Galactomannan(s) are plant-derived fiber shown to reduce post-prandial blood glucose by delaying intestinal absorption of carbohydrates and slowing down gastric emptying. We examined glucose-lowering effects of BTI320, a propriety fractionated mannan(s) administered as a chewable tablet before meal in a proof-of-concept study in Chinese subjects with prediabetes.Sixty Chinese adults aged 18–70 years with either impaired fasting glucose, impaired glucose tolerance, or glycated haemoglobin 5.7–6.4% (39-46 mmol/mol), were randomly assigned in 2:2:1 ratio to either BTI320 8 g (high dose), BTI320 4 g (low dose) or matching-placebo three times daily before meal for 16 weeks. The primary endpoint was change in fructosamine in subjects treated with BTI320 compared with placebo from baseline to week 4. Indices of glycaemic variability based on continuous glucose monitoring (CGM) and standard meal tolerance test were explored in secondary analyses.p < 0.01), 2-h (p = 0.01) and 3-h (p = 0.02) post-prandial incremental glucose area-under-curve and post-meal maximum glucose (p = 0.03) compared with placebo. Subjects receiving low dose BTI320 had greater body weight reduction than placebo group.Of 60 subjects randomized, 3 subjects discontinued study treatment prematurely. In intention-to-treat analysis, no significant differences in change in serum fructosamine between low or high dose BTI320 and placebo were observed. Using random effect models, adjusted for variability by meals, treatment with low dose BTI320 was associated with reduction in 1-h .The study was registered at The online version of this article (10.1186/s12902-018-0288-5) contains supplementary material, which is available to authorized users. One in ten Chinese adults have diabetes and recent estimates from the International Diabetes Federation indicates that there are 11 million people living with diabetes in China . IndividA simple, non-systemic pharmacological approach to disease management is a universal healthcare ideal, and extracts of natural materials represent an explored opportunity. Specifically, galactomannan(s) are the active ingredient in natural gum and are used extensively in food industry as a thickener of free water . ThroughBTI320 is a proprietary combination of fractionated mannans derived from guar gum and other plant sources and is administered in the form of a chewable tablet. In an earlier open-label study of 24 patients with type 2 diabetes, BTI320 8 g and 16 g taken before a test meal reduced 3-h post-prandial glucose area-under-curve (AUC) in 75% of patients . The mai2, history of eating disorder, and known lactose or galactose intolerance. The study was registered at www.clinicaltrials.gov, reference number NCT02358668.We undertook a randomized, double-blind, placebo-controlled, parallel arm study with the first subject enrolled on 30 March 2015 and the last subject completed the study on 19 February 2016. The study was conducted in the Diabetes and Endocrine Research Centre of the Chinese University of Hong Kong (CUHK) at the Prince of Wales Hospital, Hong Kong Special Administrative Region. Subjects were identified from non-specialist general medical or family medicine clinics at the hospital. We recruited Chinese subjects aged between 18 and 70 years inclusive, fulfilling at least two of the following three criteria: 1) fasting plasma glucose 5.6–5.9 mmol/L (IFG) and/or 2-h plasma glucose 7.8–11.0 mmol/L (IGT) during a standard 75 g oral glucose tolerance test (OGTT); 2) glycated haemoglobin (HbA1c) 5.7–6.4% (39–46 mmol/mol); and 3) at least one of the following risk factors of a) history of gestational diabetes, b) history of diabetes in first degree relatives, and c) two or more of metabolic syndrome components of triglyceride ≥1.7 mmol/L, high density-lipoprotein (HDL) cholesterol < 1.3 mmol/L in women or < 1.1 mmol/L in men, waist circumference ≥ 80 cm in women or ≥ 90 cm in men, or blood pressure (BP) ≥130/80 mmHg. Exclusion criteria included current use of dietary supplements known to affect glucose or galactose metabolism, use of anti-diabetic medications in the previous 6 weeks, cardiovascular disease in the recent 12 months, renal impairment with estimated glomerular filtration rate < 60 mL/min/1.73mThe randomization process involved the use of computer-generated random numbers. Treatment group assignment of each sequentially randomised subject were contained in individually sealed, opaque and consecutively numbered envelops, which were opened by a non-study personnel.n = 24), BTI320 8 g (n = 24) or matching placebo (n = 12) orally three times daily, 10 min before each main meal for 16 weeks. Each 4-g tablet of BTI320, administered as a chewable tablet, contained 2.0 g of the key ingredient mannan polysaccharides. Other ingredients included food grade sorbitol, magnesium stearate, malic acid, natural flavors and colors. Subjects were instructed to maintain their usual dietary pattern and physical exercise levels. Subjects were reviewed every 4 weeks for assessment of adverse events and drug compliance, the latter was established by counting the returned tablets.Subjects meeting eligibility criteria were randomly assigned to receive BTI320 4 g  using a standardized meal of 500 kcal was conducted at baseline, 4 weeks and 16 weeks measuring plasma glucose, insulin, C-peptide and glucagon-like peptide (GLP)-1 at 0, 15, 30, 60, 90 and 120 min. The standard meal consisted of two pieces of pineapple shortcakes and one carton of soymilk with nutritional breakdown as follows: carbohydrates 75.7 g , fat 21.2 g  and protein 9.3 g . Seventy-two-hour CGM using the Medtronic iPro®2 CGM and Enlite sensor was performed at baseline, 4 weeks and 16 weeks. Other metabolic parameters  and safety parameters  were measured at regular intervals.Fructosamine was measured using colorimetric test by reaction with nitroblue tetrazolium. The measuring range of the fructosamine assay was 14–1000 μmol/L, intra-assay coefficient of variations (CVs) were 0.8% and 0.5% at concentration of 275 μmol/L and 515 μmol/L, respectively, and inter-assay CVs were 1.5% and 1.2% at concentrations of 262 μmol/L and 489 μmol/L, respectively. Glycated haemoglobin was measured using immunoassay traceable to the National Glycohaemoglobin Standardisation Program and the International Federation of Clinical Chemistry standards. The measuring range of HbA1c assay was 0.3–3.4 g/dL, inter-assay CVs were 1.2% and 0.7% at concentrations of 5.3% Hb and 9.6% Hb, respectively, and inter-assay CVs were 2.2% and 1.9% at concentrations of 5.0% and 10.4% Hb, respectively. Insulin was measured by immunoassay which had a measuring range of 2–300 mIU/L with intra-assay CVs of 3.6% and 2.9% at concentrations of 11.7 mIU/L and 51.2 mIU/L, respectively, and inter-assay CVs of 6.7% and 5.3% at concentrations of 11.2 mIU/L and 47.4 mIU/L, respectively. C-peptide was measured using immunoassay which had measuring range of 0.1–20 μg/L with intra-assay CV of 2.8% and 1.7% at concentrations of 0.7 μg/L and 6.2 μg/L, respectively, and inter-assay CVs of 3.5% and 6.3% at concentrations of 0.8 μg/L and 6.3 μg/L, respectively. Glucagon-like peptide 1 was measured by enzyme-linked immunosorbent assay . The measuring range of GLP-1 was 1.25–80 pmol/L, intra-assay CVs were 9.8% and 2.2% at concentrations of 5.0 pmol/L and 7.8 pmol/L, respectively, and inter-assay CVs were 10.3% and 5.7% at concentrations of 6.1 pmol/L and 11.0 pmol/L, respectively. Glucose, total cholesterol, HDL-cholesterol and triglyceride were measured using the enzymatic colorimetric method. Insulin and C-peptide were analysed by the Siemens IMMULITE® 2000 XPi Immunoassay System, HbA1c was measured on the Roche Cobas Integra 800 System , GLP-1 was measured manually, and the rest of the assays were measured on the Roche Cobas c8000 Analytical System . All laboratory tests were performed in the Department of Chemical Pathology, the CUHK, the Prince of Wales Hospital, which was accredited by the National Association of Testing Authorities, Australia and the Royal College of Pathologists of Australasia for medical testing.All subjects completed Food Frequency Questionnaire, Hill and Blundell questionnaire on appetite, International Physical Activity Questionnaire, and World Health Organisation Quality of Life questionnaire at baseline, 4 weeks and 16 weeks.The primary endpoint was change in serum fructosamine in subjects treated with low dose and high dose BTI320 compared with placebo from baseline to 4 weeks. The main secondary endpoints were changes in calculated indices of glycaemic variability  based on CGM data in subjects treated with low dose and high dose BTI320 compared with placebo during the study. The AUCpp is the area above pre-prandial glucose starting from the beginning of each main meal to 1 h, 2 h and 3 h after the meal, obtained using the trapezoidal rule. The MPMG is the mean maximal glucose value within 3 h after each main meal. The AUC-180 is the AUC for glucose level above 180 mg/dL (10 mmol/L). The MAGE is the mean difference in glucose values between consecutive peaks and nadirs, only considering changes above and below mean glucose of more than 1 SD . The perLaboratory safety variables analyzed were renal function, liver function and complete blood counts. Self-reported adverse events including hypoglycaemic events were captured and analyzed.In our estimation of sample size, we assumed a mean serum fructosamine level of 273 μmol/L with SD of 22.5 μmol/L in the placebo arm, and a change of 10% in fructosamine level would be detected using a two-sided 5% level test with 80% power if there were 11 subjects per arm.per protocol analysis was also performed in subjects who have taken at least 70% of the treatment. Analysis of covariance (ANCOVA) was used to measure the changes in serum fructosamine, and changes in other glycaemic and metabolic indices from baseline to week 4 and week 16 between intervention arms, adjusted for age, gender and baseline measurements. The effects of low or high dose BTI320 compared with placebo on CGM glycaemic variability indices were further explored using random effect models with repeated measurements adjusted for intra-individual between-meal and between meal-day variability, age and gender. Linear mixed effect is a common statistical method to address repeated measurements [Efficacy analyses were performed in the intention-to-treat population which consisted of all randomized subjects who have received at least one dose of the assigned treatment. A urements . Post-hoper protocol analysis.A total of 77 subjects were screened and 60 subjects met eligibility for randomisation  had IFG only, 23 subjects (38.3%) had IGT only, 15 (25.0%) had both IFG and IGT, and 18 (30.0%) had normal fasting glucose and glucose tolerance but HbA1c between 5.7–6.4% (39–46 mmol/mol). Mean serum fructosamine and HbA1c were 272.1 ± 19.9 μmol/L and 6.0 ± 0.3% (42 ± 2.1 mmol/mol), respectively. Glycaemic indices were comparable among the three intervention arms at baseline (Table p = 0.57) and between high dose BTI and placebo , adjusted for gender, age, and baseline fructosamine  and − 0.1  kg in those receiving high dose BTI320 (p = 0.86)  kg in subjects receiving low dose BTI320 (2) and low (BMI < 26 kg/m2) BMI . Lastly, in subgroup analysis conducted in younger (< 59 years) and older (≥59 years) age groups, we detected reduction in AUCpp with high dose but not low dose BTI320 among younger subjects whilst reduction was observed with low dose but not high dose BTI320 in older subjects  did not reduce fructosamine levels at 4 weeks as specified in the primary endpoint but attenuated post-prandial rise in blood glucose based on CGM with modest weight loss. Future research will be required to test and confirm the glycaemic and weight effects of BTI320 in a larger sample.Additional file 1:Figure S1. Subject disposition. (DOCX 37 kb)Additional file 2:Table S1. Subgroup analysis (high and low BMI groups) on the changes in post-prandial incremental area-under-curve from baseline between low dose or high dose BTI320 and placebo using random effect models with repeated measurements adjusted for intra-individual between-meal and between meal-day variability. Table S2. Subgroup analysis  on the changes in post-prandial incremental area-under-curve from baseline between low dose or high dose BTI320 and placebo using random effect models with repeated measurements adjusted for intra-individual between-meal and between meal-day variability. Table S3. Subgroup analysis (younger and elder groups) on the changes in post-prandial incremental area-under-curve from baseline between low dose or high dose BTI320 and placebo using random effect models with repeated measurements adjusted for intra-individual between-meal and between meal-day variability. Table S4. Frequencies of gastrointestinal adverse events among subjects in low dose BTI320, high dose BTI320 and placebo groups. (DOCX 18 kb)


