Aspalathus linearis) has various health benefits. Two case studies have associated chronic Rooibos consumption with conventional prescription medications, including atorvastatin (ATV), with hepatotoxicity. Statins act by inhibiting hydroxymethylglutaryl‐coenzyme A reductase, a rate‐limiting enzyme in cholesterol synthesis. Although rare, statins are potentially hepatotoxic. The aim was to investigate interactions between aspalathin‐rich Rooibos extract GRT™ and ATV‐induced hepatotoxicity in C3A liver cells cultured with and without palmitate. Effects of co‐treatment of GRT + ATV on cell viability, oxidative stress, apoptosis, mitochondrial integrity, and cellular reactive oxygen species (ROS) production were assessed. Significantly increased ROS production was observed in cells exposed to ATV and palmitate. Combination therapy of GRT + ATV also showed significant increases in ROS production. Under palmitate‐treated conditions, ATV‐induced significant apoptosis which was not ameliorated by GRT + ATV co‐treatment. Despite studies purporting hepatoprotection from Rooibos, our study showed that GRT was unable to modulate ATV‐induced hepatotoxic effects in this model.Rooibos ( Effects of rooibos extract (GRT) and atorvastatin (ATV) treatments on reactive oxygen species (ROS) production in C3A cells without palmitate and cells treated with 500 µM palmitate for 24 hrs. The addition of GRT failed to ameliorate ROS production induced by ATV treatment. Scientific evidence confers multiple health benefits to Rooibos, such as anti‐inflammatory ; Ajuwon et al., ATV is considered a “blockbuster drug” as the best‐selling prescription drug in history, with lifetime sales of USD 148,744 million between 1996 and 2016 .22.1l‐glutamine , the combination thereof hereafter referred to as growth medium. Cells were seeded at a density of 11 × 104 cells per ml and incubated in humidified air at 37°C with 5% CO2. Replacement of culture media was done every 2–3 days. Cells were sub‐cultured to 70–80% confluence  was purchased from the American Type Culture Collection (ATCC). Eagle's modified essential medium , 10% vol/vol fetal bovine serum  and 1% vol/vol nfluence . Palmitanfluence , 2015.An aspalathin‐rich unfermented Rooibos extract (Afriplex GRT™) was obtained from Afriplex (Pty) Ltd , ATV was obtained from Sigma‐Aldrich , and palmitate was obtained from Sigma‐Aldrich . These, and all other chemicals for experimental use were of cell culture grade. Stock solutions were prepared in dimethyl sulfoxide (DMSO) and final working concentrations of treatments contained not more than 0.25% vol/vol DMSO.3‐‐2,5‐diphenyl tetrazolium bromide , 2′,7′‐dichlorofluorescein , 5,5′,6,6′‐tetrachloro‐1,1′,3,3′‐tetraethylbenzimidazolylcarbocyanine iodide , and propidium iodide  were obtained from Sigma, Stanheim, Germany.Annexin V  and caspase 3/7  were obtained from Thermo Fisher Scientific, Johannesburg, South Africa.2.2C3A liver cells, with palmitate or without palmitate, were exposed to ATV (10 and 25 µM) and Afriplex GRT™ (0.01 and 0.1 mg/ml) and a combination thereof  for 24 hr. Pretreatment with palmitate (500 µM) for 24 hr was used to simulate hyperlipidaemia in vitro  version 1.05. Three replicates per treatment were assessed in three independent experiments.Cellular metabolic activity was assessed using an MTT cell viability assay Mosmann, . C3A cel2.44 cells per ml in 200 µl growth medium per well in a black 96‐well clear bottom plate and cultured and treated as per Figure 2 in humified air). Fluorescent readings were recorded using a SpectraMax i3x multi‐mode microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 535 nm. Results were generated on SoftMax Pro Data Acquisition and Analysis Software (RRID:SCR_014240), version 7.0.2. Three replicates per treatment were assessed in three independent experiments. Reactive oxygen species production was calculated relative to cell viability which was assessed by an MTT assay.Oxidative stress was assessed by DCF staining  and data were compared using one‐way analysis of variance, followed by a Tukey post hoc test. Data are presented as mean ± standard deviation and 33.1p < .001). Combination treatment of ATV (10 and 25 µM) with GRT (0.01 and 0.1 mg/ml) did not ameliorate ATV‐induced cytotoxicity. A significant difference was noted between the monotherapy treatment groups and the combination treatment groups: GRT1 (94.05 ± 23.21%) showed significantly increased (p < .01) MTT activity compared with ATV1 + GRT1 (61.85 ± 13.18%), whilst GRT2 (97.48 ± 24.32%) showed significantly greater activity compared with ATV1 + GRT2  and ATV2 + GRT2 .Figure p < .001; 25 µM ATV = 16.58 ± 13.99%; p < .001). The adverse effect of ATV was not improved by the combination treatment with GRT at either concentration. GRT1 (91.92 ± 33.19%) increased (p < .001) MTT activity compared with ATV1 + GRT1 (39.92 ± 17.87%), and GRT2 (96.56 ± 30.92%) showed higher activity compared with ATV1 + GRT2  and ATV2 + GRT2 .In palmitate‐treated C3A cells  intracellular ROS compared with the vehicle control (100.00 ± 27.52%). Co‐treatment of GRT + ATV had a significant (p < .001) additive effect to the ATV‐induced intracellular ROS production compared with GRT2 (89.96 ± 40.96%) and ATV2 monotherapy (ATV2 + GRT2 = 845.00 ± 589.60%).In cells not exposed to palmitate as shown in Figure p < .05) and 25 µM ATV  significantly increasing intracellular ROS compared with the palmitate control (100.00 ± 19.07%). In combination, GRT at the concentrations used did not moderate ATV‐induced ROS production. A significant difference was seen between GRT monotherapy (137.80 ± 74.29% for GRT1 and 131.40 ± 87.18% for GRT2) compared with their respective combination treatment groups: ATV1 + GRT1 = 306.80 ± 192.20% (p < .05); ATV1 + GRT2 = 296.10 ± 102.00% (p < .05); ATV2 + GRT2 = 1493.00 ± 278.40% (p < .001). However, there was a significant difference (p < .001) between the ATV2 and ATV2 + GRT2 treatment groups.With palmitate treatment Figure , ATV‐ind3.3p < .05), ATV1 , and ATV2  treatment significantly reduced relative mean fluorescence intensity compared with the vehicle control (100.00 ± 8.76%). Figure p < .05).In cells not exposed to palmitate, Figure 3.4p < .01) and ATV2  significantly decreased the number of viable cells compared with the vehicle control (86.33 ± 4.63%). The addition of GRT at either concentration was unable to protect these cells against ATV toxicity. GRT1 monotherapy significantly increased the percentage of viable cells compared with ATV1 + GRT1 combination therapy . Similarly, GRT2 monotherapy significantly increased (p < .001) the percentage of viable cells compared with ATV2 + GRT2 combination therapy .In the absence of palmitate Figure , the addp < .01) in the percentage of early apoptotic cells when GRT2 (9.17 ± 3.25%) and ATV2 + GRT2 (0.33 ± 0.52%) were compared  and ATV2 group  showed significantly less prevalence of early apoptosis as compared with the vehicle control group (5.83 ± 2.04%). The combination of GRT1 and GRT2 did not significantly improve ATV1 or ATV2‐induced apoptosis.There was a significant difference  more late apoptotic cells compared the vehicle control (7.83 ± 2.79%), as did the ATV2 group , which showed the greatest percentage of late apoptotic of all the treatment groups in the normal condition. Compared with ATV1 + GRT1 (35.17 ± 19.28%), GRT1 (8.00 ± 9.90%) showed a significant (p < .01) decrease in the prevalence of late apoptotic cells. The GRT2 group (12.50 ± 6.06%) showed a significant (p < .001) decrease compared with ATV2 + GRT2 (64.83 ± 8.98%).Cells in late apoptosis, not treated with palmitate Figure , showed p < .01) decreased percentage of cells relative to the palmitate control group (13.00 ± 5.73%). Further, ATV2 + GRT2 (3.17 ± 0.98%) showed significantly decreased (p < .001) percentage of early apoptotic cells compared with the GRT2 monotherapy group (11.67 ± 2.58%). Figure In terms of the percentage of viable cells in the simulated hyperlipidaemic condition, there was no statistical differences between any of the different treatment groups Figure . Althoug3.5p < .001) caspase 3/7 activity compared with the vehicle control (100.00 ± 3.67%), as did the ATV2 group . The difference between GRT1 (100.90 ± 4.44%) and ATV1 + GRT1 (168.70 ± 45.81%) was significant (p < .001), as was the difference between GRT2 (101.80 ± 41.78%) and ATV1 + GRT2  and GRT2 and ATV2 + GRT2 (283.20 ± 135.00%). There was also a significant (p < .001) difference between ATV2 and ATV2 + GRT2.Figure p < .001) caspase 3/7 activity compared with the palmitate control group (100.00 ± 14.83%) in the hyperlipidaemic condition. The addition of GRT2 had a seemingly additive effect on the ATV monotherapy groups  as compared with their combination therapy counterparts: ATV1 + GRT1 = 155.50 ± 38.90% (p < .001); ATV1 + GRT2 = 126.20 ± 29.90% (p < .001); and ATV2 + GRT2 = 114.70 ± 21.82% (p < .01). Similarly, GRT2 monotherapy showed significantly increased (p < .001) caspase 3/7 activity compared with the ATV1 + GRT2 and ATV2 + GRT2 combination therapy groups.Figure 4Aspalathus linearis) is commonly prepared as a herbal infusion and has a number of health properties including antioxidant, hepatoprotective, and metabolic effects  and caspase activation.In cells not pretreated with palmitate, both 10 and 25 µM ATV caused a significant decrease in relative MTT activity, while GRT alone showed no difference at the concentrations used after 24 hr. In combination, GRT did not modulate the toxicity induced by ATV in terms of MTT activity. The same trend was noted in the palmitate‐treated cells. Taken together, this suggests that GRT did not have a modulating effect on ATV‐induced toxicity in terms of MTT activity.In the normal condition, as expected GRT did not induce ROS production. The higher concentration of ATV (25 µM) increased ROS production, and co‐treatment with 0.1 mg/ml GRT further exacerbated this effect. In the hyperlipidaemic condition, a similar trend was seen: GRT alone did not induce ROS production, however 25 µM ATV treatment, both alone and in combination with GRT, increased ROS. Shu et al.  attribut4− . This is an important redox component of the mitochondrial electron transport chain, responsible for synthesizing ATP. Reducing the biosynthesis of CoQ9/10 in the liver affects mitochondrial oxygen consumption and increases cellular ROS production, thought to be a major causal factor for statin‐induced adverse effects (Rundek, Naini, Sacco, Coates, & DiMauro, In contrast, other studies have demonstrated hepatoprotective effects for Rooibos against CClAlong with increased ROS, high‐dose ATV decreased mitochondrial membrane potential in both the normal and palmitate‐treated C3A cells. GRT could not attenuate the toxicity of the ATV in terms of mitochondrial membrane potential changes. The loss of mitochondrial membrane integrity and subsequent release cytochrome C from the inner membrane of the mitochondrion initiates the intrinsic apoptotic pathway culminating in the activation of caspase 3/7 (Brentnall, Rodriguez‐Menocal, De Guevara, Cepero, & Boise, 5This study found that ATV was concentration‐ and time‐dependently hepatotoxic to C3A liver cells and this hepatotoxic effect was exacerbated by the addition of palmitate pretreatment. ATV toxicity was associated with decreased mitochondrial membrane potential, increased ROS production, as well as increased caspase 3/7 activity. GRT was unable to protect against the ATV‐induced mitochondrial dysfunction and the consequent induced toxicity. The findings from the current study suggest that concurrent supplementation with GRT appears to be ineffective at protecting C3A liver cells against ATV‐induced toxicity in vitro. However, given the complexity of bioavailability and pharmacokinetics of complex mixtures, such as plant extracts, it is difficult to accurately extrapolate in vitro results to a clinical setting. Despite this, our findings suggest that it is unlikely that GRT will exhibit sufficient hepatoprotective effects in patients with ATV‐related hepatotoxicity.The authors hereby confirm that Professors Louw and Muller have a research interest in Afriplex GRT, a pharmaceutical grade GMP aspalathin‐enriched unfermented rooibos extract.


BioMed Research International has retracted the article titled “Positive Effects against UV-A Induced Damage and Oxidative Stress on an In Vitro Cell Model Using a Hyaluronic Acid Based Formulation Containing Amino Acids, Vitamins, and Minerals” [The CTR (16 h) and CTR (32 h) panels appear to be identicalThe M-HA (48 h) and M-HA (96 h) appear to be identicalSkinkò E (48 h) and Skinkò E (96 h) appear to be identicalSkinkò E (72 h) and CTR (96 h) appear to be identicalThe same cluster of cells can be seen in Skinkò E (48 h), Skinkò E (72 h), and Skinkò E (96 h).inerals” . As raisinerals” , there aThe authors said that the duplications between M-HA (48 h) and M-HA (96 h) and CTR (96) and Skinkò E (72 h) were mistakes introduced during manuscript preparation. A satisfactory explanation was not provided for the remaining concerns and the article is therefore being retracted with the agreement of the editorial board. The authors do not agree to retraction.


