Depending on whether the mean concentrations of rGSH in controls were within the accepted normal range of 0.5–5.0 µmol/L, pooled rGSH concentrations showed either a significant  or nonsignificant  difference. These results illustrate the existing and largely unaddressed methodological issues in the interpretation of the serum/plasma concentrations of tGSH and rGSH in COPD.This study aimed to review and critically appraise the current methodological issues undermining the suitability of the measurement of serum/plasma glutathione, both in the total and reduced form, as a measure of systemic oxidative stress in chronic obstructive pulmonary disease (COPD). Fourteen relevant articles published between 2001 and 2020, in 2003 subjects, 1111 COPD patients, and 892 controls, were reviewed. Nine studies, in 902 COPD patients and 660 controls, measured glutathione (GSH) in the reduced form (rGSH), while the remaining five, in 209 COPD patients and 232 controls, measured total GSH (tGSH). In the control group, tGSH ranged between 5.7 and 7.5 µmol/L, whilst in COPD patients, it ranged between 4.5 and 7.4 µmol/L. The mean tGSH was 6.6 ± 0.9 µmol/L in controls and 5.9 ± 1.4 µmol/L in patients. The concentrations of rGSH in the control group showed a wide range, between 0.47 and 415 µmol/L, and a mean value of 71.9 ± 143.1 µmol/L. Similarly, the concentrations of rGSH in COPD patients ranged between 0.49 and 279 µmol/L, with a mean value of 49.9 ± 95.9 µmol/L. Pooled tGSH concentrations were not significantly different between patients and controls (standard mean difference (SMD) = −1.92, 95% CI −1582 to 0.0219;  The references of the retrieved articles were also examined to identify potential additional studies. The articles were independently reviewed by two investigators and by a third in case of disagreement.p-value of <0.1. The I2 statistic was used to measure the magnitude of heterogeneity. For I2 < 25%, heterogeneity was considered absent; for I2 between 25 and 50%, it was considered moderate; for I2 between 50 and 75%, it was considered large; and for I2 > 75%, it was considered extreme. A random-effect model was used to summarize the results in case of substantial heterogeneity. Tests were two-tailed, and a p-value <0.05 was considered statistically significant. The study was fully compliant with the principles outlined in the PRISMA Statement [Standard mean difference (SMD) was used to pool continuous data. Heterogeneity across studies was assessed using Cochran’s Q statistic, with a tatement . Statist2 = 92.94%, p < 0.0001). As shown in p = 0.057), assessed by a random-effect model, indicated the presence of a nonsignificant difference in concentrations between controls and COPD patients. By contrast, as shown in 2 = 97.38%, p < 0.0001), and pooled SMDs for rGSH  assessed by a random-effect model indicated a significant difference in concentrations between controls and COPD patients. When considering those studies (n = 4) reporting, in control groups, a mean rGSH concentration between 0.5–5.0 µmol/L, the heterogeneity decreased ; however the pooled SMD, computed by a random-effect model, was no longer significant  (A flowchart describing the screening process is presented in  = 0.48) c.2 = 92.94%, p < 0.0001) and the slightly lower concentrations of tGSH observed in the control groups (6.6 ± 0.9 µmol/L) compared to the normally accepted values for healthy people (8.0 ± 1.4 µmol/L). Subject to these limitations, the pooled analysis using a random-effect model showed that tGSH concentrations were not significantly different in COPD patient vs. controls (p < 0.0001), which, using a random-effect model, overall shows that rGSH concentrations are significantly different in COPD patients vs. controls (p < 0.0001) and the pooled results showed that rGSH concentrations were not significantly different in COPD patients vs. controls  and red blood cells (RBCs) as a measure of systemic oxidative state in COPD ,41,42,43controls a. Unlikecontrols b. Howevecontrols c. Simila


According to physicochemical methods and monosaccharide composition analysis, LHPW was mainly composed of galactose, glucose, fructose, and arabinose. NMR and methylation analyses indicated that LHPW was a neutral polysaccharide with a backbone containing →3,6)-β-D-Galp--β-D-Galp-(1→ and →4)-α-D-Glcp--β-D-Fruf-(2→ and →6)-α-D-Galp- was extracted from Lycopi Herba and purified by DEAE-52 cellulose chromatography and G-100 sephadex Lyscopus lucidus Turcz. var. hirtus Regel is a traditional Chinese medicine that is mainly used for promoting blood circulation, removing blood stasis, and strengthening immunity  is getting back up to a crucial status in the health area . Among timmunity . Modern immunity , while iimmunity . Among timmunity . HoweverHere, we aimed to detect polysaccharides from Lycopi Herba and examine their biological activities. As the unique structural features, polysaccharides usually interact with the immune system in the human body and demonstrate immunoregulatory activities. Some polysaccharides such as lentinan have been developed as immunomodulators . TherefoGenerally, polysaccharides were extracted from Lycopi Herba and purified by DEAE-52 cellulose chromatography and G-100 sephadex. The monosaccharide composition was analyzed by high-performance liquid chromatography. The polysaccharide backbone was characterized by NMR and methylation analyses. The immunomodulatory ability of polysaccharides was determined by using the phagocytic capacity and lymphocyte proliferation assay. As a result, a neutral polysaccharide LHPW was obtained from Lycopi Herba. The structure of LHPW, including molecular weight, monosaccharide composition, and glycosyl linkages, was systematically characterized. The immunological examination showed that LHPW could activate macrophage RAW264.7 and promote splenocyte proliferation, indicating LHPW might be a potential immunomodulatory agent.Dried Lycopi Herba was purchased from Bozhou of Anhui Province in China, and identified by the Laboratory of Chinese Medicine, College of Life Sciences, Shanxi Agricultural University. A voucher specimen (No. 20200832) has been deposited at the Laboratory of Functional Polysaccharides, Shanxi Agricultural University. Bovine serum albumin and standard monosaccharides were purchased from Solarbio Science & Technology Co., Ltd. . Lipopolysaccharide (LPS), 3--2,5-diphenyltetrazolium bromide (MTT), trifluoroacetic acid (TFA), and concanavalin A (ConA) were purchased from Sigma-Aldrich. (E)-3-[(4-methylphenylsulfonyl]-2-propenenitrile (BAY 11-7082) was purchased from Beyotime Biotech. All other chemicals were of analytical grade as available.ad libitum at 21°C. All experiments were performed under the Regulations of Experimental Animal Administration issued by the State Committee of Science and Technology of the People’s Republic of China. The RAW264.7 cell line was previously preserved in our laboratory. Spleen lymphocytes were obtained by cell strainers and red blood cell lysis buffer.Kunming mice were purchased from Shanxi Medical University, China. The mice were maintained on a standard pellet diet and water − form) and sephadex G-100 which was eluted with distilled water, polysaccharide LHPW was obtained. Endotoxin contamination was routinely monitored in the laboratory using a chromogenic Limulus Amebocyte Lysate Assay Kit . LPS existed at a concentration of <0.1 ng/ml in the preparation of LHPW.The dried Lycopi Herba powder was defatted and extracted three times with distilled water at 80°C for 2 h at the ratio of 1:10 (W/V). The supernatants were concentrated and collected with three times alcohol sedimentation. The precipitate was dissolved, dialyzed, and lyophilized to obtain Lycopi Herba crude polysaccharide (LHP). After being purified by DEAE-cellulose column  measurement between 400 and 4,000 cm−1 .m-hydroxydiphenyl–sulfuric acid method, respectively. Monosaccharide compositions were analyzed by high-performance anion exchange chromatography (HPAEC) after being hydrolyzed by the 3 M trifluoroacetic acid (TFA) for 2 h  and incubated for 24 h. Then cells were treated with various concentrations of LHPW  or LPS (2 mg/ml) for 24 h. The cell supernatants were collected and stored at −80°C. TNF-α proteins were measured using the enzyme-linked immunosorbent assay (ELISA) kits (R&D) according to the instructions. Then 0.5 mg/ml MTT was added to the plates and further incubated for 4 h at 37°C. The optical density was measured at 570 nm. Also, RAW 264.7 cells were pretreated with PBS or BAY 11-7082 (3 µM) for 1 h before incubation with 200 μg/ml or 2 μg/ml LPS for 24 h. Then the TNF-α proteins were measured.RAW 264.7 cells were cultured in 96-well plates (1 × 105 cells/well) were pipetted into 96-well plates and treated with various concentrations of LHPW  for 12 h. Then 0.07% neutral red solution was added and incubated for 2 h. The medium was discarded, and cells were washed twice with PBS. Lysis buffer  was added, and the optical density of each well was measured at 540 nm. The RMPI1640 medium and LPS (2 μg/ml) were used as the blank and positive control, respectively. The phagocytosis index was calculated by the following equation:The phagocytic ability of macrophages was measured using neutral red uptake. Cells (1 × 10The RAW 264.7 cells were immunofluorescence-labeled according to the manufacturer’s instruction using a Cellular NF-κB Translocation Kit (Beyotime Biotech) . Brieflyin vitro by the MTT method. The cells (1 × 107/ml) were incubated with different concentrations of LHPW  with or without Con A (5 μg/ml) and LPS (2 μg/ml) for 48 h. MTT was added, and the samples were further incubated for 4 h at 37°C. The optical density was measured at 570 nm.Lymphocyte proliferation assays were evaluated Results were expressed as mean ± SD. Statistical analyses were performed with one-way or two-way ANOVA by using the GraphPad Prism 5.0 software. One-way ANOVA analysis was used for the statistical analysis among three or more groups of an experiment with one factor. Two-way ANOVA was used for the statistical analysis of the experiment with two factors.