Codonopsis pilosula (Campanulaceae). Their structures were determined by spectroscopic data. The regulation of Sirtuin 1 (SIRT1) activity by all the isolated compounds was evaluated.Pseudolarolides U and V, two new triterpenoids, and four biogenetically related compounds, pseudolarolides E, F, K, and P were isolated from the roots of  Codonopsis pilosula, known as “Dangshen” in Chinese, has been used in traditional Chinese medicine to treat “qi” deficiency, improve appetite, and strengthen the immune system +; HRESIMS m/z 537.3183 [M + Na]+ . Pseudolarolide U (2): Yellowish gum; c 0.15, MeOH); UV (MeOH) λmax (logε): 303 (3.76), 265 (3.80), 202 (3.74) nm; 1H- and 13C-NMR data, see m/z 537 [M + Na]+; HRESIMS m/z 537.2821 [M + Na]+ . Pseudolarolide V , 100 μM of SIRT1 peptide substrate (Enzo Life Sciences), and SIRT1 assay buffer  along with the test compound at indicated concentration. The plate was incubated at 37 °C for 30 min, and the reaction was stopped using Fluor de Lys developer II solution (Enzo Life Sciences) containing 2 mM nicotinamide. The plate was further incubated at 37 °C for additional 30 min, and the samples were read by a fluorimeter with an excitation wavelength of 360 nm and an emission wavelength of 460 nm. The SIRT1 inhibitory activity of the compounds was screened using a fluorescence-based deacetylase assay . In detaC. pilosula. Although they are not active towards SIRT1, they add new facets for the chemical profiling of C. pilosula.The present study afforded two novel triterpenoids from


N-demethylation of N-methylated (both mono- and di-methylated) arginine residues of histones and other proteins, including HSP70 (heat-shock protein 70), estrogen receptor α, and RNA helicase A. Here, we report MS- and NMR-based kinetic assays employing purified JMJD6 and multiple substrate fragment sequences, the results of which support the assignment of purified JMJD6 as a lysyl hydroxylase. By contrast, we did not observe N-methyl arginyl N-demethylation with purified JMJD6. Biophysical analyses, including crystallographic analyses of JMJD6Δ344–403 in complex with iron and 2OG, supported its assignment as a lysyl hydroxylase rather than an N-methyl arginyl-demethylase. The screening results supported some, but not all, of the assigned JMJD6 substrates and identified other potential JMJD6 substrates. We envision these results will be useful in cellular and biological work on the substrates and functions of JMJD6 and in the development of selective inhibitors of human 2OG oxygenases.JmjC domain-containing protein 6 (JMJD6) is a 2-oxoglutarate (2OG)-dependent oxygenase linked to various cellular processes, including splicing regulation, histone modification, transcriptional pause release, hypoxia sensing, and cancer. JMJD6 is reported to catalyze hydroxylation of lysine residue(s) of histones, the tumor-suppressor protein p53, and splicing regulatory proteins, including u2 small nuclear ribonucleoprotein auxiliary factor 65-kDa subunit (U2AF65). JMJD6 is also reported to catalyze  A)  .N-methyl lysyl and N-methyl arginyl demethylases  (B)  (B), but the cellular relevance of this remains unclear  B 4). Jm. JmN-metaldehyde B 5). In. InN-methyde (B) B, but thC) (R hydroxylase), JMJD7 (a lysyl C-3S hydroxylase), and the ribosomal oxygenases MINA53 and NO66 (both histidine-residue C-3S hydroxylases)  . A protoC) , 9. Othexylases) –12. Manyylases) ––12. Strulases) ––, 13, altN-methyl arginyl demethylase and lysyl C-5 hydroxylase activities (C)  with a consequently associated role in apoptosis  that is characteristic of the Fe(II) and 2OG-dependent oxygenases  , a predicted AT-hook motif (Lys283–Ser326), a potential SUMOylation site (Leu316–Asp319), and a C-terminal polyserine (poly-Ser) region   –17N-methpoptosis . Subsequpoptosis , 20, altpoptosis . Structuygenases , 20, 22.ygenases , 23. JMJygenases , 25, conFig. S1) . Human JFig. S1) .et al. (N-methyl arginyl demethylase (RDM) acting on mono- and symmetric/asymmetric di-methyl forms of arginine residues as observed in studies with isolated enzyme (B). JMJD6 is also reported to catalyze RDM reactions on N-methylated forms of heat-shock protein 70-kDa (HSP70)  , estroge (HSP70) , RNA hel (HSP70) , and the (HSP70) . Althoug (HSP70) , 31, 32.C) of the splicing regulatory (SR) proteins luc-7–like 2 (LUC7L2), cisplatin resistance-associated overexpressed protein (CROP), and u2 small nuclear ribonucleoprotein auxiliary factor 65-kDa subunit (U2AF65) (S)-stereochemistry (C) (R)-product  . JMJD6-chemistry C 16), c, cC) of -product . JMJD6 i-product . More re-product .JMJD6-knockout mice dying near birth due to growth impairment of multiple organs, including heart, brain, lung, kidney, and eye , and two C-terminally truncated variants, i.e. JMJD6 comprising residues 1–362 (JMJD6Δ363–403) and residues 1–343 (JMJD6Δ344–403) . Circular dichroism (CD) spectroscopic analyses imply similar secondary structure content for each of the three JMJD6 variants .Because our objective was to investigate the catalytic reactions catalyzed by isolated JMJD6, the activity of which can be compromised due to oligomerization , 31, we 267–278 substrate . The apparent binding constant (DKapp) of 2OG was determined using CPMG-edited NMR . mKapp values for 2OG were determined using the standard hydroxylation assay procedure and were 31, 24, and 45 μm for JMJD6FL, JMJD6Δ363–403, and JMJD6Δ344–403, respectively , consistent with Mantri et al. . In the presence of the LUC7L2267–278 substrate with JMJD6Δ344–403, JMJD6Δ363–403, and JMJD6FL, these values increased to 1.8, 8.4, and 3.6 μm succinate, respectively . Thus, under both assay conditions, succinate formation for JMJD6Δ363–403 and JMJD6FL was higher than JMJD6Δ344–403, indicating a possible role of the C-terminal poly-Ser region in enhancing catalysis and/or of active protein stability. However, given the role of the poly-Ser region in oligomerization  solution being added prior to reaction, with the same outcome, i.e. with the poly-Ser region containing proteins being more active . Given that JMJD6Δ363–403 was the most active and stable variant tested, further assays were conducted with it.Hydroxylation assays using the reported LUC7L2ubstrate  revealedited NMR ; the KDai et al.  who reporization  and the Δ363–403 under standard assay conditions: H3R2(me2s)1–25, AR(me2s)TKQTARKSTGGKAPRKQLATKAA GKGGKGLGKGGAKRHRKVLRDNIQGIT GKGGKGLGKGGAKRHK-biotin . When treated with JMJD6, hydroxylation of K4histone3, K9histone3, K14histone3, K5histone4, and K8histone4 was observed , consistent with the MALDI results. Note, we did not accrue any evidence for JMJD6 catalyzed hydroxylation of arginine residues as reported for JMJD5 1–30 .Three histone peptides with symmetrically di-methylated arginine residues, all of which have been reported as JMJD6 RDM substrates , 40, werKQLATKAA ; H4R3. Furthermore, when a histone peptide monomethylated at Lys9 (H3K9me11–17) was tested with JMJD6Δ363–403, a +16-Da mass shift was observed, implying hydroxylation and not demethylation . This observation also suggests JMJD6 does not catalyze histone lysyl demethylation, consistent with an earlier report (9(me1) compared with Lys9 was relatively low , which suggests that under our assay conditions JMJD6 prefers to hydroxylate unmodified lysine residues and provides evidence that JMJD6-catalyzed lysyl hydroxylation has the potential to be affected by Nϵ lysine modifications.Evidence for JMJD6H3K9me11– was test469) is demethylated by JMJD6 as observed by MS .HSP70 has been reported as a JMJD6 substrate wherein the mono-methylated arginine residue  . Time-course assays were then performed , with peptides displaying ≥25% hydroxylation after 6 min in kinetic studies  . To inve studies .267–278 were as follows: mKapp, 51.0 μm; kcat, 106 × 10−4 s−1; and kcatm/Kapp, 208 × 10−6 μm−1·s−1. Of the tested U2AF65 peptide fragments, U2AF6521–35  was found to be a better substrate than U2AF6530–46  and U2AF6555–70  . Other peptides tested, e.g. from the SR protein CROP, gave similar kinetic parameters . Of the SRSF11 fragments tested, JMJD6Δ363–403 hydroxylated SRSF11358–372 with a relatively high efficiency . With RBM39 (RNA-binding protein 39), hydroxylation levels were found to vary with measured mKapp values ranging within 56.0–99.0 μm . Of the tested RBM39 fragments, RBM3931–42 was substantially more efficiently hydroxylated than the others tested, with mKapp 56.0 μm and kcatm/Kapp 455 × 10−6 μm−1·s−1. These results are consistent with the proposal that JMJD6 is promiscuous with respect to its lysyl hydroxylase activity in cells , suggesting possible hydroxylation of Lys171. Assays with the alanine variant pVHL166–177K171A and with WT pVHL166–177 in the presence of a JMJD6 inhibitor, 2,4-pyridine dicarboxylic acid ,BFig. S24). LC-MS/MS analysis of JMJD6Δ363–403-treated pVHL166–177 provided further evidence for pVHL Lys171 hydroxylation .The apparent promiscuity of isolated JMJD6 with RS domains of SR proteins prompted us to test the potential of other reported JMJD6 substrates. The von Hippel-Lindau protein (pVHL) has been reported as a potential JMJD6 substrate  and was N-demethylation of methylated  Arg260 of ERα and thereby regulates methylated ERα function in response to estrogen (260 (MKGGIRKDRR(me2s/a)GG) were tested with JMJD6Δ363–403, and ERα demethylation was not observed . Interestingly, a +16-Da mass shift, corresponding to hydroxylation, was observed for both peptides . In light of the results with histone peptides, we considered it possible that JMJD6 catalyzes hydroxylation of either or both of the lysine residues (Lys252 and Lys257) in the ERα sequence. Assays using ERα peptides with unmodified Arg260 also revealed JMJD6Δ363–403-dependent hydroxylation . Hydroxylation was ablated by alanine substitution ERα251–262K252A, ERα251–262K257A, or by addition of the JMJD6 inhibitor 2,4-PDCA4 . These results suggest Lys252 and Lys257 of ERα are possible targets for JMJD6 hydroxylation and are further evidence that JMJD6-catalyzed hydroxylation is possibly regulated by other post-translational modifications.Based on Western blot analysis, it has been reported that JMJD6 catalyzes estrogen . Peptide382 .JMJD6 lysyl hydroxylase activity has also been associated with post-translational modification of the tumor-suppressor protein p53, wherein the target residue is Lys382 . Assays Δ344–403 in the presence of Fe(II) and 2OG was performed in an anaerobic glove box as reported for FIH ..