BACE1 expression and directly decreased β-secretase activity. Additionally, APP expression was reduced. In line with literature, MTXs reduced oxidative stress, decreased cholesterol and a decreased in Aβ1-42 aggregation. In conclusion, all MTXs act via the pleiotropic mechanism resulting in decreased Aβ and show beneficial properties with respect to AD in neuroblastoma cells. However, the observed effect strength was moderate, suggesting that MTXs should be integrated in a healthy diet rather than be used exclusively to treat or prevent AD.Methylxanthines (MTX) are alkaloids derived from the purine-base xanthine. Whereas especially caffeine, the most prominent known MTX, has been formerly assessed to be detrimental, this point of view has changed substantially. MTXs are discussed to have beneficial properties in neurodegenerative diseases, however, the mechanisms of action are not completely understood. Here we investigate the effect of the naturally occurring caffeine, theobromine and theophylline and the synthetic propentofylline and pentoxifylline on processes involved in Alzheimer’s disease (AD). All MTXs decreased amyloid-β (Aβ) level by shifting the amyloid precursor protein (APP) processing from the Aβ-producing amyloidogenic to the non-amyloidogenic pathway. The α-secretase activity was elevated whereas β-secretase activity was decreased. Breaking down the molecular mechanism, caffeine increased protein stability of the major α-secretase ADAM10, downregulated  AD is clinically characterized by degeneration of neurons in different brain regions, mainly cortical and subcortical areas as well as in the hippocampus, leading to deficits in memory, thinking and behavior [Alzheimer’s disease (AD), the most common form of dementia in the elderly population, affects approximately 45 million people worldwide, emphasizing AD as a major public health concern ,2. Agingbehavior . Main hibehavior , by the behavior ,8,9, genbehavior ,11, and behavior ,15,16,17behavior ,19, or cbehavior ,21. Besibehavior ,23,24. Abehavior ,26,27, abehavior . Beside behavior ,30,31. HMTXs are alkaloids and a class of compounds derived from the purine xanthine. They have at least one methyl group at the nitrogen in the xanthine structure at R1, R2, or R3 . MTXs, eInterestingly, the critical view concerning the most popular MTX caffeine has changed dramatically. Whereas the intake of caffeine was assumed to be contraindicated or having adverse effects in many diseases, recent evidences show potential beneficial effects in neurodegenerative diseases like Parkinson’s disease or AD . SeveralDue to their structural similarity to the purine adenosine, MTXs act as an antagonist of the adenosine receptor also known as purinergic P1 receptor, which is widely expressed in the body including the central nervous system . SeveralFour adenosine receptor subtypes exist  in humans. Interestingly, theobromine lacking the methyl group at the R1 position has only a weak activity as adenosine receptor antagonist whereas an elongation of the methyl group in R1 as seen in pentoxifylline and propentofylline generally increases the affinity to the adenosine receptor . In addiTherefore, the aim of the study was to (I) further elucidate the molecular mechanism especially of caffeine on the protective effect in respect to AD focusing on the Aβ metabolism and (II) compare these effects with the other two mainly naturally occurring MTXs theophylline and theobromine and two synthetic MTXs propentofylline and pentoxifylline widely used as a drug having a higher affinity to adenosine receptors compared to the naturally occurring MTXs.Caffeine and theophylline were purchased from Fisher Scientific , theobromine, pentoxifylline, propentofylline and all other chemicals used in this study were acquired from Merck former Sigma-Aldrich , if not stated otherwise.695, which overexpressed the human mainly neuronal APP isoform APP695  cells were cultivated in Dulbecco’s Modified Eagle’s Medium (DMEM), containing 10% fetal calf serum  and 0.1% non-essential amino acid solution (MEM). For the stable transfected cell line SH-SY5Y APPP695 see , 0.3 mg/2O  as a solvent control. For experiments with γ-secretase inhibitor IX, cells were pretreated 2 h with 2.5 µM γ-secretase inhibitor, before incubation with MTXs was performed as described above. For determination of cholesterol level, cells were long-term incubated for six days in DMEM reduced to 1% FCS.FCS in DMEM was reduced to 0.1%, dependent on the following experiments, 16 h prior incubation. FCS was reduced to elucidate the potential effect of MTXs on cholesterol or other lipids being also present in FCS. MTXs were incubated with a concentration of 100 µM for 24 h (8 + 16 h), whereas controls were treated with HPLC-grade HCells were lysed by using 150 mM NaCl , 50 mM Tris/HCl pH 7.4, 2 mM EDTA , 1% NP-40, 1% Triton-X 100 lysis buffer supplemented with protease inhibitor . Subsequently, the protein concentrations of the lysates were determined by bicinchoninic acid assay (BCA) as described  and adju2O. The solution was then homogenized via Minilys  for 90 s on maximum intensity. Homogenates were adjusted to 10 mg/mL by BCA (see 2.3.2.) and stored at −80 °C.Cultivated SH-SY5Y wt cells were washed two times with ice-cold PBS and collected in 150 µL HPLC-grade HPostnuclear fractions (PNFs) of SH-SY5Y wt cells were obtained by washing cells three times with ice-cold PBS and homogenizing in sucrose-buffer  via Minilys  on maximum intensity for 20 s. For the preparation of mouse brain PNFs the brains from male C57BL/6 wt mice, which were kindly provided by Prof. Dr. med. Matthias W. Laschke and frozen in liquid nitrogen directly after removing, were slowly defrosted on ice and homogenized in the same way described for SH-SY5Y wt cells before. Afterwards protein amount was adjusted to same concentration by BCA (see 2.3.2.). Samples were then centrifuged at 900 rcf for 10 min at 4 °C and supernatants were collected and stored at −80 °C. PNFs were incubated with MTXs at a concentration of 0.1 nmol MTX per 1 µg protein for 30 min before α- and β-secretase activity measurements.After BCA protein adjustment, the level of APP, ADAM10, and β-CTF were analyzed in cell lysates whereas for the determination of the total protein level of the secreted proteins Aβ, sAPPα, and sAPPβ growth media were used. The following additional loading controls were used to determine whether potential differences were due to alterations in protein loading of the western blot (WB): For secreted proteins medium was supplemented with bovine serum albumin (BSA) and detected via Ponceau staining as described in . In lysaAβ, and β-CTF level were detected by performing immunoprecipitation of equal volumes of conditioned media or cell lysates adjusted to the same protein amount as described in Grimm et al. . Precipi2O as control for 18 h. Afterwards, non-degraded human Aβ was detected by WB analysis using W02 antibody.To determine the total degradation of Aβ, human synthetic Aβ40 peptide  was supplemented (0.5 µg/mL) for an additional 6 h after cultivation of murine N2a wt cells in reduced FCS (0.1%)/DMEM for 6 h and subsequent incubation of 100 µM MTXs or HPLC-grade HThe activities of α-, β- and γ-secretases in living cells were analyzed as described in Grimm et al. .Activities of α- and β- secretase in postnuclear, cell free fractions were analyzed as described in Grimm et al. .TBP): forward 5′-CGG AGA GTT CTG GGA TTG T-3′, reverse 5′-GGT TCG TGG CTC TCT TAT C-3′; amyloid precursor protein (APP): forward 5′-GGC AGT TAT CCA GCA TTT CC-3′, reverse 5′-ATT GAG CAT GGC TTC CAC TC-3′; β-site APP-cleaving enzyme 1 (BACE1): forward 5´-GCA GGG CTA CTA CGT GGA GA-3´, reverse 5′-TAG TAG CGA TGC AGG AAG GG-3′; a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10): forward 5′-GCA AAC TGA AAC CTG GGA AA-3′, reverse 5′-TTC CTT CCC TTG CAC AGT CT-3′ . Results were normalized to TBP and the 2-(ΔΔCt) method was used to calculate expression changes.Gene expression analysis was performed as described in Grimm et al.  and the 2O. After incubation, cells were treated with 100 µM of cycloheximide for 1 h before it was replaced by incubation media also containing cycloheximide. Cells with treatment of cycloheximide were then collected after 6 h and 12 h. ADAM10 protein was detected via WB experiment as described above (see 2.4.).SH-SY5Y wt cells were cultivated in DMEM containing FCS (0.1%) for 16 h and then incubated for 8 + 16 h with 100 µM caffeine or HPLC-grade H2 Fluorometer .Incubated cells on a 96 well plate were washed twice with prewarmed (37 °C) imaging solution . Afterwards, 100 µL imaging solution containing 10 µM 3′-(p-aminophenyl)fluorescein  were added per well. Fluorescence was measured directly after APF treatment, and after 1 h incubation in the dark at 37 °C, at excitation wavelength 490 nm ± 10 nm and emission wavelength of 515 ± 10 nm using a SafireThe amount of cholesterol was measured from cell homogenates by using the Amplex Red Cholesterol Assay Kit  according to the manufacturer’s protocol.Aβ aggregation via Thioflavin T assay was done as described before  with smaTo measure the cytotoxicity of the MTXs incubation, Cytotoxicity Detection Kit (LDH) from Roche  was used according to the manufacturer’s instructions.For the determination