the ANOVA tables, we compared the three main variables, both linear , and the results are displayed in Table I], significantly affected the molecular weight of all PAM‐g‐PEOMA copolymers, as it is known that the molecular weight of a polymer is controlled by the initiator concentration, and according to Shawki and Hamielecr¯n represents the number‐average chain length of the copolymer, f is the fractional initiator efficiency, ktrm, kp, kI, and kt are the reaction rate constants for transfer to monomer, propagation, initiator decomposition, and termination, respectively, and [M] is the monomer concentration.The molecular weight distribution of all PAM‐g‐PEOMA. Figureg‐PEOMA increases as the PEOMA wt% increases, irrespective of the PEOMA length. The effect of initiator concentration on PAM‐g‐PEOMA molecular weight is observed in Figure I] increases PAM‐g‐PEOMA molecular weight, as expected for solution free‐radical polymerization. Figure I] and PEOMA wt%, and shows that polymerization with low [I] and high PEOMA wt% will produce PAM‐g‐PEOMA with the highest molecular weights.Interestingly, PEOMA wt% in the copolymer also affected the molecular weight of PAM‐g‐PEOMA molecular weight as the length of the PEOMA grafts increases, as shown in Figure g‐PEOMA copolymers with higher molecular weights; it only means that when all other variables are kept constant—namely initiator concentration, monomer/macromonomer concentration, and temperature—the molecular weight of PAM‐g‐PEOMA will decrease as the length of the hydrophobic PEOMA macromonomer increases. One could, evidently, change the polymerization conditions (such as decreasing [I]) to compensate for this effect.There seems to be a trend indicating a slight decrease in PAM‐g‐PEOMA copolymers with molecular weights ranging from 12 000 to 1 473 000 g mol−1. This wide molecular weight range will help show the effect of PAM‐g‐PEOMA molecular weight on flocculation/dewatering performance. Compared with most flocculants, the molecular weights of our PAM‐g‐PEOMA flocculants are relatively low, as it is generally accepted that high molecular weight polymers are more effective flocculants ,As seen in Table 2.4TableI], are significant factors, and the residual plots indicate a normal distribution.Figureg‐PEOMA performs best (high ISR) when the PEOMA length and wt% are high (upper right quadrant), or when the PEOMA lengths and PEOMA wt% are low (lower left quadrant). Combining this finding with the results shown in Figure g‐PEOMA copolymers lead to higher ISR when their molecular weights are higher. This agrees with our knowledge of the flocculation mechanism with these polymers, as higher molecular weight PAM‐g‐PEOMA can create larger and heavier flocs that settle faster via the bridging mechanism. Since these polymer flocculants are neutral, charge patch and charge neutralization cannot be the underlying flocculation mechanism.Once the insignificant factors are eliminated, we can create surface plots on which the effects of the manipulated variables on ISR are depicted as a function of two variables. g‐PEOMA with low PEOMA length and low PEOMA% is almost twice as high as for the PAM‐g‐PEOMA with high PEOMA length and high PEOMA%. Steric hindrance of the chains may explain these results: High molecular weight PAM‐g‐PEOMA with long hydrophobic PEOMA chains may become very large in solution, interact with one another, and hinder the entrapment of clay particles in the MFT suspension.Analyzing the numbers associated with the colors in Figure g‐PEOMA is high, and it contains high PEOMA%. The high PAM‐g‐PEOMA molecular weight and PEOMA wt% allow the flocculant to form strong bridges with multiple points of contact among the suspended particles, forming large, strong flocs that quickly settle.Figure g‐PEOMA molecular weight and ISR is stronger than for the longer PEO unit discussed in Figure I] is lowered below 0.001 mol L−1. On the other hand, the weight percent of PEOMA is not as significant, as one may achieve a high ISR for any PEOMA% when the molecular weight of PAM‐g‐PEOMA is high , although lower PEOMA% seems to be produce slightly higher ISR values.Figure 2.5TableSimilar to the ISR, the analysis of variance for turbidity values found out that all variables were significant, but we had to take the natural logarithm of the turbidity values in order to obtain a correlation with accurate prediction power. The ANOVA table shown in Figureg‐PEOMA had a high PEOMA wt% with high length (right upper corner) may be due to steric hindrance among the long hydrophobic PEOMA grafts that lower the performance of flocculation when there is a high amount of PEOMA in the copolymer. These chains may interfere with each other and are unable to trap the finest particles. On the other hand, PAM‐g‐PEOMA with shorter PEOMA grafts requires a higher PEOMA% on the copolymer to trap all of the fine particles because the PEOMA chains are shorter. Having more frequent, shorter PEOMA side chains increase the surface area of PAM‐g‐PEOMA and create more sites onto which the flocculant may adsorb to the surface of the suspended MFT particles.I] and PEOMA wt% for a constant PEOMA length of 40 and confirms our conclusions above: PAM‐g‐PEOMA with long PEOMA grafts produces supernatant with low turbidity when a low PEOMA% is present in the polymer so that there is no steric hindrance between the hydrophobic groups. Lowering the PAM‐g‐PEOMA molecular weight for a given PEOMA wt% also helps decrease the turbidity for the same reasons.Figure g‐PEOMA had high PEOMA wt% and high PEOMA length, while the turbidity analysis shows that high PEOMA length and low PEOMA% are ideal. We may suggest an explanation for this observation using the proposed flocculation mechanism for these polymers: When PAM‐g‐PEOMA polymers trap particles to form flocs, the larger flocs are heavier, and thus settle faster, but these polymers may not be able to trap all the fine particles because the larger hydrophobic PEOMA side groups interact with each other, hindering the ability to adsorb on to the suspended particles. Such copolymers would yield high ISR, but would also produce supernatants with higher turbidity. Notice that this is a discussion relative to the other PAM‐g‐PEOMA tested in this investigation. The highest turbidity measured with all copolymers was 26 NTU, which is still an acceptable value, even for the poorest performance among the PAM‐g‐PEOMA listed in Table These conclusions are different from those observed for ISR, as ISR was the highest when the PAM‐2.6TableCST data were transformed by taking the natural logarithm of the numerical data to achieve a normal residual plot  when PAM‐g‐PEOMA had a high molecular weight and PEOMA wt%. These results are easy to explain because the higher the PEOMA%, the higher the hydrophobicity of the copolymer; since CST measures the dewatering ability of flocs, a more hydrophobic flocculant is expected to retain less water inside the flocs. By the same token, a higher molecular weight PAM‐g‐PEOMA flocculant makes larger, more compact flocs that retain less water, and yield a lower CST. Although the effect of copolymer molecular weight on CST is not very significant, it contradicts the findings of our recent work where the CST of flocs produced by cationic copolymers of acrylamide and DADMAC did not depend on molecular weight.Based on the results in Table Figure 2.7Figure g‐PEOMA with long PEOMA side chains requires low PEOMA% to capture all of the fine particles, and that PAM‐g‐PEOMA with shorter PEOMA grafts needs higher PEOMA% in the copolymer to produce clear supernatants and follows the same pattern for the turbidity without centrifugation—the copolymers with a large percentage of long PEOMA grafts may hinder the entrapment of smaller particles, while the copolymers with a low percentage of short PEOMA grafts are unable to adsorb to all of the particles as there are fewer sites to form a proper bridge. Although we could obtain a strong correlation among these variables, it should be noted that the highest turbidity after centrifugation was only 16.4 NTU, which still corresponds to a very clear supernatant. For all practical purposes, after centrifugation, all PAM‐g‐PEOMA flocculants produced supernatants that were essentially free of fine particles. All polymers produced supernatants with very low turbidity. Although the differences are statistically significant and indicate that the polymer microstructure affects turbidity, these differences may have little practical significance, since all of them satisfy the condition of less than 0.5% solid residues in the supernatant indicated by Directive 085.The supernatant turbidity following centrifugation yields very similar results to the turbidity after 24 h of settling. 