Δ344–403Δ344–403·Fe·2OG crystals belong to the same space group (P21) with similar unit cell dimensions to that reported by Mantri et al. (Table S1). The structure was solved by molecular replacement using PDB code 3K2O as a search model  with no Ramachandran or side-chain conformational isomer outliers ) .The JMJD6i et al.  ) . However  core fold (B) characteristic of the 2OG oxygenases, which is located within the JmjC domain and harbours the Fe- and 2OG-binding residues. Importantly, the JMJD6Δ344–403·Fe·2OG structure reveals the geometry of the JMJD6 active-site coordination chemistry in the presence of its Fe cofactor and 2OG cosubstrate , a feature characteristic of most 2OG oxygenases (Asp is sometimes substituted with Glu or Gly) (C). JMJD6 His187 and Asp189 are located on the loop linking βII and βIII, whereas His273 is located on DSBH βVII (B). Mutagenesis studies reveal the importance of the Fe-binding residues in JMJD6 catalysis . Although the JMJD6Δ363–404·D189A variant manifests apparent low-level formation of succinate in the absence of substrate , succinate formation was not observed in the presence of LUC7L2267–278 within the limits of detection , implying the D189A variant does not support productive hydroxylation catalysis. This provides further evidence that substitution of the metal-binding residues of 2OG oxygenases does not always completely block catalysis; for example, in the case of FIH, it has been reported that substitution of the Fe binding aspartate to a glycine is insufficient to completely ablate hydroxylation activity  C. JMJD6 SBH βVII B. Mutageactivity .et al. (204 (βIV) and to make hydrogen-bonding interactions with residues Tyr131 (located immediately to the N terminus of βI), Thr184 (βII), and Asn197 (βIII) (131 and Thr184 were observed in 3K2O (204 (3LDB), as reported by Hong et al. . Note in the JMJD6Δ344–403·Fe·2OG structure, the 2OG C-1 carboxylate is trans to the distal His273 of the binding triad (His273JMJD6) rather than being trans to the proximal His187 (His187JMJD6), as is also observed in FIH, JMJD5, JMJD2A, and PHF8  structures  . Similar in 3K2O , whereinK2O . This is similar to other structurally characterized JmjC domain-containing proteins (B–E) and likely contributes to the ability of JMJD6 to bind the positively charged basic groups of its substrate lysyl side chain as well as other lysyl-/arginyl-residues in the  basic JMJD6 substrates, e.g. histones and RS domains of SR proteins.Analysis of the electrostatic surface potential  of the Je charge A. This iproteins , B–E andFig. S29), Hong et al. , with basic residues involved in these interactions, including Lys4, Lys7, Arg8, Arg48, Lys91, Arg92, Arg95, Lys111, Lys115, Lys144, Lys151, Lys154, Arg305, Lys307, and Arg310 .Following the observation of ordered sulfate molecules at conserved positions across positively-charged sites on the surface of JMJD6 , which has an additional PHD, and the JMJD2 (KDM4) JmjC KDMs . HomjC KDMs , appear e.g. due to the presence of targeting domains/proteins, knowledge of the types of reaction directly catalyzed by the 2OG oxygenase domain in vitro can assist in subsequent functional assignment work in vivo. Given its checkered history , we carried out studies on reported JMJD6 N-methyl arginyl “demethylation” and lysyl “hydroxylation” substrates . In our studies, we found no evidence that purified JMJD6 has N-methyl arginyl demethylase (RDM) activity (Table S2). Positive controls for RDM activity employing KDM4E (Table S2)  . In addig et al.  have rep . Lys382 of the tumor-suppressor protein p53 is reported to be hydroxylated by JMJD6 . Peptide fragments of two other biochemically important reported targets, i.e. pVHL and ERα . Although we did not observe JMJD6-catalyzed RDM activity on ERα as was reported .In addition to multiple SR domain substrates , we founby JMJD6 , and we  and ERα , 37, werreported , hydroxypVHL is a tumor-suppressor protein in kidneys, which has a crucial role in dioxygen dependent proteasome-mediated degradation of prolyl-hydroxylated forms of α-subunits of the transcription factor HIF in many animal cells , 51, 52.6-tagged human full-length JMJD6  was inserted into the pET28a(+) expression vector. Expression plasmids for two more variants  were prepared by inserting a stop codon after the desired site using site-directed mutagenesis (see Table S3 for primer sequences). JMJD6 variants were expressed in Escherichia coli Rosetta2(DE3) cells  and stored at −80 °C. Cell pellets were resuspended in lysis buffer , DNase I , and 0.5% Nonidet P-40). The lysates were loaded onto a 5-ml HisTrap column , equilibrated with binding buffer  using an AKTA purifier . The column was washed with wash buffer . JMJD6 was eluted with elution buffer  using a linear gradient (0–100%). Based on UV-trace (280 nm) and SDS-PAGE analyses, fractions with purified JMJD6 were pooled and concentrated to 2 ml using a membrane filter (30-kDa molecular mass cutoff membrane). Concentrated protein was loaded onto a Superdex 200 size-exclusion column , pre-equilibrated with size-exclusion buffer , for further purification. The desired fractions  were concentrated to 25–30 mg/ml, then flash-frozen in liquid nitrogen, and stored at −80 °C. Catalytic mutants of the variant JMJD6Δ363–403 were prepared by site-directed mutagenesis (see Table S3 for primer sequences) and were purified by following the same purification procedure as for WT.DNA encoding for the N-terminal His3) cells , 23. Celm phosphate buffer, pH 8.0. Spectra were acquired using a quartz cuvette with a 0.1-cm path length, between 260 and 185 nm at 0.2 nm·min−1 increments, a response time of 1 s, and a data pitch of 0.5 nm. Spectra were averaged over four scans and corrected for solvent background. To investigate secondary structure content, the CD secondary structure deconvolution method was applied to the data, which were accessed using the DICHROWEB server (CD spectra were acquired using a Jasco J-815 spectrometer (Jasco). Protein solutions were buffered with 10 mB server , 54.m JMJD6, 100 μm (NH4)2Fe(SO4)2·6H2O (Sigma), and 400 μml-sodium ascorbate (Sigma) were mixed in deuterated Tris-d11 (Sigma), pH 7.5, in a 1.5-ml Eppendorf tube. Substrate (either only 500 μm 2OG or a mixture of 500 μm 2OG and 100 μm LUC7L2267–278) was added to initiate the reaction. The mixture was immediately transferred into a 2-mm NMR tube, and the acquisition of 1H spectra was started after 3.5–5 min.2OG turnover assays employed a reported procedure . 10 μm Jm 2OG and 200 μm Zn(II) were mixed in deuterated Tris-d11, pH 7.5, and the 1H NMR signals for 2OG were recorded at 700 MHz (DKapp) were measured using obs represents change in an observable NMR parameter from the titration; Δtotal represents the total change of the monitored NMR parameter; [L]0 represents the titrated ligand concentration; and [P]0 represents the protein concentration. Typically, a 5–10% error was allowed in the curve-fitting process.20 μ 700 MHz . Titratix − (τ − 180°y − τ − 90°y − τ − 180°y − τ)n − acquisition) was applied (n = 6); acquisition time 2.94 s; relaxation delay 2 s; number of transients 128. Water suppression was achieved by presaturation 2Fe(SO4)2·6H2O (Sigma), 400 μml-sodium ascorbate (Sigma), and 500 μm disodium 2OG (Fluka). After 30 min, the reaction was quenched by an equal volume of 1% (v/v) aqueous formic acid (Sigma). In a 96-well MALDI plate (Waters), 0.8 μl of CHCA  (1 μg in 0.1 ml of CHCA solvent) and 0.6 μl of the quenched mixture were placed in a well. After drying, the samples were analyzed by MALDI-TOF MS using a reported procedure 2Fe(SO4)2·6H2O (Sigma), 500 μml-sodium ascorbate (Sigma), and 1 mm di-sodium salt of 2OG (Fluka) for 1 h or more  and subsequent CID MS/MS spectra  of the 10 most intense peaks were acquired in the Orbitrap. CID fragmentation was performed at 35% of the normalized collision energy, and the signal intensity threshold was kept at 500 counts. ETD spectra  were acquired in the ion trap. PEAKS® 8.0  was used to analyze raw data. The raw MS files were searched against the respective protein sequences. NONE was selected as the protease. Demethylation (−14 and −28 Da) and hydroxylation  were set as variable modifications. The precursor mass tolerance was 15 ppm. The fragment mass tolerances for CID and ETD were set to 0.8 Da. All spectral assignments were manually validated.Samples were desalted using a ZipTip (Millipore) and then analyzed using a NanoAcquity-ultraperformance liquid chromatography system (Waters) connected to an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific), possessing an EASY-Spray nano-electrospray ion source (Thermo Fisher Scientific). Peptides were “trapped” on an in-house packed guard column  using solvent A (0.1% (v/v) aqueous formic acid) at a pressure of 140 bar. Peptides were separated on an EASY-Spray Acclaim PepMap® analytical column  using a linear gradient , flow rate, 300 nl/min). The separated peptides were electro-sprayed directly into the spectrometer operating in a data-dependent mode using a collision-induced dissociation/electron transfer dissociation (CID/ETD)-based method. Full scan MS spectra  at a concentration of ≥25 mg/ml (500–600 μm) was used. JMJD6Δ344–403·metal·ligand complexes were formed by incubating enzyme with the other components  on ice for ∼1 h. A PhoenixTM RE crystallization robot (Art Robbins Instruments) and a Minstrel-HTTM platform with CrystalTrekTM  were used to prepare and monitor crystallization trials. Crystals were grown using the sitting-drop vapor-diffusion method . Crystals were cryo-protected using a mixture of reservoir solution diluted to 25% (v/v) glycerol and cryo-cooled by plunging into liquid nitrogen. Diffraction data were collected at 100 K at the Diamond Light Source synchrotron. PHASER was used for molecular replacement using PDB code 3K2O (Crystallization trials were performed using commercially available broad screens followed by optimization. JMJD6n method  either in method . The optode 3K2O  as the sode 3K2O , 63.Δ344–403·Fe·2OG structure are deposited in the Protein Data Bank as code 6GDY.Coordinates and structure factors for the JMJD6M. S. I., M. A. M., R. C., and C. J. S. conceptualization; M. S. I. and M. A. M. data curation; M. S. I. and M. A. M. software; M. S. I. and M. A. M. formal analysis; M. S. I. and C. J. S. funding acquisition; M. S. I. and M. A. M. validation; M. S. I., J. G., A. K., and E. P. investigation; M. S. I. and M. A. M. visualization; M. S. I. methodology; M. S. I., M. A. M., and C. J. S. writing-original draft; M. A. M., R. C., and C. J. S. supervision; M. A. M. and C. J. S. project administration; M. S. I., M. A. M. and C. J. S. writing-review and editing; C. J. S. resources.