of differences in cell proliferation dependent on particular MTX incubation, the cell proliferation II Kit (XTT) from Roche  was used according to the protocol of the manufacturer.p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001.All quantified data represent an average of at least three independent experiments. To compare the effect of caffeine with the other MTXs the average effect of all MTXs was calculated. Therefore the sum of the individual effects of the MTXs compared to solvent control was calculated and divided by the number of different MTXs  revealing the average effect strength of all MTXs. The individual effects of all MTXs are shown in the 695, the main APP isoform in neurons [2O as solvent control or caffeine, theophylline, pentoxifylline, theobromine or propentofylline in a final concentration of 100 µM. Under these conditions both cell proliferation, determined by XTT assay, and toxicity, measured by LDH release, were not affected. Effects on cell proliferation compared to control were <4% and for all MTXs not statistically significant. Cell viability differences compared to control were <4% also lacking statistically significance for all MTXs (see p = 0.0205) in our cellular system (p = 0.0310), theobromine to 83.9% ± 7.7% (p = 0.0730), although the observed decrease was not statistically significant and propentofylline incubated cells showed identical to theophylline a significant reduction to 80.2% ± 6.6% (p = 0.0168). The strongest reduction in total secreted Aβ level was obtained for pentoxifylline: 66.2% ± 3.9% (p ≤ 0.001). Averaged, all analyzed MTXs significantly reduced total Aβ level to 78.9% ± 3.3% (p = 0.0032)  , indicat695. WB analysis of sAPPα using the antibody W02 showed that caffeine significantly elevated sAPPα level to 131.3% ± 9.5% (p = 0.0070). Nearly identical to caffeine, all analyzed MTXs averaged revealed a significant increase in sAPPα level to 129.8% ± 3.7% (p = 0.0013) (p = 0.0256) and 143.1% ± 13.3% (p = 0.0117), respectively . To further elucidate non-amyloidogenic APP processing in presence of MTXs we measured α-secretase activity in living SH-SY5Y wt cells using a fluorescence-based assay. In line with the observed increase in sAPPα level, caffeine as well as all other tested MTXs significantly increased α-secretase activity. Caffeine significantly elevated α-secretase activity to 112.3% ± 3.0% (p = 0.0195), all MTXs averaged showed a significant increase to 120.0% ± 3.2% (p = 0.0035) (p = 0.0026), pentoxifylline to 114.9% ± 5.2% (p = 0.0385), theobromine to 130.9% ± 1.1% (p ≤ 0.001) and propentofylline to 119.6% ± 4.9% (p = 0.0021) (p ≤ 0.001) (p = 0.0037). In detail, all analyzed MTXs individually significantly elevated the protein level of ADAM10     , indicating that caffeine elevates ADAM10 protein stability (The determination of α-secreted APP (sAPPα) in presence of MTXs was performed in SH-SY5Y cells stably expressing APP 0.0013) a. In detectively . Pentoxi 0.0035) b. Thus,  0.0021) . The inc≤ 0.001) c and the= 0.204) . To exam 0.2186) d. Theoph 0.5321) . Caused  0.0015) d. These tability e.p = 0.1790; theophylline: 97.5% ± 3.2%, p = 0.4904; pentoxifylline: 93.6% ± 2.4%, p = 0.0628; theobromine: 97.9% ± 3.1%, p = 0.5708; propentofylline: 97.6% ± 2.3%, p = 0.3980). However, as the analyzed MTXs consistently showed a slight reduction, the average of all MTXs revealed a minor but significant direct effect on α-secretase activity   f. Howevep = 0.0110)  . Averaged, all tested MTXs tended to decrease the sAPPβ level, however, statistical analysis did not reach significance, as two out of five MTXs showed no effect on the sAPPβ level. This might be due to the fact that the observed effect strength is rater small and WB analysis of sAPPβ might not be sensitive enough to get statistical significant results. Furthermore the ratio of sAPPα/sAPPβ was calculated, because it represents the shift from amyloidogenic to non-amyloidogenic pathway , whereas theobromine showed no effect on β-secretase activity in living cells: 95.2% ± 2.6% (p = 0.1051) (p = 0.011) (p = 0.0083), when post nuclear fraction of human neuroblastoma cells were incubated with caffeine and β-secretase activity was determined in a cell free assay (p = 0.0032) (BACE1 in SH-SY5Y wt cells by RT-PCR. BACE1 gene expression was significantly reduced to 80.8% ± 6.8% (p = 0.0204) in presence of caffeine and the average of all MTXs also showed a significant reduction in BACE1 mRNA level to 84.2% ± 2.7% (p = 0.0043)  whereas propentofylline only trended to decrease BACE1 gene transcription (p = 0.0040) in caffeine-incubated SH-SY5Y cell stably expressing APP695 in the absence of γ-secretase inhibitor (p = 0.0307) compared to MTX untreated cells (p = 0.0062) in presence of caffeine whereas the average of all MTXs trended to decrease γ-secretase activity  (p ≤ 0.001) and 84.7% ± 4.6% (p ≤ 0.001), respectively, whereas theophylline and pentoxifylline showed no significant reduction  a. The sA≤ 0.001) , whereas pathway . To furt pathway c as well pathway  signific 0.1051) . Average= 0.011) c, indicaee assay d. Howeveee assay d as theoee assay  revealin 0.0032) e. To fur 0.0043) f. In detcription . To valinhibitor g. When fed cells g. The di 0.2629) h. Indivieduction .In summary, MTXs in general slightly decrease the amyloidogenic pathway by decreasing β- and/or γ-secretase activity. Interestingly, the effect on the amyloidogenic pathway is not so pronounced compared to the non-amyloidogenic pathway. Moreover, the underlying effects and mechanisms in the amyloidogenic pathway are not conserved for all MTXs. However the combined effects of both amyloidogenic and non-amyloidogenic pathways result finally for all MTXs in a consistent Aβ decrease, where no statistical significant differences between the single MTXs were observed.APP was significantly reduced to 88% ± 4.6% (p = 0.0280) in caffeine-treated SH-SY5Y wt cells (APP gene transcription to 90.8% ± 2.9% (p = 0.0349). Besides propentofylline, all tested MTXs individually significantly reduced APP gene transcription  (p = 0.0118) in presence of caffeine (APP protein level to 86.2% ± 1.2% (p ≤ 0.001), which is also in line with the observed reduced APP gene transcription. In detail, theophylline and pentoxifylline showed a reduction to 90.5% ± 4.2% (p = 0.1027) and 83.7% ± 6.7% (p = 0.0511), respectively   and of cholesterol. Gene expression of wt cells a. The av 0.9614) . In linecaffeine b. Similaectively . However 0.0437) . These rBeside the above addressed Aβ anabolism, including APP processing, the amount of Aβ strongly depends on Aβ catabolism via the main Aβ-degrading enzymes neprilysin and IDE ,19. In op ≤ 0.001) in the presence of caffeine  (p ≤ 0.001) , including hypochlorite and free hydroxyl radicals . The procaffeine d. Notabl≤ 0.001) . Therefo≤ 0.001) d, indicap ≤ 0.001), whereas the average of all MTXs showed a not significant reduction to 88.0% ± 5.4% (p = 0.0892) (p ≤ 0.001) (p = 0.0520), whereas pentoxifylline and theobromine did not affect the cholesterol level  e. Thereb≤ 0.001) . On the ol level .p ≤ 0.001) in presence of caffeine (p ≤ 0.001)   f. In det 0.0043) .BACE1 gene expression and at least for caffeine by a direct inhibition of β-secretase activity. MTXs therefore induce a shift from β-amyloidogenic to non-amyloidogenic α-secretase processing of APP preventing or reducing the formation of Aβ peptides. These findings are in line with the study by Arendash et al. showing lower hippocampal Aβ level and reduced BACE1 gene expression in APPswedish transgenic mice supplemented with 1.5 mg caffeine/day per mouse, which is the human equivalent of 500 mg caffeine per day [AD pathogenesis involves multiple pathological processes, including e.g., Aβ accumulation, tau hyperphosphorylation, oxidative stress, inflammation and alterations in lipid and energy metabolism. Despite intensive research no drug treatment is available to cure AD, only drugs that can help to mask the symptoms. Current research therefore focuses on AD prevention or delay of its progression, including dietary interventions during prodromal and early stages of the disease. Therapeutic strategies that dramatically interfere with a single pathological process involved in AD, e.g., blocking γ-secretase activity, are not reasonable, as γ-secretase dependent cleavage products of APP have important physiological functions and PS can cleave multiple substrates ,66, prob per day  present  per day . In this per day , which i per day  where β- per day ,76. Nota per day ,78, illu per day . In line per day ,80,81,82APP accompanied by a reduced APP protein level. It is important to mention that the analyzed MTXs reduced APP protein level to approximately 85%, a moderate but significant effect. Taking into consideration, that APP is ubiquitously expressed and highly conserved in any mammalian, it seems reasonable not to completely abolish APP expression and processing, which might be beneficial for treating AD, but have severe side effects by also abolishing a potential physiological function of APP or APP processing.Beside the discussed effect of MTXs to reduce the Aβ level by shifting APP processing towards the non-amyloidogenic pathway, we could show that MTXs decrease gene expression of BACE1 expression [BACE1 gene expression in presence of MTXs