The NaCS/PDMDAAC shows a synergetic enhancement effect toward the oxidation of nitrite, and the sensing performance is much better than the previous reports. Moreover, the NaCS/PDMDAAC also shows good stability and reproducibility. The NaCS/PDMDAAC/GCE was successfully applied to the determination of nitrite in ham sausage with satisfactory results. To ensure food quality and safety, developing cost-effective, rapid and precision analytical techniques for quantitative detection of nitrite is highly desirable. Herein, a novel electrochemical sensor based on the sodium cellulose sulfate/poly  (NaCS/PDMDAAC) composite film modified glass carbon electrode (NaCS/PDMDAAC/GCE) was proposed toward the detection of nitrite at sub-micromolar level, aiming to make full use of the inherent properties of individual component  and synergistic enhancement effect. The NaCS/PDMDAAC/GCE was fabricated by a simple drop-casting method. Electrochemical behaviors of nitrite at NaCS/PDMDAAC/GCE were investigated by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). Under optimum conditions, the NaCS/PDMDAAC/GCE exhibits a wide linear response region of 4.0 × 10 Nitrite is widely used as a preservative in food industry, especially in the production and processing of cured meat and fishery products . AdditioTo date, many techniques have been developed for the detection of nitrite, including chromatography ,11, UV sPoly  (PDMDAAC) is a ubiquitous linear cationic polymer that has unique properties, such as biocompatibility, low cost, high charge density, as well as good water solubility . It has 6]3−/4− as redox probe solution. The analytical parameters (including pH) were systemically explored. Finally, the content of nitrite in ham sausage was determined by differential pulse voltammetry (DPV) with satisfactory results.Herein, NaCS/PDMDAAC composite film as prepared to construct a novel electrochemical sensor for the detection of nitrite at the sub-micromolar level, aiming to make full use of the merits of individual components  and synergistic enhancement effect . NaCS/PDThe surface morphologies of NaCS film and NaCS/PDMDAAC composite film were characterized by scanning electron microscope SEM, . As show−3 mol L−1 K3[Fe(CN)6] solution. The cyclic voltamograms recorded on various electrodes are shown in Ep) of 137 mV. The anodic peak current (ipa) and cathodic peak current (ipc) is 43 μA, and 58 μA, respectively. On the NaCS modified electrode (curve b), the ∆Ep increases to 186 mV. Meanwhile, the peak currents also increase slightly, with ipa of μA and ipc of 69 μA, respectively. When modified with PDMDAAC alone (curve c), a pair of obvious and well-shaped redox peaks appears with a significant decrease on ∆Ep (101 mV). The corresponding redox peaks increase greatly, with ipa of 81 μA and ipc of 80 μA, respectively. When modified with NaCS/PDMDAAC composite film (curve d), the ∆Ep (143 mV) has a slight increase compared to that on the NaCS/GCE. The ipa and ipc is 72 μA and 89 μA, indicating that the NaCS/PDMDAAC film facilitates the electron transfer between the electrode and analyses, and eventually improves the electrochemical response. The electrochemical performance of bare GCE, NaCS/GCE, PDMDAAC/GCE, and NaCS/PDMDAAC/GCE were investigated using cyclic voltammetry (CV) recorded in 5 × 10−3 mol L−1 K3[Fe(CN)6] solution by CV. As illustrated in The influence of layers of NaCS/PDMDAAC composite film was also investigated in 5 × 10–3 mol L–1 K3[Fe(CN)6] solution. The Nyquist plots of different electrodes were shown in ct) were estimated by EIS fitting with the equivalent circuit. The Rct of bare GCE, NaCS/GCE, PDMDAAC/GCE, and NaCS/PDMDAAC/GCE are 37,830 Ω, 104,800 Ω, 36,810 Ω, and 52,190 Ω, respectively, i.e., the conductivity (1/Rct) of them are 2.6434 × 10–5 S, 9.5420 × 10–6 S, 2.7167 × 10–5 S, and 1.9161 × 10–5 S, respectively. Apparently, the conductivity of the bare GCE (2.6434 × 10–5 S) is slightly lower than that of PDMDAAC/GCE (2.7167 × 10–5 S) but much higher than that of NaCS/GCE (9.5420 × 10–6 S), indicating that when each of them is independently modified onto the bare electrode, the polycation PDMDAAC can improve the conductivity while the polyanion NaCS is not favorable for electron transfer. As is well known, both PDMDAAC and NaCS are polyelectrolytes and, theoretically, should have good conductivities. However, these polyelectrolytes are quite different in charge density, solubility, and counter-ion effects in solution , all of which are thought to be able to influence the conductivity, and the former two factors are thought to be favorable for conductivity while the last one is unfavorable. In fact, the molecular weight of PDMDAAC used in our experiment is less than 1.0 × 105, while NaCS has a much bigger average molecular weight more than 1.9 × 106, and so it is obvious that PDMDAAC contains much shorter molecular chains, which reinvests it with a higher charge density, a better solubility, as well as a weaker counter-ion effect due to narrower interfaces to attract less opposite-charged ions. Thus, although both being polyelectrolytes, PDMDAAC can improve the conductivity of the modified GCE while NaCS cannot, as the above results show. However, it is worth noting that when using the composite NaCS/PDMDAAC modifies the bare GCE, the Rct of NaCS/PDMDAAC/GCE  is only about half of that on the NaCS/GCE , suggesting the unfavorable counter-ion effect of NaCS is significantly weakened after the introduction of PDMDAAC, which actually improves the charge transfer efficiency. Obviously, the conductivity of NaCS/PDMDAAC/GCE (1.9161 × 10−5 S) is better than that of NaCS/GCE (9.5420 × 10–6 S). Thus, by considering the outstanding conductivity of PDMDAAC as well as the good performances of NaCS for film formation, the composite NaCS/PDMDAAC is the most suitable material for the modification of GCE to construct the new and effective sensor for nitrite detection, herein with a moderate Rct/conductivity value, which is consistent with the above research result conducted by the CV method. Electrochemical impedance spectroscopy (EIS) is a useful tool to acquire abundant information about electrode interface, which have been widely used in the electrocatalysis ,42, chem–1 PBS solution containing 4.0 × 10–3 mol·L–1 nitrite  firstly. Then the response peak currents of 1.0 × 10–4 mol·L–1 nitrite on these electrodes were recorded and compared. The effect of modifier loading on the response peak current was shown in The effect of the modifier loading amount is one of important factors that affect the electrochemical response of modified electrode. The bare GCEs were modified with various dosage of NaCS/PDMDAAC dispersion (w/v) is 7%, the peak current reaches the maximum. Afterwards the response peak current decreases with further increase on PDMDAAC concentration. Therefore, 7% was selected to be the suitable PDMDAAC concentration for the following measurements.The effect of the PDMDAAC concentration on the response peak current of nitrite was also explored. The results are shown in –4 mol·L–1 nitrite were measured in various pH (3.0–10.0), and the result was presented in 2- is very unstable and easily reacted with H+ to produce NO and NO2 gas. As a result, the response peak current declines as the pH decreasing. In addition, most amount of the NO2− is protonated in such acidic environment (pKa(HNO2) = 3.3). Under alkaline pH ranging of 8.0–10.0, the increase on pH leads to the decline of the response peak current due to the lack of sufficient H+. It is well known that pH plays a crucial role on the oxidation of nitrite, so it’s well worth investigating the influence of pH. The response peak currents of 1.0 × 10As we all know, accumulation time directly influences the adsorption of analyses at the surface of modified electrode. The NaCS/PDMDDA/GCE was accumulated for various times firstly, then corresponding response peak currents were record and compared D. During−8 mol·L–1 to 1.5 × 10–4 mol·L–1. The corresponding linear regression equation can expressed as i (μA) = 0.01027c (μmol·L–1) + 0.02771 . The limit of detection  is estimated to 4.3 × 10–8 mol·L–1. A comparison on sensing performances between reported electrodes and NaCS/PDMDAAC/GCE is summarized on the Under the optimized analytical conditions, the response peak currents of various concentrations of nitrite were measured by NaCS/PDMDAAC/GCE using differential pulse voltammetry (DPV). The response DPV curves of various concentrations of nitrite are shown in –5 mol·L–1 sodium nitrite mixed with the equal concentration of potential interfering substances including sodium chloride, glucose, l-cysteine, and ascorbic acid. The response peak current is hardly changed with these interfering compounds (peak current variation within 5%), indicating the nitrite is bonded specifically to the NaCS/PDMDAAC composite film and interfering substances cannot interfere with the nitrite detection. In other words, the proposed sensor has good specificity and selectivity toward nitrite. Before detection nitrite in real samples, the practicability  of the proposed NaCS/PDMDAAC/GCE were checked carefully. Firstly, the interference investigation was performed in 1 × 10−5 mol·L‒1 and 8.0 × 10‒6 mol·L–1 nitrite solution alternately. The result is shown in Then, the reproducibility of NaCS/PDMDAAC/GCE was validated by repeatedly measuring the response peak currents for 9 times in 8.0 × 10‒1 PBS solution (pH 7.0) and stored at 4 °C for 30 days. The cyclic voltammetric response of nitrite at the NaCS/PDMDAAC/GCE was measured every 3 days. As shown in Finally, the stability of the proposed sensor was investigated by continuously monitoring the response peak current for 30 days. The NaCS/PDMDAAC/GCE was always immersed in 0.1 mol·L–1 PBS (pH 7.0) using the proposed NaCS/PDMDAAC/GCE, and determination results are listed in Under the optimal analytical conditions, the pretreated ham sausage filtrate was used as the actual sample. The concentration of nitrite was detected by DPV method in 0.1 mol·L−8 mol·L–1~1.5 × 10−4 mol·L−1, and low detection limit of 43 nmol·L–1 (S/N = 3). Moreover, the proposed NaCS/PDMDAAC/GCE also has good stability and reproducibility. Finally, the NaCS/PDMDAAC/GCE was successfully used to detect the nitrite content in the ham sausage sample with satisfactory results. The proposed NaCS/PDMDAAC/GCE provided a promising sensing platform on the detection nitrite in diverse real samples at the sub-micromolar level.In this work, a novel electrochemical sensor based on NaCS/PDMDAAC composite film was proposed for the detection of nitrite at sub-micromolar level. The NaCS/PDMDAAC/GCE was fabricated by a facile drop-casting method. The surface morphology of NaCS film and NaCS/PDMDAAC composite was characterized by SEM. The electrochemical behavior of nitrite at the NaCS/PDMDAAC was investigated by CV and DPV. The NaCS/PDMDAAC not only shows the inherent properties from the individual component, but also exhibits synergetic enchantment effect toward sensing nitrite. Under optimal detection conditions, the proposed NaCS/PDMDAAC/GCE exhibits a wide linear response region of 4.0 × 10Poly (PDMDAAC) was supplied by Aldrich Co., Ltd . Cellulose sulfate sodium salt was purchased from J&K SCIENTIFIC LTD . Borax and sodium nitrite were purchased from Hunan General Chemical Reagent Works . Potassium ferricyanide, potassium hexacyanoferrate, sodium dihydrogen phosphate and disodium hydrogen phosphate were all bought from Sinopharm Chemical Reagent Co., Ltd . All the chemicals were of analytical reagent grade and were used as received without any further purification. Both PDMDAAC and NaCS were also synthesized in our experiments  PDMDAAC & (b) DMDAAC have been also plotted in the w/v)) solution was drop-casted on the surface of GCE, dried naturally to obtain PDMDAAC/GCE. Secondly, 3 μL NaCS (3.5% (w/v)) solution was dropped-casted to the surface of PDMDAAC/GCE, dried naturally to obtain NaCS/PDMDAAC/GCE. The synthesis of NaCS and PDMDAAC has been described in the –3 mol·L–1 K3[Fe(CN)6] solution. The electrochemical behavior of nitrite at the NaCS/PDMDAAC/GCE was investigated by CV. The content of nitrite was detected by differential pulse voltammetry (DPV). The supporting electrolyte for all electrochemical measurements is 0.1 mol·L–1 Na2HPO4-NaH2PO4 buffer solution  unless otherwise stated.All electrochemical experiments were performed on the CHI-760B electrochemical workstation . A standard three-electrode system is used for all electrochemical experiments, consisting of bare GCE or modified electrodes as the working electrode, platinum wire as the counter electrode, and an Ag/AgCl electrode (saturated potassium chloride) as the reference electrode. The electrochemical performance was measured by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) in 5 × 10The ham sausage samples were purchased from a local supermarket. 12.5 mL of borax saturation solution was added into 5 g ground ham sausage, and stirred to mix well. Then the ham sausage sample was washed into 50 mL volumetric flask by a small amount of hot water, and heated in boiling water bath for 15 min. Afterwards the volumetric flask was cooled to room temperature. Subsequently, 2.5 mL PBS solution was added into the volumetric flask, and then dilute with distilled water to 50 mL. After 30 min rest, the upper layer of fat or oil was removed, the spare liquor was extracted by suction filtration to obtain sample solution for quantitative analysis.


