Bayesian penalized likelihood reconstruction for PET  aims at improving convergence of lesion activity while ensuring sufficient signal-to-noise ratio (SNR). This study evaluated reconstructed spatial resolution, maximum/peak contrast recovery (CRmax/CRpeak) and SNR of Q.Clear compared to time-of-flight (TOF) OSEM with and without point spread function (PSF) modeling.4/16” , “OSEM+TOF4/16” , “PSF+TOF2/17” , “OSEM+TOF2/17” , “PSF+TOF4/8” , and “OSEM+TOF2/8” . Spatial resolution was derived from 3D sphere activity profiles. RC as (sphere activity concentration [AC]/true AC). SNR as (background mean AC/background AC standard deviation).The NEMA IEC Body phantom was scanned five times  with a GE Discovery MI PET/CT (3-ring detector) with spheres filled with 8-, 4-, or 2-fold the background activity concentration . Reconstruction included Q.Clear , “PSF+TOF150 was significantly better than all conventional algorithms at SBR 8:1 and 4:1 . At SBR 4:1 and 2:1, the spatial resolution of Q.Clear300/450 was similar or inferior to PSF+TOF4/16 and OSEM+TOF4/16. Small sphere CRpeak generally underestimated true AC, and it was similar for Q.Clear150/300/450 as with PSF+TOF4/16 or PSF+TOF2/17 . Q.Clear provided similar or higher CRpeak as OSEM+TOF4/16 and OSEM+TOF2/17 resulting in a consistently better tradeoff between CRpeak and SNR with Q.Clear. Compared to PSF+TOF4/8/OSEM+TOF2/8, Q.Clear150/300/450 showed lower SNR but higher CRpeak.Spatial resolution of Q.Clear150 compared to Q.Clear300/450 and PSF+TOF4/16/PSF+TOF2/17 while CRpeak for the small spheres did not improve considerably. This suggests that Q.Clear300/450 may be advantageous for the 3-ring detector configuration because the tradeoff between CR and SNR with Q.Clear300/450 was superior to PSF+TOF4/16, OSEM+TOF4/16, and OSEM+TOF2/17. However, it requires validation by systematic evaluation in patients at different activity and acquisition protocols.Q.Clear consistently improved reconstructed spatial resolution at high and medium SBR compared to PSF+TOF and OSEM+TOF, but only with beta = 150. However, this is at the cost of inferior SNR with Q.Clear The current clinical standard for image reconstruction in positron emission tomography (PET) is iterative algorithms, mainly ordered subset expectation maximization (OSEM). Recently developed systems commonly combine time-of-flight (TOF) capabilities and compensation for spatial variances in the scanner’s point spread function (PSF). While TOF mainly improves signal-to-noise ratio (SNR) at comparable convergence level  and leav. reported superior contrast-to-noise ratios (CNR) in a NEMA IQ phantom for Q.Clear compared to OSEM with PSF and TOF for a β of 400 and 533 but inferior CNR for a β < 300 [Bayesian penalized likelihood reconstruction, such as GE Q.Clear, has been introduced to offer full convergence of focal activity peaks and high SNR in homogenous areas within the same PET dataset . Q.Clear β < 300 . While t β < 300 , such si β < 300  and may  β < 300 . Further β < 300 , 12. PreThe aim of the current study was to generate a differentiated insight into performance benefits provided by the Q.Clear algorithm as measured by reconstructed spatial resolution, CR and SNR. This should facilitate the identification of the most appropriate algorithm for clinical use with the 3-ring digital detector configuration of the GE Discovery MI. Different β settings in Q.Clear were compared to different iterations/subsets and filter settings for OSEM with TOF with or without PSF.Three phantom measurement series were performed using the GE Discovery MI PET scanner  with a digital 3-ring detector and a reported sensitivity of 7.3 cps/kBq . Total a150, Q.Clear300, Q.Clear450). OSEM+TOF reconstruction (GE VUE Point FX) was performed as OSEM+TOF4/16 , OSEM+TOF2/17 , and OSEM+TOF2/8 . OSEM+PSF+TOF  was reconstructed as PSF+TOF4/16 , as PSF+TOF2/17  and PSF+TOF4/8 . OSEM+TOF and PSF+TOF reconstructions always included a “standard” z-axis filter. In all 9 reconstruction settings, field of view was 70 cm .PET raw data were reconstructed using Q.Clear with a β value of 150, 300, or 450, respectively  was performed with dedicated software . The mean background AC and its standard deviation (SD) were defined with a spherical background volume of interest . Maximum AC of each sphere was obtained. Using the ACCURATE tool , the peak AC of each sphere was defined as the average AC in a spherical VOI of 1.2 cm in diameter that was positioned to generate the highest peak AC of this sphere. Peak and maximum CR  were defined as the ratio of measured peak/maximum AC of the spheres to the true decay-corrected AC.SNR was defined as follows:The spatial resolution was assessed as the full width at half maximum (FWHM) of the PSF in the reconstructed images. PSF was modeled by a 3D Gaussian, and FWHM was determined by applying the method described in detail by Hofheinz et al. . This meInner volumes , inner radii , and wall thickness  of the spheres were determined based on weighting of the sphere inserts after filling with water (corrected for the volume of the tube mounting) combined with caliper measurements of the outer diameters.2) who underwent FDG-PET/CT for early response assessment for Hodgkin’s lymphoma using the same PET scanner. Injection of 250 MBq 18F-FDG was followed by acquisition in the supine position from base of the skull to proximal femora 62 min post-injection . Low-dose-CT was acquired for attenuation correction. PET raw data were reconstructed with the 9 reconstruction algorithms detailed above. SNR in the liver for each algorithm was calculated as the ratio of SUVmean to SUV standard deviation in a spherical VOI in the right liver lobe with an identical location in each dataset .A patient example Fig.  was inclU test. Statistical significance was generally assumed at p < 0.05.Statistical analysis was performed using SPSS 22 . Descriptive parameters were expressed as mean and SD. Differences in spatial resolution, CRpeak, CRmax, and SNR between reconstruction methods were compared using the Wilcoxon test. Differences in these measures between SBR were compared with either Kruskal-Wallis test or Mann-Whitney 2/8 gives a smooth sphere profile but fails to reach high CR in the small sphere. In the large sphere . OSEM+TOF4/16 and OSEM+TOF2/17 generally exceed a CR of 1.0 for the large sphere irrespective of the location along the sphere’s radius (primarily from excessive noise). In the small sphere  and from SBR 4:1 to SBR 2:1 . This decline was especially observed for the Q.Clear algorithms  significantly decreased from SBR 8:1 to SBR 4:1  while at SBR 2:1 spatial resolution was similar to PSF+TOF4/16 and OSEM+TOF4/16 (each p > 0.05). Q.Clear300 also showed significantly superior spatial resolution at SBR 8:1 compared to all conventional algorithms (each p < 0.05) except for PSF+TOF4/16 (p > 0.05). Spatial resolution for Q.Clear300 at SBR 4:1 and 2:1 was inferior to PSF+TOF4/16 (p < 0.05) and similar to OSEM+TOF4/16 (p > 0.05). Q.Clear450 was inferior to PSF+TOF4/16 (p < 0.05) and comparable to OSEM+TOF4/16 (p > 0.05) at SBR 8:1 while it was inferior to both PSF+TOF4/16 and OSEM+TOF4/16 at SBR 4:1 and 2:1 (each p < 0.05).At SBR 8:1 and 4:1, Q.Clear150 to Q.Clear300 and Q.Clear450  and PSF+TOF4/16 (p = 0.04) while Q.Clear300 showed similar SNR as PSF+TOF2/17 (p = 0.08). SNR of Q.Clear450 was significantly higher than of PSF+TOF4/16/PSF+TOF2/17 and OSEM+TOF4/16/OSEM+TOF2/17 (each p < 0.01). Figure Mean SNR significantly increased from Q.Clear150 and neither Q.Clear300 nor Q.Clear450 exceeded 10% at any SBR or sphere size . Relative CRpeak differences between Q.Clear150 or Q.Clear300 and either OSEM+TOF4/16, OSEM+TOF2/17, PSF+TOF4/8, or OSEM+TOF2/8 exceeded 10% in at least one of the three small spheres at all SBR, and differences increased with SBR (each p < 0.01). Relative differences between Q.Clear450 and OSEM+TOF4/16 and OSEM+TOF2/17 increased with SBR (p < 0.05) but did not surpass 10%. Differences in Q.Clear450 and PSF+TOF4/8 increased with SBR (p < 0.001) and exceeded 10% at SBR 8:1/4:1; differences to OSEM+TOF2/8 surpassed 10% at all SBR.None of the differences between any Q.Clear reconstruction and PSF+TOF150 showed higher CRpeak for the small spheres than OSEM+TOF2/17 (+ 10.6 ± 3.0%) while CRpeak was similar to PSF+TOF4/16 (+ 1.0 ± 2.8%) and PSF+TOF2/17 (+ 6.0 ± 2.6%) at lower SNR . Q.Clear300 resulted in both higher CRpeak and SNR than OSEM+TOF2/17 . CRpeak of Q.Clear300 and PSF+TOF2/17 were similar (+ 2.1 ± 2.2%) as was SNR (− 1.8 ± 5.9%). Q.Clear450 showed equal CRpeak as PSF+TOF4/16 (− 6.9 ± 3.9%) and PSF+TOF2/17 (− 1.5 ± 2.4%) at higher SNR . CRpeak were also similar to OSEM+TOF4/16 (+ 0.1 ± 5.3%) and OSEM+TOF2/17 (+ 3.5 ± 4.0%) at higher SNR . Compared to PSF+TOF4/8 and OSEM+TOF2/8, all Q.Clear methods showed higher CRpeak  at lower SNR .Figure 150 exceeded all other reconstruction methods by ≥ 10% for at least one of the three small spheres at all SBR  than PSF+TOF4/16, PSF+TOF2/17, OSEM+TOF4/16, and OSEM+TOF2/17 but higher CRmax compared to PSF+TOF4/8 and OSEM+TOF2/8.CRmax of Q.ClearSBR Fig. . Q.ClearThe present study assessed reconstructed spatial resolution, CR, and SNR in phantom measurements for Q.Clear in comparison to PSF+TOF and OSEM+TOF as a basis for investigations on the most appropriate algorithm for clinical use with the 3-ring-detector GE Discovery MI.4/16 and OSEM+TOF4/16), CRpeak was similar to all Q.Clear algorithms, and SNR was comparable or even lower than with Q.Clear150. Both observations are in line with Teoh et al. who showed superior CR but lower SNR for Q.Clear with β of 100 to 400 compared to OSEM+TOF with 2 iterations, 24 subsets, and 6.4 mm filter (GE Discovery 690). However, compared to PSF+TOF with 3 iterations, 24 subsets, and 2.0 mm filter, CR and SNR with Q.Clear were either similar (β of 100 or 200) or progressively lower (β ≥ 300) [4/8/OSEM+TOF2/8 may be appealing to physicians  which is less likely to reach convergence and, especially, using a smoother in-plane postprocessing filter (6.4 mm vs. 2.0 mm). Using these parameters, PSF+TOF and OSEM+TOF consistently provided significantly inferior reconstructed spatial resolution than Q.Clear as well as lower CRpeak in the small spheres . In contrast, SNR were significantly higher than with any Q.Clear algorithm. With a high product of iterations and subsets and a 2.0 mm in-plane filter , both methods achieved similar CRpeak in the small spheres  and CRpeak were higher for Q.Clear150, i.e., it offered superior image quality (tradeoff between CR and SNR). Image quality with the Q.Clear algorithms was generally superior to OSEM+TOF4/16 and OSEM+TOF2/17 but only Q.Clear450 was consistently superior to both PSF+TOF4/16 and PSF+TOF2/17 . Improving spatial resolution with increasing SBR can be seen in all reconstruction methods , Q.Clear300 and Q.Clear450 may offer the best compromise between CR and SNR for whole-body imaging. Lindström et al. used the 4-ring detector GE Discovery MI for phantom and patient examinations. The authors reported superior SNR at matched noise in a liver VOI with Q.Clear (β of 133 to 533) compared to PSF+TOF , and clinical whole-body FDG-PET data with β of 267 to 533 achieved the highest score in subjective image quality [The phantom measurements were performed as representative as possible for imaging protocols in clinical routine, i.e., typical activity concentrations in spheres and background were chosen. In addition, the effective reconstructed spatial resolution was determined using spheres with typical sizes of tumor lesions and SBR. As illustrated by the patient example in Fig.  quality . Trägård quality . The sam quality . If “AT”150 compared to Q.Clear300/450 and PSF+TOF4/16/PSF+TOF2/17 while CRpeak for the small spheres did not improve considerably. In contrast, Q.Clear300/450 showed an improved tradeoff between CR and SNR compared to PSF+TOF and OSEM+TOF with different combinations of iterations, subsets and in-plane filters. Especially compared to Q.Clear150, PSF+TOF4/16, and OSEM+TOF4/16, this may allow for an appropriately low level of image noise for whole-body PET in clinical routine while averting disadvantageous small lesion detectability and quantification. However, a dedicated systematic evaluation of patient data is required for validation at different activity and acquisition protocols.Q.Clear can provide superior reconstructed spatial resolution compared to PSF+TOF and OSEM+TOF if β is low (150) and SBR is high. However, using the 3-ring detector configuration, this is at the cost of inferior SNR with Q.Clear