Scientific Reports 10.1038/s41598-020-77199-4, published online 18 November 2020Correction to: In this Article, the legend of Figure 4 is incorrect:https://www.graphpad.com/support/faq/prism-842-release-notes/). (d) Schematic representation of the mRNA sequence of human CPT2 (hCPT2). hCPT2 has two uORFs in its 5’UTR. A luciferase-reported assay for the detection of hCPT2 5′UTR in HUVECs treated with vehicle, PERKI, TGF-β1, or TGF-β1 + PERKI for 4 h or 8 h (n = 4 per groups) was performed. P-values were determined using the unpaired t-test. In all bar graphs, the mean ± SEM are shown. In all box-and-whiskers’ plots, mean values are provided. ※p < 0.05; one-way ANOVA with Bonferroni post hoc analysis.”“Induction of EndoMT is suppressed by PERK inhibition. (a) Morphology of human umbilical vein endothelial cells (HUVECs) treated with vehicle, 1 μM PERK inhibitor (PERKI), TGF-β1, and TGF-β1 + PERKI for 8 h. (b) mRNA expression of UPR, EndoMT, and FAO-related genes in HUVECs treated with vehicle (n = 6), PERKI (n = 3), TGF-β1 (n = 3), or TGF-β1 + PERKI (n = 3). Gene expression was detected using qRT-PCR. β-actin was used as an internal control. (c) mRNA expression of UPR, EndoMT, and the FAO-related genes in HUVECs treated with vehicle, PERKI, TGF-β1, or TGF-β1 + PERKI (n = 4 per group). This graph was drawn by Graph Pad Prism8.4.2. . (d) Schematic representation of the mRNA sequence of hCPT2. hCPT2 has two uORFs in its 5’UTR. A luciferase-reported assay for the detection of hCPT2 5′UTR in HUVECs treated with vehicle, PERKI, TGF-β1, or TGF- β1 + PERKI for 4 h or 8 h (n = 4 per groups) was performed. P-values were determined using the unpaired t-test. In all bar graphs, the mean ± SEM are shown. In all box and whiskers’ plots, mean values are provided. p < 0.05; one way ANOVA with Bonferroni post hoc analysis.”“Induction of EndoMT is suppressed by PERK inhibition. (a) Morphology of human umbilical vein endothelial cells (HUVECs) treated with vehicle, 1 μM PERK inhibitor (PERKI), TGF-β1, and TGF-β1 + PERKI for 8 h. (b) mRNA expression of UPR, EndoMT, and FAO-related genes in HUVECs treated with vehicle (n = 6), PERKI (n = 3), TGF-β1 (n = 3), or TGF-β1 + PERKI (n = 3). Gene expression was detected using qRT-PCR. β-actin was used as an internal control. (c) Protein expression of human CPT2 (hCPT2) in HUVECs treated with vehicle, PERKI, TGF-β1, or TGF-β1 + PERKI (n = 4 per group). This graph was drawn by Graph Pad Prism8.4.2. . In all bar graphs, the mean ± SEM are shown. In all box-and-whiskers’ plots, mean values are provided. ※p < 0.05; one-way ANOVA with Bonferroni post hoc analysis.’’“Translation of CPT2 is induced by PERK. (a) Flow cytometry-based quantification of BODIPY 493/503 staining in HUVECs treated with vehicle (n = 8), PERKI (n = 4), TGF-β1 (n = 4), or TGF-β1 + PERKI (n = 4). (b) Quantification of the mitochondrial metabolic activity of HUVECs treated with vehicle (n = 9), PERKI (n = 4), TGF-β1 (n = 5), or TGF-β1 + PERKI (n = 5), using resazurin. These graphs were drawn by Graph Pad Prism8.4.2. . In all bar graphs, the mean ± SEM are shown. In all box-and-whiskers’ plots, mean values are provided. p < 0.05; one-way ANOVA with Bonferroni post hoc analysis.”“Transport of neutral lipids from the cytosol into the mitochondria is induced by PERK. (a) Flow cytometry-based quantification of BODIPY 493/503 staining in HUVECs treated with vehicle (n = 8), PERKI (n = 4), TGF-β1 (n = 4), or TGF-β1 + PERKI (n = 4). (b) Quantification of the mitochondrial metabolic activity of HUVECs treated with vehicle (n = 9), PERKI (n = 4), TGF-β1 (n = 5), or TGF-β1 + PERKI (n = 5), using resazurin. These graphs were drawn by Graph Pad Prism8.4.2. (freely available software after purchasing,


The three-molecule aggregates are connected into a supramolecular tape along [111] by amide-N—H⋯O(carbonyl) hydrogen bonding.In the title 1:2 co-crystal a three-molecule aggregate,  14H14N4O2·2C7H5ClO2, comprises a half-molecule of oxalamide (4LH2), being located about a centre of inversion, and a molecule of3-chlorobenzoic acid (3-ClBA) in a general position. From symmetry, the 4LH2 molecule has a (+)antiperiplanar conformation with the 4-pyridyl residues lying to either side of the central, planar C2N2O2 chromophore with the dihedral angle between the core and pyridyl ring being 74.69 (11)°; intramolecular amide-N—H⋯O(amide) hydrogen bonds are noted. The 3-ClBA molecule exhibits a small twist as seen in the C6/CO2 dihedral angle of 8.731 (12)°. In the molecular packing, three-molecule aggregates are formed via carboxylic acid-O—H⋯N(pyridyl) hydrogen bonding. These are connected into a supramolecular tape along [111] through amide-N—H⋯O(carbonyl) hydrogen bonding. Additional points of contact between molecules include pyridyl and benzoic acid-C—H⋯O(amide), methylene-C—H⋯O(carbonyl) and C—Cl⋯π(pyridyl) interactions so a three-dimensional architecture results. The contributions to the calculated Hirshfeld surface are dominated by H⋯H (28.5%), H⋯O/O⋯H (23.2%), H⋯C/C⋯H (23.3%), H⋯Cl/Cl⋯H (10.0%) and C⋯Cl/C⋯Cl (6.2%) contacts. Computational chemistry confirms the C—Cl⋯π interaction is weak, and the importance of both electrostatic and dispersion terms in sustaining the molecular packing despite the strong electrostatic term provided by the carboxylic acid-O—H⋯N(pyridyl) hydrogen bonds.The asymmetric unit of the title 1:2 co-crystal, C As is normal for nLH2 molecules  Å; symmetry code: (i) 1 − x, − y, − z] bond length is considered long, an observation ascribed to the electronegative substituents bound to the sp2-C7 atom. The conformation of the 4LH2 molecule is (+)antiperiplanar so the 4-pyridyl residues lie to either side of the planar region of the molecule. The dihedral angle between the central core and the N1-pyridyl ring is 74.69 (11)°. Owing to the anti-disposition of the amide groups intramolecular amide-N—H⋯O(amide) hydrogen bonds are formed which complete S(5) loops, Table 1The 4LH2 and two symmetry-related 3-ClBA molecules via carboxylic acid-O—H⋯N(pyridyl) hydrogen bonding, Table 1via amide-N—H⋯O(carbonyl) hydrogen bonds Fig. 2a). These give rise to 22-membered {⋯NC4NH⋯OCOH}2 synthons. Additional stability to the hydrogen-bonding arrangement is provided by supporting benzoic acid-C14—H⋯O(amide) interaction which lead to non-symmetric 10-membered {⋯HC3O⋯HNC2O}2 synthons, which flank the larger 22-membered rings. Further, a complementary C—Cl⋯π(pyridyl) contact is noted, as detailed in Table 1et al., 2008et al., 2016b). The tapes are connected into a supramolecular layer by relatively short pyridyl-C1—H⋯O(amide) contacts, Fig. 2c). A three-dimensional architecture results when benzoic acid-C12—H⋯O(amide) and methylene-C—H⋯O(carbonyl) interactions are taken into consideration, Fig. 2d). In this scheme, the amide-O1 atom participates in three pivotal C—H⋯O interactions.The most distinctive feature of the molecular packing is the association between i.e. that sustained by the carboxylic acid-O—H⋯N(pyridyl) hydrogen bonds, and for the individual components, viz. the full molecule of 4LH2 and 3-ClBA, with the use of CrystalExplorer17 , amide-N2—H2N⋯O2(carbonyl), pyridyl-C1—H1⋯O1(amide), benzene-C14—H14⋯O1(amide), benzene-C12—H12⋯O1(amide) and methylene-C6—H6A⋯O3(hydroxyl); the dnorm distances for these short contacts are given in Table 2PLATON analysis  indicating the contact distance that is slightly less than the sum of the van der Waals radii d,p), as available in CrystalExplorer17. The analysis indicates that the Cl⋯π interaction is weak in nature as evidenced from the white spot around the σ-hole region about the Cl1 atom in Fig. 4a) as well as the faint-red spot around the centre of the π-ring centre, Fig. 4b). A detailed study on the localized electrostatic charges shows that the σ-hole of Cl1 is about −0.0072 a.u. while the pyridyl π-hole is about −0.1270 a.u. indicating that the interaction is rather dispersive in nature. This observation is in contrast with other charge complementary interactions as shown from the intense blue (i.e. electropositive) and red (i.e. electronegative) regions on the electrostatic surface map. For instance, the amide-N2—H2N⋯O2(carbonyl) hydrogen bond has a point-to-point electrostatic charge of 0.1438 a.u. for H2N and −0.0622 a.u. for O2, suggestive of a strong interaction, while benzene-C14—H14⋯O1(amide) shows complementary charges of 0.0427 and −0.0486 a.u. for H14 and O1, respectively, being indicative of a relatively weaker interaction. Among all the identified close contacts, hydroxyl-O3—H3O⋯N1(pyridyl) is considered to be the strongest exhibiting a marked difference in the electrostatic charge of 0.2919 a.u. for H3O and −0.0727 a.u. for N1.To verify the nature of the Cl⋯π contact in (I)i.e. 4LH2 and 3-ClBA, were subjected to fingerprint analysis for quantification of the close contacts for each entity, Fig. 5a). Overall (I)b)–(f); others contacts amount to 6.6%, constituting contacts less than 2.0% each. Among those contacts for (I)di + de contact distances tipped at ca 1.94 and 2.08 Å, respectively, significantly less than their respective sums of van der Waals radii of 2.61 and 2.79 Å; the remaining contacts occur at distances greater than their corresponding sums of van der Waals radii.The three-molecule-aggregate of (I)4LH2 and 3-ClBA molecules. The key difference between these and that for (I)4LH2, the major contacts comprise H⋯H (34.5%), H⋯O/O⋯H (22.1%), H⋯C/C⋯H (20.3%), H⋯N/N⋯H (8.4%), H⋯Cl/Cl⋯H (6.4%) and C⋯Cl (5.0%). A detailed analysis on the corresponding contacts reveals that the -H⋯O- and -H⋯C- contacts are slightly more dominant over the -O⋯H- and -C⋯H- counterparts with the distribution of the contacts being 12.7 and 11.2% versus 9.4 and 9.1%, while the opposite is true for the -H⋯N- contact with a distribution of 0.6% as compared to 7.8% for -N⋯H-. The stark difference in the dominance for H⋯N/N⋯H is likely due to the amide-H forming a hydrogen bond to O(carbonyl) rather than to a nitrogen acceptor. Among the major contacts, -H⋯O- and -N⋯H- display minimum di + de distances of about 1.94 and 1.60 Å, respectively, which are significantly shorter than the sums of the respective van der Waals radii as compared to the -O⋯H- and -H⋯N- counterparts of 2.24 and 3.62 Å, respectively. A similar observation is noted for -H⋯C- (∼2.66 Å) despite the deviation from the sum of the van der Waals radii (2.79 Å) being less significant.A similar paw-like fingerprint profile is observed for the overall fingerprint plots of the individual X⋯Y- for some close contacts , with the distribution being 15.0, 14.4, 10.5 and 5.5% for O⋯H, C⋯H, Cl⋯H and Cl⋯C, respectively, compared to 8.5, 8.5, 1.4 and 1.0% for the corresponding H⋯O, H⋯C, H⋯Cl and C⋯Cl counterparts. In term of di + de contact distances, the key values are reciprocal to those for 4LH2 owing to the interdependency of interactions as mentioned previously.As for the individual 3-ClBA molecule, the major contacts in the overall fingerprint plot can be delineated into H⋯O/O⋯H (23.5%), H⋯C/C⋯H (22.9%), H⋯H (21.8%), H⋯Cl/Cl⋯H (11.9%), C⋯Cl/Cl⋯C (6.5%) and H⋯N/N⋯H (4.6%). The trend of dominance is more inclined towards -CrystalExplorer17  hydrogen bond has the greatest interaction energy (Eint) with the value being −48.0 kJ mol−1, and this is followed by the dimeric amide-N2—H2N⋯O2(carbonyl), benzene-C14—H14⋯O1(amide) and Cl1⋯π interactions, with a combined Eint of −38.7 kJ mol−1, the 16-membered {⋯OCNC3CH⋯} heterosynthon involving pyridyl-C1—H1⋯O1(amide) interactions (−24.6 kJ mol−1), benzene-C12—H12⋯O1(amide) and pyridyl-C4—H4⋯C11(benzene) with a combined Eint of −24.0 kJ mol−1, methylene-C6—H6A⋯O3(hydroxyl) (−15.8 