2.8TableAn interesting observation obtained through this study was the effect of centrifugation on the solids content of the sediments, compared that obtained in settling over a single day. By dividing the solid content of the centrifuged solids by that of the 24 h settled solids, we were able to compare the increase in solid content by centrifugation. Figureg‐PEOMA, the greater the increase in solids content. The centrifuge increases the g‐force on the sample, forcing the flocs to settle to the bottom of the centrifuge tubes, where the high hydrophobic PEOMA groups decrease the amount of water trapped inside of the sediments. Equally important, the lower the [I], the higher the molecular weight of PAM‐g‐PEOMA, and the greater the effect of centrifugation on the solids content. The impact of PAM‐g‐PEOMA molecular weight is shown most clearly in Figure I] values and high PEOMA%. As shown by Figure The red areas in 2.9g‐PPO copolymer, where the shorter length hydrophobic grafts combined with a high percentage of grafts performed the best.−1 macromonomer) was when they used only 5 wt% PPO. The combination of a long hydrophobic chain length and low wt% was required in our model so there was no steric hindrance. The only copolymer with the longer PPO length that performed better in some tests was a 35 wt% PPO, but it performed better in CST, where our model states that the additional hydrophobic groups would expel water faster, producing a lower value. The alignment between their results and our conclusions confirms that our model is applicable in other polymer systems where a hydrophobic chain is added to acrylamide.As mentioned in the introduction, Reis et al. explored the addition of hydrophobic groups to an acrylamide backbone. They found similar results with the PAM‐Shang et al. also added hydrophobic groups to acrylamide, making poly(acrylamide‐methacryloxyethyl trimethyl ammonium chloride‐methacryloxypropyltrymethoxy silane) (P(AM‐DMC‐MAPMS)).3As displayed through the different optimal variables for each one of the tests, there is no “perfect” polymer for treating MFT; however, it is possible to tailor a polymer for specific flocculation purposes. Depending on the required characteristics of flocculation, one is able to synthesize a polymer based on the criteria. This study allowed us to delve deeper into the reasons behind enhanced flocculation performance gained by the addition of hydrophobic chains to an acrylamide backbone. Through the analysis of the effects of initiator concentration, PEOMA length, and PEOMA wt%, we determined the effect of hydrophobic chain addition to acrylamide polymers. Adding many hydrophobic chains (>30 wt%) can effectively help in the dewatering performance of the flocculant, shown in the ISR and CST tests, but may not capture all of the fine particles, leading to a higher turbidity. A lower initiator concentration (higher molecular weight) was also shown to improve flocculation performance in most categories. The length of hydrophobic chains was observed to be of mild importance—the shorter chains required a larger percentage of hydrophobic groups to achieve the same effects as the larger chains, whereas the longer hydrophobic chains needed a low weight percent of PEOMA to prevent steric hindrance.g‐PEOMA copolymer molecular weights studied were low (<200 000 g mol−1), we still observed very good flocculation performances from all polymers tested. This contradicts the most common assumptions about flocculants, but it actually has some advantages as well. There are benefits to having low molecular weights, as high molecular weight flocculants will lead to an increase in viscosity and may cause problems such as nonuniform mixing. Reis et al. also noted that having a lower molecular weight flocculant allows for a wider window of mixing environments in which the flocculant may be used.Although most PAM‐4Materials: Monomers  methyl ether methacrylate (PEOMA)), initiator  dihydrochloride (V‐50)), and organic solvents  were purchased from Sigma Aldrich. Coanda Research and Development supplied the MFT used in this investigation.Polymerization: The copolymers were synthesized by free‐radical polymerization at 40 °C in a batch reactor. The reagents were added to the reactor in the following order: deionized water, acrylamide, and then PEOMA. Once in the reactor, the solution was purged with nitrogen for 1 h to ensure a complete nitrogen atmosphere. After that, the initiator, also purged with nitrogen, was added to begin the polymerization. The polymerization was left to run for 24 h to obtain high monomer conversion for all experiments. The total monomer concentration was kept constant at 0.1 mol L−1 for all polymerizations. Once the reaction had finished, the polymer, whose structure is shown in FigureMolecular Weight Measurement: The molecular weights of all copolymers were measured by gel permeation chromatography using an Agilent 1260 Infinity GPC and PL aquagel‐OH Mixed‐H 8 µm columns. PEO standard samples with narrow molecular weight were used for the column calibration. The concentration of all samples was 3.6 mg mL−1, diluted in an aqueous solution containing 0.2 m NaNO3, and injected at a volume of 100 µL. The analysis was performed at a flow rate of 1 mL min−1 and at a temperature of 30 °C.Experimental Design: Utilizing a central composite rotatable design allows us to compare the effects of macromonomer size, copolymer composition, and molecular weight as a function of flocculation performance. Macromonomer refers to a macromolecule with a functional group to allow further polymerization. As per the design, there were five different coded values for each variable, where 15 unique combinations and an additional three repetitions of the center point to ensure reproducibility were tested, allowing us to create a surface to map the effects of the design variables shown in TableI], was varied to obtain a range of copolymer molecular weights. All data through Statistica 13 (StatSoft Inc.) were analyzed. In Table Although Table Test Methods: In order to create the MFT slurry for testing, the MFT sample was diluted to 5 wt% solids in 160 g of slurry, using deionized water. The concentration of solids in the suspension to obtain as uniform mixing as possible when testing different polymers, and to maximize the ability to observe performance differences upon addition of polymers with different microstructures was diluted. The slurry was mixed at 610 rpm for 30 s to obtain a homogeneous distribution of solids in the slurry. At that point, 1000 ppm of a Ca2+ solution was added and mixing continued for an additional minute. The addition of calcium is necessary, as the neutral polymer flocculants here used are unable to form bridges with the suspended particles without the charge neutralization effect obtained by calcium ions. The polymer dosage was kept constant at 10 000 ppm (mg polymer per kg of dry solid) for all experiments, and the polymer mixed with the slurry at 610 rpm. The dosage of 10 000 ppm in this study was chosen based on preliminary tests with the polymers (from the highest MW to the lowest MW), and it was found out that this dosage was not too far from the working window of the polymers. It is true that 10 000 ppm would be considered a high dosage in practical application ;Once flocs formed, the mixing speed was reduced to 300 rpm and stirring continued for another 2 min. After flocculation was done, while the flocculated suspension was still under gentle mixing (to achieve uniform solid concentration upon sampling), a large pipet (0.5 cm diameter) was used and separated the flocculated suspension into three different portions: (1) A volume of 10 mL was used for testing CST with a Triton Electronics Meter (Type 319 multi‐CST) with Triton filter paper (7 cm by 9 cm); (2) An aliquot of 50 mL was transferred to a Corning centrifuge tube for centrifugation purposes; and (3) The remaining 100 mL of sample was poured into a graduated cylinder so the change in mudline level could be recorded as a function of time.After the slurry had settled in the graduated cylinder for 24 h, the supernatant was extracted for turbidity measurement in a Hach 2100 AN Turbidmeter, and additional CST measurements were performed on the solids after settling. The 5 mL of settled solids were also taken, and the solids content was calculated by weighing the sample before and after 24 h drying in an oven at 60 °C. The solids content is calculated by the following formula, where weight was measured in gramsThe authors declare no conflict of interest.


