A prospective, randomised, controlled observer-blind trial measuring the efficacy and immunogenicity of trivalent influenza vaccine (TIV) and the immunogenicity of quadrivalent meningococcal conjugate vaccine (MCV) in pregnant women and their infants up to 6 months of age was conducted in Mali. Here we reported the immunogenicity of MCV, which was used as a comparator vaccine to TIV, in this population. Third-trimester pregnant Malian women were randomized to receive TIV or MCV. Blood samples were collected from women prior to vaccination, 28 days post-vaccination, at delivery and 3 and 6 months post-delivery and from infants at birth and 3 and 6 months of age. Meningococcal-specific serogroup (Men) A, C, Y and W-specific antibodies were measured by enzyme linked immunosorbent assay in a randomly selected subset of 50 mother-infant pairs where the mother had received MCV. At birth, 94.0% (47/50) of infants had MenA specific IgG levels ≥ 2 µg/mL decreasing to 72.9% and 30.4% at 3 and 6 months of age. For MenC, 81.3% (39/48) of infants had MenC specific IgG levels ≥ 2 µg/mL at birth decreasing to 29.4% and 17.8% at 3 and 6 months of age. For MenY, 89.6% (43/48) of infants had MenY specific IgG levels ≥ 2 µg/mL at birth decreasing to 64.6% and 62.5% at 3 and 6 months of age. For MenW, 89.6% (43/48) of infants had MenW specific IgG levels ≥ 2 μg/ml at birth decreasing to 62.5% and 41.7% at 3 and 6 months of age.Maternal immunization with MCV conveyed protective levels of IgG at birth through to 3 months of age in the majority of infants. The majority of antibodies which are transferred across the placenta are IgG, and these passively acquired antibodies enter the bloodstream of the offspring providing protection in the same way as actively acquired antibodies. Following birth, these IgG antibodies are present in the bloodstream and are effective in providing protection to the neonate, however these antibodies are present in finite amounts and decline over time.Infants are not usually immunized before the age of 2–3 months (depending upon country-specific immunization schedules) because of their relative immunological immaturity. Immunization of the mother during pregnancy, at an optimal time can provide protection to the infant earlier in life. Maternally derived antibodies wane over time and the kinetics of this decline is correlated to the amount of maternal antibody present in the neonate after birth. Therefore if high levels of maternal antibodies can be achieved in infants, protection will be afforded during the most immature phase of their immune system.The principle of maternal immunization is supported by data for vaccination against tetanus, influenza and pertussis A recent post-licensure clinical trial of the safety, immunogenicity and efficacy of maternal influenza immunization for prevention of influenza in infants younger than 6 months has recently been conducted 2The full study details have been reported previously Quadrivalent meningococcal conjugate vaccine, composed of 4 μg each of Neisseria meningitidis serogroup A, C, Y, and W polysaccharides conjugated to diphtheria toxoid protein, was supplied in single-dose vials. A single 0·5 mL dose of trivalent inactivated influenza vaccine or quadrivalent meningococcal conjugate vaccine was injected into the deltoid muscle. Study vaccines were stored in secure, temperature-monitored refrigerators or cold rooms at 2–8 °C.From the cohort that received MCV, fifty women were randomly selected for assessment of the immune response to meningococcal vaccination. Blood samples were collected from the mother prior to vaccination and 28 days post vaccination, at delivery and at 3 and 6 months post-partum. Cord blood sample was collected at birth and infant blood samples collected at 3 and 6 months of age.Maternal immunization history was obtained at the time of study enrollment and 40/50 (80%) reported that they had previously received serogroup A conjugate vaccine (MenAfriVac) during the 2011 vaccination campaign conducted in Mali ClinicalTrials.gov, number NCT01430689.This trial is registered with 2.1Meningococcal (Men) serogroup A, C, W and Y-specific IgG responses were determined using a standardized enzyme-linked immunosorbent assay (ELISA) 2.2From the cohort that received MCV, fifty mother-child pairs were selected for assessment of the immune response to meningococcal vaccination, via simple random selection . Mother-3Baseline characteristics of mothers randomized to receive MCV were similar to the overall study Cohort .Table 1B3.1MenA-specific IgG concentrations at each time point measured in samples collected from mother and infants are reported in Prior to vaccination, the mothers GMC was 11.92 µg/mL (95% CI: 8.44–16.82) which increased to 40.19 µg/mL (95% CI: 32.12–50.28) 28 days post-vaccination with a decline to 29.62 µg/mL (95% CI: 23.07–38.02) at delivery and maintained over the following six months with GMCs of 27.41 µg/mL (95% CI: 20.35–36.93) and 31.69 µg/mL (95% CI: 23.81–42.16) at 3 and 6 months, respectively.The MenA-specific IgG concentration in the cord blood was approximately half of that measured in the mother at delivery, with GMCs of 16.31 µg/mL (95% CI: 11.79–22.56) and 29.62 µg/mL (95% CI: 23.07–38.02), respectively. Infant MenA-specific IgG antibody levels waned over the first months of life to 3.21 µg/mL (95% CI: 2.56–4.04) at 3 months of age with further waning to 1.41 µg/mL (95% CI: 1.0–1.98) at 6 months of age.A high proportion of mothers (88.0% (95% CI: 75.7–95.5) had MenA-specific IgG ≥ 2 µg/mL prior to receiving vaccination which increased to 100% (95% CI: 92.9–100.0) 28 days post-vaccination and remained high over the following months . A high 3.2Serogroup C, W and Y-specific IgG GMCs at each time point measured in samples collected from mother and infants are reported in Prior to vaccination, the MenC, W and Y-specific IgG GMCs were 2.79 µg/mL (95% CI: 1.87–4.17), 1.49 µg/mL (95% CI: 1.09–2.04), and 2.49 µg/mL (95% CI: 2.12–2.92) respectively. One month following vaccination, these levels increased to 12.67 µg/mL (95% CI: 9.03–17.80), 48.13 µg/mL (95% CI: 31.86–72.71), and 33.97 µg/mL (95% CI: 23.43–49.25), respectively. The MenC, W and Y-specific IgG GMCs measured in the cord blood were approximately 40–47% of that present in the mother 28 days post-vaccination with concentrations of 6.01 µg/mL (95% CI: 3.99–9.05), 19.30 µg/mL (95% CI: 12.11–30.75), and 14.73 µg/mL (95% CI: 9.89–21.95), respectively. Antibodies to all three serogroups waned over the first few months of life to GMCs of 1.01 µg/mL (95% CI: 0.73–1.40), 4.17 µg/mL (95% CI: 2.83–6.16), and 4.13 µg/mL (95% CI: 3.04–5.61), for MenC, W and Y, respectively.Prior to vaccination and 28 days post-vaccination, the proportion of mothers with MenC-specific IgG concentrations ≥ 2 µg/mL were 60.0% (95% CI: 45.2–73.6) and 92.0% (95% CI: 80.8–97.8), respectively. The proportion of cord blood samples with MenC-specific IgG concentrations ≥ 2 µg/mL were 81.3% (95% CI: 67.4–91.1) and the proportion of infants with MenC-specific IgG concentrations ≥ 2 µg/mL at 3 and 6 months of age were 29.4% (95% CI: 17.5–43.8) and 17.8% (95% CI: 8.0–32.1), respectively.Prior to vaccination and 28 days post-vaccination, the proportion of mothers with MenW-specific IgG concentrations ≥ 2 µg/mL were 32.0% (95% CI: 19.5–46.7) and 92.0% (95% CI: 80.8–97.8), respectively. The proportion of cord blood samples with MenW-specific IgG concentrations ≥ 2 µg/mL were 89.6% (95% CI: 77.3–96.5) and the proportion of infants with MenW-specific IgG concentrations ≥ 2 µg/mL at 3 and 6 months of age were 62.5% (95% CI: 47.4–76.0) and 41.7% (95% CI: 27.6–56.8), respectively.Prior to vaccination and 28 days post-vaccination, the proportion of mothers with MenY-specific IgG concentrations ≥ 2 µg/mL were 66.0% (95% CI: 51.2–78.8) and 90.0% (95% CI: 78.2–96.7), respectively. The proportion of cord blood samples with MenY-specific IgG concentrations ≥ 2 µg/mL were 89.6% (95% CI: 77.3–96.5) and the proportion of infants with MenY-specific IgG concentrations ≥ 2 µg/mL at 3 and 6 months of age were 64.6% (95% CI: 49.5–77.8) and 62.5% (95% CI: 47.5–76.0), respectively.4Malian women immunised with a serogroup A, C, W and Y conjugate vaccine during the third trimester of pregnancy showed a good antibody response to all four serogroups one month post-vaccination, with all subjects having a serogroup A-specific IgG concentrations ≥ 2 µg/mL and ≥ 90% of subjects ≥ 2 µg/mL for serogroups C, W and Y. Prior to vaccination, a high proportion of mothers had serogroup A-specific IgG concentrations ≥ 2 µg/mL, due to 40 mothers having previously received serogroup A containing vaccines. The serogroup A conjugate vaccine (MenAfriVac) campaign was performed in Mali in 2011 where vaccine was offered to all those aged 1–29 years of age Maternal antibodies contribute to the protection of neonates and infants from infectious diseases during the first months of life 1 antibodies are transferred preferentially across the placenta, with IgG2 less efficiently transferred. The response to MCV in adults has been reported to consist of both IgG1 and IgG2The concentration of antibodies placentally transferred from mother to infant depends on the type of antigen being either protein or polysaccharide, and the subclass of antibodies induced. IgGProtection from meningococcal disease is known to rely upon circulating antibodies. The serum bactericidal antibody (SBA) assay is the correlate of protection for meningococcal disease and if the infants in this study are protected is unknown, thus future studies using the SBA assay would provide this information. This study has shown the potential for maternally immunisation with a meningococcal conjugate vaccine to provide protection to the infant in the first few months of life before they are eligible for vaccination. The administration of conjugate vaccines in pregnancy also offers the potential to boost antibody responses to carrier proteins such as tetanus toxoid which would be advantageous in countries with high rates of neonatal tetanus. Implementation of maternal immunization programmes requires consideration of the potential to reduce infant responses to infant immunizations.Maternal immunization in the third trimester of pregnancy with MCV has demonstrated the increase of MenA, C, W and Y-specific IgG antibodies in the mother and infant and this approach has the potential to provide protection to the infant early in life.