Structural diversity in α-helical membrane proteins (MP) arises from variations in helix-helix crossings and contacts that may bias amino acid usage. Here, we reveal systematic changes in transmembrane amino acid frequencies (f) as a function of the number of helices (n). For eukarya, breaks in f(n) trends of packing , polar, and hydrophobic residues identify different MP assembly principles for 2≤n≤7, 8≤n≤12 and n≥13. In bacteria, the first f break already occurs after n = 6 in correlation to an earlier n peak in MP size distribution and dominance of packing over polar interactions. In contrast to the later n brackets, the integration levels of helix bundles continuously increased in the first, most populous brackets indicating the formation of single structural units (domains). The larger first bracket of eukarya relates to a balance of polar and packing interactions that enlarges helix-helix combinatorial possibilities (MP diversity). Between the evolutionary old, packing and new, polar residues f anti-correlations extend over all biological taxa, broadly ordering them according to evolutionary history and allowing f estimates for the earliest forms of life. Next to evolutionary history, the amino acid composition of MP is determined by size (n), proteome diversity, and effective amino acid cost. This puts MP in control of key physiological and pathological processes as is evident from the large number of drugs that target MP . GenomesMP traverse the lipid bilayer either as α-helices or β-sheets. To fulfill hydrogen bonding requirements when traversing the membrane core, β-sheets must form closed barrel structures , 7. In cIn the course of evolution, as organisms developed increasingly complex functional needs, the repertoire of MP structures must have expanded to implement these functions. Structural diversity in mainly parallel TM helix bundles invariably relates to helix count and length, the number and location of helix-helix crossings, and the chemical nature of helix-helix interactions. Certain aspects of these properties can be quantified from the amino acid (AA) sequence of MP. The number of TM helices, termed n, and helix length can be predicted computationally , 8. The To achieve an even coverage of TM sequence space at a resolution that is expected to include most of the currently existing MP structures while still allowing variability at non-conserved sequence positions, we studied the representative protein clusters compiled in the UniRef50 database. First, we provide an overview of MP size distribution for eukarya and bacteria. For bacteria, the distribution peaks at n = 6, which is earlier than for eukarya for which a large number of MP entries was obtained until n = 7 . When exH) is in fact lower in bacteria than in eukarya for all n  profiles that were expanded to individual residue positions along the membrane normal, termed m, with the TM helix center taken as m = 0. Studies of immersion depth preferences of AA provide valuable insight into MP architecture –18. The The f profiles of a number of residues identified pronounced f differences between peripheral and center positions. For example, the highest f values of Asn were found near the membrane border irrespective of n in confirmation of an important role in TM helix capping . In addivice versa  decreases almost linearly or remains flat up up to n = 12 after which this trend breaks down and a substantial increase in hydrophobic residues is observed . Apparenby 13.2% , which dby 13.2% . A lowerMP state . Complemecreased . Only Gld 8≤n≤12 .Gly and fPro may increase with n. Except for a slight weakness at n = 3, this expectation was fulfilled by Pro up to n = 12  profiles of eukaryotic MP suggest a functional AA grouping into hydrophobic , polar , packing , anchoring , and charged  residues. The corresponding f(n) trends indicate the existence of three n regions with distinct MP assembly principles: 2≤n≤7, 8≤n≤12 and n≥13.Pro for 1≤n≤6  that led to an organism as indicator of this process " or "Single-pass membrane protein [SL-9904]", protein existence codes 1–3, and the desired taxonomic association were initially selected. UniProt classifies protein existence in five categories  where ATP denotes the cost of synthesizing AA X de novo in E. coli [The Shannon diversity index (H) is defined as–Σ p E. coli  and the S1 Fig(a-b) f profiles of Gly for eukarya and bacteria where m denotes the residue number relative to the predicted TM helix center. Negative and positive m values indicate orientations toward the N- and C-terminus, respectively, and do not relate to extra- and intracellular orienations. In eukarya, the helix-helix association motif GxxxG  was dire(EPS)Click here for additional data file.S2 Figblack; see red), and UniProt TM borders shrunk by four residues on each side (green), respectively. Solid lines show running averages calculated with a window size of two. The n range was limited to ≤19 because of the scarcity of MP for large n  was calculated using UniProt TM borders plus Monte Carlo simulations ( large n .(EPS)Click here for additional data file.S3 FigSolid lines depict running f(n) averages calculated with a window size of two. The n range was limited to ≤19 because of the scarcity of MP for large n .(EPS)Click here for additional data file.S4 Fig(EPS)Click here for additional data file.S5 Fig(EPS)Click here for additional data file.S6 Fig(EPS)Click here for additional data file.S7 Fig(EPS)Click here for additional data file.S8 FigPhe and fX in the 2≤n≤7 range were averaged for the nine taxonomic groups shown and correlated using fX = a·fPhe + b. Fitted parameters are summarized in For AA X, f(EPS)Click here for additional data file.S9 FigPhe and fX in the 2≤n≤7 range were averaged for the nine taxonomic groups shown and correlated using fX = a·exp(b·fPhe) + c. For AA with rising fX c = 0 was assumed. Fitted parameters are summarized in For AA X, f(EPS)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


Over 1.5 million individuals in the United States are afflicted with inflammatory bowel disease (IBD). While the progression of IBD is multifactorial, chronic, unresolved inflammation certainly plays a key role. Additionally, while multiple immune mediators have been shown to affect pathogenesis, a comprehensive understanding of disease progression is lacking. Previous work has demonstrated that a member of the TNF superfamily, TNFSF14 (LIGHT), which is pro-inflammatory in several contexts, surprisingly plays an important role in protection from inflammation in mouse models of colitis, with LIGHT deficient mice having more severe disease pathogenesis. However, LIGHT is a single member of a complex signaling network. It signals through multiple receptors, including herpes virus entry mediator (HVEM) and lymphotoxin beta receptor (LTβR); these two receptors in turn can bind to other ligands. It remains unknown which receptors and competing ligands can mediate or counteract the outcome of LIGHT-signaling during colitis. Here we demonstrate that LIGHT signaling through LTβR, rather than HVEM, plays a critical role in the progression of DSS-induced colitis, as LTβR deficient mice exhibit a more severe disease phenotype. Further, mice deficient in LTαβ do not exhibit differential colitis progression compared to WT mice. However, deletion of both LIGHT and LTαβ, but not deletion of both LTαβ and LTβR, resulted in a reversal of the adverse effects associated with the loss of LIGHT. In sum, the LIGHT/LTαβ/LTβR signaling network contributes to DSS colitis, but there may be additional receptors or indirect effects, and therefore, the relationships between these receptors and ligands remains enigmatic. Inflammatory bowel disease is an immune-mediated disease in which, among other components, the microbiome, genetics and immune system all contribute to disease . Multitulymphotoxins, exhibits inducible expression, and competes with HSV glycoprotein D for HVEM, a receptor expressed by T cells]) in transgenic mice leads to colitis (+ T cells to immune deficient mice (The tumor necrosis factor (TNF) superfamily of cytokines and receptors have a diverse, but not fully defined, function in mucosal immunity and IBD pathogenesis –7. In fa colitis , 10. Als colitis , and LIG colitis , 13. On ent mice . This waLIGHT can bind two receptors, the lymphotoxin beta receptor (LTβR or TNFRSF3) and the herpes virus entry mediator (HVEM or TNFRSF14) , HVEM deHere, we report the generation and analysis of a variety of double-mutant mice to delineate the complex interplay of LIGHT/LTαβ with LTβR/HVEM signaling during DSS-induced colitis. We demonstrate that LIGHT signaling through LTβR is indispensable for protection from exacerbated DSS-induced colitis. Additionally, HVEM activation does not seem to contribute to DSS-induced colitis, even in the absence of LTβR. While LTαβ signaling by itself is not critical for altering the severity of colitis, LTαβ deletion rescued the pathogenic effect of LIGHT deletion, but not of LTβR deletion. This shows that the role of specific ligands becomes difficult to predict when multiple members of the TNF superfamily are depleted, and suggests the possibility that LTαβ has effects that extend beyond its interaction with LTβR, or alternatively, that LTβR integrates additional signals to affect the outcome in DSS-colitis.Ltbrfl/fl mice to CMV-cre for ubiquitous depletion of LTβR . Ad. AdLtbrfh Figure , typical Figures  5), ind, indLtbrLtb−/− mice exhibited weight loss and colon lengths similar to WT controls after DSS treatment  mice deficient for LTαβ and LTβR and determined if these mice exhibited augmented DSS-induced colitis progression. In these DKO mice, LIGHT-HVEM interactions occur independently of a possible HVEM competition with LTβR for binding to this ligand. Administration of DSS to s Figure . This ine Figure . Further Figures . The exaLtbr−/−Hvem−/− DKO mice. Notably, if HVEM signals drive severe inflammation in the absence of LTβR, mice deficient in both receptors should be protected from exacerbated DSS-induced colitis. However, after administration of DSS, Ltbr−/−Hvem−/− mice exhibited increased weight loss after DSS treatment, similar to Ltbr−/−Hvemhet(+/−) mice  to Ltrbfl/fl mice, that were previously described (Tnfsf14−/−) and Ltb−/− mice were provided by Dr. Klaus Pfeffer  . All mice were on the C57BL/6J background. C57BL/6J were originally purchased from the Jackson Laboratory. HVEM mice were bred and described previously . Ltbr−/−escribed . LIGHT dGermany) . Double Mice received 2.5% DSS (Affymetrix) in the drinking water for a maximum of two cycles. As previously described, 1 cycle is comprised of 5 days of water plus DSS and 2 days with regular drinking water without DSS . Given tUpon termination of an experiment, cecum and colon were isolated. Following measurement of colon length, a piece of distal colon and cecum were fixed in zinc formalin . Following paraffin embedding, fixed tissue was stained with hematoxylin and eosin. Resulting slides were then blinded and scored according to previously described criteria . Represet-test for direct comparisons when there were two groups. For determination of statistical significance for three or more groups, one-way ANOVA was employed with Tukey's post hoc test to assess differences between specific groups. All data are displayed as mean with standard error of the mean (S.E.M.).All data were analyzed using GraphPad Prism 7 software. Statistical significance was determined by unpaired Student's MK and AT contributed to the design of the study. DG, SZ, PK, EV, TR, and VM performed the experiments. DG, SZ, PK, and MK analyzed the data. DG and MK drafted 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.


