In this study, alpha-tocopherol (α-toc) and delta-tocopherol (δ-toc) levels were measured in mouse plasma and tissues following their high dosage dietary supplementation. Average α-toc levels at 5, 10 and 20 g α-toc/kg diet increased over baseline levels 6-fold in plasma, 1.6-fold in brain, and 4.9-fold in liver. These elevated α-toc concentrations remained constant from 5 to 20 g α-toc/kg diet, rather than showing further increases across these dosages. No α-toc-related toxicity occurred at these high dosages, and strain-specific differences in liver and brain α-toc levels between Balb/cJ and C57Bl/6J mice were observed. Relatively high-dosage administration of dietary δ-toc for 1 or 4 weeks resulted in 6–30-fold increases in plasma and liver levels between dosages of 0.33 and 1.67 g δ-toc/kg diet. Co-administration of sesamin with δ-toc further increased δ-toc levels between 1.3- and 14-fold in plasma, liver, and brain. These results provide valuable PK information on high dosage α-toc and δ-toc in mouse and show that supplementation of sesamin with δ-toc further increases δ-toc levels over those seen with δ-toc supplementation alone. Vitamin E isoforms are essential nutrients that are widely used as dietary supplements and therapeutic agents for a variety of diseases. However, their pharmacokinetic (PK) properties remain poorly characterized, and high dosage animal studies may provide further information on their  The molecules collectively known as vitamin E are fat-soluble compounds present in a wide variety of plant-derived foods. The 8 molecularly distinct isoforms of vitamin E are: alpha (α)-, beta (β)-, gamma (γ)-, and delta (δ)-tocopherol (toc), and α-, β-, γ-, and δ-tocotrienol t3). The α-, β-, γ-, and δ- prefixes each identify the number and position of methyl groups on the aromatic chromanol ring. In addition, all the toc isoforms possess a saturated 15-carbon tail, while t3 tails are unsaturated . The α-,,7,8,9. Ein vivo. In vitro studies show they are potent antioxidants, and also suggest they function in specific regulation of enzyme activity and gene expression, potentially stemming from roles in cell signaling and membrane fusion [Vitamin E is an essential nutrient that was first identified 90 years ago based upon its effectiveness in countering fetal reabsorption in rats . The nece fusion ,18,19,20e fusion ,20. Addie fusion ,29,30,31in vivo levels. However, even though the importance of sufficient vitamin E levels for normal human health is known from diseases of vitamin E deficiency, the effective levels of each vitamin E isoform for health benefits or disease prevention remain unclear. The interest in these potential human health benefits of vitamin E supplementation is reflected in the >25 clinical trials currently utilizing vitamin E [Alongside the continued discovery of various cellular activities affected by specific vitamin E isoforms, a growing body of data suggests that increased physiological toc/t3 levels in humans by altered dietary intake or supplementation might benefit a wide range of disease states. These include neurodegenerative disorders , cystic fibrosis, amyotrophic lateral sclerosis, cancer, ischemia-reperfusion injuries, and temporal lobe radionecrosis ,38,39,40itamin E . in vivo functions, a detailed understanding of the PK of each vitamin E isoform is needed. The most prevalent toc in vivo, α-toc, is widely used in synthetic vitamins and food supplements. Conversely, the least prevalent toc in vivo, δ-toc, has potent in vitro effects but little published in vivo data. Therefore, we performed in vivo experiments in mouse to identify PK profiles of α-toc and δ-toc. The α-toc levels in plasma, liver, and brain were measured in two mouse strains following 4 weeks of dietary supplementation with high dose α-toc. The PK profiles of single-dose δ-toc were determined, as well as its levels in plasma, liver, and brain following 1 week and 4 weeks of δ-toc dietary supplementation at relatively high doses as compared to the small amounts present in a typical diet. In addition, the sesame seed-derived lignan sesamin was co-administered with δ-toc, to determine if sesamin alters the PK of δ-toc, because sesamin has previously been shown to elevate plasma concentrations of α-toc, γ-toc, α-t3 in rat, and γ-toc and α-toc in humans [Given the potential human health benefits of vitamin E supplementation, paired with knowledge gaps concerning vitamin E n humans ,45,46,472, 100 bar. Flow rate and collection wavelength was 60 mL/min and 220 nm, respectively. Injection volume was 0.4–0.7 mL at 30 mg/mL in methanol and isopropanol. In the second method, a Chiralpak IA (2 × 15 cm) column was used with an eluent of 20% isopropanol/CO2, 100 bar. Flow rate and collection wavelength was 85 mL/min and 220 nm, respectively. Injection volume was 0.75 mL at 30 mg/mL in methanol. In the third method, a Chiralpak AS-H (2 × 15 cm) column was used with an eluent of 18% methanol/CO2, 100 bar. Flow rate and collection wavelength was 75 mL/min and 220 nm, respectively. Injection volume was 1 mL at 30 mg/mL in methanol. For analysis, a Chiralpak AS-H (25 × 0.46 cm) column was used with an eluent of 20% methanol (DEA)/CO2, 100 bar. Flow rate was 3 mL/min and detection wavelengths were 220, 254, and 280 nm. Retention time was 1.95 min. For δ-toc purification, a Chiralpak AD-H (2 × 15 cm) column was used with an eluent of 10% ethanol/CO2, 100 bar. Flow rate and collection wavelength was 75 mL/min and 220 nm, respectively. Injection volume was 0.5 mL at 30 mg/mL in methanol. For analysis, a Chiralpak AD-H (25 × 0.46 cm) column was used with an eluent of 15% ethanol/CO2, 100 bar. Flow rate was 3 mL/min and detection wavelengths were 220, 280, and 295 nm. Retention time was 2.83 min.α-Toc (vegetable oil-derived) and δ-toc were obtained from Sigma-Aldrich  as technical grade  and were subsequently purified by HPLC to >99% purity using supercritical fluid chromatography preparative systems at Lotus Separations, LLC . For α-toc purification, three separation methods were used in order to achieve the desired purity. In the first method, a Chiralpak IA (2 × 15 cm) column was used with an eluent of 15% isopropanol/COSesamin was obtained from Best PharmaTech, Inc. . Mouse diet consisted of AIN93M as control diet, or AIN93M formulated with α-toc, δ-toc, or δ-toc + sesamin . The concentrations per kg of diet or per kg of body weight of α-toc, δ-toc, and sesamin are specified for each experiment. Of note, AIN93M contains 75 mg α-toc/kg and 900 µg vitamin K/kg .ad libitum for 4 weeks, then mice were euthanized and tissues collected.All animal work was done according to NIH-approved animal care and use protocols. A total of 20 BALB/cJ and 20 C57Bl/6J  male mice were used for high dosage α-toc supplementation in the diet. Mice of each strain were divided into 4 groups of 5 mice, and each group received one of the following diet formulations: Control, 5 g α-toc/kg diet, 10 g α-toc/kg diet, and 20 g α-toc/kg diet. Mice were