Each monomer was polymerized with p-phenylenediamine and 4,4′-diaminodiphenyl ether to yield six poly(azomethine)s. Single crystal X-ray diffraction structures of 3b and 3c were determined. The structural characterization of the monomers and poly(azomethine)s was performed by FT-IR and NMR spectroscopic techniques and elemental analysis. Physicochemical properties of polymers were investigated by powder X-ray diffraction, thermogravimetric analysis (TGA), viscometry, UV–vis, spectroscopy and photoluminescence. These polymers were subjected to electrical conductivity measurements by the four-probe method, and their conductivities were found to be in the range 4.0 × 10−5 to 6.4 × 10−5 Scm−1, which was significantly higher than the values reported so far.Bis-aldehyde monomers 4-(4′-formyl-phenoxy)benzaldehyde ( Conjugated organic semiconducting polymers have emerged as new materials having potential applications in electronics, opto-electronics, and photonics ,2,3. PolThe imine bond (–CH=N–) is isoelectronic with the vinylene bond (–CH=CH–) ,5,11,19,On the other hand, synthesis of polyazomethines might be catalyzed with mineral or organic acids, and the product is purified by a simple precipitation method. The conjugated polyazomethines are widely used as a counterpart for currently employed conjugated structures in optoelectronic devices  such as As for other many conjugated polymers, various properties of polyazomethines, including conductivity, can be improved by suitable doping or protonating processes . Over thTherefore, the conductivity of wholly aromatic pristine polyazomethines is being reported by us for the first time. Herein, we report the syntheses of three bis-aldehyde monomers and their polymers. The spectroscopic characterizations accompanied by single crystal X-ray diffraction profiles are performed. The electrical conductivity is measured by using pelletized polymer samples. The expected structure–property relationship has been explained.2CO3 anhydrous , 4,4′-diaminodiphenyl ether , 1,4-phenylenediamine , and p-toluene sulfonic acid monohydrate (PTSA) Fluka  were purchased from commercial suppliers. 4-Fluorobenzaldehyde (98%), 4-hydroxybenzaldehyde , 3-methoxy-4-hydroxybenzaldehyde (m.p = 158–159 °C), and 3-ethoxy-4-hydroxybenzaldehyde (melting point = 160–163 °C) were obtained from Sigma Aldrich. The solvents n-hexane, dimethylsulfoxide, and diethyl ether were purchased from Merck , whereas ethylacetate, chloroform, and toluene were obtained from Panreac . The methanol, ethanol, and N,N-dimethylformamide were purchased from Sigma Aldrich, whereas hydrochloric acid and sulfuric acid were obtained from Riedel-de-Haen .The chemicals K3), dimethylformamide (DMF), and sulfuric acid. Thermogravimetric analyses (TGAs) were performed on a Perkin Elmer TGA instrument v.7 at heating rates of 10 and 20 °C/min in nitrogen or air up to a maximum 650–950 °C. Photoluminescence spectra of the polyazomethines were measured on a Perkin Elmer LS 55 luminescence instrument using a single glass cell. The viscosities of polyazomethines were measured at room temperature using a U-tube Ubbelhode viscometer with 20 mL of the polymer solutions. Wide-angle X-ray diffraction experiments of the powdered polymers samples were performed using Philips 3040/60 X′Pert Pro diffractometer having a Cu anode with a Kα radiation source. The electrical conductivity (σ) of polyazomethines was measured with a Keithley source meter 2400. The crystal structure data of the monomers were collected using an Oxford diffraction diffractometer (Mo radiation), structures using CrysAlisPro, and were solved and refined using the SHELXS/SHELXL14 programs and diagrams prepared by using the Mercury program. The crystal structure analysis was performed using the PLATON package and the OSCAIL suite of programs [The melting points of the synthesized aromatic bis-aldehyde monomers were determined using a Mel-Temp.  apparatus with open capillary tubes. Fourier transform infrared (FTIR) spectroscopy was performed using a Perkin Elmer 1600 series FTIR spectrophotometer . Nuclear magnetic resonance (NMR) measurements were carried out using Bruker Avance 300 digital NMR  in different solvents depending on the solubility of the samples. Tetramethylsilane was used as an internal standard. Elemental analyses were performed on a Vaio-EL instrument. The UV–visible spectra of azomethine polymers were measured on Schimadzu-1700 UV using chloroform , and stirring continued for a further 1 h. The reaction mixture was heated to reflux under a nitrogen atmosphere at 120 °C for 24 h. When the reaction completed, as indicated by TLC, the reaction mixture was poured into 200 mL of ice-cooled water, and the base was neutralized with 10% HCl solution. The precipitates were allowed to stand for 40 min, then filtered with filter paper through gentle suction. Later on, the products were dried in the oven at 40 °C before recrystallization with CHCl3/ethylacetate. After recrystallization, the precipitates were isolated for analysis and further polymerization.The syntheses of three monomers, 1) and 4-hydroxybenzaldehyde (2a) to form crystalline solid 3a, yield 90%, m.p 55–60 °C. IR (KBr): υ(Ar–H str.), 3065 cm−1; υ, 2819, 2731 cm−1; υ(C=O str.), 1675 cm−1; υ(aromatic C=C), 1581, 1494 cm−1; υ(C–O–C ether), 1236 cm−1. 1H NMR  δ 9.99 , 7.94 , 7.19 . 13C NMR  δ 190.64 (s), 161.01 (s), 132.34 , 119.37 (s). Elem. Anal. Calc.: C14H10O3: C, 74.33; H, 4.46; O, 21.22. Found: C, 75.08; H, 4.93%.The same experimental conditions were used as described above for coupling of 4-fluorobenzaldehyde  and 4-fluorobenzaldehyde (1) to yield crystalline solid with 88% yield, and m.p 67–68 °C. IR (KBr): υ(Ar–H str.), 3066 cm−1; υ, 2978, 2928 cm−1; υ, 2860, 2759 cm−1; υ(C=O str.), 1680 cm−1; υ(aromatic C=C), 1581, 1497 cm−1; υ(C–O–C ether), 1235, 1208 cm−1. 1H NMR  δ 9.98 , 9.95 , 7.88 , 7.58 , 7.52 , 7.20 , 7.07 , 3.90 . 13C NMR  δ 190.74 (s), 162.02 (s), 152.00 (s), 148.95 (s), 134.15 (s), 131.96 (s), 125.45 (s), 121.40 (s), 117.41 (s), 111.25 (s), 56.11 (s). Elem. Anal. Calc.: C15H12O4: C, 70.31; H, 4.72; O, 24.97. Found: C, 71.05; H, 4.24%.The bis-aldehyde 15H12O4, M = 256.25, orthorhombic, space group Pbcn, a = 18.4038(11), b = 8.6240(5), c = 15.7283(11) Å, V = 2496.3(3) Å3, Z = 8, Z′ = 1, R-factor (%) 5.30, 12.5. The Oak Ridge Thermal Ellipsoid Plot (ORTEP) diagram of 3b is depicted in C3c monomer was also synthesized by the above protocol using 3-ethoxy-4-hydroxybenzaldehyde (2c) and 4-fluorobenzaldehyde (1) to yield crystalline solid with 85% yield and m.p 68–70 °C. IR (KBr): υ(Ar–H str.), 3063 cm−1; υ, 2973, 2930 cm−1; υ, 2828, 2736 cm−1; υ(C=O str.), 1687 cm−1; υ(aromatic C=C), 1577, 1497 cm−1; υ(C–O–C ether), 1268, 1227 cm−1. 1H NMR  δ 9.96 , 9.95 , 7.87 , 7.54 , 7.50 , 7.22 , 7.06 , 4.11 , 1.27 . 13C NMR  δ 190.90 (s), 190.76 (s), 162.37 (s), 151.31 (s), 149.08 (s), 134.20 (s), 131.82 (s), 125.19 (s), 121.79 (s), 117.28 (s), 112.40 (s), 64.64 (s), 14.40 (s). Elem. Anal. Calc.: C16H14O4: C, 71.10; H, 5.22; O, 23.68. Found: C, 70.23; H, 4.37%.The 16H16O4, wM = 270.27, monoclinic, space group P21/c, a = 8.8926(7), b = 19.2717(11), c = 8.0635(6) Å, V = 1366.93(17) Å3, Z = 4, Z′ = 1, R-factor (%) 7.80, 24.0. The crystal exhibited twinning and aldehyde disorder in one of the aldehyde CHO groups and, therefore, was treated using the CrysAlisPro software and the SHELXL14 refinement programs.CIn 2 atmosphere by solution polycondensation. A typical procedure is as follows: All polyazomethines derived from aromatic dialdehydes were synthesized under Np-Toluenesulfonic acid (TsOH) monohydrate was added in a catalytic amount in the reaction mixture. After that, the equimolar diamine  solution in 10 mL DMF was added dropwise with continuous stirring. The reaction was distilled for azeotropic water removal with toluene using a Dean–Stark trapper. After 6 h reflux, TLC was carried out to check the presence of for any residual monomers, using CHCl3 eluent. The precipitated product was directly poured onto 300 mL of ice-cold water. It was filtered, washed with methanol, and dried at 40 °C for 6 h. The dialdehydic monomers  were dissolved in 20 mL of DMF and 10 mL of toluene. 2O. Then, the product was filtered, water washed, recrystallized from ethanol/chloroform, and subsequently dried at 40 °C for 6 h.On the other hand, if the polymers were soluble in DMF, then upon reaction completion, excess DMF was reduced to half by rotary evaporation followed by precipitation in ice cold H3a, 3b, and 3c were conveniently synthesized in the presence of anhydrous K2CO3 in DMF solvent by reaction of 4-fluorobenzaldehyde (1) with 4-hydroxybenzaldehyde (2a), 3-methoxy-4-hydroxybenzaldehyde (2b), and 3-ethoxy-4-hydroxy-benzaldehyde (2c), respectively  were found to be in accordance with the proposed structures. 3b. The resonance lines for protons integrating 2H at δ 9.98 and 9.95 ppm were assigned to the aldehydic protons of two aromatic rings because of their different electronic environment. The two doublets at δ 7.88 and 7.07 ppm each with a coupling constant 8.6 Hz were assigned to the symmetrically substituted benzene ring  were synthesized by solution phase polycondensation under N2 atmosphere. Bis-aldehyde monomer (0.1 mmol) was dissolved in 20 mL of dried DMF and 10 mL of dried toluene. Subsequently, p-toluenesulfonic acid monohydrate (PTSA) was added in catalytic amount (20 mg). Then, the diamine (0.1 mmol) solution in 10 mL of DMF was added dropwise with continuous stirring. The reaction was gently heated under reflux conditions for 6 h. Then, the product was directly precipitated by pouring it onto 300 mL of ice-cold water. Afterward, the product was filtered, washed with MeOH, and dried at 40 °C for 6 h before further characterization. The synthetic scheme of polyazomethines is shown in 1H NMR data.All polyazomethines (P-I: FT-IR (KBr): υ(end group HC=O str.), 1691 cm−1; υ(C=N str.), 1620 cm−1; υ(aromatic C=C), 1591, 1503 cm−1; υ(C–O str.), 1257 cm−1; υ(aromatic ring), 855 cm−1. 1H NMR : 8.24 , 7.93–6.70 .P-II: FT-IT (KBr): υ(end group HC=O), 1684 cm−1; υ(C=N str.), 1606 cm−1; υ(aromatic C=C), 1584, 1491 cm−1; υ(C–O str.), 1221, 1150 cm−1; υ(aromatic ring), 823 cm−1. 1H NMR : 8.31 , 7.61–6.67 .P-III: FT-IT (KBr): υ(merged peak of HC=O and C=N str.), 1691–1669 cm−1; υ(aromatic C=C), 1591, 1499 cm−1; υ(C–O str.), 1214, 1157, 1122 cm−1; υ(aromatic ring), 816 cm−1. 1H NMR : 8.43 , 7.62–6.73 .P-IV: FT-IT (KBr): υ(end group HC=O), 1684 cm−1; υ(C=N str.), 1626 cm−1; υ(aromatic C=C), 1585, 1506 cm−1; υ(C–O str.), 1214 cm−1; υ(aromatic ring), 822 cm−1. 1H NMR : 8.32 , 7.92–7.07 .P-V: FT-IT (KBr): υ(end group HC=O), 1691 cm−1; υ(C=N str.), 1620 cm−1; υ(aromatic C=C), 1581, 1491 cm−1; υ(C–O str.), 1229, 1150 cm−1; υ(aromatic ring), 829 cm−1. 1H NMR : 8.33 , 7.60–6.49 , 3.54 .P-VI: FT-IT (KBr): υ(merged peak of HC=O and C=N str.), 1699–1676 cm−1; υ(aromatic C=C), 1584, 1498 cm−1; υ(C–O str.), 1221, 1150 cm−1; υ(aromatic ring), 843 cm−1. 1H NMR : 8.25 , 7.97–6.71 .