Each blend showed a characteristic PUFA content in the lipid pool  and the content in essential and healthy n-3 PUFA is highlighted. The atherogenicity (0.54–0.72) and thrombogenicity (0.23–0.45) indices evidenced a good nutritional value of lipid fractions. As nutritional and environmentally attractive products, the consumption of the studied seaweed blends can contribute to a healthy lifestyle.Seaweeds are considered healthy and sustainable food. Although their consumption is modest in Western countries, the demand for seaweed in food markets is increasing in Europe. Each seaweed species has unique nutritional and functional features. The preparation of blends, obtained by mixing several seaweeds species, allows the obtaining of maximum benefits and ingredients with single characteristics. In this work, five seaweed blends, commercially available and produced under organic conditions in Europe, were characterized. The proximal composition included contents of ash (20.28–28.68% DW), proteins (17.79–26.61% DW), lipids (0.55–1.50% DW), and total carbohydrates (39.47–47.37% DW). Fatty acid profiles were determined by gas chromatography–mass spectrometry (GC–MS), allowing quantification of healthy fatty acids, namely  The consumption of seaweeds has been widespread in Asia for centuries, where they are consumed as food and also used in traditional medicine and in dietary therapies . Indeed,Undaria pinnatifida [Gracilaria gracilis [Palmaria palmata [n-6 and n-3 PUFA become these sea vegetables attractive [Seaweeds, also known as marine macroalgae, are photosynthetic organisms classified into three phyla, Chlorophyta (green), Rhodophyta (red) and Ochrophyta (brown), which show great variability between their chemical compositions ,8. Even natifida , Gracilagracilis , and Pal palmata . Furthertractive , namely tractive . n-3) and linoleic acid  [n-3) and arachidonic  fatty acids that play key roles in inflammatory processes, brain function, cardiovascular disease, obesity and cancer [Palmaria palmata and Porphyra sp.) are particularly rich in EPA [Gracilaria gracilis and Fucus vesiculosus, respectively [In the literature, it has been reported that some seaweeds contain essential fatty acids—alpha-linolenic acid ALA; C18:3  n-3 and 8:2 n-6) ,23—that 8:2 n-6) . In the d cancer ,26. Red ectively ,20.During the last decade, the interest and demand for seaweeds in the Western countries have been growing. Nowadays, there is also a greater offer of seaweed-based ingredients and food products such as, sausages, burgers, bread, pasta, pastries, yogurts, cheeses, etc. The incorporation of seaweeds in foodstuff allows improvement of their nutritional quality, functional properties and shelf life . SeaweedEach seaweed species has specific nutritional and functional characteristics ,29,30,31Hence, this work aimed to evaluate and compare the biochemical composition, fatty acid profile determined by gas chromatography–mass spectrometry (GC–MS) and the healthy lipids quality indices of five commercially available seaweed blends. The results reported will provide useful information on nutritional and healthy values of these mixtures, highlighting their potential as a sustainable dietary source of PUFA and healthy fats. To the best of our knowledge, this is the first study about the composition of seaweed blends.In this study, the chemical composition and the nutritional value, including the fatty acid profiles and lipid quality indices of five commercial seaweed blends, produced by the company ALGAplus Ltd. , Fucus s , Gracils (24.8% ), Palmarta , Porphy. , Ulva ra  and Unda .Protein: Crude protein content was estimated from the analysis of total nitrogen, using the nitrogen–protein conversion factor (FN-P) of 6.25, chosen because it is widely applied in the food industry. The highest protein content was obtained for blend BLD005, estimated at 26.61 ± 1.98% DW, while BLD0N1, BLD0N2, BLD001 and BLD006, showed, respectively, protein amounts of 22.27 ± 0.32, 25.97 ± 2.84, 17.79 ± 3.67 and 21.74 ± 3.40% DW , were the ones with highest protein content and similar to those reported in red seaweeds. Although the protein contents were different when comparing the five blends, all can be considered a good source of protein (EC no. 1924/2006). The amounts of protein in different seaweeds phyla are established in literature and described as follows: red ≥ green > brown . AccordiThe world’s population growth and environmental pressure have led to the need to promote the incorporation of more sustainable protein sources into the Western diet . It is aLipids: The total lipid content of the five blends was estimated by gravimetry of the lipid extracts. The blend BLD005 (0.55 ± 0.13% DW) recorded the lowest fraction, while the blend BLD0N1 (1.50 ± 0.09% DW) achieved the highest lipid fraction  and lipids) and ranged between 43.50 ± 1.95% DW in blend BLD005 to 54.73 ± 3.65% DW in blend BLD001 , palmitoleic (C16:1 n-7), oleic (C18:1 n-9), cis-vaccenic (C18:1 n-7), eicosenoic (C20:1 n-9), hexadecatetraenoic (C16:4 n-3), linoleic , gamma-linolenic (C18:3 n-6 GLA), alpha-linolenic , stearidonic , arachidonic , eicosatetraenoic (C20:4 n-3) and eicosapentaenoic  acids.A maximum of 33 fatty acids were identified in blend BLD006 and a minimum of 27 fatty acids in blend BLD001. Twenty fatty acids were common to all seaweed blends, including myristic (C14:0), palmitic (C16:0), stearic (C18:0), myristoleic (C14:1), hypogeic (C16:1 n-9 was the most abundant in blends BLD006 (22.70 ± 2.23%) and BLD0N2 (34.09 ± 2.90%); and EPA was the dominant fatty acid in blend BLD005 (39.13 ± 5.84%). Exceptionally, blend BLD001 showed two major fatty acids, FA C16:0 (27.33 ± 1.52%) and EPA (26.79 ± 1.37%), with similar relative abundances (p > 0.05).Each blend showed a different and specific major fatty acid. The FA C16:0 was the major one in blend BLD0N1 (26.83 ± 0.84%); FA C18:1 n-9 (0.33), AA (0.31), C14:1 (−0.31), C14:0 (0.29) and EPA (−0.29) were the major contributors to the variability observed along the first dimension of the PCA, and the variables with higher contributions to the second dimension included SDA (0.39), C16:1 n-7 (0.37), C16:4 n-3 (0.35), C16:1 n-9 (0.34), ALA (0.33) and C18:1 n-7 (0.30). The PCA scores plot evidenced the variability within blends, mainly in BLD005 and BLD0N2. Hierarchical clustering heatmap  and C16:1 n-7 were higher than in blend BLD0N2.Principal component analysis (PCA) was carried out to visualize similarities between blends and correlations between fatty acids. To reduce the number of descriptors associated with the dataset, while explaining the maximum amount of variability present, only the data of the 20 fatty acids common to the samples analyzed were subjected to the analysis. PCA scores plot a showed  heatmap b analysiFor each product, the sum of SFA, MUFA and PUFA were calculated . All theSaturated fatty acids: In the five blends, the majority SFA was FA C16:0 (14.34 ± 0.78%–27.33 ± 1.52%), followed by FA C18:0 (6.54 ± 0.77%–12.66 ± 4.73%) and FA C14:0 (2.27 ± 0.16%–6.40 ± 0.45%). Lauric acid (C12:0) was only detected in a trace form (<0.01%) in blends BLD006 and BLD0N2. The higher abundance of FA C16:0 in the seaweed blends was in agreement with that observed in other seaweed biomass [ biomass ,37 while biomass ,12,18,44 biomass . However biomass . Additio biomass . From a Monounsaturated fatty acids: MUFA fractions had the major contribution of FA C18:1 n-7 and FA C18:1 n-9, the latter being significantly and abundantly detected in blends BLD0N2 (34.09 ± 2.90%) and BLD006 (22.70 ± 2.23%). Another MUFA, present in all mixtures above 1%, was the FA C16:1 n-7. Similar results were found in the literature for the seaweed species that constitute the blends [n-9, that was also the most abundant MUFA in brown seaweed Fucus vesiculosus [e blends . In bleniculosus , an ingriculosus . In particulosus .Polyunsaturated fatty acids: PUFA present in the studied blends occur mainly in the form of n-6 FA  and n-3 PUFA . Of the 19 PUFA detected, only 8 were common to all blends, among which the essential fatty acids LA and ALA, the SDA and the long-chain PUFA (LC-PUFA) AA and EPA stand out. Due to their nutritional importance, these five PUFA will be discussed below.p < 0.05) and, regarding ALA values, similarities were observed between blends BLD0N1 (6.14 ± 0.22%) vs. BLD006 (5.59 ± 0.56%) and BLD0N2 (3.12 ± 0.23%) vs. BLD001 (2.91 ± 0.26%) (p > 0.05).The two essential fatty acids LA and ALA were detected in all blends at levels not higher than 7%. The relative abundance of LA was significantly different between all blends (n-3), which is a precursor of EPA, ranged between 3.79 ± 0.44% in blend BLD0N2 and 8.69 ± 0.31% in blend BLD0N1. Blend BLD0N1 was similar to BLD006 (8.64 ± 0.94%) (p > 0.05), which also did not present significant differences between BLD001 (7.04 ± 0.71%) (p > 0.05).The SDA (C18:4 p > 0.05). Blend BLD005 recorded the lowest relative amount of AA (1.03 ± 0.16%).The maximum values for AA were observed in samples BLD0N1 (10.75 ± 0.36%) and BLD0N2 (12.31 ± 0.84%). Among the PUFA identified in each blend, this was the most abundant in both BLD0N1 and BLD0N2. All the five mixtures showed significant differences from each other (p < 0.05), but different from the others (p < 0.05).Finally, EPA was the major contributor to the sum of all PUFA identified in blends BLD005 (39.13 ± 5.84%) and BLD001 (26.79 ± 1.37%), and its contribution was also appreciable, but with much less expression, in blends BLD0N2 (5.13 ± 0.50%), BLD006 (4.77 ± 0.26%) and BLD0N1 (3.70 ± 0.22%). Blends BLD005 and BLD001 were significantly similar to each other (p < 0.05). In green and red seaweeds, LA represents no more than 3% of the total FAMEs, while in brown seaweeds, the average value is 8% [Undaria pinnatifida) and, lastly, in Chlorophyta (11–15%) [n-6 LC-PUFA, like AA, from LA and n-3 LC-PUFA, as EPA and docosahexaenoic acid , from ALA [Fucus vesiculosus and Gracilaria gracilis [n-6 PUFA equivalent to or less than that of n-3 PUFA [Palmaria palmata and Porphyra spp. [n-3 LC-PUFA, supporting the current health recommendation that aims to increase the daily consumption of n-3 LC-PUFA [ue is 8% ,48. Liteorophyta –15% [11,from ALA . AA, EPAid DHA; C:6 n-3, ffrom ALA . Only AAfrom ALA ,18. AA igracilis , which wid DHA; C:6 n-3, fgracilis . Neverthid DHA; C:6 n-3, fyra spp. . With ab LC-PUFA . EPA is  LC-PUFA . Some ev LC-PUFA . The pre LC-PUFA . Particun-3 PUFA, being an excellent alternative to fish (including crustaceans and shellfish), compatible with vegetarian and vegan diets [Independently of the selected mixture, these seaweed blends are a natural source of PUFA, particularly an diets .n-6:n-3 PUFA, atherogenicity index (AI) and thrombogenicity index (TI)  . The indn-6:n-3 PUFA ratios below 1 were recorded in the remaining blends. Regarding AI and TI, the lowest values were calculated in blends BLD005 for AI (0.23 ± 0.09) and BLD006 for TI (0.57 ± 0.17), while the highest indices were determined in blend BLD0N1 . Significant differences between the seaweed blends were revealed for UFA:SFA ratio, n-6:n-3 PUFA ratio, AI and TI. For the five blends under investigation, UFA:SFA ratios between 2.59 ± 0.16 (BLD0N2) and 1.40 ± 0.15 (BLD001) were found, and PUFA:SFA ratios ranged from 0.99 ± 0.09 (BLD0N1) to 1.37 ± 0.48 (BLD005). Except for blend BLD0N2, The PUFA:SFA ratio allows assessment of the influence of diet on cardiovascular health . The UFAn-6 (pro-inflammatory) and n-3 (anti-inflammatory) fatty acids is a nutritional factor of great relevance [n-6:n-3 PUFA ratio affects the regulation of metabolic functions and the development of associated syndromes, such as insulin resistance, adiposity and inflammation [n-6:n-3 PUFA was set at 1-4:1, the Western diet is characterized by an unbalanced consumption of n-6 PUFA, with as estimated ration of 15:1, which may contribute to the development and pathogenesis of the cardiovascular diseases, cancer, inflammation and autoimmune diseases [n-6:n-3 PUFA ≤ 1, as shown for blends BLD0N1, BLD001, BLD005 and BLD006.The balance between elevance . The valammation . Althougdiseases . Hence, Additionally, the AI and TI values should preferably be less than 1, and the lower the values the healthier the seaweed blend can be considered . These an-6:n-3 PUFA ratio and small AI and TI values. This