kJ mol−1) as well as the benzene-C10⋯C10(benzene) interaction with (−15.0 kJ mol−1). Interestingly, the strongest hydroxyl-O3—H3O⋯N1(pyridyl) interaction in this crystal has an Eint value that is only slightly less than that of −49.4 and −52.0 kJ mol−1) (two independent molecules) displayed by an equivalent O—H⋯N hydrogen bond complemented by a supporting pyridyl-C—H⋯O(carbonyl) interaction in the isomeric 2:1 co-crystal of 4LH2 with 4-ClBA O⋯N1(pyridyl) interaction is the main foundation of the framework as evidenced from the thick cylindrical rods with other, relatively, thinner rods which ramify owing to various other O⋯H interactions, Fig. 6a). The O⋯H interactions together with other complementary interactions are found to contribute to the dispersion energy framework which forms a similar topology as the electrostatic energy framework, Fig. 6b). The combination of the other electrostatic and dispersion forces supersedes the strong interaction energy from the hydroxyl-O3—H3O⋯N1(pyridyl) hydrogen bonding and leads to the overall energy framework illustrated in Fig. 6c) without dominant interactions in a given direction. It is interesting to note that despite being an isomeric analogue to the 4LH2·2(4-ClBA) co-crystal .The co-crystal system is governed by a combination of electrostatic and dispersion forces leading to a three-dimensional wire mesh-like energy framework as shown in Fig. 64LH2·2(4-ClBA) 2 synthons, as highlighted in Fig. 7The aforementioned analogue of (I)et al., 2019a and IIb. This is compensated by a reduction in the H⋯Cl/Cl⋯H contacts by 4.9 and 5.6%. One possible reason for the increase in O⋯H/H⋯O contacts in (I)cf. (II) relates to the participation of the carbonyl-O atom in formal hydrogen bonding to the amide-N—H group and the prominent role of the amide-O1 atom in providing points of contact between molecules.A comparison of the percentage contributions by the most prominent contacts to the respective Hirshfeld surfaces of (I)N,N′-bis[(pyridin-4-yl)methyl]oxalamide (4LH2) was prepared according to a literature procedure: M.p. 486.3–487.6 K; lit. 486–487 K  was mixed with 3-ClBA  and the mixture was then ground for 15 min in the presence of a few drops of methanol. The procedure was repeated twice. Colourless blocks were obtained through careful layering of toluene (1 ml) on an N,N-dimethylformamide solution (1 ml) of the ground mixture. M.p. 436.6–437.7 K. IR (cm−1): 3280 ν(N—H), 3070–2919 ν(C—H), 1703–1656 ν(C=O), 1524 ν(C=C), 1415 ν(C—N), 753 ν(C—Cl).The precursor, Uiso(H) set to 1.2Ueq(C). The oxygen- and nitrogen-bound H atoms were located from a difference-Fourier map and refined with O—H = 0.84±0.01 Å and N—H = 0.86±0.01 Å, respectively, and with Uiso(H) set to 1.5Ueq(O) or 1.2Ueq(N).Crystal data, data collection and structure refinement details are summarized in Table 410.1107/S2056989020006568/wm5558sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989020006568/wm5558Isup2.hklStructure factors: contains datablock(s) I. DOI: 2004094CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Scientific Reportshttps://doi.org/10.1038/s41598-020-74140-7, published online 19 October 2020.Correction to: The Supplementary Information file that accompanies this Article contained errors in Supplementary Table 13, where values were rounded incorrectly.In the column ‘Exercise + Supplement Pre’, in the row ‘Blood Pressure’“102 (10.4)”now reads:“102.0 (10.4)”In the column ‘Exercise + Supplement Pre’, in the row ‘Heart Rate’“127 (10.2)”now reads:“127.2 (10.2)”In the column ‘Exercise + Supplement Pre’, in the row ‘Lean Muscle Mass (pounds)’“123 (22.7)”now reads:“123.1 (22.7)”In the column ‘Exercise + Supplement Pre’, in the row ‘Fluid Intelligence RT’“514 (114)”now reads:“514.5 (114)”In the column ‘Exercise + Supplement Pre’, in the row ‘Executive Function RT’“695 (133)”now reads:“693.5 (133)”In the same Supplementary Table 13, in the column ‘Exercise + Supplement Post’, in the row ‘Heart Rate’“122 (9.6)”now reads:“122.6 (9.6)”In the column ‘Exercise + Supplement Post’, in the row ‘Lean Muscle Mass (pounds)’“126 (22.8)”now reads:“126.5 (22.8)”In the column ‘Exercise + Supplement Post’, in the row ‘Fluid Intelligence RT’“493 (97.1)”now reads:“491.4 (97.1)”In the column ‘Exercise + Supplement Post’, in the row ‘Executive Function RT’“639 (124)”now reads:“638.5 (124)”In the same Supplementary Table 13, in the column ‘Exercise + Placebo Pre’, in the row ‘Heart Rate’“126 (9.6)”now reads:“126.1 (9.6)”In the column ‘Exercise + Placebo Pre’, in the row ‘Lean Muscle Mass (pounds)’“123 (23.2)”now reads:“122.8 (23.2)”In the column ‘Exercise + Placebo Pre’, in the row ‘Fluid Intelligence RT’“496 (111)”now reads:“496.4 (111)”In the column ‘Exercise + Placebo Pre’, in the row ‘Executive Function RT’“684 (136)”now reads:“683.6 (136)”Finally, in the same Supplementary Table 13, in the column ‘Exercise + Placebo Post’, in the row ‘Heart Rate’“125 (9.7)”now reads:“124.4 (9.7)”In the column ‘Exercise + Placebo Post’, in the row ‘Lean Muscle Mass (pounds)’“124 (22.1)”now reads:“124.2 (22.1)”In the column ‘Exercise + Placebo Post’, in the row ‘Fluid Intelligence RT’“505 (82)”now reads:“504.8 (82)”In the column ‘Exercise + Placebo Post’, in the row ‘Executive Function RT’“643 (143)”now reads:“643.8 (143)”Additionally, in the legend of Supplementary Table 13 the formulas for computing the values in the Abstract were omitted,“RT is reaction time in milliseconds.”now reads:“RT is reaction time in milliseconds. The values in this table were used to derive the values in the abstract of the manuscript. Exercise effects reported in the abstract were derived by computing the percent change for the Exercise + Placebo group: (Exercise + Placebo Post—Exercise + Placebo Pre)/Exercise + Placebo Pre. Multimodal fitness and nutritional intervention effects reported in the abstract were derived by computing a relative difference percent change: [(Exercise + Supplement Post—Exercise + Supplement Pre)/Exercise + Supplement Pre]-[(Exercise + Placebo Post—Exercise + Placebo Pre)/Exercise + Placebo Pre].”The original version of Supplementary Table 13 appears below as Table As a result of the errors in Supplementary Table 13, in the Abstract,“The exercise intervention alone improved several dimensions of physical fitness  and cognitive function . Relative to exercise training alone, the multimodal fitness and nutritional intervention further improved working memory (+ 9.0%), fluid intelligence reaction time (− 7.7%), processing efficiency (+ 1.8%), heart rate (− 2.4%) and lean muscle mass (+ 1.5%).”now reads:“The exercise intervention alone improved several dimensions of physical fitness  and cognitive function . Relative to exercise training alone, the multimodal fitness and nutritional intervention further improved working memory (+ 11.2%), fluid intelligence reaction time (− 6.2%), processing efficiency (+ 4.3%), heart rate (− 2.3%) and lean muscle mass (+ 1.6%).”These errors have now been corrected in the PDF and HTML versions of the Article, and in the Supplementary Information file that accompanies the Article.


Talaromyces cellulolyticus (TcXyn30B), is a bifunctional enzyme with glucuronoxylanase and xylobiohydrolase activities. In the present study, crystal structures of the native enzyme and the enzyme–product complex of TcXyn30B expressed in Pichia pastoris were determined at resolutions of 1.60 and 1.65 Å, respectively. The enzyme complexed with 22‐(4‐O‐methyl‐α‐d‐glucuronyl)‐xylobiose (U4m2X) revealed that TcXyn30B strictly recognizes both the C‐6 carboxyl group and the 4‐O‐methyl group of the 4‐O‐methyl‐α‐d‐glucuronyl side chain by the conserved residues in GH30‐7 endoxylanases. The crystal structure and site‐directed mutagenesis indicated that Asn‐93 on the β2‐α2‐loop interacts with the non‐reducing end of the xylose residue at subsite‐2 and is likely to be involved in xylobiohydrolase activity. These findings provide structural insight into the mechanisms of substrate recognition of GH30‐7 glucuronoxylanase and xylobiohydrolase.Xylanase B, a member of subfamily 7 of the GH30 (glycoside hydrolase family 30) from  TcXyn30B is a bifunctional xylanase with endo‐glucuronoxylanase and exo‐xylobiohydrolase activities. The present study determined the crystal structure of the TcXyn30B‐product complex. A 4‐O‐methyl‐α‐d‐glucuronyl moiety‐binding pocket required for glucuronoxylanase activity and Asn‐93 (which is likely involved in xylobiohydrolase activity) was structurally characterized. The present study provides insight into the mechanisms of substrate recognition of a bifunctional xylanase. GH30‐7subfamily 7 of the glycoside hydrolase family 30GH30‐8subfamily 8 of the glycoside hydrolase family 30MeGlcAO‐methyl‐d‐glucuronic acidα‐1,2‐linked‐4‐4m2XU2‐(4‐O‐methyl‐α‐d‐glucuronyl)‐xylobiose23XxylotriosenU4m2XX2‐MeGlcA‐xylooligosaccarides24m2XXU2‐MeGlcA‐xylotriose2d‐xylopyranosyl residues linked by β‐1,4‐glycosidic bonds, which are further decorated with side‐chain residues, such as α‐1,2‐ and/or α‐1,3‐linked‐l‐arabinofuranose, and α‐1,2‐linked‐4‐O‐methyl‐d‐glucuronic acid (MeGlcA). Glucuronoxylanase (https://www.qmul.ac.uk/sbcs/iubmb/) is an appendage‐dependent endoxylanase that must recognize an α‐1,2‐linked MeGlcA common to glucuronoxylans for hydrolysis. Glucuronoxylanase cleaves the glucuronoxylan main chain at the second glycosidic linkage from the MeGlcA substituent toward the reducing end to produce 22‐MeGlcA‐xylooligosaccarides . The enzyme is classified into glycoside hydrolase family (GH) 30 subfamilies 7 and 8 (GH30‐7 and 30‐8) in the CAZy database (http://www.cazy.org) , , 8, 4m2Xh XU4m2X .O‐methyl group of MeGlcA   and XYN VI glucuronoxylanase [TcXyn30B  and its mutant enzyme whose Asn‐93 was replaced by Ala (TcXyn30B‐His N93A) were prepared. The glucuronoxylanase activities of TcXyn30B‐His and TcXyn30B‐His N93A for beechwood xylan were 10.9 ± 0.5 and 12.0 ± 0.2 U·mg−1, respectively. By contrast, the xylobiohydrolase activities of TcXyn30B‐His and TcXyn30B‐His N93A for X3 were 0.290 ± 0.006 and 0.0987 ± 0.004 U·mg−1, respectively. The kinetic parameters for each enzyme activity of TcXyn30B‐His and TcXyn30B‐His N93A are shown in Table Km and kcat values for the xylobiohydrolase activity could not be determined because the initial rate of xylose production from X3 was not saturated even at a substrate concentration of 16 mm. However, the three‐fold reduction of the kcat/Km value in the N93A mutant suggests that Asn‐93 could contribute to increased catalytic efficiency for xylobiohydrolase activity but is not essential. These results also support the hypothesis that Asn‐93 interacts with the non‐reducing end of xylose residue at the subsite ‐2.To evaluate the role of Asn‐93 in nU4m2X from xylan requires the binding of substrate at subsite ‐3 onward. When the TcXyn30B model was superimposed on the EcXynA model complexed with XU4m2X, a steric clash between non‐reducing end of XU4m2X and Asn‐93 of TcXyn30B was observed . The black arrow indicates the position of TcXyn30B.Fig. S3. Crystals of TcXyn30B complexed with U4m2X.Fig. S4.Fo‐Fc omit maps (blue) contoured at 3.0 σ for sugar chains. N‐linked carbohydrate moieties. Sugar chains linked Asn‐60, Asn‐88, Asn‐334, Asn‐346 and Asn‐412 are shown. Atoms are coloured as: C of N‐linked sugar chain residues, purple; C of TcXyn30B, brown; O, red; N, blue.Fig. S5. Orientations of U4m2X bound to TcXyn30B and XU4m2X bound to EcXynA. The model of TcXyn30B with U4m2X was superimposed on EcXynA with XU4m2X (PDB ID: https://doi.org/10.2210/pdb2Y24/pdb). The distances are given in Å.Click here for additional data file.