themselves reducing oxidative stress. Beside the Aβ-reducing effect of MTXs via pleiotropic mechanisms, MTXs also reduce Aβ oligomerization and aggregation, which are essential for the formation of senile plaques. Inhibiting Aβ aggregation is considered as an effective therapeutic strategy to prevent or treat AD. All tested MTXs significantly decreased Aβ aggregation to approximately 40% to 50%. In line, Sharma and colleagues showed that the presence of caffeine in a water solution inhibited the aggregation of Aβ16-22 peptides [Interestingly, in our study caffeine and propentofylline also interfered with cholesterol homeostasis, by significantly reducing cholesterol level to 82.8% and 70.1%. Theophylline also showed a trend to decrease cholesterol level. Notably, hypercholesteremia is discussed to be a risk factor for AD and high cholesterol level have been shown to increase Aβ generation in cell culture experiments and mouse models ,62,63,64pression , which cpeptides .Limitations of the study: our present study is a pure in vitro study, utilizing human neuroblastoma SH-SY5Y cells. It aims to clarify the molecular mechanisms how MTXs, especially caffeine, affects processes involved in AD. For the direct effect on β-secretase, results were confirmed ex vivo in mouse brain. Indubitable ex vivo experiments also have their limitations, e.g., no functional blood brain barrier is present or no further metabolization in liver occurs. As discussed above, it is therefore important to emphasize that this study does not include pharmacological or pharmacokinetic aspects, which might alter the effect strength in vivo. However, our results underline that MTXs enhance the non-amyloidogenic pathway and decrease the amyloidogenic pathway resulting in a pleiotropic reduction of Aβ. This reduction has been shown to be modest under the conditions used here and might help to explain why previous in vivo studies also found modest effects in vivo. Additionally an estimation might be made about the other MTXs and their potency to treat or prevent AD, suggesting also only modest effects. However further in vivo studies are needed, to clarify if the molecular mechanisms found here for the other MTXs also lead to comparable effects in respect to AD in a human situation.APP gene transcription and APP processing are moderate, but in summary, significantly reduce Aβ generation, and provide the opportunity that APP and its cleavage products can still fulfil their physiological functions. The effect of MTXs on oxidative stress and Aβ aggregation are more pronounced. Therefore, dietary supplementation of MTXs might be a useful therapeutic strategy to prevent or treat AD, as MTXs have pleiotropic positive effects on pathological processes involved in AD pathogenesis. However, due to the observed moderate effect strength, MTXs alone in treating or preventing AD might not be sufficient. Therefore, an integration of MTXs in a dietary supplementation consisting several supplements like DHA, omega three fatty acids, vitamin D, B-vitamins etc. in combination with a healthy life style seems more reasonable and should be taken into consideration, especially due to its cost effectiveness, availability and modest side effects.In summary, natural occurring MTXs as well as synthetic xanthine derivatives positively interfere with multiple pathological processes involved in the pathogenesis of AD. The effect strengths on physiological processes like e.g., Furthermore, our results show that there was no significant difference between the single MTXs on Aβ homeostasis. If the effect on Aβ would be mediated exclusively by adenosine receptor antagonism, it could be assumed that theobromine with lowest affinity to adenosine receptors would have a significant weaker effect compared to the other MTXs, which was not the case but is in line with a pleiotropic action where several mechanisms result in a decreased Aβ level. From this point of view, a further screening for other MTXs which might have even higher affinity to adenosine receptors might have limited additional positive effects in respect to AD. However, although, natural occurring MTXs are rapidly absorbed in the gastrointestinal system, are able to cross the blood-brain-barrier and enter the brain by simple or facilitated diffusion ,89, furt


Ruxolitinib improves splenomegaly and symptoms in patients with intermediate-2 or high-risk myelofibrosis; however, nearly half develop grade 3/4 anemia and/or thrombocytopenia, necessitating dose reductions and/or transfusions. We report findings from an open-label phase 2 study exploring a dose-escalation strategy aimed at preserving clinical benefit while reducing hematological adverse events early in ruxolitinib treatment.Patients with myelofibrosis received ruxolitinib 10 mg twice daily (BID), with incremental increases of 5 mg BID at weeks 12 and 18 for lack of efficacy . Symptom severity was measured using the Myelofibrosis Symptom Assessment Form Total Symptom Score (MFSAF TSS).Forty-five patients were enrolled, 68.9% of whom had a Dynamic International Prognostic Scoring System score of 1 to 2 . Median percentage change in spleen volume from baseline to week 24 was − 17.3% (≥ 10% reduction achieved by 26 patients [57.8%]), with a clear dose response. Median percentage change in MFSAF TSS from baseline at week 24 was − 45.6%, also with a dose response. The most frequent treatment-emergent adverse events were anemia (26.7%), fatigue (22.2%), and arthralgias (20.0%). Grade 3/4 anemia (20.0%) and dose decreases due to anemia (11.1%) or thrombocytopenia (6.7%) were infrequent.A dose-escalation approach may mitigate worsening anemia during early ruxolitinib therapy in some patients with myelofibrosis.ClinicalTrials.gov identifier, NCT01445769. Registered September 23, 2011. JAK2 V617F gain-of-function mutation ) percentage reduction from baseline in spleen volume at weeks 12 and 24 was 16.3% (12.4%) and 14.9% (21.1%), respectively. A clear dose-response relationship was observed, with week 24 mean (SD) spleen volume reductions of 20.1% (18.3%) and 32.9% (12.9%) among patients receiving average total daily doses of ruxolitinib > 20 to 30 mg and > 30 to 40 mg, respectively  MFSAF TSS at baseline and study weeks 6, 12, 18, and 24 were 16.6 (10.1), 10.5 (7.2), 10.0 (7.6), 8.4 (6.4), and 9.3 (8.0), respectively. The median (range) percentage changes in MFSAF TSS from baseline at study weeks 6, 12, 18, and 24 were − 31.3% (− 89.3 to 265.7%), − 39.3% (− 96.8 to 380.0%), − 49.3% (− 98.0 to 223.8%), and − 45.6% (− 100.0 to 261.9%), respectively. At week 24, patients receiving an average daily ruxolitinib dose > 30 to 40 mg had the greatest median percentage reduction from baseline in MFSAF TSS  had a ≥ 20% reduction in MFSAF TSS from baseline. At week 24, MFSAF TSS reductions ≥ 20% were observed in 56.3% of patients who received ruxolitinib ≤ 10 mg daily and 72.4% of patients who received > 10 mg daily.At weeks 12 and 24, 17 patients (37.8%) and 18 patients (40.0%), respectively, achieved a ≥ 50% improvement in MFSAF TSS from baseline. At week 24, ≥ 50% MFSAF TSS reductions were observed in 12.5% of patients who received an average daily ruxolitinib dose ≤ 10 mg and in 55.2% of patients who received an average daily ruxolitinib dose > 10 mg.At week 24, ≥ 30% of patients had a ≥ 50% reduction of all individual components of the MFSAF TSS from baseline. At weeks 12 and 24, 19 patients (42.2%) and 20 patients (44.4%), respectively, had a ≥ 50% reduction in the abdominal symptom score  from baseline.All 45 patients received ≥ 1 dose of ruxolitinib. The median (range) duration of ruxolitinib treatment was 169 (31–257) days. The mean and median total daily doses of ruxolitinib were 19.7 and 20 mg, respectively, corresponding to approximately 10 mg BID.Overall, 42 patients (93.3%) had a treatment-emergent AE (TEAE); anemia (26.7%) was the most frequently reported, followed by fatigue (22.2%) and arthralgia (20.0%). TEAEs occurring in ≥ 10% of patients and all grade 3/4 TEAEs are summarized in Table n = 5, 11.1%) and thrombocytopenia .Nine patients (20.0%) had TEAEs leading to dose reduction. The most frequently reported TEAEs leading to dose decrease were anemia  in the first 4 weeks of treatment to 176 × 109/L, then increased and remained in a range of 193.5 × 109/L to 230.0 × 109/L from week 6 to week 20 (median 172 × 109/L at week 24). One patient each had a grade 3 and grade 4 thrombocytopenia on study. In general, median ANC counts remained stable through week 24.Median platelet count declined from baseline  maintained transfusion independence throughout the treatment phase of the study. Of the 15 patients who were red blood cell transfusion-dependent at baseline, a shift to transfusion independence occurred in one patient (6.7%) from day 1 to week 12 and in three patients each (20.0%) during weeks 6 to 18 and weeks 12 to 24.This 24-week, open-label study examined whether initiating ruxolitinib therapy at a lower dose affected the initial drop in hemoglobin that was observed during the first 8 to 12 weeks of therapy in COMFORT-I  and COMFEligibility criteria and patient populations in this study and in COMFORT-I  and COMFIn the present study, reduction in spleen volume from baseline showed a clear dose response. Mean week 24 reductions in