− form) and G-100 sephadex were applied for further purification of the LHP. Finally, a novel heteropolysaccharide designated LHPW was obtained, with the yield of 2.53 ± 0.17% from LHP.The extraction and purification scheme of polysaccharide LHPW is shown in The molecular weight of the polysaccharides is closely related to their physicochemical properties and biological activities . The mol−1 was due to the hydroxyl groups stretching vibration in the constituent sugar residues. The bands in the region of 2932.34 cm−1 were the characteristic absorption of C-H stretching vibration in the sugar ring. The absorbance peaks near 1640.32 cm−1 suggested that uronic acid content was low in LHPW. The peaks nearby 1,350–1,450 cm−1 may be caused by the variable angular vibration of C–H. The peaks at 950–1,200 cm−1 suggested the presence of C–O–C and C–O chemical bonds . As shown in After two times methylation, the infrared spectra results show that the methylation is complete . Methyla1H NMR spectrum of LHPW  spectroscopy was used to reveal the precise structural information of LHPW. The C/H chemical shifts of all monosaccharide residues were assigned as completely as possible and listed in  of LHPW , signalsectively . In agre in LHPW .1H NMR and 13C NMR. The corresponding heterocephalic hydrogen signal and heterocephalic carbon signal of residue A were 99.10 and 4.88 ppm in the HSQC. The cross peaks at δ H/H 4.88/3.76, 3.76/3.84, 3.84/4.07, and 4.07/3.98 ppm were detected in 1H–1H COSY, which suggested that the signals at δ 4.88, 3.76, 3.84, 4.07, and 3.98 ppm corresponded to H-1, H-2, H-3, H-4, and H-5 of the residue →6)-α-D-Galp-(1→ (residue A), respectively. According to HSQC, C-1, C-2, C-3, C-4, and C-5 of the residue A were δ 99.10, 69.71, 70.73, 70.08, and 70.66 ppm, respectively. The chemical shift of C-6 was 67.78 ppm.The COSY  and HSQC1H–1H COSY, which suggested that the signals at δ4.41, 3.45, 3.57, 3.96, and 3.44 ppm corresponded to H-1, H-2, H-3, H-4, and H-5 of the residue →4)-β-D-Galp-(1→ (residue F), respectively. Corresponding C-1, C-2, C-3, C-4, C-5, and C-6 were δ104.47, 73.21, 73.97, 79.03, 74.36, and 62.74, respectively. Similarly, the other proton and carbon signals of the rest of the residues were analyzed by the same method.The corresponding heterocephalic hydrogen signal of heterocephalic carbon signal δ104.47 is δ4.41 in the HSQC. The cross peaks at δH/H 4.41/3.45, 3.45/3.57, 3.57/3.96, and 3.96/3.44 ppm were detected in For fructose residues, the signal at δ105.17 ppm was the typical peak of →1)-β-D-Fruf--β-D-Fruf--β-D-Galp-(1→4)-β-D-Galp-(1→4)-α-D-Glcp--β-D-Fruf-(2→1)-β-D-Fruf- cells, and lymphocytes . MacrophOwing to the critical role of NF-κB in the progression of immunity, we detected the effect of LHPW on the activation of NF-κB. To determine whether this NF-κB activation was involved in LHPW-induced cytokine production, we treated RAW 264.7 cells with LHPW and LPS in the presence or absence of BAY 11-7082, a well-described inhibitor of NF-κB. Results showed that treatment with BAY 11-7082 at 3 µM partly suppressed TNF-α secretion induced by LHPW in RAW 264.7 cells By the use of a Cellular NF-κB Translocation Kit, p65 protein translocation into nuclei was visualized in RAW 264.7 cells after exposure to LHPW and LPS. As shown in The proliferation of spleen cells is one of the most important steps in the activation pathway of cell-mediated or humoral immunity . The effIn recent years, only some oligosaccharides have been identified from Lycopi Herba. For example, stachyose is a tetrasaccharide composed of “Galactose–Galactose–Glucose–Fructose” and has been reported to have the ability to balance the intestinal micro-ecosystem by selective proliferation of the bifidobacteria. A mixture of α-galactooligosaccharides (GOS) from the roots of Lycopi Herba could elicit a significant increase in humoral immunity, and enhancing in splenocyte proliferation . However13C NMR displayed that there were no peaks near 170–180 ppm, confirming no uronic acids in LHPW. All these results demonstrated that LHPW was a neutral polysaccharide with a backbone containing →3,6)-β-D-Galp--β-D-Galp-(1→ and →4)-α-D-Glcp--β-D-Fruf-(2→ and →6)-α-D-Galp-, and activating the NF-κB signaling pathway . Monosaccharide composition analysis showed that LHPW was mainly composed of galactose, glucose, fructose, and arabinose, and contained no uronic acids. Moreover,  actions . Previouge cells ; a neutrfunction ; a neutr pathway . InspireThe immune response plays an important role in disease development because severe inflammation and suppressed immunization can increase the risk of infections, multiple organ failure, or death . It has


Inocybe brijunica sp. nov., from sect. Hysterices , is described based on morphology and multilocus phylogenetic data. The holotype collection was found at the edge between grassland and Quercus ilex forest with a few planted Pinus pinea trees, on Veli Brijun Island, the largest island of the archipelago. It is easily recognized by a conspicuous orange to orange–red–brown membranaceous surface layer located at or just above the basal part of the stipe. Other distinctive features of I. brijunica are the medium brown, radially fibrillose to rimose pileus; pale to medium brown stipe with fugacious cortina; relatively small, amygdaliform to phaseoliform, and smooth basidiospores, measuring ca. 6.5–9 × 4–5.5 µm; thick-walled, utriform, lageniform or fusiform pleurocystidia (lamprocystidia) with crystals and mostly not yellowing in alkaline solutions; cheilocystidia of two types (lamprocystidia and leptocystidia); and the presence of abundant caulocystidia only in the upper 2–3 mm of the stipe. Phylogenetic reconstruction of a concatenated dataset of the internal transcribed spacer region (ITS), the nuclear 28S rRNA gene (nrLSU), and the second largest subunit of RNA polymerase II (rpb2) resolved I. brijunica and I. glabripes as sister species.A new ectomycorrhizal species was discovered during the first survey of fungal diversity at Brijuni National Park (Croatia), which consists of 14 islands and islets. The National Park is located in the Mediterranean Biogeographical Region, a prominent climate change hot-spot.  The islands’ surface area covers 7.4 km2; Veli Brijun is the largest island with 5.7 km2 and is devoid of permanent inhabitants. The National Park was established in 1983 to protect valuable marine and coastal (land) ecosystems and their biodiversity. The area is floristically rich, covered with evergreen vegetation and home to more than 400 native and exotic plant species mostly of Mediterranean origin. The Brijuni archipelago is characterized by a northern Mediterranean climate  34–50–65(–70) × 9–14–21 µm, Q = 2.39–3.65–5.45, predominantly utriform, lageniform, or fusiform, with obtuse apex of 6–9(–11) µm wide, sometimes (sub)clavate, conical with obtuse apex, narrowly ellipsoid or subcylindrical, in alkaline solutions mostly (sub)hyaline, less often with slightly yellowish wall, sometimes with dirty yellow cytoplasmic pigment, with strongly to poorly developed crystals at apex , thick-walled, wall most often gradually thickened towards the apex (up to 1–5.5 µm). Lamellar edge heterogeneous. Cheilocystidia of two types: (a) lamprocystidia similar to pleurocystidia , scattered to abundant, and (b) leptocystidia (paracystidia) 9–30 × 5–14 µm, mostly clavate, less often (sub)fusiform or utriform, hyaline to subhyaline, thin to moderately thick-walled (up to ca. 0.6 µm), scattered to abundant. Pileipellis a cutis, composed of repent, thin-walled, smooth to minutely encrusted, hyaline to pale yellow–brown hyphae, 1–5(–7) µm wide. Cells of the upper part of pileal context with brown, intracellular and partially also encrusted extracellular pigment (brown pigmented layer ca. 80–150 µm wide). Stipitipellis a cutis, composed of repent, thin-walled, smooth, ca. 2–10 µm wide hyphae. Caulocystidia mostly abundant (often crowded) in upper 2–3 mm of stipe length, sparsely present toward middle of the stipe; many in the form of lamprocystidia, quite similar to pleurocystidia, others very variable, narrowly utriform, lagenifom, (sub)cylindrical, clavate, urticoid or rather irregular, sometimes with subcapitate apex, some septate, hyaline, thin- to moderately thick-walled (up to ca. 1 µm); 12–100(–150) × 4–20 µm. Clamp connections present, conspicuous, rather abundant in all tissues.Basidiospores [300/3/1] (6.2–)6.6–7.5–8.8(–9.8) × (4–)4.3–4.7–5.3(–5.7) µm, averages of different basidiomata 7.3–7.6 × 4.6–4.7 µm, Q = 1.35–1.6–1.96, av. Q = 1.58–1.62, a few very large spores occasionally present (up to ca. 12 × 7 µm); in frontal view ellipsoid, oblong or ovoid with rounded to subacute base and rounded to acute apex, in side view amygdaliform to phaseoliform, rarely subellipsoid, with rounded base and rounded to acute apex, sometimes subangulate in both views ; smooth, often with small, rather indistinct, apical germ-pore, moderately thick-walled (up to 0.8 µm), pale yellow–brown in KOH, pale brown in HDistribution and ecology: Thus far only known from the holotype collection. Ectomycorrhizal, found in the Mediterranean region of Croatia (Europe), on the island of Veli Brijun in Brijuni National Park, on the edge of Quercus ilex forest, with a few planted Pinus pinea trees, edging a neighboring grassland. An ITS sequence with accession number MH310748 [Inocybe sp., from Italy shares 99% identity with I. brijunica  and may indicate a broader distribution in the Mediterranean basin.MH310748 , identifI. brijunica in sect. Hysterices [I. brijunica can be easily recognized from all other Inocybe species by a conspicuous orange to orange–red–brown membranaceous surface layer present at or just above the basal part of the stipe. Other important morphological characters are: medium brown pileus with radially fibrillose to rimose surface; pale to medium brown stipe with slightly to moderately broadened (or sometimes submarginate) base; presence of fugacious cortina in young basidiomata; spermatic odor; color of context unchanged upon bruising; relatively small, amygdaliform to phaseoliform (and sometimes subangulate), smooth basidiospores (ca. 6.5–9 × 4–5.5 µm); pleurocystidia as utriform, lageniform, or fusiform, thick-walled (up to 1–5.5 µm) lamprocystidia, mostly with crystals and not yellowing in alkaline solutions; cheilocystidia of two types (lamprocystidia and leptocystidia); and presence of abundant caulocystidia only in the upper 2–3 mm of stipe length.The results of our multilocus phylogenetic analysis and morphological study place sterices . Basidiosterices  emended I. glabripes, are very similar in size, measuring ca. 6–8 × 4–5 µm [I. brijunica. In addition, the cystidial walls of I. glabripes are thinner (up to 2(–2.5) µm thick). Inocybe glabripes is a widespread species occurring in parks and open woodlands on predominantly alkaline soils from the Mediterranean region to the boreal zone of Europe [Betula L., Fagus L., Populus L., Quercus L., Tilia L. [Castanea sativa Mill. [I. brijunica also forms ectomycorrhizal relationships only with broadleaved trees, such as Quercus ilex at the holotype locality.The basidiospores of the morphologically and phylogenetically closest species, × 4–5 µm ,50, but f Europe , which fTilia L. ,50,51, ava Mill. . It can Inocybe pseudobrunnea Alessio, which grows under Abies alba Mill. , has a similar phaseoliform, but somewhat larger basidiospores, 8.5–10.5(11) × 4.5–6 µm, and its cystidia are rather bright yellow in ammonia solution [I. brijunica. Inocybe gracilenta E. Ludw., only known from the type collection in Sweden, a damp locality under Alnus sp. , Populus tremula L. and Salix sp. , has amygdaliform but not phaseoliform basidiospores, which are otherwise similar in size compared to I. brijunica, 7–8.5(9.5) × 4.5–5.5 µm [I. barrasae Esteve-Rav., described from Spain and fruiting in spring (April–May) in thermophilous Mediterranean Quercus–Cistus forests, has amygdaliform-shaped basidiospores that are larger and more elongated , and bright yellow-colored cystidia in ammonia solution [Inocybe aerea E. Ludw., another species only known from the holotype collection in Germany [I. pyrotricha Stuntz [I. brijunica, but differs by slightly larger (7.5–10 × 4.5–6 µm) basidiospores, longer pleurocystidia (66–80 × 13.5–16.5 µm), and violaceous tinges in young lamellae and upper parts of the stipe.solution , a chara5–5.5 µm . Additio5–5.5 µm . The Medsolution . Inocybe Germany , has amya Stuntz  has oran