Salix triandra  is an established crop, grown in coppicing regimes for basket-making materials. It is known as a source of non-salicinoid phenolic glycosides, such as triandrin and salidroside. A spontaneous natural hybrid of S. triandra and S. dasyclados was subjected to metabolite profiling by high resolution LC-MS, and 22 phenolic glycosides, including 18 that are new to the Salicaceae, were identified. Structures were determined by HPLC isolation and NMR methods. The hybridisation process has introduced novel chemistry into the Salix phenolic glycoside palette, in particular, the ability to generate disaccharide conjugates where the glycosyl group is further extended by a range of sugars, including apiose, rhamnose, xylose, and arabinose. Also of note is the appearance of chavicol derivatives, also not previously seen in Salix spp. The work demonstrates the plasticity of the phenolic glycoside biosynthetic pathway, and the potential to improve established crops such as S. triandra and S. dasyclados, via high-value metabolites, for both basketry and bioenergy markets. Salix spp.) and poplars (Populus spp.). Both groups have a rich secondary chemistry based on phenolic glycosides. The genus Salix is the largest and includes over 400 species that are variable in growth form, from large trees to small shrubs, and are distributed over a wide range of habitats −. A comparison of its NMR data to those of a compound isolated from the roots of Rheum officinale Baill. (Polygonaceae) confirmed it to be p-hydroxybenzylacetone-β-d-glucopyranoside [Peak 3 at 15.2 min with [M − H]MR peaks  14]. Th. Th− at ticulata , but is triandra ,7, a parranoside   and anomeric  signals  were also detected and the structure of compound 21 was confirmed to be chavicol-β-d-glucopyranoside, a compound previously isolated from Cedronella canariensis (L.) Webb and Berth. (Lamiaceae) − at m/z 429.1767, corresponding to the molecular formula C20H30O10. The NMR data of this isolated compound was comparable to dihydrocinnamyl alcohol-α-l-arabinofuranosyl-(1→6)-β-d-glucopyranoside) (dihydro-rosarin), formerly described in Juniperus communis var. depressa [16) showed the same molecular formula, however, the NMR data indicated a different sugar moiety  and a α-arabinopyranose (anomeric proton at δH 4.53/δC 104.4). Long range correlations observed between H-7/C-1 (δH 2.71/δC 145.3), H-1/C-9 (δH 4.42/δC 72.8) in the heteronuclear multiple bond correlation (HMBC) spectrum indicated how each moiety was connected in the molecule. Thus, the structure of 16 was determined to be dihydrocinnamyl alcohol-α-l-arabinopyranosyl-(1→6)-β-d-glucopyranoside (dihydro-rosavin), and this compound has not previously been reported in the literature.Compound . In order to find more evidence of the structure of 15, a careful analysis of the 1D 1H and 2D 1H–13C-HSQC and HMBC-NMR spectra of 16 was performed. Some minor peaks corresponding to a β-apiofuranosyl moiety were observed, such as an anomeric at δH 5.07 ppm /δC 111.9 ppm (CH-1″); δH 3.96 ppm /δC 79.6 ppm (CH-2″); δC 82.3 ppm (C-3″); δH 4.00 ppm , 3.85 ppm /δC 76.7 ppm (CH2-4″); and δH 3.62 ppm (s)/δC 66.5 ppm (CH2-5″). The relative integration of these peaks to the corresponding signals of 16 was 1:3. Together with this data, the fact that 15 presented the same molecular formula and mass fragments as 16, and the proximity of them in the UHPLC run (less than 0.2 min), we can suggest that 15 is dihydrocinnamyl alcohol-β-apiofuranosyl-(1→6)-β-glucopyranoside.Although compound S. triandra and S. dasyclados (NWC1283) contains a much larger array of chemistry, particularly of phenylalkanoid disaccharides, than has previously been reported in either of the parental species. Apart from salidroside (2), which was the only phenyethanoid glycoside previously known in the Salicaceae [Salix” glycosyl moiety is further substituted at C-6 by arabinose, apiose, xylose or rhamnose. This second glycosidic substitution can also consist of either the pyranosyl form or of the furanosyl form, and evidence of both forms is present for several of the compounds isolated. As evidenced in this study, such compounds can be isolated directly from the chipped biomass with relatively straightforward extraction techniques. In this study, extraction was achieved using 20% aqueous ethanol, a solvent system that is cheap and therefore applicable to larger scale extractions. Similarly, heating steps were not required to release the compounds reported, and extraction at room temperature preserved the integrity of the compounds. This work also demonstrates the potential of plant breeding to introduce traits that result in new chemistry, potential bioactivities and provide the opportunity to add value to basketry and bioenergy crops.In summary, the hybrid of licaceae , many of1H-1D and 1H–1H and 1H–13C 2D-NMR spectra of each compound were acquired, using a 5 mm triple resonance (TCI) cryoprobe, on a Bruker Avance 600 MHz NMR spectrometer , operating at 600.05 MHz for 1H-NMR and 150.9 MHz for the 13C-NMR spectra. Typical 1-dimensional 1H spectra were obtained with an acquisition time of 4.6 s, a sweep width of 7142.9 Hz and 65,536 data points. A total of 16 scans were recorded using the zgpr pulse sequence with a 90° angle. A relaxation delay of 5 s was used to suppress the residual HOD signal. The spectra were transformed using an exponential window with a line broadening of 0.5 Hz. 1H–1H correlation spectroscopy (COSY) were run using the pulse sequence cosyprqf for 3 h, and the frequency was 600.05 MHz in both dimensions. The acquisition times were 0.1434 and 0.0896 s, and the sweep widths were 7142.9 Hz. There were 1024 data points collected in each dimension using 32 transients. 1H–13C heteronuclear single quantum coherence (HSQC) spectra were performed using the pulse sequence hsqcetgpsi2 for 10 h, at 600.05 and 150.9 MHz frequencies, with acquisition times of 0.1433 and 0.00212 s. The data were acquired using sweep widths of 7142.9 and 30,120.5 Hz. There were 2048 and 1024 data points collected using 128 transients. The 1H–13C heteronuclear multiple bond correlation (HMBC) spectra were obtained using the pulse sequence hmbcgpndqf for 22 h. The acquisition parameters were the same as stated for the HSQC data collection. For comparability with previous work [2O:CD3OD (8:2), and chemical shifts are given in δ, relative to TSP-d4 ((trimethylsilyl) propionic acid, 0.01 % w/v) added as a chemical shift reference standard. The compound concentration was typically 1 mg/mL. Phasing and baseline correction were carried out within TOPSPIN v. 2.1 . Structural assignments of carbohydrate moieties were made with reference to the authentic standards and the use of characteristic chemical shift data [The m/z 50–1500, and the source voltage, sheath gas, auxiliary gas, sweep gas and capillary temperature were set to 2.5 kV, 35 (arbitrary units), 10 (arbitrary units), 0.0 (arbitrary units) and 350 °C, respectively. Automatic MS–MS was performed on the four most abundant ions using an isolation width of m/z 2. The ions were fragmented with a normalised collision energy of 65 and an activation time of 0.1 ms, using high-energy C-trap dissociation. The data were collected and inspected using Xcalibur v. 2.2 .UHPLC–MS were recorded on an LTQ-Orbitrap Elite mass spectrometer  coupled to a Dionex UltiMate 3000 RS UHPLC system, equipped with a DAD-3000 photodiode array detector. Separation was carried out in reverse-phase using Hypersil GOLD™ column , which was maintained at 35 °C. The solvent system consisted of water/0.1% formic acid (A) and acetonitrile/0.1% formic acid (B), both Optima™ grade . The injection volume was 10 μL and separation was carried out for 40 min with a flow rate of 0.3 mL/min under the following gradient: 0–5 min, 0% B; 5–27 min, 31.6% B; 27–34 min, 45% B and 34–37.5 min, 75% B. The mass spectra were collected in negative ion mode using a heated electrospray source . The resolution was 120,000 over The compounds were isolated by repeated injection into an HPLC system  equipped with an Ascentis C-18 column . The column was maintained at 35 °C and chromatographic separation was performed using a constant flow rate of 1 mL/min. The mobile phases were water (A) and acetonitrile (B), both containing 0.1% formic acid. To achieve separation, the gradient used was as follows: 0–2 min, 5% B; 2–5 min, 12% B, 5–10 min, 12% B, 10–60 min and 40% B. The peaks were detected using UV wavelengths of 210 to 360 nm, and fractions corresponding to the target compounds were collected into glass tubes. Twelve injections (100 μL each) were performed and the fractions from repeated runs were combined and the solvent was evaporated using a Speedvac concentrator .−1, establishment year growth received no fertilisers and two pre-emergence herbicides were applied within 10 days of planting Pendimethalin (Stomp at 3.3 l/ha) and Isoxaben (Flexidor at 1.0 l/ha). The genotypes were planted in unreplicated plots each containing 80 plants (two twin rows of 40).NWC1283 is one of 16 genotypes that makes up CS/782. CS/782 was planted using winter dormant 20 cm cutting in May 2017 at Rothamsted Research  . The previous crop was winter wheat. The soil type within the field is silty clay loam with flints over clay. Planting and agronomy followed the conventional SRC best practice. Willows were planted as cuttings using the typical twin-row design at a planting density of 16,667 plants haThe winter dormant above ground biomass was harvested in January 2018. The stems were cut 5 cm above the ground, and were immediately chipped in a Petrol Shredder 100 mm (4″). The material was stored at −80 °C and then freeze-dried. A voucher specimen has been retained and is available on request.1H-NMR and UHPLC-MS and for compound isolation by HPLC.The chips (60 g) were extracted at room temperature, by soaking with 400 mL water:ethanol (4:1) for 16 h. Aliquots were taken for initial metabolite profiling by


Here we report the specific synthesis of the fructosylated dipeptide l-valyl-l-histidine  and fructosylated l-valine . Both are basic tools for the development and validation of enzymatic HbA1c assays. The two fructosamine derivatives were synthesized via a protected glucosone intermediate which was coupled to the primary amine of Val or Val-His, performing a reductive amination reaction. Overall yields starting from fructose were 36% and 34% for Fru-Val and Fru-Val-His, respectively. Both compounds were achieved in purities > 90%. A HILIC-ESI-MS/MS method was developed for routine analysis of the synthesized fructosamines, including starting materials and intermediates. The presented method provides a well-defined and efficient synthesis protocol with purification steps and characterization of the desired products. The functionality of the fructosylated dipeptide has been thoroughly tested in an enzymatic HbA1c assay, showing its concentration-dependent oxidative degradation by fructosyl-peptide oxidases (FPOX).Naturally occurring fructosamines are of high clinical significance due to their potential use in The online version of this article (10.1007/s00216-019-02186-2) contains supplementary material, which is available to authorized users. Diabetes mellitus is one of the leading medical threats with 451 million affected patients worldwide in 2017 and approximately 693 million patients expected in 2045. Global healthcare spending of 850 billion USD was associated with the disease in 2017 + calcd. 261.1333; found 261.1334; mass accuracy 0.38 ppm.2,3:4,5 di-u et al. , using 1O-Isopropylidene-aldehydo-β-d-arabinohexos-2-ulo-2,6-pyranose  was synthesized performing a Swern oxidation adapted from the published procedure by the group of Hoffmann + calcd. 276.1442; found 276.1447; mass accuracy 1.81 ppm. HRMS for iprGlu hydrate C12H20O7 (exact monoisotopic mass 276.1209 g/mol): [M+NH4]+ calcd. 294.1547; found 294.1553; mass accuracy 2.04 ppm.As further purification step as well as to clearly characterize the -workers . A twofo71–72 °C ). FT-IR iprFru-Val, 3a) and Val-His  were synthesized performing a reductive amination of iprGlu 2 and the primary amine of Val or Val-His, respectively, adapting the protocol from the group of Walton + calcd. 360.2017; found 360.2018; mass accuracy 0.28 ppm.Analytical data for iprFru-Val-His 3b: 1H NMR : δ 7.94 , 7.48 , 6.75 , 4.52 , 4.36 , 4.28 , 4.19 , 3.73 , 3.54 , 2.95 , 2.88 , 2.78 , 2.74 , 2.60 , 1.82 , 1.44 , 1.34 , 1.33 , 1.26 , 0.80 ppm . For annotation of protons, see ESM. 13C NMR : δ 172.93, 172.31, 134.38, 107.93, 107.29, 103.00, 70.77, 70.16, 69.66, 67.82, 60.36, 53.97, 52.36, 30.74, 29.00, 26.28, 25.70, 25.30, 23.97, 18.99, 18.48 ppm. For annotation of carbon nuclei, see ESM. Signal assignment of NMR data was achieved by 2D-NMR measurements. HRMS for C23H36N4O8 (exact monoisotopic mass 496.2533 g/mol): [M+H]+ calcd. 497.2606; found 497.2609; mass accuracy 0.60 ppm.Analytical data for 4a) and Val-His  were obtained by removing the isopropylidene protecting groups under acidic conditions. Therefore, 3a and 3b were stirred in a mixture of trifluoroacetic acid (TFA) and water (95 vol% TFA) as described by the group of Hoffmann + calcd. 