There is a great limitation to improving the quality and productivity of nanofibers through the conventional single-needle method. Using needleless electrospinning technology to generate multiple jets and enhance the productivity of nanofibers has attracted lots of interest for many years. This study develops a novel linear flume spinneret to fabricate nanofibers. Multiple jets with two rows can be formed simultaneously on the surface of the spinneret. The solution concentration has a significant impact on the average nanofiber diameter compared with applied voltage and collection distance. The effects of different spinning process parameters on the productivity of nanofibers are investigated. High-quality nanofibers with small nanofiber diameter and error can be fabricated successfully. The average nanofiber diameter is 108 ± 26 nm. The average error is 24%. The productivity of nanofibers can reach 4.85 ± 0.36 g/h, which is about 24 times more than that of the single-needle method. This novel linear flume spinneret needleless electrospinning technology exhibits huge potential for mass production of nanofibers in the field of industrialization. Nanofibers have attracted much attention for many years because of their outstanding properties. Compared with traditional fibers, nanofibers exhibit many advantages, such as high specific surface area, high porosity and controllable nanofiber diameter . NanofibMany methods have been attempted to improve the productivity of nanofibers. A simple approach was to increase the number of needles to develop a multi-needle electrospinning setup ,11. AlthIn this study, we presented a linear flume spinneret to fabricate nanofibers. Multiple jets could be formed on the top of the linear flume spinneret. Finite element analysis software was adopted to simulate the electric potential and the electric field distribution profiles. Both the electric potential value and the electric field intensity around the linear flume spinneret were also investigated. Later on, we further examined the effects of different spinning process parameters on the morphology and nanofiber diameter. The results demonstrated that small diameter and narrow distribution nanofibers could be achieved by adjusting the spinning process parameters. The average nanofiber diameter was 108 ± 26 nm. The average error was 24%. The productivity of nanofibers reached 4.85 ± 0.36 g/h, which was about 24 times more than that of the single-needle method. This linear flume spinneret has a great advantage to improve the quality and productivity of nanofibers. Moreover, this electrospinning setup has a significant potential for realizing the industrialization of nanofibers.N,N-Dimethylformamide (DMF) was purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd. . PAN powder was dissolved in DMF to form polymer solution with three different concentrations . The PAN solutions were stirred on a magnetic stirrer for 24 h to form a homogeneous light-yellow transparent solution.Polyacrylonitrile (PAN) with a molecular weight of 75,000 g/mol was purchased from Sinopharm Chemical Reagent Co., Ltd. . The apparatus of needleless electrospinning consisted of a linear flume spinneret, control pump, polymer solution, collector and a high voltage electrostatic generator, which are showed in A scanning electron microscope  was used to observe the morphology of nanofibers. One hundred nanofibers in the SEM image were selected to measure the nanofiber diameter and diameter distribution using Image J software . The productivity of nanofibers was weighted using electronic balance . The electric potential and the electric field distribution of the linear flume spinneret were simulated using the finite element analysis software entitled COMSOL Multiphysics 5.0 . A geometric model was established according to the actual size of the electrospinning setup. Mesh generation could be carried out with self-contained procedures. Electric potential and electric field distribution were displayed in the form of the contour.4 V. In fact, the two points are two lines for the linear flume spinneret in three-dimensional space. The polymer solution was stored in the flume. Two rows of jets could be formed from the two peak lines of the spinneret when the electric force exceeded the join forces of surface tension force and viscosity resistance. 7 V/m from the curve of the electric field intensity.The electric field of the needleless electrospinning spinneret played an important role in the formation of multiple jets during electrospinning. Therefore, we adopted finite element analysis software to simulate the electric potential and electric field intensity of the linear flume spinneret. The spinning process parameters including solution concentration, applied voltage and collection distance had a direct influence on the nanofiber diameter. Compared with the single-needle electrospinning setup, the linear flume spinneret needleless electrospinning approach could significantly enhance the productivity of nanofibers. We investigated the effect of different spinning process parameters on the productivity of nanofibers. In this study, we developed a novel linear flume spinneret to produce multiple jets and fabricate nanofibers by needleless electrospinning technology. Multiple jets with two rows were generated from the surface of the linear flume spinneret. The electric potential and electric field intensity of the linear flume spinneret were investigated. The effects of different spinning process parameters on the nanofiber diameter and error were examined in detail. The productivity of nanofibers was calculated with the different spinning process parameters. The average diameter of nanofibers was controlled between 108 and 210 nm, and the average error of nanofibers was controlled between 17% and 26%. The productivity of nanofibers reached 4.85 ± 0.36 g/h, which was about 24 times more than that of the single-needle method. Therefore, high-quality nanofibers could be fabricated by adjusting the spinning process parameters, and this novel linear flume spinneret has great potential for producing high-quality and high-productivity nanofibers.


The trade-off between reward and effort is at the heart of most behavioral theories, from ecology to economics. Compared to reward, however, effort remains poorly understood, both at the behavioral and neurophysiological levels. This is important because unwillingness to overcome effort to gain reward is a common feature of many neuropsychiatric and neurological disorders. A recent surge in interest in the neurobiological basis of effort has led to seemingly conflicting results regarding the role of dopamine. We argue here that, upon closer examination, there is actually striking consensus across studies: dopamine primarily codes for future reward but is less sensitive to anticipated effort cost. This strong association between dopamine and the incentive effects of rewards places dopamine in a key position to promote reward-directed action. Compared to reward, effort remains poorly understood, both at the behavioral and neurophysiological levels.Dopamine has been proposed as central to effort-related decision making, but its role is not clearly defined.In fact, the activity of midbrain dopamine neurons and mesolimbic dopamine levels are consistently modulated by anticipated future reward more strongly and consistently than effort, even when the weight of reward and effort on behavior are equated.These signals may promote decisions to act based on the potential gain from a future reward. Dopamine plays a central role in reward-guided learning and motivation. In general terms, there is a broad agreement on its role in appetitive motivation, namely that dopamine mediates the positive influence of potential future rewards on behavior  Box 1In neuroscientific experiments investigating effort-related choice, effort is usually treated as a cost which can diminish preference for rewards or other goals that an organism may find more desirable However, the precise nature of these effort costs is not straightforward to isolate, quantify, and compare across studies. For instance, preferences for scaling a physical barrier can be temporarily reduced by inducing physical fatigue These distinctions are important not only when considering the normative decision-making strategy in different model organisms but also because there is increasing evidence that the neural circuits necessary to assess and overcome different types of effort cost are partially distinct. For instance, whereas depletion of nucleus accumbens dopamine can cause animals to be effort-averse when choosing whether or not to scale a barrier or when needing to make multiple lever presses, they have little effect on choices between options with different force requirements Alt-text: Box 1However, beyond this initial consensus, there is much debate over the precise relationship between dopamine – particularly the rapid and transient (‘phasic’) changes in dopamine observed when animals are presented with or approach response options – and effortful choices. Part of the reason is that, to date, experiments that directly investigated effort-based decision making and dopamine have not always measured the separable influences of effort and reward. For instance, in many situations, effort has been studied in paradigms where the amount of force produced conjointly scaled with the amount of reward e.g., . In otheAnother reason for the conflicting interpretations about the relationship between dopamine and effort is that it has not always been clearly specified at which points during an effort-based decision dopamine might be acting. The reduction in willingness to overcome effort constraints observed following pharmacological manipulations could result from at least three factors: (i) changes in the way costs and benefits are valued or compared, (ii) the willingness to initiate a choice, and/or (i) the motivation to overcome the cost .To make matters even more complicated, there have been misunderstandings about how to interpret those existing data that do directly speak to the issue of the relationship between