The Langmuir, pseudo-second order, and intraparticle diffusion models could well depict the adsorption equilibrium and kinetics of CL powders toward the investigated reactive dyes. The as-prepared CL powders can be used as a potential adsorbent in the treatment of dye contaminated wastewater. Future studies will mainly focus on the application of the adsorbed CL powders for the pigment printing of textile materials.As a natural polymer, leather and its associated industries are known to be the leading economic sector in many countries. However, the huge amounts of leather waste generated from the leather industry causes severe environmental pollution. Herein, cow leather (CL) powders were prepared using a homemade machine and used as a low-cost adsorbent for the effective removal of reactive dyes from wastewater. The as-prepared CL powders exhibited dot-like, rod-like, and fiber-like morphologies. A Fourier transform infrared analysis and an x-ray diffraction analysis demonstrated that the CL powders retained the main structure of the protein contained in it. In addition, an improvement in thermal stability was also observed for the CL powders. Dye adsorption experiments indicate that the CL powders showed the highly effective removal of C.I. Reactive Red 120 (RR120), C.I. Reactive Yellow 127 (RY127), and C.I. Reactive Blue 222 (RB222) with the adsorption capacity of 167.0, 178.9, and 129.6 mg·g Water security is certain to be a major topic facing humanity. It has been reported that the freshwater ecosystem only occupies nearly 1% of the Earth’s surface . AdditioRecently, various technologies such as adsorption ,7,8,9, pLeather products in the form of protein are widely used in daily human life . HoweverSignificant research and attention in a number of publications has focused on the isolation and production of collagens from leather waste, and on more detailed reviews of the pretreatment method such as chemical treatment (oxidation of hydrolyzes) and enzymatic digestion ,19. An iPowder, a popular and low-cost technology, has been proven to be an effective and economical absorbent for removing dyes from wastewater ,16,30. IIn this paper, a homemade instrument ,35, whic2HPO4·12H2O), citric acid monohydrate (C4H2O7·H2O), Sodium hypochlorite (NaClO), and absolute ethanol were purchased from Sinopharm Chemical Reagent Co. Ltd., Shanghai, China. The RR120, RY167, and RB222 dyes were kindly supplied by Luthai Textile Co. Ltd., Shandong, China, and the chemical structures of the dyes are shown in Waste cow leather (CL) was supplied by a local tannery in Zhejiang Province, China. Acetic acid (HAC), sodium hydrogen phosphate . The parameters of pH (3–8), contact time (0–100 min), and dye concentration (50–1200 mg·L−1) were investigated to examine the adsorption properties of CL powder toward the three dyes.Typically, reactive dyes play an essential role in the textile dyeing industry , which oQe, mg·g−1) was calculated using Equations (1) and (2) [C0 and eC are the initial and equilibrium concentrations of the dye solutions (mg·L−1), respectively; V is the volume of dye solution (L); and m is the mass of the CL powder.The dye removal efficiency (% RE) and the adsorption capacity at equilibrium ( and (2) ,40, resp−1 with 256 scans at the resolution of 4 cm−1. All measurements were repeated three times from three different spots on the same sample. X-ray diffraction (XRD) was performed on an X’pert Pro, PANalytical B.V., Holland using Cu kα radiation where the irradiation conditions were 40 kV and 40 mA. Samples were scanned from 5 to 70° (2θ) with a step length of 0.02°. Thermo-gravimetric analysis (TGA) and derivative thermogravimetric (DTG) curves were obtianed by using NETZSCH TG 209 F1  in flowing nitrogen gas (20 mL·min−1). The samples were heated from 25 °C up to 800 °C with a heating rate of 10 °C·min−1.Photographs of the CL and CL powder were measured using a digital camera (Canon EOS 80D). The maximum Feret diameter (MFD) of CL powder was observed using optical microscopy . The CL powder was ultrasonically dispersed in an ethanol–water mixture (90:10 wt.%). Then, the CL powder solution was placed onto a glass slide for observation. The typical morphology of the CL powders was revealed using Image Pro Plus (edition 6.0). Morphologies of the CL and CL powder were examined using scanning electron microscopy (SEM) . Samples were sputter-coated with a thin gold layer before testing. Dynamic light scattering (DLS) measurements were used to examine the mean zeta potential (ZP) analyses of the dyes and CL powders using a Zetasizer ZEN3600 instrument  at room temperature. Fourier transform infrared spectroscopy (FTIR) of the CL and CL powder were characterized on a Bruker LUMOS FTIR microscope . FTIR was employed to record the spectra of the films from 4000 to 1000 cmThe morphologies of CL and the CL powder were examined using SEM. As shown in The measurement of particle sizes varies in complexity depending on the shape of the particle. Generally, the particle size of a spherical object can be unambiguously and quantitatively defined by its diameter, due to the same size in different orientations (0–180 degree). For irregular particles, Feret’s diameter is a frequently used descriptor for the evaluation of particle size distribution, according to previous literature ,43,44,45Optical microscope images of CL powders revealed a different morphology, shape, and size of the particles, which can be classified into three types: dot-like particles, rod-like particles, and fiber-like particles, respectively (as shown in −1 (the combined effect of N–H and O–H stretching vibrations), 2920 cm−1 (C-H asymmetric stretching vibrations), 2850 cm−1 (C–H symmetric stretching vibrations), 1633 cm−1 (amide I), and 1554 cm−1 (amide II). These bands are similar to the FTIR spectra of protein fibers such as silk, wool, and down fibers due to the similarities in the chemical structures of keratins [−1, 2926 cm−1, 2861 cm−1, 1686 cm cm−1, and 1552 cm cm−1, indicating the main structure of keratins in the CL powder. Furthermore, no new bands could be seen in the spectra of the CL powder, suggesting that the milling process does not produce new bands. The sharper peak at 3309 cm−1 shifted to 3334 cm−1 (broad peak) in −1) shifted to a higher wavenumber (1686 cm−1) after milling. These two peaks can be ascribed to the β-sheet [keratins ,35. In c β-sheet . The FTIθ angle), a broad peak at 22° (2θ angle), and a minor peak at 29.7°. The first intense peak at 7.9° for the CL and CL powder was consistent with the leather shavings in the studies of Li et al. [To investigate the effect of the developed method on the intensity of CL, x-ray diffraction was performed. As shown in i et al. . As depi2 atmosphere) [3 lost during the milling process.The TGA and DTG curves for the CL and CL powder are presented in osphere) ,51. Ther−1. The maximum adsorption efficiency occurred at a pH of 3.0 for all of the investigated dyes, which were 94.0%, 92.5%, and 99.9% for RR120, RY167, and RB222 at an initial dye concentration of 50 mg·L−1, respectively. This can be ascribed to the dissociation of these anionic dyes in the aqueous solution [Solution pH significantly affects the adsorption performance of an adsorbent, especially the surface charge of the adsorbent and the dissociation degree of the dye molecule . The cursolution . Furthersolution , resultieC is the equilibrium concentration of the reactive dyes (mg·L−1); eQ is the equilibrium adsorption capacity of the reactive dyes adsorbed onto the CL powders (mg·L−1); mQ (mg·L−1) represents the maximum adsorption capacity; and lK (L·mg−1) is a constant of the Langmuir isotherm.The adsorption isotherm is generally applied to elucidate the relationship between the adsorbent and dyes under equilibrium conditions. Moreover, the maximum adsorption capacities of the adsorbent toward dyes can be calculated according to the adsorption isotherm . In thisRl), which is defined as Equation (4) [Furthermore, the basic characteristic of the Langmuir isotherm model can be expressed by the dimensionless differentiation factor (tion (4) ,Rl = 0), favorable (0 < Rl < 1), linear (Rl = 1), or unfavorable (Rl > 1), respectively.Herein, the adsorption is typically considered irreversible  for the RR120, RY167, and RB222 were 0.9813, 0.9953, and 09823, respectively, which were higher than the Freundlich correlation coefficients (R2) for the reactive dyes. Therefore, the adsorption of RR120, RY167, and RB222 onto the CL powders can be well described by the Langmuir isotherm models. Moreover, the Rl values calculated for RR120, RY167, and RB222 were found to be 0 < Rl < 1, suggesting that the adsorption of reactive dyes onto the CL powders occurs spontaneously [−1, respectively. Hence, the excellent adsorption capacity of CL powders can mainly be ascribed to the strong electrostatic interactions between the positively charged CL powder and negatively charged dye molecules under acid conditions. As shown in As can be seen from aneously . In addi−1, which is mainly due to the strong electrostatic attraction between the adsorbate and adsorbent as well as the high dye concentration difference between the interior and exterior of the adsorbent [−1.dsorbent . Additiodsorbent . At this1k (g·mg−1·min−1) and 2k (g·mg−1·min−1) are the rate constants of the pseudo-first order and second order models; eQ (mg·g−1) and tQ (mg·g−1) are the adsorption capacities at equilibrium and at time t (min); h (mg·g−1·min−1) is the initial adsorption rate of the pseudo-second order kinetics; pk (g·mg−1·min−1) is the rate constant of the intraparticle diffusion model; and C is a constant characterizing boundary layer thickness.To further understand the adsorption mechanism of the CL powders toward the investigated dyes, three kinetic models including the pseudo-first order, pseudo-second order, and intraparticle diffusion kinetic models were considered. These kinetic models are expressed as Equations (6)–(8), respectively ,59,60,61Qe−Qt) versus t for the pseudo-first order and the plots of t/qt versus t for the pseudo-second order for the absorption of RR120, RY167, and RB222 at the initial dye concentrations ranging from 50 to 300 mg·L−1. It is obvious that the plots were better fitted in R2) values summarized in e, calQ) obtained from the pseudo-second order model was closer to the actual adsorption capacity . Therefore, the pseudo-second order model is very suitable for explaining the dye removal from the wastewater by the CL powders. On the other hand, as can be seen from Based on these results, it can be concluded that the CL powders showed excellent capability to remove reactive dyes from water. Additionally, the adsorption mechanism of the reactive dyes onto the CL powders is rather complex, as both the external surface adsorption and intraparticle diffusion occurred simultaneously ,62.−1, respectively. The adsorption of reactive dyes onto the CL powders obeyed the Langmuir isotherm, and the pseudo-second-order and intraparticle diffusion models could well depict the adsorption kinetics. Therefore, the as-prepared CL powders can be used as a potential adsorbent in the treatment of dye contaminated wastewater. Future work will mainly focus on the application of the adsorbed CL powders for the pigment printing of textile materials.In summary, fine CL powders were successfully prepared using a homemade instrument at room temperature and atmospheric pressure. Dot-like particles with an average MFD of 1.6 μm, rod-like particles with an average MFD of 17.8 μm, and fiber-like particles with an average MFD of 40.1 μm were achieved without a separation process. The CL powders exhibited a similar chemical structure and slightly lower crystallinity when compared with CL, which was confirmed using FTIR and XRD, respectively. An improvement in thermal stability was also observed for the CL powders. This method provides an alternative avenue for the reuse of primary and secondary leather waste. Moreover, dye removal experiments indicate that the prepared CL powders could be used for the effective removal of RR120, RY127, and RB222 with the adsorption capacity of 167.0, 178.9, and 129.6 mg·g