280.1390; found 280.1391; mass accuracy 0.36 ppm.Analytical data for Fru-Val 4b: 1H and 13C NMR spectra were recorded in DMSO-d6. Both measurements led to complex NMR spectra, containing signals for all four anomers present in solution (see ESM). However, the compound was clearly identified by 2D-NMR measurements and structural similarities to 3b. 1H NMR : δ 9.01–8.99 , 8.97 , 7.50–7.35 , 4.78–4.60 , 4.03–3.96 , 3.95–3.91 , 3.83–3.75 , 3.70–3.62 , 3.61–3.48 , 3.44 , 3.32–3.15 , 3.14–3.00 , 2.98–2.58 , 2.28–2.17 , 1.00–0.90 ppm . For annotation of protons, see ESM. 13C NMR : δ 171.4–171.2, 166.6–166.2, 133.9, 129.3–129.1, 117.7–116.90, 100.8, 99.2, 95.3, 83.2–81.6, 78.4, 74.9, 74.0, 70.3, 68.9, 68.7 64.9–64.3, 63.6–60.1, 51.5–51.30, 50.7, 50.0, 29.2–28.9, 26.2–26.0, 18.5–17.2 ppm. For annotation of carbon nuclei, see ESM. HRMS for C17H28N4O8 (exact monoisotopic mass 416.1907 g/mol): [M+H]+ calcd. 417.1979; found 417.1982; mass accuracy 0.72 ppm.Analytical data for Fru-Val-His l-histidine (His) were included in the HILIC-ESI-MS/MS method. For the HILIC-ESI-MS/MS analysis, an Agilent 1100 series HPLC system, consisting of a G1312A pump and a G1367A autosampler from Agilent Technologies Inc. , was used, coupled to a triple quadrupole API 2000 LC/MS/MS system with ESI source from AB SCIEX  as mass analyzer.All starting materials, intermediates and final products as well as 2O and ACN with a ratio of 70/30 (v/v), containing 5 mM ammonium formate (NH4HCO2). All substances were measured in positive ESI mode. The molecular ion with the highest abundance in positive ESI was determined, performing a manual scan. After choosing the precursor ion for routine multiple reaction monitoring (MRM) analysis, the three most abundant transition ions after collision-induced dissociation (CID) as well as the collision energies (CE) and cell exit energies (CEX) for the used instrument were determined automatically by the system.Each substance was separately injected in the MS system using a syringe pump with the flow rate of 10 μL/min. Single standard solutions for each substance with a concentration between 10 and 25 μg/mL were prepared. The substances were dissolved in a mixture of H2O/ACN in ratios of 95/5 (v/v) and 5/95 (v/v), respectively. Both eluents contained an additional 5 mM of NH4HCO2 and the pH was adjusted to 3.2 by formic acid. The optimal flow rate, starting composition of the eluents, and gradient were determined, performing various runs under different conditions. For all optimization experiments, a mix-standard in H2O/ACN in a ratio of 20/80 (v/v) containing all substances was used. Concentrations of the substances in the mix-standard used for optimization experiments varied from 6.5 to 138 μg/mL (see ESM). Ten microliters of mix-standard was injected for each run.For separation of the included compounds, a HILIC column  was used. The mobile phases A and B were mixtures of H4b was dissolved in 10 mL distilled water. Regarding the 3% of remaining Val-His, the concentration of Fru-Val-His was corrected to 10.9 mg per 10 mL. A total of five samples (S1–S5) were prepared for the assay: a blank which was hemolysis solution (S1), a blood hemolysate sample with an endogenous HbA1c value of 0.185 mmol/L (20.22 mmol/mol HbA1c/Hb (IFCC) and 14.7 g/dL Hb) (S2) (blood sample and hemolysis solution in ratio of 1/20 (v/v)), and three blood hemolysate samples which were spiked with different Fru-Val-His concentrations (S3–S5). For the spiked sample containing the highest amount of Fru-Val-His (S5), 10 μL of the stock solution was added to 990 μL hemolyzed blood, resulting in a final Fru-Val-His concentration of 10.9 mg/L (26.1 μmol/L). 100 µL and 200 μL of S5 were used and the volume adjusted to a total of 300 μL using hemolyzed blood to obtain spiked samples S3 and S4, containing 3.62 mg/L (8.69 μmol/L) and 7.24 mg/L (17.4 μmol/L) of Fru-Val-His, respectively. These three spikes represent HbA1c values of 0.359, 0.553, and 0.707 mmol/L, respectively, considering the endogenous HbA1c value of the blood sample (0.185 mmol/L) and the 1/20 (v/v) dilution of the blood sample during automated measurements of HbA1c. Addition of the reagents and sample was performed manually using the UVIKON XL spectrophotometer and automatically by the automated clinical chemistry analyzer Hitachi 917E. The absorption at 660 nm was started to be measured directly after the addition of the sample, continuing the measurement for 10 min. The temperature was maintained at 37 °C. Proportionality between absorption and the amount of Fru-Val-His was examined, determining the total change in absorption from the starting until the endpoint of the measurement (ΔAbs) which was subsequently plotted against the Fru-Val-His 4b concentration of the sample. ΔAbs of S1 was subtracted as a blank from ΔAbs of the other samples S2–S5.The enzymatic assay was performed manually with an UVIKON XL spectrophotometer from Goebel Instrumentelle Analytik GmbH  and on an automated clinical chemistry analyzer Hitachi 917E from Roche Diagnostics . Donor EDTA blood samples from internal donation were used to assess the functionality of synthetic fructosamines. As stock solution, 11.2 mg Fru-Val-His O-isopropylidene-β-d-fructopyranose  and not in the 1,2:4,5 isomer under the applied reaction conditions (see Scheme iprFru was obtained in a yield of 54% (batch size 84 mmol).To ensure a synthesis of the fructosamines Fru-Val and Fru-Val-His in sufficient yields with the formation of minor amounts of side products, an approach using a protected Fru intermediate was chosen. The hydroxyl groups on carbon atoms two to five (C2–C5) were therefore protected as isopropylidene ketals which are common protecting groups for diols –35, specO-isopropylidene-aldehydo-β-d-arabinohexos-2-ulo-2,6-pyranose which is isopropylidene ketal protected glucosone . The synthesis was conducted following a procedure from the group of Hoffmann [iprGlu was obtained. Due to ambiguous analytical data of iprGlu, which is present as the aldehyde as well as its stable hydrate from, it was converted completely into its stable hydrate form under the addition of water, which subsequently crystallized as white solid [gem-diols, are generally not stable and can therefore not be isolated, there are some exceptions [gem-diol structure was studied by Stefanowicz et al. [iprGlu into its hydrate form was confirmed by NMR and IR spectroscopy (see ESM). The complete reaction pathway of iprGlu as well as its hydrate form is shown in Scheme The remaining free hydroxyl moiety on carbon atom C1, which is the linkage position of the naturally occurring fructosamines, was subsequently oxidized to an aldehyde, allowing for the reaction with the primary amine of Val or Val-His, respectively. The oxidation was achieved by performing a Swern oxidation resulting in 2,3:4,5-di-Hoffmann , and aftte solid . Despiteceptions –41. In tz et al. , who alsiprGlu 2 can be coupled to the free primary amine of the amino acid Val or dipeptide Val-His with its aldehyde group on the C1 position, generating an aldimine which can be subsequently reduced to the desired fructosamine, as shown in Scheme 3CN) as reducing agent. Despite the fact that NaBH3CN does generally not reduce aldehydes [iprFru 1 by MS and NMR analyses as side product. We assume that the unique properties of the aldehyde of iprGlu 2, also responsible for the formation of its stable hydrate form, may be the reason for its reduction by the mild reducing agent NaBH3CN. We adapted the procedure from Walton et al. [iprGlu 2 and the respective amino acid or dipeptide were stirred for 4 h at 70 °C in a mixture of water and methanol (ratio of 1/1 (v/v)), followed by the addition of NaBH3CN and an additional 7 h stirring at 70 °C. The reaction mixture was purified by strong cation exchange (SCX) chromatography, using a DOWEX 50W X2 resin, thereby removing excessive iprGlu 2. However, remaining Val or Val-His are assumed not to be removed by the performed purification and a full conversion was aimed by the adapted reaction procedure. Yields of 97% and 86% for iprFru-Val 3a and iprFru-Val-His 3b were achieved (0.7 mmol batch size), respectively. As final step, the protecting groups of the Fru residue were removed under acidic conditions by stirring the protected fructosamines in a solution of 95 vol% trifluoroacetic acid (TFA) in water. The reaction was monitored by direct ESI-MS analysis and complete removal of the isopropylidene ketal protecting groups was achieved after 7 h. The final products were isolated with almost quantitative yields (Table 1H and 13C NMR spectra (see ESM). From NMR data, the presence of mayor impurities was able to be excluded. Especially spectra from the protected fructosamines iprFru-Val 3a and iprFru-Val-His 3b solely showed signals for nuclei from the synthesized compounds in both 1H as well as in 13C NMR analysis, with all significant signals being able to be unambiguously assigned (see ESM). Due to the multiple signals in NMR data from the final, unprotected fructosamines Fru-Val 4a and Fru-Val-His 4b, purity determination becomes more demanding. However, all signals could be assigned by 2D-NMR experiments and by comparison with NMR data of their protected counterparts. Besides the signals from nuclei of the final products, solely signals from residue solvents diethyl ether, ethanol, and TFA were observed despite the application of high vacuum. In future studies, we aim to lyophilize the final products to fully remove residue solvents. HILIC-ESI-MS/MS data revealed the presence of very slight amounts of remaining starting material Val or Val-His in the final products. This is further discussed in the following section. Combining all analytical data obtained, purities of the final fructosamines Fru-Val 4a and Fru-Val-His 4b were determined all to be above 90%.The resulting ldehydes , 43, forn et al.  by previ4a and Fru-Val-His 4b, a HILIC-ESI-MS/MS method, with the tandem MS working in multiple reaction monitoring (MRM) mode, was developed. An MRM method was chosen instead of the commonly used product ion scan in routine analysis to further increase the sensitivity for the compounds of interest. Besides the final fructosamines, all starting materials and intermediates as well as His were included in the method. For a clear identification of the included substances, three specific transition ions from the most abundant precursor ion in positive ESI mode were determined. Furthermore, optimal parameters for collision-induced dissociation (CID) were determined. The precursor ions and the three determined transitions as well as collision energies (CE) and cell exit potentials (CXP) for each substance are listed in Table iprFru and iprGlu, co-eluting within 1.6 min and 1.7 min, was not possible, as shown in Fig. tR) of the included compounds in the HILIC-ESI-MS/MS method are stated in Table 4a and Fru-Val-His 4b small amounts of remaining starting materials Val and Val-His, respectively. Besides the residual amino acid and dipeptide, no further starting material or intermediates were observed, verifying the complete removal of excessive iprGlu by SCX chromatography as well as a complete deprotection of the isopropylidene protecting groups. Since Fru-Val-His 4b was aimed to be applied in an enzymatic cleavage assay, the amount of Val-His was quantified, recording a calibration curve for Val-His at five different concentrations (see ESM). The amount of Val-His was determined to be 3%, which was taken into account for sample preparations for the subsequent enzymatic cleavage assay of synthetic Fru-Val-His by FPOX.For the routine analysis of synthesized fructosamine derivatives Fru-Val 4b as a well-defined substrate in the characterization validation of an enzymatic HbA1c assay, enzymatic oxidation was performed using reagent components of a commercially available assay (DiaSys Diagnostic Systems GmbH). During regular HbA1c quantification measurement, a hemolyzed blood sample is incubated for 5 min in the presence of reagent 1 (R1), containing FPOX and horseradish peroxidase (HRP). Since the intact protein HbA1c is not a substrate for the used FPOX, the fructosamine residue does not react with R1. However, during this incubation step, endogenous fructosamine derivatives present in the blood sample are oxidized and the released hydrogen peroxide is degraded by the HRP present, thereby increasing the specificity of HbA1c detection later [1c is enzymatically cleaved by the protease. The liberated Fru-Val-His is subsequently processed by FPOX generating hydrogen peroxide which is used by HRP to generate a dye by oxidizing the leuco dye. The operating procedure of the commercial enzymatic HbA1c assay is shown in Scheme 1c concentration in the sample.To prove the potential of the generated synthetic Fru-Val-His on later . After a4b, a