The square-planar and trigonal–bipyramidal coordination environments around platinum and arsenic, respectively, are significantly distorted with the largest outliers being 173.90 (13) and 106.98 (14)° for platinum and arsenic in (1), and 173.20 (14)° and 94.20 (9)° for (2), respectively. One intramolecular and four classical intermolecular hydrogen-bonding interactions are observed in the crystal structure of (1), which give rise to an infinite three-dimensional network. A similar situation  is observed in the crystal structure of (2). Various π-interactions are present in (1) between the platinum(II) atom and the centroid of one of the five-membered rings formed by Pt, As, C, N, O with a distance of 3.7225 (7) Å, and between the centroids of five-membered  rings of neighbouring molecules with distances of 3.7456 (4) and 3.7960 (6) Å. Likewise, weak π-interactions are observed in (2) between the platinum(II) atom and the centroid of one of the five-membered rings formed by Pt, As, C, N, O with a distance of 3.8213 (2) Å, as well as between the Cl atom and the centroid of a symmetry-related five-membered ring with a distance of 3.8252 (12) Å. Differences between (2) and the reported polymorph [Miodragović et al.  atom in the crystal structures of chlorido[dihydroxybis(1-iminoethoxy)arsanido-κ al. 2013. Angew.  Cl] (1), and [Pt(C6H14AsN2O4)Cl], (2), expand on this work and form part of an ongoing study on arsenoplatins, their solid- and solution-state behaviour and evaluation thereof.The structures reported here,  while there are slight differences for the N—Pt1—As1 and N—Pt—Cl1 bond angles: 85.18 (11) and 89.42 (11)° for (1), and 87.25 (10) and 86.05 (10)° for (2) , and 93.28 (11) and 92.34 (11)° for (1) and 94.16 (11) and 92.53 (10)° for (2) . The As1—Pt1—Cl1 bond angles also vary being 174.79 (3) and 178.51 (3)° for (1) and (2). The bond angles around arsenic are all in a similar range but have more variation in some of the angles, for instance 126.42 (10) and 130.05 (11)° (O6—As1—Pt1), 90.24 (9) and 94.20 (9)° (O2—As1—Pt1), and 95.35 (10) and 93.12 (8)° (O1—As1—Pt1) for (1) and (2), respectively. The trigonal– bipyramidal coordination environment around arsenic is distorted in both molecules with a τ5 parameter value of 0.794 and 0.711 for (1) and (2). Thus, the As atom in (2) shows a slightly higher distortion than in (1).When comparing the molecules of (1), six hydrogen-bonding interactions are observed  and one intramolecular (O6—H6⋯O2), as illustrated in Fig. 2iii = 2.750 (4) Å], which generates an infinite chain along the c-axis direction , defined by the platinum(II) atom of one molecule to the centroid of the  ring with a Pt⋯centroid distance of 3.7225 (7) Å, by the centroid of the  ring to the centroid of the   ring of an adjacent molecule with a distance of 3.7456 (4) Å, and by the centroid of the  ring to the centroid of another   ring with a distance of 3.7960 (6) Å  is likewise stabilized by one intramolecular (O6—H6⋯O1) and five intermolecular  hydrogen-bonding interactions (Table 22), one from Pt1 to the centroid of Pt1,As1,O1,C1,N1 with a distance of 3.8213 (2) Å and the other from Cl1 to a symmetry-related centroid  with a distance of 3.8252 (12) Å  and (2) pack differently due to the presence of different alkyl chains  was added to a 125 ml solution of 9:1 (v:v) CH3CN/H2O. The mixture was stirred at 363 K. Once the K2PtCl4 had dissolved, As2O3  was added to the solution and refluxed at 363 K for 48 h. The mixture was then filtered, and the filtrate was left to stand at room temperature. Crystals suitable for X-ray crystallography were obtained by slow evaporation. Yield: 301 mg (66%). 1H NMR : σ = 7.30 , 6.69 , 1.75  ppm. 13C NMR : σ =172 (CN), 23 (CH3) ppm. 195Pt NMR : σ = −3590.62 ppm. UV/Vis = λ = 285 nm, ∊ = 4029 dm3 mol−1 cm−1. Analysis calculated: C, 10.55; H, 2.21; N, 6.15. Found: C, 10.64; H, 2.22; N, 6.09%.KSynthesis of 22PtCl4  was added to a 50 ml solution of 9:1 (v:v) H2O/CH3CH2CN. The mixture was stirred at room temperature. Once the K2PtCl4 had dissolved, As2O3  was added to the solution and was stirred at room temperature for 96 h. The solution was cooled in an ice bath and then filtered. The filtrate was left to stand at room temperature. Crystals suitable for X-ray crystallography were obtained by slow evaporation. Yield: 278 mg (56%). 1H NMR : δ = 8.89 , 7.99 , 2.48 , 1.04 . 13C NMR : δ = 176.18 (CN), 24.53 (CH2), 11.70 (CH3). 195Pt NMR  = −3591.52. UV/Vis: λmax = 270 nm, ∊ = 4231 L mol−1 cm−1. Analysis calculated C, 14.90; H, 2.92; N, 5.79. Found: C, 14.82; H, 2.91; N, 5.76.KUiso(H) = 1.5Ueq(C) and 1.2Ueq(C)]. The OH and NH hydrogen atoms were located in a difference-Fourier map and their positional parameters were constrained with O—H = 0.84 (2) Å and N—H = 0.89 (2) Å for (1), and N—H = 0.87 (2) Å for (2) with O—H distances fixed at 0.82 Å and with Uiso(H) = 1.5Ueq(O). For (2), the c versus FoF plot proved ten reflections to be outliers, and they were removed from the refinement as systematic errors.Crystal data and details of data collections and structure refinements are summarized in Table 310.1107/S2056989019016463/wm5532sup1.cifCrystal structure: contains datablock(s) global, 1, 2. DOI: 1938096, 1938095CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Serotonin 2A receptors, the molecular target of psychedelics, are expressed by neuronal and vascular cells, both of which might contribute to brain haemodynamic characteristics for the psychedelic state.Aiming for a systemic understanding of psychedelic vasoactivity, here we investigated the effect of N-(2-hydroxybenzyl)-2,5-dimethoxy-4-cyanophenylethylamine – a new-generation agonist with superior serotonin 2A receptor selectivity – on brain-supplying neck-arterial blood flow.We recorded core body temperature and employed non-invasive, collar-sensor based pulse oximetry in anesthetised mice to extract parameters of local blood perfusion, oxygen saturation, heart and respiration rate. Hypothesising an overlap between serotonergic pulse- and thermoregulation, recordings were done under physiological and elevated pad temperatures.N-(2-hydroxybenzyl)-2,5-dimethoxy-4-cyanophenylethylamine  significantly increased the frequency of heart beats accompanied by a slight elevation of neck-arterial blood flow. Increasing the animal-supporting heat-pad temperature from 37°C to 41°C enhanced the drug’s effect on blood flow while counteracting tachycardia. Additionally, N-(2-hydroxybenzyl)-2,5-dimethoxy-4-cyanophenylethylamine promoted bradypnea, which, like tachycardia, quickly reversed at the elevated pad temperature. The interrelatedness of N-(2-hydroxybenzyl)-2,5-dimethoxy-4-cyanophenylethylamine’s respiro-cardiovascular effects and thermoregulation was further corroborated by the drug selectively increasing the core body temperature at the elevated pad temperature. Arterial oxygen saturation was not affected by N-(2-hydroxybenzyl)-2,5-dimethoxy-4-cyanophenylethylamine at either temperature.Our findings imply that selective serotonin 2A receptor activation modulates systemic cardiovascular functioning in orchestration with thermoregulation and with immediate relevance to brain-imminent neck (most likely carotid) arteries. As carotid branching is a critical last hub to channel cardiovascular output to or away from the brain, our results might have implications for the brain haemodynamics associated with psychedelia. Lysergic acid diethylamide (LSD) pressure . In animpressure  as well escribed . Beyond d (2ARs) , with gld (2ARs) . 5-HT2ARresearch , yet ourors e.g. . Also, iors e.g. , making 2AR agonist with 100-fold preference for 5-HT2ARs over a plethora of non-5-HT2R targets -2,5-dimethoxy-4-cyanophenylethylamine (25CN-NBOH), a recently developed selective 5-HT targets . We test targets . To avoi targets , experimion e.g. , we expeN = 41) were bred in-house at the Central Biomedical Services of Imperial College London. They were housed in individually ventilated cages with a 12:12 h light/dark cycle and maintained at an ambient temperature of 21 ± 2°C at 55 ± 10% humidity. Mice were provided with standard rodent-chow pellets  and water ad libitum.All experimental procedures performed at Imperial College London were in accordance with the United Kingdom Animal Scientific Procedures Act (1986) under Home Office Personal and Project licences , following appropriate ethical review. Adult mice of both sexes and with mixed (non-inbred) genetic background  was dissolved in isotonic saline and applied subcutaneously (<10 mL/kg). The dose used (1.5 mg/kg) was found optimal for a 5-HTtwitches . ExperimPulse oximetry was performed in anaesthetised mice using the rodent-specific MouseOx Plus collar-sensor system . Anaesthesia was induced in an anaesthesia chamber  at 2–2.5% isoflurane and, following the loss of the righting reflex, maintained via two-port mask supply at 1.5–2.0% concentration. Body temperature was maintained by a feedback-loop controlled heat-pad , which drew input from a thermosensor attached to the heat-pad’s surface, controlling for physiological and elevated (37°C and 41°C) pad temperatures, respectively. The collar-sensor was placed around the shaved necks of the animals to read oxygen saturation , heart rate (beats per minute (bpm)), respiration rate (breaths per minute (brpm)) and pulse distention of carotid arteries . Pulse dgroup and pad temperature as between-subject factors and time as within-subject factor) and analysed for main effects, interaction and contrast. Significant interactions were followed by Bonferroni-corrected multiple comparisons. For cross-parameter comparability, results were normalised to their respective baseline average and depicted as fold of baseline.Pulse oximetry 1 Hz readouts – as aggregated in 5-minute bins – and core body temperature measurements were analysed via SPSS-implemented three-factorial repeated measures analysis of variance (ANOVA) , pulse distention remained relatively stable throughout the 40-minute recording period. This contrasts with 25CN-NBOH-treated mice  = 3.51, p = 0.001), group  = 3.51, p ⩽ 0.001), time × group  = 16.37, p ⩽ 0.001), as well as time × group × temperature  = 4.99, p = 0.005): Bonferroni-corrected post-hoc analysis revealed significant group differences from 25CN-NBOH treated animals  at any of the four post-injection measurements for both temperature conditions  for 37°C: 0.98 ± 0.017 vs. 1.05 ± 0.03 (5 min), p = 0.021; 0.97 ± 0.019 vs. 1.04 ± 0.026 (10 min), p = 0.036; 0.96 ± 0.019 vs. 1.04 ± 0.026 (15 min), p = .022; 0.96 ± 0.021 vs. 1.049 ± 0.026 (20 min), p=0.028; for 41°C: 0.97 ± 0.01 vs. 1.05 ± 0.015 (5 min), p = 0.005; 0.97 ± 0.012 vs. 1.15 ± 0.027 (10 min), p ⩽ 0.001; 0.96 ± 0.012 vs. 1.15 ± 0.024 (15 min), p ⩽ 0.001; 0.96 ± 0.013 vs. 1.15 ± 0.037 (20 min), p ⩽ 0.001). At an increased pad temperature, the 25CN-NBOH-induced increase in arterial distension was much more pronounced (NBOH 37°C vs. 41°C: p = 0.026 (5 min); p = 0.031 (10 min); p = 0.029 (15 min); p = 0.036 (20 min)). For the saline-treated control mice, no such temperature effect could be found (control 37°C vs. 41°C: p = 0.93 (5 min); p = 0.85 (10 min); p = 0.92 (15 min); p = 0.99 (20 min)). Mean absolute values of arterial distension for the pre- versus post-injection intervals (given in μm) are presented in For isoflurane-anaesthetised control animals at both 37°C and 41°C pad temperatures  = 65.76, p ⩽ 0.001). Furthermore, a significant time × group interaction  = 4.59, p ⩽ 0.001) and a significant time × treatment × temperature interaction  = 3.93, p = 0.029) indicated the temporal changes in heart rate differed between both groups in a temperature-dependent