The presently increasing demand for rare earth elements (REE), particularly in high-tech and “green energy” applications, has led to global interest in the distribution, origins and formation conditions of REE deposits. The World’s first hard-rock REE sources, the polymetallic deposits of Bastnäsfältet in Bergslagen, central Sweden, were also the place of the original discovery of several REE and many of their host minerals. Similar deposits with high concentrations of REE occur along a > 100 km corridor in the region and they share a number of geological and mineralogical features; all comprising Palaeoproterozoic, skarn-hosted magnetite-REE mineralisation of ambiguous origin. Here we report oxygen isotope data for magnetite and quartz, and oxygen and carbon isotope data for carbonates from ten of these classic deposits, to model and assess their mode of origin. Combined with existing geological observations, the isotope results support an origin in a c. 1.9 Ga shallow-marine back-arc, sub-seafloor setting, where felsic magmatic-sourced, high-temperature fluids reacted with pre-existing limestone interlayers, leading to localised skarn formation and magnetite-REE-mineral precipitation. These findings help us to better understand the geological processes that have produced economic REE mineralisation and may assist future exploration for these critical commodities. Economic projections suggest an even greater demand for REE in the immediate future, especially since numerous countries aim at phasing out fossil fuel-based transportation and energy production7. This demand, combined with the present lead of China as the main supplier of REE, has led to a renewed global interest in REE deposits of various types and geological origins. In Europe, one of the regions with the largest exploration potential for critical metals such as the REE is the Fennoscandian Shield  are critical components in a vast range of modern high-tech products and technologies. These include many types of industrial, research and consumer electronics, as well as increasingly abundant “green energy” applications such as wind power and electric vehicleseld Fig. , where aden Fig. . While nthe Bastnäs field) in Bergslagen , was from the mid-19th century where the first hard-rock underground mining was performed to extract REE9. The main REE minerals in the ore are cerite-(Ce) [9[SiO4]6[SiO3OH](OH)3] and ferriallanite-(Ce) [CaFe3+AlFe2+[SiO4][Si2O7]O(OH)]. Moreover, Bastnäsfältet was also the location of the original discovery of cerium, lanthanum, and “didymium” (a mixture from which praseodymium and neodymium were later isolated)12, as well as numerous minerals, including the already mentioned cerite and the eponymous bastnäsite-(Ce) [(CO3)F]13. After the initial discovery at Bastnäsfältet by Hisinger14, bastnäsite was to become one of the World’s major sources of REE4. As additional mineralisations were recognised to have a similar character as those at Bastnäsfältet, the term “Bastnäs-type deposit” was coined16. The Bastnäs-type deposits are characterised by skarn-hosted magnetite-REE mineralisation, with or without additional polymetallic assemblages17. These deposits occur as clusters along the so-called “REE-line”18; a more than 100 km long, narrow belt in the western Bergslagen region of variably Na, K and/or Mg altered, c. 1.90–1.88 G.y. old felsic metavolcanic rocks with intercalated marble layers , fluorbritholite-(Ce), törnebohmite-(Ce)], and the carbonate bastnäsite-(Ce), besides numerous low-volume accessory phases17. The skarns hosting the magnetite-REE mineralisations are calc-silicate aggregates, typically amphibole-dominated, featuring actinolitic to tremolitic, as well as anthophyllitic compositions30. Magnetite was always the main ore mineral mined in these deposits, besides the REE mineralisations, but copper as well as cobalt ores have also been locally exploited30.The mines of Bastnäsfältet  metamorphism27. Later studies have rendered this theory obsolete32, and they mostly revolve around an essentially syn-volcanic hydrothermal formation scenario at c. 1.90–1.88 Ga, or somewhat later. Here we employ mineral oxygen and carbon isotopes to unravel the fundamental process involved in the formation of the Bastnäs-type REE deposits, and to provide robust constraints on the temperature and source(s) of ore-forming fluids.The genesis of the Bastnäs-type REE deposits has been critically debated with respect to their formation process, and their temporal relationships to host rocks and to other mineralisation types18O values ranging from −2.3 to +2.6‰, whereas co-existing quartz (n = 3) separated from the BIF-hosted magnetite-skarn assemblages have δ18O values between +7.2 and +8.3‰ (Table 3] and dolomite [CaMg(CO3)2], as well as mixtures between the two, and have δ18O values from +5.8 to +10.0‰ and δ13C values from −5.4 to −2.8‰  and show (uncorrected) homogenisation temperatures of 370 to 460 °C31. Considering that significant lithostatic pressures are likely to have prevailed at the depths of skarn formation (c.f.19), and that, in general, bastnäsite-(Ce) is paragenetically late relative to magnetite, the fluid inclusion homogenisation temperatures should represent lower-end estimates of the range of mineralisation temperatures. Thus, the combined oxygen isotope and fluid inclusion evidence suggest that primary magnetite-REE mineralisation in Bastnäs-type deposits formed, at least initially, from fluids with temperatures of ≥450 °C.Thermometric calculations for magnetite-quartz oxygen isotope pairs (n = 3), using the fractionation factor of Zheng & Simon°C Table . Althoug18O and δ13C values that are significantly lower than those of non-mineralised, Palaeoproterozoic marine carbonate rocks in the Bergslagen province :18Oi and δ18Of are the initial and final oxygen isotope compositions of the rock, respectively35. This model is based on the so-called “calc-silicate limit“, which assumes that all carbon is released as CO2 while c. 60% of the oxygen remains in the rock if the reaction goes to completion35. Unlike what has locally been observed in other marble-skarn environments in Bergslagen (e.g.36), the analysed carbonates from the Bastnäs-type deposits do not plot on the steep or near-vertical trends of pure thermal decarbonation 41, using different end-member compositions. Mixing is defined as:M is the isotope ratio of element X in a mixture of compositions A and B; XA and XB are the concentration of X in A and B, respectively; and f is the weight fraction of A defined as f = A/(A + B)41. The calculated magmatic fluid-carbonate mixing trajectories envelop essentially all of the analysed carbonates from the Bastnäs-type deposits  carbonates, as well as re-precipitation of secondary  carbonates of similar compositions (cf.44). Such processes have, notably, been recognised at magnetite skarn deposits elsewhere  and at several magma-carbonate contacts .The carbonates from Bastnäs-type deposits have δ18O values measured in magnetite, respectively, and established fractionation factors33. For the proposed temperatures of mineralisation (c. 370 to 545 °C), fluids in equilibrium with magnetite of average composition have δ18O values ranging from +6.4 to +7.6‰ 41. If the upper end of the temperature range is used, fluids in equilibrium with magnetite with the lowest δ18O value are slightly lower than primary magmatic waters  of the analysed carbonates , the reactive carbonates are more easily affected by both high-temperature processes that could lead to, e.g., de-volatilisation, and low-temperature fluid overprinting, either during primary skarn and ore formation or during subsequent phases of regional metamorphism and granitoid intrusion. Such retrograde isotopic modification may be more marked in some carbonates than in others, explaining the variation in carbonate equilibrium fluid δ18O values  from external, non-magmatic water sources.Overall, the gradual transition from higher to lower equilibrium fluid δ18O) water sources, such as seawater. Combined with available geological, geochronological and textural observations32, the new results are most easily reconciled with a scenario involving sub-seafloor, felsic magmatic activity at c. 1.90–1.88 Ga, within a shallow-marine back-arc setting  feature of high-grade REE concentration in the Bergslagen province . In selected cases, subsequent field emission electron microprobe analyses (FE-EPMA) were performed on a JEOL JXA-8530F Hyperprobe at the Department of Earth Sciences, Uppsala University.To determine the composition of carbonate separates prior to stable isotope measurements, samples were subjected to powder X-ray diffraction (XRD) analysis at the Swedish Museum of Natural History, Stockholm. The samples were first crushed to a fine powder and placed in a silicon holder. They were then analysed using a PANalytical X’pert PRO automated diffractometer, utilising an acceleration voltage of 45 kV and a beam current of 40 mA. The 2θ angles were measured in the interval 5–70° for 11 minutes, and mineral identification was done off-line using the Highscore Plus software.52. Each sample was reacted in the presence of 10 kPa BrF5, after which purified O2 gas was collected onto a 5 Å molecular sieve within a glass storage bottle. Carbonate separates were analysed using a conventional carbonate line, where purified CO2 gas was extracted after reacting the samples with 100% phosphoric acid53. Oxygen isotope ratios of magnetite, quartz and carbonates, and carbon isotope ratios of carbonates, were measured off-line using a Finnigan Delta XP mass spectrometer in dual-inlet mode. For magnetite and quartz analysed using laser fluorination, the Monastery Garnet standard54 was used to normalise the raw data and to correct for drift. Carbonates were analysed alongside the Namaqualand Marble standard55, and the raw data was corrected based on the calcite:dolomite ratio determined previously for each sample by XRD. The δ18O data are reported in per mil (‰) relative to the Standard Mean Ocean Water (SMOW) standard, and the δ13C data in per mil relative to the Pee Dee Belemnite (PDB) standard. Both types of analyses gave 2σ errors of 0.2‰.Stable isotope data were produced at the University of Cape Town, South Africa. Magnetite and quartz separates were analysed following the laser fluorination technique described in Harris & Vogeli