Psittaciphilus Fain, Bochkov & Mironov, 2000 (Acariformes: Prostigmata: Syringophilidae) collected from columbiform birds (Columbiformes) are described: Psittaciphilus montanus n. sp. form the ruddy quail-dove Geotrygon montana Gosse from Trinidad and Tobago, Brazil and Panama, and Psittaciphilus patagioenas n. sp. from the band-tailed pigeon Patagioenas fasciata (Say) from Colombia and the scaled pigeon Patagioenas speciosa (Gmelin) from Surinam. A key to the species of the genus Psittaciphilus is provided. Our finding is the first record of the representatives of this genus on columbiform birds.Two new quill mite species of the genus  The quill mites of the family Syringophilidae Lavoipierre, 1953 (Acariformes: Prostigmata) are permanent and highly specialised ectoparasites, infesting quills of different types of feathers in the plumage of their avian hosts Kethley, . These mPsittaciphilus Fain, Bochkov & Mironov, 2000 is one of less known genera in the family Syringophilidae. Until now, only two species have been described, P. amazonae Fain, Bochkov & Mironov, 2000 and P. fritschi Fain, Bochkov & Mironov, 2000, both associated with parrots  and P. patagioenas n. sp. from Patagioenas fasciata (Say) and Patagioenas speciosa (Gmelin).In this paper we describe two new species of the genus The mite material used in the present study was collected from dry bird skins housed in the ornithological collection of the Bavarian State Collection of Zoology, Munich, Germany (ZSM). Feathers were examined using a dissecting microscope and opened with a fine scalpel. Before mounting, mites were softened and cleared in Nesbitt’s solution at room temperature for three days. Then, mites were mounted on slides in Hoyer’s medium. Taxonomic analysis of mite specimens was carried out with an Olympus BH-2 light microscope , equipped with DIC optics and a camera lucida. All measurements are given in micrometres. Measurements (ranges) of paratypes are given in parentheses following data for the holotype. In the descriptions below, the idiosomal chaetotaxy follows Grandjean  as adaptSpecimen depositories are cited using the following abbreviations: AMU, A. Mickiewicz University, Department of Animal Morphology, Poznan, Poland; ZSM, Bavarian State Collection of Zoology, Munich, Germany.Family Syringophilidae Lavoipierre, 1953Subfamily Syringophilinae Lavoipierre, 1953GenusPsittaciphilusFain, Bochkov & Mironov, 2000Psittaciphilus montanusn. sp.Type-host: Geotrygon montana (Linnaeus) (Columbiformes: Columbidae), ruddy quail-dove.Type-locality: Trinidad Island, Aripo Berge, Trinidad and Tobago.Type-material: Female holotype and 19 female paratypes from quills of under-tail coverts, 25.viii.1912, coll. Klages. The holotype and 17 paratypes were deposited in the AMU (Reg. no. AMU-SYR.571A); 2 female paratypes were deposited in the ZSM (Reg. no. ZSM20112080).Additional material examined: Six females from quills of tail coverts of the same host species; Brazil, Para and Rio Negro, 1844, collector unknown; all mite specimens deposited in the AMU (Reg. no. AMU-SYR.571B). Eleven females from quills of tail coverts of the same host species; Panama, Chiriqui, 1895, coll. Dalmas; all mite specimens deposited in the AMU (Reg. no. AMU-SYR.571C), except 2 females in the ZSM (Reg. no. ZSM20112081).ZooBank registration: To comply with the regulations set out in article 8.5 of the amended 2012 version of the International Code of Zoological Nomenclature . Gnathosoma. Infracapitulum apunctate. Stylophore apunctate, 190 (160–190) long. Each medial branch of peritremes with 2 chambers, each lateral branch with 4 or 5 chambers ; si 60 (40–60); se 205 (210–245); c1 215 (220–230); c2 195 (180–205); d1 180 (170–190); d2 220 (230–255); e2 170 (170–195); f1 25 (30–35); f2 150 (150–180); h1 25 (25–30); h2 330 (310–315); ag1 125 (120–145); ag2 40 (35–55); ag3 165 (160–185); g1 and g2 45 (30–45); ps1 and ps2 10 (10); tc′III–IV 20 (15–30); tc″III–IV 40 (35–55); l′RIII 55 (55–60); l′RIV 45 (45); 3b 60 (65–75); 3c 75 (90–100). Length ratios of setae: ve:si:se 1.2–1.6:1:4–5.3; d2:d1:e2 1.3–1.4:1:1; ag1:ag2:ag3 2.4–3.1:1:3.2–4.1; f1:f2 1:5–6; h1:f1 1:1–1.2; 3b:3c 1:1.2–1.3; ps:g 1:3–4.5.Differential diagnosisPsittaciphilus montanus n. sp. is morphologically similar to P. fritschi, collected from unidentified parrot from Zoo of Anverp (Belgium)  (Columbiformes: Columbidae), band-tailed pigeon.Type-locality: Near San Juan River, Chocó Department Lama Hermosa, Colombia.Type-material: Female holotype, 14 female paratypes from quill of under-tail coverts, 19.ix.1909, coll. Palmer. The holotype and 12 paratypes were deposited in the AMU (Reg. no. AMU-SYR.572); 2 female paratypes were deposited in the ZSM (Reg. no. ZSM20112082).Additional material examined: Twenty-seven females from quills of wing coverts of the scaled pigeon Patagioenas speciosa (Gmelin) (Columbiformes: Columbidae); Surinam, Kraka, 23.ii.1963, coll. Haverschmidt; all mite specimens deposited in the AMU (Reg. no. AMU-SYR.573), except 2 females in the ZSM (Reg. no. ZSM20112083).ZooBank registration: To comply with the regulations set out in article 8.5 of the amended 2012 version of the International Code of Zoological Nomenclature . Gnathosoma. Infracapitulum apunctate. Stylophore apunctate, 230 (220–230) long. Each medial branch of peritremes with 2 chambers, each lateral branch with 5 or 6 chambers ; si 40 (30–35); se 245 (245–260); c1 260 (260–285); c2 245 (235–250); d1 250 (240–250); d2 255 (260–280); e2 (230–260); f1 45 (30–40); f2 280 (250–260); h1 30 (30–35); h2 360 (370–405); ag1 195 (190–210); ag2 70 (50–60); ag3 275 (280–295); g1 and g2 45 (35–45); ps1 and ps2 20 (10–15); tc′III–IV 35 (30–35); tc″III–IV 70 (70–80); l′RIII 55 (55–60); l′RIV 50 (50–70); 3b 100 (70–95); 3c 160 (130–145). Length ratios of setae: ve:si:se 3.4–4.3:1:6–8.5; d2:d1:e2 1:1:1; ag1:ag2:ag3 2.7–4.2:1:3.9–5.6; f1:f2 1:5.7–8.3; h1:f1 1:1; 3b:3c 1:1.6–1.8; ps:g 1:2.2–3.5.Differential diagnosisPsittaciphilus patagioenas n. sp. is morphological similar to P. amazonae, collected from Amazona amazonica (Linnaeus) (Psittaciformes: Psittacidae) from Colombia (Fain et al., ve and si are 110–120 and 30–47 µm, respectively, and coxal fields I-IV are apunctate. This new species differs from P. amazonae by the following features: in females of P. patagioenas n. sp., the propodonotal and the pygidial shields are apunctate, the hysteronotal shields surrounding bases of setae d2 are present, the lengths of hysteronotal setae d1, d2, and e2 are 240–250, 255–280 and 230–260 µm, respectively. In females of P. amazonae, the propodonotal and the pygidial shields are punctate, the hysteronotal shields surrounding bases of setae d2 are absent, and the lengths of hysteronotal setae d1, d2, and e2 are 144–155, 179–192 and 179–184 µm, respectively.Key to the species ofPsittaciphilusve 1.2–1.6 times longer than si ……………………………………………………………………………… P. montanus n. sp.Propodonotal shield punctate with three patches, setae ve at least 2.4 times longer than si …………………………… 2Propodonotal shield apunctate without patches, setae ve and si 83–101 and 18–22 µm, respectively ………………………………………………….. P. fritschi Fain, Bochkov & Mironov, 2000Lengths of setae ve and si 110–123 and 30–47 µm, respectively …………………………… 3Lengths of setae d1, d2, and e2 144–155, 179–192, and 179–184 µm, respectively. Hysteronotal shields surrounding bases of setae d2 absent ………………………………………………… P. amazonae Fain, Bochkov & Mironov, 2000Propodonotal and pygidial shields punctate. Length of setae d1, d2, and e2 240–250, 260–280, and 230–260 µm, respectively. Hysteronotal shields surrounding bases of setae d2 present …………………………………………………………………… P. patagioenas n. sp.Propodonotal and pygidial shields apunctate. Length of setae


The title compound was isolated in 76% yield from a reaction of N,N′-diisopropylcarbodiimide with potassium hydride in DME. The single-crystal X-ray structure determination of the title compound revealed a polymeric chain structure comprising cage-like dimeric units, with the amidinoguanidinate ligand displaying a mixed σ-/π-coordination mode.The first potassium amidinoguanidinate complex,  N,N′-chelating donor ligands such as amidinate anions [RC(NR)2]− and guanidinate anions [R2NC(NR)2]− are of significant importance in various fields of organometallic and coordination chemistry. It is generally accepted that both types of N,N′-chelating ligands can be regarded as ‘steric cyclopentadienyl equivalents’  as well as the design of highly active homogeneous catalysts  (NR)C(NR)2]  and the holmium(III) complex [nBuC(=NCy)(NCy)C(NCy)2]Ho[nBuC(NCy)2](μ-Cl)2Li(THF)2 dipotassium]-μ-1,2-dimethoxyethane-κ2O:O′] [{iPrN=CHN(iPr)N(NiPr)2K}2(μ-DME)]n.We recently reported in this journal that, under certain conditions, seemingly straightforward reactions of lithium alkyls with N,N′-diisopropylcarbodiimide was added to a suspension of potassium hydride in 1,2-dimethoxyethane (DME). With the attempt to prepare the corresponding amidinate K[HC(NiPr)2], the reactants were used in a molar ratio 1:1. After filtration and concentration of the filtrate to a small volume, the product crystallized directly at room temperature and could be isolated as colorless, plate-like, moisture-sensitive crystals in 76% yield . The compound was characterized through elemental analysis as well as IR, NMR (1H and 13C) and mass spectra. However, the usual set of analytical and spectroscopic methods did not allow for an unequivocal elucidation of the molecular structure. NMR data clearly indicated the presence of coordinated DME. However, both the 1H and 13C NMR spectra showed two sets of iso-propyl resonances, thereby ruling out the formation of a simple potassium formamidinate salt of the composition ‘(DME)K[HC(NiPr)2]’. Fortunately, the single crystals obtained directly from the filtered and concentrated reaction solution were suitable for X-ray diffraction analysis. This study confirmed the formation of a new amidinoguanidinate complex through dimerization of N,N′-diisopropylcarbodiimide in the coordination sphere of potassium.As illustrated in Fig. 1N,N′-chelating mode, with the K atom in the N3C plane of the guanidinate. The same guanidinate moiety is linked to the symmetry-equivalent K atom in an η3-diazaallyl mode, i.e. the metal atom is situated above the N1/C1/N2 plane. The exposed nitrogen donor of the amidinate backbone (N4) in the title compound is attached to the metal center in a simple monodentate coordination, with the N atom having a perfectly planar environment (sum of bond angles = 360.0°). This is in agreement with the expected sp2 hybridization of atom N4  Å (N1) and 2.814 (2) Å (N2), while the bond to the single amidinate nitrogen donor (N4) is considerably longer at 2.939 (2) Å. All these values are within the range usually observed for K—N bonds  Å (C5⋯C14) between the methyl carbon atoms of isopropyl groups.et al. formamidinates, see: Junk & Cole 2007. For oth al. 1999, Stalke  al. 1992, Cole et al. 2003, Aharono al. 2008, Chlupat al. 2011, Cole &  al. 2011, Junk &  al. 2011, Benndor al. 2011, Nevoral al. 2013 and Hong al. 2013.General Procedures: The reaction was carried out under an inert atmosphere of dry argon employing standard Schlenk and glove-box techniques. The solvent dimethoxyethane (DME) was distilled from sodium/benzophenone under nitrogen atmosphere prior to use. All glassware was oven-dried at 393 K for at least 24 h, assembled while hot, and cooled under high vacuum prior to use. The starting material N,N′-diisopropylcarbodiimide was obtained from Sigma–Aldrich and used as received. Commercially available potassium hydride was freed from protecting paraffin oil by thoroughly washing with n-pentane and stored in a glove-box. The 1H and 13C NMR spectra were recorded in solutions on a Bruker Biospin AVIII 400 MHz spectrometer at 298 K. Chemical shifts are referenced to tetramethylsilane. The IR spectrum was measured with a Bruker Optics VERTEX 70v spectrometer, and the electron impact mass spectrum was recorded using a MAT95 spectrometer with an ionization energy of 70 eV. Microanalysis of the title compound was performed using a ‘vario EL cube’ apparatus from Elementar Analysensysteme GmbH. The melting/decomposition point was measured on a Büchi Melting Point B-540 apparatus.Synthesis of [{iPrN=CHN(iPr)N(Ni2K}2(Pr)μ-DME)]n: 1.6 mL  of N,N′-diisopropylcarbodiimide were added to a stirred suspension of 0.41 g (10 mmol) of KH in 50 ml of DME. The reaction mixture was stirred for two days and refluxed for an additional 2 h. After cooling to room temperature, all insoluble solid parts were filtered off and the volume of the resulting clear solution was reduced to ca 25 ml. After three days at room temperature, the title compound crystallized as colorless, plate-like crystals suitable for single-crystal X-ray diffraction. Yield: 1.3 g (76%). M.p. 378 K (dec.). C32H68K2N8O2 (M = 675.15 g mol−1): calculated C 56.93, H 10.15, N 16.60; found: C 56.81, H 10.24, N 16.33%. IR (ATR): ν = 2952 m, 2858 m, 2824 w, 1626 m, 1538 s, 1465 m, 1453 m, 1383 m, 1369 m, 1358 m, 1343 m, 1318 m, 1298 m, 1196 m, 1162 m, 1125 m, 1111 m 1048 w, 993 m, 955 w, 946 w, 858 w, 815 w, 674 w, 575 w, 516 w, 442 m 373 w, 338 m, 295 w, 262 m cm−1. 1H NMR : δ = 7.90 , 3.47 , 3.43 , 3.27 , 3.01 , 1.15 , 0.94  ppm. 13C NMR : δ = 166.0 (N—CH=N), 150.0 (N—CN—N), 72.6 (DME), 58.9 (DME), 55.5 [CH(CH3)2], 49.4 [CH(CH3)2], 28.2 [CH(CH3)2], 25.0 [CH(CH3)2] ppm. MS : m/z = 254 (5) [C14H30N4]+, 211 (30) [C14H30N4 − iPr]+, 184 (32), 170 (38), 144 (82), 129 (100).3 groups were allowed to rotate freely around the C—C vector, and the corresponding C—H distances were constrained to 0.98 Å. C—H distances within CH2 groups were constrained to 0.99 Å, C—H distances within the iPr CH groups to 1.00 Å, and the C—H distance within the amidinate group (i.e. at C2) to 0.95 Å. The Uiso(H) values were set at 1.5Ueq(C) for methyl groups and at 1.2Ueq(C) in all other cases. The reflections (001) and (010) disagreed strongly with the structural model and were therefore omitted from the refinement.Crystal data, data collection and structure refinement details are summarized in Table 110.1107/S2056989018015980/zl2742sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989018015980/zl2742Isup2.hklStructure factors: contains datablock(s) I. DOI: 1862025CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Scientific Reports 10.1038/s41598-019-44090-w, published online 22 May 2019Correction to: This Article contains a typographical error in the Results section under subheading ‘Application of modified GBS EDX criterion to the cohort’ where,“When using our proposed GBS EDX criterion, 39 (35.8%), 47 (43.1%) and 13 (11.9%) of all GBS patients were classified as “Definite”, “Probable” and “Possible” GBS respectively .”should read:“When using our proposed GBS EDX criterion, 39 (35.8%), 47 (43.1%) and 13 (11.9%) of all GBS patients were classified as “Definite”, “Probable” and “Possible” GBS respectively .”