spleen volume in COMFORT-I and COMFORT-II were 31.6 and 29.2%, respectively, among patients taking initial ruxolitinib doses of 15 or 20 mg BID (decreased to 10–15 mg BID during the first 8–12 weeks in some cases) , 8, 10. Compared with changes in spleen volume, changes in MFSAF TSS depended less on ruxolitinib dose. The median percentage reduction (improvement) in MFSAF TSS was 45.6% at 24 weeks. This finding was similar to 24-week data from COMFORT-I, in which median reduction in MFSAF TSS was 56.2% . NotablyThe alternative dosing scheme explored in this study (ruxolitinib 10 mg BID) provided benefit with regard to symptoms and reduced palpable spleen length in most patients; however, improvements in efficacy outcomes observed here with the 10-mg BID dose were smaller compared with those observed in COMFORT-I, in which the mean doses at the end of 24 weeks were ~ 10 mg (for a starting dose of 15 mg BID) and 15 to 20 mg BID (for a starting dose of 20 mg BID) [Current guidelines for the use of ruxolitinib to manage symptoms consider dose-response relationships and dose adjustments in the development of specific recommendations for the clinical management of MF. The European LeukemiaNet and the Italian Society of Hematology have strongly recommended ruxolitinib for improving symptomatic or severe splenomegaly  in patients with intermediate-2 or high-risk disease, as well as for improving systemic symptoms in patients with a score of ≥ 44 on the MPN10 , refractory severe itching, unintended weight loss not attributable to other causes, or unexplained fever . HoweverConcerns of progressive cytopenias in patients with MF treated with ruxolitinib are well established. Results from this open-label, multicenter, single-arm, phase 2 study suggest that initiating therapy at lower doses can be performed safely and may provide clinical benefit, including improvements in splenomegaly and symptoms, in patients with MF for whom anemia is, or is likely to become, a concern while receiving treatment with ruxolitinib. Current recommendations, including those detailed in the NCCN Clinical Practice Guidelines in Oncology: Myeloproliferative Neoplasms , continu


Here, we describe the characteristics of the Innovative Medicines Initiative (IMI) Diabetes Research on Patient Stratification (DIRECT) epidemiological cohorts at baseline and follow-up examinations .n = 2127) . We also recruited people from clinical registries with type 2 diabetes diagnosed 6–24 months previously (n = 789) into a second cohort study . Follow-up examinations took place at ~18 months (both cohorts) and at ~48 months (cohort 1) or ~36 months (cohort 2) after baseline examinations. The cohorts were studied in parallel using matched protocols across seven clinical centres in northern Europe.From a sampling frame of 24,682 adults of European ancestry enrolled in population-based cohorts across Europe, participants at varying risk of glycaemic deterioration were identified using a risk prediction algorithm  and enrolled into a prospective cohort study (n = 693) of cohort 1 (prediabetes risk) had normal glucose regulation and 67% (n = 1419) had impaired glucose regulation. Seventy-six per cent of participants in cohort 1 was male. Cohort 1 participants had the following characteristics (mean ± SD) at baseline: age 62 (6.2) years; BMI 27.9 (4.0) kg/m2; fasting glucose 5.7 (0.6) mmol/l; 2 h glucose 5.9 (1.6) mmol/l. At the final follow-up examination the participants’ clinical characteristics were as follows: fasting glucose 6.0 (0.6) mmol/l; 2 h OGTT glucose 6.5 (2.0) mmol/l. In cohort 2 (diabetes), 66% (n = 517) were treated by lifestyle modification and 34% (n = 272) were treated with metformin plus lifestyle modification at enrolment. Fifty-eight per cent of participants in cohort 2 was male. Cohort 2 participants had the following characteristics at baseline: age 62 (8.1) years; BMI 30.5 (5.0) kg/m2; fasting glucose 7.2 (1.4) mmol/l; 2 h glucose 8.6 (2.8) mmol/l. At the final follow-up examination, the participants’ clinical characteristics were as follows: fasting glucose 7.9 (2.0) mmol/l; 2 h mixed-meal tolerance test glucose 9.9 (3.4) mmol/l.Using ADA 2011 glycaemic categories, 33%  contains peer-reviewed but unedited supplementary material, which is available to authorised users. The global prevalence of type 2 diabetes is burgeoning. There is no cure, nor are there treatments effective enough to halt the progression of the disease. The burden the disease conveys at a societal and personal level is enormous, with an estimated world prevalence in 2017 of around 425 million people with type 2 diabetes and a further 352 million at risk of developing the disease ) ) of the total cohort 1 baseline sample. The other centres in the Netherlands, Denmark and Sweden enrolled 22% (n = 473), 13% (n = 275) and 7% (n = 139) of the total cohort, respectively.The number of participants enrolled into cohort 1 varied between centres, with the Finnish subcohort being the largest  mmol/mol [5.5 (0.27)%], fasting glucose 5.7 (0.6) mmol/l, 2 h glucose 5.9 (1.6) mmol/l, fasting insulin 10.9 (7.6) pmol/l, glucose sensitivity 113 (55) pmol min−1 m−2 (mmol/l)−1 and insulin sensitivity  381 (59) ml min−1 m−2. Participants had a 0–48 month mean (SD) difference in fasting plasma glucose levels of 0.3 (0.2) mmol/l.Briefly, at baseline, participants had a mean (SD) age of 62 (6.2) years, BMI 27.9 (4.0) kg/mn = 41).Of 830 individuals in cohort 2 enrolled to attend the screening visit, 789 passed all inclusion, exclusion and quality control criteria. Of these, 272 were treated with lifestyle modification plus metformin and 517 were treated with lifestyle intervention only. Participants who withdrew consent, who were receiving any other oral hypoglycaemic agent or who reported ever receiving insulin treatment were excluded (n = 167), 18% (n = 141), 21% (n = 166), 21% (n = 167), 12% (n = 96) and 7% (n = 52) of the total cohort, respectively; 52–63% of the subcohort participants were male.Of the participants in cohort 2 at baseline, the UK , Dutch (Amsterdam), Swedish (Lund) and Danish (Copenhagen) study centres enrolled 21%  mmol/mol [6.4 (0.53)%], fasting glucose 7.2 (1.4) mmol/l, 2 h glucose 8.6 (2.8) mmol/l, fasting insulin 107 (71) pmol/l, glucose sensitivity 83 (55) pmol min−1 m−2 (mmol/l)−1 and insulin sensitivity (2 h OGIS) 298 (69) ml min−1 m−2. Participants had a 0 months to 18 months mean difference in fasting plasma glucose levels of 0.8 (1.9) mmol/l.Briefly, at baseline, participants had a mean (SD) age 62 (8.1) years, BMI 30.5 (5.0) kg/mAs some study centres enrolled participants into both cohorts, we elected to characterise the genetic population substructure across the cohorts by study centre (i.e. pooling both cohorts at a given centre where possible). Genetic substructures closely map to the geographic location of the populations , indicatHere, we report the characteristics of the IMI DIRECT cohorts at baseline, at 18 months follow-up, and at 48 or 36 months follow-up (for cohorts 1 and 2 respectively) for glycaemic deterioration and consider these results in the context of the implemented protocols and the plans outlined in the design and rationale paper published previously . The desMajor advances in technologies and methods over the past decade make high-resolution quantification of disease phenotypes and processes possible in large sample collections. Applying modern assays to historical biosamples is particularly useful when studying processes that take decades to unfold. However, biosamples often degrade during long-term storage and many older studies did not deploy the advanced phenotyping methods available today. Recognising these limitations, we designed and initiated two state-of-the-art prospective cohort studies as part of the IMI DIRECT Consortium. Designed for biomarker discovery in glycaemic deterioration and diabetes progression, the IMI DIRECT cohorts include conventional and cutting-edge phenotyping techniques and technologies that are repeated on multiple occasions during a follow-up period of up to 48 months (currently ongoing). We note that the subsequent biomarker discovery analyses using these cohorts will combine the clinical phenotypic data described in this paper with omic measures such as transcriptomics (RNA sequencing from fasting whole blood), microbiomics , proteomics (targeted array in fasting plasma) and metabolomics (targeted and untargeted assays in fasting plasma).N = 24,682) derived from established prospective cohort studies, whereas cohort 2 used clinical registries to identify eligible participants. The strategy for recruiting participants from existing prospective studies for cohort 1 facilitated access to data that were used to predict risk of rapid glycaemic deterioration. However, despite the relatively large sampling frame, it was necessary to enrol lower-risk participants in order to achieve the target sample size; in doing so, we recognised that this would likely reduce the overall rate of glycaemic deterioration in the cohort, although the generalisability of the study’s findings will be greater. In cohort 2, we fell slightly short of the target sample size of 1000 participants . This reflects the difficulties in engaging some general practices, which was necessary to access diabetes registries in some regions.The recruitment strategies for the two IMI DIRECT cohorts differed in that cohort 1 focused on recruiting participants from an existing large sample frame (We stratified cohorts 1 and 2 by broad glucose-control category (overtly normoglycaemic or impaired glycaemic regulation in any ADA category) or treatment category (lifestyle only vs lifestyle plus metformin), respectively, to reflect the basic stages of progression at baseline for descriptive purposes.The two IMI DIRECT cohorts are not identical. However, they share many methodology parallels that permit complimentary analyses to be performed, such as determining whether biomarkers for glycaemic deterioration are conditional on disease state. Nevertheless, several key differences in the protocols (e.g. fsOGTT vs MMTT) should be considered when interpreting results. We also note a difference in missing accelerometry  data between cohort 1 (19%) and cohort 2 (8.5%); we were unable to definitively explain this discrepancy. Partitioning change from error is very challenging when variables are assessed at only two time points owing to regression to the mean. Notwithstanding this, we note a modest 0 month to 48 month difference in mean (SD) fasting plasma glucose levels, 0.3 (0.5) mmol/l and 0.8 (1.9) mmol/l in cohort 1 and cohort 2, respectively