Expression and pharmacological interventional studies identified an imbalance of c-Jun N-terminal kinase (JNK) and p38 MAPK induced by AA, which mediate an activation of angiogenesis. Moreover, increased AA in aldh2.1−/− zebrafish did not induce hyperglycemia, instead AA inhibited the expression of glucokinase (gck) and glucose-6-phosphatase (g6pc), which led to an impaired glucose metabolism. In conclusion, the data have identified AA as the preferred substrate for Aldh2.1's detoxification ability, which subsequently causes microvascular organ damage and impaired glucose metabolism.Reactive carbonyl species (RCS) are spontaneously formed in the metabolism and modify and impair the function of DNA, proteins and lipids leading to several organ complications. In zebrafish, knockout of the RCS detoxifying enzymes glyoxalase 1 (Glo 1), aldehyde dehydrogenase 3a1 (Aldh3a1) and aldo-ketoreductase 1a1a (Akr1a1a) showed a signature of elevated RCS which specifically regulated glucose metabolism, hyperglycemia and diabetic organ damage.   •ALDH2.1 was compensatory upregulated in glyoxalase 1 zebrafish mutants.•Loss of ALDH2.1 increases acetaldehyde leading to vascular retinal alterations.•Acetaldehyde controls glucose metabolism via glucose-6-phosphate and glucokinase.•Altered JNK and p38 cause microvascular complications. Theses for MG ,9. Subse animals ,11. In caldh2.1 expression was also increased in glo1−/− zebrafish mutants [aldh2.1 is the zebrafish homolog to human aldh2, which can detoxify a variety of reactive metabolites, including acetaldehyde (AA), 4-HNE, malondialdehyde (MDA) and MG [ldh2 can increase reactive oxygen species (ROS), which leads to mitochondria dysfunction and upregulation of cytochrome P450 2E1 (CYP2E1) [ mutants . aldh2.1) and MG . Because) and MG . In addi(CYP2E1) . Yet, Alaldh2.1 in zebrafish caused an increased endogenous AA concentration, which subsequently impaired the glucose metabolism and induced microvascular damages in retinal blood vessels in zebrafish larvae and adults.Therefore, the study aimed to evaluate the detoxification ability of Aldh2.1 for different RCS in zebrafish and to identify a potential regulatory function of Aldh2.1 on glucose metabolism, organ physiology and diseases. Our data identified that the loss of 22.1aldh2.1 knockout zebrafish model as previous studies [aldh2.1 in reactive metabolite detoxification.Recent studies in vertebrates and mammals indicated a major role of Aldh enzymes in detoxifying short and long chained RCS ,15,22,23 studies ,24 suggealdh2.1−/− zebrafish line has not been generated, thus a knockout for aldh2.1 by using CRISPR/Cas9 technology was established. First, CRISPR-guideRNA (gRNA) targeting exon 3 of aldh2.1 was synthesized and injected together with Cas9 mRNA into one-cell stage Tg(fli1:EGPF) zebrafish embryos. Sequencing identified a reading frame shift mutant due to a deletion of five nucleotides, which was then utilized for further breeding and studies  was not altered compared to aldh2.1+/+ littermates  were aldh2.1+/+, 122 (43.2%) were aldh2.1+/− and only 61 (21.7%) had the Δ5 bp version aldh2.1−/−  followed by brain (6.8%) and eyes (5%) as compared to the expression of the house keeping gene b2m. Overall, these results confirmed expression of aldh2.1 in zebrafish organs comparable to other vertebrates [aldh2.1 knockout resulted in a functional reduction of total Aldh enzyme activity. A significant reduction of total Aldh enzyme activity in lysates from aldh2.1−/− larvae could be observed with the following substrates: AA  pathway analysis revealed several pathways significantly regulated by the aldh2.1 knockout, including but not limited upregulated stress signaling and a downregulated energy metabolism  , that mediates the first step in glycolysis by phosphorylation of glucose to glucose-6-phosphate, was observed in aldh2.1−/− mutants. Moreover, phosphoenolpyruvate decarboxylase expression (pepck) (g6pd)  I, a key ) (g6pd) H, which egulated . Togethe2.6aldh2.1−/− mutants identified microvascular complications in retinal vessels, but it remained unclear whether these alterations were directly induced by the aldh2.1−/− mutant or indirectly by increased endogenous AA and subsequently impairing glucose metabolism and stress signaling. In order to address this question, we incubated wildtype zebrafish larvae with AA and repeated prior analysis of retinal hyaloid structures and RT-qPCR for selected gene expressions. Toxicity tests for AA in zebrafish larvae were done beforehand and revealed a tolerance of up to 500 μM AA -2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole. Analysis of hyaloid vessels  and carried out in accordance with the approved guidelines.4.2Tg(fli1:EGFP) was used [In this study the zebrafish line was used . Embryoswas used . Feeding4.3aldh2.1−/− fishline was generated using CRISPR/Cas9 as previously described. Briefly: the technique used one guide RNA (gRNA) targeting exon 3 of aldh2.1, which was designed using ZiFiT Targeter 4.2 and cloned into a T7-driven promoter expression vector  (m) containing gRNA (200 pg/L) and Cas9 mRNA was injected into one-cell stage Zebrafish embryos [The Addgene) . Additio embryos . The res embryos .4.4m) before further handling.Adult Zebrafish were transferred to single boxes and fasted one day prior to preparation. After 16–18 h, fish were used either directly or first fed with 0.5 g flakes for 1 h followed by another hour in fresh water for postprandial measurement. Before preparation and blood glucose measurement could take place, fish were euthanized in ice water for 2 min. Subsequently blood was extracted from caudal vessels and measured by a glucometer (Freestyle Abbott) . Immedia4.5Confocal images for phenotype evaluation were acquired using a confocal fluorescence microscope (DM6000 B) with a scanner (Leica TCS SP5 DS) utilizing a 20 × 0.7 objective, 1024× 1024 pixels and 1.5 μm Z-steps. For evaluation Leica Application Suite X, Gimp 2 and ImageJ were used.m, Roth) for 80 min at 37 °C. Afterwards larvae were washed three times for 15 min and stored in PBS until preparation. The larvae retinal hyaloid vasculature was dissected under a stereoscope and visualized with the confocal fluorescence microscope as described above [Alterations in larvae retinal hyaloid vasculature was imaged at 5 dpf, at which point larvae were anaesthetized in 0.003% tricaine and fixed in 4% PFA/PBS for 24 h at 4 °C. Fixed larvae were washed three times for 15 min in PBS at RT before incubation in 0.25% Trypsin/EDTA solution (Gibco) buffered at pH 7.8 with TRIS  in Tris-HCl (0.2 m) and incubated again at 37 °C for 1.5 h. After incubation in trypsin the retina was transferred to a microscope slide and the retinal cells were removed from the vasculature by dropping ddH2O from a syringe on top of the retina. The cells were removed from the slide through water aspiration and the vasculature was left to air-dry.Retinal digest preparations were performed according an established protocol with slight alterations . After e2O and then moved to fresh undiluted Mayer's hemalum solution for 7 min. Afterwards, they were placed in lukewarm tap-water for 2 min and then moved shortly to 70% and then 80% ethanol before being put in first 96% and then 99.8% ethanol and kept there for 5 min each. After the last change of ethanol, the slides were placed in two changes of xylene and kept there for 5 min each before the slides were covered with cover slips using DPX mounting medium (Thermo Fisher Scientific).The retinal digest preparations were stained using Mayer's hemalum solution (Millipore). The slides were briefly placed in ddH2 of capillary area.Images of the stained digest preparations were taken at 200× magnification using the B×51 upright microscope (Olympus Life Science) with the XC10 camera (Olympus Life Science). Analysis of the vessel diameter and the number of endothelial cells and pericytes was performed using the Cell-F software . The cells were counted in six to eight randomly selected areas in a circular area of the intermediate third of the retina, leaving out the area close to the entrance of the optic artery into the retina and the peripheral area. Endothelial cells and pericytes were identified by their distinct location and morphology. The cells were counted over 200 μm and the vessel diameter was measured. The cell numbers were then calculated as number of cells per mm4.7Imaging and Processing of zebrafish kidneys for Electron Microscopy (EM) was prepared in cooperation with the institute of Pathology IPH at Heidelberg University Hospital. Preparation was done as previously described .4.8m) in 1 × PBS on ice to inhibit phosphatases. Then they were lysed in NP40 lysis buffer (NaCl (150 mmol L−1), Tris-HCl (50 mmol L−1), pH 7.4, 1% NP40, EDTA (10 mmol L−1), 10% glycerol, and protease inhibitors) using a 1 mL Syringe and a 25 G needle. Followed by incubation on ice for 30 min on a shaker. The supernatant containing the protein lysate was diluted 5:1 with Laemmli sample buffer and boiled at 95 °C for 5 min, separated via SDS-PAGE, and then transferred to a nitrocellulose membrane for antibody incubation. Visualization by enhanced chemiluminescence (ECL) was acquired after incubation with Horseradish Peroxides substrate .For Western blot analysis, larvae/adult organs were taken, incubated for 10 min with Natrium-Vanadate  was designed, synthesized, and coupled to KLH (Keyhole Limpet Hemocyanin) by PSL GmbH, Heidelberg, Germany and subsequently injected into guinea pigs for immunization following standard procedures from GPCF Unit Antibodies, DKFZ Heidelberg, Germany.4.10m–5 mm, Sigma-Aldrich) or p38 MAPK inhibitor  started at 4 hpf and was refreshed daily at 24, 48, 72, 96 hpf.Fertilized zebrafish embryos were transferred into 5 cm petri dishes. Each Petri dish held 30 embryos in 10 mL egg water. At 24 hpf, the chorion was removed using tweezers. Treatment with either AA  containing DL-2-amino-1-propanol (10 mm), NAD (0.5 mm) and one of the following: MG (2 mm) or 4-HNE (4 mm) or AA (5 mm) or MDA or ACR by measuring the rate of NAD formation at 340 nm [d-lactoylglutathione [Total ALDH activity was measured with larvae at 96 hpf at 25 °C in Tris-HCl (0.5 mt 340 nm . Glo 1-atathione .m), DL-glyceraldehyde (10 mm)/ACR (5 mm), and NADPH (0.1 mm).AKR activity was determined by measuring the rate of reduction of NADPH at 340 nm, pH 7.0, and 25 °C. The assay mixture contained potassium phosphate  with 96 hpf larvae lysates.4.14MG, 3-DG, and glyoxal were measured as previously described ,77,78.4.15AA was measured in a LC-MSMS experimental setup, using acetaldehyde-d4 as internal standard .4.164-HNE amount was determined according to the manufacturer's instruction  with 96 hpf larvae lysates.4.17Protein-bound ACR was determined according to manufacturer's instruction  with 96 hpf larvae lysates.4.18Total RNA was isolated from homogenized zebrafish larvae (96 hpf/120 hpf) or organs using RNeasy Mini Kit according to the manufacturer's instruction (Qiagen). Subsequently cDNA was generated following the instructions Maxima First Strand cDNA Synthesis Kit (Thermo Scientific). Primer design was done using the NCBI primer blast tool and are listed in 4.19www.bgi.com, BGI). Gene expression analysis were conducted by the Core-Lab for microarray analysis, centre for medical research (ZMF) as previously described [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE189416.Total RNA was isolated from homogenized zebrafish larvae (120 hpf) or organs using RNeasy Mini Kit according to the manufacturer's instruction (Qiagen). Library construction and sequencing were performed with BGISEQ-500 .The amino acid sequences of Aldh2.1 protein from zebrafish, human and mouse were accessed by Uniprot database. For comparison the selected sequences were aligned using Clustal Omega Multiple Sequence Alignment (4.22Experimental results are expressed median using box plots with whiskers. Statistical significance between different groups was analyzed using Student's t-test. GraphPad Prism 8.3.0 was used for analyses and p values of 0.05 were considered as significant: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.aldh2.1 knockout zebrafish, C.S.M. performed retina digests and analysis. J.M. and T.F. implemented and performed biochemical experiments and gave conceptual and technological advice. C.S. analyzed RNA-seq data. I.H. performed histological analysis and electron microscopy of adult zebrafish kidneys. J.S., R.H., H.P.H. and P.P.N. gave conceptual and technological advice and revised the manuscript. J.K. conceived and designed the study and wrote the manuscript. J.K. is the guarantor of this work and has full access to all data and takes responsibility for the integrity of the data and accuracy of the data analysis.D.P.W. performed experiments, analyzed data and wrote the manuscript. B.L. generated The authors declare no conflict of interest.