This article describes the effect of the oxidative stress inducers Angiotensin II and 6-hydroxydopamine (6-OHDA) on different cell lines. The levels of expression Angiotensin type 1 and type 2 receptors in different dopaminergic cell lines are shown. The data indicate that treatment with Angiotensin II and 6-OHDA increases the production of reactive oxygen species (ROS) and decreases cell viability. NRF2 is a transcription factor induced by ROS. We provide data that NRF2 overexpression increases cell viability in response to oxidative stress inducers compared to control cells, and that these inducers can, both separately and in combination, enhance the expression of NRF2-regulated genes heme oxygenase 1 (Hmox1), NAD(P)H quinone dehydrogenase 1 (Nqo1) and Kruppel like factor 9 (Klf9). Interpretation of these data and additional information is presented in the research article “Angiotensin II induces oxidative stress and upregulates neuroprotective signaling from the NRF2 and KLF9 pathway in dopaminergic cells“   We also22.1Dopaminergic Mes23.5 cell line, dopaminergic N27 cell line and SH-SY5Y cell line were cultured as previously described HA–NRF2, kindly donated by Dr. Donna D. Zhang  was used to infect cell lines. Vectors were transfected into HEK 293 T cells with the corresponding viral packaging plasmids. Viruses were collected from the supernatant and used to infect neuronal cell lines in the presence hexadimethrine bromide . Cells were selected with puromycin  starting 72 h after infection. Stable cell lines were established after 2 weeks of selection and were continuously grown in 5 µg/ml of puromycin afterwards.2.2Cell viability was assessed 24 h after the last treatment: cultures were incubated with MTT  for 4 h at 37 °C. Culture supernatant was then carefully removed and the formazan crystals resulting from cellular dehydrogenases were dissolved in acidic isopropanol and quantified at 570 nm using a plate reader  after gentle shaking. The absorbances were expressed as percentage of control.2.3Nrf2, Nqo1, Hmox1 or Klf9, AT1 or AT2 using Actin as housekeeping gene. Primer sequences are for rat Hmox1 F 5′-TCTATCGTGCTCGCATGAAC-3′, R 5′-CCTCTGGCGAAGAAACTCTG-3′; mouse Hmox1 F 5′-CCGCCTTCCTGCTCAACAT-3′, R 5′-CTGACGAAGTGACGCCATCT-3′; rat Nqo1 F 5′-GCCCGGATATTGTAGCTGAA-3′, R 5′- GTGGTGATGGAAAGCAAGGT-3′; rodent Klf9 F 5′-AGTGCATACAGGTGAACGGC-3′, R 5′-AACGGAACTGCTTTTCCCCA-3′; rat Nrf2 F 5′-CCAGCACATCCAGACAGACA-3′, R 5′-ATATCCAGGGCAAGCGACTC-3′; rodent Actb F 5′-TCGTGCGTGACATTAAAGAG-3′, R 5′-TGCCACAGGATTCCATACC-3’; human HMOX1 F 5′-GAGCCTGAATCGAGCAGAAC-3′, R 5′-AGCCTTCTCTGGACACCTGA-3′; human NQO1 F 5′-AAAGGACCCTTCCGGAGTAA-3′, R 5′-CCATCCTTCCAGGATTTGAA-3′; human KLF9 F 5′-GGAAACACGCCTCCGAAAAG-3′, R 5′-GCCGTTCACCTGTATGCACT-3′; human ACTB F 5′-GGACTTCGAGCAAGAGATGG-3′, R 5′-AGCACTGTGTTGGCGTACAG-3′. Total RNA (without reverse transcription) was used as a negative control. Relative mRNA expression was calculated by using comparative cycle threshold (2−ΔΔCt) method and the relative amount of mRNA was presented in the form of fold change over control.Total ribonucleic acid (RNA) was extracted from cell lines using Trizol . Equal amounts of RNA (2 µg) were reverse-transcribed to complementary DNA with Moloney murine leukemia virus reverse transcriptase  following manufacturer׳s instructions. PCR using real-time iCycler PCR platform  in combination with iQ SYBR Green Supermix  were used to assess expression of 2.4L-cysteine  was used as an antioxidant and control for low ROS generation, and hydrogen peroxide  was used as a control for high ROS generation. Fluorescent intensity was expressed as relative units (RU) calculated as the difference from time 0, normalized relative to control at 15 min.Cellular ROS measurement was determined using 2′,7′-dichlorofluorescin-diacetate (DCFDA). Cultured cells were washed and loaded with DCFDA  for 30 min at 37 °C. Basal fluorescence was measured at this time (time = 0) using a plate reader (Tecan Infinite M200) at excitation 485 nm/emission 520 nm. Fluorescence was again measured 15 min, 30 min, 45 min, 60 min, 75 min and 120 min after addition of different treatments. AngII (100 nM) and 6-OHDA (40 µM) were added at the same time in these experiments. N-Acetyl-2.5two groups were analyzed by Mann-Whitney Rank sum test and multiple comparisons were analyzed with one-way ANOVA or two-way ANOVA followed by Holm Sidak test. Differences at p < 0.05 were considered as statistically significant. Statistical analyses and graph design were carried out with SigmaPlot 11.0  except otherwise stated.All data were obtained from at least three separated experiments, with a minimum sample size of four wells per group and per experiment. Data were expressed as percentage of the control group of the same batch  and expressed as a mean±standard error of the mean (SEM). Data from


The dihedral angle between the bromo- and the methyl-substituted benzene rings is 16.1 (3)°. In the crystal, molecules are connected by N—H⋯O and weak C—H⋯O hydrogen bonds, forming a–axis direction.The title molecule displays a  15H13BrN2O, displays a trans configuration with respect to the C=N double bond. The dihedral angle between the bromo- and methyl-substituted benzene rings is 16.1 (3)°. In the crystal, molecules are connected by N—H⋯O and weak C—H⋯O hydrogen bonds, forming R21(6) ring motifs and generating chains along the a–axis direction. The optimized structure generated theoretically via density functional theory (DFT) using standard B3LYP functional and 6–311 G basis-set calculations renders good support to the experimental data. The HOMO–LUMO behaviour was elucidated to determine the energy gap. The intermolecular interactions were quantified and analysed using Hirshfeld surface analysis.The title molecule, C R1R2C=NNH2 structural motif. They are related to ketones and aldehydes in which oxygen has been replaced with an NNH2 group -4-bromo-N′ -benzohydrazide and (E)-4-toluic -N′-benzohydrazide have been recently reported . The molecule adopts an (E) configuration across the C=N bond, joining the hydrazide group and the benzene ring. In the crystal, the dihedral angle between the bromo- and methyl-substituted benzene rings is 16.1 (3)°. The structure was optimized with the Gaussian09W software . The geometrical parameters  is essentially planar, with a maximum deviation from the least-squares plane of 0.099 (4) Å for atom C10. The O1=C8 bond length  indicates single-bond character. The N1—N2 bond length [1.379 (5) Å for XRD and 1.364 Å for B3LYP] is in good agreement with other experimental values  and fingerprint plots were generated based on the di and de distances using Crystal Explorer3.1  are the indicators of hydrogen-bonding contacts whereas other visible spots are due to H⋯H contacts. The dominant H⋯O interaction in the title compound is evident as a bright-red area in Fig. 3a)] conveys information about each donor–acceptor pair and while the curvedness surface [Fig. 4b)] is effectively divided into sets of patches, respectively. The tiny extent of area and light colour on the surface indicates weaker and longer contacts other than hydrogen bonds. The 2D fingerprint plots in Fig. 5Hirshfeld surface analysis  of the molecule is about 4.42 eV.The highest-occupied molecular orbital (HOMO), which acts as an electron donor, and the lowest-unoccupied molecular orbital (LUMO), which acts as an electron acceptor, are very important parameters for quantum chemistry. If the energy gap is small, then the molecule is highly polarizable and has high chemical reactivity. The energy levels were computed by the DFTB3LYP/6-311G method  region is spread around the carbonyl oxygen atom whereas the hydrogen atom attached to nitrogen is positively charged (blue).The molecular electrostatic potential is related to the electron density and molecular electrostatic potential (MESP) maps are very useful descriptors for understanding reactive sites for electrophilic and nucleophilic reactions as well as hydrogen-bonding interactions -2-hydroxybenzohydrazide -N′-[(pyridin-2-yl)methylene]benzohydrazide -N′-(4-methoxybenzylidene)pyridine-3-carbohydrazide dihydrate -4-bromo-N′-(4-methoxybenzylidene)benzohydrazide-3-(1H-indol-2-yl)-1-(4-nitrophenyl)prop-2-en-1-one hemihydrate -4-bromo-N′-benzohydrazide and (E)-4-toluic-N′-benzohydrazide  and intermolecular N—H⋯O hydrogen bonds (between the amide hydrogen and the carbonyl oxygen atoms).A search of the Cambridge Structural Database  =1.2Ueq(C) or 1.5Ueq(C-methyl).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989018017978/lh5887sup1.cifCrystal structure: contains datablock(s) global, I, 80R. DOI: Click here for additional data file.10.1107/S2056989018017978/lh5887Isup2.cmlSupporting information file. DOI: 1580647CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Plasmodium falciparum from the Greater Mekong Sub region (GMS). However, the anxiety regarding PQ-induced acute haemolytic anaemia in glucose-6-phosphate dehydrogenase deficiency (G6PDd) has hindered its use. Therefore, we assessed the tolerability of SLDPQ in Cambodia to inform national policy.The WHO recommends single low-dose primaquine  in falciparum-infected patients to block malaria transmission and contribute to eliminating multidrug resistant P. falciparum. Randomisation was 4:1 DHAPP+SLDPQ: DHAPP for G6PDd patients and 1:1 for G6PDn patients, according to the results of the qualitative fluorescent spot test. Definitive G6PD status was determined by genotyping. Day (D) 7 haemoglobin (Hb) concentration was the primary outcome measure.This open randomised trial of dihydroartemisinin-piperaquine (DHAPP) + SLDPQ vs. DHAPP alone recruited Cambodians aged ≥1 year with acute uncomplicated p = 0.040) in G6PDd (n = 9) vs. G6PDn (n = 46) DHAPP+SLDPQ recipients: 10.9 vs. 12.05 g/dL, Δ = -1.15 (95% CI: -2.24 ─ -0.05) g/dL. Three G6PDn patients had D7 Hb concentrations < 8 g/dL; D7-D0 Hbs were 6.4 ─ 6.9, 7.4 ─ 7.4, and 7.5 ─ 8.2 g/dL.One hundred nine patients , aged 4–76 years (median 23) were enrolled; 12 were G6PDd Viangchan . Mean nadir Hb occurred on D7 [11.6 (range 6.4 ─ 15.6) g/dL] and was significantly lower  D7-D0 Hb decline was -1.45 (-4.8 ─ 2.4) g/dL, associated significantly with higher D0 Hb, higher D0 parasitaemia, and receiving DHAPP; G6PDd was not a factor. No patient required a blood transfusion.DHAPP+SLDPQ was associated with modest Hb declines in G6PD Viangchan, a moderately severe variant. Our data augment growing evidence that SLDPQ in SE Asia is well tolerated and appears safe in G6PDd patients. Cambodia is now deploying SLDPQ and this should encourage other GMS countries to follow suit.NCT02434952.The clinicaltrials.gov reference number is The online version of this article (10.1186/s12879-019-3862-1) contains supplementary material, which is available to authorized users. Plasmodium falciparum in sick patients, few countries have introduced SDLPQ in their treatment guidelines. Reluctance has centred on the risk of precipitating dose-dependent, acute haemolytic anaemia (AHA) in patients with glucose-6-phosphate dehydrogenase deficiency (G6PDd), the logistical difficulty in identifying G6PDd patients at the point of care, and the lack of suitable, paediatric PQ tablet strengths and formulations  who underwent mass drug administration with SLDPQ. Mean  fractional falls of 5 (9) % and 1.7 (4) % were seen in the G6PDd and G6PDn individuals, respectively .p = 0.14) of a greater fractional D7-D0 Hb decline in six G6PDd DHAPP+PQ vs. two G6PDd DHAPP recipients; maximal declines were 29.8 and 4.4%, respectively  and showed a possible trend  cases fell from ~69 ─ ~16/1000 population and P. vivax (Pv) from ~16 ─ ~11/1000 , (iii) Hb , (iv) Giemsa-stained malaria blood films, read under × 1000 magnification; a negative thick film was declared after reading 8000 white blood cells, (v) thin blood films for red cell morphology and reticulocyte count, and (vi) dried blood spots for: (a) malaria genotype (Other samples taken were for: (i) in vitro parasite drug sensitivity (D0), (ii) full blood count: D0, 7, 14, 28 . Fresh samples were sent on ice from the site to Phnom Penh where the Hb type was also determined by Hb electrophoresis .Common SE Asian G6PD gene mutations were sought in selected patients with G6PD enzyme activities ≤12.3 U/g Hb : (i) inThe primary outcome was the D7 Hb concentration. The main secondary outcomes included: (i) D7-D0 absolute and fractional falls, (ii) modelled Hb changes over time, (iii) total malaria attributable fall (MAFt): D28Hb-nadirHb, (iv) Hb recovery (D28 Hb > D0 Hb concentration), (v) G6PD geno- and phenotype, thalassemia type, ), (vi) Dhttps://rsc.tech-res.com/docs/default-source/safety/table_for_grading_severity_of_adult_pediatric_adverse_events.pdf).These were assessed by an open question, symptom checklist, and a visual analogue scale graded 1 (“I/my child feel/s very bad”) to 5 (“I/my child feel/s very well”). We recorded the following AEs and SAEs: (i) early (≤ 1 h) or late (> 1 h) abdominal pain, nausea or vomiting, (ii) Hb fractional fall ≥25% vs. D0, (iii) passing dark urine graded ≥5 (Hillmen colour chart ), (iv) iSeparate computer-generated randomisation lists were used for the G6PDd and G6PDn groups. G6PDd patients were randomised 4:1 to receive DHAPP+SLDPQ and DHAPP (i.e. 4 DHAPP+SLDPQ for every one DHAPP); the G6PDn group was randomised on a 1:1 basis. The randomisation lists showed the treatment allocation (i.e. DHAPP+SLDPQ or DHAPP) and were openly accessible to the senior research team member responsible for drug allocation.The mean [standard deviation (SD)] D7 Hb in ~6800 ACT-treated, falciparum-infected patients of all ages from SE Asia, was 11.27 (1.74) g/dL . For a two-sided alpha of 0.05, power of 80%, SD of 1.74 g/dL, and assuming mean D7 Hb Δ of − 1 g/dL in the G6PDd DHAPP+SLDPQ arm vs. G6PDn DHAPP+SLDPQ arm, the sample size was 48/arm, rounded up to 50. We also decided to recruit 50 G6PDd and 50 G6PDn patients to receive DHAPP (‘control’ group).Data collected on paper case record forms were double entered into Epi Data v3.1, cleaned, then analysed in Stata v14 . All patients who received ≥1 dose of DHAPP +/− SLDPQ were analysed.ABX Pentra XL 80 measured Hb concentrations were considered the ‘gold’ standard and used for analyses involving D0, D7 and D7-D0 declines. Because HemoCue Hb measurements were more frequent, we used these results for: (i) defining the Hb nadir day, (ii) modelling Hb dynamics, (iii) MAFt, and (iv) Hb recovery, cognizant that HemoCue Hb concentrations were generally higher than those of the ABX Pentra XL 80.Proportional data were compared using chi-squared or Fisher’s exact test, as appropriate. Student’s ‘t’ and non-parametric tests were used to assess mean and distribution differences in normally or skewed distributed continuous data, respectively. Multiple linear regression determined factors  for the D7-D0 Hb fall and MAFt (backward-stepwise approach). A linear mixed effects regression model determined factors associated with changes over time in the mean reticulocyte counts, and mean Hb concentrations; analysis of covariance was adopted to adjust for the D0 Hb concentration by including it as a model covariate. Factors influencing Hb recovery were determined by logistic regression.Of 134 patients volunteering for the trial, 109 were recruited . LTF rates were not significantly different (p = 0.18) between DHAPP+SLDPQ: 9/56 (16.1%) vs. DHAPP 14/53 (26.4%) recipients.Following treatment, 5 patients were lost to follow up, 2 withdrew early, and 23 had recurrent Genotyping confirmed 12 G6PD Viangchan patients: 9 hemizygous males and 3 heterozygous females. Eight males and one female were diagnosed correctly by the FST but one male (enzyme activity 3.1 U /g Hb) and two females (7.9 & 8.4 U / g Hb) were diagnosed as G6PDn. Three patients  who were all FST and RDT G6PDn did not have G6PD activity measurements; the male was classed as G6PDn and the two females were genotyped as G6PD wild type. Additional file p = 0.040) lower in the G6PDd patients 10.9 (8.5 ─ 13.2) vs. G6PDn 12.05 (8.7 ─ 14.5) g/dL. Within the G6PDn group, D7 mean Hbs were significantly lower (p = 0.01) in the DHAPP vs. DHAPP+SLDPQ recipients: 11.19 vs. 12.05 g/dL, Δ = -0.86 (-1.56 ─ -0.15).For all patients, the nadir Hb occurred on D7: mean (range) 11.6 (6.4 ─ 15.6) g/dL. In the DHAPP+SLDPQ treated patients, the mean Hb was significantly (p = 0.63) different in the G6PDd vs. G6PDn arms: -1.46 (-3.3 ─ 0.4) g/dL vs. -1.24 (-4.6 ─ 2.4) g/dL, for fractional falls of -11.2 (-25.6 ─ 3.9) % vs. -8.6 (-25.6 ─ 20.7) %.The absolute D7-D0 Hb declines were consistent between the G6PDd and G6PDn patients and inversely related to D0 Hb Fig. : median p = 0.009. These three significant factors explain 42.4% of the variation in the D7-D0 Hb decline (adjusted R2 = 0.4247).The multiple linear regression model demonstrated an inverse relationship between the D7-D0 decline and (i) D0 Hb, and (ii) D0 parasite count Table , but DHAFor all patients, the mean HemoCue measured nadir Hb was 12.4 g/dL (D7) and rose to 13.1 g/dL by D28; Hb dynamics according to G6PD status are shown in Fig. p = 0.063): 1.81 vs. 0.57 g/dL, Δ = -1.25 (-0.75 ─ 2.56) g/dL. MAFt explanatory factors were D7 Hb concentration, treatment failure, both associated with lower a MAFt, and male sex (increased MAFt). The D28 Hb recovery rate was low, 35.7% (25/98), and more likely in males and older patients but less likely with higher D0 Hb concentrations  higher in G6PDd (2.9%) vs. G6PDn (1.8%) patients. There was an inverse relationship between the D0 Hb and reticulocyte dynamics , and (ii) D7 [1.6 (0.9–2.4)], p = 0.007.There was little change in the median metHb concentrations over time in both treatment arms; the highest value was 3.6% in a DHAPP-SLDPQ treated G6PDn patient  were aged 7 or 8 with grade 1 (mild) vomiting, and one was a DHAPP+SLDPQ treated, 35 years old male with grade 2 (moderate) vomiting. There were no AEs of clinical concern and no drug-related SAEs. Reported symptoms over time are shown in Additional file Six (5.9%) of 102 patients had fractional falls in Hb ≥ 25% ; five were G6PDn (one on DHAPP+SLDPQ) and one was G6PDd on DHAPP+SLDPQ.We evaluated the WHO recommended dose of SLDPQ in DHAPP-treated Cambodian patients and have shown it was well tolerated and did not result in clinically significant haemolysis in moderately severe G6PDd Viangchan. Initial declines in Hb were not significantly different by G6PD status but the DHAPP+SLDPQ treated G6PDd patients had significantly lower mean D7 Hb by just over 1 g/dL.The initial fall in Hb is the most critical period that determines whether a patient develops clinically severe anaemia to warrant a blood transfusion; in SE Asia, that risk in uncomplicated falciparum malaria is 1% . The anxp = 0.01) between their G6PDd and G6PDn ACT+SLDPQ recipients was ~0.9 g/dL, a value similar to our study.Factors associated with a greater initial D7-D0 fall in our patients Hb were higher baseline Hb concentrations and parasitaemia, consistent with previous studies , 11, 30.P. falciparum in the GMS  vs. D0 (p=0.007). (DOCX 13 kb)Methaemoglobin concentration over time, expressed as the % of total haemoglobin. Within the G6PDn group, metHb distributions were significantly different for: (i) D3 [1.6 (0.6-3.4)] vs. D0 [1.3 (0.6-2)] Additional file 3:Symptoms reported and selected signs detected during follow up and selected signs suggestive of anaemia. (DOCX 30 kb)


Their structures were identified based on spectroscopic data. Especially, the absolute configurations of fructose and glucose residues in 1 and 2 were assigned by acid hydrolysis, followed by derivatization and gas chromatography (GC) analysis. Among the known compounds, (−)-hopeaphenol (3), (+)-syringaresinol (4), and quercetin (5), were isolated from B. chinensis for the first time. In addition, biological evaluation of 1 and 2 against cholesterol synthesis and metabolism at the gene level was carried out. The results showed that compounds 1 and 2 could regulate the expression of cholesterol synthesis and metabolism-associated genes, including 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), squalene epoxidase (SQLE), low density lipoprotein receptor (LDLR), and sortilin (SORT1) genes in HepG2 cells.Two new sucrose derivatives, namely, belamcanosides A ( Belamcanda chinensis (L.) DC., is an ornamental plant in the Iridaceae family. The dried rhizomes of B. chinensis have been used for the treatment of sore throat and pharyngitis in traditional Chinese medicine +  , 6.46 , 7.57 , 6.19 , and 7.67 , 6.38 ], and three benzyl moieties with an ABX spin system , as well as three methoxyl groups at δH 3.80 , 3.83 , 3.84 , respectively. The 13C-NMR and DEPT spectra of 1 indicate that it is comprised of 42 carbons ascribed to three methyls, 3 methylenes, 23 methines, and 13 quaternary carbons. These data indicate that 1 might contain three cinnamic acid residues. The 1H-1H COSY correlations of H-7′′/H-8′′, H-7′′’/H-8′′’, H-7′′′′/H-8′′′′, HMBC correlations of H-7′′, H-8′′/C-1′′, H-7′′’, H-8′′’/C-1′′’, H-7′′′′′, H-8′′′′/C-1′′′′, and ROESY correlations of three methoxyl groups respectively with H-2′′, H-2′′′, and H-2′′′′, secure the presence of three feruloyl moieties. The large coupling constants (15.9 Hz) of three pairs of olefinic protons suggested the trans-form of α,β-unsaturated carbonyl systems. Apart from three feruloyl residues, the 1H- and 13C-NMR spectra also show an α-anomeric resonance at δH 5.60, with a small coupling constant  due to a gauche conformation, according to the Karplus relation +  , δC 172.3 and δC 20.9 (OAc-3′)] in 2. Moreover, the carbon signals for C-3′ (δC 74.5) and C-4′ (δC 70.2) in 2 are shifted downfield and upfield, respectively, relative to those in 1. These data, together with a long range correlation between H-3' and the ester carbonyl group in the HMBC spectrum -β-d-fructofuranosyl-(2→1)-(3-acetyl-4-O-feruloyl)-α-d-glucopyraoside, and named belamcanoside B.Belamcanoside B (35.2580) . The 1H- 1 and 2  are simispectrum , imply t3) +, HRESIMS m/z 893.2478 [M + Na]+ ; 1H- and 13C-NMR data, see Belamcanoside B (2): light yellowish gum; c 0.80, MeOH); UV (MeOH) λmax (logε) 195 (4.13), 218 (4.27), 235 (4.21), 302 (4.26), 326 (4.41) nm; CD (MeOH) ∆ε (nm) + 2.8 (346); ESIMS m/z 935 [M + Na]+, HRESIMS m/z 935.2585 [M + Na]+ ; 1H- and 13C-NMR data, see 1 or 2  in 1 N HCl was stirred at 70 °C for 5 h. After cooling, the mixtures were extracted with EtOAc. The aqueous layer was neutralized with 1 N NaOH and concentrated in vacuo, which was subsequently dissolved in anhydrous pyridine (2 mL). To these solutions, l-cysteine methyl ester hydrochloride (2.0 mg) was added, and the mixtures were stirred at 60 °C for 2 h, followed by concentrating in vacuo at 0 °C. Slow addition of 