fed each diet N = 3 at each timepoint. Plasma was collected at 0 min, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h. Liver, kidney, lung, and brain were collected at 0 min, 4 h, 8 h, 12 h, and 24 h. A total of 30 male CD-1 mice were used for studies where single-dose δ-toc at 100 mg δ-toc/kg body weight was administered by intraperitoneal (IP) injection. For tissue collection, N = 3 per group at each of 11 timepoints: 0 min, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, and 48 h. A total of 231 male C57Bl/6 mice  were used for studies in which single-dose δ-toc and sesamin in 100% corn oil were administered by oral gavage at a dose volume of 10 mL/kg. Of note, tocs are a natural component of corn oil, and thus small additional amounts of tocs were administered in each corn oil gavage at the following approximate concentrations (mg/kg body weight): α-toc = 0.002, β-toc = 0.00025, γ-toc = 0.004, δ-toc = 0.00025 ,49. MiceA total of 60 Balb/cJ mice (Jackson Laboratories) were used for high dosage dietary supplementation of δ-toc and δ-toc + sesamin, with 30 mice each for the 1-week and 4-week studies. In all cases of sesamin supplementation, concentration was 2 g sesamin/kg diet. Six groups of 5 mice were each given diets supplemented with one of the following δ-toc/sesamin concentrations : (1) Control (AIN93M), (2) 0.33 g δ-toc, (3) 1.67 g δ-toc, (4) sesamin + 0.067 g δ-toc, (5) sesamin + 0.33 g δ-toc, and (6) sesamin + 1.67 g δ-toc.For serum collection, blood samples were collected into chilled, heparinized tubes by retro-orbital sinus bleed or terminal cardiac puncture. After cooling to 4 °C, blood samples were centrifuged to separate serum, and isolated serum was snap frozen at −80 °C. For all other tissue collections, mice were euthanized, and subsequently tissues were collected and immediately snap frozen at −80 °C.18 reverse phase HPLC column  using a methanol-water gradient system, peaks were analyzed by mass spectrometry using electrospray ionization in multiple reaction monitoring mode. Mobile phase A was 7.5 mM ammonium acetate in water, mobile phase B was 0.1% formic acid in methanol, and the flow rate was 1 mL/min. The gradient program included a 0.5 min hold at 20% B (the starting conditions), followed by a gradient to 99% B over 1.25 min and a 1 min hold at 99% B. The column was then returned to starting conditions and equilibrated over 1.25 min. Calibration curves for each tissue were determined by preparing a 50× stock of α-toc or δ-toc in 1-propanol, then generating serial dilutions. These serial dilution samples were then diluted 50-fold into homogenized tissues.Analytical samples were prepared by homogenizing tissue in 3 volumes of 1-propanol per gram of tissue, then centrifuging to remove precipitated proteins. Samples were analyzed using an Agilent 6410 mass spectrometer coupled with an Agilent 1200 HPLC and a CTC PAL chilled autosampler, all regulated by MassHunter software (Agilent). After separation on a Cp < 0.05 deemed significant. Values below the lower limit of quantitation (<LLOQ) were estimated at LLOQ/2 [0–24) or 0 to 48 h (AUC0–48) were determined using the trapezoidal rule, and mean AUCs were compared by two-tailed t-tests.One-way or two-way ANOVA tests with post-hoc Tukey-Kramer multiple comparisons tests were performed to compare mean α-toc and δ-toc levels, with t LLOQ/2 . Area unp < 0.05 for each strain). In contrast, adipose α-toc levels showed no significant differences from controls for either strain at any dosage (p < 0.05). Modest α-toc increases of less than 2-fold were seen in brain . These data indicated that α-toc concentrations remained constant in these tissues even though dietary dose escalated from 5 to 20 g α-toc/kg diet, and suggested the existence of an in vivo α-toc concentration plateau at or below 5 g α-toc/kg diet. Adult Balb/cJ and C57Bl/6J mice were given high dosage dietary α-toc for 4 weeks, at concentrations of 0 (control), 5, 10 and 20 g α-toc /kg diet. Subsequently, the mice were examined for pathological abnormalities, and the α-toc concentrations in plasma, brain, liver, and adipose tissue were measured . Patholoy dosage B. Post-hp ≤ 0.002; for mouse strain, p ≤ 0.009). Mean plasma α-toc concentrations were higher in Balb/cJ mice, while brain and liver α-toc concentrations were higher in C57Bl/6J mice. Post-hoc analyses comparing these two mouse strains revealed statistically significant differences in mean α-toc concentrations at some dosages (indicated by † in p < 0.05). Taken together, these results indicate that α-toc concentrations increase in mouse plasma, brain, and liver following dietary supplementation of large dosage α-toc, and these α-toc concentrations in these tissues plateau at a dosage at or below 5 g α-toc/kg diet. In addition, C57Bl/6J and Balb/cJ mouse strains show quantitatively different in vivo α-toc levels following α-toc supplementation, suggesting allelic variation between these strains might contribute to altered efficiency of the metabolic pathways regulating α-toc storage and removal.Interestingly, two-way ANOVA revealed significantly different mean plasma, liver, and brain α-toc concentrations when comparing not only α-toc dosage groups, but also mouse strains  at 10, 50, or 250 mg/kg body weight. Subsequently, plasma, liver, and brain concentrations of δ-toc were measured at timepoints from 0 to 48 h , but significantly increased liver δ-toc concentration . These data suggest co-administration of sesamin with δ-toc has the potential to increase in vivo δ-toc concentrations.Sesamin, a component of sesame seeds, has been reported to increase plasma and tissue concentrations of α-toc, γ-toc, α-t3, and γ-t3 by inhibiting their metabolism ,47,51,52To further investigate sesamin’s effect on δ-toc levels, and to generate PK data for long-term δ-toc supplementation, plasma and tissue δ-toc concentrations were determined following long-term dietary supplementation with high dosage δ-toc and high dosage δ-toc with sesamin. Balb/cJ mice were exclusively fed chow containing high dosage δ-toc (at 0.33 or 1.67 g/kg diet) or high dosage δ-toc with sesamin  for 1 week or 4 weeks. Pathological analyses of livers from mice receiving long-term dietary supplementation of δ-toc or δ-toc with sesamin showed no notable abnormalities that differed from those of controls , indicatp ≤ 0.0004 for δ-toc supplementation in plasma, brain, and liver, and with p ≤ 0.02 for sesamin co-supplementation in brain and liver. Similarly, 4 weeks of supplemental high dosage δ-toc alone or with sesamin . After 1 week , signifi sesamin  caused svs.p = 0.03) and liver (p = 0.00004). While post-hoc analyses showed no differences at the 0.067 g or 0.33 g δ-toc dosages between the two time periods, either with or without sesamin, significant changes were seen at the 1.67 g δ-toc dosages (p = 0.01), and also maintained plasma δ-toc levels. These data suggest that over long time periods of administration, sesamin can allow increased in vivo δ-toc