strategy could be useful to improve the human diet quality, preventing inflammatory, cardiovascular and nervous system disorders. It is important to clarify that the impact of a given food on human health is not governed by its fatty acid composition, but by the set of all its nutritional parameters.Overall, the evaluation of fatty acid profile provides evidence of the proper choice of seaweed species for the blend formulation, given its high PUFA content, low Five seaweed blends  were provided by the company ALGAplus Ltd. , a seaweed farmer in a land-based integrated multi-trophic aquaculture system (IMTA) in Portugal . Each blend consisted of two to four dried and ground seaweed species produced under EU organic standards . AccordiFor moisture content determination, the samples (250 mg × 5 replicates (n = 5)) were dried in crucibles on an oven at 105 °C for 8 h. Moisture content (%) was obtained by Equation (1), as follows:Ash content was quantified gravimetrically after direct incineration at 575 °C for 22 h of the dry sample (250 mg × 4 replicates (n = 4)) on a muffle furnace equipped with a ramp and landing program , in accordance with that described by Van Wychen and Laurens . Ash conCrude protein content was indirectly estimated multiplying the elemental nitrogen content of the sample (2 mg × 3 replicates (n = 3)), previously determined by thermal conductivity using a Leco TruSpec 630-200-200 CNHS analyzer , by 2 nitrogen–protein conversion factors of 6.25  and 5 3. The resv/v) in a glass PYREX tube, followed by homogenization in a vortex for 2 min and ultrasound bath for 1 min. After incubation on ice on a rocking platform shaker  for 2 h, the suspension was centrifuged  for 10 min at 2000 rpm, and the organic phase was collected. To promote phase separation and wash the lipid extract, 1.25 mL of dichloromethane and 2.25 mL of Milli-Q water were added to the collected organic phase, centrifuged for 10 min at 2000 rpm, and the lower organic phase was recovered. Re-extraction steps using the same solvent proportions were repeated two more times. The organic phases collected were dried under a stream of nitrogen. Extracts were weighed to determine lipid content and stored at −20 °C. The results were expressed in % of dry weight of sample (DW).Lipid content was determined gravimetrically after total lipids extraction was per-formed according to a modified Bligh and Dyer method, as previously described in studies carried out by the research team ,61. The m/z in a one second cycle in full scan mode acquisition. The oven temperature was programmed from an initial temperature of 80 °C for 2 min, a linear increase to 160 °C at 25 °C min−1, followed by linear increase to 210 °C at 2 °C min−1, then to 250 °C at 20 °C min−1, standing at 250 °C for 20 min. The injector and detector temperatures were 220 °C and 230 °C, respectively. Helium was the carrier gas at a flow rate of 1.4 mL min−1. The software GCMS5977B/Enhanced MassHunter was used for data acquisition. The acquired data were analyzed using the qualitative data analysis software Agilent MassHunter Qualitative Analysis 10.0, and FA identification was performed by MS spectrum comparison with the chemical database NIST library and confirmed with the literature. The relative amounts of each FA were calculated by the percent relative area method with proper normalization using internal standard methyl nonadecanoate , considering the sum of all relative areas of identified fatty acids.Fatty acid profiling was performed by gas chromatography–mass spectrometry (GC–MS), as described in previous studies ,61. Fattn-6:n-3 ratios, and by the atherogenicity (Equation (3) AI) and thrombogenicity (Equation (4) TI) indices. The indices of AI and TI were calculated according to the equations reviewed by Chen and Liu [Nutritional quality of the lipid fraction was evaluated based on the fatty acid profile, through the UFA:SFA, PUFA:SFA and PUFA  and Liu , listed The total carbohydrate content of each sample was calculated by difference with the remaining parameters of the nutritional profile . The resp < 0.05) level were considered significant.All the experimental data are shown as mean ± standard deviation. Univariate statistical analysis was carried out using GraphPad Prism 9 software . Tests of normality (Shapiro–Wilk test or Kolmogorov–Smirnov test) and homogeneity of variance (Brown–Forsythe test or Bartlett test) were applied before the analysis. Kruskal–Wallis test, followed by Dunn’s post hoc comparisons, were performed in the absence of normality. One-way ANOVA followed by Tukey’s post hoc comparison was applied when checking for normality. Differences between means at the 5%  to analyze dissimilarities among the samples in terms of fatty acids components [After normalization  of the data, principal components analysis (PCA) and heatmap were performed using MetaboAnalyst 5.0 (Xia Lab @ McGill, Quebec, Canada; mponents . This study evaluated and compared the nutritional values of five commercially available seaweed blends, sustainably produced in Europe, with emphasis on the lipid fraction. To our knowledge, this is the first report assessing the composition of seaweed blends.n-3 PUFA. The nutritional and healthy indicators of the blends showed well-balanced n-6:n-3 ratios, with the AI and TI indices suggesting that they have the potential as key ingredients for sustainable and healthy diets contributing to the prevention and treatment of cardiovascular and inflammatory diseases. The blends can contribute to overcoming n-3 PUFA deficiency and replace fish oil in diets of Western populations and at the same time, can aid in the avoidance of the overexploitation of fish and shellfish. The blends were characterized by high ash levels, corresponding to high mineral loads, and shown to be good sources of protein of vegetable origin with low lipid contents. Lipidomic analysis based on GC–MS methodology showed that each blend has a characteristic fatty acid profile. In the five blends, the most abundant fatty acids were unsaturated, including These seaweed blends can be considered natural sources of valuable nutrients for human health, with a relevant role in the prevention of chronic and inflammatory diseases. Although our results indicate the presence of bioactive lipids, it is important to evaluate bioactive activities to apply these products in functional food. Further investigations must be carried out to prove the health-promoting role of the consumption of the seaweed blends in various diseases, including NCDs.


Structural characterization revealed that both K+/Cl− and K+/F− co-doping reduced the LixNi1−xO impurities and enlarged the lattice parameters compared to those of pure LNMO. Besides this, the K+/F− co-doping decreased the Mn3+ ion content, which could inhibit the Jahn–Teller distortion and was beneficial to the cycling performance. Furthermore, both the K+/Cl− and the K+/F− co-doping reduced the particle size and made the particles more uniform. The K+/Cl− co-doped particles possessed a similar octahedral structure to that of pure LNMO. In contrast, as the K+/F− co-doping amount increased, the crystal structure became a truncated octahedral shape. The Li+ diffusion coefficient calculated from the CV tests showed that both K+/Cl− and K+/F− co-doping facilitated Li+ diffusion in the LNMO. The impedance tests showed that the charge transfer resistances were reduced by the co-doping. These results indicated that both the K+/Cl− and the K+/F− co-doping stabilized the crystal structures, facilitated Li+ diffusion, modified the particle morphologies, and increased the electrochemical kinetics. Benefiting from the unique advantages of the co-doping, the K+/Cl− and K+/F− co-doped samples exhibited improved rate and cycling performances. The K+/Cl− co-doped Li0.97K0.03Ni0.5Mn1.5O3.97Cl0.03 (LNMO-KCl0.03) exhibited the best rate capability with discharge capacities of 116.1, 109.3, and 93.9 mAh g−1 at high C-rates of 5C, 7C, and 10C, respectively. Moreover, the K+/F− co-doped Li0.98K0.02Ni0.5Mn1.5O3.98F0.02 (LNMO-KF0.02) delivered excellent cycling stability, maintaining 85.8% of its initial discharge capacity after circulation for 500 cycles at 5C. Therefore, the K+/Cl− or K+/F− co-doping strategy proposed herein will play a significant role in the further construction of other high-voltage cathodes for high-energy LIBs.K Furthermore, LNMO possesses a three-dimensional framework structure, and the cost of the raw materials is relatively low, making it suitable for large-scale applications [xNi1−xO or NiO impurity phases and side reactions at the electrode interface at high potential, leading to structural instability and severe capacity fading during cycling [Currently, high-energy-density lithium-ion batteries (LIBs) are in urgent demand due to their wide applications in plug-in hybrid electric vehicles, portable electronic devices, and renewable energy storage devices ,2,3,4. T cycling .+ and Na+ ions have been incorporated into the Li site to synthesize Lix1+Ni0.5Mn1.5O4 and Lix1−NaxNi0.5Mn1.5O4 materials [3+ [2+ [3+ [3+ [3+ [4+ [5+ [3+ [4+ [5+ [− [− [3+, Cr3+, and F− co-doped LNMO [+ and F− co-doped LNMO [2+ and F− co-doped LNMO [2+ and Al3+ co-doped LNMO [2+, Al3+, and Ti4+ co-doped LNMO [2+ and Si4+ co-doped LNMO [4+ and La3+ co-doped LNMO [0.475Al0.01Cr0.04Mn1.475O3.95F0.05 cathode through a sol–gel method. After a series of tests, the Al3+/Cr3+/F− co-doped sample possessed excellent rate performance and cycling stability. Moreover, the co-doping also enhanced the cycling stability at room temperature (20 °C) and an elevated temperature (55 °C) [2+ and F− ions were incorporated into LNMO. The Mg2+/F− co-doping increased the quantities of Mn3+ ions and the lattice parameters. In addition, the Mg2+/F− co-doping increased the particle size, which could reduce the number of side reactions to some extent. The electrochemical results showed that the Mg2+/F− co-doped LNMO-MF sample obtained excellent rate performance and cycling stability at a high C-rate of 5C [To address the problems mentioned above, researchers have attempted to introduce metal cations or anions with similar radii into LNMO at different sites . Most of the previous studies were restricted to single-ion doping. For example, Liaterials ,8,9. Forials [3+ , Cu2+ [1 [3+ [2+ , Y3+ [12 [3+ [3+ , Cr3+ [1 [3+ [3+ , Zr4+ [1 [3+ [4+ , P5+ [16 [5+ [3+ , Ti4+ [1 [3+ [4+ , and V5+ [4+ [5+ , have be+ [5+ [−  and Cl− 5+ [− [−  ions havped LNMO ; Li+ andped LNMO ,24; Mg2+ped LNMO ; Cu2+ anped LNMO ,27; Cu2+ped LNMO ; Mg2+ anped LNMO ; and Ti4ped LNMO . Among t (55 °C) . In our te of 5C .+ possesses a larger radius (0.133 nm) than Li+ (0.076 nm), it is often used as a doping ion to replace Li+ in several cathode materials, including LiMn2O4 [0.5Co0.2Mn0.3O2 [1.2Ni0.2Mn0.6O2 [1.2Ni0.13Co0.13Mn0.54O2 [+ doping decreases the cation mixing and expands the Li layer spacing, thereby enhancing the structural stability and accelerating the Li+ diffusion in the bulk lattice. As reported by Yang et al., K+-doped Li1.2Ni0.2Mn0.6O2 was prepared via a sol–gel method. The K+ doping stabilized the surface O2− and reduced the Mn3+ ion content. The electrochemical results showed that the electrode capacity retention in K+-doped Li1.2Ni0.2Mn0.6O2 was 99.96% after 100 cycles, and it exhibited outstanding cycling stability [− (328 kJ mol−1) and Cl− (349 kJ mol−1) ions, with larger electron affinities, are commonly used elements for O2− (141 kJ mol−1) site substitution in LNMO cathodes, and they can stabilize the crystal structure and reduce the LixNi1−xO or NiO impurity generation due to the stronger Mn-F/Ni-F bonds or Mn-Cl/Ni-Cl bonds as compared to Mn-O/Ni-O bonds [As K LiMn2O4 , LiNi0.52Mn0.3O2 , Li1.2Niadius 0.1 nm than Mn0.54O2 . The K+ adius 0.1 nm than -O bonds .