Thymopentin (TP5) is an immunomodulatory pentapeptide that has been widely used in malignancy patients with immunodeficiency due to radiotherapy and chemotherapy. Here, we propose that TP5 directly inhibits the stemness of colon cancer cells HCT116 and therefore enhances the cytotoxicity of oxaliplatin (OXA) in HCT116 cells. In the absence of serum, TP5 was able to induce cancer stemness reduction in cultured HCT116 cells and significantly reduced stemness-related signals, such as the expression of surface molecular markers  and stemness-related genes , and resulted in altered Wnt/β-catenin signaling. Acetylcholine receptors (AchRs) are implicated in this process. OXA is a common chemotherapeutic agent with therapeutic effects in various cancers. Although TP5 had no direct effect on the proliferation of HCT116, this pentapeptide significantly increased the sensitivity of HCT116 to OXA, where the effect of TP5 on the stemness of colon cancer cells through stimulation of AchRs may contribute to this process. Our results provide a promising strategy for increasing the sensitivity of colon cancer cells to chemotherapeutic agents by incorporating immunomodulatory peptides. Colorectal cancer (CRC) is one of the common malignant tumors of the gastrointestinal system, and the current 5-year survival rate of colorectal cancer is 66% worldwide, but the 5-year survival rate of patients with advanced disease is only 13% . CurrentDuring tumor treatment, cancer stem cells exhibit a high degree of insensitivity to chemotherapy and radiotherapy . It has in vitro assays of human cells , which exhibits very strong immunomodulatory activity in many animal models and an cells . Numeroufections . The comfections . In addifections . Our prefections .In the present study, we found that TP5 can directly inhibit cultured colon cancer stem cells in medium without serum, and reduce the expression of cancer stem cell markers: CD44, CD24 and CD133, and stem-cell related genes: ALDH2, SOX2, OCT4 and Nanog. Additionally, although TP5 could not inhibit the proliferation of colon cancer cells directly, this peptide could promote the anti-proliferative effect of chemotherapeutic drug OXA on colon cancer cells HCT116, and acetylcholine receptors (AchRs) are implicated in this process.in vitro  promote the growth and metastasis of certain malignant tumor cells  and the percentage of CD44-positive cells decreased from 48.0 ± 0.3% to 38.4 ± 0.7% (p < 0.001); whereas the early addition of 10 μg/ml or 100 μg/ml TUB, the proportion of CD24-positive cells reversed to 25.9 ± 0.2%  and 24.9 ± 0.8% ; the proportion of CD44-positive cells reversed to 39.7 ± 1.4% (p > 0.05 vs. 38.4 ± 0.7%) and 45.0 ± 0.9% (p < 0.01 vs. 38.4 ± 0.7%).Our previous studies suggested that D-tubocurarine (TUB), an antagonist of nAchRs , signifip < 0.01, vs. 19.3 ± 0.3%), and the proportion of CD44-positive cells to 34.3 ± 1.6%, and 33.5 ± 1.2% (p < 0.05 vs. 38.4 ± 0.7%).In contrast, the cholinergic agonist the nicotine L-Nicotine (L-Nico) further promoted the inhibitory effect of TP5 on cancer stem cells in colon cancer cells . PretreaWe further found that the inhibitor TUB (1–100 μg/ml) significantly blocked the TP5-promoted proliferation inhibition of HCT116 by OXA, whereas the agonist L-Nico promoted this effect of TP5 , implyinAccording to a large number of clinical studies , TP5 is First, the ability of sphere formation is an important method to identify cancer stem cells in vitro . Oxaliplatin (OXA), D-tufosine pentahydrate (TUB) and L-nicotine (L-Nico) were purchased from MedChemExpress (USA). HCT116 cells were cultured in RPMI-1640 medium  supplemented with 10% fetal bovine serum , penicillin (100 IU/ml) and streptomycin . LoVo cells were cultured in F-12K Nutrient Mixture  supplemented with 10% fetal bovine serum , penicillin (100 IU/ml) and streptomycin . Cells were incubated in a humidified incubator containing 5% COThe serum-free medium for spheroid culture consisted of DMEM/F12  medium supplemented with 0.4% BSA , 20 ng/ml human recombinant epidermal growth factor , 20 ng/ml human recombinant basic fibroblast growth factor , 25 μg/ml human insulin , 2% B27 supplement (50×) , and 0.1% 2-mercaptoethanol . Cells were plated in Corning Ultra-Low Attachment 24-well plates  at a density of 5,000 viable cells/well and grown in serum-free medium. Cells were grown for 6 days and the number of spheroids was counted under the microscope  as previously described .5 cells/well) were seeded in 12-well plates and exposed to different concentrations of TP5 for 24 h. The supernatant was then removed and cells were digested with 0.25% trypsin . After washing, cells were resuspended in 100 ul of PBS . For surface staining, cells were labeled with PE-CD24 , FITC-CD44 , and PE-CD133  (HCT116 cells or LoVo cells (1.5 × 10ciences) . Cell suTotal RNA was isolated and reverse transcribed using EZ-press RNA purification kit  and PrimeScript RT kit , respectively, according to the manufacturer’s instructions , and qPC5 cells/well) were seeded in 6-well plates with different treatments of TP5  for 48 h at 37°C. Cells were lysed with ultrasound, centrifuged and the supernatant (100 μL/well) was transferred to UV-Star 96-well plates  and incubated at 37°C for 10 min. ALDH activity was measured at 340 nm using a microplate reader .ALDH activity was assessed using an ALDH activity detection kit  according to the manufacturer’s instructions . HCT116 After treatment with TP5  for 48 h, total proteins from HCT116 cells were lysed in lysis buffer  containing 1% cocktail  . Cell ly2+-free concentration was assessed using the fluorescent Ca2+-probe Fluo-4 AM . Briefly, cells were incubated in PBS at 37°C with 2 μM Fluo-4 AM for 30 min in the dark, followed by three washes with PBS. Binding buffer (500 μL) containing 1% FBS  was added and incubated for 30 min in the dark at room temperature. The results were then evaluated by flow cytometry. The excitation wavelength was 488 nm and the emission wavelength was 520 nm. The experiment was repeated at least three times.HCT116 cells were incubated in 12-well plates with different treatments of TP5  for 24 h at 37°C. To determine the cellular calcium concentration , the celCell viability was assessed using the MTT assay kit  according to the manufacturer’s protocol . HCT116 5 cells/well) were seeded in 12-well plates, exposed to different concentrations of TP5 for 48 h, and stained with 5 μl of FITC-labeled Annexin V and 5 μl of PI simultaneously at room temperature, protected from light. Stained cells were analyzed using a fluorescence-activated cell sorter flow cytometer . A minimum of 10,000 cells were used for each analysis, and experiments were performed in triplicate.The extent of apoptosis in treated HCT116 cells was assessed using the Annexin V-FITC/PI Apoptosis Assay Kit  according to the manufacturer’s instructions . HCT116 p less than 0.05 was considered statistically significant.Data are expressed as mean ± SD. All experiments were performed independently, at least 3 times. Statistical analyses were performed as described in each corresponding legend. Differences between two groups were assessed by unpaired two-sided Student’s t-test, and differences between multiple groups were assessed by one- or two-way ANOVA and Dunnett’s post hoc test.


When sintered for 4 h, no grain growth occurred for the x = 0.000 composition over a wide temperature range, demonstrating that the existence of initial vacancies is essential for grain growth. As x increased, that is, as the vacancy concentration increased, the critical driving force (ΔGC) for 2D nucleation decreased, and abnormal grain growth was promoted. The number and size of these abnormal grains increased as the sintering temperature increased, but at sintering temperatures above 1100 °C, they decreased again owing to a large drop in ΔGC. The x = 0.005 specimen sintered at 1085 °C exhibited excellent piezoelectric properties of d33 = 498 pC/N and kp = 0.45 due to the large number of large abnormal grains with an 83% tetragonal phase fraction. The x = 0.000 specimen sintered at 1130 °C with suppressed grain growth exhibited good energy storage properties because of its very high relative density and small grain size of 300 to 400 nm.This study investigated the causes of microstructural changes and the resultant electrical properties according to the sintering temperature of 0.96(K However, recently tightened environmental regulations around the world have urged the development of lead-free piezoelectric ceramics that do not contain lead oxide, which leads to harmful effects such as high toxicity and high vapor pressure in manufacturing and disposal processes [3 (KNN)-based ceramics are ferroelectric materials with ABO3–type perovskite structures and are actively being studied as eco-friendly lead-free piezoelectric materials because of their high Curie temperatures and good piezoelectric properties [O-T and TR-O) to near room temperature [O-T and TR-O meet near room temperature by doping Li, Bi, Ag, etc. at the A-site and Sb, Zr, Hf, etc. in the B-site of KNN, excellent piezoelectric properties comparable to those of PZT-based ceramics are obtained because of the coexistence of rhombohedral and tetragonal phases [Piezoelectric ceramics are smart materials that enable the interconversion between mechanical and electrical energy and are used in various electronic devices such as actuators, transducers, sensors, and motors ,2,3,4,5.rocesses ,7,8.  for the formation of 2D nuclei on the grain surface is expressed assl is the solid-liquid interfacial energy, r is the radius of the growing grain, and r∗ is the average radius of the grains around the growing grain. Only grains that satisfy the condition ΔG ≥ ΔGC can grow via 2D nucleation. The variable ε in Equation (1) is inversely proportional to the configurational entropy; therefore, it can be decreased as the vacancy increases [The grains of KNN-based ceramics exhibit a faceted cube shape, signifying their very high interfacial energy anisotropy ,29,30. T0.46−xNa0.54−x)Nb0.95Sb0.05O3-0.04Bi0.5(Na0.82K0.18)0.5ZrO3 compositions with intentionally reduced alkali element content were designed, and the effect of vacancy concentration on the microstructure and electrical properties of sintered ceramics was investigated. It was found that grain growth was suppressed in compositions in which the alkali element content was not controlled, and there were specific compositions and sintering temperatures to obtain a microstructure exhibiting excellent piezoelectric properties. In addition, we report that when a highly dense microstructure with suppressed grain growth is secured, the energy storage performance is enhanced, even if the ferroelectricity is weakened owing to the small grain size.In this study, 0.96(K0.46−xNa0.54−x)Nb0.95Sb0.05O3-0.04Bi0.5(Na0.82K0.18)0.5ZrO3 compositions  were prepared using K2CO3 (99% purity), Na2CO3 (99% purity), Nb2O5 (99.9% purity), Sb2O3 (99.9% purity), Bi2O3 (99.9% purity), and ZrO2 (98% purity) raw powders  by solid state synthesis. K2CO3 and Na2CO3 powders were heated at 400 °C for 24 h under a vacuum to achieve complete dehydration. The raw powders were mixed according to their compositions and milled with ethanol and yttria-stabilized zirconia balls in a polyethylene jar for 24 h. The powder mixtures were calcined at 950 °C for 6 h with a heating rate of 4 °C/min and naturally cooled down to room temperature in a furnace (furnace-cooling). The calcined powders were re-milled for 24 h and pressed into disk-shaped powder compacts at 100 MPa. The powder compacts were sintered for 4 h at temperatures ranging from 1070 °C to 1130 °C with a heating rate of 4 °C/min and naturally cooled down to room temperature in a furnace.The 0.96 and impedance analyzer . Polarization vs. electric field (P-E) curves were measured using a