Motor symptoms in Gilles de la Tourette syndrome (GTS) have been related to changes in frontostriatal brain networks. These changes may also give rise to alterations in cognitive flexibility. However, conclusive evidence for altered cognitive flexibility in patients with GTS is still lacking. Here, we meta-analyzed data from 20 neuropsychological studies that investigated cognitive flexibility in GTS using the Wisconsin Card Sorting Test (WCST). Results revealed medium-sized GTS-related performance deficits, which were significantly modulated by age: Whilst being substantial in children and adolescents with GTS, WCST deficits seem to dissolve in adult patients with GTS. This age-related normalization of WCST performance might result from the compensatory recruitment of cognitive control in adult patients with GTS. We addressed this possibility by examining neural correlates of proactive and reactive cognitive control in an event-related potential (ERP) study. We analyzed cue- and target-locked ERPs from 23 adult patients with GTS and 26 matched controls who completed a computerized version of the WCST. Compared to controls, patients with GTS showed a marked increase in parietal cue-locked P3 activity, indicating enhanced proactive cognitive control. We conclude that the additional recruitment of proactive cognitive control might ensure flexible cognitive functioning in adult patients with GTS. Many ofOne central aspect of executive functioning is cognitive flexibility . CognitiA number of neuropsychological tests have been developed for the assessment of cognitive flexibility, the most popular of which is the Wisconsin Card Sorting Test WCST; . On the With respect to GTS, In the following, we present a meta-analysis that aimed at obtaining a reliable effect-size estimate for potential WCST deficits in patients with GTS. In addition, this meta-analytical approach enabled us to investigate whether the extent of GTS-related alterations in WCST performance is moderated by patient characteristics such as age and gender. Following the description of this meta-analysis, we present an event-related potential (ERP) study that we conducted to examine the electrophysiological correlates of WCST performance in patients with GTS. In this study, we analyzed the ERP waveforms evoked by stimuli in a computerized version of the WCST. This approach allowed us to further elucidate the neural underpinnings of WCST performance in patients with GTS. Specifically, we were able to investigate whether patients with GTS differ from healthy control participants with regard to the neural correlates of the cognitive control processes that they recruit while performing the WCST. Hence, our two studies complement each other in providing (a) a powerful test of potential GTS-related WCST performance deficits  and (b) an in-depth analysis of the mechanisms that underlie WCST performance in patients with GTS (ERP study).22.1A systematic literature review was conducted in April 2015 and updated in July 2017 using the databases PubMed, ScienceDirect, PsychInfo, and Scopus as well as Google Scholar. In a first step, we screened the results of a Google-Scholar search involving the combination of the keywords “Tourette” and “Wisconsin Card Sorting Test” (2345 hits). We then looked for studies that did not explicitly mention the term “Wisconsin Card Sorting Test”, but that involved the keyword “Tourette” as well as either “Card Sort”; “Card Sorting”; “WCST”; or “MCST” (952 hits). When screening these 3297 records for eligibility; we excluded a record as soon as we were able to determine that it does not fulfill all the inclusion criteria of our meta-analysis see . The fol1) The study had to administer a standard version of the WCST to a sample of patients with GTS. This implies that studies reporting WCST data from single patients with GTS were not considered.2) The study had to involve WCST performance data from a sample of healthy control participants or it had to report standard scores that describe the performance of patients with GTS in comparison to normative data.3) The study had to report data for at least one of the best-established WCST measures  at a level of detail that allows for the calculation of effect sizes  that allow estimating means and standard deviations according to the procedure described by When the title of a record did not allow determining that one of these criteria was not fulfilled, we screened the abstract. When the abstract did not allow excluding the record, we accessed the full text. After screening the potentially eligible full texts, we retained 20 records that fulfilled the criteria listed above. We repeated the same procedure using the databases PubMed, ScienceDirect, PsychInfo, and Scopus, but theses searches did not render any additional studies to be included.t-statistic for the between-group comparison was calculated using the two-sample t-test provided by GraphPad QuickCalcs . For the studies reporting standard scores, the t-statistic was obtained by computing a one-sample t-test  comparing the mean standard score in the patient sample to the mean in the normative sample.Hence, we performed our meta-analysis on 20 studies reporting WCST performance data from patients with GTS. Fourteen studies involved a direct comparison between patients with GTS and healthy controls, whereas the other six studies reported standard scores based on normative data. For the studies comparing patients with GTS and healthy controls, the d) and their confidence intervals were calculated from t-statistics using the syntaxes provided by Effect sizes . When more than one measure of perseveration was reported  and showed a more pronounced tendency to perseverate (d = 0.28) in comparison to healthy controls or to normative values. Both effect sizes remained significant when we excluded those studies that did not involve a control group. Effect-size heterogeneity for the number of completed categories was significant and moderate in size (I2 = 50%). Effect-size heterogeneity for the number of perseverations was not significant and moderate-to-small in size (I2 = 32%). Rank-correlation analysis did not reveal any signs of publication bias .2(1) = 13.46, p = 0.001. The effect size in the adult samples is small (d = 0.17) with a 95% confidence interval that includes zero. The effect size in children and adolescents is large (d = 0.70) with a 95% confidence interval that neither includes zero, nor overlaps with the 95% confidence interval around the effect size in the adult population. Note further that effect-size heterogeneity (as measured by the 2I statistic) was considerably smaller in these two subgroups than in the overall sample of studies. The effect size also appeared to be moderated by the proportion of female participants in the patient sample, β = −1.90, p = 0.043. However, when both predictors were entered simultaneously, only age, χ2(1) = 6.02, p = 0.014, but not gender, β = −0.53, p = 0.573, emerged as a significant moderator of the GTS-related decrease in the number of completed WCST categories. Neither age, χ2(1) = 0.03, p = 0.866, nor gender β = −0.50, p = 0.524, significantly moderated the GTS-related increase in perseveration on the WCST.As can be seen from inspection of One question that cannot be adequately addressed by our meta-analysis relates to the association between WCST performance deficits and tic severity in patients with GTS. We did not find any reports of a significant correlation between these two variables in the published literature. Seven of the studies reviewed above described the absence of such a correlation and only three of them reported the magnitude of the correlation coefficients. To further complicate the interpretation of these results, the signs of the published correlation coefficient are not unambiguous, thus rendering the sensible aggregation of effect sizes across studies infeasible.k = 5, d = 0.35, 95% CI = [0.03–0.68]; perseverations: k = 4, d = 0.29, 95% CI = [-0.21–0.78]).We initially planned to also analyze whether WCST performance deficits in patients with GTS are moderated by the presence of comorbid disorders (such as attention deficit (hyperactivity) disorder, AD(H)D, or OCD). However, information with regard to these comorbidities was not reported consistently across the studies involved in our meta-analysis and only five studies explicitly excluded patients with comorbidities or provided data from a subgroup of patients without comorbidities. Notably, the above-described GTS-related decrease in the number of completed WCST categories also emerged in this subset of studies including patients without comorbid disorders , indicating that there is no evidence for a contribution of ADHD symptoms to the WCST performance deficits that can be found in patients with GTS.In addition, our literature review revealed six studies that directly compared patients with GTS with and without ADD/ADHD . The stuWe did not find a similar number of studies comparing patients with GTS with and without OCD. Patients with GTS and comorbid OCD showed larger WCST performance deficits than patients without this comorbidity in the study by 3Our meta-analysis revealed medium-sized WCST performance deficits in patients with GTS, which seem to be most pronounced with regard to the number of completed categories. However, these deficits appeared to be largely restricted to the underage population. These findings raise the question of why the pronounced WCST performance deficits in children and adolescents with GTS are markedly reduced in studies including older patients. We approached this question by analyzing the electrophysiological correlates of WCST performance in a sample of adult patients with GTS. Specifically, we applied the event-related potential (ERP) technique to explore whether adult individuals with and without GTS differ with regard to the neural processes they recruit when completing the WCST.ERPs can be obtained by averaging EEG activity that is time-locked to the stimuli or responses occurring during the course of a laboratory task. The voltage deflections comprising the ERP are commonly thought to be linked to the neural mechanisms underlying cognitive processes . In the In contrast to the broad interest in the executive functions listed above, no study has yet investigated the electrophysiological correlates of cognitive flexibility as it is required on the WCST in patients with GTS. One possibility to measure these correlates is to combine the ERP technique with a computerized version of the WCST . When usIn shifting paradigms (such as the cWCST), proactive and reactive control processes have been proposed to manifest themselves in modulations of ERP deflections in the latency range of the P3  were tested between June 2014 and July 2015. The diagnosis of GTS was confirmed by an experienced psychiatrist (KMV) according to DSM-5. Eight patients were on psychiatric medication on the day of testing .Twenty-three patients with GTS  without psychiatric and neurological diseases served as control participants. One additional control participant was tested but excluded from all analyses due to extremely slow RTs  on the cWCST. The group of control participants did not differ significantly from the GTS group in terms of age, gender, or the number of education years, all p > 0.79, see Twenty-six adults  and the Adult Tic Questionnaire . Patients also completed the Premonitory Urge for Tics Scale . The Yale-Brown Obsessive Compulsive Scale  was administered to confirm the clinical diagnosis of OCD . Three patients had been diagnosed with OCD based on the clinical assessment of an experienced psychiatrist (KMV). These diagnoses were confirmed by Y-BOCS scores ≥ 16 in all these three patients.A number of clinical and (neuro-) psychological assessments were administered to further characterize our sample of patients with GTS and healthy controls. Some measures were only administered to patients with GTS. Disease severity was quantified using the Yale Global Tic Severity Scale .In accordance with previous studies , we usedParticipants were required to match cards according to one of three possible sorting rules. Target displays consisted of four key cards which appeared invariantly above one stimulus card, all configured around the center of the computer screen . StimuluParticipants were instructed that their task would be to match the stimulus card with one of the four key cards in accordance with the appropriate rule. They sorted the cards by pressing one of four keys on the response pad that were mapped to the spatial position of the key cards on the screen. Target displays remained on screen until a key was pressed.After an interval of 800 ms following participants’ response, a feedback cue was presented for 400 ms, indicating whether the applied sorting rule should be maintained or changed. The German words for “REPEAT” (“BLEIBEN”) and “SHIFT” (“WECHSELN”), displayed in 28 point Arial, were used as feedback cues. Subsequent target stimuli appeared 1200 ms after feedback-cue onset.M = 211.96, SD = 19.01) and controls . Prior to the test sequence, five practice runs were administered. Participants were explicitly informed