manner. Bonferroni-corrected post-hoc comparisons revealed the significant time × group interaction was due to an 25CN-NBOH-induced tachycardia at all four post-injection measurements for the 37°C condition : 1.05 ± 0.018 vs. 1.12 ± 0.018 (5 min), p = 0.041; 1.07 ± 0.02 vs. 1.18 ± 0.027 (10 min), p ⩽ 0.001; 1.08 ± 0.026 vs. 1.19 ± 0.031 (15 min), p = 0.01; 1.08 ± 0.03 vs. 1.19 ± 0.032 (20 min), p = 0.016) and at the first post-injection measurement for the 41°C condition : 1.07 ± 0.016 vs. 1.13 ± 0.026 (5 min), p = 0.044). Accordingly, temperature-dependent heart rate differences in 25CN-NBOH-treated animals could be detected at the 15- and 20-minute measurements and as a trend for the 10-minute measurement (NBOH 37°C vs. 41°C: p = 0.095 (10 min); p = 0.015; 40 min (15 min): p = 0.029; p = 0.014 (20 min)). Mean absolute values for the pre- versus post-injection intervals (given in bpm) are shown in Isoflurane-anesthetised mice overall exhibited a tendency for an increase of heart rate over time  = 47.46, p ⩽ 0.001). There was a significant main effect group  = 8.27, p = 0.007) and a significant time × group interaction  = 3.21, p = 0.03). Following up on the time × group interaction, 25CN-NBOH significantly decreased the respiration rate at all four post-injection measurements of the 37°C condition : 0.96 ± 0.041 vs. 0.88 ± 0.038 (5 min), p = 0.01; 0.91 ± 0.041 vs. 0.78 ± 0.053 (10 min), p = 0.014; 0.87 ± 0.048 vs. 0.72 ± 0.046 (15 min), p = 0.025; 0.87 ± 0.051 vs. 0.7 ± 0.048 (20 min), p = 0.01), but only as a trend at the 5-minute post-injection measurement of the 41°C condition (0.94 ± 0.015 vs. 0.86 ± 0.025 (5 min), p = 0.073). Mean absolute values for the pre- versus post-injection intervals (given in brpm) are listed in Isoflurane-anesthetised mice overall exhibited a tendency for a decrease of respiration rate over time  = 3.24, p = 0.05) (group and time × group (× temperature) interactions turned out insignificant, indicating that blood oxygenation was not affected by 25CN-NBOH or its interaction with the pad temperature. Mean absolute values for the pre- versus post-injection intervals (given in % saturation) are presented in Overall, there was a slight tendency for blood oxygenation to decrease during anaesthesia, as implied by a significant main effect  = 0.05) . Main efn = 6–9 per group), the core body temperature was quantified via a rectal probe. Over the 40-minute observation period, the rectal temperature was noted to slightly increase across study groups  = 36.53, p ⩽ 0.001). Time × treatment interaction likewise was significant  = 9.13, p = 0.006), with a marginal contribution from pad temperature  = 1.85, p = 0.095). Follow-up analysis revealed the time × group interaction was driven by 25-NBOH favouring body warming during the last intervals of recording at 42°C : 1.006 ± 0.0019 vs. 1.014 ± 0.005 (16 min), 1.0053 ± 0.003 vs. 1.014 ± 0.005 (18 min), and 1.005 ± 0.002 vs. 1.02 ± 0.005 (20 min), each p ⩽ 0.05)  . Mean abime, F1, 5 = 36.532AR agonist 25CN-NBOH and investigated its effect on the parameters of neck-arterial blood flow.Serotonin secreted into the blood stream is a major tissue hormone with a variety of actions on the cardiovascular system and, when released from the brain stem’s raphe nuclei, also mediates the neuronal regulation of blood pressure . Assumin2AR-related vasoconstriction in the cutaneous bed, which interferes with heat dissipation  dynamic characteristics for psychedelia.In summary, our results show that 25CN-NBOH induces a temperature-dependant increase in heart rate, a decrease in respiration rate and an increase in arterial blood flow, as measured by pulse oximetry drawn from brain-supplying (most likely carotid) arteries. At present, 25CN-NBOH is the most selective 5-HTeceptors . Given t


Trypanosoma. While the common cap‐0 (m7GpppN) shows a rather simple methylation pattern, the Trypanosoma cap‐4 displays seven distinguished additional methylations within the first four nucleotides. The study of essential biological functions mediated by these unique structural features of the cap‐4 and thereby of the metabolism of an important class of human pathogenic parasites is hindered by the lack of reliable preparation methods. Herein we describe the synthesis of custom‐made nucleoside phosphoramidite building blocks for m62Am and m3Um, their incorporation into short RNAs, the efficient construction of the 5′‐to‐5′ triphosphate bridge to guanosine by using a solid‐phase approach, the selective enzymatic methylation at position N7 of the inverted guanosine, and enzymatic ligation to generate trypanosomatid mRNAs of up to 40 nucleotides in length. This study introduces a reliable synthetic strategy to the much‐needed cap‐4 RNA probes for integrated structural biology studies, using a combination of chemical and enzymatic steps.Eukaryotic mRNAs possess 5′ caps that are determinants for their function. A structural characteristic of 5′ caps is methylation, with this feature already present in early eukaryotes such as  Highly methylated mRNA caps are encountered in Trypanosoma. A solid‐phase‐based chemoenzymatic approach makes these RNAs synthetically accessible, thereby facilitating the study of the metabolism of, and essential biological functions mediated by these unique structural features found in this important class of human pathogenic parasites. This is an essential step for export out of the nucleus.Posttranscriptional processing adds another layer of information to RNA. This affects mRNA splicing,Our growing understanding of the functionality of 5′ caps and the consequences of disrupting cap‐dependent biochemical processes that result in severe medical disorders make 5′ caps attractive for the development of novel pharmaceutic and therapeutic approaches.7GpppN), which is typically for lower eukaryotes, cap‐1 (m7GpppNm) and cap‐2 (m7GpppNmNm), which are typically for higher eukaryotes.O‐methyl group. Recent studies have shown that 2′‐O‐methylations play a key role in cellular discrimination of endogenous from pathogenic RNA.Trypanosomatidae family from the Trypanosomatida order and the Kinetoplastida class.Significant structural diversity is encountered for mRNA 5′ caps. The most common caps in eukaryotes are cap‐0 (mp‐(m62Am)(Am)(Cm)(m3Um)) first by the phosphoramidite solid‐phase method. The 5′‐phosphorylated tetranucleotide was then chemically coupled with m7GDP to yield the cap‐4 structure.Trypanosoma cruzi trans‐spliced leader (SL) RNA of a specific 39‐nucleotide (nt) sequence that harbors the unique hypermethylated cap‐4 structure.The study of essential biological functions mediated by these unique structural features of cap‐4 and thereby of the metabolism of an important class of human pathogenic parasites is hindered by the lack of reliable preparation methods. To the best of our knowledge, only the chemical synthesis of the terminal four‐nucleotide fragment of cap‐4 has been published thus far. This was accomplished by obtaining the tetranucleotide fragment  in pyridine. Finally, phosphitylation was executed with 2‐cyanoethyl‐N,N‐diisopropylchlorophosphoramidite (CEP‐Cl) in the presence of N,N‐diisopropylethylamine (DIPEA). Starting from nucleoside 1, our route provides phosphoramidite 3 with 45 % overall yield in three transformations involving two chromatographic purifications; in total, 0.85 g of compound 3 was prepared over the course of this study.The functionalization of commercially available 2′‐O‐methyladenosine 4 into the desired m6Am phosphoramidite 9 involved six reactions, which are summarized in Scheme 5 with N,N′‐bis[(dimethylamino)methylene]hydrazine (BDMAMH) dihydrochlorided‐ribofuranosyl)‐6‐purine 6, which was converted quantitatively into N,N,2′‐O‐trimethyladenosine 7 with ethanolic dimethylamine. Compound 7 was tritylated to give compound 8, and finally phosphitylated with CEP‐Cl in the presence of DIPEA. Starting from nucleoside 4, our route provides phosphoramidite 9 in 46 % overall yield over five steps involving five chromatographic purifications; in total, 0.75 g of compound 9 was prepared during the course of this study.The functionalization of commercially available 2′‐O‐pivaloyloxymethyl (PivOM)‐protected nucleoside phosphoramidites, we optimized our protocol for the implementation of standard 2′‐O‐tert‐butyldimethylsilyl (TBDMS) and/or 2′‐O‐[(triisopropylsilyl)oxy]methyl (TOM) phosphoramidites  methyltransferase) following a protocol previously established by us  and S‐ribosyl homocysteine lyase (LuxS) were added to the reaction mixture to enzymatically degrade the byproduct S‐adenosylhomocysteine, which is a strong inhibitor of methyltransferases.The N7‐methyl group was efficiently enzymatically added by means of the methyltransferase Ecm1 (Am)(Cm)(m3Um) synthetic oligonucleotide.We developed a practical route toward hypermethylated cap‐4 RNAs. This included the three‐ and five‐step syntheses of mO‐‐2′‐O‐methyl‐3‐N‐methyluridine (2)5′‐: 2′‐O‐Methyluridine (1)  was dissolved in dry dimethyl sulfoxide (2 mL). Sodium hydride  was added to the reaction mixture and stirred at room temperature under argon atmosphere until bubbling stopped. Then, methyl iodide  was added dropwise. The solution was stirred for another 5 h. All volatiles were evaporated under high vacuum. The residue was co‐evaporated once with methanol and twice with pyridine before it was dissolved in anhydrous pyridine (1 mL). Dimethoxytrityl chloride  and 4‐dimethylaminopyridine  were added. The reaction solution was stirred under argon at room temperature overnight before the reaction was quenched by adding methanol. All volatiles were evaporated, the residue was diluted in methylene chloride and was washed three times with a solution of 5 % citric acid, once with a saturated sodium bicarbonate solution, and once with a saturated sodium chloride solution. The product was isolated after column chromatography (100:0.1 to 100:0.5 methylene chloride/methanol). Yield: 0.24 g (54 %) of 2 as white foam. 1H NMR : δ=3.15 , 3.24–3.36 ), 3.44 ), 3.75 ), 3.80–3.58 ), 3.94–4.00 ), 4.19–4.255 ), 5.22 ), 5.39 ), 5.84 ), 6.90 ), 7.21–737 ), 7.80 ppm ); 13C NMR : δ=27.3 , 55.0 ), 58.0 ), 62.3 (C(5′)), 68.4 ), 82.4 ), 82.8 ), 88.7 ), 100.5 ), 113.4 ), 127.0 ), 127.61 ), 127.80 ), 129.9 , 138.4 ); HRMS (ESI) m/z calcd: 597.2207 [M+Na]+; found: 597.2219.O‐Dimethoxytrityl‐2′‐O‐methyl‐3‐N‐methyluridine‐3′‐(2‐cyanoethyl‐diisopropylphosporamidite) (3)5′‐: Compound 2  was dried under high vacuum and vented with argon. The solid was dissolved in dry methylene chloride (6 mL) and treated with N,N‐diisopropylethylamine  and 2‐cyanoethyl‐N,N‐diisopropyl‐chlorophosphoramidite . The reaction solution was stirred for 4 h at room temperature. Afterward, the reaction was quenched by adding 1 mL of methanol. It then was diluted with methylene chloride (1:10), washed with saturated sodium bicarbonate solution and with a saturated sodium chloride solution. The product was isolated by column chromatography . Yield: 0.27 g (84 %) of 3 as white foam. 1H NMR : δ=0.96 , 1.08–1.12 , 2.33 , 2.57 , 3.25 , 3.35–3.6 , NCH‐(CH3)2, PO‐CH), 3.53 ), 3.73 ), 3.68–3.71 ), 4.15–4.19 ), 4.36–4.42 ), 4.51–4.56 ), 5.21–5.26 ), 5.93–5.97 ), 6.74–6.79 ), 7.16–7.35 ), 7.88–7.99 ppm ); 31P NMR : δ=150.17; 150.73 ppm; HRMS (ESI): m/z calcd: 775.3466 [M+H]+; found: 775.3472.O‐methyladenosine (5)3′,5′‐Diacetyl‐2′‐: 2′‐O‐Methyladenosine  was co‐evaporated with pyridine, before dimethylformamide (1.4 mL), dry pyridine (0.7 mL), acetic anhydride  and 4‐dimethylaminopyridine  were added. The reaction solution was stirred at 0 °C for 3 hours. The reaction progress was controlled by TLC (5 % methanol in methylene chloride). Methanol (0.5 mL) was added to quench the reaction. Then all volatiles were removed by distillation under high vacuum (70 °C) before the residue was co‐evaporated once with pyridine and three times with toluene. This crude product was directly used for the next reaction. For analytical analysis the product was further purified by column chromatography (1 % methanol in methylene chloride). Yield: 0.64 g (98 %) of 5 as white solid. 