different increase in absorbance could be observed  into account . The chosen Fru-Val-His concentrations in the enzymatic degradation reflect the HbA1c concentration in human blood which varies between about 0.06 and 0.46 mmol/L [R2) of 0.9870 and a slope of 101 mAU/mmol/L Fru-Val-His . Premixed R1 and R2 components of this assay are subsequently added to the hemolysate, as shown in Scheme ved Fig. . Furtherunt Fig. . The cho6 mmol/L , 49. PerHis Fig. . Comparal/L Fig. , therebyl/L Fig. , synthet1c quantification, proving its functionality as a specific enzymatic substrate. Taken together, we here provide a new, fast, and efficient protocol for the synthesis and purification of well-defined fructosamines in suitable yield and purity that can be used for the validation and standardization of enzymatic assays for HbA1c quantification. Furthermore, the presented synthetic approach can be adapted also for the synthesis of different fructosamines, studying their harmfulness on the organism or the formation of advanced glycation end-products (AGEs). A solid-phase synthesis approach as well as an additional purification of fructosamines by preparative HPLC can be performed if needed in future studies. Synthetic Fru-Val-His material can also be formulated in specific blood-based matrices to be potentially used as an independent control material and evaluated using IFCC-based reference material and quantification methods in a next step. In addition, it could also open new ways to characterize the enzymes processing fructosamines  in various applications.In the presented work, we introduced a synthetic approach towards the two clinically relevant fructosamines Fru-Val and Fru-Val-His via a protected glucosone intermediate. The two synthetic fructosamines were obtained in suitable quantity and purities > 90%. The structures of the synthesized products were fully characterized by different NMR experiments. Besides their synthesis, a routine HILIC-ESI-MS/MS method was developed, including starting materials and intermediates, allowing for high-throughput quality control of synthesized reference fructosamines. Despite their partial co-elution, all compounds were specifically identified by tandem MS operating in MRM mode. Synthetic fructosamine Fru-Val-His was further applied in an established enzymatic oxidative degradation assay using FPOX for HbAESM 1(PDF 4234 kb)


Context: A previous study demonstrated that the chloroform extract of Salvia connivens Epling (Lamiaceae) has anti-inflammatory activity.Objective: Identification of the active components in the dicholorometane extract (DESC), and, standardization of the extract based in ursolic acid.Material and methods: DESC was prepared by percolation with dichlromethane and after washed with hot hexane, its composition was determined by CG-MS and NMR, and standardized by HPLC. The anti-inflammatory activity was tested on acute TPA-induced mouse ear oedema at doses of 2.0 mg/ear. The cell viability of macrophages was evaluated by MTT method, and pro- and anti-inflammatory interleukin levels were measured using an ELISA kit.Results: Ursolic acid, oleanolic acid, dihydroursolic acid and eupatorin were identified in DESC, which was standardized based on the ursolic acid concentration (126 mg/g). The anti-inflammatory activities of DESC, the acid mixture, and eupatorin (2 mg/ear) were 60.55, 57.20 and 56.40% inhibition, respectively, on TPA-induced ear oedema. The IC50 of DESC on macrophages was 149.4 μg/mL. DESC (25 μg/mL) significantly reduced TNF-α (2.0-fold), IL-1β (2.2-fold) and IL-6 (2.0-fold) in macrophages stimulated with LPS and increased the production of IL-10 (1.9-fold).Discussion: Inflammation is a basic response to injuries, and macrophages are involved in triggering inflammation. Macrophage cells exhibit a response to LPS, inducing inflammatory mediators, and DESC inhibits the biosynthesis of the pro-inflammatory and promote anti-inflammatory cytokines.Conclusion: DESC has an anti-inflammatory effect; reduced the levels of IL-1β, Il-6 and TNF-α; and increases IL-10 in macrophages stimulated with LPS. Ursolic acid is a good phytochemical marker. Inflammation is a response to an injury as part of the body’s immune response Weiss . InitialSalvia (Lamiaceae) includes more than 900 species. Several plants from this genus have been used for their beneficial healing properties . These compounds possess significant anti-inflammatory activity  was deposited in the Herbarium Isidro Palacios of the Universidad Autónoma de San Luis Potosí.Fresh aerial parts of Dried, ground aerial parts (100 g) were extracted by percolation with dichloromethane and washed with hot hexane. The solvent was then evaporated to dryness in a rotatory evaporator under reduced pressure; the yield was 5.1%.O-tetradecanoylphorbol-13-acetate (TPA), and the composition of the fractions with anti-inflammatory activity was determined.The extract (5 g) was separated by column chromatography. The column was packed with silica gel , which was prepared using hexane as the mobile phase. Then, the polarity was increased with ethyl acetate. Fractions of 100 mL were collected and compared by thin-layer chromatography; fractions with the same chromatographic pattern were pooled. The resulting fractions were tested on ear oedema in mice induced by 12-O-bis(trimethylsilyl)trifluoroacetamide with 10% of trimethylchlorosilane was added to the samples and heated to 100 °C for 10 min in a CEM Discover microwave at 150 W and 290 psi .Fraction 7, which was obtained with hexane/ethyl acetate (FHA) (90:10 v/v), produced a white solid (465 mg). Prior to the GC-MS analysis, the mixture was derivatized into trimethylsililethers as follows: 7 mg of the solid or 2 mg of the standard samples were dissolved in 2 mL isooctane. For trimethylsilylation, 100 μL of N,GC-MS analysis was performed on a gas chromatograph coupled to a mass spectrometer , which was coupled to a mass selective detector (model 5973) with an HP-5MS capillary column . The injector temperature was 250 °C. This temperature program started at 150 °C for 2 min with a heating rate of 15 °C/min up to 250 °C; this temperature was maintained for 1 min. A splitless injection was performed at a ratio of 1:100, and the injector temperature was 320 °C. The spectra were measured at 70 eV. The compounds were identified based on their mass spectra by comparison to the spectra of the standard samples.After derivatization, the white solid was identified as a mixture of three compounds, which were the following trimethylsililethers:m/z (%): 600 , 585 (6.6), 482 (21.7); 320 (31.1), 279 (4.7), 203 (100.0), (35.8), 133 (54.9), 73 (80.4);41.8% oleanolic acid . MS (EI)m/z (%): 600 , 585 (8.8), 482 (15.7), 320 (93.1), 279 (11.8), 203 (100.0), (40.2), 133 (54.9), 73 (80.4); and47.69% ursolic acid . MS (EI)m/z (%): 598 , 583 (3.9), 481 (12.6), 305 (14.6), 201 (41.7), (36.9), 73 (100.0).11.13% dihydroursolic acid . MS (EI)max = 3433, 3070, 2945, 1652, 1603, 1271, 841 cm−1; 1H-NMR; Agillent NMR Spectrometer DD2, Palo Alto, CA : δ 3.73 , 3.86 , 3.91 , 6.76 , 6.86 , 7.07 , 7.44 , 7.54 , 12.87 . 13C-NMR (150 MHz), δ: 56.12, 56.66, 60.28, 91.99, 103.75, 105.59, 112.65, 113.72, 119.49, 123.70, 132.61, 147.71, 152.26, 152.99, 153.62, 159.57, 164.79, 183.15. MS (EI): m/z (%): 344 , 329 (88.9), 315 (24.7), 298 (24.7), 153 (24.7).A yellow solid was obtained from fraction 13 with hexane/ethyl acetate  and identified as eupatorin : 76.5 g;2 with a flow of 1.7 L/min at 67 °C evaporation temperature. The chromatographic conditions were evaluated and optimized in a Grace Smart RP C18 column . The column temperature was 40 °C. The flow rate was set to 0.7 mL/min. The linear gradient elution of the mobile phase was initiated with 0.5% (v/v) acetic acid in a water-methanol-acetonitrile solution with a ratio of 5:30:65 (v/v) and completed after 20 min with a composition of 0.5% acetic acid in a water-methanol-acetonitrile solution with a ratio of 5:10:85 (v/v). The curve calibration was realized with six points  of ursolic acid standard . Each point was obtained from the average of three injections; the volume was adjusted to 20 μL for all samples. The integral chromatographic peak, which was obtained at 6.21 min of retention time, was used to generate the standard calibration curve (R2 = 0.99637). DESC (6 mg) was dissolved in 1 mL of methanol (0.6 mg/mL), and 20 μL of the sample were injected by triplicate.The DESC was analyzed by HPLC  with an Evaporative Light Scattering Detector (ELSD)  using Nad libitum. The experiments were conducted according to the Official Mexican Norm NOM 062-ZOO-1999 . All animal procedures were approved by the Research Bioethics Committee of Universidad Autónoma Metropolitana-Xochimilco.Male CD-1 mice (20–25 g) from the Universidad Autónoma Metropolitana-Xochimilco Animal Facility were housed in isolated cages at 24 °C under 12 h light/dark cycle. The animals were supplied with Lab Diet 5001 food and water The method for TPA-induced mouse ear oedema has been described previously -2,5-diphenyl tetrazolium bromide (MTT) at 5 mg/mL in a PBS buffer were added, and the plates were incubated for 4 h at 37 °C. Then, the medium was removed, and the formazan crystals were dissolved in 100 μL of DMSO. The optical density (OD) was determined at 540 nm in an ELISA plate reader from BioRad. Six replicate wells were used to determine the viability using the following equation.J774A.1 macrophages were washed with a PBS buffer. The solution was decanted, and the cells were seeded in a Dulbecco’s Modified Eagle’s Medium (DMEM) in 96-well microplates at a density of 8 × 10TNF-α, IL-6, IL-10, and IL-1β serum levels were measured using a commercially available ELISA kit according to the manufacturer’s instructions. The OD was measured in a microplate reader at 405 nm with a wavelength correction set at 650 nm.6. Then, the cells were treated separately with the DESC at a concentration of 25 μg/mL, whereas another group was treated with IND at a concentration of 50 μM. The cells were incubated for 2 h. After treatment with LPS (1 μg/mL) and incubated for 24 h, the cell-free supernatants were collected and stored at −20 °C until they were analyzed by immunoassay for the quantification of cytokines. Specifically, the concentrations of IL-6, IL-1β, IL-10 and TNF-α in the supernatants of the J774A.1 cell culture were determined using a commercial immunoenzymatic kit PEPROTECH . J774A.1 macrophages were grown at a density of 1 × 10t-test and ANOVA, which was followed by Tukey’s test. Statistical significance was set at p < 0.05.The results are expressed as the means ± SEM. Statistical analyses were performed using Student’s The white solid obtained in fraction 7 was identified by GC-MS analysis as a mixture of the following three compounds: oleanolic acid (41.18%), ursolic acid (47.69%) and hydroursolic acid (11.13%)  as the flavonoid eupatorin (Lantana montevidensis (Spreng) Briq. . The results showed that the DESC contains 126 mg/g (12.6%) of ursolic acid.The DESC was standardized using ursolic acid as phytochemical marker. This compound displayed a retention time of 6.1 min; a representative chromatogram of ursolic acid and DESC is presented in DESC, the acids mixture and eupatorin at doses of 2 mg/ear reduced TPA-induced ear oedema by 60.55 ± 1.8%, 57.2 ± 1.1% and 56.4 ± 1.4%, respectively. The inhibitory activities of the three of them were similar to that observed when IND at a dose of 2 mg/ear IND (64.5 ± 1.4%).50 was found as 149.4 μg/mL. The effect of DESC and IND on the production of the pro-inflammatory cytokines TNF-α, IL-1β and IL-6 and the anti-inflammatory cytokine IL-10 was measured in the culture medium of macrophages stimulated with 1 μg/mL of LPS alone or in combination with 25 μg/mL of DESC . Thus, we suggest that the anti-inflammatory effect of DESC is due to ursolic acid (Baricevic et al. Ursolic acid suppresses the expression of COX-2 and iNOS (Suh et al. The effect of DESC and IND on the production of the pro-inflammatory cytokines TNF-α, IL-1β and IL-6 and the anti-inflammatory cytokine IL-10 was measured in the culture medium of macrophages stimulated with 1 μg/mL of LPS alone or in combination with 25 μg/mL of DESC . DESC siInflammation is a basic response to injuries and protects the body from inflammatory stimuli, and macrophages are involved in triggering inflammation in pathological conditions. Macrophage cells exhibit a response to LPS, which is an endotoxin that induces a variety of inflammatory mediators, such as TNF-α, IL-1β and IL-6 Rankin . Thus, tDESC has an anti-inflammatory effect; reduced the levels of IL-1β, Il-6 and TNF-α; and increases the production of IL-10 in macrophages stimulated with LPS. One of the compounds responsible for this activity is ursolic acid, which was used for DESC standardization by HPLC. This method was adequate.