about the three possible sorting rules, and they were told that the valid rule would change from time to time.Rules changed in an unpredictable manner  after ru3.1.4Continuous electroencephalogram (EEG) was recorded from 30 active Ag-AgCl electrodes mounted on an actiCap  according to the international 10–20 system. BrainVision Recorder version 1.20 software was used . Electrode impedance was kept below 10 kΩ. Electrodes were referenced to FCz electrode. Vertical and horizontal electrooculogram were recorded with two additional electrodes positioned at the suborbital ridge and the external ocular canthus of the right eye, respectively. All channels were amplified  and digitized at 250 Hz using a BrainAmp amplifier .3.1.5For the analyses of performance data and ERP amplitudes, we distinguished between four different trial types . After aOnly trials with correct responses were included for RT analysis. RTs shorter than 100 ms or longer than three standard deviations above the mean for each participant were excluded. Mean RTs were subjected to a 2 × 4 mixed ANOVA involving the factors group (GTS vs. controls) and trial type (first shift vs. second shift vs. first repeat vs. second repeat).For the analysis of response accuracy, we computed the percentages of erroneous responses for each of the four trial types. Error rates were subjected to a 2 × 4 mixed ANOVA involving the factors group (GTS vs. controls) and trial type (first shift vs. second shift vs. first repeat vs. second repeat). Note that errors on the cWCST can occur for a variety of reasons. For example, on the first shift trial, participants might make an erroneous response because they fail to shift away from the previously applied rule or because they switch to the wrong rule. Whereas the first type or error (a perseverative error) is typically regarded as an indicator of set-shifting deficits, the second type of error is rather a sign of an efficient trial-and-error process required by the demands of the cWCST . SimilarEEG data were evaluated using BrainVision Analyzer 2.0 . After offline filtering , data were screened for nonstereotyped artifacts . Ocular and muscle artifacts were removed using independent component analysis . EEGs weIn line with previous studies investigating the P3 deflections in shifting paradigms, we analyzed the P3 at a frontal (Fz) and a parietal (Pz) electrode to dissociate the anterior and posterior portions of this component . At eachpη2).Significant main effects or interactions involving the factor group were followed up by rerunning the respective analysis with the inclusion of clinical scales assessing comorbid symptoms as covariates. Specifically, we added the BDI-II sum score, the three BSI-18 scales, the DSM-IV symptom list for ADHD, the eight CAARS scales, and the WURS-k as covariates to assess whether the GTS diagnosis significantly contributes to the respective group differences over and above the presence of affective or ADHD symptoms. All analyses were carried out using SPSS 23.0 . The level of significance was set to 0.05. Effect sizes for ANOVAs were calculated as partial eta squared , MoCA , or M-WCST see . In cont3.2.2F = 0.43, p = 0.518, pη2 = 0.01, nor the trial type × group interaction, F = 2.11, p = 0.128, pη2 = 0.04, was statistically significant. There was a significant main effect of trial type, F = 228.55, p < 0.001, pη2 = 0.83, with low accuracy on first shift trials, high accuracy on first repeat trials, and intermediate levels of accuracy on second shift trials and second repeat trials  × 2 (group) ANOVA on participants’ error rates did not reveal any evidence for response accuracy differences between patients with GTS and controls. Neither the main effect of group, ials see . Note thials see .Table 4PF = 42.21, p < 0.001, pη2 = 0.47, in a 3 (error type) × 2 (group) ANOVA. However, the main effect of group, F = 0.73, p = 0.397, pη2 = 0.02, and the error type × group interaction, F = 1.14, p = 0.322, pη2 = 0.02, were not significant.The focused analysis of particular error types yielded similar results. Participants committed more perseverative and integration errors than set-loss errors, as indicated by a significant main effect of error type, F = 6.13, p = 0.017, pη2 = 0.12 in a 4  × 2 (group) ANOVA on participants’ RTs. On average, patients with GTS responded 556 ms more slowly than controls. This main effect of group did not remain significant when we included the sum scores on the BDI-II (p = 0.092), the three BSI-18 scales (p = 0.187), the DSM-IV symptom list for ADHD (p = 0.272), the eight CAARS scales (p = 0.063), and the WURS-k (p = 0.056) as covariates. The main effect of trial type was significant as well, F = 37.01, p < 0.001, pη2 = 0.44. Response latencies gradually decreased from first shift to second repetition trials . Most crucially, the ANOVA revealed a significant three-way interaction between locking event, recording site, and group, F = 7.60, p = 0.008, pη2 = 0.14 (p = 0.026), the three BSI-18 scales (p = 0.027), the DSM-IV symptom list for ADHD (p = 0.040), the eight CAARS scales (p = 0.012), and the WURS-k (p = 0.021) as covariates. Follow-up analyses revealed that cue-locked, F = 5.22, p = 0.027, pη2 = 0.10, but not target-locked, F = 0.84, p = 0.364, pη2 = 0.02, P3 amplitudes were significantly modulated by the interaction of recording site and group. At electrode Fz, cue-locked P3 amplitudes did not differ significantly between patients with GTS and HC, t[47] = −1.25, p = 0.221. At electrode Pz, cue-locked P3 amplitudes were significantly larger in patients with GTS than in HC, t[47] = 2.56, p = 0.014.Cue-locked and target-locked ERP activity is displayed in 2 = 0.14 . This in3.2.4We provide a table depicting the bivariate correlations between the rating scales administered during background assessment and the performance and ERP measures obtained from the cWCST in the supplementary materials. Although the exploratory nature of these analyses and the large number of examined associations do not permit drawing strong inferences, some aspects of the data might be of interest for future follow-up investigations. First, RTs in patients with GTS were positively related to inattentive symptoms as measured with the DSM-IV symptom list for ADHD. Second, the number of perseveration, integration, and set-loss errors committed by patients with GTS on the cWCST increased with increasing BIS scores. Third, cue-locked P3 amplitudes at electrode Pz were particularly large in patients with GTS and increased YBOCS scores.4Our meta-analysis revealed the first conclusive evidence for significant WCST deficits in patients with GTS. However, this effect was mainly driven by the studies examining WCST performance in underage individuals. While performance deficits were of substantial magnitude in children and adolescents with GTS, they appeared to dissolve in samples of adult patients. Our analysis of event-related neural activity in adult patients with GTS provided first insights into potential neural mechanisms underlying this age-dependent normalization of WCST performance. We observed a marked increase of cue-locked parietal P3 amplitudes in adult patients with GTS as compared to matched controls, which is suggestive of the recruitment of additional proactive control resources. The GTS-related enhancement of cue-locked P3 activity might thus be a correlate of those neural adaptations that allow adult patients with GTS to successfully complete complex neuropsychological tests of cognitive flexibility.At first sight, our meta-analytical results contrast with the conclusion of In line with our meta-analytical results, the adult patients with GTS participating in our ERP study did not differ significantly from a matched control group with regard to M-WCST performance. Similarly, patients with GTS did not commit an increased number of errors on the computerized version of the WCST. In contrast, patients’ RTs were significantly prolonged, which might suggest that patients with GTS require more time to adequately respond to the rule-shifting demands of the cWCST. However, GTS-related RT prolongation was found on all trial types, indicating that patients did not have specific difficulties with shifting cognitive sets. Instead, the speed of motor output in general seems to be slowed in patients with GTS. Similar data were observed by The most important result of our ERP study is the increase in cue-locked parietal P3 activity in patients with GTS. Cue-related P3 deflections are commonly considered as a neural correlate of proactive cognitive control processes . Recall This interpretation is consistent with the findings reported by Jackson and colleagues , who infThe study of executive functions in general or cognitive flexibility in particular in patients with GTS might have important implications for the design and administration of behavioral treatments for tic disorders. For example, habit reversal training techniques involve learning to execute an alternative response when patients detect a premonitory urge to tic . Intact 4.1One important limitation of our present analysis is its exclusive reliance on cross-sectional data. While our meta-analysis showed a clear moderating effect of age on GTS-related WCST deficits, interpreting this effect as evidence for an age-related normalization of cognitive flexibility in GTS does involve some degree of speculation. Due to the lack of longitudinal studies that monitor patients’ WCST performance as they mature, we cannot conclusively demonstrate that the WCST deficits shown by a particular patient disappear when this patient reaches adulthood. Similarly, our approach of studying the ERP correlates of WCST performance in adult patients with GTS to shed light on the neural mechanisms that underlie the proposed age-related normalization is clearly limited. It would certainly be desirable to complement our results with comparable data obtained from children with GTS in the future. Ideally, such a future investigation could follow the example of a study recently published by Another aspect of our results that requires further investigation using optimized research designs is the potential relationship between clinical variables and behavioral and electrophysiological cWCST measures. Our post-hoc correlation analysis revealed a potentially interesting correlation between cue-locked parietal P3 amplitudes and OCD symptoms in patients with GTS. However, as our study was not designed to characterize the relationship between ERPs and clinical variables, the size of our sample does not allow for a conclusive test of this correlation. Specifically, it is not possible to clarify whether the observed P3 alteration is more closely related to OCD symptoms than to tic severity. Large-sample replication studies are required to answer this particular question and, more generally, to determine the role of comorbid OCD symptoms for WCST performance deficits in GTS. These studies might also benefit from focusing on the role of impulsiveness, a trait that was related to patients’ performance on the cWCST in the present study  and in a previous study involving patients with dystonia . By contFinally, it is important to acknowledge that cognitive flexibility is likely to be a multifaceted construct, and different aspects of cognitive flexibility might differ in their relevance for patients with GTS. In addition to the WCST results we analyzed here, GTS has been reported to be associated with significantly improved  and sign5GTS appears to involve deficits in cognitive flexibility, which are significantly modulated by age-related changes. Adults with GTS may be able to successfully complete complex tasks of cognitive flexibility by enhanced recruitment of proactive cognitive control mechanisms. The study of the electrophysiological correlates of these cognitive alterations opens a new window onto patterns of neural reorganization in GTS.None.