ferroelectric test system .An Ag paste was printed on both surfaces of the sintered specimens and fired at 600 °C for 30 min with a heating rate of 4 °C/min and naturally cooled down to room temperature in a furnace. The specimens were electrically poled by applying a DC electric field of 4 kV/mm for 30 min at room temperature in a silicone oil bath. Structural analyses were performed using X-ray diffraction , Raman spectroscopy , energy-dispersive X-ray spectroscopy , and scanning electron microscopy . The piezoelectric and dielectric properties of the sintered specimens were measured using a d0.46−xNa0.54−x)Nb0.95Sb0.05O3-0.04Bi0.5(Na0.82K0.18)0.5ZrO3 powders calcined at 950 °C, and the ratio decreased as x increased. As all of the compositions exhibit perovskite structures without a secondary phase , respectively, and the v1 peak shifts to a higher wavenumber when an alkali ion deficiency (or vacancy) exists or when ions with a small ionic radius, such as lithium, are substituted for the A-site [0.46−xNa0.54−x)Nb0.95Sb0.05O3-0.04Bi0.5(Na0.82K0.18)0.5ZrO3 grains increases with an increase in x.The ve A-site ,33,34. T0.46−xNa0.54−x)Nb0.95Sb0.05O3-0.04Bi0.5(Na0.82K0.18)0.5ZrO3 ceramics sintered for 4 h at temperatures ranging from 1070 to 1085 °C. C because the vacancy concentration of grains was too low. However, for x = 0.005, 0.010, and 0.015 specimens sintered at 1070 °C, abnormal grains of several tens of micrometers in size were observed in the small matrix grains that did not grow. This can be interpreted as 2D nucleation and growth progressing in some grains, satisfying the ΔG ≥ ΔGC condition as ε decreases owing to the increased vacancy concentration. Moreover, the number and size of these abnormal grains increased as the sintering temperature increased. This implies that the higher the sintering temperature, the more active the alkali element volatilization, which increases the number of grains satisfying the ΔG ≥ ΔGC condition and advances the onset of 2D nucleation during sintering. Matrix grains of the x = 0.005, 0.010, and 0.015 specimens sintered at temperatures below 1080 °C did not grow. However, the x = 0.005 specimen sintered at 1085 °C exhibited a microstructure composed of large grains of several tens of micrometers and small grains of several micrometers, suggesting that the ΔGC continued to decrease with time at 1085 °C, and the growth of small-sized grains was also initiated.Unusually, no grain growth was observed in the x = 0 specimen under all sintering temperatures, signifying that 2D nucleation was suppressed in all grains owing to the high ΔG0.46−xNa0.54−x)Nb0.95Sb0.05O3-0.04Bi0.5(Na0.82K0.18)0.5ZrO3 ceramics sintered at 1085 °C for 4 h. The x = 0.000 specimen, in which grain growth did not occur, exhibited poor piezoelectric properties of d33 < 100 pC/N and kp < 0.2, whereas the x = 0.005 specimen, in which large grain growth was observed, exhibited excellent piezoelectric properties of d33 = 498 pC/N and kp = 0.45. A large grain size makes domain rotation easier, thereby increasing the piezoelectric properties [operties ,35. Howe0.46−xNa0.54−x)Nb0.95Sb0.05O3-0.04Bi0.5(Na0.82K0.18)0.5ZrO3 ceramics sintered at 1085 °C for 4 h showed a pure perovskite structure without a secondary phase, as shown in C peak was observed for the x = 0.005, 0.010, and 0.015 specimens composed of large grains. Deconvolution analysis of the (200)C peaks in R-T) and Curie temperature (TC) shifted toward lower temperatures Nb0.95Sb0.05O3-0.04Bi0.5(Na0.82K0.18)0.5ZrO3 ceramics sintered at temperatures higher than 1085 °C. For the x = 0.000 specimen, grain growth was not observed even at a very high sintering temperature of 1130 °C, which indicates that the ΔGC value of the stoichiometric composition, in which the content of the initial alkali element was not reduced, could be maintained at a very high level even at a high sintering temperature. In the case of the x = 0.005 specimen, as the sintering temperature increased, the number of large abnormal grains decreased, and the matrix grains grew to a size of several micrometers. This phenomenon was also observed in the x = 0.010 specimen, but the number of abnormal grains began to decrease at a lower sintering temperature (1100 °C) than that of the x = 0.005 specimen (1110 °C). The x = 0.010 specimen sintered at 1130 °C displayed a microstructure in which all grains were grown to a size of several micrometers without large abnormal grains.C decreases as the concentration of vacancies increases, and if the number of grains satisfying the ΔG ≥ ΔGC condition rapidly increases during the sintering process, the growth of large abnormal grains is inevitably suppressed, and the number of abnormal grains is also limited. In the case of the x = 0.010 specimen, because the initial vacancy concentration was higher than that of the x = 0.005 specimen, ΔGC decreased more rapidly, and the abnormal grain growth started to be suppressed at lower temperatures.The decrease in the number of abnormal grains as the sintering temperature increased is considered to be due to the increase in the vacancy concentration due to the enhanced volatilization of the alkali element. ΔG33 and kp sharply increased as the sintering temperature increased up to 1085 °C but decreased again at higher sintering temperatures. By correlating the results of R-T due to the increased vacancy concentration (similar to the explanation in As shown in st), recoverable energy density (Wrec), and efficiency (η) of the dielectric capacitor are expressed as follows:Furthermore, the weakening of ferroelectricity owing to the fine grains with a pseudo-cubic structure led to a slim P-E curve with very little hysteresis loss. The stored energy density (Wmax) and low remnant polarization (Pr) of the P-E curve in st = 0.66 J/cm3, Wrec = 0.52 J/cm3, and η = 79%. Therefore, by designing a dense microstructure composed of small grains with a low vacancy concentration, excellent energy storage characteristics can be obtained, even if the piezoelectric characteristics deteriorate.Due to the high maximum polarization (P0.46−xNa0.54−x)Nb0.95Sb0.05O3-0.04Bi0.5(Na0.82K0.18)0.5ZrO3 ceramics were investigated. For the x = 0.000 specimen, grain growth was not observed at any sintering temperature from 1070 °C to 1130 °C because the high ΔGC owing to the very low vacancy concentration was maintained even at high sintering temperatures. In the case of the x = 0.005, 0.010, and 0.015 specimens, the grains satisfying the ΔG ≥ ΔGC condition owing to the increased vacancy concentration grew into abnormal grains by 2D nucleation. The x = 0.005 specimen sintered at 1085 °C exhibited a microstructure composed of large grains of several tens of micrometers and small grains of several micrometers with excellent piezoelectric properties of d33 = 498 pC/N and kp = 0.45. For the x = 0.010 and 0.015 specimens sintered at 1085 °C, the piezoelectric properties deteriorated compared to those of the x = 0.005 specimen because the TR-T of x = 0.010 and 0.015 specimens moved to lower temperatures than that of the x = 0.005 specimen. For the x = 0.005 specimen sintered at temperatures higher than 1085 °C, the number of large abnormal grains decreased again as the number of grains satisfying the ΔG ≥ ΔGC condition rapidly increased, owing to the further increased vacancy concentration; accordingly, the values of the piezoelectric coefficients also decreased. The relative density of the x = 0.000 specimen rapidly increased with an increase in the sintering temperature, and the x = 0.000 specimen sintered at 1130 °C with a high relative density of 98% and a fine grain size of 300–400 nm exhibited good energy storage performance of Wst = 0.66 J/cm3, Wrec = 0.52 J/cm3, and η = 79%. The results of this study demonstrate that the microstructure of 0.96(K0.46−xNa0.54−x)Nb0.95Sb0.05O3-0.04Bi0.5(Na0.82K0.18)0.5ZrO3 ceramics highly depends on the vacancy concentration, and their applications can be diversified depending on the microstructure. These findings are expected to be applied to the study of microstructure control of other KNN-based ceramics.In this study, the effects of alkali element deficiency and sintering temperature on the microstructure and electrical properties of 0.96(K


Maclura tricuspidata fruit to prevent TNF-α-induced skin inflammation in normal human dermal fibroblasts (HDFs). It focused on alpinumisoflavone (AIF) that suppressed the accumulation of ROS and nitric oxide (NO) in tumor necrosis factor-alpha (TNF-α)-treated HDFs. AIF inhibited the TNF-α-induced increase in matrix metalloproteinase-1, decreased procollagen I α1, and suppressed pro-inflammatory mediators and pro-inflammatory cytokines, including NO synthase, cyclooxygenase-2, interleukin (IL)-1β, IL-6, and IL-8 that trigger inflammatory responses. AIF inhibited nuclear factor-κB and activating protein 1 mitogen-activated protein kinases that were increased by TNF-α stimulation. These results suggest that AIF may protect skin from aging and various cutaneous lesions.The skin is an important organ in the human body that protects the body from environmentally hazardous substances. Reactive oxygen species (ROS) cause inflammatory reactions and degradation of the extracellular matrix leading to skin aging and various cutaneous lesions. This study evaluated the potential of isoflavones isolated from  Skin is the primary protector of the human ectoderm system, is in direct contact with potentially harmful factors, and performs three major functions: skin sensation, control, and protection . IntrinsThe production of ROS has been reported to cause oxidative damage and affect biological functions, such as cell membrane destruction, DNA denaturation, inflammatory response, and immunodeficiency ,9,10. ROExposure of the skin to UV radiation produces cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, IL-6, and IL-8 ,16,17 thMaclura tricuspidata Carr. (syn. Cudrania tricuspidata) is a deciduous tree belonging to the family Moraceae; it has been found in various medicinal and nutritional applications in East Asia. Prenylated isoflavones and xanthones are considered the major constituents of M. tricuspidata and have been shown to have various biological activities, including anti-inflammatory, antioxidant, antiatherosclerotic, hepatoprotective, and neuroprotective effects.Maclura tricuspidatan possessed anti-oxidative and 2,2-diphenyl-1-picrylhydrazyl (DPPH)-scavenging properties. Previous studies reported that the antioxidant activity of isoflavones may regulate oxidative damage within cells, implying that isoflavones may prevent oxidative stress-related damage in the skin ECM [M. tricuspidata fruit. In the present study, we describe the antioxidant and anti-aging effects of isoflavones on TNF-α-stimulated HDFs and identify the mechanisms of active compounds.Our preliminary experiment showed that compounds isolated from skin ECM ,24. We uM. tricuspidata fruit extract (MTF), alpinumisoflavone (AIF), and 4′-O-methyl alpinumisoflavone (MAIF) were prepared from the fruits of M. tricuspidata as previously described [A voucher specimen (accession number KH1-4-090814) was deposited at the Department of Plant Biotechnology, Korea University, Seoul, Korea. escribed .The indicated concentrations of samples were dissolved in dimethyl sulfoxide (DMSO) and mixed with 100 µL of DPPH solution in a 96-well plate. After reacting for 30 min in dark conditions, the reaction results were read on a microplate reader  at 517 nm. Data are presented as means ± standard error of mean (SEM) of three independent experiments performed in triplicate.2 atmosphere in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum  and penicillin/streptomycin  antibiotics.Human dermal fibroblast HDFs were obtained from PromoCell GmbH . Cells were maintained at 37 °C in a humidified, 5% CO4 cells/well in 96-well plates, allowed