−1) were ascribed to –CH=N– bonds of azomethine. Similarly, the absence of –NH2 absorption peaks also had additive effects to the aforesaid formation of polymers. Introduction of ethoxy side groups into the polyimine structure shifted the azomethine peak toward higher wavenumbers, as indicated by the broad merged peaks for P-III and P-VI, which showed that the end group HC=O and CH=N bond stretching frequencies overlapped, whereas the methoxy side groups did not affect the imine peaks in comparison to the structure containing no side groups.The disappearance and/or only weak absorption peaks of the carbonyl group of aldehydes confirmed the synthesis of polymers. The weak absorptions were due to the carbonyl end groups on the macrochains. This infers that the aldehydic carbonyl groups were utilized in the successful formation of azomethine linkages. On the other hand, the presence of medium intensity absorption peaks in the range of υ  showed the highest crystallinity, those synthesized from 3b  showed little or no crystallinity, whereas those synthesized from 3c  showed a totally amorphous nature. This clearly shows the role of symmetry of the polymeric macrochain in determining the semicrystallinity of the polymer. The polymers of 3b and 3c having –OCH3 and –OC2H5 side groups to the main chain hindered their close packing and, thus, made them amorphous.Powdered X-ray diffraction is the most important technique to determine the degree of crystallinity in polymers. The intensities, peak positions, shapes, and widths give important information about the structure of the polymeric material. The X-ray diffraction profiles of powdered polyazomethines samples were conducted at room temperature to get information about the semicrystallinity of polyazomethines. The X-ray diffraction patterns of  reports ,42. The 3  were shown in terms of wavelength and occurred in the range from 54 to 92 nm, indicating an energy decline during electronic transitions. This energy loss can be due to the excimer formation as demonstrated by the bathochromic effect and enlargement of the emission bands (see Photoluminescence (PL) measurements d were peands see d.Ton, T20%, Tmax, and TF in degrees, and percent char yield showing onset of degradation, 20% weight loss, maximum degradation temperature, and final degradation temperatures, respectively. The onset temperature of degradation (Ton) for these polymers was in the range from 150 to 450 °C. The maximum stability to initial heating was shown by polymers synthesized from monomer 3a  viz 300 and 450 °C and minimum stability showed by polymers synthesized from 3c having the –OC2H5 side group. The maximum degradation temperature (Tmax) for polyazomethines was between the range of 475 and 650 °C. The high thermal stability of these polymers can be ascribed to the rigidity of the monomers and effective staking of aromatic moieties in macrochains.The thermal stability of the polyazomethines was investigated by thermogravimetric analysis (TGA), and the data are tabulated in r) = 0.625 cm, area (A) = 1.22718 cm2, and distance between two probes (L) = 0.3 cm.The electrical conductivity measurements of the synthesized pristine polyazomethines were measured in air atmosphere in solid state on a Keithley 2400 instrument using pelletized powdered samples. The pellets of the polymer samples were prepared by pressing the powdered sample for 5 min at 4.9 metric ton pressure using a hydraulic press. Electrical connections on pellets were made with Cu wires, which were abraded with fine grit silicon carbide sandpaper, and placed equidistant from each other. The Cu connections on pellets were fixed with a conducting paste of silver. The current (ranging 1–3 µA) was forced through the two points of the probe, labelled as 1 and 4, and measured the voltage (V) as the output signal through points 2 and 3 . Subsequ−11 Scm−1, while those of the doped samples with iodine were 10−6 Scm−1 [−5 to 6.4 × 10−5 Scm−1, which is ten times higher than the conductivities of any pristine polyazomethines reported so far. Although the effect of doping on the electric properties of polyazomethines have been presented in numerous papers, according to our best knowledge, no one has reported the conductivity of undoped polyazomethines as high as 6.4 × 10−5 Scm−1. The low conductivity values were attributed to the low degree of conjugation in the polymers caused by nonplanarity of the polymer chains. But in actual cases, the nonplanarity of the CH=N bond is not the cause of low conductivity, as was demonstrated by our synthesized polymers—they showed conductivity in the 10−5 Scm−1 range. Rather, the low conductivity was caused by different unconjugated aliphatic spacers. And when the polymers were doped with iodine in the doping process, the electron emitting imine nitrogen and the electron pulling iodine coordinated to form a radical cation (polaron) structure. The electron vacancy formed because this polaron facilitates the electron transfer from one chain to another, thus causing an increase in the electrical conductivity. Still, the maximum conductivity of iodine-doped polymers is reported to be 10−6 Scm−1 [P-III has the highest conductivity value of 6.4 × 10−5 Scm−1, which probably is due the straight pathway of electronic conduction provided by azomethine linkages between 3c and PP monomers. In general, the electrical conductivities of the polymers synthesized from the PP monomer were higher than those synthesized from DA. The lower values of conductivities of polymers of DA may be ascribed to the presence of an additional ether linkage, which is less conductive as compared to the azomethine group. Overall, the conductivities of all the polymers were comparable to each other, which indicate the same electronic environment in all these polymers.The electrical conductivities of the undoped polyimines have been reported to be 10−6 Scm−1 ,44. Howe−6 Scm−1 ,34,44,45−6 Scm−1 , which s1H NMR, FT-IR, UV–vis spectroscopy, and photoluminescence. The results suggest that although the solubility of polymers might slightly hinder their applications, they showed good optical properties. The blue light emitting polymers could be structurally engineered to use in optoelectronics. The polyazomethines exhibited lower band gaps, good emission properties, and high strokes shift values, so they can be used in the preparation of fluorescence sensor material. Wide-angle X-ray diffraction showed their semicrystallinity, while thermal analysis showed good thermal resistance of all polyazomethines. High percent char residue in thermogravimetric analysis is suggestive that the synthesized polyimines have an excellent thermal stability and can be used as potential candidates for high-temperature applications. It was observed that the conductivities of polyazomethines in the solid state were from 4.0 × 10−5 to 6.4 × 10−5. The semiconducting polyazomethines might be used in electronic, optoelectronic, and photovoltaic applications. We believe that these conjugated polyazomethines will be key functional materials for future applications. In this paper, we have presented three monomers  and six wholly aromatic polyazomethines, which were tested in pristine form for their electrical conductivity. Methods of synthesis and spectroscopic characterizations of monomers and polyazomethines have been discussed. Synthesis of each polymer was conducted via polycondensation in solution form. The synthesized polymers were orange-yellow or dark brown-yellow powders. Obtained polymers were investigated by


Recently, we reported that PM2.5 induced cellular macromolecule disruption of lipids, proteins, and DNA, via reactive oxygen species, eventually causing cellular apoptosis of human keratinocytes. In this study, the ethanol extract of Cornus officinalis fruit (EECF) showed anti-oxidant effect against PM2.5-induced cellular oxidative stress. EECF protected cells against PM2.5-induced DNA damage, lipid peroxidation, and protein carbonylation. PM2.5 up-regulated intracellular and mitochondrial Ca2+ levels excessively, which led to mitochondrial depolarization and cellular apoptosis. However, EECF suppressed the PM2.5-induced excessive Ca2+ accumulation and inhibited apoptosis. The data confirmed that EECF greatly protected human HaCaT keratinocytes from PM2.5-induced oxidative stress.The skin is one of the large organs in the human body and the most exposed to outdoor contaminants such as particulate matter < 2.5 µm (PM Particularly, iridoid glycosides are one of the active ingredients in the C. officinalis fruit 2.5-induced oxidative stress in human keratinocytes. Therefore, this study was conducted to investigate the potential of EECF to cure the PM2.5-induced cell damage.There are more than 65 species classified under the genus C. officinalis collected from an area around the city of Gurye , were provided by Gurye Sansuyu Farming Association Corporation. For the preparation of EECF, the dried fruit (20 g) were cut into small pieces and extracted three times with 400 mL 70% ethanol at 4°C for 3 h. After filtering, the filtrate was concentrated using a vacuum rotary evaporator . The residue was then freeze-dried using a freeze dryer and stored at -80°C. The powder (EECF) was dissolved in dimethyl sulfoxide  to obtain a final concentration of 100 mg/mL (extract stock solution), and was stored at 4°C. The stock solution was diluted with culture medium to the desired concentrations prior to use. EECF was dissolved in DMSO. Diesel PM2.5  was purchased from Sigma‑Aldrich Chemical Co. and was dissolved in DMSO to prepare the stock solution (25 mg/mL). To avoid agglomeration of the suspended PM2.5, the solution was sonicated for 30 min The dried fruit of 2.The human HaCaT keratinocytes  were cultured in Dulbecco's modified Eagle's medium . The medium was supplemented with antibiotic solution consisting of 100 units/mL penicillin, 100 µg/mL streptomycin, and 0.25 µg/mL amphotericin B . In addition, the medium was supplemented with 10% fetal bovine serum. The cultured cells were incubated in a 100% humidified atmosphere at 37°C with 5% CO5 cells per well and specific wells were separately treated with EECF at final concentrations of 25, 50, 100, 200, 300, 400, and 500 µg/mL. The MTT stock solution (2 mg/mL) was incubated with the cells for 4 h until formazan crystals were formed. The crystals were then dissolved in DMSO and the absorbance of the reaction solution was detected using a multi-well spectrophotometer at a wavelength of 540 nm.The cytotoxicity of the EECF on HaCaT cells was measured using the 3--2,5-diphenyl tetrazolium bromide (MTT) assay. Cells were cultured in a 96-well plate at a density of 1.0 × 10EECF  was mixed with 0.15 mM 2,2-diphenyl-1-picrylhydrazyl (DPPH), shaken gently, and kept in the dark for 3 h. The residual DPPH was determined at 520 nm using a spectrophotometer.5 cell density and intracellular ROS levels those generated via 1 mM hydrogen peroxide (H2O2), were measured using the 2',7'-dichlorofluorescein diacetate  assay. Cells were seeded on chamber slides at a 1.5 × 105 cell density and incubated with PM2.5 (50 µg/mL) for 1 h. The cells were stained with DCF-DA for 30 min and the fluorescence emission was detected using confocal microscopy .Cells were seeded in 96 well palate at a 1.5 × 10Superoxide anion was generated though the reaction between 5,5-dimethylpyrroline-N-oxide (DMPO) and the xanthine/xanthine oxidase system. The generated DMPO/·OOH adduct was detected using electron spin resonance (ESR). Then, 20 µL xanthine oxidase (0.25 U/mL) was mixed with 20 µL each of xanthine (10 mM), EECF (200 µg/mL), and 3 M DMPO, and after 2.5 min, the ESR signaling was measured. ESR spectrophotometer settings were set as follows: power, 1.00 mW; central magnetic field, 336.8 mT; frequency, 9.4380 GHz; amplitude, 600; modulation width, 0.2 mT; sweep width, 10 mT; sweep time, 30 sec; gain, 500; time constant, 0.03 sec; temperature, 25°C 2.5 (50 µg/mL) for 24 h and staining with diphenyl-1-pyrenylphosphine (DPPP) for 30 min in the dark. Images were analyzed using a confocal microscope A four-well chamber slide was used to plate the cells in the presence of 200 µg/mL EECF, followed by exposure to PM2.5 (50 µg/mL) for 24 h. Protein oxidation was assessed using an OxiSelectTM protein carbonyl enzyme-linked immunosorbent assay kit  according to the manufacturer's instructions.Cells were incubated with 200 µg/mL EECF for 1 h and treated with PM2.5 (50 µg/mL) for another 30 min. After coating with 110 µL 0.7% low-melting agarose, the cells were immersed in lysis buffer  for 1 h at 4°C. An electrical field  was used for electrophoresis. Slides were stained with 40 µL ethidium bromide (10 µg/mL) and analyzed using the comet 5.5 image analyzer . The percentage total fluorescence and tail lengths were recorded (50 cells per slide).Cells were seeded in the medium with 200 µg/mL EECF in a 1 mL micro tube for 30 min and treated with PM5 cell density and images of fluorescence-visualized cells were captured using a confocal microscope ROS-induced DNA damage was assessed using the avidin-tetramethylrhodamine isothiocyanate  conjugate (Sigma-Aldrich) assay, based on fluorescence-binding activity. Initially, the cells were fixed on chamber slides at a 1.5 × 102+ level and mitochondrial Ca2+, respectively. Fluorescence was measured using confocal microscopy Treated cells were loaded with 10 µM fluoro-4-acetoxymethyl ester (Fluo-4-AM) or rhod-2-acetoxymethyl ester  for 30 min to detect intracellular Ca5 cells. After treatment with EECF (200 µg/mL), cells were exposed to PM2.5 (50 µg/mL) for 24 h, stained with 5 μM 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide , and analyzed using confocal microscopy.Cells were seeded on chamber slides at a density of 1.5 × 10Harvested cells were lysed