+/Cl− or K+/F− co-doping effects on the crystal structures and electrochemical performances of LNMO are still lacking. Hence, pure LNMO, K+/Cl− co-doped Lix1−KxNi0.5Mn1.5Ox4−Clx (x = 0.02 and 0.03), and K+/F− co-doped Lix1−KxNi0.5Mn1.5Ox4−Fx (x = 0.01 and 0.02) materials were prepared via a solid-state method. The characterization indicated that the influences of K+/Cl− co-doping on the structures and morphologies were different from those of K+/F− co-doping. The electrochemical results substantiated that both the K+/Cl− and K+/F− co-doped LNMO materials exhibited better rate and cycling performances than pure LNMO. The K+/Cl− co-doping was more conducive to improvement in the rate properties of LNMO, while the K+/F− co-doping tended to enhance the cycling stability. Hence, the structures, morphologies, and electrochemical performances of pure LNMO, K+/Cl− co-doped Lix1−KxNi0.5Mn1.5Ox4−Clx, and K+/F− co-doped Lix1−KxNi0.5Mn1.5Ox4−Fx were comprehensively compared in this study.Based on the studies described above, investigations into K+/Cl− co-doped, and K+/F− co-doped samples. The synthesis process was as follows. First, stoichiometric amounts of Li2CO3 (5% excess), NiO, MnO2, and KCl (0.525:0.5:1.5:x by mole) were dispersed in ethanol by ball-milling  for 10 min at 200 rpm and then for 210 min at 400 rpm to generate a homogeneous mixture. The obtained mixture was dried at 100 °C for 3 h to evaporate the ethanol. Finally, the obtained powder was annealed at 500 °C for 250 min and 850 °C for 8 h in air to form the K+/Cl− co-doped Lix1−KxNi1.5Mn0.5Ox4−Clx  samples . The K+/F− co-doped Lix1−KxNi1.5Mn0.5Ox4−Fx  samples, using KF as the dopant, were obtained using the same synthesis process as that used for the Lix1−KxNi1.5Mn0.5Ox4−Clx samples. The obtained samples were referred to as LNMO-KF0.01 and LNMO-KF0.02. The LNMO-KCl0.04, LNMO-KF0.005, and LNMO-KF0.03 samples shown in the A simple solid-state ball-milling process followed by a high-temperature calcination procedure was used to synthesize pure LNMO, KThe crystalline structures and lattice parameters of all samples were characterized by X-ray diffraction  with Cu Kα radiation in the 2θ range of 10–90°. The Rietveld refinement was carried out through TOPAS 4.2 software. The functional groups of the as-prepared samples were obtained by examining their Raman spectra . The surface elemental states and distributions of the samples were investigated via X-ray photoelectron spectroscopy  with λ = 633 nm. The material morphological characteristics were evaluated by scanning electron microscopy  and transmission electron microscopy . Energy-dispersive spectroscopy  mappings were used for surface element characterization of the co-doped LNMO samples.−2 on each electrode. In an Ar-filled glovebox, the coin cells were assembled with metallic lithium as the negative electrode, a porous polypropylene separator , and a carbonate-based electrolyte (1 M LiPF6/EC+EMC+DMC (1:1:1 by volume)). The charge and discharge measurements of the cells were carried out in the voltage range of 3.5−5.0 V (1C = 140 mA g−1) using a testing system  at 25 °C. Cyclic voltammetry  and electrochemical impedance spectroscopy  tests were conducted using an electrochemical workstation  between 3.5 and 5.0 V.Electrochemical tests of all the samples were performed by assembling CR2032-type coin cells . The positive electrodes were prepared by blending the co-doped LNMO powders, poly (vinylidene fluoride), and super-P carbon (8:1:1 by wt.%). The mixture was dispersed in an N-methyl-2-pyrrolidone solvent, and the resulting slurries were coated onto Al foil and then dried at 105 °C for 12 h under vacuum. The mass loading of the corresponding active materials was ≈1.5 mg cmxNi1−xO phases generated by LNMO losing oxygen when its calcination temperature exceeds 700 °C, as shown in xNi1−xO contents and lattice parameters are listed in xNi1−xO for the pure LNMO, LNMO-KCl0.02, LNMO-KCl0.03, LNMO-KF0.01, and LNMO-KF0.02 samples were 3.9, 3.3, 1.5, 2.4, and 2.0 wt.%, respectively. The contents of LixNi1−xO impurities were reduced by the K+/Cl− and K+/F− co-doping because Mn-Cl/Ni-Cl and Mn-F/Ni-F bonds are stronger than Mn-O/Ni-O bonds, which prevented oxygen release from the host lattice [+/Cl− co-doping caused the lattice parameters to become larger as the K+/Cl− co-doping content increased due to the larger ionic radius of K+ (0.133 nm) than that of Li+ (0.076 nm) and the larger ionic radius of Cl− (0.181 nm) than that of O2− (0.140 nm). This expanded the Li+ transport channels and facilitated Li+ diffusion [+/F− co-doping content increased, the lattice parameters of LNMO-KF0.005 (8.17508), LNMO-KF0.01 (8.17686), LNMO-KF0.02 (8.17540), and LNMO-KF0.03 (8.16980) increased first and then decreased  and Mn4+ (643.4 eV). The proportions of Mn3+ calculated based on the peak areas were 61.4%, 61.3%, 61.4%, 56.5%, and 53.5% for pure LNMO, LNMO-KCl0.02, LNMO-KCl0.03, LNMO-KF0.01, and LNMO-KF0.02, respectively. The K+/Cl− co-doping did not affect the amount of Mn3+. However, the content of Mn3+ was inclined to decrease with increasing K+/F− co-doping content, which agrees with the CV and the charge–discharge curves at the 4 V plateau discussed below. The Mn3+ contents of the five samples calculated by XPS were much higher than those calculated at the 4 V plateau during the charge–discharge tests, which may have been due to the existence of more Mn3+ on the surfaces of the samples instead of inside the structures [1/2) is evident in both samples after a major peak at ~292.0 eV (ascribed to K 2p3/2). This is consistent with the characteristics of the valence state of K+ [3/2 , the particle sizes of LNMO-KCl0.02, LNMO-KCl0.03, LNMO-KF0.01, and LNMO-KF0.02 were smaller at 0.794, 0.724, 0.717 and 0.460 µm, respectively. The K+/Cl− and K+/F− co-doping both narrowed the particle size distribution, and LNMO-KF0.02 exhibited the smallest particle size. +/Cl− or K+/F− co-doped samples were lower than that of the pure LNMO, and LNMO-KF0.02 had the smallest standard deviation, suggesting that the K+/Cl− and K+/F− co-doping resulted in a more uniform particle distribution [+ diffusion length. However, the larger surface area of the co-doped samples could accelerate the side reactions between the cathode and electrolyte, leading to capacity fading [formance ,43. To fribution . The smay fading . Figures+/Cl− co-doped LNMO-KCl0.02 and LNMO-KCl0.03 samples possessed the same octahedral structures as the pure LNMO, and the particle sizes of the LNMO-KCl0.02 and LNMO-KCl0.03 were slightly smaller than those of the pure LNMO. In addition, +/F− co-doped LNMO-KF0.01 and LNMO-KF0.02 samples also possessed smaller particle sizes compared to the pure LNMO  between the cathodic and anodic peaks for all the samples. As the scan rate increased, pure LNMO showed the largest potential difference among the five samples. Furthermore, LNMO-KCl0.03 showed the smallest potential difference, indicating that it exhibited the smallest electrode polarization and the best electrochemical reversibility. The lithium-ion diffusion coefficient can be calculated by the following equation:pI is the peak current, A is the electrode area (1.13 cm2), n is the number of electrons of each molecule in the electronic transfer reaction, C is linked to the concentration of Li+, and v is the scan rate (v s−1) [pI ∼ v1/2, and the diffusion coefficients of lithium ions for the five samples are given in LiD for pure LNMO (5.77 × 10−11/6.70 × 10−11) was smaller than those for LNMO-KCl0.02 (9.08 × 10−11/1.06 × 10−10), LNMO-KCl0.03 (1.14 × 10−10/1.64 × 10−10), LNMO-KF0.01 (6.34 × 10−11/8.79 × 10−11), and LNMO-KF0.02 (8.33 × 10−11/7.60 × 10−11). The LNMO-KCl0.03 exhibited the largest LiD because the K+/Cl− co-doping expanded the Li+ diffusion channels and improved the structural stability. The enhanced LiD for the LNMO-KF0.02 may have been due to it having the smallest particle size and more positive {100} crystal faces, which shortened the Li+ diffusion path and benefitted Li+ diffusion. The improved Li+ diffusion coefficients for the K+/Cl− and K+/F− co-doped samples were favorable for enhancing the rate performance.To investigate the effects of Ktructure , in accoformance . To eval (v s−1) . Figure +/Cl− and K+/F− co-doping effectively enhanced the discharge capacities of the electrode materials compared to those of pure LNMO at high C-rates, as shown in −1 at high C-rates of 5C, 7C, and 10C, respectively, compared with the values of 95.2, 73.0, and 27.6 mAh g−1 for pure LNMO. +/Cl− co-doped LNMO  and K+/F− co-doped LNMO  samples. After an appropriate amount of K+/Cl− or K+/F− co-doping, the LNMO-KCl0.03 and LNMO-KF0.01 samples delivered optimal rate performances. The rate capability of LNMO-KF0.01 was better than that of the pure LNMO but worse than that of LNMO-KCl0.03. The improved rate capability of LNMO-KCl0.03 was attributed to the synergistic effect of K+ and Cl− co-doping, which gives it the highest Li+ diffusion coefficient and a stable structure. The charge–discharge curves of all the samples exhibited three plateaus  after 500 cycles at a high rate of 5C. However, increased discharge capacity of 85.2 mAh g−1 for LNMO-KCl0.02, 96.3 mAh g−1 for LNMO-KCl0.03, 91.7 mAh g−1 for LNMO-KF0.01, and 90.2 mAh g−1 for LNMO-KF0.02 was observed, corresponding to improved capacity retentions of 77.7%, 82.7%, 82.5%, and 85.8%, respectively. To further understand the cycling behavior, the 300th and 500th charge/discharge curves of the five samples are presented in +/Cl− and K+/F− co-doping could alleviate the electrode polarization and improve the electrochemical kinetics. The LNMO-KCl0.03 and LNMO-KF0.02 exhibited the smallest polarization degrees of all the samples. The synergistic effect of the appropriate K+/Cl− and K+/F− co-doping was responsible for the improved cycling performances of LNMO-KCl0.03 and LNMO-KF0.02. Both K+/Cl− and K+/F− co-doping reduced the formation of LixNi1−xO impurities, decreased the particle sizes, expanded the Li+ diffusion channels, accelerated the electron migration, and improved the structural stability. Moreover, LNMO-KF0.02 showed the best cycling stability. In addition, the synergistic effect of K+ and F− co-doping not only exhibited a truncated octahedral morphology with more {100} faces but also exhibited the smallest particle size and lowest Mn3+ ion contents among the five samples. This allowed the Li+ diffusion between the active electrode particles to occur more easily, inhibited the Jahn–Teller distortion, and was therefore beneficial to improving the cycling stability.+/Cl− and K+/F− co-doping on the electrochemical properties of LNMO, EIS plots of the five electrodes after 200 cycles at 2C were obtained, as shown in Rs represents the electrolyte resistance, Rct is the contribution of the charge transfer resistance at the interface of the electrode and electrolyte, and CPE is the double-layer capacitance [w), related to the solid diffusion of Li+ into the LNMO material. The corresponding fitting results are listed in Rct value of pure LNMO (121.8 Ω) was higher than those of LNMO-KCl0.02 (100.5 Ω), LNMO-KCl0.03 (82.6 Ω), LNMO-KF0.01 (81.8 Ω), and LNMO-KF0.02 (77.1 Ω), suggesting that both K+/Cl− co-doping and K+/F− co-doping could reduce the charge transfer resistance, maintain stable interface structures, and promote charge transfer during the Li+ extraction/insertion processes.To further understand the detailed effects of the Kacitance . The sho+/Cl− and K+/F− co-doping on the structures, morphologies, and electrochemical performances of LNMO were systematically studied. Both K+/Cl− co-doping and K+/F− co-doping increased the lattice parameters and reduced the LixNi1−xO impurities, which expanded the Li+ transport channels and improved the structural stability. Furthermore, the K+/F− co-doping could also decrease the Mn3+ ion content and inhibit Jahn–Teller distortion. Besides this, both the K+/Cl− and the K+/F− co-doping decreased the particle size and made the particles more uniform, resulting in a shorter Li+ ion diffusion distance. Especially at a high C-rate, the LNMO-KCl0.02, LNMO-KCl0.03, LNMO-KF0.01, and LNMO-KF0.02 samples exhibited enhanced rate and cycling performances compared to pure LNMO. The LNMO-KCl0.03 showed the best rate capability with a discharge capacity of 93.9 mAh g−1 at 10C and superior cycling performance with capacity retention of 82.7% after 500 cycles at 5C. However, LNMO-KF0.02 delivered the best cycling stability and retained 85.8% of its initial capacity. The excellent electrochemical performances of the K+/Cl− and K+/F− co-doped LNMO samples will meet the practical application demands of LIBs.The effects of K