Bacillus licheniformis strain BS-3 was purified to homogeneity by ammonium sulfate precipitation, DEAE-52 column chromatography and Sephadex G-75 column chromatography. The purified protein was designated as F2 protein, inhibited the growth of Aspergillus niger, Magnaporthe oryzae and Rhizoctonia solani. F2 protein was a monomer with approximately molecular weight of 31 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gave a single peak on High Performance Liquid Chromatography (HPLC). Using Rhizoctonia solani as the indicator strain, the EC50 of F2 protein was 35.82 µg/mL, displaying a higher antifungal activity in a range of pH 6.0 to pH 10.0, and at a temperature below 70 °C for 30 min. F2 protein was moderately resistant to hydrolysis by trypsin, proteinase K, after which its relative activities were 41.7% and 59.5%, respectively. F2 protein was assayed using various substrates to determine the enzymatic activities, the results showed the hydrolyzing activity on casein, however, no enzymatic activities on colloidal chitin, CM-cellulose, xylan, M. lysodeikticus, and p-nitrophenyl-N-acetylglucosaminide.An antifungal protein produced by  Bacillus licheniformis is a saprophytic bacterium that is widespread in Nature. It has been applied widely to produce proteases . The mobile phase consisted of 0.2% trifluoroacetic acid (TFA) in water (solvent A) and 0.2% TFA in acetonitrile (solvent B) using the following gradient program: 0–45 min, 10%–90% B. The flow rate was 2.0 mL/min, and the sample injection volume was 20 μL. The pattern of the eluent was monitored at 280 nm.The concentrated F2 sample obtained from Sephadex G-75 column chromatography was filtered through a 0.45-μm polytetrafluoroethylene (PTFE) membrane  and injected onto a Agilent Zorbax 300SB-CRhizoctonia solani as the indicator strain. To determine the thermal stability by two methods, the purified antifungal protein was treated at pH 7.0 and different temperature  for 30 min. After cooling to room temperature, the residual antifungal activity was assayed. To assay the resistance of the purified antifungal protein to pH changes, the pH was adjusted to 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 with 0.1 mol/L HCl and 0.1 mol/L NaOH and kept at room temperature for 30 min. Then each sample was adjusted to pH 7.0 and assayed for the residual activity.The sensitivity of the antifungal protein to enzymes was tested using the purified antifungal protein, which was treated at 37 °C and pH 7.2 for 2 h with trypsin and proteinase K. The final concentration of each enzyme was 1 mg/mL. After incubation, the compounds were assayed for the residual activity using For measuring the following several enzyme activities, F2 protein solution was prepared by distilled water to 0.1 mg/mL. Chitinase activity was measured using colloidal chitin as substrate . F2 protCellulase xylanase or activity was measured using carboxymethyl cellulose (CMC) or larch wood xylan as substrates. F2 protein solution (0.1 mL) was added to substrate solution (0.4 mL), which contained 0.5% xylan or 1.25% CMC in an acetate buffer solution . Following incubation at 37 °C for 10 min and centrifuging, the amount of reducing sugar produced in the supernatant was measured using the dinitrosalicylic acid (DNS) method. One unit of xylanase or cellulase activity was defined as the quantity of enzyme required to release 1 µmol of reducing sugars per minute at 37 °C and pH 5.0.To assay protease activity, F2 protein solution (0.2 mL) was mixed with 1% (w/v) casein in phosphate buffer (pH 7.0) (2.5 mL) and incubated at 37 °C for 10 min. The reaction was stopped by the addition of 0.19 mol/L trichloroacetic acid (5 mL). The reaction mixture was then centrifuged and the soluble peptide in the supernatant was measured using tyrosine as the reference compound. One unit of protease activity was defined as the quantity of enzyme required to produce 1 µmol of tyrosine per minute.M. lysodeikticus cells. The reaction mixture contained a M. lysodeikticus cell suspension  in 50 mmol/L phosphate buffer (pH 7.0) and F2 protein solution (1.5 mL). Cell suspensions of M. lysodeikticus were prepared as described previously [Lysozyme activity was determined using a spectrophotometer by measuring the rate of lysis of eviously . The mixeviously .N-acetylglucosaminidase was assayed with p-nitrophenyl-N-acetyl-β-D-glucosaminide (p-NP-GlcNAc) as substrate. The reaction mixture contained 5 mmol/L p-NP-GlcNAc (0.1 mL) in 50 mmol/L sodium phosphate buffer (pH 7.0) and F2 protein solution (0.1 mL) with a total volume of 0.2 mL. The reaction was stopped by adding 0.25 mol/L Na2CO3 (2 mL) following incubation for 10 min at 37 °C and the absorbance of the p-nitrophenol released was measured at 405 nm. One unit of enzyme activity was defined as the quantity of enzyme required to release 1 µmol of p-nitrophenol per minute at 37 °C.licheniformis BS-3 was purified and characterized. The purification to homogeneity to F2 protein by mmonium sulfate precipitation, DEAE-52 column chromatography and Sephadex G-75 column chromatography. F2 protein was purified 18.80-fold and recovery was 43.15%. The relative molecule weight of F2 protein was estimated to be 31.0 kDa on SDS-PAGE. F2 protein had better inhibitory effects on Aspergillus niger, Magnaporthe oryzae, Rhizoctonia solani and Fusarium oxysporum (schl.) f. sp. benincasae. Using Rhizoctonia solani as the indicator, F2 protein displayed higher antifungal activity, retaining more than 97% activity at temperature below 70 °C and above 90% in pH 6 to pH 10 conditions, the optimal pH was pH 8.0. F2 protein was assayed using various substrates to determine the enzymatic activities, it showed the hydrolyzing activity on casein, however, no enzymatic activities on colloidal chitin, CM-cellulose, xylan, M. lysodeikticus, p-nitrophenyl-N-acetylglucosaminide. This antifungal protein is expected to develop as a biological prevention agent for farm crops.In the present study, an antifungal F2 protein from B.