to adhere overnight, and then starved for 24 h under serum-free conditions. HDFs were challenged with 20 ng/mL TNF-α  in the presence or absence of MTF (12.5 and 25 µg/mL), AIF (25 and 50 µM), MAIF (25 and 50 µM), and 10 µM DCFDA . The reaction results were read on a microplate reader  at an excitation and emission spectra of 495 nm and 529 nm. Data are presented as means ± SEM of three independent experiments performed in triplicate.Experiments were conducted as previously described . Briefly4 cells/well in 96-well plates, allowed to adhere overnight, and then starved for 24 h under serum-free conditions. HDFs were challenged with 20 ng/mL TNF-α in the presence or absence of MTF (12.5 and 25 µg/mL), AIF (25 and 50 µM), and MAIF (25 and 50 µM) for 24 h. The nitrite content of the supernatant was quantified using the Griess assay. The supernatant was incubated with 1% sulfanilamide, 0.1% N-(1-naphthyl)-ethylenediamine, and 5% phosphoric acid at room temperature for 10 min. Thereafter, nitrite content was read on a microplate reader using 540 nm wavelength. NO production in each sample was then calculated using a standard sodium nitrite (NaNO2) curve.Experiments were conducted as previously described . Briefly4 cells/well in 48-well plates, allowed to adhere overnight, and then starved for 24 h under serum-free conditions. HDFs were challenged with 20 ng/mL TNF-α in the presence or absence of MTF (12.5 and 25 µg/mL), AIF (25 and 50 µM), and MAIF (25 and 50 µM) for 12 h  and 24 h (for MMP-1 and COLIA1). Proteins in supernatants were quantified by enzyme-linked immunosorbent assay (ELISA) using a corresponding ELISA kit , according to the manufacturer’s instructions.Experiments were conducted as previously described . Briefly5 cells/well in 6-well plates, allowed to adhere overnight, and then starved for 24 h under serum-free conditions. HDFs were challenged with 20 ng/mL TNF-α in the presence or absence of MTF (12.5 and 25 µg/mL), AIF (25 and 50 µM), and MAIF (25 and 50 µM) for 4 h , and 24 h (for MMP-1 and COLIA1). Cells’ mRNA expression was quantified by quantitative real-time polymerase chain reaction (qRT-PCR). Cells’ RNA was extracted using the RNeasy Mini Kit  according to the manufacturer’s instructions. The cDNA was synthesized from RNA using the RevertAid First Strand cDNA Synthesis kit . The PCR reaction was conducted with PowerUp SYBR PCR Master Mix , each corresponding primer, and synthesized cDNA using the following thermal conditions: initial denaturation at 95 °C for 10 min, followed by 40 cycles with denaturation at 95 °C for 1 s, annealing and elongation at 60 °C for 30 s. The mRNA expression was normalized to that of the β-actin reference gene. The primers used are shown in t method.Experiments were conducted as previously described . Briefly5 cells/well in 6-well plates, allowed to adhere overnight, and then starved for 24 h under serum-free conditions. HDFs were challenged with 20 ng/mL TNF-α in the presence or absence of MTF (12.5 and 25 µg/mL), AIF (25 and 50 µM), and MAIF (25 and 50 µM) for 15 min , ERK, phospho-C-Jun, T-terminal kinase (p-JNK), JNK, p-p38, p38, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH)), 4 h (for nuclear factor-kappa B (NF-κB) and activator protein-1 (AP-1)), and 6 h (for iNOS and COX-2). The cells’ protein expression was quantified by Western blot analysis. Cell lysates were extracted using radioimmunoprecipitation assay buffer . Protein concentration was determined using the Pierce™ BCA Protein Assay Kit . Proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Thereafter, proteins were transferred to a polyvinylidene difluoride membrane  and blocked with 5% skim milk. The membrane was incubated at room temperature with diluted primary antibodies, including iNOS, COX-2, NF-κB (p65), AP-1, ERK1/2, phospho-ERK1/2, p38, phospho-p38, JNK, phospho-JNK, and GAPDH , for 4 h. The membrane was washed and incubated at room temperature with rabbit IgG secondary antibody for 1 h. The protein signal was measured using SuperSignal® West Femto Maximum Sensitivity Chemiluminescent Substrate (Pierce) and the Fusion Solo Chemiluminescence System . The protein expression was normalized to that of GAPDH reference protein. The analysis was performed using a Fusion Solo Chemiluminescence System . The relative protein expression was calculated compared to an untreated group using the ImageJ software .Experiments were conducted as previously described . Brieflyp < 0.05, p < 0.01, and p < 0.001 were considered significant.All data are expressed as the mean ± standard error of the mean. Statistical analyses were conducted using one-way analysis of variance (ANOVA) and Tukey’s post-test to evaluate differences among the experimental groups . M. tricuspidata fruit extract (MTF), alpinumisoflavone (AIF), and 4′-O-methyl alpinumisoflavone (MAIF) (50) of MTF was 17.0 µg/mL. The EC50 of AIF and MAIF were at 9.2 and 17.8 μM, respectively. Previous studies have reported that various isoflavones acted as antioxidants and inhibited oxidative stress within cells [In preliminary experiments, e (MAIF)  displayee (MAIF) . The conin cells ,31,32,33As described above, exposure to UV radiation increases the accumulation of intercellular ROS and the secretion of pro-inflammatory cytokines such as TNF-α. Additionally, ROS produced by mitochondria serve as signaling molecules that upregulate inflammatory cytokines including TNF-α. Excessively elevated TNF and ROS regulate and activate the elevated levels of each other that lead to collagen cleavage and various inflammatory responses. Therefore, ROS and TNF-α can be used as substitute inducers because they act by a mechanism similar to skin aging and inflammatory response resulting from UV exposure. We used TNF-α as a marker to evaluate the inhibitory effects of MTF, AIF, and MAIF on ROS accumulation and NO production.p < 0.001) compared to the untreated cells (p < 0.05) by MTF at 12.5 and 25 µg/mL concentrations, respectively. Treatment with AIF alone suppressed the intracellular ROS production in TNF-α-treated HDFs to significantly lower levels , whereas MAIF did not. These results showed that MTF extract and compound AIF inhibited ROS accumulation in TNF-α-treated HDFs, indicating a possibility that these two agents might attenuate the skin damage caused by oxidative stress.To investigate the antioxidant potential of MTF, AIF, and MAIF, the inhibition of intracellular ROS production after TNF-α treatment in HDFs was quantified with DCFDA fluorogenic dye. Because the extract and two isoflavones were not cytotoxic to HDFs at 25 μg/mL and 50 μM, respectively (data not shown), experiments were performed using these concentrations. Intracellular ROS production after 30 min of adding 20 ng/mL TNF-α revealed that the basal ROS production in HDFs was 2.48 ± 0.06-fold (ed cells A. The prp < 0.001) compared to the untreated cells  , whereas MAIF did not lower NO secretion. These results indicated that NO production was inhibited in TNF-α-treated HDFs by compound AIF that may inhibit skin damage caused by the inflammatory response.To assess the anti-inflammatory potential, the inhibition of pro-inflammatory NO production in HDFs after TNF-α treatment was quantified by the Griess assay. NO production after 24 h of 20 ng/mL TNF-α administration revealed that the NO secretion in HDF was 4.24 ± 0.18 µM  B. SimilaTaken together, these results indicate that AIF removes excess ROS and suppresses NO production but MAIF does not. AIF and MAIF presented a typical pyranoisoflavone structure, which is a type of isoflavone. In moieties of isoflavone, they equally attached with the one a pyran group to carbon positions 6 and 7 . They substituted the hydroxyl group or the methoxy group at C-4′. In detail, AIF was attached with the hydroxyl groups, in contrast, MAIF was attached with the methoxy group. Therefore, the potential of AIF against ROS scavenging and NO inhibition was considered in correlation with hydroxyl group substitution of C-4′. Base on these results, we focused on AIF in the subsequent experiments.Because the activation of COX-2 and iNOS plays an important role in NO production, we evaluated the effect of AIF on COX-2 and iNOS protein expression increased by TNF-α stimulation in HDFs.p < 0.01) compared to the untreated cells  but the change was not concentration-dependent (p < 0.01) compared to the untreated group (p < 0.01) and 7.19 ± 0.48-fold (p < 0.01) by 25 and 50 µM of AIF, respectively  compared with the untreated cells  in a concentration-dependent manner. Analogously, after TNF-α treatment, protein secretion of MMP-1  was significantly increased compared with the untreated group (2.31 ± 0.22 ng/mL), whereas it was significantly suppressed to 7.21 ± 0.72 ng/mL (p < 0.05) and 5.43 ± 0.53 ng/mL (p < 0.01) by 25 and 50 µM of AIF, respectively  compared to the untreated cells (p < 0.01) was significantly increased after TNF-α treatment compared with the untreated group (14.01 ± 0.46 ng/mL) (TNF-α administration inhibited the mRNA expression of COLIA1 after 4 h of 20 ng/mL 0.42 ± 0.08-fold (ed cells A. AIF ad6 ng/mL) A. Howeve6 ng/mL) B. These Pro-inflammatory cytokines, such as TNF-α, IL-1β, IL-6, and IL-8, are sensitized by cellular oxidative stress and upregulate the inflammatory response ,40. ThesIL-1β, IL-6, and IL-8 after 4 h of 20 ng/mL TNF-α administration increased 5.78 ± 0.72-fold (p < 0.05), 4.50 ± 0.53-fold (p < 0.01), and 4.32 ± 0.36-fold (p < 0.01), respectively, compared to untreated cells , IL-6 , and IL-8 .The mRNA expression and secretion of IL-1β, IL-6, and IL-8 proteins in TNF-α-treated HDFs treated with AIF are shown in ed cells A. Comparp < 0.05), 43.4 ± 0.84 ng/mL (p < 0.001), and 31.4 ± 1.89 ng/mL (p < 0.001) compared to untreated cells  and 7.87 ± 0.74-fold (p < 0.001) by 25 and 50 µM of AIF, respectively. The secretion of IL-8 was also reduced by AIF, however, only at 50 µM  concentration. These results indicate that AIF may suppress skin inflammatory responses against TNF-α stimulation by inhibiting pro-inflammatory cytokines. Therefore, AIF may attenuate inflammation-related skin aging and diseases.To evaluate the change in the actual secretion of pro-inflammatory cytokines, ELISA was performed. Analogously with mRNA results, secretion of IL-1β, IL-6, and IL-8 proteins in HDFs after 12 h of 20 ng/mL TNF-α administration increased to 12.4 ± 1.46 ng/mL  and 11.3 ± 0.60-fold (p < 0.001) by 20 ng/mL TNF-α administration, respectively. The protein expression of NF-κB (p65) was dramatically inhibited by AIF; however, the inhibition was not concentration-dependent . Expression of AP-1 was suppressed by AIF in a concentration-dependent manner .NF-κB (p65) and AP-1 protein expression after 12 h of 20 ng/mL TNF-α administration increased compared to untreated cells A. As shoIn The AP-1 and NF-κB pathways regulate MMP-1 and pro-inflammatory cytokines, and these pathways are upregulated by MAPK activation . To assep < 0.01, 5.94 ± 0.34-fold , and 3.02 ± 0.12-fold  by 20 ng/mL TNF-α administration, respectively. The phosphorylation of ERK was decreased in the AIF-treated group to 2.87 ± 0.07-fold  and 1.97 ± 0.01-fold . The phosphorylation of JNK was significantly inhibited in the AIF-treated group to 4.76 ± 0.21-fold  and 0.60 ± 0.19-fold . The phosphorylation of p38 was reduced in the AIF-treated group to 2.41 ± 0.25-fold  and 1.07 ± 0.16-fold . These results indicate that AIF may suppress AP-1 and NF-κB activation induced by TNF-α by suppressing the phosphorylation of MAPK.The MAPK protein expression after 15 min of 20 ng/mL TNF-α administration increased, compared to that in untreated cells A. TreatmUV light directly or indirectly induces ROS and pro-inflammatory mediators, such as NO, iNOS, COX-2, IL-1β, IL-6, and IL-8 ,16,17. TTaken together, AIF isolated from MTF has antioxidant and inflammatory effects through the inhibition of ROS and NO accumulation in TNF-α-induced HDFs. AIF can inhibit the degradation of skin ECM by increasing MMP-1 collagenase and decreasing collagen synthesis. AIF has been associated with the inhibition of iNOS and activation of COX-2 and pro-inflammatory cytokines IL-1β, IL-6, and IL-8. Inhibition of TNF-α-induced activation of NF-κB, AP-1, and MAPKs in HDFs by AIF suggests its possible utility in attenuating skin aging.M. tricuspidata fruit inhibits TNF-α-induced ROS, NO, and MMP-1 expression and increased collagen synthesis. AIF reduces the expression of TNF-α-induced pro-inflammatory cytokine mediators, including iNOS and COX-2, and the pro-inflammatory cytokines IL-1β, IL-6, and IL-8. The mechanism by which AIF inhibits TNF-α-induced responses in HDF is mediated through inhibition of NF-κB, AP-1, and MAPK activation. Our findings provide the first evidence that AIF may be effective in attenuating skin damage caused by oxidative stress. Although further research is needed fully to understand the mechanisms of AIF’s activity, the compound is a potential agent for inhibiting skin aging and various cutaneous lesions.ROS is a major part of the inflammatory response and ECM degradation that leads to skin aging and various cutaneous lesions. Therefore, ROS inhibitors can be used to attenuate skin aging and disease. This study shows that alpinumisoflavone (AIF) isolated from