levels, by limiting the removal of circulating δ-toc and also by increasing accumulation of δ-toc in tissues such as liver and brain. Comparison of δ-toc concentrations between the 1- and 4-week supplemental dietary δ-toc groups vs. revealed dosages . When 1.a,b,c). In all tissues, only the 1.67 g δ-toc dosages achieved significant increases in δ-toc concentrations. In regards to sesamin co-administration, only at the 1.67 g dosages did co-administration of sesamin cause a significant increase over the levels achieved for δ-toc alone . Similarly, high brain δ-toc levels were achieved when sesamin was administered with the 1.67 g δ-toc dose for 4 weeks (0.285 µM). Taken together, these data suggest measurable effects by sesamin on δ-toc concentration cannot be achieved at low dosages, on the order of 0.067–0.33 g/kg diet, but instead require higher δ-toc dosages, on the order of 1.67 g δ-toc/kg diet. Additional post-hoc analyses comparing mean δ-toc concentrations at each dosage within plasma, brain, or liver showed that only a subset of δ-toc levels significantly differed from one another  can be administered to human patients with rare disorders that cause vitamin E deficiency, as well as to patients with amyotrophic lateral sclerosis ,33,64,65in vivo α-toc levels differ between normal animals and those exhibiting various diseases.Mouse brain showed small increases in α-toc levels in response to high dosage α-toc supplementation when compared to those of plasma and liver. On average, the brain showed a 1.6-fold increase to 2 µM, as compared to the 6-fold increase in plasma to 4.9 µM and the 4.9-fold increase in liver to 240 µM. This suggests the presence of brain-specific mechanisms that limit α-toc uptake into the brain, potentially at the blood-brain barrier. An understanding of the brain’s response to elevated intake of tocs is important for interpretation of neurological studies, which have suggested elevated dietary intake and plasma levels of tocs/t3s may play a role in delaying neurological disease in humans and mouse models ,70,71,72in vivo δ-toc levels in mouse plasma, liver, and brain. Sesamin is a naturally occurring component of sesame seeds, and functions in vivo as an antioxidant [in vitro have shown that it blocks CYP oxidation/excretion of tocs and t3s [in vivo effect of sesamin on each toc/t3 are required.This study revealed that the oral administration of high dosage δ-toc increased δ-toc levels throughout the body, and these δ-toc levels further increased with co-administration of sesamin at the highest dosage of 1.67 mg δ-toc/kg diet. To our knowledge, this is the first report showing sesamin can increase ioxidant , anti-caioxidant ,83,84, aioxidant ,89,90,91ioxidant ,94,95,96 and t3s ,98, thus and t3s ,43,44,45 and t3s ,47. Howe and t3s ,46,47. Tin vivo δ-toc levels achieved following high dosage ingestion are different between normal and disease states.The brain exhibited lower δ-toc concentrations than those in plasma and liver, similar to the lower brain α-toc concentrations seen following α-toc supplementation. Therefore it appears that the ability to elevate δ-toc levels in the brain and the resulting potential to alleviate phenotypes of neurological disease is severely restricted. However, it remains possible that, even at low concentrations, the unique chemical properties of δ-toc based upon its structure may result in beneficial physiological effects in some parts of the body. For example, some animal studies suggest δ-toc may display properties that can be used to prevent cancer progression . Since ein vivo functions of δ-toc, it also suggests that high dosage of δ-toc or δ-toc with sesamin did not radically change the rate of normal δ-toc metabolism. High concentrations of α-toc are maintained by the preferential binding and release of α-toc to the plasma by α-toc transfer protein (α-TTP), while lower concentrations of the other seven vitamin E isoforms result from their rapid, CYP-mediated breakdown [in vivo is unknown, although functional importance is supported by the conservation of α-toc enrichment across vertebrates [in vivo levels of other tocs. Supplemental α-toc decreased levels of δ-toc and γ-toc [in vivo. This will allow correlation of the different biological functions of each isoform, often determined from in vitro studies, with their potential for in vivo therapeutic applications. The efficient achievement of therapeutic levels may require adjustment of the in vivo ratios of individual vitamin E isoforms. In these studies, δ-toc plasma and tissue concentrations never reached levels that were similar to those of α-toc, even when co-administered with sesamin. While this suggests that mouse may not be a tractable animal model for studying the reakdown ,102,103.tebrates . It doestebrates ,109,110 nd γ-toc ,114,115,nd γ-toc ,110. ThiThe baseline α-toc and δ-toc levels reported here correlate with a subset of previous mouse studies. In mice fed standard diets, previously reported plasma levels were 5.0–20 µM for α-toc ,55,56,57Similarly, the α-toc and δ-toc levels reported in this study resulting from high dosage α-toc and δ-toc showed both similarities and differences to previous mouse studies. Supplemental α-toc at 5 g α-toc/kg diet resulted in liver concentrations similar to those found in our study 156–207 µM), but greater α-toc concentrations in brain (25–29 µM) . Supplem–207 µM, vs. 4 weeks in current study), or may reflect strain-specific variation affecting toc metabolism, as these studies used a variety of mouse strains. The strain-specific differences in α-toc levels between Balb/cJ and C57Bl/6J identified in the current study support the latter hypothesis, as does the existence of human polymorphisms that are associated with functional differences in vitamin E metabolism. These include missense mutations in the toc/t3 metabolic enzyme CYP4F2 that result in differing enzymatic activity [The higher brain and plasma levels in these previous studies could reflect the usage of longer time periods of diet supplementation (8–48 weeks activity , as wellactivity ,122,123.activity . in vivo studies analyzing the effects of these two vitamin E isoforms on mammalian health as well as their potential for treatment of disease.This study provides essential quantitative and strain-specific PK information on administration of high dosage α-toc and δ-toc in the mouse. High dosage dietary α-toc and δ-toc can result in their increased tissue concentrations in plasma, liver, and brain. Furthermore, co-supplementation of sesamin with high dosage δ-toc can increase δ-toc concentrations in plasma and liver to concentrations greater than those reached with high dosage dietary δ-toc alone. These findings will be valuable resources for future