A damaged cell model was established using 2.6 mmol/L oxalate to injure HK-2 cells. Then, different concentrations of TPSs were used to repair the damaged cells. Index changes of subcellular organelles of HK-2 cells were detected before and after repair. The four kinds of TPSs possessed radical scavenging activity and reducing power, wherein TPS2 with moderate Mw presented the strongest antioxidant activity. After repair by TPSs, cell morphology of damaged HK-2 cells was gradually restored to normal conditions. Reactive oxygen species production decreased, and mitochondrial membrane potential (Δψm) of repaired cells increased. Cells of G1 phase arrest were inhibited, and cell proportion in the S phase increased. Lysosome integrity improved, and cell apoptotic rates significantly reduced in the repaired group. The four kinds of TPSs with varying Mw displayed antioxidant activity and repair effect on the mitochondria, lysosomes, and intracellular DNA. TPS2, with moderate Mw, showed the strongest antioxidant activity and repair effect; it may become a potential drug for prevention and treatment of kidney stones.This study aims at investigating the antioxidant activity and repair effect of green tea polysaccharide (TPS) with different molecular weights (Mw) on damaged human kidney proximal tubular epithelial cells (HK-2). Scavenging activities on hydroxyl radical ( TPS exerts protective effects against cellular damage induced by oxidative stress.Tea originated from China features a long history of over 4000 years; it is also the most popular nonalcoholic beverage and common food ingredient in Asia . Tea polMany studies have shown that tea polyphenol has inhibitory effects on calcium oxalate urolithiasis due to its antioxidative effects , 6. Tea 4 Da. 7WA mainly contains arabinose and galactose at a molar ratio of 1.0 : 0.96 and possesses a backbone consisting of 1,3- and 1,6-linked galactopyranosyl residues, with branches attached to O-3 of 1,6-linked galactose residues and O-4 and O-6 of 1,3-linked galactose residues. Wang et al. .For fluorescence quantitative detection by a microplate reader, cells  was used as a positive control. After the repair was completed, the cells were harvested and then stained using Annexin V-FITC/PI cell death assay kit according to the manufacturer's instructions. The cells were resuspended in 200 μL of binding buffer. Then, 5 μL of Annexin V-FITC was added, followed by incubation in the dark for 10 min at room temperature. The cells were resuspended in 200 μL of binding buffer and stained with 5 μL of PI. The prepared cells were then analyzed using a flow cytometer.According to our previous study , apoptosp < 0.05, there was a significant difference; if p < 0.01, the difference was extremely significant; if p > 0.05, there was no significant difference.The experimental data were expressed by mean ± standard deviation  at 4%, 8%, and 14% concentrations, respectively, of Hectively . TPSs ar2O2 is moderate, and the extent of degradation can be controlled without changing the structure of the main chain of polysaccharides [2O2 to obtain four polysaccharide fractions with Mw of 550, 347, 285, and 21 kDa. All degraded polysaccharide fractions had basically similar structure of the functional group. Hou et al. [Laminaria japonica fucoidan by changing H2O2 concentration, reaction temperature, and pH and obtained seven degraded fractions with Mw of 1.0, 3.8, 8.3, 13.2, 35.5, 64.3, and 144.5 kDa. No significant changes were observed in the major backbone structure and sulfate group content of all polysaccharide fractions.Degradation reaction of Hcharides –37. For charides  performe2O2 totaled 4% and 8%, carboxyl contents of degraded TPS1 and TPS2 products reached 12.3% and 12.7%, which were slightly higher than that of TPS0 (11.2%) before degradation. The above results were attributed to the increased solubility of degraded polysaccharides  and –COOH (1602.1 and 1610.5 cm−1) in degraded TPS1 and TPS2 fractions were notably stronger, respectively, indicating that TPS1 and TPS2 exposed numerous –OH and –COOH groups [Each polysaccharide sample manifested the same amount (2.0 mg). Thus, absorption peak intensity can reflect the content of characteristic functional groups . CompareH groups . The abs2O2 degradation. Therefore, as representative for TPS0, 1H and 13C NMR spectra of polysaccharide were characterized, and the spectra are shown in 13C NMR and 1H NMR signal assignment of TPS0 is shown in As shown in FT-IR spectra, the basic structure of TPS remained undamaged during H1H NMR spectrum, signals in the range of δ 4.5–5.5 ppm were assigned to the sugar ring of polysaccharides. H−1 proton signals derived from α-configuration sugar ring are detected at more than 4.95 ppm, whereas most of β-configuration protons will appear at less than 4.95 ppm. Thus, β- and α-configuration existed in TPS simultaneously [δ 5.0, 3.74, 3.95, 4.25, 4.37, and 4.37 ppm corresponded to H-1 to H-6, respectively, of (1 → 4)-α-GalpA in TPS [δ 4.63, 3.75, 3.56, 3.77, 3.60, and 3.91 ppm were attributed to H-1 to H-6 of (1 → 6)-β-Galp, whereas the signals at δ 5.24, 4.17, 4.09, 4.11, and 3.85 ppm were assigned to H-1 to H-5 of -Araf, respectively [δ 3.08, 3.10, 3.17, and 3.45 ppm were assigned to H-2, H-4, H-5, and H-6 of (1→)-β-D-Glcp, respectively [δ 4.89 and 3.64 ppm were assigned to H-1 and H-6 of (1 → 4)-α-D-Glcp, respectively [In aneously . The sigA in TPS . The sigectively . The sigectively . The sigectively .13C NMR spectrum, the signals in the region of δ 100–104 ppm indicated that monosaccharides existed in the form of pyranose ring. The signals at δ 170–180 ppm were attributed to the uronic acid of polysaccharides [δ 98.9, 68.2, 68.9, 77.8, 72.7, and 173.4 ppm were assigned to C-2 to C-6 of (1 → 4)-α-GalpA, respectively. The signals at δ 107.1, 73.1, 74.7, 71.5, 75.4, and 70.5 ppm corresponded to C-1 to C-6 of (1 → 6)-β-Galp, respectively [δ 109.5, 81.3, 77.8, 85.3, and 70.1 ppm were assigned to C-1 to C-5 of -Araf, whereas δ 74.3, 76.6, 70.3, 76.7, and 60.8 ppm were assigned to C-2 to C-6 of (1→)- β-D-Glcp, respectively [δ 99.5 and 61.2 ppm were attributed to C-1 and C-6 of (1 → 4)-α-D-Glcp, respectively [In the charides . The sigectively . The sigectively .·OH radical scavenging ability of TPS. As shown in ·OH scavenging rates of TPS0, TPS1, TPS2, and TPS3 reached 51%, 62%, 79.4%, and 37%, respectively. These values were all lower than 95.3% of Vc.Fenton reactions were used to investigate the As shown in The reducing power of four TPSs followed the order TPS2 > TPS1 > TPS0 > TPS3 . TPS2 stμg/mL, and TPS2 exhibited the strongest promotion effect. The above results showed that these TPSs caused no cytotoxicity on HK-2 cells and promoted cell growth.Antioxidant activities of TPS1 (8.16 kDa) and TPS0 (10.8 kDa) showed minimal difference due to their similar Mw .ThereforThe effects of oxalate concentration and injury time on the viability of HK-2 cells were shown in μg/mL , therefore mediating the occurrence of a series of inflammatory responses and causing cell dysfunction or death , 47. Theψm of the damaged HK-2 cells before and after repair. The red/green fluorescence intensity ratio in the mitochondria of the normal control group reached 67.5. When cells were damaged by 2.6 mmol/L oxalate, the red/green fluorescence ratio reduced to 3.8, indicating that Δψm was reduced evidently. However, after the damaged cells were repaired by TPS0, TPS2, and TPS3, Δψm increased at different degrees. After the damaged cells were repaired by TPS2, the red/green fluorescence ratio in the mitochondria reached 22.9, which was higher than that of the TPS0 treatment group (19.8) and TPS3 treatment group (9.3). Thus, TPS2 showed the strongest repair effect on damaged mitochondria.Acridine orange (AO), a metachromatic fluoroprobe, is a lysosomotropic component that accumulates in lysosomes by proton trapping. AO accumulation changes fluorescence emission from green in the cytoplasm to red