dopamine and effort. We have undertaken studies to systemically examine how both reward and effort influence dopamine activity Motivated by these discrepancies, controversies, and conflicting interpretations, we review here the evidence for a role for phasic changes in dopamine during effort-related decision making. We outline areas where there seems to be a consensus and areas where disagreements remain. Finally, we address the open questions that we see as being crucial for further progress in this area.There is an extensive literature, both in primates and rodents, showing that the responses of many midbrain dopamine neurons elicited by predictive cues reflect the expected value of a future reward. Such signals are consistently larger when the benefits will be greater Box 2The trade-off between costs and benefits is central to many disciplines interested in behavior. In economics, decision making is captured by a comparison between the utility of potential items (goods or services) on offer .In biology, the trade-off between costs and benefits is also central, but what animals are thought to optimize in their behavior is the allocation of energy, rather than utility. This has been captured by the theory of optimal foraging, which predicts that the energy spent to obtain food should not exceed the energy provided by the food obtained Alt-text: Box 2However, because we all know from daily life situations  that any decision relies not only on the anticipated value of future reward but also on predictions of the effort that must be exerted to gain this benefit  et al. Although effort costs did influence performance parameters such as reaction times, it could be argued that this discrepancy between reward and effort coding came from the fact that the weights of the cost and benefit parameters on value were not completely equated, particularly because the willingness of the monkeys to work was only influenced by the effort costs on a small minority of sessions. However, a subsequent study by Varazzani One possible reason for the limited evidence for effort encoding by dopamine, outlined above, is that the studies to date have mostly targeted neurons in the substantia nigra pars compacta, which projects to dorsal parts of the striatum. By contrast, manipulations of dopamine transmission in rodents has strongly implicated mesolimbic pathways from the ventral tegmental area (VTA), particularly to the core of the nucleus accumbens (NAcC), as being necessary to allow animals to select options requiring additional effort for greater reward over a lower-reward alternative To test this, Gan and colleagues However, it is not the case, as has been sometimes described, that there was no influence of effort costs on dopamine levels; the modulation by effort was present, but was weaker and transient  B. As witA complementary picture emerges from a companion study by Hollon and colleagues Box 3For the sake of simplicity, we have chosen to treat the dopamine system as a functional unit. Indeed, our deliberate aim has been to focus on the concordance in the literature regarding dopamine and effort, highlighting the common features that were reliable enough to be described regardless of the species, the task, or the method used to measure activity. Nonetheless, we do not wish to underplay the widespread evidence for diversity of molecular features, anatomical connections, and coding across dopamine neurons. For instance:(i) At the anatomical level, the projections of dopamine neurons vary as a function of their ventromedial to dorsolateral location in the midbrain and of their molecular properties (ii) At a physiological level, a significant proportion of dopamine neurons within particular midbrain regions respond in a stereotyped way to rewards and their predictors (iii) At a functional level, experiments have shown that artificial activation of dopamine neurons or their projections originating from distinct midbrain regions can have distinct functional consequences Although this evidence converges to suggest that the dopamine system can be broken down into several functional units, the exact boundaries between these units and the exact nature of their functions remain to be elucidated. Crucially, the level of description remains strongly dependent upon the question of interest and the scale at which it is studied . A useful comparison may be with primary visual cortex which, for some purposes, can be considered as a functional entity even though it can be broken down into several patches based on ocular dominance or the position of the visual input, and is also part of an interconnected set of cortical regions involved in visual processing.Alt-text: Box 3The emerging picture from the studies discussed above is one of convergence: dopamine is reliably modulated by expectations of future reward, whereas the negative influence of effort costs is much more limited and is only observed in restricted task conditions .How then can one reconcile these data with the careful, elegant experiments showing a strong relationship between the activity of dopamine neurons and quantitative utility prediction error However, although it is indisputable that dopamine encodes parameters about expected future outcomes relevant to a decision, there is also increasing evidence that its function may not be to directly guide action selection between simultaneously available options. Indeed, even when choices are defined solely by differences in outcomes, midbrain dopamine activity and NAcC dopamine levels in response to individual options are not necessarily a reliable predictor of preference because they often signal the value of an upcoming choice even when it is not reward-maximizing What then might be the functions of transient increases in dopamine before effort-related actions? One possibility is that dopamine does not signal predictions of future reward to guide what action to take, but instead provides a signal to shape whether  to act given the potential benefits of taking a presented opportunity in a particular environment. In naturalistic settings, potential rewards are often encountered sequentially rather than simultaneously. This implies that a key computation, recurring across species, is whether or not to engage with a presented opportunity For instance, NAcC dopamine levels are dynamically modulated not only by reward prediction errors but also by whether or not an action should be made to gain the reward We would speculate that this signal incorporates effort costs only if the pursuit of the reward has an influence on the net income or reward rate of the animal. This close connection with rates of reward might also explain the important link between dopamine and time judgments The ideas discussed in earlier sections about the relationship between dopamine and effort emerged primarily from recent correlative recording studies in behaving animals. In addition, there is a history of studies examining the effects of pharmacological manipulations of dopamine transmission during effort-based decision making A frequent observation, for instance, is that blocking dopamine receptors reduces the likelihood and speed of engagement as a function of future reward We have emphasized what we see as a consensus in the literature on the point that effort costs have a limited influence on dopamine activity. This may seem, at first glance, contradictory to the fact that pharmacological manipulations of dopamine often cause pronounced changes in allocation of effort. There are some subtleties here, however, that can perhaps easily be missed, particularly when comparing correlative and interventional approaches. One subtle distinction is that, even if dopamine can promote energy expenditure, it only does so as a function of the upcoming reward, and not as a function of the upcoming (energy) cost itself. Crucially, the few recent experiments that examined the influence of dopamine treatments in tasks where efforts costs and reward benefits were dissociated, also found a stronger influence on reward-based over effort-based decisions If effort costs are not directly encoded by dopamine signals, an obvious question remains: which circuits signal effort costs, and how these might interact with dopamine? As described earlier, there is general agreement that anterior cingulate cortex and dorsomedial motor areas play some role in representing effort, and the former has recently been shown to modulate VTA activity during an effort task The function of dopamine has long generated a great deal of debate, and will likely continue to do so. We highlight here what we believe to be a conspicuous point of consensus. Crucially, this consensus is only arrived at when the data are considered for what they specifically show, putting aside any attempt to fit them into one or other established position. To us, it seems that the data converge to a picture where dopamine signals display strong and consistent reward encoding, but limited and sometimes transient effort encoding.cfOutstanding QuestionsWhat is the timescale of dopamine action? Does the average background (‘tonic’) dopamine level have a role distinct from that of the transient cue-evoked signals in motivating animals to overcome effort, or has the distinction between its action at fast and slow timescales been overstated?Does dopamine also influence movement parameters? NAcC dopamine levels do not straightforwardly correlate with movement times. However, several studies have suggested a link between nigrostriatal dopamine, at least, and the vigor with which an action is performed.What is the relationship between dopamine and other structures, such as anterior cingulate cortex, and other neurochemical systems during effort-related choices?Clearly, there are many issues that remain to be addressed (see Outstanding Questions) and we would be surprised if there are no new points of divergence once various aspects of effort and different dopamine pathways are examined in more detail. For instance, our starting position is that the convergence we observe suggests there may be a unitary, or at least general, function for dopamine in promoting action based on predictions of future benefits cf, 70, 71,