Necator americanus, an intestinal blood-feeding parasite of humans. Sequence-specific 1H, 13C and 15N resonance assignments have been obtained for the recombinant 170 amino acid protein, using three-dimensional triple-resonance heteronuclear magnetic resonance experiments. Backbone assignments have been obtained for 99.3 % of the non-proline HN/N pairs (146 out of 147). The amide resonance of T45 was not observed, probably due to rapid exchange with solvent water. A total of 96.9 % of backbone resonances were identified, while 97.7 % assignment of amino acid sidechain protons is complete. All Hα(166), Hβ(250) and Hγ(160) and 98.4 % of the Hδ (126 out of 128) atoms were assigned. In addition, 99.4 % Cα (154 out of 155) and 99.3 % Cβ (143 out of 144) resonances have been assigned. No resonances were observed for the NHn groups of R93 NεHε, arginine, Nη1H2, Nη2H2, histidine Nδ1Hδ1, Nε1Hε1 and lysine Nζ3H3. Na-FAR-1 has a similar overall arrangement of α-helices to Ce-FAR-7 of the free-living Caeorhabditis elegans, but with an extra C-terminal helix.The fatty acid and retinol-binding (FAR) proteins are a family of unusual helix-rich lipid binding proteins found exclusively in nematodes, and are secreted by a range of parasites of humans, animals and plants. Na-FAR-1 is from the parasitic nematode  Necator americanus being the most prevalent. The infective larvae burrow into skin and develop to adulthood in the intestine, where they feed on blood and tissue. The infection causes anaemia and growth stunting, the effects being most severe in children and women of child bearing age  proteins are α-helix-rich proteins of around 20 kDa that are produced at different life cycle stages of nematodes and have been observed to be secreted by adult parasites  with a C8 stationary phase and water/acetonitrile/trifluoroacetic acid mobile phase. The protein was refolded in aqueous buffer and concentrated to approximately 0.5 mM in 50 mM sodium phosphate pH 7.2, 50 mM NaCl. D2O was added to a final concentration of 10 % (v/v) prior to data acquisition. All spectra were recorded at 311 K on a Bruker Avance 600 MHz spectrometer equipped with a TCI cryoprobe.Recombinant Na-FAR-1 was expressed in BL21 (λDE3) 15N-HSQC, 3D HNCACB, 3D CBCA(CO)NH  were assigned using 2D HBCBCGCDHD and 2D HBCBCGCDCEHE spectra  and analysed with CCPN analysis . Excluding the His-tag residues, backbone resonance assignments have been obtained for 99.3 % of the non-proline HN/N pairs (146 out of 147). The amide resonance of T45 was not observed, probably due to rapid exchange with solvent water. Figure 1H–15N HSQC spectrum with the assigned crosspeaks. A total of 96.9 % of backbone resonances were identified, while 97.7 % assignment of amino acid sidechain protons is complete. All Hα(166), Hβ(250) and Hγ(160) and 98.4 % of the Hδ (126 out of 128) atoms were assigned. In addition, 99.4 % Cα (154 out of 155) and 99.3 % Cβ (143 out of 144) resonances have been assigned. No resonances were observed for the following NHn groups: R93 NεHε, arginine, Nη1H2, Nη2H2, histidine Nδ1Hδ1, Nε1Hε1 and lysine Nζ3H3.All Na-FAR-1 polypeptide backbone resonances were assigned, with the exception of the 15 His-tag C. elegans FAR protein, with an additional α-helix segment at the C-terminus  , accession number 18637.The


The ErIII atom in the cation is coordinated by eight O atoms from two bidentate nitrate anions and four hexamethylphosphoramide ligands in a distorted square-antiprismatic geometry. Together with the two nitrate ligands, the cation is monovalent, which leads to the anionic chain having a [WS4Ag] repeat unit. The polymeric anionic chain has a distorted linear configuration, with W—Ag—W and Ag—W—Ag angles of 161.37 (2) and 153.548 (12)°, respectively. The title complex is isotypic with the Y, Yb, Eu, Nd, La, Dy, Sm, Lu and Tb analogues.In the title compound, {[Er(NO For the al. 2007. For iso al. 2007; Cao et  al. 2007; Zhang,  al. 2007; Tang, Z al. 2007; Tang, Z al. 2007; Zhang 2(C6H18N3OP)4][AgWS4] = 0.047wR(F2) = 0.092S = 1.089597 reflections532 parametersH-atom parameters constrainedmax = 0.91 e Å−3Δρmin = −1.04 e Å−3ΔρCrystalClear  used to solve structure: SHELXTL  I, global. DOI: Click here for additional data file.10.1107/S1600536812049987/rz5031Isup2.hklStructure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:


The CoII ion is six-coordinated by two O atoms from water molecules, two O atoms and two N atoms from two imidazole-4,5-dicarboxylate ligands, giving a slightly distorted octahedral geometry. The GdIII ions exhibit three types of coordination environments. One Gd ion is eight-coordinated in a bicapped trigonal–prismatic geometry by four O atoms from two imidazole-4,5-dicarboxylate ligands, two O atoms from two SO4               2− anions and two coordinated water molecules. The other Gd ions are nine-coordinated in a tricapped trigonal–prismatic geometry; one of these Gd ions is bonded to four O atoms from two imidazole-4,5-dicarboxylate ligands, three O atoms from three SO4               2− anions and two water O atoms and the other Gd ion is coordinated by one O atom and one N atom from one imidazole-4, 5-dicarboxylate ligand, five O atoms from three SO4               2− anions as well as two coordinated water molecules. These metal coordination units are connected by bridging imidazole-4,5-dicarboxylate and sulfate ligands, generating a three-dimensional network. The crystal structure is further stabilized by N—H⋯O, O—H⋯O, and C—H⋯O hydrogen-bonding interactions between water molecules, SO4               2− anions, and imidazole-4,5-dicarboxylate ligands.The asymmetric unit of the title compound, [CoGd DOI: 10.1107/S1600536811026821/pv2424Isup2.hkl            Structure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The molecule is roughly planar, with the dihedral angles between the thienyl and benzene rings being 6.24 (16) and 9.7 (11)° for the major and minor components, respectively. The central fragment is almost planar [r.m.s. deviation = 0.0275 (2) Å for the ten non-H atoms]. The mean plane through this middle unit makes a dihedral angle of 2.71 (7)° with the benzene ring, whereas these values are 4.46 (15) and 7.7 (11)° for the major and minor components of the thienyl ring, respectively. In the crystal, molecules are linked into dimers by pairs of N—H⋯O hydrogen bonds, forming R22(8) ring motifs. These dimers are arranged into sheets parallel to the ac plane.In the title compound, C DOI: 10.1107/S1600536812017114/bq2351Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S1600536812017114/bq2351Isup3.cmlSupplementary material file. DOI:  crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:


C/EBPβ-LIP:LAP ratio is regulated by endoplasmic reticulum (ER) stress and mTOR signalling, both of which are altered in obesity. In 3T3-L1 adipocytes, 11β-HSD1 mRNA levels were down-regulated following induction of ER stress by tunicamycin but were up-regulated following inhibition of mTOR by rapamycin. These data point to a central role for C/EBPβ and its processing to LIP and LAP in transcriptional regulation of 11β-HSD1 in adipose tissue. Down-regulation of 11β-HSD1 by increased C/EBPβ-LIP:LAP in adipocytes may be part of a nutrient-sensing mechanism counteracting nutritional stress generated by HF diet.11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) catalyses intracellular regeneration of active glucocorticoids, notably in liver and adipose tissue. 11β-HSD1 is increased selectively in adipose tissue in human obesity, a change implicated in the pathogenesis of metabolic syndrome. With high fat (HF)-feeding, adipose tissue 11β-HSD1 is down-regulated in mice, plausibly to counteract metabolic disease. Transcription of 11β-HSD1 is directly regulated by members of the CCAAT/enhancer binding protein (C/EBP) family. Here we show that while total C/EBPβ in adipose tissue is unaltered by HF diet, the ratio of the C/EBPβ isoforms liver-enriched inhibitor protein (LIP) and liver-enriched activator protein (LAP) (C/EBPβ-LIP:LAP) is increased in subcutaneous adipose. This may cause changes in 11β-HSD1 expression since genetically modified C/EBPβ Adult male mice (n = 16/group) were fed control diet  or HF diet  for 6 weeks. Adult male C/EBPβ mutant mice, heterozygous C/EBPβ5 cells per well in a 12-well plate then transfected the next day with siRNA (32 pmol) in serum-free medium for 3 h followed by 21 h incubation in growth medium. All siRNAs were purchased from Applied Biosystems  and were scrambled , C/EBPα (ID 101889), C/EBPβ (ID 288793) and CHOP (ID 288792). In the experiments designed to induce ER stress, 3T3-L1 adipocytes were treated with tunicamyin (4 µg/ml). Rapamycin  was used to inhibit the mammalian target of rapamycin (mTOR) in 3T3-L1 adipocytes.3T3-L1 murine preadipocytes  were maintained and differentiated into mature adipocytes as previously described Following dissection, subcutaneous adipose tissue  was frozen on dry ice and stored at −70°C. Tissues were weighted on a balance model AA-160 . RNA was extracted using Qiagen RNeasy Lipid Tissue Mini kit . 3T3-L1 cells were harvested in Trizol. RNA (2 µg) was reverse transcribed using SuperScript III (Invitrogen) and quantified by real-time PCR on a LightCycler 480 (Roche) as described Subcutaneous adipose tissue  was homogenized in lysis buffer  in the presence of a protease inhibitor cocktail  and heated at 100°C. 3T3-L1 adipocytes were harvested directly into lysis buffer and processed similarly. Electrophoresis was carried out on 4–12% NuPage Bis-Tris gels (Invitrogen). After transfer, blots were probed with antibodies to C/EBPα, -β, -δ , CHOP  and β-tubulin  and secondary antibodies anti-rabbit IgG-HRP (1∶2000 dilution from stock 400 µg/ml Santa Cruz Biotechnology Inc.), anti-mouse IgG-HRP , anti-rabbit IgG Alexa Fluor 700 (Invitrogen) and anti-mouse IgG . The resulting bands were analyzed by ImageJ  or the Odyssey Infrared Imaging System , as appropriate.5′-CTAGTGCTGCCTGAGACTACTCC-3′ and 5′-TTTTCATAACTGCCATCAAACAG-3′), FP3 and FP4 (5′- CAGTAGGAGATGCTCAGGAACC-3′ and 5′-AGAGCAACGATTGGCTTTGG-3′), FP9 (5′-ACTTGCCTGAGAGTCTTGAACTG-3′ and 5′-AGCAAAATAAGCTCAAGGGAAAA-3′) and a C/EBPβ binding site in the C/EBPα promoter (5′-GGTGACTTAGAGGCTTAAAGGAG-3′ and 5′-GTGACTTTCCAAGGCGGTGAGT-3′). Primers spanning an 11β-HSD1 intronic sequence not predicted to bind C/EBP were used as a control (5′-TCAAACATCCAGGTTCTTTCATT-3′ and 5′-CATACCACATAAACCACACATGG-3′). Data are expressed relative to levels of input DNA in immunoprecipitations.ChIP assays were carried out using an Upstate EZ ChIP kit  according to the manufacturer’s protocol. Briefly, differentiating 3T3-L1 cells (8 h after induction of differentiation) or fully mature adipocytes (differentiated for 9d) in 15 cm tissue culture dishes were incubated with dimethyl adipimidate.2 HCL  for 30 min in the dark followed by 1% (v/v) formaldehyde. Cells were lysed in sodium dodecyl sulphate buffer (provided in the kit). Chromatin was sheared by sonication using a Soniprep 150  with eight 10 s pulses, at maximum amplitude, keeping cells on ice between pulses. Immunoprecipitations were carried out with 5 µg C/EBP or control rabbit IgG antibody (Santa Cruz). After reversal of cross-links, purified DNA was amplified by standard PCR  or real-time PCR  as above, using primers spanning the following C/EBP-binding sites in the 11β-HSD1 promoter t test or ANOVA followed by post hoc Tukey, Fisher LSD or Dunnet tests using SigmaStat 2.03 statistical software. Significance was set at p≤0.05.All data were analyzed by Student’s vs control diet, 26.3±0.34 g; p<0.01) with increased subcutaneous adipose tissue weight  corrected for body weight; p<0.01), while liver weight was unchanged  corrected for body weight). 11β-HSD1 mRNA was down-regulated in adipose tissue by HF diet both in subcutaneous . The results also showed that reduced expression of one of the C/EBPs in mature 3T3-L1 adipocytes did not affect mRNA levels of the other C/EBPs tested  and C/EBPβΔuORF mice which have increased and decreased C/EBPβ-LIP:LAP ratio, respectively. In C/EBPβ(+/L) mice, a “knock-in” of C/EBPβ-LIP  replaces the normal C/EBPβ gene ΔuORF mice, deletion of the upstream open reading frame  prevents translation of C/EBPβ-LIP (+/L) mice (increased C/EBPβ-LIP:LAP ratio) than in wild-type controls  and C/EBPβΔuORF mice, have altered levels of the C/EBPβ isoforms. C/EBPβΔuORF mice show activation of acute-phase response genes after partial hepatectomy, suggesting that C/EBPβ-LIP may be a natural restraint of inflammation Alterations in C/EBPβ-LIP:LAP ratio are likely to impact upon other metabolically-important genes, many of which, like 11β-HSD1, are regulated by C/EBPβ


The pen ligands chelate the metal ion, forming a six-membered ring which adopts a half-chair conformation, while the sac ligands adopt the most common coordination mode. The crystal packing is stabilized by N—H⋯O hydrogen bonds, which form a one-dimensional network along [010]. It is also supported by an N—H⋯S hydrogen bond between the amine group of the pen ligand and the sulfonyl group of the sac ligand.In the title complex, [Ni(C For hyd al. 1995. For a r al. 2007.7H4NO3S)2(C3H10N2)2] = 0.033wR(F2) = 0.101S = 0.832590 reflections177 parametersH atoms treated by a mixture of independent and constrained refinementmax = 0.74 e Å−3Δρmin = −0.37 e Å−3ΔρX-AREA  I, global. DOI: 10.1107/S1600536812010525/ds2179Isup2.hklStructure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:


E. coli isolates from Norway(n = 37), Sweden (n = 24), UK(n = 20) and Spain (n = 19), producingCTX-M-type - (n = 84), or SHV-12(n = 4) extended spectrum β-lactamases, or the plasmidmediated AmpC, CMY-2 (n = 12), were typed using multi-locussequence typing (MLST) and multi-locus variable number of tandem repeat analysis(MLVA). Isolates clustered into 33 Sequence Types (STs) and 14 Sequence TypeComplexes (STCs), and 58 MLVA-Types (MTs) and 25 different MLVA-Type Complexes(MTCs). A strong agreement between the MLST profile and MLVA typing results wasobserved, in which all ST131-isolates (n = 39) and most ofthe STC-648 (n = 10), STC-38 (n = 9),STC-10 (n = 9), STC-405 (n = 8) andSTC-23 (n = 6) isolates were clustered distinctly intoMTC-29, -36, -20, -14, -10 and -39, respectively. MLVA is a rapid and accuratetool for genotyping isolates of globally disseminated virulent multidrugresistant E. coli lineages, including ST131.One hundred  Antibiotic resistance is a globally interrelated public health problem, the threat ofwhich is ever increasing. Dispersion of large mobile genetic elements carryingmultiple resistance determinants coupled with dissemination of successful cloneshave been chronicled in most bacterial species E. coli co-resistant totrimethoprim-sulfamethoxazole and ciprofloxacin are increasingly reported β-lactams belong to our most important and widely used class of antibiotics.Bacteria belonging to the Enterobacteriaceae family have during the last decadesevolved to enable carriage of multiple β-lactamases, including extended-spectrumβ-lactamases (ESBLs), plasmid mediated AmpC enzymes as well as carbapenemasessuch as VIM, NDM, KPC and OXA-48, which has compromised the clinical utility ofβ-lactam drugs mdh and gyrB combined with O25brfbpabBST131 was originally identified using the Achtman multi-locus sequence typing (MLST)scheme Bacillus,Yersinia, Mycobacterium,Enterococcus, Staphylococcus,Neisseria, Salmonella and E. coli O157 E. coli.Several studies have explored the use of tandem repeated DNA as mean foridentification of different bacterial strains. Multi-locus variable number of tandemrepeat analysis (MLVA) has been successfully applied for rapid and interlaboratorytyping of various bacteria such as List of abbreviations and resistance genes given in E. coli isolatesproducing ESBLs- or plasmid-mediated AmpC were tested. They had been collectedbetween year 2000–2008, from Norway (n = 37), Sweden(n = 24), UK (n = 20) and Spain(n = 19), and of these, 84 isolates were CTX-M producers, 12 were CMY-2 producers and 4 were SHV-12producers  MLST was performed according to the scheme developed by Achtmans group targetingseven housekeeping genes; E. coli was prepared from overnight cultures usingthe RT Spin Bacteria DNA minikit . MLVA was performedin a single laboratory by targeting nine tandem repeats  identified and described byLindstedt et al. Total DNA of The 100 isolates were typed to 33 different Sequence Types (STs) and 14 differentSequence Type Complexes (STCs). A total of 81 of the 100 isolates wereidentified from 6 globally dispersed ST or STCs; i) ST131(n = 39), ii) STC-648 (n = 10)spanning 3 different STs, iii) STC-38 (n = 9), spanning 2different STs, iv) STC-10 (n = 9), spanning 4 differentSTs, v) STC-405 (n = 8), spanning 2 different STs, and vi)STC-23 (n = 6), spanning 4 different STs . The remThe 100 isolates were typed to 58 different MLVA-types (MTs) and 25 differentMLVA Complexes (MTCs) of related isolates. Isolates belonging to the six majorSTs or STCs were clustered as; i) ST131 (n = 39/39) andST62 (n = 1/1) -isolates into MTC labelled 29, spanning 14MTs, ii) STC-648 (n = 8/10) into MTC labelled 36, spanning6 MTs, iii) STC-38 (n = 8/9) into MTC labelled 20, spanning6 MTs, iv) STC-10 (n = 7/9) into MTC labelled 14, spanning4 MTs v) STC-405 (n = 7/8) into MTC labelled 10, spanning 5MTs, and vi) STC-23 (n = 5/6) into MTC labelled 39,spanning 4 MTs .The remST131 isolates (n = 39) were collected from Sweden(n = 14), UK (n = 12), Norway(n = 11), and Spain (n = 2), encodingβ-lactamases CTX-M-1 (n = 11), CTX-M-3(n = 7), CTX-M-15 (n = 14), CTX-M-9(n = 3), and CMY-2 (n = 4). All 39isolates were typed into a single MTC-29, with 24 identical isolates labelledMT29, 12 SLVs, 1 DLV, 1 TLV and 1 QLV . InteresSTC-648 isolates (n = 10) were collected from Sweden(n = 4), Norway (n = 3), UK(n = 2) and Spain (n = 1), encodingβ-lactamases SHV-12 (n = 1), CTX-M-1(n = 3), CTX-M-15 (n = 2), CTX-M-9(n = 2), CTX-M-14 (n = 1), and CMY-2(n = 1). The majority of the strains(n = 8) were grouped into a single MTC-36, consisting ofsix different MTs, varying exclusively at loci CVN002 and/or CVN014.The STC-38 isolates (n = 9) were collected only from Norway(n = 7) and Sweden (n = 2), encodingβ-lactamases CTX-M-1 (n = 1), CTX-M-9(n = 2), CTX-M-14 (n = 2), CTX-M-27(n = 1) and CMY-2 (n = 3). Eight ofthese isolates were typed into MTC-20, consisting of six different MTs, varyingat loci CVN014 and/or CVN016. All CTX-M-9 and CTX-M-14 isolates from Norway weregrouped into a single genotype MT20.Isolates belonging to the STC-405 (n = 8) were collectedfrom Norway (n = 5), Spain (n = 2) andUK (n = 1), encoding β-lactamases CTX-M-3(n = 1), CTX-M-15 (n = 5), CTX-M-14(n = 1) and CMY-2 (n = 1). Seven ofthe isolates were grouped into MTC-10, consisting of five different MTs, mainlyvarying at loci CCR001 and CVN016.The STC-10 isolates (n = 9) were collected from Spain(n = 5) and Norway (n = 4), encodingβ-lactamases CTX-M-14 (n = 6), CTX-M-15(n = 2) and CMY-2 (n = 1). Seven ofthese isolates were typed into MTC-14, and the remaining two CTX-M-14 producingisolates from Spain in MTC-5. The MTC-14 was consisting of four different MTs.Six isolates were collected from Spain (n = 3), Norway(n = 2) and UK (n = 1), encodingβ-lactamases SHV-12 (n = 2), CTX-M-3(n = 1), CTX-M-15 (n = 1), CTX-M-14(n = 1), and CMY-2 (n = 1) of theSTC-23. Five of the isolates were grouped into a single MTC-39, varying at lociCVN002, CVN014 and CVN016.E. coli lineages,including ST131. Independently collected isolates from four countries during 9 yearsdisplayed high concordance between MLST types and MLVA types with an increasedresolution (E. coli of diverse serotypes E. coli isolates (n = 3417),allele 10 at locus CVN004 is only present in 4% of the isolates(n = 139) including the strains from this study . Thus, the particular alleles 8 and 10, at locus CVN004 seemto be important indicators for the presence of the major STCs and ST131.In this study we document MLVA as a tool for rapid and accurate typing ofinternationally-dispersed virulent ESBL producing solution .InteresE.coli producing CTX-M-15 are usually co-resistant to quinolones andaminoglycosides E. coli in addition to MLVA include DNA microarrayE. coli serotypes, they hold the potential foralso typing MDR lineages of E. coli. Indeed, as we havesuccessfully shown with the existing MLVA scheme; a potential for rapid, accurateand high resolution genotyping of ST131 and other internationally-dispersed,virulent multidrug resistant E. coli lineages.Currently, phenotypic detection of the ST131 clone is not possible andmolecular-based techniques are required. Accurate detection is an important firststep towards monitoring and controlling ST131 dissemination. ST131 E. coli in the future We have identified MLVA type complexes that correspond to the major MLST-definedcomplexes. Assuming extracted DNA and availability of all required equipment, theexpected turnover time for MLVA of a single isolate is close to five hours, whereasit may require a couple of days for its complete MLST analysis. As such MLVA is agood alternative to MLST for epidemiological surveillance of virulent multidrugresistant Table S1MLST and MLVA data on all 100 isolates included in thisstudy.(XLSX)Click here for additional data file.