1-(trimethylsiyl) imidazole to the mixtures was followed by stirring at 60 °C for 1 h. Aliquots (2 µL) of the supernatants were subjected to chiral GC analysis to determine that d-fructose and d-glucose units present in 1 and 2 [A solution of  1 and 2 .v/v) foetal bovine serum (FBS) and incubated at 37 °C with 5% CO2 atmosphere.Human liver carcinoma cells HepG2 were maintained in Dulbecco’s modifed Eagle’s medium (DMEM) supplemented with 10%  was added to dissolve the formazan crystals. The samples were detected at 490 nm by an enzyme-labeled microplate reader.Cell viability was determined by MTT assay. HepG2 cells were plated in 96-well plates at a density of 5.0 × 10TM RNA MiniPrep kit (ZYMO RESEARCH) according to the manufacturer’s instructions. cDNA was synthesized and amplifed from 1 μg of total RNA using the PrimeScriptTM RT Master Mix (TaKaRa), as described in the manufacturer’s protocol. qPCR analyses were performed with SYBR® Premix Ex Taq™ II (TaKaRa) under the following conditions: 30 s at 95 °C, 40 cycles at 95 °C for 5 s and 60 °C for 35 s. Gene expression levels were determined and normalized to the expression level of 36B4. The sequences of the primer sets used were h36B4, 5′-TCTAC AACCCTGAAGTGCTTGAT-3′ and 5′-CAATCTGCAGACAGACACTGG; hHMGCR, 5′-GAA TGGCCCTAGAACTGTGC-3′ and 5′-CAAAGAGCCCTGTGTGAATG-3′; hSQLE, 5′-CCTGA ATCAGAAAATAAGGAGCA-3′ and 5′-GCTTGTTTCTGAAATATTGGTTCC-3′; hLDLR, 5′-G CTTGTCTGTCACCTGCAAAT-3′ and 5′-AACTGCCGAGAGATGCACTT-3′; hSORT1, 5′-GG CATCATTGTGGCCATT-3′ and 5′-TTGACCTTCGTCTGTGGAGA-3′.Total RNA was extracted from HepG2 cells using Direct-zolB. chinensis. Biological determination of the two new sucrose derivatives reveals that belamcanosides A and B at 20 μM could significantly reduce HMGCR and SQLE mRNA expression in HepG2 cells. Moreover, belamcanoside A could also promote LDLR mRNA expression in HepG2 cells at 20 μM. Both of them show good potency in regulating the cholesterol synthesis pathway, and exhibit low cytotoxicity. The present investigation adds new facets for the chemical composition of the seeds of B. chinensis and provides a foundation for further research of the two new sucrose derivatives.To conclude, this study led to the isolation of two new sucrose derivatives and three compounds firstly isolated from


An intramolecular O—H⋯N hydrogen bond forms an S(6) ring motif. In the crystal, the molecules are connected by C—H⋯π interactionsThe molecule has mirror symmetry with the all non-H atoms, except  22H28ClNO, shows mirror symmetry with all its non-H atoms, except the tert-butyl groups, located on the mirror plane. There is an intramolecular O—H⋯N hydrogen bond present forming an S(6) ring motif. In the crystal, the molecules are connected by C—H⋯π interactions, generating a three-dimensional supramolecular structure. Hirshfeld surface analysis and two dimensional fingerprint plots were used to analyse the intermolecular interactions present in the crystal, indicating that the most important contributions for the crystal packing are from H⋯H (68.9%) and C⋯H/H⋯C (11.7%) interactions.The title Schiff base compound, C There is an intramolecular O—H⋯N hydrogen bond present -1-[(3-chloro-4-methylphenylimino)methyl]naphthalen-2-olate-N,O}copper(II) -[(3-chloro-4-methylphenyl)imino]methyl}-4-(trifluoromethoxy)phenol ]diphenolato}nickel(II) -(3-chloro-4-methylphenyl)iminomethyl]-2-methoxy-3-nitrophenyl acetate  to 2.139 (blue) a.u. The red spots identified in Fig. 3The Hirshfeld surface analysis was performed using a) (H⋯H) shows the two-dimensional fingerprint of the  points associated with hydrogen atoms. It is characterized by an end point that points to the origin and corresponds to di = de = 1.08 Å, which indicates the presence of the H⋯H contacts in this study (68.9%). The graph shown in Fig. 6b) shows the (C⋯H/H⋯C) contacts between the carbon atoms inside the surface and the hydrogen atoms outside the Hirshfeld surface and vice versa, which contribute 11.7%. There are two symmetrical wings on the left and right sides. Furthermore, there are also Cl⋯H/H⋯Cl (11%), C⋯C (4.5%), C⋯N/N⋯C (2.2%), O⋯H/H⋯O (1.3%) and N⋯H/H⋯N (0.4%) contacts.Fig. 5A view of the three-dimensional Hirshfeld surface of the title compound plotted over electrostatic potential energy in the range −0.030 to 0.044 a.u. using the STO-3G basis set at the Hartree–Fock level of theory is shown in Fig. 7tert-butyl-2-hydroxybenzaldehyde  in ethanol (30 mL) and a solution containing 3-chloro-4-methylaniline  in ethanol (20 mL). The reaction mixture was stirred for 4 h under reflux. Crystals suitable for X-ray analysis were obtained by slow evaporation of an ethanol solution .The title compound was prepared by refluxing a mixture of a solution containing 3,5-di-Uiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989018016377/xu5948sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989018016377/xu5948Isup2.hklStructure factors: contains datablock(s) I. DOI: 1873612CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Scientific Reports 10.1038/s41598-019-49717-6, published online 13 September 2019Correction to: µM’, it should read ‘mM’. The correct Figure In Figure 1D, the y-axis is incorrectly labelled as ‘


The thiophene ring in each crystal structure has an envelope conformation. The largest differences between the two molecular structures is in the bond lengths about the quaternary C atom of the thiophene ring. In the spirocyclic structure, the C—C bonds to the spiro C atom in the cyclobutane ring are around 1.60 Å and weak intermolecular C—H⋯X  interactions link the molecules into extended ribbons. In the other structure, weak C—H⋯π interactions link the molecules into sheets.The title compounds were prepared  19H21F3O2S and C24H19F3OS, were prepared via chemo- and regioselective [3 + 2]-cycloadditions of the respective thiocarbonyl ylides (thiocarbonyl S-methanides), generated in situ, with (E)-4,4,4-trifluoro-1-phenylbut-2-en-1-one. The thiophene ring in the crystal structure of each compound has an envelope conformation. The largest differences between the two molecular structures is in the bond lengths about the quaternary C atom of the thiophene ring; in the spirocyclic structure, the C—C bonds to the spiro C atom in the cyclobutane ring are around 1.60 Å, although this is also observed in related structures. In the same structure, weak intermolecular C—H⋯X  interactions link the molecules into extended ribbons running parallel to the [001] direction. In the other structure, weak C—H⋯π interactions link the molecules into sheets parallel to the (010) plane.The title compounds, C On the other hand, the isomeric (E)-4-aryl-1,1,1-trifluorobut-3-en-2-ones undergo cycloaddition with the same thiocarbonyl S-methanide to afford 1,3-oxathiole derivatives exclusively -4,4,4-trifluoro-1-phenylbut-2-en-1-one with thiobenzophenone S-methanide as well as with 3-thioxo-2,2,4,4-tetramethylcyclobutan-3-one S-methanide led to the corresponding title tetrahydrothiophene derivatives, trans-8-benzoyl-1,1,3,3-tetramethyl-7-trifluoromethyl-5-thiaspiro[3.4]octan-2-one, 1a, and trans-3-benzoyl-2,2-diphenyl-4-(trifluoromethyl)tetrahydrothiophene, 1b, respectively, as crystalline products in high yields. Single crystals were grown from petroleum ether and used for single-crystal X-ray diffraction analyses, the results of which are reported here.1,4-Disubstitued α,β-unsaturated ketones are known as reactive dipolarophiles, and in the case of aryl,trifluormethyl-substituted representatives, the [3 + 2]-cycloadditions with electron-rich thiocarbonyl ylides occur chemoselectively either on the C=C or C=O bond, depending on the location of the CF1a and 1b crystallized as racemates with the benzoyl and trifluoromethyl substituents on the thiophene ring in a trans configuration  = 0.5164 (14) Å, φ(2) = 359.73 (18)° and atom S1 is 0.853 (1) Å from the mean plane through the other four ring atoms. The corresponding puckering parameters for 1b are Q(2) = 0.5714 (16) Å, φ(2) = 349.86 (19)° with atom S1 being 0.921 (1) Å from the mean plane through the other four ring atoms. These parameters show that the thiophene ring is slightly more distorted from an ideal envelope conformation in 1b than in 1a.Compounds n Figs. 1 and 2 ▸.1a, at around 1.60 Å, are significantly longer than is usual for an alkyl C—C bond and 0.058 (3) and 0.072 (3) Å, respectively, longer than the corresponding bonds to the phenyl rings in 1b. In concert, the S1—C2 and C2—C3 bonds are around 0.034 (2) Å shorter and the C3—C4 bond 0.018 (3) Å longer in 1a than in 1b; all other related bond lengths in the two molecules are comparable. Despite these variations and the acute ‘bite angle’ of the cyclobutane ring at C2 of the thiophene ring [89.45 (12)° compared with 110.44 (14)° for the diphenyl-substituted 1b], the intra-ring bond angles in the thiophene rings of the two compounds are not very different. The above-mentioned differences in ring puckering presumably allow the bond-length variations not to impinge on the intra-ring angles. The Cambridge Structural Database  and −29.5 (3)° in 1a and 1b, respectively. The O1—C6—C3—C4 torsion angles also differ by about 41°, so that the carbonyl group is more slanted with respect to the mean plane of the thiophene ring in 1b than in 1a.The carbonyl group in 1a, there are three unique potentially significant weak supramolecular contacts . The combination of these interactions leads to double-stranded chains of molecules, or ribbons, running parallel to the [001] direction , albeit with a rather sharp C—H⋯F angle of 121° -cycloaddition of the intermediate thiocarbonyl S-methanides 4 with the activated C=C bond. Whereas the more stable 2a, derived from 3-thioxo-2,2,4,4-tetramethylcyclobutanone, decomposes at 318 K, the less stable precursor 2b, derived from thiobenzophenone, already extrudes N2 at 228 K. The 1H NMR analysis showed that only one product was formed in each case. After chromatographic purification, the isolated products were crystallized from petroleum ether by slow evaporation of the solvent.The title compounds were prepared according to the reaction sequence presented in Fig. 5 al. 2016. ThermalUiso(H) = 1.5Ueq(C) while each group was allowed to rotate freely about its parent C—C bond. All other H atoms were placed in geometrically idealized positions and constrained to ride on their parent atoms with C—H distances in the range 0.95–1.00 Å and Uiso(H) = 1.2Ueq(C). For 1a, one low angle reflection was omitted from the final cycles of refinement because its observed intensity was much lower than the calculated value.Crystal data, data collection and structure refinement details are summarized in Table 410.1107/S2056989018015335/vm2212sup1.cifCrystal structure: contains datablock(s) 1a, 1b, global. DOI: 10.1107/S2056989018015335/vm22121asup2.hklStructure factors: contains datablock(s) 1a. DOI: Click here for additional data file.10.1107/S2056989018015335/vm22121asup4.cdxSupporting information file. DOI: 10.1107/S2056989018015335/vm22121bsup3.hklStructure factors: contains datablock(s) 1b. DOI: Click here for additional data file.10.1107/S2056989018015335/vm22121bsup5.cdxSupporting information file. DOI: 1876143, 1876144CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