At 30 min prior to the start of in vivo pharmacological testing, administration of 2-AP , morphine (6 mg/kg i.p.), dexamethasone (2 mg/kg s.c.), or vehicle alone was performed. In the acetic acid-induced abdominal writhing tests, pretreatment with 2-AP significantly reduced the number of abdominal writhes, as well as decreased licking times in the glutamate and formalin tests. Investigation of the mechanism of action using the formalin model led to the conclusion that the opioid system does not participate in its activity. However, the adenosinergic system is involved. In the peritonitis tests, 2-AP inhibited leukocyte migration and reduced releases of proinflammatory mediators TNF-α and IL-1β. In vitro antioxidant assays demonstrated that 2-AP presents significant ability to sequester superoxide radicals. In silico docking studies confirmed interaction between 2-AP and the adenosine A2a receptor through hydrogen bonds with the critical asparagine 253 residues present in the active site. Investigation of 2-AP demonstrated its nociception inhibition and ability to reduce reactive oxygen species. Its interaction with A2a receptors may well be related to proinflammatory cytokines TNF-α and IL-1β reduction activity, corroborating its antinociceptive effect.2-Allylphenol (2-AP) is a synthetic phenylpropanoid, structurally related to cardanol, thymol, and ortho-eugenol. Phenylpropanoids are described in the literature as being capable of promoting biological activity. Due to the similarity between 2-AP and other bioactive phenylpropanoids, the present research aims at evaluating the antioxidant, antinociceptive, and anti-inflammatory potential of 2-AP in silico,  This sensitization initially occurs in nociceptive neurons near injured tissue, leading to increases in pain sensation at the site  to canonize the structure, add hydrogens, perform aromatic form conversions, clean the molecular graph in three dimensions, and save the compounds in sdf format [The structure of 2-AP was used as input data for Marvin 14.9.1.0, 2014, ChemAxon (f format .http://www.rcsb.org/pdb/home/home.do) [The human adenosine A2a receptor structures in complex with endogen ligand (PDB ID 2YDO) and agonist UKA (PDB ID 3QAK) were downloaded from the Protein Data Bank (home.do) , 21. Thehome.do) . All of home.do) .Three-dimensional structures (3D) were used as input data in the Volsurf+ program v. 1.0.7 and were subjected to molecular interaction fields (MIF)  to generp) of less than 0.05.The results were analyzed using one-way analysis of variance (ANOVA) method followed by the Dunnett test for comparison between the means. Data were expressed as mean±SEM (standard error of the mean), and values were considered significant when they presented a level of significance (p < 0.001), with respective inhibition percentages of 49.4%, 80.1%, 88.3%, and 90.0%. Morphine as expected reduced the contortion response by 89.3%  (2-AP at doses of 25 (14.3 ± 2.9), 50 (5.3 ± 1.5), 75 (3.3 ± 1.9), and 100 (2.8 ± 2.2) mg/kg presented a significant reduction in the number of contortions induced by acetic acid as compared to the control group (28.5 ± 2.3) (< 0.001) .p < 0.001). The two lower doses of 2-AP (50 and 75 mg/kg) were not effective in the glutamate test. MK-801, an NMDA receptor antagonist, reduced glutamate-induced nociceptive behavior by 93.3%  (Pretreatment with 2-AP at a dose of 100 (25.5 ± 9.1 s) mg/kg reduced the glutamate-induced licking time, with an inhibition value of 82.5%, compared to the control group (< 0.001) .p < 0.05), with respective inhibition rates of 37.4% (50.3 ± 7.3 s) and 35.8% (51.6 ± 11.1 s) in the first phase and 69.0% (68.7 ± 32.2 s) and 93.0% (15.4 ± 10.0 s) in the second phase. Morphine reduced the behavior in both phases, respectively, 70.2% (23.9 ± 8.4 s) and 98.6% (3.2 ± 3.2 s) (p < 0.001)  (< 0.001) .p < 0.001). The effect produced by morphine was significantly reversed  was not reversed as a function of prior naloxone administration (reversed .p < 0.05)  expressively in the second phase of the formalin test ( < 0.05) .μL) inhibited leukocyte migration at doses of 50 and 100 mg/kg by 35.9% (7.9 ± 1.6 × 106 leukocytes/mL) and 51.75% (5.8 ± 0.8 × 106 leukocytes/mL), respectively, when compared to the controls (12.0 ± 1.1 × 106 leukocytes/mL) (p < 0.001). As expected, dexamethasone reduced leukocyte migration by 64.6% (4.3 ± 1.4 × 106 leukocytes/mL) (p < 0.001) (2-AP administered 30 min before administration of carrageenan (1% 300 < 0.001) .α levels by 74.0% (172.2 ± 93.2) compared to the controls (662.5 ± 42.5) (p < 0.0001). To assess IL-1β levels, 2-AP at doses of 50 and 100 mg/kg reduced IL-1β by 45.8% (319.5 ± 69.1) and 63.4% (215.6 ± 35.4), respectively, when compared to the control group (589.4 ± 42.2) (p < 0.0001). Dexamethasone reduced levels of TNF-α by 99.8% (0.7 ± 0.8) (p < 0.0001) and IL-1β by 79.9% (117.9 ± 115.0) (p < 0.001)  .The present study demonstrated DPPH radical sequestering activity by 2-allylphenol in differing concentrations . At the concentration of 0.25 mg/mL, sequestering activity was 34.3%, greater than the activity occurring at the larger concentrations which did not present significant differences between them .In the hydroxyl radical sequestering activity tests, for 2-allylphenol at the concentration of 0.5 mg/mL, there was no inhibition of the hydroxyl radical; however, in the other concentrations , there were activity increases, with significant differences between the concentrations tested .2-Allylphenol presented high percentages of superoxide radical sequestration at concentrations of 0.1 (88.40 ± 4.2%), 0.25 (96.73 ± 0.19%), and 0.5 mg/mL (96.08 ± 1.11%), with no significant differences between them .The total antioxidant capacity was expressed in gallic acid equivalents . 2-AP presented a TAC of 319.97 ± 0.38 mg/g.In 2YDO crystallography, 2-AP presented interactions with critical residues of threonine (Thr) 88 and asparagine (Asn) 253 Figures  forming The analgesic activity study was initiated with acetic acid-induced contortion testing, a classic model of pain used for screening analgesic and anti-inflammatory substances . AdminisGinkgo biloba extract reduces the influx of calcium in hippocampal neurons, preventing the onset of noxious stimulus.Glutamate is the principal neurotransmitter of the central nervous system, involved in various physiological processes such as memory, learning, and excitotoxicity . GlutamaAs a model of neurogenic and inflammatory pain, the formalin test was performed to evaluate the antinociceptive properties of 2-AP. The neurogenic phase, the first phase, is directly related to chemical stimulation of receptors with probable involvement of substance P, glutamate, and bradykinin. The second phase corresponds to inflammatory pain caused by the release of serotonin, histamine, and prostaglandins . CentralInvestigation of mechanisms of action is based on the use of known pharmacological antagonists, which, if previously administered, block effects resulting from specific receptor activation and prevent the substance under study from binding to its receptor and promote its activity.The first system to be investigated was the opioid system. Opioid receptors are expressed in nerves involved in pain transmission (sensory ascending trajectory) and modulation (descending inhibitory trajectory) in sites located at the spinal and supraspinal levels .In order to verify participation of the opioid system in the antinociceptive effect of 2-AP, the animals were previously treated with naloxone (an anatomopathic opioid). The results presented no reversal of 2-AP antinociceptive effect and indicated that such effect is not due to opioid receptor activation. This preliminary result demonstrates a certain advantage over morphine because 2-AP is able to exert similar antinociceptive activity, yet for not acting on the opioid system, it is possible that 2-AP would not cause respiratory depression or other side effects characteristic of opioids.2+ channels followed by postsynaptic hyperpolarization occurs. In supraspinal regions, the mechanism involves interaction with K+ channels, increasing their activity and consequent hyperpolarization [Participation of the adenosinergic system in the 2-AP mechanism of action was then investigated. Adenosine acts as an endogenous signaling agent, acting on four distinct extracellular G protein-coupled receptors  . Preclinrization –41. A2A rization . A3 recerization .In interaction with caffeine, 2-AP presented a reversal of its antinociceptive effects in both phases of the formalin test. From these results, it is suggested that A1, A2A, and/or A3 receptors may be involved in the mechanisms of central and peripheral antinociceptive activity exerted by 2-AP.In the docking study using crystallography of the A2a adenosine receptor, hydrogen-binding (H-bond) interactions between 2-AP and the critical Asn 253 residue were observed. From the crystallographic structure of adenosine (2YDO), the ligand is capable of forming two hydrogen bonds with the Asn 253 residue, suggesting the relevance of this receptor interaction. The synthetic agonist (UKA), which was designed specifically for high A2a receptor selectivity , interacIn terms of polarity (LogP 2.77), 2-AP presents an intermediate character; its lipophilic/hydrophilic balance can be associated with its residue interactions since Asn, Thr, and Ser are polar group holders and form hydrogens with the hydrophilic region of the molecular surface, represented by the hydroxyl group; whereas the conformation of 2-AP within the active site indicates that its hydrophobic region is accommodated within the protein.α. Corroborating these findings, Ravani et al. [The A2A receptor is peripherally expressed in cells of the immune system and plays a key role in the mechanism of inflammation; its activation is commonly associated with reduced inflammatory status . Antonioi et al.  considerα and IL-1β, corroborating its antinociceptive effect.The docking studies confirmed an affinity between 2-AP and the A2a receptor; this interaction may be related to a reduction in proinflammatory cytokines TNF-Administration of carrageenan i.p. generates an inflammatory process characterized by leukocyte aggregation and fluid extravasation; with participation of cytokines, nitric oxide, leukotrienes, and PGE2 . Our datα and IL-1β, which are found at high levels in animal models of neuropathic pain. Reduction of such cytokines, both in animal models and clinical studies, has been shown to decrease painful sensation [Central and peripheral nervous system inflammatory processes play an important role in the development of various persistent pathological conditions of pain . Inflammensation , 50.α and IL-1β proinflammatory cytokines, suggesting that it achieves antinociception by reducing nociceptor sensitization as promoted by these inflammatory mediators.In the present study, it was verified that 2-AP decreases levels of both TNF-Ginkgo biloba extract have demonstrated its effectiveness in reducing inflammation by acting as a free radical scavenger against reactive oxygen species involved in inflammatory processes [Taylor et al.  demonstrrocesses .Oxidative stress occurs as a result of deregulation of the antioxidant/oxidant balance, favoring increased production of reactive oxygen or nitrogen species and leading to inflammation-related processes . The antThe total antioxidant capacity (TAC) test determines the amount of free radicals sequestered by a sample and allows evaluation of the antioxidant capacities of natural compounds . A low Tμg mL-1, which is higher than that presented by cardanol, and which in the same concentration was also able to sequester 15.3% of the DPPH radical [50) at 3.22 μg/mL) was very active in the DPPH test and highlighted its antioxidant potential as compared to the other liquid constituents tested in cashew nut shells. Though 2-AP demonstrated DPPH radical sequestering activity, it was much more discreet within its parameters of comparison.The DPPH test is widely accepted as a tool for assessing antiradical activity of antioxidants. Due to the harmful effects of free radicals on biological membranes, this is extremely important . However radical . Oliveir radical  has repoThe hydroxyl radical inhibition test was performed using the Fenton system which generates reaction between ferrous iron and hydrogen peroxide . The hyd2-AP sequestrated hydroxyl radicals in a concentration-dependent manner at the final three concentrations tested, reaching a maximum of 6.15%. This feature suggests that phenylpropanoids might be used in skin wounds, reducing OH radicals generated by UV rays . The resμg/mL. A similar study involving Ginkgo biloba essential oil observed inhibition of this superoxide radical by 72.24% [Superoxide radicals are produced normally in cells and exert a catalytic function towards formation of several other species of radicals. Due to the direct influence of the radical superoxide in various diseases, inhibition becomes important . 2-AP pry 72.24% .The superoxide anion is a ROS released by macrophages and neutrophils and is responsible for activating and sensitizing nociceptive neurons, amplifying the experience of pain . It was α and IL-1β reductions, being relevant data that corroborate its antinociceptive effect. 2-AP was able to sequestrate the superoxide radical, demonstrating its antioxidant activity. Docking, interactions between 2-AP and critical residues present in the active site of the A2a adenosine receptor were identified, suggesting agonistic action for this receptor subtype. Given our results, the clinical potential of 2-allyphenol to treat pain and inflammation is evident (2-AP presents antinociceptive activity via adenosinergic system participation. The anti-inflammatory properties of 2-AP involve TNF- evident .