The inhibitory capacity of the phenolic extracts could be associated with the content of secoiridoids, lignans and phenolic acids.The increasing interest in the Mediterranean diet is based on the protective effects against several diseases, including neurodegenerative disorders. Polyphenol-rich functional foods have been proposed to be unique supplementary and nutraceutical treatments for these disorders. Extra-virgin olive oils (EVOOs) obtained from ′Brava′ and ′Mansa′, varieties recently identified from Galicia (northwestern Spain), were selected for in vitro screening to evaluate their capacity to inhibit key enzymes involved in Alzheimer′s disease (AD) (acetylcholinesterase (AChE), butyrylcholinesterase (BuChE) and 5-lipoxygenase (5-LOX)), major depressive disorder (MDD) and Parkinson′s disease (PD) (monoamine oxidases:  The recognized beneficial properties of plant polyphenols are used in the design of drugs as neuroactive multitarget directed ligands (MTDLs) designed to combat the progression of major depressive disorder (MDD), and of neurodegenerative disorders, such as Alzheimer′s (AD) and Parkinson′s (PD) diseases, between others . There iRegarding the last mechanism, it is known that reduced levels of the neurotransmitters in the synaptic cleft are engaged in the pathophysiology of several CNS disorders. Two major cholinesterase (ChEs), acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) play an important role in AD. Based on the cholinergic hypothesis, high levels of these enzymes reduce the levels of the neurotransmitter acetylcholine (ACh) in the synaptic gap, consequently, an increase of the AD progression occurs. Therefore, restoring the levels of the neurotransmitter acetylcholine (ACh) neurotransmitter via inhibition of AChE and BuChE enzymes is actually the most useful therapeutic approach to treat AD and other forms of dementia ,8.hMAOs) are located on the outer mitochondrial membrane and are present in all type of cells at different proportions and concentrations. hMAO-A and hMAO-B catalyze the oxidative deamination of a variety of neurotransmitters. hMAO-A inhibitors are used as antidepressant agents for MDD with a central serotonin and noradrenaline deficiency and hMAO-B inhibitors are applied in PD therapy, where a central dopamine deficiency is responsible for the characteristic motor deficits [Monoamine oxidase isoforms  enzyme has an important role in inflammation catalyzing the conversion of arachidonic acid released from membranes, into an oxidized compound, which is further metabolized into different leukotrienes considered as potent inflammatory mediators. Thus, 5-LOX inhibitors could effectively reduce inflammation associated with neurodegenerative disorders ,12,13. p-coumaric acid, luteolin and lignans including pinoresinol and syringaresinol as owners of anticholinesterase activities [p-coumaric acid, and luteolin with inhibitory activity against 5-LOX [h-MAOs [Some phenolic compounds have already addressed to exhibit several remarkable biological activities on inhibition of the previously described enzymes: tivities ,15,16,17st 5-LOX ,19,20 an [h-MAOs ,22. These phenolic compounds are found in the virgin olive oil (EVOO), the major source of energy in the Mediterranean diet. Recent epidemiological evidence and clinical trials connect the Mediterranean diet (Med Diet) with protective effects against the health. The latest reports have highlighted that the molecular effect of olive polyphenols counteract others diseases via its anti-inflammatory properties, including cancer, atherosclerosis, liver steatosis and/or other liver tissue damage, obesity, type 2 diabetes, metabolic syndrome, cardiovascular diseases, amyloid and neurological diseases, between others ,26,27,28Therefore, the phenolic compounds can be considered as a nutraceutical-encompassing prophylaxis and cure of diseases . It is pThe phenolic profiles of monovarietal ′Brava′ and ′Mansa′ EVOOs are displayed in −1 in ′Mansa′ EVOO) and oleuropein aglycone (Ol Agl)  (74 ± 5–23 ± 10 mg·kg−1). Among the ligstroside derivatives, ligstroside aglycone (Lig Agl) (p-HPEA-EA) (214 ± 2 mg·kg−1) was predominant in ′Brava′ oil while oleocanthal (d-Lig Agl) (p-HPEA-EDA) (167 ± 8 mg·kg−1) was in ′Mansa′ oil. In addition, elenolic acid, (EA) (nonphenolic compound) was three times higher in ′Mansa′ than in ′Brava′ oils. Secoiridoids (oleuropein and ligstroside derivatives) were the main phenolic group comprising 67–83% of the total phenolic compounds in the studied EVOOs. Quantitative differences between both olive oils can be observed when the individual secoiridoids are considered a. The ma−1 in Mansa′ EVOO) and tyrosol, (Ty) (11 ± 0.51–4.2 ± 0.12 mg·kg−1) . The main phenyl alcohols were hydroxytyrosol (Hyt) (8 ± 0.1 in ′Brava′ EVOO–1.5 ± 0.5 mg·kgmg·kg−1) b. In addmg·kg−1) c.In the current work, the activities of the phenol-rich extracts obtained from ′Brava′ and ′Mansa′ oils were investigated against these enzymes. In addition, they were compared to that of RSO, which lacks phenolic compounds. As far as we know, this is the first report in evaluating the ability to inhibit CNS-related enzymes of phenol-rich extracts from any EVOOs extracted from ′Brava′ and ′Mansa′ Galician varieties. Only one previous work reported in vitro neuroprotector effect of phenol-rich EVOO extracts from ′Cornicabra′ and ′Picual′ Spanish varieties . BuChE inhibition. As it is shown in 50 and IC25 values were calculated (lculated . 50 = 298 ± 6 μg of dry extract·mL−1) than ′Mansa′ EVOO (IC50 = 668 ± 26 μg of dry extract·mL−1). Different concentrations of the main reference drug, namely galanthamine, were used as positive control and ran in parallel, being the effect stronger than that obtained with any extract (IC50 = 7 ± 0.5 μg·mL−1). Similar results to those reported herein were found by Figueiredo-González et al. [50 = 249 ± 6 μg of dry extract·mL−1) and ′Picual′ (IC50 = 357 ± 38 μg of dry extract·mL−1) Spanish varieties. ′Brava′ EVOO revealed to be more effective against BuChE . Only ′Brava′ EVOO was able to inhibit this enzyme in a concentration dependent manner  (50 = 2 ± 0.3 μg·mL−1) was more effective than that exerted by ′Brava′ EVOO. These results are in accordance with those reported by Figueiredo-González et al. [25 values of 503 ± 36 mg EVOO·mL−1 for ′Cornicabra′ variety. t manner  and in act·mL−1) . In the z et al. , who fouLOX inhibition. ′Brava′ and ′Mansa′ oils were both able to inhibit this enzyme, although the effect was lower than that one showed by the positive control . The IC50 values were of 50 ± 8 and 124 ± 17 μg of dry extract·mL−1, respectively. Our results are in agreement with those recently published by Figueiredo-González et al. [−1 for hydromethanol-based extracts obtained from ′Cornicabra′ and ′Picual′ oils, respectively. On the contrary, RSO did not displayed activity against 5-LOX up to the highest tested concentration (2 mg of dry extract·g−1). z et al. , who obshMAO inhibition. Data from the present study provide evidence of the concentration-dependent inhibitory effect of the tested extracts from ′Brava′ and ′Mansa′ oils on hMAO-A and on hMAO-B activities. Although the observed activity for both EVOOs against both hMAOs was significantly lower than that of clorgyline , IC50 values were calculated . lculated . ′Brava′z et al.  who founThe reduction of acetylcholine (ACh) levels is associated with the progression of different neurological disorders including AD. Since ACh is hydrolyzed by the two major forms of cholinesterases (BuChE and AChE), their inhibition is a valuable tool to improve cognitive functions .′Brava′ EVOO was the most potent against both enzymes, revealing dual ChE inhibition. There are reports suggesting that the use of compounds with dual ChE inhibition can effectively increase the efficacy of the treatment . On the The phenolic compounds of the EVOOs could be responsible of cholinesterase inhibition. In fact, RSO without any phenolic compounds did not show. Likewise, Collado-González et al.  did not p-coumaric acid (p-Cou) and luteolin (Lut) [Phenolic compounds reported in literature as owners of anticholinesterase activity are in (Lut) ,16 in adin (Lut) , and pinin (Lut) . The conp-HPEA-EDA (23 ± 1–167 ± 8 mg·kg−1), Hyt (8 ± 0.1–2 ± 0.5 mg·kg−1) p-Cou (0.2 ± ˂0.01–0.1 ± ˂0.01 mg·kg−1) and Lut (2.1 ± 0.1–1.7 ± 0.1 mg·kg−1) in ′Brava′ and in ′Mansa′, respectively, already have been described as potential anti-inflammatories by 5-LOX inhibition. The certain synergistic effect between all olive oil phenols might also be responsible for the observed activity [5-LOX has been associated with AD and other aging-related events . Thus, 5activity ,19,20,32hMAOs. The activity observed may result from synergism and/or antagonism phenomena that occur among the several phenolic compounds and/or the presence of flavonoid compounds, such as apigenin (Apig) and Lut, [hMAOs than towards cholinesterase enzymes; the last ones present the highest level of human homology. In addition, the design and synthesis of novel dual monoamine-cholinesterase inhibitors is a hot-topic in neuropharmacology research [Extracts obtained from EVOOs displayed inhibitory activity against and Lut, ,22 descrresearch . TherefoRelationship