Dnmt1, −3a, and −3b mRNA levels and did not affect Tet family transcripts in the AA-ethanol group when compared with AA- and Wistar-water rats. DNMT and TET enzymatic activity measurements showed similar results to qPCR, where DNMT activity was increased in AA-ethanol group compared with AA-water and Wistar-water groups, with no statistically significant difference between groups in TET enzyme activity. In line with previous data, we found an increased percentage of global DNA methylation and hydroxymethylation in the AA-ethanol group compared with control rats. Finally, we investigated changes of selected candidate genes from dynorphin/kappa-opioid receptor system  and Dnmt3a genes that might be important in AUD-related behaviour. Our gene expression and promoter methylation analysis revealed a significant increase in the mRNA levels of Pdyn, Kor, and Dnmt3a in the AA-ethanol group, however, these changes can only be partially associate with the aberrant DNA methylation in promoter areas of the selected candidate genes. Thus, our findings suggest that the aberrant DNA methylation is rather one of the several mechanisms involved in gene expression regulation in AA rat model.Growing evidence suggests that epigenetic mechanisms, such as DNA methylation and demethylation, and histone modifications, are involved in the development of alcohol and drug addiction. However, studies of alcohol use disorder (AUD) that are focused on epigenetic DNA modifications and gene expression changes remain conflicting. Our aim was to study the effect of repeated ethanol consumption on epigenetic regulatory enzymes such as DNA methyltransferase and demethylase enzymes and whether those changes affected dynorphin/kappa-opioid receptor system in the Nucleus Accumbens (NAc). Two groups of male alcohol-preferring Alko Alcohol (AA) rats, rats which are selectively bred for high voluntary alcohol consumption and one group of male Wistar rats were used. The first group of AA rats had access to alcohol (10% ethanol solution) for 90 min on Mondays, Wednesdays and Fridays over a period of 3 weeks to establish a stable baseline of ethanol intake (AA-ethanol). The second group of AA rats (AA-water) and the Wistar rats (Wistar-water) were provided with water. Using qPCR, we found that voluntary alcohol drinking increased  Alcohol use disorder (AUD) is complex psychiatric disorder that is characterized by excessive alcohol drinking, alcohol dependence and relapses even after long periods of abstinence . It is ade novo DNMT)  . To estavo DNMT) .DNA demethylation caused by removal of the 5mC modification, can be performed in several ways. First, the modification can be removed by passive demethylation, where methylation maintenance process is impaired and no new 5mC marks are copied during DNA replication. Second, ten-eleven translocation enzymes (TET1–3) actively demethylate DNA. TET enzymes can mediate the oxidation of 5mC in order to generate hydroxymethylcytosine (5hmC) that can be further modified . Previouµ) opioid receptor (MOR), the enkephalins (ENK), which binds to delta (δ) opioid receptors (DOR) and the dynorphins (DYN), which binds to kappa (κ) opioid receptor (KOR) (Alcohol affects many parts of the brain and exerts its effects through different neurotransmitter systems. One of the systems affected by ethanol is the endogenous opioid system (EOS), that has been shown to play critical role in development of alcohol and other drug addictions . In alcoor (KOR) . These evia epigenetic modifications, underlie molecular mechanisms of AUD and relapses after abstinence. To test this hypothesis, we used two different rat strains, the standard laboratory Wistar rat and the alcohol preferring Alko Alcohol (AA) rat. The AA rat line is produced by selective breeding based on high voluntary alcohol consumption, and therefore, these rats are naturally prone to high alcohol consumption, which is hypothesized to occur due to abnormal function of opioidergic mechanisms , and it was suggested that may mediate their rewarding and reinforcing properties . Additiochanisms . Alteratad libitum. Animal experiments were conducted according to the 3R principles of the EU directive 2010/63/EU governing the care and use of experimental animals and following local laws and regulations . The protocols were authorized by the national Animal Experiment Board of Finland (license: ESAVI/10030/2018).30 male alcohol-preferring  rats  from generations F124.1 and 10 regular Wistar male rats  were used in the study. Before ethanol intake treatment AA rats were separated into two groups, first AA-ethanol group  and second AA-water . The rats were 3 months old and weighed at least 250 g at the onset of the experiments. The rats were housed in standard individually ventilated cages in groups of 2–3 rats per cage and the experiments were performed in reversed 12/12 h light/dark cycle (lights off at 8 am). Temperature was maintained at 22 ± 1°C. Water and rat chow were available The AA-ethanol group rats were trained to drink 10% ethanol solution using an intermittent, time-restricted two-bottle choice ethanol access paradigm [modified from ]. To monThe brains were harvested and snap frozen in isopentane chilled with dry ice. Immediately after freezing, the brains were stored at −80°C until RNA, DNA or nuclear extracts were prepared. The NAc was dissected on ice using a rounded puncher (inner diameter of 1.5 mm) with a rat brain atlas as a guide .RNeasy Mini Kit  was used according to the manufacturer’s instructions. For the generation of cDNA, 0.2 μg RNA and RevertAid First Strand cDNA Synthesis Kit  was used according to the manufacturer’s instructions. qPCR was performed on a QuantStudio 12K Flex Real-Time PCR system  using SYBR Green RT-PCR Master Mix  and previously designed primers  using the comparative CT (2−ΔΔcT) method  method .Nuclear proteins were extracted from NAc tissue according to the manufacturer’s protocol , and 5 μg of nuclear protein extracts were used for assays. DNMT activity was determined using an EpiQuik DNMT Activity/Inhibition Assay Ultra Kit and TET activity was measured using Epigenase 5mC-Hydroxylase TET Activity/Inhibition assay kit  according to manufacturer’s instructions. DNMT and TET activity levels (OD/h/mg) were calculated according to the manufacturer’s protocol.Genomic DNA was extracted from NAc tissue using DNeasy Blood & Tissue Kit  according to the manufacturer’s protocol and 100 ng of DNA were used for further assays. Global DNA methylation and hydroxymethylation analysis were performed using a Global DNA Methylation Assay Kit  and a Global DNA Hydroxymethylation Assay Kit  according to the manufacturer’s instructions. The percentage of methylated DNA (5mC%) and hydroxymethylated DNA (5hmC%) in total DNA was calculated according to the manufacturer’s protocol and formula.Dnmt3a, Kor, Pdyn promoter regions were detected according to the manufacturer’s instructions using the EpiMark 5hmC and 5mC Analysis Kit . Briefly, 5 μg of NAc genomic DNA were treated with T4 Phage b-glucosyltransferase and incubated at 37°C for 16 h. Glucosylated genomic DNA was digested either with 100 U of MspI or 50 U of HpaII or no enzyme (negative control) at 37°C for 5 h. Digestion was terminated using Proteinase K at 40°C for 30 min and inactivated at 95°C for 10 min. Real-time PCR was then performed on 1 μl of the glycosylated/digested DNA using site-specific primers listed in t method.Genomic DNA from new NAc tissue were extracted as above. The 5hmC and 5mC levels at CCGG sites in p-value of less than 0.05 was considered statistically significant. One-way ANOVA with repeated measures followed by Tukey’s multiple comparisons test were used for comparisons using GraphPad Prism 9 software .All data are expressed as the mean ± standard error of the mean (SEM). The significance level was established at 0.05 and a Dnmt and Tet genes in NAc. Our data showed  = 5.324, p = 0.0169; Dnmt3a, F = 14.30, p = 0.0003; Dnmt3b, F = 6.070, p = 0.0109; followed by Tukey’s post-hoc test; Dnmt1, p < 0.05 AA-water vs. AA-ethanol; mean ± SEM values: Wistar-water 1.00 ± 0.06; AA-water 0.98 ± 0.06; AA-ethanol 1.21 ± 0.05; Dnmt3a, p < 0.01, p < 0.001 AA-water vs. AA-ethanol; mean ± SEM values: Wistar-water 1.00 ± 0.05; AA-water 1.01 ± 0.05; AA-ethanol 1.33 ± 0.05; Dnmt3b, p < 0.05, p < 0.05 AA-water vs. AA-ethanol; mean ± SEM values: Wistar-water 1.00 ± 0.06; AA-water 1.00 ± 0.05; AA-ethanol 1.22 ± 0.05, n = 5–7). A trend towards a reduction in mRNA levels of Tet genes in the NAc after ethanol treatment was observed, but these changes did not reach statistical significance .First, we used qPCR to evaluate the effect of intermittent alcohol exposure on expression levels of a showed , that etDnmt1, Dnmt3a, and Dnmt3b were altered following ethanol exposure, we measured the activity of DNMT and TET enzymes in the NAc. As illustrated in  = 7.757, p = 0.0061; mean ± SEM values: Wistar-water 10.82 ± 1.53; AA-water 17.42 ± 5.37; AA-ethanol 32.04 ± 4.53, n = 5–6) in AA-ethanol compared with Wistar-water group (p < 0.01). No significant changes were observed in TET activity  = 0.5209, p = 0.6059; mean ± SEM values: Wistar-water 10.48 ± 0.41; AA-water 9.49 ± 0.78; AA-ethanol 9.89 ± 0.76, n = 5–6) in response to ethanol intake. These results are similar to our qPCR observations where ethanol altered Dnmt genes and did not affect Tet genes.As the transcription levels of  = 17.98, p = 0.0001; followed by Tukey’s post-hoc test; p < 0.001 compared with Wistar-water group, p < 0.001 AA-water vs. AA-ethanol; mean ± SEM values: Wistar-water 2.16 ± 0.17; AA-water 2.34 ± 0.11; AA-ethanol 3.24 ± 0.14, n = 5–7). Interestingly, the global level of 5hmC was also increased in the AA-ethanol group  = 8.372, p = 0.0036; followed by Tukey’s post-hoc test; p < 0.01compared with Wistar-water group, p < 0.05 AA-water vs. AA-ethanol; mean ± SEM values: Wistar-water 0.10 ± 0.01; AA-water 0.16 ± 0.02; AA-ethanol 0.28 ± 0.05, n = 5–7). These data demonstrate that ethanol treatment increases both DNA methylation and hydroxymethylation in the NAc.Next, we analysed global DNA 5mC and 5hmC levels in the NAc in response to ethanol treatment. We observed a significant increase in the relative percentage of 5mC  in the APdyn) and kappa opioid receptor (Kor) because these candidate genes have been demonstrated to be affected by ethanol  = 7.609, p = 0.0058; followed by Tukey’s post-hoc test; p < 0.01, p < 0.05 AA-water vs. AA-ethanol; mean ± SEM values: Wistar-water 1.00 ± 0.10; AA-water 1.26 ± 0.28; AA-ethanol 2.07 ± 0.13, n = 5–7). Kor mRNA (p < 0.01) after alcohol exposure  = 6.884, p = 0.0076; mean ± SEM values: Wistar-water 1.00 ± 0.10; AA-water 1.33 ± 0.14; AA-ethanol 1.79 ± 0.13, n = 5–7).Next, we studied the effect of ethanol treatment on mRNA levels of selected candidate genes related to ethanol addiction in NAc. We selected prodynorphin  = 3.623, p = 0.0588; mean ± SEM values: Wistar-water 12.87 ± 0.82; AA-water 9.04 ± 0.81; AA-ethanol 8.93 ± 1.67, n = 5–7; 5hmC%, F = 1.625, p = 0.2375; mean ± SEM values: Wistar-water 10.84 ± 1.03; AA-water 8.96 ± 0.72; AA-ethanol 8.51 ± 1.11, n = 5–7), but these changes did not reach statistical significance.Altered global changes in DNA methylation and hydroxymethylation could cause changes at certain gene promoters enriched with CpG islands and consequently influence the expression of these genes. In this work, we evaluated the transcription of several genes involved in opioid- and dopaminergic systems and related to drug addiction . We chosor genes  followiner, 5mC%  and 5hmCer, 5mC% , were slPdyn promoter methylation status we observed a significant reduction in 5mC%  = 11.06, p = 0.0019; followed by Tukey’s post-hoc test p < 0.01 Wistar-water vs. AA-water, p < 0.01 Wistar-water vs. AA-ethanol; mean ± SEM values: Wistar-water 65.23 ± 6.43; AA-water 37.26 ± 3.92; AA-ethanol 34.11 ± 4.77, n = 5–7). We also observed a slight increase in promoter 5hmC%  = 1.526, p = 0.2568; mean ± SEM values: Wistar-water 9.19 ± 6.33; AA-water 18.11 ± 4.53; AA-ethanol 20.48 ± 3.09, n = 5–7), but due to variability between animals, these changes did not reach statistical significance.Regarding the  in 5mC%  in AA-waer 5hmC%  in AA-waKor promoter methylation and observed that 5mC% was reduced slightly  = 3.621, p = 0.0581; mean ± SEM values: Wistar-water 27.21 ± 1.55; AA-water 20.91 ± 1.42; AA-ethanol 20.18 ± 2.81, n = 5–7) whereas 5hmC% was increased  = 8.793, p = 0.0045; followed by Tukey’s post-hoc test p < 0.01 Wistar-water vs. AA-ethanol, mean ± SEM values: Wistar-water 11.01 ± 2.35; AA-water 16.35 ± 0.62; AA-ethanol 20.30 ± 1.24, n = 5–7).Finally, we analysed slightly  in AA-wancreased  in the AIn this work, we investigated whether intermittent ethanol exposure affects the expression and enzymatic activity of the epigenetic DNA modifiers DNMT and TET using the alcohol-preferring rat model. Previously, we have shown that addictive substances and environmental factors, may affect the transcription of epigenetic DNA modifiers in different brain regions . Thus, iPdyn, Kor, and Dnmt3a; however, we failed to convincingly demonstrate that the aberrant DNA methylation in promoter areas are responsible for the changed gene expression of these genes.We report three key findings. First, we show that intermittent ethanol exposure increased significantly the mRNA levels of DNMTs and these changes were associated with significant increases in enzymatic activity in AA-ethanol rats compared with control rats in the NAc. No significant changes were observed in TET mRNA levels or enzymatic activity. Second, we demonstrate a significant increase in the relative percentage of global methylation and hydroxymethylation in the AA-ethanol group compared with the control groups. Finally, our selected gene expression and promoter methylation status analysis revealed a significant increase in the mRNA levels of Dnmt3a and to a lesser extent both Dnmt1 and Dnmt3b mRNA levels, and the increase in expression of these genes was associated with increased DNMT enzymatic activity in NAc. Our data are consistent with previously published results, for instance, excessive alcohol drinking increased Dnmt1 expression and reduced histone H4 acetylation in NAc of rodents and reducing DNA methylation by inhibiting the activity of DNMT with systemic administration of 5-azacitidine prevented excessive alcohol use in mice  leads to increases in 5hmC even if TET enzymatic activity is not changed. Thus, an increase in 5hmC levels may be a compensatory process to ensure optimal DNA demethylation in the cell. Changes in the global levels of 5mC and 5hmC also suggest that a new DNA methylation and demethylation balance develops following repeated ethanol exposure in the NAc. We have found similar phenomena also after repeated psychostimulant exposure . TherefoIn addition to the changes in ethanol-induced global methylation, we were interested in whether repeated ethanol drinking also affects the methylation state of promoters of selected genes in the AA rat model. There is currently no consensus on genes that could serve as marker genes in AUD models. Therefore, we selected candidate genes that have been studied in various alcohol models.Dat gene, but we found significant increases in Drd1, Drd2, Kor, Pdyn, and DNMT3a mRNA levels in AA-ethanol rats. The results of the methylation status in the promoter areas of selected genes were more complex. The Pdyn promoter region under study was significantly hypomethylated, while in the Kor and Dnmt3a promoter regions we saw a trend towards hypomethylation, but it did not reach statistical significance. Previous studies of promoter methylation status had revealed several genes that are affected by the interplay between alcohol and epigenetic regulation and that may play a role in alcohol addiction  in infant and adolescent rats and Kor mRNA levels in the prefrontal cortex in infant rats. The changes in gene expression in the VTA were accompanied by a reduction of DNA methylation at the Pdyn gene promoter, and by a reduction of DNA methylation at the Kor gene promoter. These results suggest that different patterns of alcohol consumption in experimental animals may similarly affect the methylation and transcription of Pdyn and Kor gene promoters.We examined the transcription of several genes from dopaminergic and opioidergic systems  previousddiction . Some ofddiction  upregulaKor, Dnmt3a) showed that the increase in transcription after repeated alcohol consumption in AA rats was only partially explained by a 5mC decrease in promoter, suggesting that other regulatory mechanisms may be involved. Accumulating data suggest that the DNA methylation and histone modification interact and influence each other and together may fine-tune gene expression . However, no definitive conclusions about 5hmC changes can be drawn from the analysis of the three genes.Interestingly, the changes in the 5hmC level of the selected gene promoters were variable. This may indicate that after repeated alcohol administration a new balance between 5mC and 5hmC levels may be formed in the promoter region of some genes (e.g., Finally, the results of our study indicate that the epigenetic changes also contribute to the AA-rat model susceptibility to ethanol drinking rather than being a pure response to alcohol exposure. This speculation is supported by the differences in DNMT enzymatic activity and the marker genes promoter methylation in AA-water rats compared to Wistar-water rats. The underlying mechanism for these epigenetic changes is unclear, but we hypotheses that the stress level of an individual rat in the early stages of development may affect epigenetic mechanisms of basal level and some of these epigenetic changes may persist into adulthood  and causOur results, using alcohol preferring AA-rat model, support previous findings that repeated alcohol exposure tends to increase DNMT activity and may induce the aberrant DNA methylation in the NAc. The aberrant DNA methylation together with other epigenetic mechanism, such as histone modification can be the specific molecular mechanisms mediating dependence-induced neuroadaptations induced by alcohol consumption.


MAF-9 exhibits very large N2-induced breathing and colossal positive and negative thermal expansions like FMOF-1, but the lower molecular weight and smaller volume of MAF-9 give 16% and 4% higher gravimetric and volumetric N2 uptakes, respectively. In contrast, MAF-2F is quite rigid and does not show the inversed temperature-dependent N2 adsorption and large guest-induced expansion like MAF-2. Further, despite the higher molecular weight and larger volume, MAF-2F possesses 6% and 25% higher gravimetric and volumetric CO2 uptakes, respectively. These results can be explained by the different pore sizes and side group arrangements in the two classic framework prototypes, which demonstrate the delicate roles of ligand side groups in controlling porosity, surface characteristic and flexibility.3-Ethyl-5-trifluoromethyl-1,2,4-triazole is synthesized by a one-pot reaction. Using this asymmetric triazole ligand bearing one trifluoromethyl and one ethyl as side groups, we construct two new porous coordination polymers, MAF-9 and MAF-2F, being isostructural with the classic hydrophobic and flexible materials, FMOF-1 and MAF-2, based on symmetric triazole ligands bearing two trifluoromethyl groups or two ethyl groups, respectively. MAF-9 and MAF-2F can adsorb large amounts of organic solvents but completely exclude water, showing superhydrophobicity with water contact angles of 152 By partial replacement of the non-coordinative ligand side groups, the classic porous framework materials can be easily synthesized with their main adsorption properties retained or even improved. As a new type of adsorbent showing high structural regularity and extremely rich structural diversity, porous coordination polymers (PCPs) or metal-organic frameworks (MOFs) have attracted great attention for achieving extraordinary properties . PCPs ca2-induced framework breathing, colossal positive/negative thermal expansion, and low dielectric constant [[Ag(bftz)] -1,2,4-triazole) is a classic PCP constructed by a perfluorinated organic ligand , which cconstant ,35. Neveconstant ,36, whic2 adsorption –: 164.04, found: 164.12. EA calcd for C5F3H6N3 (%): C, 36.37; N, 25.45; H, 3.66. Found. C, 36.90; N, 25.52; H, 3.62.The synthesis method reported for Hfmtz was used . A mixtu3  in water (2.0 mL). After about two weeks, colorless crystals were collected for single-crystal X-ray diffraction analyses. Microcrystalline powders: a solution of Hfetz  in toluene (20 mL) was poured into an aqueous solution (20 mL) of AgNO3 . After the suspension was stirred for 2 h at room temperature, the white crystalline powder was filtered and washed by methanol (40 mL) three times, and then dried in air for 2 h . EA calcd(%) for AgC5F3H5N3: C 22.08, N 15.45, H 1.85; found: C 22.34, N 15.26, H 1.77.Single crystals: toluene (2.5 mL) and a solution of Hfetz  in methanol (2.0 mL) were sequentially layered onto a solution of AgNO3)2 · 3H2O  in water (3 mL) and a solution of Hfetz  in toluene (3 mL) were mixed and sealed in a 15-mL Teflon-lined reactor, heated at 160°C for 72 h, and then slowly cooled to room temperature to give colorless crystals. Microcrystalline powders: a solution of Hfetz  in ethanol (10 mL) was added into a suspension of Cu2O nanoparticle (0.5 mmol) in ethanol (10 mL). N2 was bubbled into the mixture for 2 min to evacuate O2. The solution was sealed in a glass bottle, refluxed for 30 min, and then slowly cooled to room temperature. The resultant white crystalline powders were filtrated, washed with ethanol three times, and then dried in air for 2 h . EA calcd(%) for C5CuF3H5N3: C 26.38, N 18.46, H 2.21; found: C 26.81, N 18.40, H 2.37.Single crystals: a solution of Cu(NOP/P0 = 0.95), using the liquid N2 density of 0.804 g cm−3 or liquid CO2 density of 1.104 g cm−3. Volumetric uptake was converted from the gravimetric uptake using the crystal density, supposing that the material did not change volume after adsorption, which was basically valid for MAF-2/MAF-2F during N2/CO2 adsorption. FMOF-1/MAF-9 breathed significantly during N2 adsorption with very similar amplitudes, so that the absolute volumetric uptakes have relatively large errors, but they can be compared with each other.Gas sorption isotherms of FMOF-1 and MAF-2 were adopted from the literature ,46. Gas F2 with SHELXTL-2014 package. Anisotropic thermal parameters were applied to all non-hydrogen atoms. The hydrogen atoms were generated geometrically. To keep the anisotropic thermal parameters of the disordered trifluoromethyl and ethyl groups of MAF-9 within reasonable limits, ISOR restrictions were used in the refinements. Crystal data were summarized in Table S1.Single-crystal X-ray diffraction intensities of MAF-9, MAF-2F and MAF-2 were collected on a Pilatus XtaLAB P300DS or a Rigaku Oxford SuperNova single-crystal diffractometer by using graphite monochromated Cu-Kα radiation. Absorption corrections were applied by using the multi-scan program REQAB or CrysAlisPro. The structures were solved by the direct method and refined by the full-matrix least-squares method on Description/analysis of the crystal structures used the true C−H bond length of 1.1 Å. The void ratio was calculated by the SOLV route of PLATON 130220, using the default setting (probe radius of 1.2 Å). The void ratio of MAF-9 containing disordered ethyl groups was calculated as the average value of the two values supposing the material adopts two extreme structures.θ range of 5−40° on unit-cell parameters, zero point and background terms with Pseudo-Voigt profile function and Berar-Baldinozzi asymmetry correction function. All the indexing and refinements were performed by the Reflex plus module of Materials Studio 5.5.PXRD patterns were collected on a Bruker D8 DAVANCI X-ray powder diffractometer with CuK-α radiation in the transmission mode at room temperature. Pawley refinements of PXRD data were performed in the 22 molecules were both regarded as rigid. The simulation box contained one unit cell, and the Metropolis method based on the universal force field (UFF) was used. Mulliken charges calculated from Density Functional Theory (DFT) were adopted for all the atoms of the host frameworks and CO2 molecules [6.All simulations/calculations were performed using the Materials Studio 5.5 package. All the gas adsorption sites were generated from Grand Canonical Monte Carlo (GCMC) simulations with the fixed pressure task (at 195 K and 1 atm) in the Sorption module. The host frameworks and COolecules , with th−5 Ha, 4 × 10−3 Å and 5 × 10−3 Å, respectively.Before the GCMC simulations, full geometry optimizations were performed according to the literature . The widnwaa094_Supplemental_FilesClick here for additional data file.