using 150 μL of protein lysis buffer and the collected cell lysates were centrifuged at 13,000 rpm for 5 min. The resulting suspensions were collected and protein levels were analyzed as previously described 2.5 (50 µg/mL) for 18 h. The cells were stained with Hoechst 33342 (20 µM) and DNA-specific fluorescence was visualized using a fluorescence microscope. Nuclear condensation levels were evaluated and quantified for the apoptotic cells.Cells were treated with 200 µg/mL EECF for 1 h, followed by PMP < 0.05 was considered statistically significant.All experiments were performed in triplicate. Data are presented as the means ± standard error and were analyzed using the Sigma Stat 3.5 version software  using Tukey's test and analysis of variance (ANOVA). A 2O2, respectively) scavenging activity and, therefore, 200 µg/mL was selected as the optimal concentration for further experiments  and B-cell lymphoma-2-associated X protein (Bax) and decreased the expression of B-cell lymphoma-2 (Bcl-2)  Figure A. This fl Figure B.2.5 is considered an air pollutant, which has harmful effects on the skin, such as skin aging and inflammatory skin diseases, mediated by the generation of intracellular ROS C. officinalis consists of 11 highly polar compounds, particularly, iridoid isomers  C. officinalis fruit; however, their content could vary with the state of the fruit, depending on whether they are processed or crude. Particularly, loganin possesses immune-regulatory and anti-inflammatory activities, while morroniside is involved in the prevention of diabetic angiopathy , and ameliorated intracellular ROS generation, as revealed by DCF-DA staining  2.5-induced lipid peroxidation (Figure 2.5-induced DNA damage. EECF significantly attenuated the DNA strand breaking and at 200 µg/mL, also reduced the elevated 8-oxoG level in PM2.5-treated cells.PM2+ accumulation, while the endoplasmic reticulum releases Ca2+. As previously reported, ROS degrade the Δψm2.5-induced excessive Ca2+ accumulation in the cell and mitochondria. This effect restored cellular Ca2+ homeostasis and EECF restored the Δψm. In conclusion, our results confirmed that EECF has considerable antioxidant activity against PM2.5-induced skin damage.A previous study reported that cellular oxidative stress causes mitochondria stress, which eventually results in cell apoptosis


The effect of two anti-fungal treatments  on the color and phenolic profile of Tempranillo and Graciano red wines has been studied. To evaluate possible modifications in color and phenolic composition of wines, control and wines elaborated with treated grapes under good agricultural practices were analyzed. Color was assessed by Glories and CIELab parameters. Color changes were observed for treated wines with boscalid + kresoxim-methyl, leading to the production of wines with less color vividness. Phenolic profile was characterized by HPLC analysis. Boscalid + kresoxim-methyl treatment promoted the greatest decrease on the phenolic content in wines. Botrytis cinerea), powdery mildew (Uncinula necator) and downy mildew (Plasmopara viticola). Although practicing different traditional techniques it can be possible to minimize the incidence of these fungi during cultivation of the grape, the most effective means to combat them is the application of fungicides. With time, fungi can develop resistance to the most frequently applied fungicides, making it necessary to replace the traditionally used fungicides by others that include new generation active substances or new fungicides  × 100% copigmented = [(A20 − ASO2)/Aacet] × 100% monomeric = − ion at m/z 859. MS/MS fragmentation of m/z 429 produced a daughter ion at m/z 303 [M−H−C6H6O3]−, which was indicative for a loss of phloroglucinol (126 Da) and the retro-Diels-Alder (RDA) product at m/z 261 [M−H−C8H8O4]−. The MS analysis of catechin and epicatechin adducts showed a [M−H]− ion at m/z 413 and a [2M−H]− ion at m/z 827. MS/MS fragmentation product ions of m/z 413 were detected at m/z 287 [M−H−C6H6O3]− (loss of phloroglucinol) and at m/z 261 [M−H−C8H8O3]− (RDA fission).Due to the lack of the corresponding standards, extension subunits, nd Jones . In any Anthocyanins. Ethanolic extract (20 µL) was injected into the column and eluted at 35 °C. Mobile phase A and B were 5% formic acid aqueous solution and methanol, respectively, and the flow rate was 1 mL∙min−1. The following linear gradient was used: 0–5 min, 90% A and 10% B; 15 min, 80% A and 20% B; 30 min, 70% A and 30% B; 40–85 min, 68% A and 32% B; 87 min, 60% A and 40% B; 96 min, 50% A and 50% B; 98–108 min, 5% A and 95% B; 110–120 min, 90% A and 10% B. DAD chromatograms were registered at 520 nm.Phenolic acids and resveratrol. Ethanolic extract (20 µL) was injected into the column and eluted at 35 °C. Mobile phase A and B were 0.2% formic acid aqueous solution and methanol, respectively, and the flow rate was 0.8 mL∙min−1. The following linear gradient was used: 0 min, 97% A and 3% B; 40 min, 70% A and 30% B; 50–53 min, 50% A and 50% B; 55–65 min, 5% A and 95% B; 67–77 min, 97% A and 3% B. DAD detection wavelengths of 280, 320 and 309 nm were selected for phenolic acids, hydroxycinnamic acids and resveratrol, respectively.Flavonols. Ethanolic extract (20 µL) was injected into the column and eluted at 35 °C. Mobile phase A and B were 2.5% formic acid aqueous solution and methanol, respectively, and the flow rate was 1 mL∙min−1. The following linear gradient was used: 0 min, 80% A and 20% B; 10 min, 75% A and 25% B; 30 min, 65% A and 35% B; 40–42 min, 60% A and 40% B; 45 min, 50% A and 50% B, 48 min, 40% A and 60% B; 50–60 min, 5% A and 95% B; 62–72 min, 80% A and 20% B. DAD chromatograms were registered at 370 nm.t-student test. Data analyses were performed using the STATGRAPHICS Centurion XV 15.2.05 Software . For sample comparison, the data are presented as means ± standard deviation (SD) of analyses performed in triplicate. Significant differences among treated wines and control wines for each variety and compound were assessed by the ab parameter confirmed that these CIELab changes could be perceived by wine consumers. Although significant differences were observed for all determined phenolic compounds in wines, for both treatments and varieties, the results showed again that G and T wines obtained from grapes treated with boscalid + kresoxim-methyl were the most affected.Results showed that the wines obtained from grapes treated under good agricultural practices with boscalid + kresoxim-methyl had lower chroma and higher hue than control wines, resulting in less colorful wines. The ΔE*


N-[8-(2-hydroxybenzoyl)amino]caprylate (SNAC) is a new kind of absorption enhancer that has good safety. To investigate the absorption enhancement effect of SNAC on non-polar charged and polar charged drugs and establish the absorption enhancement mechanism of SNAC, SNAC was synthesized and characterized. Two representative hydrophilic drugs—notoginsenoside R1 (R1) and salvianolic acids (SAs)—were selected as model drugs. In vitro Caco-2 cells transport and in vivo rat pharmacokinetics studies were conducted to examine the permeation effect of SNAC on R1 and SAs. R1, rosmarinic acid (RA), salvianolic acid B (SA-B) and salvianolic acid B (SA-A) were determined to compare the permeation enhancement of different drugs. The MTT assay results showed that SNAC had no toxicity to Caco-2 cells. The transepithelial electrical resistance (TEER) of Caco-2 cell monolayer displayed that SNAC facilitated passive transport of polar charged SAs through the membrane of epithelial enterocytes. The pharmacokinetics results demonstrated that area under the curve (AUC) of RA, SA-B and SA-A with administration of SAs containing SNAC was 35.27, 8.72 and 9.23 times than administration of SAs. Tmax of RA, SA-B and SA-A were also prolonged. The AUC of R1 with administration of R1 containing SNAC was 2.24-times than administration of R1. SNAC is more effective in promoting absorption of SAs than R1. The study demonstrated that SNAC significantly improved bioavailability of R1 and SAs. What’s more, the effect of SNAC on absorption enhancement of charged drugs was larger than that of non-charged drugs. The current findings not only confirm the usefulness of SNAC for the improved delivery of R1 and SAs but also demonstrate the importance of biopharmaceutics characterization in the dosage form development of drugs.For drugs with high hydrophilicity and poor membrane permeability, absorption enhancers can promote membrane permeability and improve oral bioavailability. Sodium  There is considerable interest in delivery platforms that can improve the oral bioavailability of poorly absorbed drugs, because the oral route can improve patient compliance, and oral formulations can also reduce the costs associated with sterile manufacturing and use of healthcare professionals. The intestinal absorption of hydrophilic drugs is usually limited by their poor membrane permeability. Therefore, absorption enhancers (AEs) have often been studied to improve the absorption of these poorly permeable drugs. These AEs include surfactants, bile salts, chelating agents, fatty acids and coco-glucoside ,2,3.N-[8-(2-hydroxybenzoyl)amino]caprylate (SNAC) is a delivery agent that has been reported to enhance the permeability of a diverse spectrum of molecules, including proteins, such as insulin caprylate (SNAC) was provided by Shanghai Synmedia Chemical Co., Ltd. . Acetonitrile and methanol (UPLC-MS grade) were purchased from Fisher Scientific . Heparin sodium was purchased from Beijing Yaobei Biological and Chemical Reagents Company .Saikosaponin A and notoginsenoside R1were bought from the National Institute of the Control of Pharmaceutical and Biological Products . Salvianolic acid A was bought from Biopurify Phytochemicals Ltd. . Rosmarinic acid was bought from Shanghai Yuanye Biotechnology Co., Ltd. . Salvianolic acid B was bought from Shanghai Ronghe Pharmaceutical Technology Co. Ltd. . Salvianolic acid extract of Sprague-Dawley rats, male, healthy, weighing 250 ± 20 g, were purchased from Vital River Laboratory Animal Technology Co. Ltd.  for the animal experiments. Experimental animals were maintained in accordance with internationally guidelines for laboratory animal use, and the study was approved by the Ethical Committee of Experimental Animal Center of Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College .In brief, the raw material was dissolved in methanol, thionyl chloride was added dropwise into the solution that was then stirred overnight. The intermediate product was dissolved in dichloromethane and an accurate amount of trimethylamine was added. Aspirin was dissolved in dichloromethane and oxalyl chloride was added dropwise, followed by addition of triethylamine. Then dissolving in methanol, sodium hydroxide solution was added. Then the acidity was adjusted to excessive acid. Dissolving in the ethanol and then adding concentrated sodium hydroxide solution. Drying overnight and getting the final product.1H-NMR instrument .SNAC was analyzed with DMSO-d6 as solvent using an Avance II (400 MHz) 5 cells per well in 96-well plates and incubated at 37 ± 1 °C in an atmosphere of 5% CO2. After 24 h of culture, the medium was replaced with SNAC, R1 or SA. After 24 h, the medium was discarded and the wells were washed twice with hanks balanced salt solutions (HBSS). 