In the presence of Et2AlCl and Cl3CCOOEt (ETA), 1a–1e exhibit good activities for the polymerization of ethylene, affording linear, semicrystalline PEs with a melting temperature of approximately 130 °C. Mainly alternating copolymers with high comonomer incorporation were obtained in the E/NB copolymerization. A relationship was found between the electronic and steric properties of the phosphine ligands and the catalytic performance. Overall, the presence of electron-withdrawing ligand substituents increases the productivity, complexes with aryl phosphine (weaker σ–donor character) exhibiting a higher (co)polymerization initiation rate than those with alkyl phosphines (stronger σ–donor character). Steric effects also seem to play a key role since 1d and 1e, having large size phosphines  are more active than 1a (PMe2Ph θ = 122°). In this case, the larger size of PtBu3 and PCy3 likely compensates for their higher donor strength compared to PMe2Ph.Herein, we report the homo- and co-polymerization of ethylene (E) with norbornene (NB) catalyzed by vanadium(III) phosphine complexes of the type VCl Phosphines are one of the most important families of ligands in organometallic chemistry ,2. The sVanadium is an element of growing importance. With multiple oxidation states and coordination number and flexible geometry, applications of vanadium complexes in catalysis and polymer science continue to grow ,7,8,9,103(PMePh2)2, in combination with Et2AlCl and ETA, catalyzed the E/NB copolymerization  and steric properties  as defined by Tolman [3(THF)3 employed in this study for comparison. First, than MAO . Indeed, Et2AlCl ,10, whil Et2AlCl . The poly Tolman . Table 13(THF)3, likely due to the faster displacement of the THF ligand (indeed THF is expected to be a weak donor ligand and thus to dissociate faster), but also due to different ratios of the dissociated ligand re-insertion rate relative to the ethylene insertion/coordination rate [All the vanadium phosphine complexes exhibited good activities for the polymerization of ethylene, A affordiion rate .Generally, both the activity and the molecular weight of the resulting PEs were strongly affected by the substituents on the phosphorus atom, although the relationship between the catalytic activity, the polymer molecular weight and the phosphine ligand is not straightforward. This is because phosphine ligands are involved in dynamic “on/off” events, which include dissociative and associative pathways, and competitive reactions , and ele3(PMenPh3-n)2 and VCl3(PPh3)2, the activity increased in the order 1a (PMe2Ph) < 1b (PMePh2) ≤ 1c (PPh3) with decreasing ligand donor strength  gave an activity close to that of 1b : in this case, the bigger size of 1e compared to 1b more than compensates for its higher donor strength (1d (PCy3) and 1e (PtBu3). These two complexes have similar electronic properties , but 1e exhibited an activity higher than 1d likely due to the larger cone angle of PtBu3 (θ = 182°) compared to that of PCy3 (θ = 170°) (Changing the substituents on the phosphorus atoms yielded large changes in the ligand donor properties: aryl phosphines (weaker σ-donor character) being weakly coordinated to the metal compared to alkyl phosphines (stronger σ-donor character). However, according to the parameters defined by Tolman , a generstrength A. Stericstrength A. Moreov = 170°) A.3(THF)3, and in some cases even two times more. This is likely because VCl3(THF)3 is more prone to β–H elimination, followed by a fast displacement of a vinyl-terminated polymer, than the vanadium phosphine complexes. Complexes containing phosphines with an increased σ-donor character gave PEs with higher molecular weight: 1e  gave the polymer with the highest molecular weight , while PPh3 (the weakest σ-donor) gave the polymer with the lowest molecular weight , close to that obtained from VCl3(THF)3 . It is worthwhile to note that the molecular weight of PEs obtained from complexes having aryl phosphines increased in the reverse order with respect to the activity; that is 1c  < 1b  < 1a . PEs obtained from the vanadium phosphine complexes had higher molecular weight than the polymer obtained from VCl1a–1e were used for the E/NB copolymerization under the same set of conditions of 1 atm of ethylene, at 20 °C, in toluene, with Et2AlCl (500 equiv to V), ETA (50 equiv to V) and for a NB/E feed ratio of 4. The copolymerizations were performed at short reaction times to ensure the uniformity of the catalytic process, even in the presence of a very high comonomer consumption in the early instants of the reaction, and thus to obtain copolymers with uniform composition [Then, position ,17,24. TNone of the investigated complexes was active in the homopolymerization of NB under the conditions employed, while high activities were obtained for the copolymerization of E/NB in the first seconds of the reaction. When both the comonomers are available for the active intermediates, all the complexes are instantaneously activated and the copolymerization proceeds at significant rates, particularly at the initial stage. The increased activity could be due to the coordination of the nucleophilic and sterically demanding NB that, in some cases, is expected to stabilize the active site .1a–1c, the activity increased in the order 1a (PMe2Ph) < 1b (PMePh2) < 1c (PPh3), 1c being the most active according to the lower donor strength of PPh3  despite the higher donor strength of PtBu3 and PCy3 compared to PMe2Ph (θ = 145°). In this case, the bigger size of 1d and 1e compared to 1b more than compensates for their higher donor strength  giving the copolymer with the lowest molecular weight  close to that obtained with VCl3(THF)3 . Overall, the increase in the ligand σ–donor ability has a positive effect on the (co)polymer molecular weight: a stronger electron-donating ligand may stabilize the electron-deficient vanadium intermediate and reduce the rate of β–H elimination and subsequent chain-transfer [As observed in the case of ethylene polymerization, the average molecular weight of the copolymers generally increased with increasing the ligand σ-donor character, in the order transfer ,41. ConvOn the other hand, the type of phosphine ligand had no significant effect on the NB incorporation and copolymer microstructure. All the copolymers have a NB content ranging from 37.8 to 39.3 mol%, regardless of the steric and electronic properties of the phosphine ligand. The same result was found by some of us ,17, and 13C-NMR. The signals of each chemical shift region were assigned, according to the literature [2  in the range from 73 to 93 °C, thus suggesting that the copolymers are amorphous possibly with a homogeneous composition [Mw, Tg increases with increasing NB incorporation in the copolymers [3(THF)3 , despite the highest content of NB (41.2 mol%), has the lowest Tg (73 °C) even when compared with sample 9 of the same molecular weight . We attribute this result to the fact that sample 12 is probably less homogenous in terms of composition distribution, likely due to a faster initiation efficiency of VCl3(THF)3 and hence to a more pronounced compositional drift. This leads to the formation of NB-rich copolymer chains at the beginning of the copolymerization and E-rich chains at the end, causing a non-homogeneous distribution of the comonomer that helps lower the Tg [The analysis of copolymer thermal properties revealed no melting events, but only a unique glass transition temperature  was refluxed over Na for 8 h and then distilled and stored over molecular sieves. ETA  was stirred over CaH2 for 4 h and then distilled trap-to-trap. Et2AlCl (Aldrich), and VCl3(THF)3 (Aldrich) were used as received without further purification. NB  was stirred over molten potassium at 80 °C under nitrogen for 4 h and then distilled. A stock solution was prepared by dissolving 50 g of freshly distilled NB in 86.2 mL of toluene. A deuterated solvent for NMR measurements (C2D2Cl4)  was used as received. All the vanadium phosphine complexes were synthesized according to that already reported in the literature [The manipulation of air- and/or moisture-sensitive reagents was performed under an inert atmosphere using a dual vacuum/nitrogen line and standard Schlenk-line techniques. Ethylene and nitrogen were purified by passage over columns of CaClterature .2AlCl in that order, and then the solution was degassed, and ethylene was added until saturation. Polymerization was started by adding a toluene solution (2 mg/mL) of a vanadium complex via syringe under continuous flow of ethylene. Polymerizations were stopped with methanol containing a small amount of hydrochloric acid; the precipitated polymers were collected by filtration, repeatedly washed with fresh methanol and finally dried in vacuum at room temperature to constant weight.Polymerizations were carried out in a 25-mL round-bottomed Schlenk flask. Prior to starting polymerization, the reactor was heated to 110 °C under vacuum for 1 h and backfilled with nitrogen. The reactor was charged with toluene, NB, ETA and Et1H) and 100.58 MHz (13C), working in the PFT mode at 103 °C. The 13C experiments were performed with a 10-mm probe in C2D2Cl4 and referred to hexamethyldisiloxane as the internal standard. Thermal analysis was performed with a differential scanning calorimeter (DSC) Perkin Elmer 8000 ). The scans were recorded in a flowing nitrogen atmosphere at scanning rates of 10 °C/min. The molecular weight average (Mw) and the molecular weight distribution (Mw/Mn) were obtained by a high temperature Waters GPCV2000  size-exclusion chromatography (SEC) system equipped with a refractometer detector. The experimental conditions consisted of three PL Gel Olexis columns, ortho-dichlorobenzene (o-DCB) as the mobile phase, a 0.8-mL/min flow rate, and 145 °C temperature. The calibration of the SEC system was constructed using eighteen narrow Mw/Mn poly(styrene) standards with Mws ranging from 162 to 5.6 × 106 g/mol. For SEC analysis, approximately 12 mg of polymer was dissolved in 5 mL of DCB with 0.05% of BHT as antioxidant.NMR spectra were recorded on a Bruker  NMR advance 400 Spectrometer operating at 400 MHz  complexes bearing commercially available monodentate phosphines exhibit good activity for the polymerization of ethylene and the E/NB copolymerization in the presence of Et3 and PtBu3 ligands, exhibiting a higher initiation rate than PMe2Ph (weaker σ–donor). In this case, the larger cone angle of PCy3 and PtBu3 more than compensates for their higher donor strength compared to PMe2Ph. The type of phosphine ligand also strongly affects the (co)polymer molecular weight. Complexes with electron-donating phosphines afford (co)polymers with higher molecular weight, likely because an electron-donating ligand stabilizes the electron-deficient catalytic intermediate, reducing the rate of chain transfer.A relationship was found to exist between the phosphine electronic and steric properties and the catalytic performance. Overall, electron-withdrawing ligand substituents significantly enhance catalytic performance, vanadium complexes with aryl phosphine (weaker σ–donor character) exhibiting a higher initiation rate than those with alkyl phosphines (stronger σ–donor character). Steric properties also have a relevant effect, with complexes with great steric hindrance, such as PCy