V atom in the polymeric title compound, [NaV(C15H13N3O3)O2(H2O)2]n, is O,N,O′-chelated by the Schiff base dianion and is five-coordinated in a trigonal–bipramidal coordination geometry. The oxide O atoms occupy the equatorial sites and one oxide O atom is connected to the NaI atom. The ligand simultaneously O,O′-chelates to the water-coordinated NaI atom; its coordination number is seven owing to an Na⋯Npyridyl bond. The two independent formula units, which are disposed about a false center of inversion, are connected into a layer. Adjacent layers are consolidated into a three-dimensional network by O—H⋯O and O—H⋯N hydrogen bonds.The V For rel al. 2000; Plass e al. 2000; Plass & al. 2000.       15H13N3O3)O2(H2O)2] = 0.038                           wR(F                           2) = 0.120                           S = 1.04                           8084 reflections489 parametersH-atom parameters constrainedmax = 0.56 e Å−3                        Δρmin = −0.74 e Å−3                        Δρ                                 APEX2  used to solve structure: SHELXS97 (Sheldrick, 2008SHELXL97 (Sheldrick, 2008X-SEED (Barbour, 2001publCIF (Westrip, 2010Data collection: 10.1107/S1600536811019106/im2289sup1.cif            Crystal structure: contains datablocks global, I. DOI: 10.1107/S1600536811019106/im2289Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


T and AB-11, were isolated from Aiding and Manasi salt lakes in Xinjiang of China, respectively. The strains were found to be able to grow at NaCl concentrations of 0–21 % (w/v), with optimum growth occurring at 6–8 % (w/v) NaCl. The optimal temperature and pH for growth were determined to be 33–37 °C and pH 7.0–7.5. Cells of the strains are motile by means of polar flagella. Both strains can produce ellipsoidal spores. The major cellular fatty acids were identified as anteiso-C15:0, iso-C15:0, iso-C14:0, anteiso-C17:0 and iso-C16:0. The diamino acid in the peptidoglycan and the major quinone system were determined to be meso-diaminopimelic acid (meso-DAP) and MK-7, respectively. The DNA G+C contents of stains AD7-25T and AB-11 were 39.8 and 40.0 mol%, respectively. Phylogenetic analysis based on 16S rRNA gene sequences revealed that these two novel strains are closely related to the genus Oceanobacillus showing 90–99.5 % similarity with respect to type strains. These two novel strains were most closely related to Oceanobacillus oncorhynchi subsp. incaldanensis DSM 16557T (99.1 and 99.5 %), followed by O. oncorhynchi subsp. oncorhynchi JCM 12661T (99.1 and 99.4 %), Oceanobacillus neutriphilus CGMCC 1.7693T (97.0 and 97.5 %), Oceanobacillus sojae JCM 15792T (97.6 and 98.0 %) and Oceanobacillus locisalsi KCTC 13253T (96.5 and 96.9 %). The DNA–DNA hybridization data indicated that DNA relatedness between strains AD7-25T and AB-11 was 91.0 %, and the genomic homology of representative strain AD7-25T with O. oncorhynchi subsp. incaldanensis DSM 16557T, O. oncorhynchi subsp. oncorhynchi JCM 12661T, O. neutriphilus CGMCC 1.7693T, O. sojae JCM 15792T and O. locisalsi KCTC 13253T were 41, 39, 20, 23 and 17 %, respectively. On the basis of phenotypic and phylogenetic distinctiveness, strains AD7-25T and AB-11 should be assigned to the genus Oceanobacillus as a new species, for which the name Oceanobacillus aidingensis sp. nov. was proposed. The type strain is AD7-25T (=CGMCC 1.9106 T = NBRC 105904T).Two Gram-positive, rod-shaped moderately halophilic bacterial strains, designated AD7-25The online version of this article (doi:10.1007/s10482-014-0128-1) contains supplementary material, which is available to authorized users. Bacillaceae. Many new genera of this kind of bacteria have been reported along with more research ongoing. The genus Oceanobacillus was first described by Lu et al.  and Manasi salt lake  of Xinjiang in China, respectively. For isolation, the samples were suspended in sterilized water supplied with 2 % (w/v) NaCl, serially diluted, spread on improved Gibbson medium . Transmission electron microscopy (JEM 1230) was used for observing bacterial flagellation. Gram reaction was determined according to the methodology used by Gregersen  were compared with sequences from the GenBank, EMBL, DDBJ and PDB databases using the BLAST program via the National Center for Biotechnology Information (NCBI). Pairwise sequence similarities were calculated using the BioEdit software package  and maximum-parsimony methods  between the two novel strains was 99.1 %. The 16S rRNA gene sequence comparison indicated that the two strains showed 90–99.5 % similarity with respect to the sequences of type strains of validly named Oceanobacillus species. Strains AD7-25T and AB-11 were found to be most closely related to O. oncorhynchi subsp. incaldanensis DSM 16557T (99.1 and 99.5 % sequence similarity), followed by O. oncorhynchi subsp. oncorhynchi JCM 12661T (99.1 and 99.4 %), O. neutriphilus CGMCC 1.7693T (97.0 and 97.5 %), O. sojae JCM 15792T (97.6 and 98.0 %) and O. locisalsi KCTC 13253T (96.5 and 96.9 %). The sequence similarities with the other Oceanobacillus species were 90–95.2 %. Phylogenetic analysis revealed that strains AD7-25T and AB-11 were on the same phylogenetic branch within the genus Oceanobacillus . On the basis of sequence analysis, it is evident that the novel strains should be assigned to the genus Oceanobacillus.Two Gram-positive moderately halophilic bacterial strains, designated AD7-25T and AB-11 was 91 ± 5 %. The representative strain AD7-25T and AB-11 shared 41 ± 9 and 42 ± 10 % similarity with O. oncorhynchi subsp. incaldanensis DSM 16557T, 39 ± 3 and 39 ± 7 % with O. oncorhynchi subsp. oncorhynchi JCM 12661T, 20 ± 4 and 21 ± 4 % with O. neutriphilus CGMCC 1.7693T, 23 ± 6 and 25 ± 7 % with O. sojae JCM 15792T and 17 ± 7 and 22 ± 9 % with O. locisalsi KCTC 13253T at the genomic level. The DNA G+C contents of strains AD7-25T and AB-11 were determined to be 39.8 and 40.0 