in lysosomes . TherefoAs shown in Cell cycle mainly includes early DNA synthetic phase (G1 phase), DNA synthetic phase (S phase), and late DNA synthetic phase (G2 phase). Arrest of the cell cycle reflects the degree of DNA damage .As shown in We performed flow cytometric analysis to quantify apoptotic and necrotic cells using Annexin V/propidium iodide (PI) double staining. Annexin V staining was applied to reveal surface exposure of phosphatidylserine (apoptosis) and PI to reveal the loss of plasma membrane integrity (necrosis) , 51.1H NMR and 13C NMR spectrum -α-D-Glcp, (1→)-β-D-Glcp, (1 → 6)-β-Galp, and -Araf, which were consistent with the results clarified by Wang et al. [From the results of spectrum , TPS comg et al.  and Scopg et al. .2O2 degradation can attack glucosidic linkages. Oxidative scission produced diverse termini, which can be further oxidized to produce carboxyl acid. Splitting of C–C bonds within sugar residues also occurred, leading to ring-opening oxidation and formation of carboxyl groups [2O2. FT-IR and NMR suggested no distinct change in the structures of 1,4-β-D-glucose main chain, whereas changes only occurred in the side chain of LWCS.FT-IR results revealed that the four polysaccharide fractions featured a similar backbone structure, but characteristic absorption peak intensities of –COOH and –OH of polysaccharide differed. This result may explain why the hydroxyl radicals produced by Hl groups . For insl groups  prepared2·-scavenging effect of LWCS (9 kDa) was more effective than that of high-molecular-weight chitosan (760 kDa). However, Ma et al. [Pleurotus eryngii polysaccharide (413 kDa) against HepG-2 cells was better than that of low-Mw fraction (12 kDa).For different types of plant polysaccharides, the best bioactivity is attributed to different ranges of Mw. For example, Xing et al.  reporteda et al.  observed·OH, ABTS scavenging rates, and reducing power of low-Mw TPS3 (2.31 kDa) were weaker than those of moderate Mw TPS2 (4.82 kDa). This phenomenon possibly caused the significant destruction in the chain structure of TPS3 (with the lowest Mw); TPS3 featured the most loose molecular structure. The number of effective hydroxyl groups capable of chelating metal ions reduced [The antioxidant activity of polysaccharides is closely correlated with Mw. As shown in  reduced , 59. TheAthyrium multidentatum (Doll.) Ching polysaccharide and obtained four polysaccharide fractions with Mw of 14,528 (CPA-1), 12,370 (CPA-2), 11,548 (CPA-3), and 6403 Da (CPA-4). At a concentration of 0.2 mg/mL, DPPH-free radical scavenging rate of CPA-1, CPA-2, CPA-3, and CPA-4 totaled 0.687, 0.605, 0.429, and 0.420, respectively. In other words, polysaccharides with low Mw show weak antioxidant activity. Lai et al. [Many studies have also reported similar results. Sheng and Sun  performePolysaccharides can supply single electrons or hydrogen atoms to terminate free radical chain reaction and achieve radical scavenging activity , 62. In Polysaccharide bioactivity depends on helical structure of the main chain and the presence of hydrophilic groups (hydroxyl group) located on the outside surface of the polysaccharide helix . High-Mw5 Da to 6.3 × 105 Da (PW1), 3.5 × 104 Da to 7.4 × 104 Da (PW2), and 5.3 × 103 Da to 2.3 × 104 Da (PW3). IC50 values of scavenging ABTS radical of PW1, PW2, and PW3 measured 0.35, 0.2, and 0.04 mg/mL, respectively. The above results indicate the weak antioxidant activity of high-Mw polysaccharides. Sun et al. [2O2 degradation of k-carrageenan polysaccharide with Mw of 350,000 Da and obtained four fractions with Mw of 3.25, 5.82, 15.08, and 20.9 kDa. IC50 values of scavenging superoxide anion free radicals of the four degraded fractions totaled 2.65, 3.22, 6.66, and 8.13 mg/mL. As for hydroxyl radical scavenging, IC50 values reached 0.014, 0.049, 0.062, and 0.110 mg/mL.Many studies have reported the weak antioxidant activity of polysaccharides with high Mw , 68. For2+ and Cu2+) that are necessary in producing ·OH radicals to form complexes. Therefore, generation of radicals and initiation or progress of lipid chain reaction is inhibited [When high-Mw polysaccharides are degraded into a certain range of Mw, they can achieve optimal bioactivity. Polysaccharides with moderate Mw can not only possess sufficient spatial scale to form three-helical polymerization structure , 59, 64 nhibited .Camellia seed cake (COP-C) using ultrasonic wave and obtained four polysaccharide fractions, namely, COP-1, COP-2, COP-3, and COP-4, with molecular weights of 7.9, 36, 83, and 225 kDa, respectively. At the concentration of 2 mg/mL, radical scavenging capacity and reducing power order followed the order of COP-2 > COP-3 > COP-4 > COP-1. Only the polysaccharide with moderate Mw of 36–83 kDa exhibited the strongest antioxidant activity. Trombetta et al. [Opuntia ficus-indica (L.) cladodes also benefits enhancement of ability to repair damaged cells. The polysaccharide fraction with Mw higher than 104 Da showed a wound-healing effect on damaged skin epithelium cells in mice. Wound-healing effect is more remarkable for polysaccharides with Mw ranging 104–106 Da than those with Mw > 106 Da.Xu et al.  degradeda et al.  have shoThe four studied TPSs exhibited repair effect on damaged HK-2 cells induced by oxalate, and repair ability was positively related with antioxidant activity. TPS2, which presented the strongest antioxidant activity, also showed the strongest cell repair ability.Bacteroides fragilis with the molecular weight of larger than 100 kDa is endocytosed by antigen-presenting cells (APCs) and localizes to the conventional MHCII compartment (MIIC). This observation was also confirmed in primary mouse splenocytes, human THP-1 monocytes, and mouse B1 B cells. Time course studies indicated that entry and surface localization of PS-A was visible in 30 min and peaked at 6 h. Therefore, the TPS polysaccharide (2.31 ~ 10.88 kDa) used in this study can access to the HK-2 cells.In living organisms, small molecules can pass through the plasma membrane directly or through the help of carrier proteins or ion channels , while sLycium barbarum polysaccharides can protect tissue cells from DNA damage induced by oxidative stress [Hericium erinaceus polysaccharides can scavenge DPPH free radical, reduce ROS production, improve cell viability, inhibit reduction of mitochondrial membrane potential, and exhibit protective effects on amyloid beta-induced neurotoxicity in PC12 cells [Salvia brachyantha extract reduces H9C2 cell apoptosis induced by xanthine oxidase/xanthine by preventing generation of toxic-free radicals and by enhancing the intracellular antioxidant defense system [Psidium guajava leaf polysaccharides can scavenge radicals to relieve H2O2-induced oxidative stress and DNA injury in Vero cells and inhibit lipid peroxidation. After cell repair by 12.5, 25, and 50 μg/mL of Psidium guajava leaf polysaccharides, intracellular ROS production decreased from 129.5% in the damage group to 118.9%, 109.7%, and 99.7%, respectively. A polysaccharide from Lonicera japonica flowers remarkably reduced malondialdehyde levels, elevated superoxide dismutase and glutathione peroxidase activities, and protected the rat brain against ischemia/reperfusion injury [Numerous studies have shown that accumulation of ROS in vivo can attack cells and cause protein oxidation, lipid peroxidation, and nucleic acid fracture, which can affect normal cell functions and result in the occurrence of chronic diseases , 47, 76.e stress . Hericiu12 cells . Salvia e system . Kim et e system  revealedn injury .ψm. TPS can repair the membrane potential of cells and increase Δψm. In damaged cells, the percentage of cells in the S phase decreased but that in the G1 phase increased. After treatment with TPS, TPS promoted cell cycle progression from the G1 phase to the S phase and repaired DNA replication. Finally, TPS alleviated cell apoptosis induced by oxidative stress and decreased the underlying risk of stone formation.On the basis of our research results, a proposed repair mechanism of damaged HK-2 cells by TPS is illustrated in Four TPS fractions  with Mw of 10.88, 8.16, 4.82, and 2.31 kDa, respectively, were obtained. All TPS fractions exhibited antioxidant activity. The order of hydroxyl radical scavenging, ABTS radical scavenging activity, and reducing power was as follows: TPS2 > TPS1 > TPS0 > TPS3. The four TPSs also showed repair effects on HK-2 cells with damage induced by 2.6 mmol/L oxalate. Repair effect of TPSs was positively related with antioxidant activity. TPS2, featuring a moderate Mw, displayed the strongest antioxidant activity and cell repair ability. Compared with the damage group, cell morphology in the repaired group was closer to that of normal cells. The treated groups also yielded the following results: cell viability strengthened, mitochondrial membrane potential and integrity of lysosome improved, ROS production decreased, cells were arrested in the G1 phase, and cell apoptosis rate was reduced. All these findings indicate that these TPSs show repair effect on cell morphology, mitochondria, DNA, and other subcellular organelles in damaged HK-2 cells. Our results suggest that these TPS fractions, especially TPS2, may become potential drugs for prevention and cure of kidney stones.