The incidence of obesity is increasing worldwide, and it is a risk factor for diabetes, dyslipidemia, and nonalcoholic fatty liver disease. Our previous study had demonstrated that high-fat diet induced increased weight gain, fat weight, serum cholesterol, triglyceride, and ATL levels in liver, and influenced the diversity and composition of cecal microbiota in mice. Hence, this study aimed to investigate the roles of the gut microbially derived metabolites and liver metabolites between the obese and lean mice, focusing on their association with the progression of obesity induced by high-fat diet (HFD).An obesity model in mice was established with HFD for 16 weeks. Cecal contents and liver tissues metabolomics based on ultraperformance liquid chromatography-quadrupole-time-of-flight mass spectrometry and orthogonal partial least squares discriminant analyses (OPLS-DA) was performed to identify the alterations in metabolites associated with obese mice.Obese and lean groups were clearly discriminated from each other on OPLS-DA score plot and major metabolites contributing to the discrimination were mainly involved in glycerophospholipid metabolism, primary bile acid biosynthesis, and biosynthesis of unsaturated fatty acids pathways. HFD-induced alterations of 19 metabolites in liver and 43 metabolites in cecum contents were identified as potential biomarkers related to obesity. Specifically, chenodeoxycholic acid, taurochenodeoxycholate, and tauroursodeoxycholic acid in liver were elevated 35.94, 24.36, and 18.71-fold, respectively. PI(P-16:0/18:1(9Z)), PG(19:0/16:0), PS), PI(22:1(11Z)/12:0), and PE(21:0/0:0) in cecum were enhanced 884, 640.96, 226.63, 210.10, 45.13-fold in comparison with the lean mice. These metabolites were the most important biomarkers for discriminating between the obese and lean mice. In addition, cecum contents metabolites were strongly correlated with hepatic metabolites through gut-liver axis analysis.HFD increased lipid profiles  and total bile acid (primary and secondary bile acid) in liver and cecum, suggesting that they may play an important role in the progression of obesity. These metabolites can be used to better understand obesity and related disease induced by HFD. Furthermore, the level alterations of these metabolites can be used to assess the risk of obesity and the therapeutic effect of obesity management. The obesity epidemic and its metabolic complications are a major public health problem around the world . ObesityGut microbiota is now considered to be an important factor affecting human health and disease . EmerginDeciphering signatures specific to liver alterations would be most useful for future obesity diagnostic biomarkers. Several metabolites, including fatty acids, lysophophatidylcholines (lysoPCs), and branched-amino acids (BCAAs) have been identified as potential biomarkers of obesity and related diseases through human and animal studies , but undEighteen female 7-week-old Kunming mice were obtained from Vital River Laboratory Animal Technology Co. Ltd , and housed in cages under a 12 h light–dark cycle. After 1-week acclimation period, the mice were randomly divided into two treatment groups (n = 9) and fed with (1) normal-chow diet, (2) high fat diet (HFD) composed of 67% (w/w) normal chow, 10% lard, 20% sucrose, 2.5% cholesterol, and 0.5% sodium cholate  according to the previous report with slight modifications .g, 10 min), and stored at − 80 °C for further analysis. Liver tissues were washed with ice-bold buffered saline, weighed and homogenized in saline with a homogenizer. The homogenates were then centrifuged  for further analysis. The Livers and cecum contents were extracted and snap-frozen in liquid nitrogen, and then stored at ‒ 80 °C for metabolomics analysis.After 16 weeks of experimental administration, the mice were fasted 6 h. The blood samples were collected from eighteen mice and placed at room temperature for 1 h. Serum was obtained by centrifuged (3000g for 20 min at 4 °C. The supernatants were collected for ultraperformance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UHPLC-Q-TOF/MS) analysis. In parallel to the preparation of the test samples prepare a bulk quality control (QC) sample, which is made by mixing equal volumes from each of the samples. The QC samples were used to monitor the LC–MS response.The Liver tissues (60 mg) were homogenized, vortexed for 1 min and dissolved in 800 μL solvent mixture containing methanol/acetonitrile . The samples were vortexed for 1 min, and sonicated for 30 min on ice and the sonication cycle was repeated for twice, and then placed at − 20 °C for 1 h, and then centrifugation at 14,000Metabolic analysis of liver samples were carried out by an Agilent 1290 Infinity LC ultra-high pressure lipid chromatograph (UHPLC) system  equipped with an electrospray ionization source operating in positive and negative ion mode. For the metabolomics analysis, an ACQUITY UPLC HSS T3 column  was used. The column was maintained at 25 °C and eluted at a flow rate of 300 μL/min. The mobile phase was composed of A (25 mM ammonium acetate and 25 mM ammonium hydroxide in water) and B (acetonitrile). The process of linear gradient elution as follows: 0–1.0 min, 95% B; 1.0–14.0 min, 95–65% B; 14.0–16.0 min, 65–40% B; 16.0–18.0 min, 40% B; 19.0–18.1 min, 40–95% B; 18.1–23.0 min, 95% B. The auto sampler was maintained at 4 °C.m/z of 60–1000 Da; product ion scan m/z range, 25–1000 Da; TOF MS scan accumulation time 0.20 s/spectra; product ion scan accumulation time, 0.05 s/spectra; Ion Source Gas1(Gas1), 60 psi; Ion Source Gas2 (Gas2), 60 psi; Curtain gas (CUR): 30 psi; source temperature, 600 °C; Ion Sapary Voltage Floating (ISVF), ± 5500 V; Declustering potential (DP): ± 60 V; Collision energy, 35 ± 15 eV. MS/MS data were acquired in the information dependent acquisition (IDA) mode and using high sensitivity modes. The settings of IDA as follows: exclude isotopes within 4 Da, candidate ions to monitor per cycle, 6. Meanwhile the QC samples were analyzed to monitor the system stability and data quality.The mass spectrometer (MS) was a Triple TOFTM 5600 system  equipped with an electrospray (ESI) as ionization source in positive (ESI+) and negative (ESI−) ion mode. The MS properties were set as follows: scan range, g for 15 min at 4 °C. The resulting supernatant was transferred to a fresh glass vial for analysis. The quality control (QC) sample was prepared by mixing an equal aliquot of the supernatants from all of the samples.50 mg of cecal sample was weighted to an EP tube, and 1000 μL extract solution (acetonitrile: methanol: water = 2: 2: 1) with 1 g/mL internal standard was added. After 30 s vortex, the samples were homogenized at 35 Hz for 4 min and sonicated for 5 min on ice. The homogenization and sonication cycle was repeated for 3 times. Then the samples were incubated for 1 h at − 40 °C and centrifuged at 12,000LC–MS/MS analyses were performed using an UHPLC system  with a UPLC HSS T3 column  coupled to Q Exactive mass spectrometer . The mobile phase A was 0.1% formic acid in water for positive mode, and 5 mmol/L ammonium acetate in water for negative mode, and the mobile phase B was acetonitrile. The elution gradient was set as follows: 0–1.0 min, 1% B; 1.0–8.0 min, 1–99% B; 8.0–10.0 min, 99% B; 10.0–10.1 min, 99% ~ 1% B; 10.1–12 min, 1% B. The flow rate was 500 μL/min. The injected volume was 2 μL. The QE mass spectrometer was used for its ability to acquire MS/MS spectra on information-dependent acquisition (IDA) mode in the control of the acquisition software . In this mode, the acquisition software continuously evaluates the full scan MS spectrum. The ESI source conditions were set as following: sheath gas flow rate as 45 Arb, Aux gas flow rate as 15 Arb, capillary temperature 400 °C, full MS resolution as 70,000, MS/MS resolution as 17,500, collision energy as 20/40/60 in NCE mode, spray Voltage as 4.0 kV (positive) or − 3.6 kV (negative), respectively.The raw UHPLC-Q-TOF/MS data were converted to the mzXML format using ProteoWizard and processed with an in-house program, which was developed using R and based on XCMS, for peak detection, extraction, alignment, and integration. Then an in-house MS2 database (BiotreeDB) was applied in metabolite annotation. The cutoff for annotation was set at 0.3.p < 0.05 (student t test) were considered as significantly changed metabolites. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used to analyze the differential metabolites. In addition, all differentially abundant metabolites were queried against KEGG (http://www.kegg.jp/) and mapped to KEGG pathways. Enrichment analysis was performed to further explore the impact of differentially expressed metabolites and to analyze the internal relationships between differentially expressed metabolites using the Fisher’s exact test. Only functional categories and pathways with p < 0.05 were considered to have significant enrichment.The final dataset was imported to SIMCA15.0.2 software package  for multivariate statistical analysis. Orthogonal partial least squares discriminant analyses (OPLS-DA) was performed to distinguish the overall differences in metabolic profiles between obese and lean samples and to find differential metabolites between the two groups. Then, a sevenfold cross validation was performed to calculate the value of R2 and Q2. R2 indicates how well the variation of a variable is explained and Q2 means how well a variable could be predicted. To check the robustness and predictive ability of the OPLS-DA model, 200 times permutations was further conducted. Afterward, the R2 and Q2 intercept values were obtained. Furthermore, the value of variable importance in the projection (VIP) of the first principal component in OPLS-DA analysis was obtained. It summarizes the contribution of each variable to the model. The metabolites with VIP > 1 and 2X = 0.808, R2Y = 1, Q2 = 0.796 for positive data, and R2X = 0.572, R2Y = 0.993, Q2 = 0.842 for negative data were obtained. The permutation test was used to guard against overfitting of the OPLS-DA models. As shown in Fig. 2 = 0.999 and Q2 = − 0.148 for positive data, and R2 = 0.944 and Q2 = − 0.473 for negative data, which demonstrated that the OPLS-DA models were robust and reliable without overfitting.The hepatocytes are most exposed to gut-derived toxic factors through gut-liver axis, including bacteria and bacterial products. Examination of the metabolites composition in the liver will provide more accurate understanding about liver lipid metabolism and signaling. UHPLC-Q-TOF MS liver metabolomics analysis discovered the different metabolomics features in the obese and lean groups. As shown in Fig. p < 0.05. UHPLC-Q-TOF MS metabolomics analysis dis-covered 19 differentially enriched metabolites between the obese and lean groups. Twenty-six of the metabolites were elevated in the obese group (fold change > 1.2) while Twenty-eight of them were decreased (fold change < 0.7). As shown in Fig. p < 0.05), while PC (16:0/16:0) and 1-stearoyl-2-oleoyl-sn-glycerol 3-phosphocholine were significantly decreased with fold changes of 0.46 and 0.6, respectively (p < 0.05), which indicates high relevance to the difference between normal and high-fat diet fed mice. Compared to the lean group, HFD intervention significantly increased the amounts of oleic acid (3.89-fold), while the levels of eicosapentaenoic acid, -4,7,10,13,16,19-docosahexaenoic acid, and alpha-linolenic acid involving in biosynthesis of unsaturated fatty acid pathway, were significantly decreased with fold changes of 0.30, 0.62, and 0.67 (p < 0.05), respectively.The significantly differential metabolites were selected