Craniopharyngiomas are one of the most frequently diagnosed hypothalamo-pituitary tumors in childhood. The adamantinomatous histological subtype accounts for most pediatric cases, while the papillary variant is almost exclusively diagnosed in adults. Here, we report a case of papillary craniopharyngioma in a very young child, confirmed by molecular tissue analysis.A 4-year-old girl was being investigated for symptomatic central hypothyroidism. Brain MR imaging revealed a large solid/cystic suprasellar mass, splaying the optic chiasm and measuring 3 × 1.9 × 2.3 cm. The patient underwent a transsphenoidal near total resection of the lesion, which was encased within a tumor capsule. Post-operatively, the patient developed transient diabetes insipidus but otherwise recovered well. The pathology of the lesion was consistent with a papillary craniopharyngioma with regions of stratified squamous epithelium accompanied by superficial goblet cells and ciliated cells. Subsequent next-generation sequencing analysis of the lesion confirmed the presence of a BRAF V600E mutation . To date, she remains free from progression 1 year following surgery.This is the youngest case published to date of papillary craniopharyngioma with a confirmed BRAF V600E mutation. The case encourages discussion about the most appropriate adjuvant therapy for tumor progression in such cases, given the risks of radiotherapy to the developing brain and the increasing availability of oral BRAF inhibitor therapy. Craniopharyngiomas are frequently diagnosed hypothalamo-pituitary tumors of childhood . DespiteTwo histopathological variants are recognized . AdamantHere, we report one of the youngest patients to date with a papillary craniopharyngioma, confirmed by histological and molecular analysis. We demonstrate its rarity by literature review and discuss adjuvant therapeutic implications for affected young children, given the concerns regarding conventional radiotherapy and the advent of novel targeted therapy.A 4-year-old girl presented with symptomatic central hypothyroidism and short stature. Initial examination was unremarkable, including normal visual field assessment on confrontation testing.Assessment of pituitary functioning confirmed central hypothyroidism, but also identified cortisol and growth hormone deficiency. Subsequent gadolinium-enhanced magnetic resonance imaging (MRI) of the brain identified a solid/cystic suprasellar lesion, measuring 3 × 1.9 × 2.3 cm , and Rathke’s cleft cyst (RCC) with squamous metaplasia. Histological and clinical assessment discounted LCH, while the absence of Wnt pathway activation excluded an adamantinomatous craniopharyngioma. Likewise, despite sharing histological similarities to a papillary craniopharyngioma, RCC was excluded by the absence of a BRAF point mutation , 19, 27.Papillary craniopharyngiomas are extremely rare in children. This is highlighted by a literature review of published pediatric cases (aged below 16 years), using OVID and Pubmed search with clearly defined terms (Table Our patient remains stable almost 1 year following tumor surgery. Nevertheless, in adults, papillary craniopharyngiomas are often refractory to initial treatment. Therefore, the case encourages discussion on the most appropriate adjuvant therapy for young children with papillary craniopharyngiomas who have significant post-operative tumor residuum or progression. While stereotactic radiotherapy remains the recognized standard, concerns persist regarding neurocognitive, vascular, metabolic, and endocrinological sequelae, even with state-of-the-art proton beam radiotherapy , 20, 21.Moreover, since most papillary craniopharyngiomas harbor the BRAF V600E oncogenic mutation , 24, 25.This case represents the youngest patient reported to date with a papillary craniopharyngioma that has been validated by molecular analysis, revealing a BRAF V600E mutation. While a rare occurrence, such a molecular test, is advocated if, following histopathological analysis, papillary craniopharyngioma and Rathke’s cleft cyst remain possible diagnoses. Given the burgeoning data on the efficacy and tolerability of MAPK pathway drug inhibition, this adjuvant therapeutic option warrants consideration against conventional stereotactic radiotherapy for recurrent or refractory residual pediatric papillary craniopharyngiomas.


Conductive nanofiber mats can be used in a broad variety of applications, such as electromagnetic shielding, sensors, multifunctional textile surfaces, organic photovoltaics, or biomedicine. While nanofibers or nanofiber from pure or blended polymers can in many cases unambiguously be prepared by electrospinning, creating conductive nanofibers is often more challenging. Integration of conductive nano-fillers often needs a calcination step to evaporate the non-conductive polymer matrix which is necessary for the electrospinning process, while conductive polymers have often relatively low molecular weights and are hard to dissolve in common solvents, both factors impeding spinning them solely and making a spinning agent necessary. On the other hand, conductive coatings may disturb the desired porous structure and possibly cause problems with biocompatibility or other necessary properties of the original nanofiber mats. Here we give an overview of the most recent developments in the growing field of conductive electrospun nanofiber mats, based on electrospinning blends of spinning agents with conductive polymers or nanoparticles, alternatively applying conductive coatings, and the possible applications of such conductive electrospun nanofiber mats. Electrospinning is a relatively simple method to produce nanofibers from diverse polymers or polymer blends ,2. EmbedGenerally, electrospinning is performed by pressing a polymer solution or a melt through a needle ,10 or byOne of the possibilities to nevertheless prepare conductive nanofiber mats is based on the carbonization of polyacrylonitrile, the most often used precursor for carbon nanofibers , lignin The number of studies on electrospun conductive nanofiber mats has strongly increased during recent years due to their large field of applications, as visible in One possibility to create conductive nanofibers by electrospinning is based on including conducting nanoparticles or nano-sheets and sintering after electrospinning to remove the non-conductive polymeric matrix . Li et aWang et al. prepared conductive core-shell nanofiber mats by needle-based electrospinning a solution from multi-wall carbon nanotubes (CNTs) in polycaprolactone (PCL) and silk fibroin. While the conductivity of these nanofiber mats is not reported in their paper, the nanofiber mats were found to allow for neurite extension and cell migration along the nanofibers .−5 and 10−2 S/cm, as compared to the conductivity of the pure isolating PU of 10−10 S/cm . Th. Th82]. 3 nanoparticles on electrospun PAN/PAni nanofibers to prepare counter electrodes of DSSCs ..2C nanof3 nanofibers coated with RuO2 nanorods as a sensor for H2O2 and L-ascorbic acid. They could show that by the addition of the RuO2 nanorods, the electrocatalytic activity was increased, and the sensing abilities were significantly improved in comparison with pure WO3 nanofibers, as shown in Lee et al. used electrospun WOTo sense dopamine, Ozoemena et al. used electrospun PAN/onion-like carbon nanofibers and found a high conductivity and sensitivity of the resulting nanofibers . By elecShaker et al. developed a polyurethane/PEDOT:PSS electrospun nanofiber mat which exhibited a resistance of approximately 3 kΩ and could be used as a reliable strain gauge sensor . Yang etHarjo et al. developed conductive fiber scaffolds by coating electrospun glucose-gelatin nanofiber mats with polypyrrole and investigated their electro-chemo-mechanical response, showing stable actuation for more than 100 cycles as well as reasonable sensor properties [Finally, In this review, we report we give an overview of the most recent developments in the research area of conductive electrospun nanofiber mats. As well as the possible applications, varying from biomedicine to sensors to batteries to hydrogen evolution, the range of conductivities achievable with different methods is wide. While conductivities in the range of some 10 µS/cm are sufficient for some biotechnological applications, some techniques such as embedding silver nanowires into the electrospinning solution or coating nanofiber mats with diverse conductive materials result in high conductivities of some 100 to some 1000 S/cm. In many cases, the authors of the cited studies report on additional advantageous findings, such as enhanced biocompatibility or improved fiber diameters.While this review can only give a short overview of the most recent development, it aims at supporting the growing number of researchers working in this highly interesting field of conductive nanofiber mats to find the best solutions for their own applications.