N,N′-dioctyl perylene diimide (C8PDI). In this study, we combined P3HT and polyethylene glycol (PEG) to amphiphilic block copolymers and studied the crystallization modification effect of these block copolymers. It is found that the crystallization modification effect of the block copolymers is retained and gradually enhanced with P3HT content. The length of C8PDI crystals were well controlled from 2 to 0.4 μm, and the width from 210 to 35 nm. On the other hand, due to the water solubility of PEG block, crystalline PEG-b-P3HT/C8PDI micelles in water were successfully prepared, and this water phase colloid could be stable for more than 2 weeks, which provides a new way to prepare pollution-free aqueous organic semiconductor inks for printing electronic devices.The preparation of micron- to nanometer-sized functional materials with well-defined shapes and packing is a key process to their applications. There are many ways to control the crystal growth of organic semiconductors. Adding polymer additives has been proven a robust strategy to optimize semiconductor crystal structure and the corresponding optoelectronic properties. We have found that poly(3-hexylthiophene) (P3HT) can effectively regulate the crystallization behavior of  The addSMDPPEH) . Huang erivative . By adjurivative . Therefob-PEG/C8PDI micelles in water by extracting their micelles in toluene (Tol) with water, which provides a new way to prepare pollution-free aqueous organic semiconductor inks for printing electronic devices.Block copolymers can combine the superior properties of different polymers, and also produce various phase structures and self-assembly morphologies due to microphase separation . PEG hasSynthesis of block copolymers. Three block copolymers were synthesized by cycloaddition click reaction between monoethynyl-terminated P3HT (EP3HT) and azide-terminated PEG (azide-PEG) by copper-catalyzed azide-alkyne, shown in 1H nuclear magnetic resonance (NMR) spectroscopy shows the regioregularity of EP3HT is 90% (n) of EP3HT is 2,800 g/mol and the polymer dispersity index (PDI) is 1.15  (w). The azide-PEGs were purified by sedimentation in ether. Their structures were characterized by 1H NMR and fourier transform infrared spectroscopy (FT-IR) spectroscopy  respectively by copper-catalyzed azide-alkyne cycloaddition click reaction. The success of block copolymers synthesis was confirmed by 1H NMR spectroscopy and the molecular weights measured by GPC.T is 90% . Accordi is 1.15 . The Mn -TOF-MS) . The rattroscopy  and theio-dichlorobenzene (o-DCB).Self-Assembly in  To investigate the crystallization modification effect of block copolymers, we compared the crystallization behavior of C8PDI without and with block copolymers. 1.0 mg/ml C8PDI supersaturated solution in o-DCB was prepared by heating at 120°C for hours. Before precipitation, the solution was put into ultrasound for 2 h. Yellow clear solution changed to brick red suspension soon after putting in the sonication bath. The resulted crystals show an average length of 2.91 μm and an average width of 0.33 μm, as shown in b-PEG and 1.0 mg/ml C8PDI in o-DCB. The supersaturated solution of C8PDI with P3HT-b-PEG is in orange color and clear for hours after cooled to room temperature, while the C8PDI supersaturated solution without block copolymers precipitates soon after cooling. During sonication, the C8PDI with P3HT-b-PEG solution changes to wine red color and remains clear. By radiating the solution with a red laser, distinct Tyndall scattering was observed which means colloid is formed  and b , respectively  experiments were performed on the films corresponding to the four samples in ectively . And theirection . Two priirection , correspectively . After tPreparation of micelles of diblock copolymer and C8PDI in water. By extracting the Tol solution of diblock copolymer P3HT-b-PEG and C8PDI (10:1) with water, crystalline P3HT-b-P3HT/C8PDI micelles in water were successfully prepared. The specific method is first adding water to the Tol solution after ultrasonic crystallization and vigorously shaking the solution. After letting the solution stand for a period of time, the solution could be clearly divided into two layers. The upper layer, Tol phase, was emulsion, and the lower water phase was light yellow, clear, transparent  shows an absorption band ranging from 300 to 550 nm with a maximum at about 420 nm  is redshifted by 10 nm compared with that in Tol. C8PDI nanocrystals in sonicated Tol solution (the red line) shows absorption between 400–700 nm (b-PEG and C8PDI colloid in sonicated Tol solution (the green line) shows an addition absorption of C8PDI and P3HT-b-PEG. The absorption at 580 nm corresponds to the absorption at 590 nm of the C8PDI nanocrystals in sonicated Tol solution. P3HT-b-PEG and C8PDI colloid in water extracted from their Tol solution (the orange line) shows an absorption band ranging from 400 to 650 nm with the maximum absorption peak at 480 nm and two shoulder peaks at 530 and 580 nm, respectively. The shoulder peak at 580 nm resulting from the assembly of C8PDI proves the success of extracting C8PDI from the Tol solution into the water.The UV-vis absorption spectra of solutions or colloidal solutions  also prot 420 nm . The abs0–700 nm . The peab-PEG and C8PDI. We prepared C8PDI (0.2 mg/ml), P3HT-b-PEG (2 mg/ml) and P3HT-b-PEG: C8PDI (2:0.2) Tol solutions because pure C8PDI has low solubility in Tol even at high temperature. Similarly, heat was applied to completely dissolve the solute and then the solutions were exposure to ultrasound. After the ultrasound, the PL spectra of the three samples were measured. And the P3HT-b-PEG: C8PDI (2:0.2) colloidal solution and P3HT-b-PEG (2 mg/ml) colloidal solution were extracted by water, then the PL of these two water phases were also measured. The results were shown in b-PEG has a maximum PL intensity at ∼576 nm which is characteristic of regioregular P3HT in good solvent  measurement was also used to explore the interaction between P3HT- solvent . The PL N,N,N′,N′,N″-pentamethyldiethylenetriamine (PMDETA) was purchased from Innochem. 1,3-bis(diphenylphosphino)-propanenickel (II) chloride [Ni(dppp)Cl2] was purchased from Acros. Neutral alumina was purchased from Shanghai Weiting. Deionized water was purchased from Xi’an Dongguan. Tetrahydrofuran (THF), dichloromethane, methanol, diethyl ether, hexane, chloroform, Tol, o-DCB were purchased from Chengdu Kelong. THF was freshly distilled prior to use from sodium/benzophenone under argon. Dichloromethane was freshly distilled prior to use from calcium hydride under argon. Diethyl ether was dried with anhydrous sodium sulfate. Azide-PEGs mPEG-N3 , N3-PEG-N3  and 4Arm-PEG-N3  were purchased from Meiluo Technology. C8PDI was purchased from Ogantec.2,5-dibromo-3-hexylthiophene, isopropylmagnesium chloride (2.0 M solution in THF), ethynylmagnesium bromide (0.5 M solution in THF) and deuterated chloroform were purchased from Energy Chemical. Copper (I) bromide was purchased from aladdin. 1H-NMR spectra were obtained on an Ascend™ 400 spectrometer at room temperature. The solvent in measurement was deuterated chloroform. GPC measurements were carried out at room temperature on a PL-GPC 220 using THF as eluent at a flow rate of 1ml/min at room temperature. MALDI-TOF mass measurements were performed on a Bruker autoflex III smartbeam MALDI-TOF/TOF mass spectrometer using 2-[(2E)-3-(4-tert-butylphenyl)-2-methylprop-2-enylidene] malononitrile (DCTB) as matrix. UV-vis spectra were recorded using a Lambda 35 UV-vis spectrometer. FT-IR spectra were obtained on a Nicolet 6700 FT-IR spectrometer. The PL measurement was performed on a QM40 spectrometer. The recycling preparative liquid chromatography is LC-9104. The flow rate was 12 ml/min. Ultrasound treatment was performed on a 20 kHz KC-D40A ultrasonic cleaner at 12–18°C. TEM was performed on a HITACHI HT 7700 electron microscope operating at 95kV accelerating voltage. XRD analysis was performed on a SMARTLAB(3) diffractometer. Cryo-TEM image was obtained at a FEI Talos F200C TEM.Synthesis of EP3HT. 2,5-dibromo-3-hexylthiophene  and THF (48 ml) were added to a 200 ml Schlenk flask. Then isopropylmagnesium chloride  was added to the flask at 0°C and the mixture was stirred for 2 h. After the reaction, the solution was transferred to another 500 ml flask containing 433.6 mg Ni(dppp)Cl2 and 96 ml THF. The mixture was then stirred overnight at room temperature. 6.72 ml ethynylmagnesium bromide  was added to the flask and the solution was stirred for another 6 h. The reaction was quenched by adding methanol. Filtering could be used to get crude product and the product was purified by Soxhlet extractions with methanol, hexane and chloroform. The hexane extraction was purified by column chromatography  and circulating preparative liquid chromatography (eluent: Tol) to get the final product. 1H NMR , δ (ppm): 0.91 , 1.26–1.43 , 1.61 , 2.74–2.56 , 3.53 , 6.89–7.06 . GPC: Mn = 2,795, PDI = 1.15. MALDI-MS: Mn = 1832, PDI = 1.01.General procedure for the synthesis of block copolymers. All block copolymers were prepared by the similar procedure. EP3HT , mPEG-N3  and CuBr  were placed in a 50 ml Schlenk flask. 15 ml THF was added to the flask and the mixture was purged with argon for 20 min. After adding 85.2 μl PMDETA (0.4 mmol) to the flask and the solution was purged with argon for another 10 min. The reaction was kept for 2 days at 45°C. After the reaction, the solution was purified by neutral Al2O3 column twice .b-PEGP3HT-: 1H NMR , δ (ppm): Triazole ring: 7.82 . P3HT block: 6.98 , 2.79–2.57 , 1.68 , 1.25–1.41 , 0.89 . PEG block: 4.5 , 3.79–3.46 , 3.37 . GPC: Mn = 9761, PDI = 1.77.b-PEG-b-P3HT:P3HT-1H NMR , δ (ppm): Triazole ring: 7.82 . P3HT block: 6.98 , 2.79–2.57 , 1.68 , 1.25–1.41 , 0.89 . PEG block: 4.5 , 3.79–3.46 . GPC: Mn = 15,582, PDI = 1.56.b-PEG:4P3HT-1H NMR , δ (ppm): Triazole ring: 7.82 . P3HT block: 6.98 , 2.79–2.57 , 1.68 , 1.25–1.41 , 0.89 . PEG block: 4.5 , 3.79–3.46 . GPC: Mn = 15,585, PDI = 1.61. The GPC results were shown in The blend solution of block copolymer and C8PDI was heated at 120°C to make the solute dissolve completely. After cooled to room temperature, the solution was exposed to ultrasound for 2 h. The solution was shaken by hand for 0.5 h, and then sonicated for another 1.5 h without shaking. The water temperature of ultrasonic cleaner was maintained at 12–18°C by cooling circulation pump. The nanocrystal suspension of pure block copolymer or C8PDI was prepared by the same method.Drop cast 2 μl of suspension on copper grid coated with carbon film and the excess suspension was removed by filter paper. After drying in air, the samples were checked by TEM.XRD samples were prepared by drop casting 50–150 μl solutions or colloidal solutions respectively on freshly washed silicon wafers.b-PEG/C8PDI micelles in water were successfully prepared, which provides a new way to make pollution-free aqueous organic semiconductor inks for printing electronic devices.A series of P3HT and PEG block copolymers containing linear diblock, triblock and dendritic star block copolymers were synthesized and found to be able to regulate the crystallization of C8PDI. The regulation effect increases as the P3HT block increases. On the other hand, stable crystalline P3HT-