RS)-4--2-methylpiperazine, Lf] with 5-fluoroisophthalic acid leads to a charge-assistant hydrogen-bonding network between HLf+ cations and 3-carboxy-5-fluorobenzoate anions.Reaction of lomefloxacin , indicating the presence of both enantiomers of the cation in the structure.In the title organic salt, C RS)-4--2-methylpiperazine] belongs to the fluoroquinolones that represent an important family of highly effective broad-spectrum antibacterial agents  for a proton-transfer reaction, and report here synthesis and crystal structure of the produced salt (HLf)+·(5-F-Hip−), (I)In this context, we have used 3-carboxy-5-fluorobenzoic acid ° between the aromatic ring plane and the piperazinium ring (the latter exhibits a chair conformation). An intramolecular S(6) hydrogen-bonding pattern , with the highest deviation of 0.2645 (13) Å for the carboxylate O6 atom.The structures of the molecular entities of (I)A⋯O3iii interactions between the amino function of the piperazinium moiety and the non-protonated O atom of the carboxylic group of a neighboring HLf+ cation result in a head-to-tail chain motif with descriptor C(13). Adjacent 5-F-Hip− moieties also form a head-to-tail chain, based on a C(8) pattern, involving O5—H5⋯O7i bonds between the carboxylic acid function and the carboxylate function. The two kinds of chains interlink with each other through N1—H1B⋯O6ii interactions between the second H atom of the amino group of the cation and one of the carboxylate O atoms of the anion to form a three-dimensional network structure. Within this array  was mixed with a slurry of lomefloxacin (Lf)  in 5 ml water under stirring. The mixture was exposed to ultrasound for ca 20 min, and was then filtered and left to slowly evaporate. Colourless block-like single crystals suitable for X-ray analysis were obtained after several weeks. Yield: 65% . Analysis calculated for C25H24F3N3O7: C, 56.08; H, 4.52; N, 7.85%. Found: C, 56.06; H, 4.50; N, 7.82%. FT–IR : 3431b, 3070 (w), 2475 (w), 1718 (s), 1620 , 1456 (s), 1371 (m), 1275 (s), 1090 (m), 959 (m), 901 (w), 766 (m), 689 (m), 521 (w).A methanol solution (6 ml) of 5-fluoroisophthalic acid (5-F-HUiso(H) = 1.2Ueq(C) or 1.5Ueq(C-methyl). All O-bound and N-bound H atoms were initially found in difference electron-density maps, and then refined using a riding model . The methyl group bound to the piperazinium ring is disordered over two positions with occupancies of 0.645 (5) and 0.355 (5).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989018016961/wm5472sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989018016961/wm5472Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989018016961/wm5472Isup3.cmlSupporting information file. DOI: 1025160CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


R,2S,4r)-1,2,4-triphenylcyclopentane-1,2-diol and -4-(2-methoxyphenyl)-1,2-diphenylcyclopentane-1,2-diol form one-dimensional chains via O—H⋯O hydrogen bonding in their crystals. The diols may serve as precatalyst activators for ring-opening polymerization of cyclic esters.Achiral 2, C23H22O2, (I)] and 4-(2-methoxyphenyl)-1,2-diphenylcyclopentane-1,2-diol . Their single crystals have been obtained by crystallization from a THF/hexane solvent mixture. Diols (I) and (II) crystallize in orthorhombic (Pbca) and triclinic (PI)] and three [in the case of (II)] crystallographically independent molecules of the achiral -diol isomer. Each hydroxyl group is involved in one intramolecular and one intermolecular O—H⋯O hydrogen bond, forming one-dimensional chains. Compounds (I) and (II) have been used successfully as precatalyst activators for the ring-opening polymerization of ∊-caprolactone.Reductive cyclization of 1,3,5-triphenyl- and 3-(2-methoxyphenyl)-1,5-diphenylpentane-1,5-diones by zinc in acetic acid medium leads to the formation of 1,2,4-triphenylcyclopentane-1,2-diol [1,2,4-Ph The corresponding diones are formed by condensation of acetophenone with benzaldehyde/2-methoxybenzaldehyde under basic conditions  may sometimes be present (up to 20%), but it does not crystallize under the conditions used here.1,2-Diphenyl-4-arylcyclopentane-1,2-diols can be readily synthesized by the reductive cyclization of 1,5-diphenyl-3-arylpentane-1,5-diones with zinc in an acetic acid medium (THF)n]2  are active in ring-opening polymerization (ROP) of cyclic esters 2 is catalytically inactive, but displays relatively high catalytic activity upon activation by a primary alcohol 2 activated even by various bulky alcohols  and (II) as activators of the Mg(BHT)2(THF)2 precatalyst for polymerization of ∊-CL  found by 1H NMR spectroscopy and by size-exclusion chromatography (SEC) are very close to the calculated value . However, when the ratio [diol]/[Mg(BHT)2] = 1:2, and two chains are growing at one diol, the nP values (entries 2 and 4) are somewhat higher than expected , which might be explained by a longer reaction time of the second [Mg(BHT)2(THF)2] molecule with the same initiator molecule with respect to the time of polymer-chain propagation. This is also supported by larger polydispersity index (Đ) values (compare entries 2 and 4 with entries 1 and 3), pointing to unequal growth of the two chains.In the case of the ratio [diol]/[Mg(BHT)2(THF)2] and (I) or (II) are capable of catalysing ROP of ∊-CL, providing 100% monomer conversion. When using the diol/Mg(BHT)2 ratio equal to 1:1, the ROP can be carried out in a more controlled manner.Therefore, catalytic tests have shown that systems based on . The intermolecular O—H⋯O bond angles lie in the expected range of 160 (3) to 173.2 (19)°. The chains are oriented along the b-axis direction in (I) and approximately along the ab diagonal in (II). It might be also mentioned that for both the inter- and intramolecular hydrogen bonds, the O⋯O and consequently O—H⋯O distances are slightly elongated in (II) compared to (I), likely as a result of crystal-packing effects.Regardless of some structural differences, diols 2 was prepared as described earlier 2 were performed under a purified argon atmosphere in a dry box in absolute solvent media. Tetrahydrofuran was pre-dried over NaOH and distilled from potassium/benzophenone ketyl. Hexane was distilled from an Na/K alloy. Toluene was distilled from sodium/benzophenone ketyl. ∊-Caprolactone (∊-CL) was distilled from CaH2 under reduced pressure of argon. CDCl3  was used as purchased. The NMR spectra were recorded on Bruker AV400 and AV600 spectrometers at 300 K; chemical shifts are reported in ppm relative to the solvent residual peak. The SEC analysis of polymer samples was performed at 323 K using an Agilent PL-GPC 220 gel permeation chromatograph equipped with a PLgel column, with DMF as eluent (1 ml min−1) and poly(ethylene oxide) standards.The starting compounds 1,3,5-triphenylpentane-1,5-dione and 3-(2-methoxyphenyl)-1,5-diphenylpentane-1,5-dione were obtained in high yields by the previously described procedure -1,2,4-Triphenylcyclopentane-1,2-diol, (I), was prepared as described previously : δ = 2.61 , 2.84 , 3.58 , 4.11 , 6.97–7.12 , 7.15–7.36 , 7.43 .(1I) was dissolved in a warm mixture of THF/hexane (1:10 v/v) to provide a saturated solution. Single crystals formed in two weeks.A small portion of -4-(2-Methoxyphenyl)-1,2-diphenylcyclopentane-1,2-diol, (II), was prepared analogously to (I) but with some minor modifications. Zinc powder  was added by small portions over 5 h to a vigorously stirred solution of 1,5-diphenyl-3-(2-methoxyphenyl)pentane-1,5-dione  in 900 ml of glacial acetic acid at 363 K. The formed hot mixture was filtered. The resulting solution was cooled to room temperature and poured into 5000 ml of water. The formed yellowish precipitate was collected, washed with water (2 × 100 ml) and dried under vacuum. The solid was recrystallized from a hot mixture of petroleum ether (boiling temperature range of 343–373 K) and toluene . The white microcrystals were dried under dynamic vacuum. The yield was 17.42 g , : δ = 2.56 , 2.81 , 3.46 , 3.90 , 4.27 , 6.96 , 7.02–7.07 , 7.09–7.13 , 7.31 , 7.45 . 13C{1H} NMR : δ = 34.07, 44.21, 55.40, 85.58, 110.67, 120.52, 126.34, 126.47, 126.82, 127.34, 127.48, 132.17, 143.51, 158.26.(1II), suitable for X-ray diffraction analysis, were grown from a THF/hexane mixture (1:10 v/v) over two weeks.Single crystals of (I) or 36 mg of (II)] in 1 ml of THF was added to a stirred solution of Mg(BHT)2(THF)2  in 1 ml of THF. The resulting solution was stirred for 20 min. A solution of ∊-CL  in 1 ml of THF was then added at once to the formed catalyst solution. The solution was stirred for 30 min and then a sample was taken to determine conversion of the monomer by 1H NMR spectroscopy. A 100% conversion was established in all cases based on the absence of a resonance signal at 4.22 ppm (∊-CL) and the presence of a signal at 4.05 ppm (PCL), both corresponding to the –C2HO(CO)– fragment. The remaining viscous solution was poured into methanol (50 ml) containing a drop of acetic acid. The resulting precipitate was separated by centrifugation, washed with methanol (3 × 25 ml) and hexane (2 × 10 ml) and dried under vacuum. Polymer samples were further studied by SEC and 1H NMR analysis. The degree of polymerization was determined by integration of a PCL terminal group signal at 3.63 ppm (–CH2—C2H—OH).In a typical polymerization experiment, a solution of 0.1 mmol of a diol [33 mg of (I) and the hydroxy H atoms in (II) were found from the difference maps. These H atoms were refined independently with individual isotropic displacement parameters. The other H atoms in (II) were positioned geometrically  and refined as riding atoms with relative isotropic displacement parameters Uiso(H)= 1.5Ueq(C) for methyl H atoms and 1.2Ueq(C) otherwise. A rotating group model was applied for methyl groups. For (II), reflections SHELXL was used for (II)  I, II, New_Global_Publ_Block. DOI: 10.1107/S2056989019008673/fy2139Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989019008673/fy2139IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989019008673/fy2139Isup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989019008673/fy2139IIsup5.cmlSupporting information file. DOI: 1929065, 1929064CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Activated invariant natural killer T (iNKT) cells rapidly produce large amounts of cytokines, but how cytokine mRNAs are induced, stabilized and mobilized following iNKT activation is still unclear. Here we show that an endoplasmic reticulum stress sensor, inositol-requiring enzyme 1α (IRE1α), links key cellular processes required for iNKT cell effector functions in specific iNKT subsets, in which TCR-dependent activation of IRE1α is associated with downstream activation of p38 MAPK and the stabilization of preformed cytokine mRNAs. Importantly, genetic deletion of IRE1α in iNKT cells reduces cytokine production and protects mice from oxazolone colitis. We therefore propose that an IRE1α-dependent signaling cascade couples constitutive cytokine mRNA expression to the rapid induction of cytokine secretion and effector functions in activated iNKT cells. Invariant natural killer T (iNKT) cells rapidly enhance cytokine secretion and effector function following activation, but the underlying mechanism is still unclear. Here the authors show that an endoplasmic reticulum stress sensor, inositol-requiring enzyme 1α, activates the p38 kinase to stabilize cytokine mRNA for enhanced iNKT functions. A hallmark feature of iNKT cells is their ability to rapidly produce and secrete immunomodulatory cytokines following T-cell receptor (TCR) ligation, implicating them in