200 μL MTT solution (0.5 mg·mL−1 in PBS) was added to each well, and incubated 4 h at 37 ± 1 °C for MTT formazan formation. Subsequently, the supernatant was carefully removed and the wells were washed twice with PBS. DMSO (200 μL) was added to each well and the plates were then mildly shaken for 15 min to ensure the dissolution of formazan crystals. The optical density values were measured by using MQX200 microplate reader  at wavelength 570 nm. Six replicates were read for each sample and mean value was used as the final result. The spectrophotometer was calibrated to zero absorbance using culture medium without cells.Caco-2 cells were plated at a cell density of 4 × 10−1 R1 or 50 μg·mL−1 SAs  as the test solutions. A 1.5 mL volume of sample was taken from the basolateral side at 2 h. Sample aliquots of 500 μL were mixed with 500 μL of methanol, shaken for 1 min using a vortex mixer, and centrifuged at 8000 rpm for 10 min. The supernatant was injected into the HPLC system for measuring R1, RA, SA-B and SA-A content. The apparent permeability coeffcient (Papp) was calculated according to the following equation: Papp = dQ/dt × 1/(AC0), where dQ/dt is the permeability rate, C0 is the initial concentration at the apical side, and A is the surface area of a monolayer. TEER was determined using a Millicell-ER system  before and after membrane absorption. After membrane absorption, the cells were washed three times with HBSS solution, complete media was added for 2 h or 24 h, and the TEER was then determined, the cell toxicity of test drugs and regeneration of cell membrane were also investigated.To investigate the influence of SNAC on the absorption properties of R1 and SA-A, SA-B and RA were used as an in vitro model of the gastrointestinal epithelium. To evaluate the transport, SNAC with R1 or SAs (1:1) were diluted with HBSS solution to a final concentration of 200 μg·mL−1 consisted of 0.1% formic acid in water (A) and 0.1% formic acid in methanol (B). The elution was carried out as follows: 20% A at 0–1.0 min; 20% A to 100% A at 1.0–3.0 min; 100% A at 3.0–4.0 min; 100% A to 20%A at 4.0–4.10 min, 20% A at 4.1–7 min. The injection volume was 10 μL and the partial loop with needle overfill mode was used for sample injection. The mass spectrometer was operated in negative ionization mode using MRM to assess the R1: m/z 931.9→637.5 for R1 and m/z 779.7→617.4 for saikosaponin (IS). The optimized cone voltage and collision energy were 295 V and 52 eV for R1, 290 V and 48 eV for IS, respectively. A spray voltage of 4500 V was used, and the capillary temperature was 550 °C. The scanning range was selected as m/z 100→1200: no interference was observed around target compound peaks. Data acquisition and processing were accomplished on a 4500 Q TRAP® mass spectrometer .The assay was performed on a Waters UPLC-MS system . A Phenomenex Kineter EVO C18  was used. The temperature was 30 °C. The mobile phase with the flow rate of 0.4 mL·min−1. The system was run with a gradient program of 83% B to 60% B at 0–10 min, 60% B to 50% B at 10–20 min, 50% B to 35% B at 20–30 min, 35% B to 83% B at 30–31 min, 83% B at 31–41 min. The detection wavelength was 286 nm.The HPLC system consisted of a LC-10AT series quaternary LC pump from Shimadzu Technology , with a SPD-10 AVP ultraviolet detector, CTO-10ASV column oven, SCL-10AVP controller and CLASS-VP work station. The mobile phase consisted of methanol: acetonitrile (3:1) (A) and 0.3% phosphoric acid in water (B). The flow rate was kept at 1.0 mL·min−1 were prepared by spiking the appropriate amount of saikosaponin A (50 ng·mL−1) standard solutions in blank plasma obtained from healthy rats. Similarly, quality control samples (QC) at low concentration (6 ng·mL−1), medium concentration (60 ng·mL−1) and high concentration (800 ng·mL−1) were also prepared as described.Calibration standard of R1 at concentrations of 1.97, 5.15, 10.6, 18.2, 51.9, 94.8, 211, 505, and 964 ng·mL−1 were prepared in blank plasma obtained from healthy rats. Similarly, quality control samples (QC) at low concentration (0.19 µg·mL−1), medium concentration (0.76 µg·mL−1) and high concentration (3.04 µg·mL−1) were also prepared as described. Calibration standard of SA-A at concentrations of 0.058, 0.12, 0.23, 0.47, 0.93, 1.87, 3.74, 7.48, 14.95, 29.9 µg·mL−1 were prepared in blank plasma obtained from healthy rats. Similarly, quality control samples (QC) at low concentration (7.48 µg·mL−1), medium concentration (3.74 µg·mL−1) and high concentration (0.93 µg·mL−1) were also prepared as described. Calibration standard of rosmarinic acid at concentrations of 0.055, 0.11, 0.22, 0.44, 0.89, 1.78, 3.55, 7.1, 14.2, 28.4 µg·mL−1 were prepared in blank plasma obtained from healthy rats. Similarly, quality control samples (QC) at low concentration (7.1 µg·mL−1), medium concentration (3.55 µg·mL−1) and high concentration (0.89 µg·mL−1) were also prepared as described.Calibration standard of SA-B at concentrations of 0.19, 0.38, 0.76, 1.52, 3.04, 6.08, 12.16 µg·mL−1 in acetonitrile, IS) was added to 100 μL of blank blood. Then, the analytical sample was vortexed with 0.39 mL acetonitrile for 5 min and centrifuged at 14,000 r × min−1 for 10 min. The supernatant was collected, transferred to a clean centrifuge tube, and evaporated to dryness using a vacuum centrifugal thickener . The resulting residue was dissolved in 100 μL 20% acetonitrile, vortexed for 3 min and centrifuged at 14,000 r·min−1 for 10 min for twice, and injected into UPLC-MS/MS system for analysis.Ten μL saikosaponin A (50 ng·mL−1) was added to 200 μL of blank blood. Then, the analytical sample was vortexed with 2 mL ethyl acetate for 90 s and centrifuged at 12,000 r·min−1 for 5 min. The supernatant was collected, transferred to a clean centrifuge tube, and evaporated to dryness. The resulting residue was dissolved in 100 μL methanol, vortexed for 20 s and centrifuged at 12,000 r·min−1 for 5 min, and injected into HPLC system for analysis.Thirty μL hydrochloric acid  was determined. The absolute recovery was determined in three replicates by comparing the peak areas of the extracted samples to the peak areas obtained from the organic solutions at the same concentration.The precision of the method was determined by analyzing six replicates at low, medium and high concentrations with the same analytical run on three consecutive days, respectively.The LOQ of R1, RA, SA-B and SA-A under chromatographic conditions were determined at an S/N  of about 10, respectivelyn = 6). The animals were fasted 12 h prior to oral administration and had free access to water during the experiment. All groups were treated by oral administration via oral gavage and dosing volume of 10 mL·kg−1. R1 was oral administrated with 200 mg·kg−1. SAs was oral administrated with 500 mg·kg−1. Blood samples of 200 μL were withdrawn from the eye socket according to the specific time intervals of at 0, 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 12 h after administration and immediately mixed with 10 μL of 1% heparin sodium to prevent clotting. Blood samples were centrifuged  and plasma was collected and stored at 20 °C until analysis.Male Sprague-Dawley rats with a mean body weight of 250 ± 20 g were randomly divided into 6 groups . Statistical analyses were performed using SPSS16.0. Statistical significance was considered to be reached at In this article, a novel absorption enhancer (SNAC) was synthesized and its absorption enhancement effect on drugs with different polarity properties were studied. Caco-2 cell transport in vitro and pharmacokinetics in vivo experiments were conducted. The administration of SAs and R1 in the presence of SNAC resulted in an enhancement of membrane transport. TEER values during the exposure of Caco-2 cells to SNAC suggested that the absorption-enhancing effect of SNAC may not involve tight junction complex opening. The in vivo results demonstrated that SNAC could promote oral absorption of both R1 and SAs. What’s more, it could prolong the retention time in vivo and promote oral absorption of polar molecules to a greater extent. The possible mechanism is ions interact to form complexes between the polar molecules and SNAC. The current work demonstrates the capability of SNAC to increase the epithelial permeability of small hydrophilic molecules, especially for polar molecules.


Irpex lacteus, one from Phanerochaete chrysosporium, one from Ceriporiopsis subvermispora, and another from Nematoloma frowardii) could all degrade four major mycotoxins  only in the presence of a dicarboxylic acid malonate, in which free radicals play an important role. The I. lacteus and C. subvermispora MnPs behaved similarly in mycotoxins transformation, outperforming the P. chrysosporium and N. frowardii MnPs. The large evolutionary diversity of these MnPs suggests that mycotoxin degradation tends to be a common feature shared by MnPs. MnP can, therefore, serve as a candidate enzyme for the degradation of multiple mycotoxins in food and feed if careful surveillance of the residual toxicity of degradation products is properly carried out.Enzymatic treatment is an attractive method for mycotoxin detoxification, which ideally prefers the use of one or a few enzymes. However, this is challenged by the diverse structures and co-contamination of multiple mycotoxins in food and feed. Lignin-degrading fungi have been discovered to detoxify organics including mycotoxins. Manganese peroxidase (MnP) is a major enzyme responsible for lignin oxidative depolymerization in such fungi. Here, we demonstrate that eight MnPs from different lignocellulose-degrading fungi (five from  See Mycotoxins are a diverse, large group of fungal secondary metabolites that exert severe toxic effects on human and animals worldwide. A recent study revealed that, among the nearly 20,000 feed and feed raw material samples which were collected from Asia, Europe, Americas, Africa, and Middle East, 72% contained at least one of the five major mycotoxins   × 100, where Ai and At are the absorbance at the initial and a given time [The reactions were carried out at 30 °C in a total volume of 200 μL containing 50 mM malonate buffer (pH 5.0), 0.1 mM Hven time .Saccharomyces cerevisiae BLYES strain was inoculated into 30 mL YMM ((NH4)2SO4) 1.7 g/L, CuSO4 12 mg/L, FeSO4 684 µg/L, KH2PO4 11.6 g/L, KOH 3.6 g/L, MgSO4 171 µg/L, D-(+)-glucose 20 g/L, biotin 20 µg/L, pantothenic acid 400 µg/L, inositol 1 mg/L, pyridoxine 400 µg/L, thiamine 400 µg/L, adenine 42.7 mg/L, arginine HCl 17.1 mg/L, aspartic acid 100 mg/L, glutamic acid 85.5 mg/L, histidine 42.73 mg/L, isoleucine 25.64 mg/L, lysine HCl 25.64 mg/L, methionine 17.1 mg/L, phenylalanine 21.4 mg/L, serine 320.4 mg/L, threonine 192 mg/L, tyrosine 25.7 mg/L) in a baked 250 mL glass flask. The cells were cultured at 28 °C with constant shaking at 200 rpm to an OD600 of 0.6. ZEN was dissolved in 70 mM malonate buffer to 5 μg/mL supplemented with 1 mM MnSO4 and 0.1 mM H2O2. Twenty microliter of appropriately diluted ZEN pre-treated or non-treated with IlMnP5 or IlMnP6 were mixed with 200 µL BLYES and the estrogenicity was checked by measuring the bioluminescence of the cells collected 6-h post treatment for analysis [The 1 treated or nontreated with an MnP. Fifty μg/mL of AFB1 were incubated with IlMnP5 or IlMnP6 (0.5 U/mL each) in 70 mM malonate buffer supplemented with 1 mM MnSO4 and 0.1 mM H2O2. The reaction was carried out at 30 °C for 10 h. Each test dish contained 1 mL of test solution and three normal healthy hydra. The hydra were examined for signs of toxicity at 20 h and 40 h, respectively. The toxic endpoint was determined by the “tulip” or “disintegration” stage of the hydra. In each test, experimental treatments were compared with untreated and solvent controls [The hydra was maintained clean and free from bacteria and fungi contamination by treating with diluted iodine solution (2.7 ppm) periodically. The assay was performed by exposing the hydra to AFBcontrols .1, ZEN, DON, and OTA was performed using a SHIMADZU 20A series instrument  with an Agilent ZORBAX SB-C18 column  . The elution condition for AFB1 and ZEN was set as: no acetonitrile (ACN), 4 min; 0–100% ACN, 25 min; 100% ACN, 6 min, at a flow rate of 0.8 mL/min. AFB1 and ZEN were monitored at 365 nm or 316 nm [2O:HAc (99:99:2) and the flow rate was 1.0 mL/min. OTA was monitored for its absorbance at 333 nm.HPLC analysis of AFBr 316 nm , respect1, ZEN transformation products were analyzed by using LC-MS/MS, which was carried out by coupling a SHIMADZU Nexera UHPLC system  to an AB-SCIEX 5600+ Triple TOF mass spectrometer . The solvent A for LC is mixed ACN:methanol (1:1) and solvent B is 0.1% fomic acid. The program was set as: 30–70% solvent A, 10 min; 70% solvent A, 8 min; 100% solvent A, 2 min; 30% solvent A, 5 min. The parameters for MS analysis were: positive and high-sensitivity mode; GS1, 50 psi; GS2, 50 psi; curtain gas, 25 psi; temperature, 500 °C; ion spray voltage floating, 5, 500 V; CE energy, 35 V ± 15 V. Degradation of FB1 was analyzed by using UHPLC-MS/MS, which was carried out by the same method as above.AFBThe alignment of the amino acid sequences of 8 MnPs were conducted using the clustalW algorithm of MEGA-X. Phylogenetic tree constructions were performed using the neighbor-joining method of MEGA-X. The reliability of the trees was tested by bootstrap analysis and the parameter was set to 1000. The other parameters used default values.