M phenylborate) complexes of cobalt(II) with bidentate ligands were synthesized. Both Tp* and ToM coordinate to cobalt(II) in a tridentate fashion when the bidentate ligand is the less hindered acetylacetonate. In crystal structures, the geometry of cobalt(II) supported by the N3O2 donor set in the Tp* complex is a square-pyramid, whereas that in the ToM complex is close to a trigonal-bipyramid. Both Tp*- and ToM-acac complexes exhibit solvatochromic behavior, although the changing structural equilibria of these complexes in MeCN are quite different. In the bis(1-methylimidazol-2-yl)methylphenylborate (LPh) complexes, Tp* retains the tridentate (к3) mode, whereas ToM functions as the bidentate (к2) ligand, giving the tetrahedral cobalt(II) complex. The bowl-shaped cavity derived from the six methyl groups on ToM lead to susceptibility to the bulkiness of the opposite bidentate ligand. The entitled scorpionate compounds mediate hydrocarbon oxidation with organic peroxides. Allylic oxidation of cyclohexene occurs mainly on the reaction with tert-butyl hydroperoxide (TBHP), although the catalytic efficiency of the scorpionate ligand complexes is lower than that of Co(OAc)2 and Co(acac)2. On cyclohexane oxidation with meta-chloroperbenzoic acid (mCPBA), both ToM and Tp* complexes function as catalysts for hydroxylation. The higher electron-donating ToM complexes show faster initial reaction rates compared to the corresponding Tp* complexes.Scorpionate ligands Tp* borate) and To The third, fourth, and fifth positions of pyrazole can be modified by introducing various groups, and the resulting substituted pyrazoles are also applied to borate ligands such as [HB(pzR)3]‒ . Hydrotrisborate, called Tp* , is classified as “the first generation of scorpionates” along with the nonsubstituted Tp. Its moderate bulkiness, derived from the methyl groups, allows it to form both homo- and heteroleptic complexes 2] and [M(к3-Tp*)Lx]; L denotes ligands other than Tp*), in the latter of which the metal centers have various coordination numbers. Therefore, various metal complexes with Tp* have been investigated extensively so far  and [MI(к3-ToM)(CO)3] are, however, very close, because no steric repulsions exist between the metal-surrounding methyl groups on the scorpionates and the liner diatomic CO ligands. The electron-donating ability of ToM is higher than that of Tp* estimated from νCO of IR measurement. From electrochemical investigations, however, the donating ability of Tp* is higher than that of ToM according to the order of the oxidation potentials, [MI(к3-Tp*)(CO)3] < [MI(к3-ToM)(CO)3]. Such different interpretations arise from differences in the oxidation state. The former estimation based on IR data is attributed to the single oxidation state of the metal centers, while the latter results from changing the oxidation state of the metal centers on the rigid pseudo-octahedral complexes  and [NiII(к3-ToM)Cl]. The Tp* complex is very close to the symmetric structure of C3v, as evidenced by an almost linear arrangement of Cl-Ni---BTp. In the ToM complex, however, an arrangement of Cl-Ni---BTo deviates from a straight line. Therefore, ToM does not work as a “tetrahedral enforcer,” although solid angle analysis . We focused on the catalytic performance of the synthesized mixed ligand complexes toward alkene oxidation with tert-BuOOH (TBHP) and alkane oxidation with meta-chloroperbenzoic acid (mCPBA), as the estimation of the functions depends on the scorpionate ligands. These aspects provide useful information for the design of functional metallocomplexes with appropriate ligands.In this work, we investigated coordination behaviors of Tp* and ToPh; [B(ImN-Me)2MePh]‒) as a nitrogen-donating ligand  (1) and [CoII(ToM)(acac)] (2), respectively. It is known that homoleptic complexes [CoII(к3-Tp*)2] [II(к2-ToM)2]  is also formed in the presence of proton sources [The acac complexes were synthesized by ligand metathesis reactions between Co3-Tp*)2]  and [CoI2-ToM)2]  are form sources . In the 1H-NMR, UV-vis) and X-ray crystallography. Molecular structures of an MeCN adduct of 1 and 2 are shown in 1 (when the coordinating MeCN molecule is omitted) and a distorted trigonal-bipyramid in 2. In Tp* complex 1, both oxygen donors of acac were located on equatorial positions. In ToM complex 2, however, one of the two oxygen donors from acac sat on the trigonal plane and another was on the axial position. Such difference of locations of the acac ligands in the static solid state arose from steric hindrance derived by cobalt-surrounding methyl groups on the azole rings of the scorpionates. In 2, steric repulsion between two equatorial 4,4-dimethyl oxazoline groups of ToM and a virtual equatorial acac ligand would occur. Similar structural variations depending on the bulkiness of substituent groups of TpR are also observed in [CoII(к3-Tp3R)( к3-acac)] (3R denotes substituent groups on the third position of the pyrazolyl rings)   . In the ture see . In cont of MeCN . The sperent see . TherefoPh ([B(ImN-Me)2MePh]‒) ‒) ,17,18,193 had already been reported as an analogue of a penta-coordinated iron(II) complex [Fe(Tp*)(LPh)], which showed O2-binding capability to yield the corresponding mononuclear iron(III)-superoxo compound   . As descM complex 4 was a tetrahedral cobalt(II) complex with bidentate ToM and LPh ligands, as shown in Ph in 4 were located almost coplanar , and an N–Co–N bite angle (97.7°) was larger than a right angle. Because of the steric hindrance derived from the methyl groups on the oxazoline rings of ToM, it was impossible to form a penta-coordinated cobalt(II) complex with к3-ToM and к2-LPh. One of three oxazolynyl groups of ToM in 2 was left from the cobalt center through the ligand substitution from acac to LPh such that the bidentate ToM was observed in a homoleptic complex, [CoII(к2-ToM)2] [M were also reflected on spectroscopic properties. IR spectra of the solid samples of 4 (II(к2-ToM)2] exhibited characteristic peaks around 1600 and 1575 cm−1 and 1603 and 1554 cm−1, respectively. The bands around 1600 cm−1 would be attributed to νC–N of the noncoordinating oxazolynyl groups of к2-ToM. UV-vis spectra of CH2Cl2 solutions showed intense band characteristics to the tetrahedral cobalt(II) d–d transition at λmax = 525 (ε ≈ 440 M–1 cm–1) and 554 nm (ε ≈ 580 M–1 cm–1) for 4 (λmax = 562 (ε = 539 M−1 cm−1) and 576 nm (ε = 552 M−1 cm−1) for [CoII(k2-ToM)2]. The observed blue shift of the d–d transition bands from those of [CoII(k2-ToM)2] to 4 might imply that the strength of the ligand fields is LPh > ToM. In fact, a homoleptic LPh complex [CoII(LPh)2]  and 548 nm (ε ≈ 420 M–1 cm–1) 2] . Similarles of 4  and , showed reversible O2 sorption/desorption ability at low temperature, although its oxidation potential was higher (0.52 V) than that of 4   . In the R  borate), and mesoporous silica-supported TpCF3Co complexes, was examined without any additive, and the activity of CoII(OAc)2·4H2O and [CoIIBr] under homogeneous conditions was lower than that of the immobilized complexes under heterogeneous reactions  (6)  (6) ) were loII(acac)2 reacts with excess TBHP in the presence of N-donating L ligands such as pyridine or 1-methyimidazole to yield the corresponding cobalt(III)-OOtBu complex [CoIII(OOtBu)(acac)2(L)]  [It is known that Coac)2(L)] ,30. In cu)(TpR)] . These rM, [CoII(ToM)(OAc)], functions as an efficient catalyst precursor for cyclohexane hydroxylation with mCPBA [IITp*)2(μ-OH)2], did not function as a catalyst precursor for alkane oxidation with mCPBA when the ratio of cobalt(II) to mCPBA to cyclohexane was 1:50:2500  + Na+]+. Elemental analysis calcd (%) for [CoII(Tp*)(acac)] (C20H29N6BO2Co): calc. C 52.77 H 6.42, N 18.46; found: C 52.55, H 6.06, N 18.79.FT-IR (KBr): M  was slowly added to a THF solution (30 mL) of CoII(acac)2  at room temperature. The purple slurry solution was stirred for 1 h, and the reaction was heated at reflux for 2 h. The volatile solvent of the solution was evaporated, and the residue was redissolved in CH2Cl2. The solution was then passed through a filter with Celite to remove any inorganic salts. After evaporation of CH2Cl2, the residue was redissolved in pentane and the solution was passed through a filter with Celite. After reduction of solvent volume by evaporation, the solution was subject to recrystallization at −30 °C to give the title complex as red purple powder . Recrystallization from the pentane solution at −30 °C for 1 day gave a purple block crystal suitable for X-ray crystallography.A THF solution (60 mL) of NaToν = 3077 (w), 3042 (w), 2965 (vs), 2894 (w), 1589 , 1520 cm−1 . UV-vis : λ = 450 nm (ε = 38.4 M−1 cm−1), 555 nm (ε = 65.4 M−1 cm−1). UV-vis : λ = 513 nm (ε = 123.2 M−1 cm−1), 557 nm (ε = 162.3 M−1 cm−1), 584 nm (ε = 167.6 M−1 cm−1). 