based on the criteria of an OPLS-DA model VIP > 1 and p < 0.05), respectively, while the levels of cholic acid (CA), taurocholic acid (TCA) and taurine were decreased with fold changes of 0.62, 0.35, and 0.41 (p < 0.05), respectively, which were potential markers related to obesity. Additionally, HFD intervention significantly increased taurodeoxycholic acid (TDCA) and taurolithocholic acid (TLCA) belonging to secondary bile acid with the fold changes of 5.88 and 4.36 (p < 0.05), respectively. Besides on these results, we observed that several metabolites were also significantly changed, including the levels of purines, nicotinamide, and amino acids (data not shown).Changes in circulating bile acids are associated with obesity and related diseases. In mice, more than 95% conjugated bile acids are taurine-conjugated bile acids, while glycine-conjugated bile acids are more abundant in human . Consist2X = 0.384, R2Y = 0.992, Q2 = 0.92 for positive data, and R2X = 0.409, R2Y = 0.996, Q2 = 0.967 for negative data were obtained. In addition, the OPLS-DA models validated by the permutation test with 200 random permutation tests generated intercepts R2 = 0.85 and Q2 = − 0.74 for positive data, and R2 = 0.78 and Q2 = -0.88 for negative data , 9,10-epoxyoctadecanoic acid, 13-OxoODE), and glycerolipids /0:0), gingerglycolipid A) were filtered and identified as potential biomarkers. Twenty-three of the metabolites involving in glycerophospholipid metabolism were significantly changed (p < 0.05), twenty of which were elevated, while three metabolites were reduced after HFD intervention. Nine phosphatidylcholines (PC) species, including PC(0:0/14:0), PC(O-16:0/2:0), PC(2:0/O-16:0)[U], PC(7:0/8:0)[U], PC(O-15:0/0:0), PC(O-16:1(11Z)/2:0), PC(O-8:0/O-8:0), PC(8:0/7:0)[U], and PC(8:0/6:0), were significantly increased 18.18, 17.86,17.04, 9.20, 6.40, 4.15, 2.77, 1.56, and 1.52-fold, respectively, which contributed to evaluate the difference between normal and high-fat diet mice. In addition, the levels of PG(19:0/16:0), PS), PI(P-16:0/18:1(9Z)), PI(22:1(11Z)/12:0), PE (21:0/0:0), and PE (13:0/0:0) were increased 640.96, 226.63, 884.00, 210.10, 45.13, and 8.17-fold with VIP > 1.5, respectively, which were the most important hepatic metabolites for discriminating between lean and obese mice. However, the levels of LysoPC(18:1(9Z)), LysoPE(15:0/0:0), and LysoPE) were negatively affected with 0.32, 0.44, and 0.30-fold, respectively. L-palmitoylcarnitine involved in fatty acid metabolism was enhanced 16.16-fold. The levels of three metabolites involving in biosynthesis of unsaturated fatty acid, including adrenic acid, icosenoic acid, and stearic acid were elevated 1.39, 2.31, and 1.44-fold, respectively.As shown in Table Total bile acid metabolites were enhanced in comparison with the lean mice. The increased concentration of bile acid in obese mice might be associated with the disorder of liver function. As shown in Table The Metabolites produced by the gut microbiota play important roles in mediating the complex interaction between the gut microbiota and the human holobiont . In our Disorders of glycerophospholipid and fatty acid metabolism have been found to be directly related to the initiation and development of hyperlipidemia. In hepatic metabolites, compared to the lean mice, the overall levels of glycerophospholipids were increased, indicating that HFD led to lipid disorders. The levels of most glycerophospholipids were enhanced in the liver of obese mice, including choline, sn-glycerol phosphoethanolaminesn, CDP-choline, O-phosphoethanolamine, glycerophosphocholine, and phosphorylcholine. Excess glycerophospholipids were positively associated with fat accumulation, which agrees with the finding that an increased glycerophospholipid metabolites level in obese mice was positively correlated with fat accumulation . CholineWe found that there was a possible link between the altered microbial metabolites and liver metabolites. Most of glycerophospholipid metabolites were also enhanced in cecum contents. In addition to PC species, we also found many other glycerophospholipids, such as phosphoethanolamine (PE), phosphatidyl inositol (PI), and phosphatidylserine (PS) species, were significantly increased in the HFD group. These findings are in accordance with the notion that glycerophospholipids play several important roles in the development of NAFLD , 30. NinThere exists highly efficient bile acid preservation and recycling system within the body, which is termed the enterohepatic circulation. Bile acids, which are biosynthesized by the catabolism of cholesterol in the liver, are involved in regulating lipid absorption and metabolism , 41, andA few changes to primary and secondary metabolites in both liver and cecum contents between the lean and obese mice confirmed that lipids primarily drove this metabolic pathology. Cholic acid and chenodexycholic acid, known as primary bile acids, are synthesized from cholesterol in the liver and enter the small intestine where they are conjugated to glycine and taurine. Cholic acid (CA), the major bile acid, was significantly decreased 0.62-fold in the liver, which is inconsistent with the study that CA was increased 68-fold after high fat, high cholesterol, and cholate diet in mice . The resFurthermore, the level of oleic acid was also affected by HFD. Oleic acid is a monounsaturated fatty acid, which was enhanced 3.89-fold in the liver of obese mice. This result is consistent with the finding that oleic acid was significantly enhanced in serum of liver cirrhosis and hepatocellular carcinoma patients . Alpha-lMost studies used male mice to construct high-fat diet-induced obesity model , 65, 66.The UPLC-Q-TOF MS-based liver and cecum contents studies have been performed to reveal the complex interactions between liver and gut of obese mice induced by a high-fat diet and provided important metabolic information for obesity and related diseases. A high-fat diet increased lipid profiles  and total bile acid (primary and secondary bile acid) in liver and cecum, indicating that HFD regulated glycerophospholipid metabolism, primary bile acid synthesis, and fatty metabolism pathways. This study provides evidence regarding the important regulatory functions of metabolites produced by gut commensal bacteria as well as insights into the interaction between the microbial products and host. The differences in these metabolic profiles between the obese and lean mice may provide a better understanding of the metabolic changes of obesity, which could be used for future clinical diagnosis and treatment.


Scientific Reports 10.1038/s41598-021-93521-0, published online 09 July 2021Correction to: The original version of this Article contained errors.In Figure 10, the slip color scale for Mw 6.4 foreshock (lower panel) was incorrect. As a result, the chroma subsampling was incorrect.The original Figure Additionally, in the Conclusions section,“On the F1 fault, The maximum slip was 1.5 m.”now reads:“On the F2 fault, the maximum slip was 1.5 m.”The original Article has been corrected.


The target copolymers CCP1–5 were isolated in excellent yield and characterized by various instrumental analysis techniques. Interestingly, investigation of the copolymers' porosity properties discloses BET surface areas up to 337 m2 g−1 for the target compounds bearing fluorinated iron(ii) clathrochelate units CCP2,5. Moreover, the fluorinated copolymers display an outstanding uptake capacity of iodine with a maximum adsorption of 200 wt%. The target metalorganic copolymers CCP1–5 reveal very good adsorption of organic dyes, namely, methyl blue and methylene blue, from aqueous media.We report the synthesis of metalorganic copolymers made from the palladium catalyzed Sonogashira cross-coupling reaction between various iron( ii) clathrochelate building blocks with diethynyl–triptycene and fluorene derivatives.We report the synthesis of metalorganic copolymers made from the palladium catalyzed Sonogashira cross-coupling reaction between various iron( In addition, the abovementioned cross-coupling reactions permit a noticeable control over the polymerization reaction, thus, yielding highly structured copolymers. On top that, Suzuki and Sonogashira cross-coupling reactions are powerful synthetic tools for the preparation of porous materials with additional properties, such as, making conjugated polymers, introducing functional groups into the macromolecules' backbones, etc.1,25 Typically, palladium complexes in combination with copper(i) salts are employed as catalysts in Sonogashira cross-coupling reactions although alternative catalytic systems were also found to be very effective.26–28In the past two decades, there has been growing interest in designing amorphous polymers with contorted structures that induce poor three-dimensional packing in the solid state, therefore, leading to the formation of intrinsic pores which bestow the resulting materials with prominent properties allowing for their use in various applications, namely, gas storage and separation, sensing, and catalysis.ii) clathrochelate is a robust metalorganic unit with a considerable internal free volume (IFV) and which can be easily synthesized and functionalized, especially when employed as a building block in cross-coupling reactions.29–31 Iron(ii) clathrochelate complexes were tested as biosensors,32,33 catalysts for hydrogen generation,34,35 materials for electronic transport,36 organogels,37 and to make supramolecular structures of definite shapes and sizes.38–41 Recently, several iron(ii) clathrochelate based polymers were reported disclosing prominent porous properties.42–44 In this prospect, we report herein the synthesis and characterization of five new iron(ii) clathrochelate copolymers CCP1–5 using Sonogashira cross-coupling reaction conditions. Interestingly, the target copolymers exhibit Brunauer–Emmett–Teller (BET) surface area up to 337 m2 g−1 as well as high iodine uptake and excellent adsorption of organic dyes, namely, methyl blue and methylene blue.Iron was performed on aluminum sheets coated with silica gel 60 F254 and revealed using a UV lamp. NMR  spectra were recorded on Bruker BioSpin GmbH 600 MHz spectrometer using CD2Cl2 as a solvent with the chemical shifts (δ) given in ppm and referred to tetramethylsilane (TMS). FT-IR spectra were recorded on Agilent Cary 630 FTIR instrument. UV-Vis spectra were recorded on Shimadzu UV1800 spectrophotometer. Brunauer–Emmett–Teller (BET) surface area and porosity measurements were evaluated using a Surface Area and Pore Size Analyzer  at the boiling point of liquid nitrogen (−196 °C). Samples were degassed in VacuPrep 061 sample degassing system at a temperature of 105 °C for overnight, before the experiments. Surface areas (SBET) were calculated using the Brunauer–Emmet–Teller (BET) model of isotherms, and the adsorption of N2 at small relative pressures. Total pore volume (Vt) was determined from the specific adsorption of N2 at a p/p0 = 0.99. The t-plot method was used to estimate micropore volume (Vmic) and external surface area (Sext). Quantachrome Autosorb iQ2 analyzer was used to collect gas adsorption data. In a typical gas uptake setup, samples (120–150 mg) were loaded in a 9 mm cell and were subjected to degassing at 120 °C for 5–8 h by attaching to the degassing unit. The cells with the degassed materials were refilled with helium gas and weighed accurately. Subsequently, cells were reattached to the analysis unit of the instrument for measurements. Various temperatures of the analysis unit sample cell were maintained using KGW isotherm bath that was filled with liquid N2 (77 K), or using a temperature-controlled bath (298 K and 273 K). Polymers molecular weights were recorded on Agilent 1260 infinity II gel permeation chromatograph (GPC) against with a refractive index (RI) detector at room temperature and two columns (PL mixed-C) which are calibrated twelve monodisperse polystyrene (PS) standards with THF employed as eluent at a flow rate of 1.0 mL min−1 UV-Vis spectra were recorded on Shimadzu UV1800 spectrophotometer. X-ray Photoelectron spectroscopy (XPS) data were recorded with a Thermo ESCALAB 250 Xi using a monochromatic Al Kα-radiation source (1486.6 eV) with a spot size of 850 μm. Spectra acquisition and processing were carried out using the software Thermo Advantage Version 4.87. The base pressure in the XPS analysis chamber was in the range 10−10 to 10−9 torr. The analyzer was operated with pass energy of 20 eV, dwell time of 50 min and with a step size of 0.1 eV.All the reactions were carried out under inert atmosphere using dry argon. All chemical reagents were used without further purification as purchased from Aldrich, Merck, and HiMedia unless otherwise specified. 