a range of inflammatory, allergic, and autoimmune diseases1. Although this functional aspect of iNKT cell biology is not fully understood, it has been suggested that the presence of preformed cytokine mRNAs as well as histone acetylation of distinct cytokine loci facilitate rapid iNKT cell cytokine production3. However, beyond such studies, it has proved difficult to investigate the potential regulatory mechanisms involved in iNKT cell cytokine production as many of these signaling pathways also control iNKT cell development, maturation, and survival4. We therefore sought to investigate whether iNKT cells utilize components of the unfolded protein response (UPR) to accommodate the rapid increase in cytokine production following activation as has been observed for the production of antibodies during plasma cell differentiation6.Invariant natural killer T (iNKT) cells are characterized as an innate T-cell subset that recognizes glycolipid antigens presented by CD1d, an MHC class I-related molecule7. Prolonged or severe ER stress that cannot be resolved by induction of the UPR is widely considered to trigger apoptosis and inflammation and is involved in the development of a number of human diseases characterized by a metabolic or inflammatory pathology8.UPR is an intracellular signal transduction pathway conserved from yeast to mammals that senses perturbations in protein folding, protein synthesis and/or calcium homeostasis within the endoplasmic reticulum (ER). In mammals, the UPR consists of the three proximal ER stress sensors; inositol-requiring enzyme 1α (IRE1α), ER-resident protein kinase R-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6) that collectively function to promote ER homeostasis by increasing protein folding capacity and protein biosynthesis within the ER during stress9. During ER stress, oligomerization of the luminal domain of IRE1α results in autophosphorylation of the cytosolic domain and activation of a sequence-specific endoribonuclease (RNase) which recognizes and cleaves an intron from pre-mRNA encoding the bZIP transcription factor XBP110. Translocation of cleaved or spliced XBP1 (XBP1s) to the nucleus is associated with the upregulation of ER chaperone proteins and enzymes which function to increase protein folding capacity and quality control within the ER12. The RNase domain of IRE1α also targets and degrades distinct mRNAs containing a consensus sequence in a process termed regulated IRE1α-dependent decay (RIDD)13, further reducing protein translocational load during ER stress. In addition to these functions, autophosphorylation of IRE1α during UPR is also associated with downstream c-Jun kinase (JNK) phosphorylation14, which is proposed to promote apoptosis in cells unable to resolve ER stress15. ER stress however also activates additional ER stress sensors including the protein kinase PERK and the transcription factor ATF617. Here, the substrates for the protein kinase activity of PERK have been identified, namely the eukaryotic translation initiation factor 2α (eIF2α). EIF2α has been shown to counteract the formation of reactive oxygen species and to inhibit cap-dependent mRNA translation18. ER stress-mediated proteolysis of the ER luminal domain of ATF6 results in the liberation of a bZIP transcription factor that induces genes involved in ER chaperone function and ER-associated protein degradation (ERAD)19. Collectively, UPR therefore promotes ER homeostasis and cell survival by regulating an adaptive response at both the transcriptional as well as translational level. Irremediable ER stress is however associated with inflammatory signaling and the initiation of apoptosis15.IRE1α is a type I ER-resident transmembrane protein that comprises an ER luminal and cytosolic domain with both serine-threonine kinase and endoribonuclease activity20. Here, XBP1s is required for the increased synthesis and secretion of Ig chains by plasma cells6, suggesting that IRE1α is directly activated during B-cell differentiation. Similarly, T-cell differentiation following TCR ligation is associated with the activation of IRE1α21, whereas PERK has been shown to control T-cell effector functions by regulating the translation of distinct cytokine mRNAs22.Although it is widely acknowledged that UPR regulates cellular survival during ER stress, several studies have shown that UPR is also required by B lymphocytes during plasma cell differentiationIn this study, we demonstrate that defined iNKT sublineages constitutively express components of the UPR, and require IRE1α to stabilize cytokine mRNAs following activation both in vitro and in vivo. We propose that these findings represent a novel mechanism whereby IRE1α functions as a central regulator of cytokine production for specific iNKT subsets in vivo.FP/WT) mice23. We utilized ERAIFP/WT mice as a UPR-specific reporter as they express a partial sequence of human XBP-1 that includes the sites at which IRE-1α splices XBP-1, fused to Venus fluorescent protein (VenusFP), which allows reporting of the IRE-1α arm of the UPR23. Subsequent flow cytometric analysis of the T-cell compartment using a Self-Organizing Map (FlowSOM)24 visually defined specific T-cell lineages as a minimal spanning (MS) tree in which clusters of similar cells were represented as nodes within each T-cell subset , CD8+ T cells (red), CD1d tetramer-negative NKT-like cells25 (light blue), iNKT cells (dark blue) and γδ T cells (purple), respectively  mice. As is shown in Fig. VenusFP expression was found to be restricted to iNKT cells present in the spleen and liver; however, reporter expression was also detected within peripheral iNKT and γδ T-cell lineages  and mediastinal lymph node (MLN) did not  in T cells isolated from the thymus, spleen and liver of naïve ERAI-transgenic  agonists such as lipopolysaccharide (LPS) or Poly (I:C) induced significant IRE1α RNase activity above that observed for medium controls  by intercrossing CD4-cre transgenic mice with mice carrying a LoxP flanked IRE1α allele. A subsequent analysis of the frequency and absolute cell counts of iNKT cells present in the thymus, spleen, and liver of CD4cre;IRE1αΔ/Δ mice revealed no significant differences when compared to littermate IRE1αF/F controls  to stage 3 (CD44hiNK1.1hi) iNKT cells occurred normally in the absence of IRE1α  during plasma cell differentiationase Fig. . To examcre;IRE1αΔ/Δ mice were backcrossed onto a CXCR6gfp/− background as this mouse line has previously been shown to be a reporter specific for hepatic iNKT cells1. CXCR6gfp/−IRE1αF/F (IRE1αWT) and CXCR6gfp/−CD4cre;IRE1αΔ/Δ (IRE1αKO) mice were subsequently imaged for 2 h at steady-state until they received an i.v. injection with α-GalCer, after which they were imaged for two more hours to capture the TCR-mediated cell arrest that follows iNKT cell activation32. The overall appearance and behavior of IRE1αWT and IRE1αKO mice were very similar  and p38 mitogen-activated protein kinase (MAPK), respectively39. Both JNK and p38 MAPK have been shown to regulate gene expression within the T-cell compartment by regulating cytokine mRNA stability41, suggesting that IRE1α might function within activated iNKT cells in a similar manner.During ER stress, oligomerization of the luminal domain of IRE1α is associated with increased serine-threonine kinase activity within the cytosolic domain of the proteinF/F and CD4cre;IRE1αΔ/Δ mice and expanded in vitro. Resting control and IRE1α-deficient iNKT cells were then restimulated with anti-CD3/CD28 and analyzed for intracellular expression of JNK, phosphorylated JNK (p-JNK), p38, and p-p38 by FACS. As shown in Fig. 42, or the p38 MAPK inhibitor SB20358043, prior to actinomycin D addition. Here, incubation of restimulated iNKT cells with the JNK inhibitor BI-78D3 had a negligible effect on cytokine mRNA stability when compared to vehicle controls -2 have been previously associated with the stabilization of inflammatory mRNAs51. In this context, a common characteristic of inflammatory mRNAs is the presence of AU-rich sequences in their 3′ untranslated regions. AU-rich elements (ARE) can act as potent mRNA destabilizing sequences as they can target mRNA for rapid deadenylation in vivo as well as promote 3′-5′ exonuclease decay52. It has been proposed that p38-mediated stabilization of inflammatory mRNAs is facilitated by a class of ARE-binding proteins (AREBP), such as TIA-1, which are functionally altered following activation of the p38 MAPK signaling pathway53. It remains to be determined whether IRE1α-p38-mediated stabilization of cytokine mRNAs within iNKT cells requires downstream modulation of AREBP function; however, it is interesting to note that TIA-1 has previously been shown to modulate IL-4 production within CD4+ T cells22.Our findings are in line with a previous report describing a role for MAPK kinase (MKK) 3, an upstream activator of the p38 MAPK pathway, in regulating cytokine production by iNKT cellscre;IRE1αΔ/Δ mice are protected from oxazolone colitis suggests that IRE1α-dependent signaling contributes to iNKT cell effector function in vivo as well. These results are significant as it suggests that IRE1α-mediated stabilization of cytokine mRNAs may be a general intracellular signaling cascade required for modulating iNKT cell effector function in vivo. Currently, a range of inflammatory, allergic and autoimmune diseases are believed to be modulated by cytokines secreted by iNKT cells in mouse and human. Although we were able to show that inhibition of IRE1α kinase function using APY29 inhibited cytokine mRNA stability in cultured iNKT cells, in vivo administration of APY29 to target iNKT cell effector function may have unwanted off-target side-effects such as inhibition of exocrine gland and liver function49. We therefore propose that a more detailed understanding of the mechanism by which IRE1α regulates cytokine production in iNKT cells may yield novel insights into signaling pathways that can be pharmacologically targeted to either inhibit or augment iNKT cell-driven disease.Finally, the finding that CD4#Miur (ERAI-transgenic) mice have been previously described23. T-cell-specific IRE1α-deficient mice  were generated by intercrossing mice carrying a LoxP flanked IRE1α allele33 with CD4-cre transgenic mice34. These transgenic mice were intercrossed with CXCR6gfp mice for intravital imaging of iNKT cells as previously described32 All mice were housed and bred according to the guidelines of the Ghent University vivarium. All animal procedures were approved by the Institutional Animal Care and Ethics Committee.Tg(CAG-XBP1*/Venus)+ provided by NIH tetramer core facility, USA. Stainings for intracellular proteins were performed using the FoxP3/Transcription Factor staining buffer set (eBioscience) for iNKT cell transcription factors or methanol for protein kinases according to the manufacturers’ recommendations. T lymphocyte subsets are defined as DN (CD3ε−CD4−CD8−); DP (CD3ε−CD4+CD8+); CD4+ T (CD4+CD8−CD1d-αGC−TCRβ+); CD8+ T (CD4−CD8+CD1d-αGC−TCRβ+); iNKT (TCRβ+CD1d-αGC+); NKT-Like (TCRβ+CD1d-αGC−NK1.1+), MAIT (MR1–5-OP-RU tetramer+ TCRβ+) and γδ+ T (TCRγδ+TCRβ−). TCRβ+CD1d-αGC+ NKT sublineages are defined as NKT1 (CD27+/−IL-2Rβ+), NKT2 (CD27+IL-2Rβ−), and NKT17 (CD27−IL-2Rβ−).Cells were analyzed on FACSCantoII (BD Biosciences) and FlowJo software (Tristar), and sorted on FACS Aria III (BD Biosciences). Antibodies used were