1H NMR : δ=2.04, 2.14 CO‐CH3, O(3′)CO‐CH3)), 3.28 ), 4.21–4.38 ‐H, Ha,b‐C(5′)), 4.88 ), 5.49–5.51 ), 6.03 ), 7.34 , 8.16  or H‐C(2)), 8.38 ppm  or H‐C(8)); 13C NMR : δ=20.9, 21.0 ‐CO‐CH3, O(3′)‐CO‐CH3), 59.5 ), 63.2 ), 80.3 ), 81.2 ), 87.7 ), 139.5  or C(2)), 153.5 ppm  or C(8)); HRMS (ESI): m/z calcd: 366.1408 [M+H]+; found: 366.1414.O‐methylfuranosyl)‐6‐purine (6)9‐ was co‐evaporated with pyridine and treated with N,N‐bis[(dimethylamino)methylene]hydrazine dihydrochloride . Yield: 0.47 g (76 %) of 6 as white foam. 1H NMR : δ=2.05, 2.16 , H3C‐CO‐OO(3′)), 3.31 ), 4.28–4.44 , Ha,b‐C(5′)), 4.92 ), 5.53–5.56 ), 6.24 ), 7.34 , 8.96  or H‐C(2)), 9.03 or H‐C(8)), 9.63 ppm ; 13C NMR : δ=21.16, 21.19 ‐CO‐CH3, O(3′)‐CO‐CH3), 59.0 ), 63.7 ), 71.3 ), 80.4 ), 80.8 ), 86.8 ), 141.9 , 146.8  or C(8)), 152.8 ppm  or C(2)); HRMS (ESI): m/z calcd: 418.1470 [M+H]+; found: 418.1475.N6‐‐Dimethyl)‐2′‐: Compound 6  was suspended in 33 % dimethylamine solution in ethanol (3 mL) and stirred under argon atmosphere at room temperature for 48 h. During that time, the white suspension turned into a clear solution. All volatiles were removed under vacuum. Further processing was done without further purification. For analytical reasons the crude product was purified by column chromatography . Yield: 0.34 g (98 %) of 7 as white foam. 1H NMR : δ=3.31 ), 3.46 2‐N), 3.53–3.71 , Ha,b‐C(5′)), 3.98 ), 4.33–4.34 , H‐O(5′)), 5.24 ), 5.33–537 ), 6.03 ), 8.22  or H‐C(2)), 8.40 ppm  or H‐C(8)); 13C NMR : δ=37.8 , 57.4 ), 61.3 ), 68.7 ), 82.4 ), 85.6 ), 86.3 ), 138.3  or C(8)), 151.9 ppm  or C(2)); HRMS (ESI): m/z calcd: 310.1510 [M+H]+; found: 310.1515.O‐Dimethoxytrityl‐6‐‐dimethyl)‐2′‐: Compound 7  together with 4‐(dimethylamino)‐pyridine  were co‐evaporated with pyridine. The residue was diluted in dry pyridine (8 mL). 4,4′‐O‐Dimethoxytrityl chloride  was dried for 2 h on high vacuum before it was added to the reaction solution, and the mixture was stirred overnight at room temperature together with molecular sieves under argon atmosphere. The reaction was quenched with methanol (0.5 mL) before all volatiles were removed under reduced pressure. The residue was co‐evaporated three times with toluene. Afterward the product was purified by column chromatography . Another column chromatography (12–25 % acetone in toluene) can be necessary to further improve purity. Yield: 0.382 g (81 %) of 8 as white foam. 1H NMR : δ=3.20 ‐Ha,b), 3.31–3.59 , 3.73 ), 4.06 ), 4.41 , H‐C(2′)), 5.25 ), 6.06 ), 6.78–6.88 ), 7.16–7.40 ), 8.18  or H‐C(2)), 8.26 ppm  or H‐C(8)); 13C NMR : δ=38.8 , 55.5 ), 59.3 ), 63.4 ), 70.1 ), 83.5 ), 83.9 ), 86.6 ), 113.4 ), 127.0 ), 128.1 ), 128.4 ), 130.4 ), 136.0  or C(8)), 152.8 ppm  or C(2)); HRMS (ESI): m/z calcd: 612.2817 [M+H]+; found: 612.2822.O‐Dimethoxytrityl‐6‐‐N‐dimethyl)‐2′‐,N‐diisopropylphosphoramidite) (9): Compound 8  was dried under high vacuum for several hours and ventilated with argon. The solid was dissolved in dry methylene chloride (10 mL), and treated with N,N‐diisopropylethylamine  and 2‐cyanoethyl N,N‐diisopropylchlorophosphoramidite . The reaction solution was stirred under argon atmosphere for 3.5 hours at room temperature. Then methanol (2 mL) was added and the solution was stirred for another 20 minutes. The solution was diluted with methylene chloride (1:10) and washed with saturated sodium bicarbonate solution and saturated sodium chloride solution. The product was isolated by column chromatography . Yield: 551 mg (77 %) of white foam. 1H NMR : δ=1.06–1.07 2), 1.19–1.22 2), 2.38–2.40 , 2.65–2.68 , 3.31–3.35 ), 3.48–3.69 , H3C‐O(2′), N‐(CH3)2, N‐CH, P‐O‐CH2), 3.80–3.79 ), 3.87–3.98 (mP‐O‐CH2‐), 4.33–4.39 ), 4.55–4.67 ), H‐C(3′)), 6.13–6.15 ), 6.80–6.84 ), 7.20–7.35 ), 7.44–7.47 ), 7.92–7.97  or H‐C(2)), 8.30–8.28 ppm  or H‐C(8)); 31P NMR : δ=150.21, 150.91 ppm; HRMS (ESI): m/z calcd: 812.3895 [M+H]+; found: 812.3901.N,N‐bis[(Dimethylamino)methylene]hydrazine dihydrochloride:1H NMR : δ=3.00 , 8.36 ppm .Guanosine 5′‐diphosphate di‐tributylammonium salt: DOWEX‐50WX8 200–400 resin (H+ form) was filtered with nanopure water using a glass frit (pore size 16–40 μm) to remove small particles. Afterward, guanosine 5′‐diphosphate disodium salt  was dissolved in water (4 mL) and eluted through a column  packed with 2 cm of wet DOWEX‐50WX8 200–400 for 10 minutes. After filtration into a flask with 25 mL ethanol at 0 °C, tributylamine  was added. The solution was stirred while the column was rinsed with water until pH was about 7–8 before all volatiles were removed under high vacuum. To preserve a dry powder further co‐evaporations (three times with ethanol and two times with dioxane) were performed. The resulting white solid was directly used without further purifications. Yield: 0.70 g (86 %) of a white solid. 1H NMR : δ=0.89 , 1.29 , 1.55 , 2.86 , 3.92–4.05 , H2‐C(5′)), 4.27 ), 4.47 ), 5.68 ), 6.62 , 7.89 ), 10.6 ppm ; 31P NMR : δ=−10.20 (d), −10.85 ppm (d).RNA solid‐phase synthesis: All RNAs were assembled on an ABI392 synthesizer using 2′‐O‐TOM nucleoside phosphoramidites (ChemGenes Corporation) and CPG supports (2′‐tBDSilyl Guanosine (n‐PAC) 3′‐lcaa CPG 1000 Å, 2′‐tBDSilyl Adenosine (n‐PAC) 3′‐lcaa CPG 1000 Å, ≈40 μmol g−1 ChemGenes Corporation). Standard RNA synthesis cycle: detritylation (120 s) with dichloroacetic acid/1,2‐dichloroethane (4:96); coupling (2.0 min) with phosphoramidites/acetonitrile (0.1 m) and benzylthiotetrazole/acetonitrile (0.3 m); capping  with Cap A: 4‐(dimethylamino)pyridine in acetonitrile (0.5 m) and Cap B: acetic anhydride/sym‐collidine/acetonitrile (2:3:5); oxidation (1.0 min) with iodine (20 mm) in tetrahydrofuran (THF)/pyridine/H2O (35:10:5). Commercially available modified nucleoside building blocks: 2′‐O‐methyl adenosine and 2′‐O‐methyl cytidine (ChemGenes Corporation), were incorporated using modified synthesis cycles with longer coupling times (6 min).RNA phosphitylation, hydrolysis, oxidative amidation was performed in analogy to ref. m aqueous triethylammonium bicarbonate, 5 mL water and 4 mL acetonitrile) over a period of 5 min. This treatment was repeated four times. The solid support was then rinsed with 20 mL of acetonitrile before being dried under vacuum for several hours. Oxidative amidation was done by incubation with 0.5 mL of a solution containing 600 mg imidazole, 2 mL N,O‐bis(trimethylsilyl)acetamide, 4 mL anhydrous acetonitrile, 4 mL bromotrichloromethane and 0.4 mL trimethylamine for 30 min. This treatment was repeated four times. The solid support was intensively rinsed with dry acetonitrile. Finally, Gpp attachment was achieved by application of 0.5 mL of coupling solution  over 8 h at room temperature. This treatment was repeated three times.Gppp‐capped RNA deprotection: The solid supports with the Gppp‐capped and otherwise fully protected oligos were rinsed with DBU in acetonitrile , then washed with acetonitrile and dried. Cleavage from the support and base deprotection was effected by treating the solid support with a 1:1 mixture of 40 % aqueous methylamine and 30 % aqueous ammonia (AMA) in a screw‐cap vial for 2 h at 40 °C. The resulting suspensions were filtered and the filtrates dried. Removal of 2′‐O silyl protecting groups was carried out with of 1 m tetrabutylammonium fluoride trihydrate in THF (1 mL) for 6 h at 40 °C. The reaction was quenched by the addition of 1 m triethylammonium acetate solution  and then concentrated to approximately 1 mL. This viscous solution was desalted by size exclusion chromatography on a HiPrep 26/10 Sephadex G25 column . The crude products were evaporated and re‐dissolved in water (1 mL). Quality assessment of the crude Gppp‐capped RNAs via anion‐exchange HPLC on a Dionex DNAPac PA‐100 column (4×250 mm); conditions: flow 1 mL min−1; eluent A: 25 mm Tris hydrochloride, 6 m urea, pH 8.0, eluent B: 500 mm sodium perchlorate, 25 mm Tris hydrochloride, 6 m urea, pH 8.0; gradient: 0–60 % B in 45 minutes; temperature: 40 °C, UV detection at 260 nm. The Gppp‐capped RNAs were isolated by anion‐exchange HPLC on a Dionex DNAPac PA‐100 column (4×250 mm); Conditions: flow 1 mL min−1; eluents: see above; temperature 40 °C; UV detection at 260 nm. The product fractions were diluted with an equal amount of triethylammonium bicarbonate buffer  and loaded onto equilibrated C18 Sep‐Pak (Waters Corporation). The cartridge was washed with water and the RNA was eluted with acetonitrile/water (1:1). All volatiles were evaporated and the residue re‐dissolved in water (1 mL). Yields were determined UV photometrically. The quality of the product was analyzed via anion‐exchange HPLC as described above and by reversed‐phase LC–ESI‐MS.Enzymatic N7 methylation of Gppp‐RNA: The enzymatic transformation was conducted in analogy to refs. 10 (4 nmol) was dissolved in buffer  followed by the addition of an aqueous solution of S‐adenosylmethionine , and the addition of water to obtain a total volume of 68.8 μL. To the mixed solution, enzymes were added successively ). The mixture was incubated for 45 min at 37 °C. The solution was extracted twice with an equal volume of chloroform/isoamyl alcohol solution . The organic layers were rewashed twice with an equal volume of water. The aqueous layers were combined and lyophilized, to eliminate remaining organic solvents. Analysis of the methylation reaction and purification of the product was performed by anion‐exchange chromatography (conditions see above). The integrity of the product was confirmed by LC–ESI mass spectrometry (conditions see below).Enzymatic ligation of cap‐4 RNA. The 38‐nt T. cruzi cap‐4 RNA was prepared by splinted enzymatic ligation of an 11‐nt cap‐4 RNA and a chemically synthesized 5′‐phosphorylated 27‐nt RNA by using T4 DNA ligase (Thermo Fisher) in analogy to ref. m of RNA fragment 10 (8 nmol), 12.5 μm of RNA fragment 11 (10 nmol), and 12.5 μm of a 20‐nt DNA splint oligonucleotide  were heated at 70 °C for 2 min and passively cooled to room temperature. Afterward water was added up to a total volume of 560 μL before 10× ligation buffer  and PEG  was added. T4 DNA ligase  was added to a final concentration of 0.5 U μL−1 in a total volume of 0.8 mL before the mixture was incubated for 2 h at 37 °C. Ligation was stopped by chloroform/isoamyl alcohol  extraction. After lyophilization and re‐dissolving, analysis of the ligation reaction and purification of the ligation products were performed by anion‐exchange chromatography (conditions see above). The integrity of product was confirmed by LC–ESI mass spectrometry (conditions see below).Mass spectrometry of cap‐4 RNA. All experiments were performed on a Finnigan LCQ Advantage MAX ion trap instrument connected to a Thermo Fisher Ultimate 3000 system. RNA sequences were analyzed in negative‐ion mode with a potential of −4 kV applied to the spray needle. LC: sample , column  at 21 °C; flow rate: 0.1 mL min−1; eluent A: Et3N (8.6 mm), 1,1,1,3,3,3‐hexafluoroisopropanol (100 mm) in H2O (pH 8.0); eluent B: MeOH; gradient: 0–100 % B in A within 20 min; UV detection at 254 nm.The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


In this paper,The corrected footnote for Table 2 is as follows:Solution 1:1000 mL volume; 20 mg/1000 mL concentration.Solution 2:100 mL volume; 20 mg/100 mL concentration.