Initial data suggest that prevalence of leprosy infection is variable and often low in different squirrel populations. Nothing is known about the presence of leprosy bacilli in other wild squirrel species despite two others  having been reported to be susceptible to experimental infection with M. leprae. Rats, a food-source in some countries where human leprosy occurs, have been suggested as potential reservoirs for leprosy bacilli, but no evidence supporting this hypothesis is currently available. We screened 301 squirrel samples covering four species  from Europe and 72 Mexican white-throated woodrats  for the presence of M. leprae and M. lepromatosis using validated PCR protocols. No DNA from leprosy bacilli was detected in any of the samples tested. Given our sample-size, the pathogen should have been detected if the prevalence and/or bacillary load in the populations investigated were similar to those found for British red squirrels.Eurasian red squirrels  Mycobacterium leprae and Mycobacterium lepromatosis  in the southern United States of America and Mexico  and sooty mangabey monkeys (Cercocebus atys) in Africa and several primate species have been infected experimentally with leprosy bacilli  in the British Isles where autochthonous human cases have not occurred for centuries  and Thirteen-lined ground squirrels (Ictidomys tridecemlineatus)  , 16, butM. lepromatosis, two human cases of M. lepromatosis infection in central America were linked to the possible consumption of meat from rata de campo or “field rat”  from the Matehuala region were bought at Monterrey meat market. Sample types included pinnae, footpads, liver, and lymph nodes  and M. leprae (RLEP 7 and 8)  or the Qiamp UCP Pathogen kit (Mexican samples) as previously described . PCR amp7 and 8)  and 3 μlM. leprae DNA  into each sample followed by RLEP PCR.Total DNA was extracted from 10 to 100 mg of pinna tissue using the Extracta kit (Quanta Bioscience). PCR amplification was performed using the RLEP and LPM244 primer sets as described above. In addition, a primer set targeting the mitochondrial 12S ribosomal RNA gene of the Eurasian red squirrel genome was used to assess the performance of the Extracta DNA extraction method and to exclude the possibility of PCR inhibitors . For thehttp://www.brixtonhealth.com/pepi4windows.html) DESCRIBE, we calculated the sample size necessary to find at least one case with 95% confidence if leprosy occurred at a prevalence of 21% in a population based on the estimated values currently available for clinically healthy squirrels in the British Isles , was recently published (TLR1 polymorphisms were found more frequently in healthy red squirrels infected with leprosy bacilli compared to diseased animals implying that they may prevent the development of clinical disease. Further investigation of the functional genomics of different squirrel populations is required to understand why some Eurasian red squirrels are susceptible to leprosy bacilli while others are not, and whether higher genetic diversity on the European continent (Immunogenetics, which plays an important role in human and nine banded armadillo leprosy , 38, mayublished . Indeed,ontinent  preventsThis is the first study to investigate the presence of leprosy bacilli in wild populations of squirrels outside the British Isles and in Mexican white-throated woodrats. The lack of detection of leprosy bacilli or clinical signs of leprosy was unexpected given the prevalence of leprosy in red squirrel populations in the British Isles. It shows that further targeted surveillance efforts and increased knowledge of the genetic determinants of leprosy resistance in animal hosts are necessary to fully understand the disease dynamics of leprosy in wildlife populations.A-KS, CA, SC, J-LC, LV-C, AM, KS, PL, and JD-P conceived and designed the study. RU, J-LC, NF, CR, MM, BP, A-KS, WE-F, PL, CS, and JO-C collected samples. A-KS, CA, JM, PB, WE-F, and JO-C extracted DNA. A-KS, CA, JM, KS, PB, WE-F, and JO-C conducted molecular analysis. A-KS and JD-P conducted histopathological analysis. A-KS, CA, SC, KS, AM, PL, and JD-P analyzed and interpreted the data. A-KS, KS, CA, SC, and AM wrote the first draft of the manuscript. RU, CS, and PL wrote sections of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.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.