1,4-Diethynyl-9,10-dihydro-9,10-benzenoanthracene 1a, 2,7-diethynyl-9,9-dimethyl-9Electron impact high-resolution mass spectra (EI-HRMS) were recorded on a Thermo (DFS) with a standard PFK (perfluorokerosene) as lock mass. The analyzed data is converted to accurate mass employing X-Calibur accurate mass calculation software.2.22.2.12  in MeOH (20 mL) and the mixture was refluxed for 3 h under argon. The reaction mixture was allowed to cool to RT, and the resulting precipitate was isolated by filtration, washed with MeOH, diethyl ether, and dried under vacuum to yield a red solid ; 1H-NMR : δ 7.05–7.04 , 2.92 , 1.83 ; 13C-NMR : δ 167.98, 166.31, 152.96, 122.24,115.69, 26.75, 22.11; HRMS: m/z calculated for (M˙+) C30H28B2Br2F4FeN6O6 881.9883 found 881.9885.A Schlenk tube was charged with nioxime , 4-bromo-2,6-difluorophenylboronic acid  and anhydrous FeCl2.2.22  in MeOH (10 mL). Red solid ; 1H-NMR : δ 7.03–7.02 , 2.82–2.79 , 1.56–1.53 , 1.34 1.30 , 0.89–0.86 ; 13C-NMR : δ 168.15, 166.48, 157.94, 121.92, 115.60, 29.66, 27.69, 23.00 14.06; HRMS: m/z calculated for (M˙+) C42H58B2Br2F4FeN6O6 1056.2230 found 1056.2236.CC3 was prepared following procedure A with: butyl dioxime , 4-bromo-2,6-difluorophenylboronic acid  and anhydrous FeCl2.2.32NH  which was bubbled with argon for 30 min. Before the addition of CC1  and 1a . The mixture was purged with argon for 10 min. Before adding CuI  and Pd(PPh3)4  under a positive stream of argon. The reaction was stirred for 48 h at 110 °C and the precipitate was isolated by filtration while hot and washed with DMF (50 mL), water (50 mL), THF (50 mL), DCM (50 mL), acetone (50 mL), methanol (50 mL) and diethyl ether (50 mL) to yield a red solid ; FTIR (cm−1): 2955 , 2201  scale" fill="currentColor" stroke="none"><path d="M80 600 l0 -40 600 0 600 0 0 40 0 40 -600 0 -600 0 0 -40z M80 440 l0 -40 600 0 600 0 0 40 0 40 -600 0 -600 0 0 -40z M80 280 l0 -40 600 0 600 0 0 40 0 40 -600 0 -600 0 0 -40z"/></g></svg>C stretch.), 1664  scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>N stretch.), 1550 (N–O stretch.), 1431 , and 954 (aromatic CC bend.); UV-vis: , λmax [nm] = 329 and 450.A Schlenk tube was charged with a mixture of DMF and iPr2.2.43)4  and CuI  in 9 mL of a 1 : 1 degassed mixture of DMF and iPr2NH. Red solid . FTIR (cm−1): 2944 , 2201 (CC stretch.), 1618 (CN stretch.), 1545 (N–O stretch.), 1458 , 1395 (C–F stretch.), and 964 (aromatic CC bend.); UV-vis: , λmax [nm] = 329 and 450.CCP2 was prepared following procedure B with: CC2 , 1a , Pd(PPh2.2.53)4  and CuI  in 10 mL of a 1 : 1 degassed mixture of DMF and iPr2NH. The solvent was evaporated under reduced pressure and the resulting residue was extracted with ethyl acetate from an aqueous solution of 10% LiCl (100 mL). The organic layer was washed with deionized water (100 mL × 3), concentrated, and the product was precipitated by adding hexane. The precipitate was isolated by filtration under reduced pressure and washed exhaustively with hexane. Red solid ;1H-NMR : δ 7.60–7.53 , 7.31–7.09 , 6.03 , 2.87 , 1.60 , 1.38 , 0.93 ; 13C-NMR : δ 167.96, 166.32, 157.94, 148.21, 145.02, 129.68, 128.68, 126.23, 124.63, 115.58, 115.35, 114.98, 114.76, 52.62, 29.70, 27.69, 23.03, 14.11; FTIR (cm−1): 2958 , 2210 (CC stretch.), 1618 (CN stretch.), 1544 (N–O stretch.), 1458 , 1400 (C–F stretch.), and 716 (aromatic CC bend.); GPC (THF): Mw (g mol−1): 14 106, Mn (g mol−1): 7388, Đ: 1.9; UV-vis: , λmax [nm] = 329 and 450.CCP3 was prepared following procedure B with: CC3 , 1a , Pd(PPh2.2.63)4  and CuI  in 15 mL of a 1 : 1 degassed mixture of DMF and iPr2NH. Red solid ; FTIR (cm−1): 2955 , 2201 (CC stretch.), 1602 (CN stretch.), 1492 (N–O stretch.), 1434 , and 960 (aromatic CC bend.); UV-vis: , λmax [nm] = 354, 371, 400 and 450.CCP4 was prepared following procedure B with: CC1 , 1b , Pd(PPh2.2.73)4  and CuI  in 8 mL of a 1 : 1 degassed mixture of DMF and iPr2NH. Red solid ; FTIR (cm−1): 2942 , 2207 (CC stretch.), 1618 (CN stretch.), 1542 (N–O stretch.), 1436 , 1396 (C–F stretch.), and 959 (aromatic CC bend.); UV-vis: , λmax [nm] = 354, 371, 400 and 450.CCP5 was prepared following procedure B with: CC2 , 1b , Pd clathrochelate synthons CC1–3 with each of 1,4-diethynyl triptycene 1a and 2,7-diethynyl-9,9-dimethyl fluorene 1b. The desired copolymers CCP1–5 were isolated after 2 days of reaction at 110 °C by simple filtration while hot followed by exhaustive sequential washings of the precipitates with hot solvents, thus, affording the desired target materials in excellent yields (87–95%). The copolymers whose clathrochelate units bear cyclohexyl side groups  were found to be highly insoluble in common organic solvents. Consequently, the hitherto mentioned four copolymers were characterized using FTIR and XPS spectroscopy , therefore, allowing for its thorough structural analysis by 1H- and 13C- NMR, FTIR, and XPS spectroscopy  clathrochelate unit whereas the four peaks observed at 2.87 ppm, 1.6 ppm, 1.38 ppm, and 0.93 ppm are assigned to the butyl side chains. Furthermore, the characteristic chemical shift of the sp3 protons in the triptycene unit is detected at 6.03 ppm whereas the aromatic protons of this latter are identified in the range of 7.31–7.09 ppm. In addition, 13C-NMR spectrum of CCP3 displays all the characteristic chemical shifts, which further confirms its successful formation in high purity  clathrochelate copolymers with triptycene units CCP1–3 display analogous absorption bands with two peaks maxima at ∼329 nm and 450 nm, where the former can be attributed to the absorption of triptycene47 while the latter corresponds to the clathrochelate moiety.47 On the other hand, clathrochelate copolymers with fluorene units CCP4,5 display three main absorption bands maxima, two of which are attributed to fluorene at ∼354 nm and 371 nm,48 whereas the one detected at 450 nm corresponds to the clathrochelate unit.The photophysical properties of CCP1–5 were investigated by means of UV-Vis absorption spectroscopy using THF as a solvent . Iron whereas the latter corresponds to that of imine carbons (CN). The binding energy for oxygen detected at ∼532.3 eV relates to a hydrocarbon bonded to boron and nitrogen. On the other hand, N1s spectrum exhibits two peaks at 400.63 eV and 401.52 eV, which correspond to carbon–nitrogen (C–N) and nitrogen–oxygen (N–O), respectively. F1s core-level spectrum was detected at 685.65 eV, thus, indicating the presence of C–F.50 B1s core-level spectrum was detected at 190.65 eV, which clearly divulges the presence of boron oxide (B–O) usually observed above 190 eV.51ii)–N compounds.52 It is noteworthy that the other target copolymers CCP2–5 portray similar XPS binding energy values, which undoubtedly confirm their structures  was utilized to analyze the elemental composition of copolymers CCP1–5. XPS survey-scan spectrum of CCP3, shown in Mw of ∼14 kDa and number average molar mass Mn of ∼7 kDa, thus, showing a polydispersity index (PDI = Mw/Mn) of ∼1.9. Nevertheless, the scarce solubility of the triptycene-containing copolymers CCP1,2 and those bearing dimethyl fluorene units CCP4,5 in common organic solvents prevented recording their GPC chromatograms.As it could be noticed from 4.2 adsorption experiments at 77 K and low relative pressure (2 g−1) which can be explained by the presence of the flexible butyl side chains which block the intrinsic pores.44 The clathrochelate copolymers bearing triptycene CCP1 and dimethyl fluorene CCP4 units portray surface areas of ∼79 m2 g−1 and 111 m2 g−1CCP1,3, respectively. Interestingly, the replacement of the two hydrogen atoms located at the ortho positions with respect to boron with the bulkier and more electronegative fluorine atoms in the iron(ii) clathrochelate moiety of both copolymers i.e. the one with dimethyl fluorene units CCP5 and that with triptycene derivatives CCP2 resulted in a noticeable increase of the surface areas of 279 m2 g−1, for the former and 337 m2 g−1, for the latter. All five target copolymers were found to have pore volumes ranging between 0.039 cm3 g−1 and 0.222 cm3 g−1  clathrochelate copolymers which revealed superior BET surface areas, i.e.CCP2 and CCP5, display the highest iodine uptake reaching a maximum of 200 wt%. It is noteworthy that iodine adsorption of CCP2 reached ∼160 wt% after 6 hours of exposure while that of CCP5 attained 150 wt% for the same period time. On the other hand, the maximum uptake capacities of copolymers CCP1 and CCP4 were found to reach 160 wt% and 140 wt%, respectively, after 24 hours of exposure to I2  underwent desorption by simple heating in air at 120 °C. The complete wt% of iodine released from I2@CCP1–5 samples was recorded at different time intervals , ∼290 nm and ∼357 nm (due to polyiodide ions), which confirms the release of the adsorbate from CCP1–5 samples under ambient conditions. It is noteworthy that the amount of iodine released from CCP1–5 reached equilibrium after 45 minutes with a clear change in the color intensity of the solution from colorless to yellow  and methylene blue . The former is an important reagent for various biological and industrial applications whereas the latter is considered to be a primary source of pollution in water resources.ntervals  file. Inntervals . Target ntervals  file.7.via a one-step Sonogashira cross-coupling reaction between various dibrominated iron(ii) clathrochelate units with 1,4-diethynyl triptycene and 2,7-diethynyl-9,9-dimethyl fluorene synthons. Gel permeation chromatography of the soluble target copolymer CCP3 disclosed weight average molar mass Mw of ∼14 kDa and number average molar mass, Mn of ∼7 kDa. Porosity investigation divulged BET surface areas ranging from 45 to 337 m2 g−1 with greater porosity values for the fluorinated copolymers CCP5 bearing fluorene derivatives and CCP2 containing triptycene units, which disclosed surface areas of 279 m2 g−1 for the former copolymer and 337 m2 g−1 for the latter. Likewise, iodine uptake studies depicted adsorption capacities up to 200 wt% for the fluorinated copolymers CCP2,5. Furthermore, CCP1–5 demonstrate efficient adsorption of both methyl blue and methylene blue from aqueous solution with a higher preference towards the former. It is worth mentioning that the metalorganic copolymers CCP1–5 present several advantages, among others, their ease of synthesis and isolation, superior stability, in addition to the possibility for their utilization in the treatment of emission of toxic iodine vapors and organic dyes.Five metalorganic copolymers CCP1–5 were prepared in very good yields There are no conflicts to declare.RA-011-D1RA02357H-s001