N,N-dimethylformamide molecules are linked to the cations by C—H⋯O contacts.In the crystal, each cation is connected to two anions by N—H⋯ Br hydrogen bonds, forming an  17H18N3S+·Br−·C3H7NO, the central thiazolidine ring adopts an envelope conformation with puckering parameters Q(2) = 0.310 (3) Å and φ(2) = 42.2 (6)°. In the crystal, each cation is connected to two anions by N—H⋯ Br hydrogen bonds, forming an R42(8) motif parallel to the , C⋯H/H⋯C (17.9%) and Br⋯H/H⋯Br (7.0%) interactions, as concluded from a Hirshfeld analysis.In the cation of the title salt, C The C=N double bond [N1=C4 = 1.272 (4) Å] is in a Z configuration. The dihedral angle between the mean planes of the benzene (C5–C10) and phenyl (C12–C17) rings is 83.95 (18)° and they make dihedral angles of 16.60 (17) and 87.42 (17)°, respectively, with the mean plane of the thiazolidine ring. The N2—N1—C4—C5 bridge that links the thiazolidine and 4-methylbenzene rings has a torsion angle of −176.8 (3)°.As shown in Fig. 1N,N-dimethylformamide molecules are linked to the cations by C—H⋯O contacts  shows the 2D fingerprint plot of the sum of the contacts contributing to the Hirshfeld surface represented in normal mode while those delineated into H⋯H, C⋯H/H⋯C and Br⋯H/H⋯Br contacts are given in Fig. 7b–d, respectively. The most significant intermolecular interactions are the H⋯H interactions (55.6%) . The reciprocal C⋯H/H⋯C interactions appear as two symmetrical broad wings with de + di ≃ 2.6 Å and contribute 17.9% to the Hirshfeld surface . The reciprocal Br⋯H/H⋯Br interaction with a 7.0% contribution is seen as branch of sharp symmetrical spikes at diagonal axes de + di ≃ 2.2 Å . Furthermore, there are also O⋯H/H⋯O (3.2%), S⋯H/H⋯S (4.6%), N⋯C/C⋯N (3.8%), N⋯H/H⋯N (2.9%), S⋯C/C⋯S (2.4%), C⋯C (1.5%), Br⋯C/C⋯Br (0.2%), Br⋯S/S⋯Br (0.2%), N⋯N (0.4%) and N⋯S/S⋯N (0.5%) contacts  Fig. 7b. The rce Fig. 7c. The r Å Fig. 7d. Furths Table 3.et al., 2016et al., 1994et al., 1994et al., 1994et al., 1993aet al., 1993bet al., 1995et al., 1994p-toluenesulfonate. In all three structures, the most disordered fragment of these molecules is the asymmetric C atom and the Cl atom attached to it. The disorder of the cation in the racemate corresponds to the presence of both enantiomers at each site in the ratio 0.821 (3):0.179 (3). The system of hydrogen bonds connecting two cations and two anions into 12-membered rings is identical in the racemic and in the optically active crystals. YITCEJ  contacts also contribute to the molecular packing. In the crystal of ZIJQAN, the cations, anions and water molecules are linked into a three-dimensional network, which forms cross layers parallel to the (120) and  in ethanol (20 ml) was added 4-methylbenzaldehyde (1 mmol). The mixture was refluxed for 2 h and then cooled. The reaction product precipitated from the reaction mixture as colorless crystals, was collected by filtration, washed with cold acetone , and recrystallized from dimethylformamide to obtain single crystals.1H NMR  : 2.33 ; 4.55 ; 4,88 ; 5.60 ; 7.28–7.98 ; 8.41 ; 10.33 . 13C NMR : 21.27; 45.36; 55.90; 127.79; 128.69; 128.86; 129.09; 129.46; 130.21; 137.50; 141.68; 151.04; 167.50. MS (ESI), m/z: 296.40 [C17H18N3S]+ and 79.88 Br−.Uiso(H) = 1.2 or 1.5Ueq.Crystal data, data collection and structure refinement details are summarized in Table 410.1107/S2056989020012712/jy2001sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989020012712/jy2001Isup2.hklStructure factors: contains datablock(s) I. DOI: 1837125CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In the crystal, corrugated layers of molecules extending along the ab plane are generated by C—H⋯O hydrogen bonds. The intermolecular interactions were quantified by Hirshfeld surface analysis and two-dimensional fingerprint plots. The most significant contributions to the crystal packing are from H⋯H (42.3%), H⋯O/O⋯H (34.5%) and H⋯C/ C⋯H (17.6%) contacts. Molecular orbital calculations providing electron-density plots of the HOMO and LUMO as well as molecular electrostatic potentials (MEP) were computed, both with the DFT/B3LYP/6–311 G++ basis set. A molecular docking study between the title molecule and the COVID-19 main protease (PDB ID: 6LU7) was performed, showing that it is a good agent because of its affinity and ability to adhere to the active sites of the protein.In the molecular structure of the title compound, C The C14 atom is at the envelope flap position, and it deviates from the least-square plane through the remaining four atoms by 0.070 (2) Å. The other oxazolidine ring (N3/O7/C18–C20) has a twisted conformation along the C20—C19 bond, with puckering parameters Q(2) = 0.1732 (18) Å and φ(2) = 299.7 (6)°. The dihedral angles between the mean planes of the oxazolidine rings and the quinoline ring systems are 38.04 (9)° for (N2/O2/C12–C14) and 57.34 (8)° for (N3/O7/C18–C20). The molecular conformation is stabilized by an intramolecular C20—H20B⋯O4 contact  ring motif.In the title molecule Fig. 1, the phect Fig. 1, produciA⋯O3i and C17—H17A⋯O6ii hydrogen bonds between methylene groups and carbonyl O atoms as well as C19—H19B⋯O3iii hydrogen bonds lead to the formation of corrugated layers extending parallel to (001)  Fig. 2. Notableet al., 2016et al., 2015et al., 2015et al., 2019bS(6) ring. In the crystal structure, inversion dimers linked by pairs of weak C—H⋯O interactions generate R22(6) loops. In UHUHAI, the two rings of the quinoline system have a dihedral angle of 2.28 (8)° between their mean planes. The plane of the attached benzene ring is inclined to the plane of the quinoline system by 7.65 (7)°. There is a short intramolecular C—H⋯O contact involving the carbonyl group. In XOFGAD, the molecule is essentially planar with the mean plane of the ethyl acetate group making a dihedral angle of 5.02 (3)° with the ethyl 6-chloro-2-ethoxyquinoline mean plane. There is an intramolecular C— H⋯O hydrogen bond forming an S(6) graph-set motif. Weak intermolecular π–π interactions are observed in this crystal structure.A search of the Cambridge Structural Database  to 1.1337 (blue) a.u. . The pale-red spots symbolize short contacts and negative dnorm values on the surface and correspond to the C—H⋯O interactions (Table 1b). The convex blue regions symbolize hydrogen-donor groups and the concave red regions hydrogen-acceptor groups. The absence of adjacent red and blue triangles in the shape-index map, which generally indicate π–π interactions, reveals that this kind of interaction is not present in the title compound. The curvedness map was generated in the range −4.0 to 4.0 Å . It shows large regions of green with a relatively flat (i.e. planar) surface area while the blue regions demonstrate areas of curvature. The overall two-dimensional fingerprint plot is illustrated in Fig. 4a, with those delineated into H⋯H, H⋯O/O⋯H, H⋯C/ C⋯H, H⋯N/N⋯H and C⋯N/N⋯C contacts associated with their relative contributions to the Hirshfeld surface given in Fig. 4a–f, respectively. The most important intermolecular interactions are H⋯H, contributing 42.3% to the overall crystal packing. H⋯O/O⋯H contacts arising from intermolecular C—H⋯O hydrogen bonding . The pair of wings in the fingerprint plot delineated into H⋯C/ C⋯H contacts (17.6% contribution to the Hirshfeld surface) have a nearly symmetrical distribution of points, with the tips at de + di ∼2.54 Å. The contributions of the other contacts to the Hirshfeld surface are negligible, i.e. H⋯N/N⋯H of 2.0% and C⋯N/N⋯C of 1.2%.Hirshfeld surface analysis was used to quantify the intermolecular contacts of the title compound, using u. Fig. 3a. The ps Table 1. The shas Table 1b. The c Å Fig. 3c. It shg Table 1 make a 3 Å Fig. 4c. The pGAUSSIAN 09  using the standard B3LYP functional and 6–311G++  basis-set calculations  map Fig. 6 was calcet al., 2020PyRx AutoDock Vina Wizard. The intermolecular interactions between the title compound and the target protein were visualized by using the Discovery Studio 2020 Client program  values for nine different poses of the ligand docked onto receptor 6LU7 are listed in Table 3−1) energy and nine active hydrogen-bonding sites. The 2D and 3D visuals of the intermolecular interactions for the best binding pose of the title compound docked into macromolecule 6LU7 can be seen in Fig. 7A molecular docking study was performed to determine possible intermolecular interactions between the COVID-19 main protease (PDB ID: 6LU7) and the title molecule. The crystal structure of COVID-19 main protease in a complex with an inhibitor N3 was taken from the RSCB Protein Data Bank  tetra-n-butylammonium bromide (TBAB). The reaction mixture was stirred at 363 K for 9 h in DMF. After removal of formed salts by filtration, DMF was evaporated under reduced pressure, and the residue obtained was dissolved in dichloromethane. The organic phase was dried over Na2SO4 and then concentrated in vacuo. The resulting mixture was chromatographed on a silica gel column [eluent: ethyl acetate/hexane (2/8 v/v)]. Colourless single crystals of the title compound were obtained by slow evaporation of an ethanol solution.A solution of 0.8 g (4.23 mmol) of 2-oxo-1,2-dihydroquinoline-4-carboxylic acid in 30 ml of DMF was mixed with 1.5 g (8.46 mmol) bis(2-chloroethyl)amine hydrochloride, 2.33 g  KCrystal data, data collection and structure refinement details are summarized in Table 510.1107/S2056989020015960/wm5588sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989020015960/wm5588Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989020015960/wm5588Isup3.cmlSupporting information file. DOI: 2048734CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


II complex with 1,2-bi(4-pyridyl)ethane is reported. The Zn cation is coordinated by two N atoms from the 1,2-bi(4-pyridyl)ethane unit and four O atoms from two mandelate anions in a slight distorted octahedral coordination geometry.The synthesis and characterization of a new mandelato Zn II compound, [Zn(C8H7O3)2(C12H12N2)]n, the Zn cation is coordinated by two N atoms from 1,2-bis(pyridin-4-yl)ethane unit and four O atoms from two mandelate [or hydroxy(phenyl)acetate] anions in a slightly distorted octahedral coordination geometry. The 1,2-bis(pyridin-4-yl)ethane unit bridges two ZnII cations, related by an inversion centre, to form a polymeric chain along [110]. The crystal structure features extensive O—H⋯O and weak C—H ⋯O hydrogen bonds, with C—H ⋯ π interactions and π–π interactions also being present. The centroid–centroid distance between the phenyl ring of the mandelate group and the 1,2-bis(pyridine-4-yl)ethane moiety is 4.951 (2) Å. The 1,2-bis(pyridin-4-yl)ethane ligand is disordered over two positions, with a refined occupancy of 0.578 (14) for the major component.In the title polymeric Zn There is an inversion centre located at the mid-point of the ethane C—C bond in the 1,2-bis(4-pyridin-4-yl)ethane molecule. Each ZnII cation is coordinated by two N atoms from two 1,2-bis(4-pyridin-4-yl)ethane molecules in a trans arrangement and four O atoms from two mandelate anions in a slightly distorted octahedral coordination geometry, as shown in Table 12+ cation form five-membered chelate rings via an oxygen atom of the OH group [Zn—O3 = 2.1013 (15) Å] and an oxygen atom of the carboxyl group [Zn—O1= 2.0290 (14) Å]. The ZnII cations are linked together via 1,2-bis(4-pyridin-4-yl)ethane bridges, forming a polymeric chain along [110].The asymmetric unit of the title compound comprises one Znii =2.572 (2) Å] , 3.378 (3), and 3.064 (3) Å, respectively (Table 2A⋯Cg5iv = 3.781 (2) Å and C12′–H12B⋯Cg5ii = 3.649 (8) Å, Table 2Cg5⋯Cg3iv between the centroids of the phenyl ring (C3–C8) of the mandelate group and of the 1,2-bis(pyridine-4-yl)ethane moiety (C9–C13) [symmetry code: (iv) −x + y + z + The crystal structure features extensive O—H⋯O hydrogen bonding [O3⋯O2Å] Fig. 2, establiy Table 2. In addiet al., 2016catena-[[μ-oxido(phenyl)acetato]zinc(II) perchlorate monohydrate] (phenyl)acetato]{μ-4-[2-(pyridin-4-yl)ethyl]pyridine}nickel(II)], isostructural with the title compound, has also been reported ethane]bis(2-hydroxy-2-phenylacetato)zinc(II)] ethane moiety were found in the Cambridge Structural Database 2 , 1,2-bi(4-pyridyl)ethane  and mandelic acid  were mixed in deionized water. The mixture was placed in a 25 mL Teflon linear reactor and heated at 423 K in an autoclave for 24 h. The resulting solution was slowly cooled to room temperature. Yellow transparent single crystals of the title compound were obtained in 75% yield (based on Zn).Zn(NOUiso(H) = 1.5Ueq(C-methyl) and 1.2Ueq(C) for other H atoms·The hydroxy H atoms, which could not be located in a difference-Fourier map, were included in idealized calculated positions that gave the most sensible geometry.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989020014322/cq2039sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2056989020014322/cq2039Isup2.hklStructure factors: contains datablock(s) I. DOI: 2040978CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