Body mass index (BMI) overweight/obesity thresholds in South Asian (SA) adults, at equivalent type-2 diabetes risk are lower than for white Europeans (WE). We aimed to define adjusted overweight/obesity thresholds for UK–SA children based on equivalent insulin resistance (HOMA-IR) to WE children.In 1138 WE and 1292 SA children aged 9.0–10.9 years, multi-level regression models quantified associations between BMI and HOMA-IR by ethnic group. HOMA-IR levels for WE children were calculated at established overweight/obesity thresholds (at 9.5 years and 10.5 years), based on UK90 BMI cut-offs. Quantified associations in SA children were then used to estimate adjusted SA weight-status thresholds at the calculated HOMA-IR levels.2, 21.3 kg/m2 (boys) and 20.0 kg/m2, 22.5 kg/m2 (girls). At equivalent HOMA-IR, SA overweight and obesity thresholds were lower by 2.9 kg/m2 (95% CI: 2.5–3.3 kg/m2) and 3.2 kg/m2 (95% CI: 2.7–3.6 kg/m2) in boys and 3.0 kg/m2 (95% CI: 2.6–3.4 kg/m2) and 3.3 kg/m2 (95% CI: 2.8–3.8 kg/m2) in girls, respectively. At these lower thresholds, overweight/obesity prevalences in SA children were approximately doubled . Patterns at 10.5 years were similar.At 9.5 years, current WE BMI overweight and obesity thresholds were 19.2 kg/mSA adjusted overweight/obesity thresholds based on equivalent IR were markedly lower than BMI thresholds for WE children, and defined more than half of SA children as overweight/obese. Body mass index (BMI), the most widely used method for assessing adiposity, underestimates total body fat (BF) in South Asian (SA) people . SeveralIn the Child Heart and Health Study England (CHASE), a school-based study of 9.0–10.9 year UK children of different ethnic origins  (HOMA-IRinteraction = 0.01), which was included in the model as a fixed effect. Standardised residual plots were used to assess the model fit.Statistical analyses were performed using Stata v14. HOMA-IR was positively skewed and log transformed  for analysis. Multi-level regression models were fitted using the residual maximum likelihood approach to assess the associations between log HOMA-IR (dependent variable) and BMI in both ethnic groups for both boys and girls. Models were adjusted for age (continuous), sex, ethnic group and month of measurement (fixed effects) with a random effect to allow for clustering of children at school level. Two-way interaction terms between BMI and both sex and ethnicity were tested using the Wald test at the 5% significant level. A statistically significant interaction was detected between BMI and ethnic group  aged 9–10 years without diabetes provided complete data, including 1138 WE and 1292 SA children. Characteristics of the analysis sample are summarised in Supplementary Table 2 increase in BMI was associated with a 9.2% (95% CI: 8.2–10.1%) increase in HOMA-IR in WE and a 10.8% (95% CI: 10.0–11.7%) increase in SA children. Histograms and normal plots of the standardised residuals from the regression models did not show any departures from normality, and there was no evidence of residual curvature when standardised residuals were plotted against fitted values.After adjusting for age, sex and month of measurement, each 1 kg/m2 and 21.3 kg/m2, respectively. The equivalent HOMA-IR levels corresponding to these thresholds were 0.8 and 0.9, respectively. At these HOMA-IR levels, overweight and obesity thresholds for SA boys were lower by 2.9 kg/m2 (95% BCI: 2.5–3.3 kg/m2) and 3.2 kg/m2 (95% BCI: 2.7–3.6 kg/m2), respectively  and 3.3 kg/m2 (95% BCI: 2.8–3.8 kg/m2), respectively  and 3.3 kg/m2 (95% BCI: 2.8–3.8 kg/m2), respectively and for SA girls were lower by 3.1 kg/m2 (95% BCI: 2.7–3.6 kg/m2) and 3.5 kg/m2 (95% BCI: 2.9–4.0 kg/m2), respectively  to examine the feasibility of developing ethnic-specific BMI thresholds for overweight and obesity for UK–SA children based on equivalent HOMA-IR to those in WE children. In SAs, HOMA-IR levels were markedly higher and the associations between HOMA-IR and BMI were stronger, so that BMI thresholds for overweight and obesity at equivalent HOMA-IR were markedly lower.2 lower. These differences in BMI thresholds between SA and WE children are appreciably larger than those in our earlier UK study based on equivalent total BF (~1.1 kg/m2 for 4–12 year olds) [2 [2 lower [To our knowledge, this is the first report to estimate overweight and obesity thresholds in UK–SA children based on equivalent HOMA-IR. On this basis, BMI thresholds defining overweight and obesity in UK–SA children aged 9–10 years  would be ~3 kg/mar olds) . Howeverolds) [2  or 3–4 k[2 lower .Thus derivation of ethnic-specific BMI thresholds for SA children based on equivalent HOMA-IR (reflecting their higher emerging T2D risk at any given BMI level) would be feasible, though further validation over a wider age-range would be needed. However, the observed prevalences of overweight and obesity in SA children using these adjusted thresholds are extremely high, with more than half of SAs aged 9–10 years identified as overweight or obese. These findings emphasize the major population health challenge represented by overweight, obesity and T2D in the UK–SA population. Although individually based identification and management of overweight and obese children could play a role in prevention, focussed population-wide strategies for controlling weight gain in SA children, emphasizing the maintenance of healthy diets with appropriate energy intakes coupled with sustained physical activity through childhood and beyond, will be particularly important.Supplementary Material


Increasing age negatively affects different phases of bone fracture healing. The present study aimed to explore underlying mechanisms related to bone fracture repair in the elderly. GSE17825 public transcriptome data from the Gene Expression Omnibus database were used for analysis. First, raw data were normalized and differentially expressed genes (DEGs) were identified. Next, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses were implemented to evaluate pathways and DEGs. A protein–protein interaction (PPI) network was then constructed. A total of 726, 861, and 432 DEGs were identified between the young and elderly individuals at 1, 3, and 5 days after fracture, respectively. The results of GO, KEGG, and PPI network analyses suggested that the inflammatory response, Wnt signaling pathway, vascularization-associated processes, and synaptic-related functions of the identified DEGs are markedly enriched, which may account for delayed fracture healing in the elderly. These findings provide valuable clues for investigating the effects of aging on fracture healing but should be validated through further experiments. The eldFracture healing is a complex but well-orchestrated process that can be temporally divided into three partially overlapping processes, namely, inflammation, repair, and remodeling –9. In thHowever, changes in the inflammatory system occur with age. In older animal models, prolonged inflammation results in delayed chondrogenesis and smaller callus size , 13. AgeIn this study, transcriptional analysis of GSE17825 was used to illustrate molecular mechanisms by which age affects fracture healing. Specifically, differentially expressed genes (DEGs) at three different time-points, namely, 1, 3, and 5 days after fracture, were evaluated by pathway and functional enrichment analysis. A protein–protein interaction (PPI) network was then constructed using these DEGs. These analyses revealed several molecular mechanisms that may contribute to age-related changes in fracture healing.Transcriptome profiles of GSE17825, which was submitted by Liang et al., were obtained from the National Centre of Biotechnology Information (NCBI) Gene Expression Omnibus database . GSE17825, which comprises a total of 18 chips, including nine old group (8-month-old mice) and nine young group (6-week-old mice) samples, was based on the platform of the PL1261 Affymetrix Mouse Genome 430 2.0 Array. For each group, fracture calluses were dissected and total RNA was isolated at each of the three time-points (n = 3 per time-point).The robust multichip average algorithm from the Oligo package  was used to preprocess non-normalized raw data by background correction normalization, probe summary, and log2 transformation . Probe iIn this study, several paired groups were compared as follows: (i) old vs. young group at 1 day; (ii) old vs. young group at 3 days; and (iii) old vs. young group at 5 days. Student's t-test was used for comparisons. Genes with fold changes > 2.0 and P < 0.05 were considered DEGs.The Database for Annotation, Visualization, and Integrated Discovery  can provide functional annotation of enormous quantities of genes derived from different genomic resources. In this study, DAVID was used to conduct Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) analyses to determine significant DEGs . GO termThe Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database (http://www.string-db.org) was used to evaluate potential protein–protein interactions. Cytoscape is an open-source tool for network visualization of genes, proteins, and other types of BPs . The defBox plots of GSE17825 value distribution before and after data normalization were generated . Data fr−4), taurine and hypotaurine metabolism (P = 5.02 × 10−3), fat digestion and absorption (P = 7.08 × 10−3), glycerolipid metabolism (P = 2.13 × 10−2), and dorsoventral axis formation (P = 2.69 × 10−2). At 3 days, the upregulated DEGs were primarily involved in ECM–receptor interaction (P = 4.38 × 10−5), focal adhesion (P = 3.76 × 10−4), regulation of actin cytoskeleton (P = 5.50 × 10−4), PI3K-Akt signaling pathway (P = 1.23 × 10−3), and phagosome (P = 2.13 × 10−3). Finally, at 5 days after fracture, these were involved in complement and coagulation cascades (P = 4.25 × 10−3), neuroactive ligand–receptor interaction (P = 2.19 × 10−2), cell adhesion molecules (P = 2.86 × 10−2), rheumatoid arthritis (P = 4.11 × 10−2), and leukocyte transendothelial migration (P = 7.69 × 10−2).The five most enriched KEGG pathways of up- and downregulated DEGs at each time-point  are presented in Figures −8), ECM–receptor interaction (P = 1.33 × 10−5), focal adhesion (P = 9.59 × 10−5), platelet activation (P = 1.13 × 10−3), and PI3K-Akt signaling pathway (P = 1.76 × 10−3). At 3 days, these were primarily enriched in pathways related to ABC transporters (P = 1.12 × 10−2), morphine addiction (P = 1.45 × 10−2), Wnt signaling pathway (P = 1.61 × 10−2), phototransduction (P = 2.82 × 10−2), and glycosphingolipid biosynthesis: lacto and neolacto series (P = 2.82 × 10−2). Finally, at 5 days after fracture, these were enriched in vascularization-associated pathways including platelet activation (P = 1.43 × 10−5), hematopoietic cell lineage (P = 3.86 × 10−5), and VEGF signaling pathway (P = 2.89 × 10−2).At 1 day, the downregulated DEGs were involved in protein digestion and absorption (P = 3.28 × 10−6), tissue development (P = 5.61 × 10−6), negative regulation of multicellular organismal process (P = 6.69 × 10−6), negative regulation of cell development (P = 3.32 × 10−6), and trabecular formation (P = 1.04 × 10−4). At 3 days after fracture, these were the most enriched in antigen processing and peptide or polysaccharide antigen presentation via MHC class II (P = 5.58 × 10−7). At 5 days after fracture, these were predominantly associated with synaptic-related functions, such as anterograde trans-synaptic signaling (P = 5.80 × 10−7), trans-synaptic signaling (P = 5.98 × 10−7), synaptic signaling (P = 7.67 × 10−7), neuron–neuron synaptic transmission (P = 1.00 × 10−5), and chemical synaptic transmission (P = 4.16 × 10−5).The 10 most significantly enriched GO terms (BPs) for the identified DEGs are listed in PPI network analysis was performed using Cytoscape software. Results revealed that, at 1 day after fracture, DEGs were enriched in platelet-derived growth factors binding, extracellular matrix structural constituent, and the Wnt signaling pathway . At 3 daFracture healing involves multiple biological phases that are characterized by both anabolic and catabolic processes . While aBecause inflammation is a critical step in fracture healing, any disruption of this process can negatively affect overall healing . Our GO β-catenin signaling must be precisely regulated to facilitate the differentiation of osteoblasts; dysregulation of this pathway alters the normal bone healing response [MSC differentiation is another key process in fracture healing, and understanding the mechanisms leading to delayed cell differentiation is important for understanding the cause of reduced skeletal regeneration with age , 31. Herresponse , 36. In KEGG pathway enrichment and PPI network analysis revealed that the downregulated DEGs were primarily related to vascularization-associated functions  at 5 days after fracture. Vascularization is a complex process that is essential for successful bone repair . DecreasNotably, DEGs at day 5 after fracture were primarily enriched in synaptic-related functions. The exact role of sensory nerves in fracture healing remains elusive –39 becauOne limitation of the present study is that the analyzed raw dataset did not include transcriptome data from 2, 3, 4, or 5 weeks following fracture, limiting conclusions about changes in molecular processes after bone fracture over time. Taken together, our findings demonstrate that elevated inflammatory status, a dysregulated Wnt signaling pathway, and the downregulation of vascularization-associated functions and synaptic-related processes play essential roles in fracture healing in the elderly. Further studies are required to determine the mechanisms by which these molecular events affect fracture healing.