1H-NMR : δ = 46.69 , 43.74 , 27.57 , 21.57 , 18.04 , 5.81 , −60.42 . 1H-NMR : δ = 41.43 , 40.86 , 23.83 , 20.36 , 17.08 , 5.53 , −56.73 . Magnetic susceptibility: μeff = 4.4 μB. ESI-MS+ (MeOH): m/z = 540 [[CoII(ToM)(acac)] + H+]+. Elemental analysis calcd (%) for [CoII(ToM)(acac)] (C26H36N3BO5Co): calc. C 57.80, H 6.72, N 7.78; found: C 57.46, H 6.68, N 8.02.FT/IR (KBr): n-butyllithium hexane solution  was added to a THF solution (20 mL) of HLPh  at −80 °C to give LiLPh solution. The solution was then gradually warmed to room temperature with stirring. This LiLPh solution was slowly added to a THF solution (20 mL) of 2  at −80 °C. The solution was then gradually warmed to room temperature and stirred for 2 h. The volatile solvent of the solution was evaporated, and the residue was redissolved in CH2Cl2. The solution was then passed through a filter with Celite to remove any inorganic salts. After evaporation of CH2Cl2, the red purple solid was washed with pentane to give the title complex as light pink powder . Recrystallization from the Et2O/hexane solution at room temperature gave a purple block crystal suitable for X-ray crystallography. We were unable to obtain satisfactory elemental analysis data for this complex.An ν = 3062 (w), 2965 (w), 2927 (vs), 2889 (m), 1602 , 1575 , 1283 cm−1 . UV-vis : λ = 525 nm (ε = 441 M−1 cm−1), 554 nm (ε = 580 M−1 cm−1). Magnetic susceptibility: μeff = 4.0 μB. ESI-MS+ (MeOH): m/z = 707 [[CoII(ToM)(LPh)] + H+]+.FT-IR (KBr): n-butyllithium hexane solution  was added to a THF solution (25 mL) of HLPh  at −80 °C to give LiLPh solution. The solution was then gradually warmed to room temperature with stirring for 1 h. To 10 mL MeOH solution of CoCl2 , THF solution of LiLPh was added, and the resulting reaction mixture was stirred for 1 h. The volatile solvent of the solution was evaporated, and the residue was redissolved in CH2Cl2. The solution was then passed through a filter with Celite to remove any inorganic salts. After evaporation of CH2Cl2, the resulting solid was dissolved in MeCN, and refrigeration yielded a pink powder of [CoII(LPh)2] . Recrystallization from the MeCN solution at room temperature gave a red block crystal suitable for X-ray crystallography.An ν = 3142 (m), 3118 (s), 2917 (vs), 2905 (vs), 2890 (vs), 2836 (vs), 2828 (vs), 2760 (s), 1286 cm−1 . UV-vis : λ = 520 nm (ε = 303 M−1 cm−1), 548 nm (ε = 419 M−1 cm−1). Magnetic susceptibility: μeff = 3.7 μB. Elemental analysis calcd (%) for [CoII(LPh)2] (C30H36N8B2Co): calc. C 61.15, H 6.16, N 19.02; found: C 60.92, H 6.17, N 19.04.FT-IR (KBr): 6 [II(OAc)2]·4H2O  was added to a THF solution (70 mL) of KTpMe2  at room temperature and the yellow solution was stirred for 1.5 h. The volatile solvent of the solution was evaporated, and the residue was redissolved in CH2Cl2. The solution was then passed through a filter with Celite to remove any inorganic salts. After evaporation of CH2Cl2, the residue was redissolved in MeCN and the solution was filtered to give the title complex as yellow powder .The previously reported synthetic procedure for 6  was modiν = 3123 (w), 2976 (w), 2925 (s), 2863 (w), 2733 (s), 2507 . UV-vis : λ = 470 nm (ε = 130.9 M−1 cm−1). 1H-NMR : δ = 45.16 , 43.97 , −81.56 . Magnetic susceptibility: μeff = 4.7 μB. ESI-MS+ (MeOH): m/z = 653 [CoIII(Tp*)2]+. Elemental analysis calcd (%) for [CoII(Tp*)2] (C30H44N12B2Co): calc. C 55.15, H 6.79, N 25.73; found: C 54.84, H 6.73, N 25.78.FT/IR (KBr): 1, 2, and 5) or 133 K (for 4) by cold N2 gas flow on the goniometer. For complex 1, three sets of sweeps  of data were done using ω oscillations from −115.0 to 65.0° in 0.3° steps at χ = 45° with an exposure rate of 13.3 s/°, a detector swing angle of −25°, and a crystal-to-detector distance of 55 mm . For complex 2, two sets of sweeps (at φ = 0 and 90°) of data were done using ω oscillations from −110.0 to 70.0° in 0.5° steps at χ = 45° with an exposure rate of 20 s/°, a detector swing angle of −20°, and a crystal-to-detector distance of 45 mm . For complex 4, two sets of sweeps (at φ = 0 and 90°) of data were done using ω oscillations from −110.0 to 70.0° in 0.5° steps at χ = 45° with an exposure rate of 66.7 s/°, a detector swing angle of −20°, and a crystal-to-detector distance of 45 mm . For 5, two sets of sweeps (at φ = 0 and 90°) of data were done using ω oscillations from −110.0 to 70.0° in 0.25° steps at χ = 45° with an exposure rate of 80 s/°, a detector swing angle of −20°, and a crystal-to-detector distance of 45 mm .Diffraction data for single crystals were collected using a Saturn 70 CDD area detector system  with graphite monochromated Mo-Kα radiation. The block-shaped crystals were mounted on a CryoLoop with liquid paraffin and flash-cooled to 113 K , 21,792 reflections , 13,756 reflections , and 13m918 reflections  were collected. Equivalent reflections were merged. The linear absorption coefficient μ for Mo-Kα radiation was 7.02 cm−1 for 1, 6.05 cm−1 for 2, 5.00 cm−1 for 4, and 5.66 cm−1 for 5. A numerical absorption correction was applied, which resulted in transmission factors ranging from 0.7770 to 0.9077 for 1, 0.8138 to 0.9041 for 2, 0.9106 to 0.9708 for 4, and 0.8349 to 0.9375 for 5. The data were corrected for Lorentz and polarization effects. Structural solution by a direct method (SIR-92) [F2 with all reflections were performed on WinGX software  [1) CCDC-1843691 (2), CCDC-1843692 (4), and CCDC-1893693 (5). These data can be obtained free of charge from the Cambridge Crystallographic Data Centre at www.ccdc.cam.ac.uk/data_request/cif. The molecular structures, bond lengths, and angles as well as crystallographic data and structure refinement parameters of 1, 2, 4, and 5 obtained in this work are given in 1 and 2) and 4) and 5) and Data collection and processing were performed using Rigaku CrystalClear software  . In all,(SIR-92)  and refigow, UK) . Crystal3N (10 μmol), and cyclohexene (2.0 mmol) in MeCN (5 mL) was placed in a Schlenk and degassed with Ar gas. Next, 70% aqueous solution of TBHP (2.0 mmol) was added under Ar atmosphere and stirred at room temperature or 60 °C. The reaction products were analyzed using GC .A solution of the cobalt(II) compounds (2.0 μmol), Et2Cl2 (1 mL) was placed in a Schlenk and degassed with Ar gas. Next, a CH2Cl2 (2 mL) solution of mCPBA (2.0 mmol) was added under Ar atmosphere and stirred at 35 °C. To monitor product formation, 0.2 mL of the reaction mixture was corrected at certain times  and quenched with a dichloromethane solution (0.5 mL) of triphenylphosphine (10 mg), and then the solution was subjected to GC analysis.A solution of cobalt(II) compounds (2.0 μmol), cyclohexane (15.0 mmol) in MeCN (1 mL), and CHM toward cobalt(II) with bidentate ligands were compared. Both Tp* and ToM coordinate to cobalt(II) with tridentate fashion when the bidentate ligand is the less hindered acetylacetonate. In the bis(1-methylimidazol-2-yl)methylphenylborate (LPh) complexes, Tp* retains the tridentate (к3) mode, whereas ToM functions as the bidentate (к2) ligand, giving the tetrahedral cobalt(II) complex. The bowl-shaped cavity derived from the six methyl groups on ToM leads to susceptibility to the bulkiness of the opposite bidentate ligand. The entitled scorpionate compounds catalyze degradation of TBHP to induce the peroxidation of cyclohexene, although their catalytic efficiencies are lower than those of Co(OAc)2 and Co(acac)2. On the cyclohexane oxidation with mCPBA, both ToM and Tp* complexes function as the catalysts. The difference in the electron-donating abilities of the scorpionates, that is, ToM > Tp*, is reflected in the initial reaction rates.The coordination behaviors of Tp* and To