The fabrication of tissue-engineered vascular grafts to replace damaged vessels is a promising therapy for cardiovascular diseases. Endothelial remodeling in the lumen of TEVGs is critical for successful revascularization. However, the construction of well-functioning TEVGs remains a fundamental challenge. Herein, chiral hybrid scaffolds were prepared by electrospinning using D/L-phenylalanine based gelators [D(L)PHEG] and poly-ε-caprolactone (PCL). The chirality of scaffolds significantly affected the endothelial remodeling progress of TEVGs. Compared with L-phenylalanine based gelators/poly-ε-caprolactone (L/PCL) and PCL, D-phenylalanine based gelators/poly-ε-caprolactone (D/PCL) scaffolds enhanced cell adhesion, and proliferation and upregulated the expression of fibronectin-1, and vinculin. These results suggests that chiral hybrid scaffolds can promote endothelial remodeling of TEVGs by upregulating adhesion-associated protein levels. This study offers an innovative strategy for endothelial remodeling of TEVGs by fabricating chiral hybrid scaffolds, and provides new insight for the treatment of cardiovascular diseases. Cardiovascular diseases are associated with extremely high morbidity and mortality rates in various countries worldwide, and the primary threat to human health globally . Owing tThe speed of endothelial remodeling in the lumen primarily depends on the physiochemical properties and bioactivity of the materials used to fabricate TEVGs. Synthetic polymers such as poly-ε-caprolactone (PCL) have been extensively employed in electrospinning for the fabrication of tissue-engineering vessels because of their excellent biocompatibility and mechanical properties . ElectroThe endothelial remodeling process is directed by the extracellular microenvironment, including proteins, cytokines, structures, and mechanical and chiral properties . Among tIn this study, chiral hybrid scaffolds were fabricated by electrospinning using solvent-dependent chiral assemblies of C2-symmetric D(L)PHEG and PCL, that mimic the extracellular microenvironment of human vascular endothelial cells (HUVECs) . First, 1H NMR  results are as follows: δ = 3.1 , 3.4 , 4.2 , 4.7 , 7.3 , 7.8 , 8.9  ppm. EI-MS for L(D)PFEG calcd. 636.71; found 637.28 (M + H)+.All starting materials and solvents were obtained from commercial suppliers and used without further purification. L/D-phenylalanine methyl ester hydrochloride, 1,4-benzenedicarbonyl dichloride and diglycol were purchased from Aladdin Chemicals. LPHEG and its enantiomer DPHEG were synthesized according to a previous report . 1H NMR The D(L)PHEG powder was suspended in deionized water (2 mg/ml). The solution was then heated until a transparent solution was obtained. The hydrogel was formed when the solution was cooled down to 25°C.PCL  and D(L)PHEG were dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) to prepare the D(L)PHEG and PCL mixture. PCL was dissolved at a concentration of 12% w/v at different LPHEG or DPHEG concentrations at 1, 2, 3, and 4 mg/ml. Moreover, 12% w/v PCL/HFIP was prepared as a control. An electrospinning platform was used to generate chiral hybrid scaffolds, which consisted of a grounded collector covered by an aluminum foil, a high-voltage power supply and a syringe driven by a syringe pump. Electrospinning was conducted at an applied voltage of 10.0 kV, solution flow rate of 1.0 ml/h, needle tip-to-collector gap distance of 20 cm, and a collection speed of 200 revolutions per minute. Each of the scaffolds was collected on the aluminum foil after 6 h of electrospinning followed by drying in vacuum.First, the DPHEG and LPHEG powders were suspended in deionized water (2 mg/ml) to form hydrogels. The DPHEG and LPHEG powders were also dissolved in HFIP (2 mg/ml). CD and UV-Vis signals of DPHEG hydrogel, LPHEG hydrogel, DPHEG HFIP solution, and LPHEG HFIP solution were collected using the JACSO J-815 CD spectrometer. The CD spectra of all samples were recorded in the UV region (200–400 nm) with a bandwidth of 0.5 nm.−1 at intervals of 0.4821 cm−1. To obtain ATR-FTIR spectra, chiral hybrid scaffolds were scanned in the wavelength range of 4,000–600 cm−1 at an interval of 1.9285 cm−1.FTIR spectra of the DPHEG and LPHEG powders and ATR-FTIR spectra of all chiral hybrid scaffolds were obtained using a Bruck EQUINOX55 instrument. The PCL scaffold was used as a control. The KBr disk technique was used for solid-state measurements to collect FTIR spectra from D(L)PHEG powder samples. Samples were scanned in the wavelength range between 4,000 and 600 cmn = 3).The hydrophilicity of different chiral hybrid scaffolds and PCL was assessed based on water contact angle measurements using a JC 2000D optical contact angle meter . Results are indicative of the surface wettability of the scaffold. The PCL scaffold was used as a control. The water droplet (volume: 2.0 μl) was placed on the surfaces of different scaffolds for 1 and 10 s, and the shapes of the water droplet were recorded by a camera. The static water contact angles of the different chiral hybrid scaffolds were also recorded. For each scaffold, the measurements were conducted three times at room temperature (25°C) and the results were averaged .The mechanical properties of the chiral hybrid scaffolds and PCL scaffolds were characterized by tensile tests performed on a universal tester . The samples used for tensile testing were cut into 1 cm wide and 5 cm long stripes, and the thickness of each sample was measured using an electronic digital micrometer. The samples were stretched at a constant tensile rate of 90 mm/min. The stress–strain curves of all scaffolds were plotted. Young’s modulus, fracture strain, and ultimate tensile stress were analyzed statistically (2 in a humidified atmosphere. HUVECs were trypsinized for cell seeding. The 24-well plates were coated with DPHEG and LPHEG (100 μl). The plates and scaffolds were pre-sterilized by exposure to ultraviolet (UV) light, with the plates exposed for 40 min.Primary HUVECs were obtained from Shanghai Jiao Tong University in Shanghai. They were cultured in DEME/F12  and supplemented with 5% (v/v) fetal bovine serum and 1% (v/v) antibiotics at 37°C in an incubator with 5% CO5 cells per well) were seeded on 96-well plates, which contained chiral hybrid scaffolds and PCL scaffolds. CCK-8 reagent was added according to the recommended measurements after 1–5 days of incubation. After incubation for 2 h, a microplate reader  was used to measure the optical density at 450 nm (630 nm was used as a reference). The results were analyzed statistically (n = 3).Cell proliferation was evaluated based on the cell counting kit-8 assay . HUVEC suspensions (1 × 105 cells per well) were seeded on 24-well plates coated with DPHEG hydrogel and LPHEG hydrogel cultured in an incubator for 2 days. In addition, 24-well plates containing chiral hybrid scaffolds and PCL scaffolds at the same cell number were cultured for 5 days. Subsequently, according to the recommended measurements, each well was stained using a Calcein/PI Live/Dead Viability/Cytotoxicity Assay Kit  for 30 min. A fluorescence microscope  was used to acquire images of the DPHEG hydrogel and LPHEG hydrogel for 2 days. Chiral hybrid scaffolds were observed using a confocal laser scanning microscope  on days 1 and 5.HUVECs (1 × 104 cells per well) were seeded in 48-well plates with chiral hybrid and PCL scaffolds cultured for 1 day. The cytoskeletal organization and cell nuclei of HUVECs on the different scaffolds after 1 day of culture were analyzed by fluorescent staining. Samples for fluorescent staining were washed three times with phosphate buffer solution (PBS). HUVECs were then fixed with 4% (v/v) paraformaldehyde solution for 30 min. Subsequently, according to the recommended measurements, each well was stained with FITC–phalloidin  (that stains the actin cytoskeleton) for 120 min at room temperature, and then incubated with 4,6-diamidino-2-phenylindole (DAPI)  (that stains live nuclei) at 25°C for 10 min. The stained samples were washed and visualized using a confocal laser-scanning microscope. Image J was used to calculate the number of cells.HUVECs  was used to extract ribonucleic acid from HUVECs cultured on the D/PCL-2 and PCL scaffolds according to the manufacturer’s specifications. A blank control (tissue culture plastic) was used. Reverse transcription was achieved using the PrimeScript™ RT reagent synthesis kit with gDNA Eraser   by MiniOptiCon real time PCR detection system . All operations were performed according to the manufacturer’s instructions. The 2× SYBR Green qPCR Master Mix (Low Rox)  and ABI 7300 real-time PCR system  were used for RT-qPCR. The primer sequences used are listed in p < 0.05 accepted as statistically significant.All quantitative data are expressed as mean ± standard deviation. Statistical analysis was conducted using one-way analysis of variance (ANOVA) and Student’s t-test with 2O showed Cotton effects at 226 and 265 nm  × 104/cm2, respectively, PHEG  were named as D/PCL-1, D/PCL-2, D/PCL-3, and D/PCL-4 scaffolds, and L/PCL-1, L/PCL-2, L/PCL-3, and L/PCL-4 scaffolds, respectively . The PCLII bands . The strands  × 104/cm2, (1.95 ± 0.16) × 104/cm2, (1.71 ± 0.19) × 104/cm2, (1.07 ± 0.10) × 104/cm2, and (1.64 ± 0.08) × 104/cm2, respectively,  × 104/cm2, (5.74 ± 0.24) × 104/cm2, (5.20 ± 0.38) × 104/cm2, (2.59 ± 0.21) × 104/cm2 and (2.97 ± 0.23) × 104/cm2, respectively,  and vinculin are two well-known proteins that promote cell adhesion . FN-1 anTissue engineering for transplantation is a rapidly developing regenerative therapy field . ImproveSubsequently, we explored the effect of chirality on the endothelial remodeling process induced by TEVGs. The effect of chiral hybrid scaffolds on the regulatory mechanism of cell behaviors and endothelium remodeling are complicated . All theWhile the topographies of different scaffolds exhibited minor differences, the wettability and bioactivity properties were tuned based on the scaffold components. The successful integration of PCL and D(L)PHEG hydrogels increased the hydrophilicity of the electrospun scaffolds owing to the water solubility of the chiral hydrogel. The adhesion and proliferation of cells on scaffolds may be enhanced by increased hydrophilicity PHEG and PCL by blending electrospinning to detect the moderating effect of chiral structures that play a crucial role in vascular endothelium remodeling. Compared with L/PCL scaffolds, D/PCL scaffolds showed better regulation of cell adhesion, spreading, and proliferation. The D/PCL-2 scaffold significantly promoted endothelial remodeling by enhancing FN-1 and vinculin. The chiral hybrid scaffolds are promising materials used to aid desirable endothelium remodeling, which is a critical step for the success of TEVGs. This study was an attempt to utilize chiral hybrid scaffolds to fabricate tissue-engineered vessels for the treatment of cardiovascular diseases. The study also revealed that chirality is a vital factor that ought to be considered in tissue engineering.