Saitoella complicata and those in enlarged S. complicata cells after zymolyase treatment. TSA-treated and enlarged cells showed similar changes in nucleosome occupancy in five out of six positions in the gene promoters. These results suggest that changes in nucleosome formation at the gene promoters could serve as stress response mechanisms elicited in response to spheroplast (zymolyase treatment) and TSA treatment. In addition, we demonstrated that changes in nucleosome position occurred mainly in cells treated with 1 µg/mL TSA, whereas cells treated with 2 and 3 µg/mL TSA did not exhibit significant changes in the degree of nucleosome formation.We measured the degree of nucleosome formation at the gene promoters in trichostatin A-treated  cells of the archiascomycete  Thus, we measured the extent of nucleosome formation at the gene promoters in enlarged S. complicata cells and compared them with nucleosome formation levels in TSA-treated cells.In addition, the ascomycetous yeast 2.2.1.Saitoella complicata NBRC 10748  was cultivated in YM broth  at 25 °C for 24 h as a control sample. Afterwards, TSA  was added to the S. complicata culture; cells were subsequently incubated at 25 °C for 24 h. For the enlarged spheroplast generation, S. complicata was grown in minimal SD broth  at 25 °C for 30 h. Harvested cells were centrifuged for 5 min at 3000 rpm and suspended in buffer containing 0.8 M sorbitol and 25 mM phosphate at 25 °C for 20 min. Zymolyase 20T  was added to the cell suspension; the cells were incubated at 37 °C for 60 min. S. complicata cells were harvested, centrifuged for 5 min at 3000 rpm, and cultured in minimal SD broth (adjusted to pH 7.5) at 25 °C for 4–7 days.2.2.S. complicata culture and 2% formaldehyde were mixed and incubated for 10 min. Next, 5 mL of 1.25 M glycine was added to the resulting solution. S. complicata cells were collected, washed with 50 mM Tris-EDTA buffer (pH 8), and then suspended in zymolyase buffer . Zymolyase  (50 U) was added to the cell suspension, and the resulting solution was incubated at 37 °C for 1 h. Cells were collected by centrifugation and suspended in 2.5 mL of zymolyase buffer, after which 1 U of MNase  was added. The resulting digestion solution was incubated at 37 °C for 30 min, and the reaction was stopped by adding sodium dodecyl sulfate to a final concentration of 1% and EDTA to a final concentration of 10 mM. Proteinase K solution (5 µL) was added to the solution, and the mixture was incubated at 56 °C for 1 h. DNA was phenol/chloroform-extracted, ethanol-precipitated, and treated with RNase . Nucleosomal DNA fragments were isolated via electrophoresis on 2% agarose gel. The mononucleosomal DNA band was excised and purified using the QIAquick Gel Extraction Kit .Equal volumes of 2.3.Schizosaccharomyces pombe protein. G7K_3456-t1 and G7K_5676-t1 are genes that are both upregulated in response to TSA treatment in a concentration-dependent manner .In this study, we selected six nucleosome positions in the gene promoters  of the following four locus tags: G7K_2351-t1, G7K_2810-t1, G7K_3456-t1, and G7K_5676-t1. G7K_2351-t1 and G7K_2810-t1 encode homologs to 19S proteasome regulatory subunit Rpn3 and 20S proteasome-component α6 subunit Pre5, respectively, and are known to be increasingly downregulated upon treatment with increasing concentrations of TSA Click here for additional data file.t test with Holm's adjustment using R statistical software (http://www.r-project.org/).We performed analysis of variance (ANOVA) and a pairwise 3.Saitoella complicata cell is approximately 5 µm, whereas that of an enlarged spheroplast cultured in minimal SD broth after zymolyase treatment was measured to be approximately 15 µm  different at position 2351 but significantly different (p < 0.05) at the five other positions, namely, 2810_1, 2810_2, 3456, 5676_1, and 5676_2 (ANOVA results showed that nucleosome formation levels were not significantly (d 5676_2 .t test with Holm's adjustment showed no significant differences in terms of the degree of nucleosome formation at position 5676_1 (p > 0.05) between normal budding cells (0 µg/mL TSA) and enlarged cells . However, significant differences (p < 0.05) in nucleosome formation levels were observed in the four other positions   . In addi 5676_2) . The abo 5676_2) . HoweverChanges in the degree of nucleosome formation appeared to occur in a TSA concentration-dependent manner at positions 3456 (decreasing) and 5676_1 (increasing) . Howeverp values, cells treated with 1 µg/mL TSA evidently showed nucleosome movement (Nucleosome formation at position 5676_1 increased after TSA-treatment . On the movement . Interesmovement .In positions 2810_1 and 2810_2 (neighboring regions), nucleosome formation levels decreased as a result of TSA-treatment . This suExcept for position 2351, nucleosome formation levels in all other positions were significantly different between cells treated with 0 and 1 µg/mL TSA. However, no significant differences were observed between cells treated with 2 and 3 µg/mL TSA. The above results indicate that changes in the nucleosome formation occurred mainly in cells treated with 1 µg/mL TSA but not in cells treated with 2 and 3 µg/mL TSA.4.Saitoella complicata showed similar changes in nucleosome formation in five out of six gene promoter positions examined in the present study. These results strongly suggest that changes in nucleosome formation could serve as a stress response mechanism of S. complicata cells. Different stressors (TSA and zymolyase treatments) induce similar changes in the patterns of nucleosome formation in gene promoters in S. complicata.We demonstrated that although TSA-treatment and zymolyase-treatment are completely different stimulus, TSA-treated cells and enlarged spheroplasts of


QT78 in a project targeted to identify new non-hepatotoxic tacrine derivatives for Alzheimer’s disease therapy. We have found that QT78 is less toxic than tacrine at high concentrations (from 100 μM to 1 mM), less potent than tacrine as a ChE inhibitor, but shows selective BuChE inhibition (IC50 (hAChE) = 22.0 ± 1.3 μM; IC50 (hBuChE) = 6.79 ± 0.33 μM). Moreover, QT78 showed effective and strong neuroprotection against diverse toxic stimuli, such as rotenone plus oligomycin-A or okadaic acid, of biological significance for Alzheimer’s disease.We report the synthesis and relevant pharmacological properties of the quinoxalinetacrine (QT) hybrid  Alzheimer’s disease (AD) is the most common neurodegenerative disorder. Many biochemical hallmarks are involved in the progress and development of this pathology: extracellular senile plaques that appear by amyloid-beta peptide deposition and aggregation, hyperphosphorylated tau-protein, neuroinflammation, cell death, oxidative stress, and a strong deficit of neurotransmitters such as acetylcholine, dopamine, and serotonin . These fI, In this context, and pursuing our search for non-hepatotoxic tacrine  analogueQT78   , as the QT78 has been carried out as shown in QT78 showed spectroscopic data in good agreement with its structure and excellent analytical data  in a 24 h period. The HepG2 viability was significantly decreased when QT78 was incubated at the highest concentrations (300 and 1000 μM)  . However tacrine .50 values highlighted that QT78 acts as a modest but selective hBuChE inhibitor . Thus, it is obvious that the enlargement of the three-membered tetrahydroacridine scaffold to the four-membered tetrahydroquinolino quinoxaline nucleus was detrimental regarding its activity towards both ChE enzymes, leading to a decrease of the inhibitory potency of two orders of magnitude (tacrine: IC50 (hAChE) = 0.374 ± 0.053 μM; IC50 (hBuChE) = 0.044 ± 0.002 μM) quinoxalin-12-amine  δ 8.17 , 8.01–7.70 , 7.10 , 2.98–2.96 , 2.69–2.56 , 1.88–1.85 ; 13C-NMR  δ 165.8 , 148.9 (C5a), 147.5 (C11a), 144.2 (C10a)*, 140.0 (C6a)*, 131.0 (C9)**, 130.6 (C8)**, 129.3 (C7)***, 129.0 (C10)***, 110.3 (C12a), 34.5 (C4), 23.9 (C1), 22.3 (C2)*, 21.9 (C3)*; MS (EI) m/z (%): 250 [M+] (100), 235 (18). HRMS. Calc. for C15H14N4 (M + H)+: 251.12912. Found: 251.12989. Anal. Cald. for C15H14N4: C, 71.98; H, 5.64; N, 22.38. Found: C, 71.75; H, 5.61; N, 22.15.A mixture of 3-amino-2-quinoxalinecarbonitrile   and Derek Nexus  knowledge-based expert systems  were employed for metabolism and toxicity predictions . HepatotThe metabolism was predicted by Meteor Nexus version 3.1.0, , a SAR (structure-activity relationship) tool used to predict the likely metabolic fate of a chemical structure through a knowledge-base composed of a biotransformation dictionary, rules and example metabolic pathways. Meteor was setup for human Phase I biotransformation reactions, since in most cases the reactive metabolites are generated particularly through Phase I metabolic reactions . An abso2 and 95% air. Culture media were changed every 2 days. Cells were sub-cultured after partial digestion with 0.25% trypsin-EDTA. For assays, cells were subcultured in 96-well plates at a seeding density of 1 × 105 cells per well. When the cells reached 80% confluence, the medium was replaced with fresh medium containing 1–1000 µM QT78 and tacrine, or 0.1% DMSO as a vehicle control.The human hepatoma HepG2 cell line was kindly provided by the IdiPAZ Institute for Health Research . They were cultured in Eagle´s minimum essential medium (EMEM) supplemented with 15 nonessential amino acids, 1 mM sodium pyruvate, 10 % heat-inactivated FBS, 100 units/mL penicillin, and 100 µg/mL streptomycin . Cultures were seeded into flasks containing supplemented medium and maintained at 37 °C in a humidified atmosphere of 5% COQT78 to inhibit human ChE activity was assessed using Ellman’s assay [50 extrapolated.The ability of ’s assay . A JascoQT78 was built using Discovery Studio 2.1 [QT78 was energy-minimized using the adopted-based Newton–Raphson algorithm with the CHARMm force field [−1. The ligand was set up for docking with the help of AutoDockTools  to define the torsional degrees of freedom to be considered during the docking process. All the rotatable bonds of the ligand were allowed to rotate freely. The enzyme structure used for the calculations was the human AChE (Protein Data Band code 1B41). To give flexibility to the binding site, residues lining the site were allowed to move. These residues were Trp286, Tyr124, Tyr337, Tyr341, Tyr72, Asp74, Thr75, Trp86. Docking calculations for hBuChE were performed following a similar protocol to that described before for hAChE. The coordinates of hBuChE were obtained from the Protein Data Bank (PDB ID: 4BDS). For docking purposes, initial proteins were prepared by removing all water molecules, heteroatoms, any co-crystallized solvent and the ligand. Proper bonds, bond orders, hybridization and charges were assigned using protein model tool in the Discovery Studio, version 2.1, software package. A CHARMm force field was applied using the receptor ligand interactions tool in the Discovery Studio software package. ADT was used to add hydrogens and partial charges for proteins using Gasteiger charges and to generate the docking input files. AutoDock Vina software was employed for all the protein-ligand calculations and they were applied to the whole protein target (blind docking). For hAChE, the grid box was built with a resolution of 1 Å and 60 × 60 × 72 Å and it was positioned at the middle of the protein . For hBuChE, a grid box of 66 × 60 × 74 with grid points separated 1 Å was positioned in the middle of the protein . The resultant structure files were analyzed using Discovery Studio.The structure of compound udio 2.1 . The molce field  with a c2 and 95% air. For assays, SH-SY5Y cells were sub-cultured in 96-well plates at a seeding density of 5 × 104 cells per well for 2 days. The cells were then incubated with 30 μM rotenone and 10 μM oligomycin-A (R/O) or okadaic acid (20 nM)  according to previous studies [QT78 at different concentrations for 24 h. In this study, all cells were used at a low passage number (<14).Human SH-SY5Y cells were maintained in a 1:1 mixture of Nutrient Mixture F-12 and Eagle´s minimum essential medium (EMEM) supplemented with 15 nonessential amino acids, 1 mM sodium pyruvate, 10% heat-inactivated FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin. Cultures were seeded into flasks containing supplemented medium and maintained at 37 °C in a humidified atmosphere of 5% CO studies ,19 with 2 and 95% air (v/v), the supernatant was removed, the obtained purple formazan product was re-suspended in 100 μL of DMSO. Colorimetric determination of MTT reduction was measured in an ELISA microplate reader at 540 nm. Non-treated cells were considered controls and were taken as 100% viability. MTT reduction was performed as described  for botht-test or ANOVA with Tukey’s multiple comparison test, as indicated. P values less than 0.05 were considered significant.Statistical analyses were performed using GraphPad Prism software . The data were analyzed by QT78, the parent compound of a new family of tacrine derivatives bearing the quinoxaline heterocyclic ring core, was investigated. As a result, we have identified QT78 as a promising hit compound, which is less toxic than tacrine at high concentrations (from 100 µM to 1 mM), but less potent than tacrine as an inhibitor of both ChEs, with a modest selectivity towards BuChE. At 0.1 and 0.3 μM, QT78 exhibited a significant neuroprotective effect against different toxic stimuli, including ROS generation and hyperphosphorylation of tau protein. In addition, computational analyses shed light into the toxicological profile, binding and ADME properties of QT78. Taken together, the reported data are relevant to the search for new anti-AD agents. Our work is now in progress to prepare and evaluate new, related QTs based on QT78. The results will be reported elsewhere in due course.In the search for a new MTSM  for AD ,26,27, t