SPIO is effective in sentinel node (SN) detection. No nuclear medicine department is needed, and no allergic reactions have occurred. This study aimed to compare retro-areolar and peri-tumoral SPIO injections regarding skin staining, detection rates and number of SNs.Data on staining size, intensity and cosmetic outcome  were collected by telephone interviews with 258 women undergoing breast conservation. SN detection and the number of SNs were prospectively registered in 332 women.p < 0.001) developed skin staining, with remaining staining in 46.2 vs. 9.4% after 36 months (p < 0.001). Initial mean size was 16.3 vs. 6.8 cm (p < 0.001) and after 36 months, 6.6 vs. 1.8 cm2 (p < 0.001). At 75.1% of 738 interviews, staining was reported paler. After retro-areolar injections, cosmetic outcome scored worse for 2 years. The mean (median) scores were 1.3(0) vs. 0.5(0) points, and 0.2(0) vs. 0.1(0) points, at 12 and 36 months, respectively. Overall detection rates were 98.3% and 97.4% (p = 0.43) and the number of SNs 1.35 vs. 1.57 (p = 0.02) after retro-areolar and peri-tumoral injections. Injection, regardless of type, 1–27 days before surgery increased detection rates with SPIO, 98.0% vs. 94.2% (p = 0.06) ,and SN numbers, 1.56 vs. 1.27 (p = 0.003).After retro-areolar and peri-tumoral injections, 67.3% and 37.8% (SPIO is effective and facilitates planning for surgery. Peri-tumoral injection reduced staining with a similar detection rate. Staining was not considered a cosmetic problem among most women. Injecting SPIO 1–27 days before surgery increased the detection rate by 3.8% and increased the number of SNs by 0.3. However, SPIO may be injected up to 4 weeks before a SNB. At Uppsala University Hospital, SPIO has been used as the sole SN detection method for 2 years in most cases.3Superparamagnetic iron oxide nanoparticles (SPIO) have comparable detection rates as the dual technique using TechnetiumAs with BD, SPIO injection may cause skin staining for more than a year. This is seen almost exclusively after breast-conserving surgery (BCS). For BD, 41% of patients have been reported with staining after 12 months and up to 8.6% after 36 months.1The purpose of the study was to compare the rate of skin staining after retro-areolar and peri-tumoral injections and how the different injection techniques related to patient-experienced cosmetic outcome. The detection rates and the number of removed SNs also were studied.® in 3 ml of NaCl) was given at least 20 min before surgery followed by a 5-min massage. Later, when SPIO was injected up to 4 weeks before surgery, massage was optional. During the latter part of the study period, SPIO was injected close to palpable tumours or in the peritumoral area for nonpalpable tumours. If the transcutaneous magnetic signal in the axilla was low, BD was added according to the surgeon’s decision. If no SN was found, the decision to perform an axillary clearance, an axillary biopsy, or no staging was left to the surgeon. Age, body mass index (BMI), type of surgery, tumour size and grade, number of SNs, and lymph node status were documented. The size of skin staining was recorded at the first postoperative visit, 3 weeks after surgery. Women with a skin stain were thereafter telephone interviewed every third month. The size of the staining, intensity, and cosmetic outcome was self-assessed. At first, only change of intensity was described, but later women classified the intensity of the staining according to a Likert item scale from 0 to 5, based on photos of selected cases mailed to the women  were tested in a multivariate regression model. SPSS® version 23.0  was used for statistical analyses.Staining was analysed only in women with BCS, but analyses of SN detection were done in all women, BCS, and mastectomies. Comparisons between cohorts were conducted using parametric tests, when appropriate. Data measured on a Likert scale were analysed using nonparametric procedures. Univariate analyses of correlation were performed; variables with significance or trend to significance .Breast-conserving surgery was performed in 257 women . Six women had conversion to mastectomy, and staining was only registered at the first postoperative visit. Seven women  did not have a SN biopsy (SNB), even though SPIO was injected, because they were part of the SentiNot trial.p < 0.001). Including all women, the mean size of staining was 16.3 (range 2-100) cm2 and 6.8 (range 1–100) cm2 after retro-areolar and peritumoral injections (p < 0.001), at the first visit. Including only those 130 with an actual staining, the mean size was 24.2 and 17.9 cm2 (p = 0.02), respectively. After 6, 12, 24, and 36 months, 65.4%, 63.6%, 58.1%, and 46.2% had a remaining staining after retro-areolar injections and 34.0%, 31.3%, 14.0%, and 9.4% after peritumoral (p < 0.001 at 36 months). Size diminished successively over time .After BCS, 74 of 110 (67.3%) had a skin staining after a retro-areolar and 56 of 148 (37.8%) after a peritumoral injection (.3 range -100 cm2 p < 0.001) and 0.6 (0) vs. 0.2 (0) points (p = 0.02). However, the difference was gone after 36 months: 0.2 (0) vs. 0.1 (0) for retro-areolar and peritumoral injections, respectively (p = 0.49). Analysing women with an actual stain at each time point showed no statistically significant differences between the two injection types (data not shown). Women with a higher BMI scored lower at all time points, regardless of injection type, but the differences were not statistically significant (data not shown). Younger women (< 60 years) scored worse than older women (≥ 60 years) during the first 2 years, but the difference was gone at year 3. Mean (median) for < 60 years were 2.4 (3), 2.4 (3), 1.3 (1), and 0.2 (0) points and for ≥ 60 years 1.4 (1), 1.4 (1), 0.7 (0), and 0.4 (0) points at 6, 12, 24, and 36 months, respectively . There was no statistically significant difference in self-assessed cosmetic outcome comparing 27 women with small and less intensive staining or large and more intensive staining after 12 months .Self-assessed cosmetic outcome (0-5 points) was worse after retro-areolar compared with peritumoral injections at 12 and 24 months: mean (median) 1.3 (0) vs. 0.5 (0) points (p = 0.43), respectively. Excluding those with palpable metastasis and just looking at SN detection by SPIO showed no significantly different detection rates for retro-areolar and peritumoral injections: 95.9% (164/171) versus 97.4% (149/153) (p = 0.50). Nonpalpable SN metastasis were all detected by SPIO, regardless of injection type or timing: 32/32 for retro-areolar and 19/19 for peritumoral injections.There were 333 operations with a goal for a SNB. Four of those had had earlier axillary surgery and were excluded from analyses. The overall SN detection rate was 97.9% (322/329). In four women, SNs were detected by BD only. All of these had a retro-areolar injection of SPIO on the day of surgery. Five women had palpable macrometastatic lymph nodes when entering the axilla, despite a negative axillary ultrasound, and intraoperative frozen section confirmed metastases. Detection rates for those with retro-areolar and peritumoral injections were 98.3% and 97.4% (p = 0.06); 1–7 days before surgery 97.5%, 8–14 days 100% and 15–27 days 97.5%. No difference in detection rates was found between injection types, comparing women who had their injection at similar timepoints before surgery.The SN detection rate with SPIO was 98.0% (199/203) after injection 1-27 days before surgery and 94.2% (113/120) after an injection on the day of surgery (p = 0.02). In those injected at the day of surgery, the number of SNs were 1.21 after retro-areolar and 1.53 after peritumoral injections (p = 0.07). The mean number of SNs was significantly higher in those injected 1 to 27 days before surgery regardless of injection type: 1.56 and 1.27, respectively (p = 0.003). The mean number of SNs after injections at days 1–7, 8–14, and 15–27 before surgery was 1.74, 1.53, and 1.52, respectively.The mean number of removed SNs were 1.35 and 1.57 for those with retro-areolar and peritumoral injections, respectively (2) had higher SN detection rate with a similar number of SNs, regardless of injection type: 100% versus 93.3% (p < 0.02), and 1.45 versus 1.46, respectively (p = 0.31). Women with mastectomy or BCS had similar SN detection rates: 98.7% versus 97.5% (p = 0.53). The mean number of SNs removed was 1.29 at mastectomy and 1.51 at BCS (p = 0.04), but numbers were similar if SPIO was injected at similar time points .Women with a lower BMI (< 25 kg/m99 and BD.p = 0.06) with 0.3 more removed SNs. Most women did not consider skin staining to be a cosmetic problem.Sentinel node identification using SPIO is comparable to TCMore than 300 women were followed for up to 3 years. All data were registered prospectively, and the cosmetic outcome of skin staining was patient-assessed. Patient reported outcomes have been shown to be more sensitive in reflecting patient satisfaction than outcomes reported by clinicians.A peritumoral injection reduced the incidence of staining, and the cosmetic outcome was initially better compared with retro-areolar injections. The staining disappeared earlier after a peritumoral injection, and after 12 months the mean score was 0.5 (median 0). The BD also results in staining of the skin, but little is reported about patient-experienced outcome. Govaert et al. noticed that none of 33 women followed for 18 months reported the blue staining a cosmetic or psychological problem.4In concordance with what has been reported for the dual technique, different injection sites resulted in similar SN detection rates.®) is now tested in volumes of 1.5 and 1.0 ml without dilution.22An updated version of SPIO (SiennaXPSPIO is a tracer with comparable results with the standard dual technique. Its benefits lie in the flexibility that it provides, which is an important property in the global setting where access to the isotope is difficult and in interesting clinical applications that are currently investigated. Refinement of the technique is expected to lead to optimal results and address issues with MRI follow-up compatibility, providing the possibility for a new standard in axillary mapping for breast cancer.