Modulated differential scanning calorimetry (MDSC), hot stage polarized microscopy (HSPM), and real-time wide-angle X-ray diffraction (WAXD) were used for studying the crystallization and melting behaviors, fiber morphology, and crystalline structure evolution of the LDA and LDH fibers’ homocrystals and stereocomplex crystals during heating. The molecular chain orientations of the LDA and LDH fibers were obtained through spinning and improved through the hot drawing processes. When the molecular chain was oriented on the fiber axis, the homocrystals and stereocomplex crystals of the fibers began to form in turn as the heating temperature exceeded the glass transition temperature of the fiber. The side-by-side packing of the molecular chains was promoted by mixing the molecular chains with the extrusion screw during the spinning process, facilitating stereocomplex crystallization. When the LDA fiber was heated above the glass transition temperature of the fiber, movement of the fiber molecular chain—including molecular chain orientation and relaxation, as well as crystallization, melting, and recrystallization of homocrystals and stereocomplex crystals—were investigated through HSPM. MDSC and real-time WAXD were used to observe the molecular chains of the melted poly(l-lactide) and poly(d-lactide) homocrystals of the fibers rearranging and transiting to form stereocomplex crystals during heating.A series of poly( Blending poly(l-lactide) (PLLA) with poly(d-lactide) (PDLA) results in l- and d- molecular chains packed side by side to form stereocomplex crystals or consisting solely of l- or d- molecular chains to form homocrystals [l-lactide)/poly(d-lactide) (LD) blends was determined to be 165 °C. The growth of concentric spherulites composed of homocrystals and stereocomplex crystals revealed that stereocomplex crystals formed in the first-stage growth acted as a nucleating agent.Polylactide (PLA) is an aliphatic polyester with excellent biocompatibility and biodegradability. Monomer, lactic acid exists in two enantiomeric forms: crystals ,2. The scrystals ,4 reportcrystals  also repThe homocrystal (α-form) to stereocomplex crystal transition was identified as a melting and recrystallization process. Fujita et al.  reportedModulated differential scanning calorimetry (MDSC) has been proven to be a useful technique for understanding various thermal events and for analyzing them more precisely where standard DSC fails to separate some overlapping processes . SolarskA fiber was obtained at a spinning temperature of 250 °C with a take-up velocity of 2 km/min and a 36-hole spinneret. The hot-drawn LDH fiber was prepared in a stretching machine of a set of drawing rolls and a heating chamber at a draw ratio of 1.6 to 2.1. The drawing temperature was 105 °C. The compositions of the LD blends used in this study are listed in PLLA (Synterra PLLA 1510) and PDLA (Synterra PDLA 1010) were supplied in chip form by Synbra Technology BV . The initial thermal degradation temperatures of PLLA and PDLA were 305 °C and 307 °C, respectively, as measured by thermogravimetric analysis . A series of PLLA/PDLA blends were prepared through melt-blending various PLLA:PDLA chip weight ratios in a mixer at 210 °C with a rotation speed of 100 rpm and a throughput speed of 8 k/h. The feed ratios of PLLA:PDLA weights were 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, and 10:90. Using a laboratory-scale melt spinning system, the as-spun LD2 was used as the purge gas at a flow rate of 50 cm3/min. The sample weight was 4–6 mg.The thermal properties of the samples were investigated using a differential scanning calorimeter  with a modulation and a thermal analysis system . In and Pb were used for temperature calibration. Sapphire was used for heat capacity calibration. N2 was 120 cm3/min. The heating rate was 10 °C/min. The birefringence of the fibers was measured using a polarizing microscope  with a Berek compensator  and a monochromatic light absorption filter .The morphology and structure evolution of the fibers during heating were observed using a polarizing microscope  with a heater , an electric microscope controller , and a digital camera . The flow rate of NC chips, as-spun LDA fibers, and hot-drawn LDH fibers. The diffraction patterns were recorded using a Mar345 imaging plate area detector . The reflection profile was deconvoluted using Jade 6 curve-fitting software  with the peak search method, and a full width at half maximum value of 3 was applied to separate the amorphous phase and crystal reflections.Endstation BL17A1  of the Nation Synchrotron Radiation Research Center, Hsinchu, Taiwan, was used to observe the crystalline structures of LDC, DC, and LDC chips were first melted at 290 °C for 3 min, then quickly cooled to 140 °C, and then subjected to isothermal crystallization for 120 min. A heater  equipped with an electric microscope controller  was used for sample preparation. A series of 1D WAXD profiles with deconvoluted reflections and 2D WAXD patterns for the LC, DC, and LDC chips are provided in C and DC exhibited four 2θ diffraction peaks in the 1D profiles and four diffraction rings in the 2D patterns, which related to (010), (110/200), (203), and (015) planes of the homocrystals. For the LDC chips, seven 2θ diffraction peaks were seen, including (110), (010), (110/200), (203), (300/030), (015), and (220) planes from the center of the 2D profile outwards. Here, (110), (300/030), and (220) planes belonged to a stereocomplex crystal.For WAXD crystalline structure analysis, the LA and DA were not significantly different from those of LDA. This means that whether molecular chains were packed side by side or in the solely L or D form did not affect the orientation of molecular chains during spinning at a temperature of 250 °C at a take-up velocity of 2 km/min. The molecular chain orientation of the as-spun fiber was further improved during the hot drawing process  and (203) related to homocrystals were observed on all of the hot-drawn fibers  and (203) reflections related to the homocrystal appear in the L5DA fiber. At temperatures of 70 °C to 105 °C, the diffraction intensities of the reflections (110/200) and (203) increased, and a new weak (110) reflection associated with the stereocomplex crystal appeared. This indicates that the temperature was higher than the Tg of L5DA, the molecular chain of the fiber relaxed and then crystallized, and the homocrystal and stereocomplex crystal were sequentially formed. As the temperature was further increased to 150 °C, (300/030) and (220) reflections associated with the stereocomplex crystals appeared; however, the diffraction intensities of the (110/200) and (203) peaks decreased. The melting of homocrystals was combined with the formation of stereocomplex crystals. When the temperature reached 181 °C, the homocrystal completely melted. The diffraction intensities of (110), (300/030), and (220) peaks increased to 181 °C and then gradually decreased. All reflections almost disappeared at 250 °C. When the temperature exceeded Tg, the homocrystal and stereocomplex crystal formed sequentially; and during the heating at approximately 105 °C to 180 °C, stereocomplex crystal formation occurred simultaneously with the melting of the homocrystal.The homocrystallization and stereocomplex crystallization of the as-spun and hot-drawn fibers during heating was further investigated using a real-time WAXD measurement. The as-spun L5DH fiber obtained at the set temperatures and the corresponding intensity curves are depicted in H fiber had two (110/200) and (203) reflection arcs associated with the homocrystal. When the fiber was heated to 75 °C, the diffraction intensities of (110/200) and (203) peaks increased. At temperatures of 75 °C to 105 °C, the diffraction intensities of (110/200) and (203) peaks increased, and a new weak (110) reflection associated with the stereocomplex crystal appeared. When the temperature was further increased to 162 °C, (300/030) and (220) reflections associated with the stereocomplex crystals appeared; however, the diffraction intensities of (110/200) and (203) peaks were significantly lowered. The (110/200) and (203) reflections related to the homocrystal continued to exist from 162 °C to 181 °C. The diffraction intensities of (110), (300/030), and (220) peaks increased to 181 °C, then gradually decreased and all reflections disappeared completely at 250 °C. The results demonstrate that stereocomplex crystallization of L5DH was almost the same as that of L5DA. All L5DH fibers exhibited arc reflections, and all L5DA fibers exhibited ring reflections. This indicates that the molecular chain orientation of the L5DA fiber was improved after the hot-drawing process.The 2D WAXD patterns of the L5DA fibers obtained at a take-up velocity of 2 km/min did not crystallize during spinning at 250 °C. All of the as-spun LDA fibers were in an amorphous state with a slight molecular chain orientation along the fiber axis. Compared to the chips, the oriented molecular chains of the corresponding as-spun fiber are induced to form homocrystal and stereocomplex crystals earlier in the subsequent heating process. The cold crystallization related to the homocrystal occurred just after the molecular chain relaxation of the as-spun fiber, and the recrystallization and melting behaviors of the homocrystal of the as-spun fiber then overlapped in the range of 130 °C to 180 °C. As the molecular chains were further mixed by the extrusion screw during the spinning process, side-by-side packing of the molecular chains was promoted. This resulted in the melting enthalpy of the homocrystal of the as-spun LDA fiber being lower than that of the corresponding LDC chip, and the melting enthalpy of the stereocomplex crystal of the as-spun LDA fiber was higher than that of the corresponding LDC chip. The cold crystallization of the stereocomplex occurred just after the cold crystallization of the homocrystal of the as-spun fiber. The MDSC results indicated that the stereocomplex crystal was formed mainly during recrystallization, which means that the molecular chains of the molten PLLA and PDLA were rearranged and transited to form stereocomplex crystals; the recrystallization and melting behaviors of the stereocomplex crystal overlapped in the range of 170 °C to 220 °C.In this research, a series of the LD chips, as-spun fibers, and hot-drawn fibers were prepared for studying homocrystallization and stereocomplex crystallization during heating. WAXD analysis showed that the as-spun LDTg. The homocrystal and stereocomplex crystal were sequentially formed just after the relaxation phenomenon and subsequent melting and recrystallization of the homocrystal. The molecular chains of the molten PLLA and PDLA were rearranged and transited to form the stereocomplex crystal, and then melting and recrystallization of the stereocomplex crystal occurred. Compared with standard DSC, MDSC more accurately separated the homocrystallization and stereocomplex crystallization behaviors, and HSPM photographs closely matched to MDSC results.HSPM observations, MDSC analysis, and real-time WAXD measurements indicated that the molecular chain of the fiber relaxed when the temperature exceeded