between neuroprotective potential and phenolics for EVOOs differentiation. Phenolic compounds found in the oil extracts seem to play a role in the inhibition of neuroprotective enzymes. Thus, we have contemplated as a relevant step in our study to carry out a Pearson´s correlation test, considering individual phenolics and IC50 values obtained for these enzymes. In order to increase sample size, phenolic data from the studied Galician EVOO extracts as well as phenolics from Spanish ′Cornicabra′ and ′Picual′ EVOO extracts [extracts  were joir = −0.5000) between the phenolic compounds and IC50 values are shown. These correlations, inversely proportional, means that EVOO extracts with high phenolic content are more active.r = −0.9069 for BuChE, r = −0.9263 for 5-LOX and r = −0.8765 for h-MAO-A). This fact could be mainly ascribed to the DOA  and in less extension to Olg Agl . On the contrary, for hMAO-B the strongly negative correlation between IC50 value for this enzyme and total ligstroside derivatives content  was observed. On the other hand, lignans show strongly negative correlations with IC50 values calculated for BuChE (r = −0.7006), 5-LOX (r = 0.7220), hMAO-A (r = −0.6611) and hMAO-B (r = −0.6570) enzymes being Syr and Pin responsible (except for Pin in the case of hMAO-B inhibition). The different levels of correlation found point to the influence, at least partial, of phenolics from olive oil and/or synergistic/antagonistic interactions between them as responsible of tested activities. Regarding secoiridoids, a Pearson test showed a strongly negative correlation of total oleuropein derivatives content , IC50 values were sign-changed. In this sense, it is easy to see positive correlations in bidimensional spaces since the variables will occupy a close position.As the interesting correlations are negative, linking high phenolic concentrations with low levels of IC50 and phenolics could be divided into two main clusters or groups. The first group cluster on the left could be further divided at the linkage distance of 100 into 2 subgroups. The first subgroup on the left contains all IC50 values connected to a few phenolics concentration values. If we further split the first subgroup on the left at a distance of 50, the IC50 values of BuChE, hMAO-A and 5-LOX enzymes are clustered at a very small distance with Val, Syr, DOA and Pin. In the same way, hMAO-B is associated with acetoxypinoresinol (AcetPin) and d-Lig Agl phenolics. Choosing a relatively large and safe cutting value at the linkage distance of 200 in the dendrogram , the var50 enzymatic data. PLS2 modelling provided a two-factor model explaining 98% of the variance in X (phenolic profiles) and 51% of that in Y (IC50 enzymatic data)  and explained variation almost the EVOOs samples. In the same way as for hMAO-A and 5-LOX were highly correlated with phenolic variables of Val, Syr, DOA and Pin. All of them were closely connected to hMAO-B, which was highly correlated with AcetPin and d-Lig Agl. All these four enzymatic IC50 values and six phenolic compounds have a higher weight in PC1 to separate ′Cornicabra′ EVOOs from the rest. To separate the rest of EVOOs, PC2 is very useful, connecting ′Picual′ with mainly Lig Agl and Ol Agl, which are in the same direction of the bidimensional space. ′Brava′ is associated with the phenolics in the same up-left quadrant, and ′Mansa′ with the phenolics in the down-left quadrant. All this supports the idea of ′Cornicabra′ being the best of the EVOOs, followed by ′Picual′ and then ′Mansa′ and ′Brava′, according to their effects on the inhibition of enzymes related with neurodegenerative processes, but also all these EVOOs can be separated by a specific profile of phenolics.A PLS2 was also used to correlate phenolic profiles with ICic data) . The ens50 values can be used alone to separate oils, a discriminant analysis was performed using only this kind of data , bovine serum albumin (BSA), 5,5-dithiobis (2-nitrobenzoic acid) (DTNB), Trizma®hydrochloride (Tris–HCl), N-Methyl-N-propargyl-3-propylamine hydrochloride or clorgyline, kynuramine dihydrobromide crystalline, monoamine oxidase A and B , lipoxydase (from glycine max), linoleic acid, quercetin, sodium hydroxide, potassium dihydrogen phosphate and sodium chloride were obtained from Sigma-Aldrich . Magnesium chloride hexahydrate (MgCl2·6H2O) was purchased from Merck .Galantamine, AChE (from electric eel), acetylthiocholine iodide (ATCI), BuChE (from equine serum), ′Brava′ and ′Mansa′ are two ancient cultivars recently identified from northwestern Spain . Olives Two monovarietal olive oils from ′Brava′ and ′Mansa′ cultivars, separately, were obtained under optimal conditions with an OLIOMIO 50 , equipped with a knife crusher, a horizontal malaxator and a two-phase decanter. The malaxation trials were carried out for 45 minutes, and a temperature of 18 ± 2 °C for both varieties on two consecutive days. Once in the laboratory, three different bottles of 500 mL from each variety were pooled and homogenized to obtain a final representative sample prior to analysis. The samples were kept at a constant temperature of 4 °C until analysis using amber bottles without headspace.Both monovarietal oils were classified as extra virgin, considering that their quality indices  fell within the ranges established by legislation .Phenolic compounds were extracted, by duplicate, from EVOOs according to the methodology proposed by Bajoub et al. . IdentifIn parallel with rich-phenol extracts from EVOOs, the activity of a refined sunflower oil (RSO) was also assessed. RSO does not contain phenolic compounds and can be considered as a blank matrix to evaluate the effect of bioactive compounds.Cholinesterases. Inhibition of AChE and BuChE was assessed according to a previously described methodology [−1 in buffer B) or of BuChE (0.10 U mL−1 in buffer B). Galantamine was used as positive control. hodology . BrieflyLipoxygenase. The inhibitory effect on 5-LOX was assessed in 96-well plates, using a previously documented procedure [rocedure . BrieflyMonoamine oxidases. Inhibition of hMAO-A and hMAO-B was assessed according to a methodology that has been described before by [hMAOs (17 U mL−1) and the mixture was further incubated at 37 °C for 70 min. Afterwards, the reaction was stopped by the addition of NaOH 2 N and the absorbance was measured at 314 nm using a spectrophotometer . Clorgyline was tested as positive control. efore by . Concise50 values were calculated from three independent assays, each of them performed in triplicate, and all results are presented as mean values. Values obtained were compared using unpaired t-test . Differences at p < 0.05 were considered statistically significant, as well as Pearson partial correlations between both sets of variables: IC50 and phenolic concentrations.The IC50 and phenolic concentrations, based on the squared Euclidean similitude distance by Ward′s method [50 values were also performed at a p < 0.05 with the statistical software package Statgraphics Centurion XVI from StatPoint Technologies Inc. . A cluster analysis to group variables, both ICs method  together50 (Y matrix) and phenolic concentrations (X matrix), through a linear multivariate model, performed with The Unscrambler soft from CAMO. The idea is to relate a matrix of responses Y to the predictor variables of matrix X. To this end, matrix X is successively deflated; PLS seeks the directions in the X and Y-spaces corresponding to the maximum covariance. In this way, PLS forms principal components as linear combinations of the original variables, which are then related to the samples scores via a linear model. By plotting the principal components, one can view interrelationships between different variables sets (IC50 and phenolic concentrations), and detect and interpret sample patterns, groupings, similarities or differences.PLS is a method for relating two data matrices, IChMAO-A and hMAO-B, in a dose-dependent manner. Only ′Brava′ oil also showed a dual inhibition against both cholinesterase enzymes, which could imply increase the efficacy of the treatment of several disorders affecting the CNS. Therefore, the oils could be potential candidates in developing medicinal preparations and nutraceutical or functional foods for these disorders. This work successfully demonstrates, for the first time, that rich-phenolic extracts obtained from Galician EVOOs could act as multi-target ligands directly inhibiting CNS-related enzymes. Although both EVOOs exhibited a weaker activity than that of the positive controls, they were able to inhibit simultaneously BuChE, 5-LOX, hMAOs inhibition were some secoiridoids derivatives, lignans and vanillic acid. Although the cell-free model assays cannot be simply extrapolated to in vivo, they must be interpreted as easy and fast tools for a first approach. Thus, this work provides an approximation more on the nutraceutical properties of the EVOO phenolics, a valuable potential yet to be explored. In this regard, clinical trials are needed to provide a broader insight on the structure–activity relationship of olive oil polyphenols.According to Pearson′s correlation test, the phenolic compounds with a greater influence on BuChE, 5-LOX and