CD122- PerCP-eFluor710 (TM-B1/46-1222-82/1:200), CD19-eFluor 450 (eBio1D3/48-0193-82/1:300), CD24-FITC (M1-69/11-0242-82/1:250), CD25-PE (PC61.5/12-0251-82/1:400), CD27-APC (LG.7F9/47-0271-82/1:150), CD3ε-V500 (500A2/560771/1:100/BD Biosciences), CD4-FITC/APC-eFluor780 (RM4-5/11-0042-82/17-00471-83/1:280), CD44-FITC/APC-eFluor780 (IM7/11-0441-85/25-0441-U100/1:275), CD62L-PE-Cy7 (MEL-14/60-0621-50U/1:600/TONBO), NK1.1-PE-Cy7 (PK136/25-5941-82/1:400), CD8α-V500 (53-6.7/560776/1:300/ BD Biosciences), βTCR-APC-eFluor780 (H57-597/47-5961-82/1:400), γδ ΤCR-PerCP-eFluor710 (eBioGL3/46-5711-82/1:250), IL2-PE-Cy7 (JES6-5H4/25-7021-80/0.25 μg), IL4-APC (11B11/17-7041-82/0.25 μg), IL6-FITC (MP5-20F3/561363/0.25 μg), IL13-PE-Cy7 (eBio13A//25-1733-80/0.25 μg), IFNγ-FITC (XMG1.2/11-7311-81/0.25 μg), TNFα-APC (MP6-XT22/17-7321-81/0.25 μg), T-bet-PE (eBio4B10/12-5825-82/0.25 μg), Gata-3-AlexaFluor488 (TWAJ/14-9966-82/0.25 μg), RORγt-PE (B2D/12-6981-80/0.25 μg), PLZF-AlexaFluor488 (Mags.21F7/53-9320-82/0.25 μg) . p38α/β (A-12), JNK (D-2), β-Actin , p-JNK-AlexaFluor 647 (G9), IRE-1α (14C10/3294/1:1000), and EIF2α (9722/1ː1000) from Cell Signaling). α-GalCer-loaded CD1d tetramers (1:400) and MR1-5-OP-RU-APC (NIH/1:800). iNKT cells were stained at 4 °C using α-GalCer-loaded CD1d tetramers. MAIT cells were stained at room temperature using MR1–5-OP-RU tetramer24. Briefly, T-cell subsets were manually gated and the data for all the selected cells were aggregated per tissue. FlowSOM analysis used intensity measurements for NK1.1, CD4, α-GalCer-loaded CD1d tetramer, βTCR, γδ ΤCRand CD8α to group the cells into 100 nodes where node size indicates the mean percentage of cells assigned to the nodes. For analysis of VenusFP expression, the median VenusFP expression was computed for each node per sample. The final color value assigned to the node corresponds to the difference between the mean of the values obtained for ERAI-transgenic and wild-type mice, respectively.FlowSOM analysis was performed as described previouslySingle cell suspensions from the thymus were resuspended in phosphate-buffered saline (PBS) containing 1 mM ethylenediaminetetraacetic acid (EDTA) and 0.5% bovine serum albumin (BSA) while spleen and liver suspensions were layered over Ficoll or 33% Percoll gradients, respectively. Peripheral blood mononuclear cells (MNCs) were isolated by cardiac puncture in 20 U/ml PBS/heparin. iNKT thymocytes were enriched for using mouse CD8-Dynabeads (Invitrogen) and resuspended in 2 ml PBS containing 1 mM EDTA and 0.5% BSA.F/F or CD4cre;IRE1αΔ/Δ mice as described earlier54. Briefly, α-GalCer/CD1d tetramer+ TCRβ+ iNKT cells present within the lymphocyte were gated after eliminating cell doublets, dead cells, CD19+ and γδT lymphocytes from the analysis by plotting FSC-H versus FCS-W, DAPI, CD19 (eFluor450), γδTCR (PerCp-Cy5.5) versus FSC-A, respectively  and stimulated with anti-CD3ε/CD28 . For in vitro expansion experiments, sorted splenic bulk iNKT or NKT sublineages were resuspended in complete RPMI 1640 medium containing murine IL-2 (10 ng/ml), IL-12 (1 ng/ml), and soluble anti-CD28  and stimulated with plate-bound anti-CD3ε (3 µg/ml) for 2 days. iNKT cells were then rested for 2 days and resuspended in complete medium containing murine IL-7  for 4 days. iNKT cells were then restimulated with plate-bound α-CD3ε (3 µg/ml) for a further 3 days and subsequently expanded for an additional 10 days in the presence of IL-7 and plate-bound anti-CD3ε, as described above. For restimulation experiments, 1 × 106 iNKT cells were resuspended in complete RPMI 1640 medium , 100 U/ml penicillin/streptomycin, 2 mM glutamine, 0.1 mM nonessential amino acids, 5.5 × 102 µM µ-ME, all from GIBCO) and stimulated with either PMA/Ionomycin , recombinant murine IL-12p70 , recombinant murine IFNβ , LPS , Poly I:C , 10 µg/ml murine CD1d  loaded with 100 ng/ml α-GalCer or anti-CD3ε/CD28 . For cytokine measurements, supernatants were collected after 72 h of stimulation and measured by ELISA.iNKT cells were FACS sorted from CD5-enriched MNCs isolated from the spleens or livers of pooled C57Bl/6, IRE1α6 cells) were stimulated with anti-CD3ε/CD28  for 0, 15, 30 and 60 min. Cells were spun for 10 min at 400 × g at 4 °C. After centrifugation, the medium was removed and washed with cold PBS. Cells were lysed for 15 min on ice in E1A buffer . Insoluble material was discarded by cold centrifugation, and a fixed amount of lysate was mixed with sample buffer before separation by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Samples were separated using 6% SDS-PAGE containing 50 μM Phos-tag (NARD Institute) and 50 μM MnCl2. Gels were soaked in 1 mM EDTA before transfer for 10 min and transferred onto 0.45 μM polyvinyldifluoride membranes (Millipore). After transfer to a membrane, proteins were visualized by enhanced chemiluminescence kit (Thermo scientific).Phosphorylation of IRE1α was monitored by Phos-tag SDS gels according to the manufacturer’s instructions (NARD Institute). Expanded bulk splenic iNKT cells (2 × 10+ T cells (CD4+TCRβ+CD1d-αGC−NK1.1−CD44−CD62Lhi) were FACS sorted from the pooled spleens of IRE1αF/F (n = 5) and CD4cre;IRE1αΔ/Δ mice (n = 5), resuspended in complete RPMI 1640 media containing polarizing cytokines and plated at 1 × 105 cells/well in 96-well plates coated with anti-CD3ε/α-CD28 . Plated CD4+ T cells were differentiated under TH1-polarizing conditions using recombinant murine IL-12 , recombinant murine IL-2 , and anti-IL-4 ; TH2-polarizing conditions using recombinant murine IL-4 , recombinant murine IL-2 (10 ng/ml) and anti-IFNγ ; and TH17-polarizing conditions using recombinant murine IL-6 , recombinant murine IL-23 (10 ng/ml),55, recombinant murine IL-1β , recombinant human TGFβ1 , anti-IL-4 (10 μg/ml), anti-IFNγ (10 μg/ml) and anti-IL-2 , respectively. Cells were then removed from TCR stimuli after 3 days, resuspended at 1 × 106/ml in fresh media and cultured in a new 96-well plate without TCR stimuli for 2 days. 1 × 106 polarized T cells were then resuspended in complete RPMI 1640 medium and restimulated using anti-CD3ε/CD28 .Naïve CD4For serum cytokine production, 5 μg of α-GalCer in 200 ml PBS was injected intraperitoneally. Blood was subsequently collected at by retro-orbital puncture at 4 h postinjection and assayed for cytokine content by ELISA. For intracellular cytokine measurements, 2 μg of α-GalCer in 200 ml PBS was injected intraperitoneally and splenic iNKT cells were analyzed 2 h later by flow cytometry. For intravital imaging, 1 μg of α-GalCer in 20 μl PBS was injected intravenously. For in vivo labeling with BrdU (Sigma-Aldrich), the mice were injected i.p. with 1 mg BrdU (in PBS), followed by injection of 2 μg of α-GalCer intraperitoneally. At the same time, BrdU (1 mg/ml) was placed in the drinking water till the end of the experiment. Five days later, splenic iNKT cells were analyzed for BrdU incorporation using a BrdU flow kit (BD Biosciences) according to the manufacturer’s instructions.56. Briefly, to establish a baseline iNKT cell speed, 2 h of steady-state images were recorded prior to injection with α-GalCer. Imaging was continued for 2 h postinjection. Images were analyzed with Imaris (Bitplane) using ImarisTrack to quantify iNKT cell speeds over time. Cell velocities were plotted as a moving average of ten frames for each timepoint. Tracks of individual iNKT cells, normalized to their starting coordinates, were plotted with Matlab 9.1 (MathWorks). The square of the displacement distance over time was shown at steady-state and 1 h postinjection. Finally, a confinement index was calculated by multiplying cell track straightness by the square root of the track duration, both at steady-state and 1 h postinjection.Multiphoton imaging of iNKT cells in the liver was performed as described beforeRpl13a, Atp5b, Eif4b, Cyc1, B2m, Ubc, Actb, Gapdh, Sdha or Tubb4a. Primer sequences for housekeeping, cytokine, ER chaperone and UPR signature genes are listed in Supplementary Table Total RNA was extracted from T lymphocytes analyzed in this study using the miRNeasy Mini kit . cDNA was produced and amplified using the QuantiTect Whole Transcriptome Kit . PCR products were amplified with the Fast Start SYBR Green Master mix  and detected with a LightCycler 480 System . Samples were normalized using qBasePlus  against at least three of the following housekeeping genes: + T-cell subsets were stimulated with α-CD3ε/CD28  for 3 h after which 3 µg/ml of actinomycin D (Sigma-Aldrich) was added. RNA was subsequently isolated from actinomycin D-treated iNKT cells using the miRNeasy Mini kit . Cytokine mRNAs were then normalized against at least three housekeeping genes expressed as the % mRNA remaining after the zero timepoint of actinomycin D addition. The slope of the line for % mRNA remaining between 0 and 90 min post-actinomycin D addition was determined by linear regression for each group or treatment condition. For inhibitor studies, anti-CD3ε/α-CD28-stimulated iNKT cells were incubated in the presence of 20 µM APY29 , 20 µM B-109 (kindly provided by Andrew’s lab), 12.5 µM B1-78D3 (Sigma-Aldrich), 2.5 µM SB-203580 (Sigma-Aldrich) or equivalent vehicle controls prior to the addition of actinomycin D.RNA stability assays iNKT and differentiated CD4Eight-week-old mice were presensitized with 200 µl of a 3% (w/v) solution of oxazolone  in 100% ethanol or 100% ethanol alone (vehicle) on the abdominal skin. Five days later mice were anesthetized with a mixture of ketalar, rompun and PBS and rechallenged intrarectally with 150 µl of either 1% (w/v) oxazolone in 50% ethanol or 50% ethanol alone (vehicle).Histology: Mice were euthanized 10 days after induction of oxazolone colitis. The colons were subsequently removed and fixed with 4% formalin (Sigma-Aldrich). After paraffin embedding 7 µm sections were cut and stained with hematoxylin–eosin.Samples of the mid-to-distal colon (adjacent to the samples used for histology) were rinsed with cold PBS, blotted dry, and immediately frozen in liquid nitrogen for storage at − 80 °C. Myeloperoxidase (MPO) activity was determined using the O-dianisidine method.t test unless otherwise indicated. The Mann–Whitney U test was applied when data were demonstrated to not follow a Gaussian distribution, which is indicated in the respective figures legends. Linear regression analysis and ANOVA were performed for mRNA stability assays. Mixed model analysis was performed to assess differences in weight loss. All p values were two-tailed, and statistical significance was assumed at p < 0.05. For intravital imaging, linear mixed models for repeated measures were used except for the speed distribution, for which binomial logistic regression was performed.Statistical testing was performed using the unpaired Student’s Supplementary InformationDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2


The correct name is: Tamara Escrivá-Martinez. The correct citation is: Ferrer-Pérez C, Escrivá-Martinez T, Montagud-Romero S, Ballestín R, Reguilón MD, Miñarro J, et al. (2018) Indomethacin blocks the increased conditioned rewarding effects of cocaine induced by repeated social defeat. PLoS ONE 13(12): e0209291. The affiliation for the second author is incorrect. Tamara Escrivá-Martinez is not affiliated with #1 but with #2 Department of Personality, Evaluation and Psychological Treatment, Faculty of Psychology, Universitat de València, Valencia, Spain.