After H2O2 treatment, SHIP2 inhibition enhanced phosphatase and tensin homologue activity, which, in turn, affected phosphoinositide 3‐kinase/protein kinase B and mitogen‐activated protein kinase/extracellular signal‐regulated kinase activation.Azzi investigated the effects of SH2‐domain containing phosphatidylinositol‐3,4,5‐trisphosphate 5‐phosphatase (SHIP2) inhibition by AS1938909 on H 2O2‐mediated AKT and mitogen‐activated protein kinase/extracellular signal‐regulated kinase pathway activation. In addition, SHIP2 inhibition enhances reactive oxygen species generation. Interestingly, I found that SHIP2 inhibition and H2O2 treatment enhance lipid and protein phosphatase activity of PTEN. Pharmacological targeting or RNA interference(RNAi) mediated knockdown of PTEN rescues extracellular signal‐regulated kinase and AKT activation. Using a series of pharmacological and biochemical approaches, I provide evidence that crosstalk between SHIP2 and PTEN occurs upon an increase in oxidative stress to modulate the activity of mitogen‐activated protein kinase and phosphoinositide 3/ATK pathways.Phosphatidylinositol ‐trisphosphate P3) is required for protein kinase B (AKT) activation. The level of PIP3 is constantly regulated through balanced synthesis by phosphoinositide 3‐kinase (PI3K) and degradation by phosphoinositide phosphatases phosphatase and tensin homologue (PTEN) and SH2‐domain containing phosphatidylinositol‐3,4,5‐trisphosphate 5‐phosphatase 2 (SHIP2), known as negative regulators of AKT. Here, I show that SHIP2 inhibition in cervical cancer cell lines alters H ΔΨmmitochondrial membrane potentialAKTprotein kinase BAS19AS1938909CCK‐8Cell Counting Kit‐8DAPI4′,6‐diamidino‐2‐phenylindole dihydrochlorideDCF2',7'‐dichlorofluoresceinDCFDAdichlorofluorescein diacetateDMEMDulbecco's modified Eagle's mediumERendoplasmic reticulumERKextracellular signal‐regulated kinase2O2Hhydrogen peroxideMAPKmitogen‐activated protein kinasePARPPoly (ADP‐ribose) polymerasep‐phosphorylatedPHpleckstrin homologyPIP3Phosphatidylinositol ‐trisphosphatePPIP2Phosphatidylinositol ‐bisphosphatePI3Kphosphoinositide 3‐kinasePLCδphospholipase C, deltaPTENphosphatase and tensin homologueRNAiRNA interferenceROSreactive oxygen speciesSEMstandard error of the meanSHCSrc homology 2 domain‐containing proteinSHIP2SH2‐domain containing phosphatidylinositol‐3,4,5‐trisphosphate 5‐phosphatase 22O2) is the main substance used to study oxidative stress at the cellular level. Experimental evidence exists for H2O2‐mediated ROS production that activates several signaling pathways, among which are mitogen‐activated protein kinase (MAPK) and phosphatidylinositol 3‐kinase (PI3K)/protein kinase B (Akt), which promote cell growth and survival ‐2‐thiophene‐carboxamide (AS1949490), a small‐molecule drug that was previously characterized from a high‐throughput screen  (Fisher Scientific), SHIP2 inhibitor AS19 from Sigma (Cat. No. SML1022), HHEK293T and HeLa cells were obtained from S. Schurmans . SiHa cells were obtained from S. Kateryna (GIGA‐University of Liege).−1d‐glucose supplemented with 10% FBS, 100 U·mL−1 penicillin, 100 U·mL−1 streptomycin and 1% l‐glutamine (Gibco). Cells between 3 and 10 passages were used. All cultures were maintained at 37 °C in a humidified 5% CO2 incubator. Unless otherwise stated, each cell was treated with 10 µm SHIP2 inhibitor AS1949490 or equivalent volume of DMSO as a control for 24 h.Cells were grown as recommended in DMEM containing 4.5 g·Lm. Cells were seeded overnight at equal density, 15 000 cells per well in a 96‐well plate. Upon 24 h treatment with DMSO as vehicle or SHIP2 inhibitor (10 μm), DCFDA was supplemented to the growth medium and incubated for 30 min at 37 °C. Stained cells were rinsed with warm basal DMEM and incubated again for 1 h in fresh medium. Where indicated, pyruvate pretreatment (5 mm) for 1 h was followed by H2O2 (1 mm). Fluorescent signal detection was measured using Tecan Infinite M200 PRO Multi‐Detection Microplate Reader (Tecan Life Science) or VICTOR™ X Series Multilabel Plate Readers (PerkinElmer) set to 485‐nm excitation and 530‐nm emission wavelength.Cytoplasmic ROS levels were measured using the fluorescent probe 2′,7′‐DCFDA in a final concentration of 25 μm MitoTracker Red for 30 min at 37 °C. With different treatments, cells were fixed using ice‐cold methanol at −20 °C during 10 min. After three washes with PBS, cells were then incubated with DAPI (0.1 µg·mL−1). Images were captured using Leica TCS SP5 microscope and processed with imagej .Cells were plated overnight on glass coverslips in 24‐well plates (150 000 cells/well) followed by treatment with vehicle or SHIP2 inhibitor for 24 h. For mitochondria staining, live cells were supplemented with 100 nm siRNA. Twenty‐four hours later, the medium was replaced, then treated with vehicle or SHIP2 inhibitor for 24 h where indicated. The following siRNAs were used, ON‐TARGET plus SMART pool siRNA against PTEN (#L003023‐00‐0005) and a control siRNA (#D‐001810‐10‐05).siRNA transfection was performed using Dharmafect 4 according to the manufacturer's instruction (Dharmacon). In brief, cells were plated a day before at 50% confluence. They were then transfected with 75 nPlasmid transfection was performed using Lipofectamine 3000 according to the manufacturer's instructions. The following plasmid constructs GFP–SHIP2, GFP–BTK–PH, GFP–TAPP1–PH and GFP–phospholipase C, delta (PLCδ)–PH were obtained from S. Schurmans .m NaCl, 1.0% Nonidet P‐40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mm Tris–HCl, pH 8.0), containing protease and phosphatase inhibitors (ROCHE). Protein samples were collected by centrifugation  at 4 °C. Sample concentrations were quantified using Bio‐Rad Protein Assay according to the manufacturer's instructions. Equal amounts of protein extract were loaded and separated by SDS/PAGE and then transferred to nitrocellulose membranes. The membranes were blocked for 60 min at room temperature with 5% BSA in Tris‐buffered saline supplemented with 0.05% Tween 20. Membranes were then incubated with primary antibodies overnight at 4 °C, followed by horseradish peroxidase‐labeled secondary antibodies. Labeled proteins were visualized with an enhanced chemiluminescence system (Fisher Scientific). Optical densities of proteins were determined using imagej.Cells were first washed with PBS and then resuspended in radioimmunoprecipitation assay buffer  #4377, ERK #4695, PARP #9542, Phospho‐MEK1/2 (Ser217/221) #9121, MEK1/2 #9122, phosphor‐Pten S380 #9551, Pten #5959, vinculin #13901 and phospho‐SHC (Tyr239/240) #2434]. Horseradish peroxidase‐linked secondary antibodies, Rabbit A0545 and Mouse A9044, were from Sigma. Actin and SHC were from Santa Cruz Biotechnology . Anti‐SHIP2 Ig was a gift from C. Erneux .m H2O2 for 4 h and treated with WST‐8 for 1 h at 37 °C. The absorbance at 450 nm (A450 nm) was then measured using Tecan Infinite M200 PRO Multi‐Detection Microplate Reader.Cell viability was measured using Cell Counting Kit‐8 (CCK‐8) . Cells were first seeded overnight at equal density, 15 000 cells per well in a 96‐well plate. They were then treated with vehicle or SHIP2 inhibitor for 24 h followed by 1 mm SHIP2 inhibitor for 24 h. Upon 1 mm H2O2 treatment during 90 min, MitoTell™ Orange solution was added and incubated for 30 min at 37 °C. Assay buffer B was then added. Fluorescence was then measured using Infinite M200 PRO Multi‐Detection Microplate Reader. Excitation wavelength was set at 540 nm and emission wavelength at 590 nm. Quantification was performed relative to control untreated cells. HeLa cells treated with carbonyl cyanide m‐chlorophenyl hydrazone 20 µm  were used as a control.ΔΨm measurement was performed using Cell Meter™ Mitochondrion Membrane Potential Assay Kit (AAT Bioquest #22805). HeLa cells were seeded overnight at equal density, 10 000 cells per well in a 96‐well plate, then treated with vehicle or 10 µgraphpad prism 7 Software . Data were presented as mean ± SD. For comparisons of three or more conditions, one‐way ANOVA with Tukey's honestly significant difference post hoc test was used. Differences less than 0.05 were considered statistically significant.Statistical analyses were performed using The author declares no conflict of interest.AA conceived and designed the experiments; acquired, analyzed, and interpreted the data; and wrote the manuscript.Fig. S1.  RNA levels of SHIP2 and PTEN in different cell lines . See also The Human Protein Atlas (https://www.proteinatlas.org/ENSG00000165458‐INPPL1/cell and https://www.proteinatlas.org/ENSG00000171862‐PTEN/cell).Fig. S2. SHIP2 inhibition alters AKT activation upon H2O2 treatment in SiHa cells. (A) SiHa cells were first treated with vehicle or SHIP2 inhibitor for 48 h, followed by 1 mM H2O2 for 6 h. Cell lysates were analyzed for the indicated antibodies. H2O2 treatment was performed in biological triplicates for vehicle and SHIP2‐inhibited cells. (B) The ROS scavenger pyruvate prevents H2O2‐induced cell death. SiHa cells were first treated with vehicle or SHIP2 inhibitor for 48 h, followed by pyruvate (5 mM) for 2 h, and then treated with 1 mM H2O2 for 4 h. Cell lysates were analyzed for the indicated antibodies. (C) SiHa cells were treated as in (B); then cell viability was measured with CCK‐8 assays. After 5 h of 1 mM H2O2, cells were treated with WST‐8 for 1 h at 37 °C. The A450 nm was measured with a microplate reader. Data are the means ± SEM from two independent experiments, each in four replicates. One‐way ANOVA, F = 92.1, P < 0.0001. Tukey's multiple comparisons test, **P ˂ 0.01.Fig. S3. H2O2 induces SHIP2 clusters formation. (A) HeLa cells were first transfected with GFP plasmid alone as a control or with GFP–SHIP2. Twenty‐four hours later, cells were treated with vehicle or SHIP2 inhibitor for 24 h, then followed by 1 mM H2O2 for 1 h. Cells were then fixed and mounted. Scale bar: 10 μM. (B) HeLa cells were treated with a vehicle or AS19 for 24 h and subsequently followed by AKT activator (SC79), 20 µM for 2 h and 1 mM H2O2 for 4 h. Total lysates were analyzed for the indicated antibodies.Fig. S4. SHIP2 inhibition enhances PIP2 accumulation. HeLa cells were first transfected with plasmid GFP‐tagged PLCδ‐PH domain; 6 h later they were treated with vehicle or AS19 for 24 h, then fixed and mounted. Fluorescent images of PIP2 levels are shown. Scale bar: 10 μM. C‐, vehicle‐treated cells.Click here for additional data file.Appendix S1. Original uncropped western blot figures. Some pictures are with additional samples data. However, these samples are not related to the results of this article (please see where indicated).Click here for additional data file.