mol%, respectively, which are in the range for the genus Oceanobacillus.The DNA–DNA hybridization data revealed that DNA relatedness between strains AD7-25T and AB-11 are Gram-positive and rod-shaped with a size range of 0.5–0.6 × 1.2–2.0 μm. Ellipsoidal endospores were found be formed and the cells to be motile by means of polar flagella . Growth was determined to occur at 4–45 °C, pH 6–10 and 0–21 % (w/v) NaCl. Catalase, urease and oxidase tests were found to be positive, but β-galactosidase was negative. Tween 80 and aesculin were found to be hydrolyzed. H2S and indole were not produced. Nitrate was found to be reduced to nitrite. The Voges–Proskauer test was negative. Acids were found to be produced from d-maltose, d-fructose, d-glucose, d-mannitol, d-galactose, d-lyxose, starch, melibiose and sucrose. The morphological, physiological and biochemical characteristics in detail are given in the species description below. The phenotypic differences between the two novel strains and Oceanobacillus species are shown in Table Phenotypic analysis showed that cells of the strains AD7-252) (3.0 %), respectively. The diamino acid in the peptidoglycan was identified as meso-diaminopimelic acid (meso-DAP). The polar lipids of strain AD7-25T were found to consist of diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol . The major cellular fatty acids of strains AD7-25T and AB-11 were identified as anteiso-C15:0 , iso-C15:0 , iso-C14:0 , iso-C16:0  and anteiso-C17:0 , respectively. Detailed information on the cellular fatty acid composition, the difference between strains AD7-25T, AB-11 and the reference type strains are provided in Table The menaquinones were determined to be MK-7 (93.3 %), MK-6 (3.7 %) and MK-6H (3.0 %),T and AB-11 belong to the genus Oceanobacillus. However, the DNA–DNA hybridization, DNA base composition and phylogenetic analysis clearly revealed that the two strains are of the same species but are different from the members of this genus. Furthermore, the two strains can be differentiated from the recognized species according to some phenotypic characteristics, such as enzyme activities, growth conditions, acid production from sugars, hydrolysis reactions and nitrate reduction .Phylogenetic and chemotaxonomic analyses suggested that strains AD7-25Oceanobacillus aidingensis .2S and indole can’t be produced. Methyl red and Voges–Proskauer reaction (acetoin) are negative. According to the API 50CHB system, acids are produced from d-maltose, d-fructose, d-glucose, d-mannitol, d-galactose, d-lyxose, starch, melibiose and sucrose but not from N-acetylglucosamine, raffinose, arbutine, gentiobiose, cellobiose, d-trehalose, amygdalin, d-mannose, glycerol, l-rhamnose, salicin, d-tagatose, erythritol, adonitol, methyl beta-d-xyloside, l-sorbose, dulcitol, methyl alpha-d-mannoside, methyl alpha-d-glucoside, melezitose, glycogen, xylitol, d-turanose, dl-fucose, 2-ketogluconate, 5-ketogluconate, dl-arabinose, d-ribose, dl-xylose, inositol, d-sorbitol, d-adonito, dl-arabitol or inulin. Acid production from lactose and gluconate is variable (the type strain is negative). Susceptible to (μg/ml): rifampicin , erythromycin (50), kanamycin (100), chloramphenicol (20) and gentamicin (40). But not susceptible to streptomycin , ampicillin , gentamicin (10), chloramphenicol (10), kanamycin (50), erythromycin (30), tetracycline , spectinomycin  and nalidixic acid . The diamino acid in murein is meso-DAP and the major menaquinone is MK-7, with minor amounts of MK-6 and MK-6(H2). Polar lipids of the type strain consist of diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. The major cellular fatty acids  are anteiso-C15:0, iso-C15:0, iso-C14:0, iso-C16:0, anteiso-C17:0, C17:0, iso-C17:0 and C16:0, respectively. The DNA G+C content of the type strain is 39.8 mol%.Cells are rod-shaped with a size range of 0.5–0.6 × 1.2–2.0 μm, Gram-positive, occurring singly or in pairs and motile by means of polar flagella. Ellipsoidal endospores are located at a central or subterminal position. Colonies are circular, smooth, entire, cream coloured and 1–3 mm in diameter after incubating 3 days. Growth occurs at 0–21 % (w/v) total salts, and the optimum is 6–8 % (w/v). The optimal temperature range for growth is 33–37 °C, and growth occurs at 4–45 °C. The optimal pH value for growth is 7.0–7.5 and pH range is 6–10. Catalase, urease and oxidase are produced but β-galactosidase, arginine dihydrolase, lysine decarboxylase, tryptophan deaminase and ornithine decarboxylase are absent. Tween 80 and aesculin are hydrolyzed but tyrosine, gelatin, xanthine, hypoxanthine, starch and casein are not hydrolyzed. Nitrate can be reduced to nitrite. Citrate cannot be utilized. HT (= CGMCC 1.9106T = NBRC 105904T), was isolated from the Aiding Salt Lake in Xinjiang, China. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain AD7-25T is FJ428529.The type strain, AD7-25Supplementary material 1 (DOCX 121 kb)Supplementary material 2 (DOCX 644 kb)Supplementary material 3 (PPTX 88 kb)Below is the link to the electronic supplementary material.


AbstractEriophyidae, Theaphyesrapaneaegen. n. and sp. n. which is found on the type host Rapaneaneriifolia (Sieb. et Zucc.) Mez (Myrsinaceae) and Paracaphyllisatheaceasp. n., are described and illustrated. They are vagrants on the tea plant Camelliasinensis (L.) Kuntze and no apparent symptoms were detected. A key to the eriophyoid mites including thirteen species associated with tea plants all over the world is provided.A new genus and two new species of mites in the family  Camellia) are perennial evergreen plants in the family Theaceae. They constitute a highly diverse taxon, presently composed of approximately 625 species  have a worldwide distribution. Eriophyoidea is a large mite superfamily with more than 4,000 described species (Eriophyoid mites ( species . They ar species . Nearly  species .Rapaneaneriifolia (Sieb. et Zucc.) Mez (Myrsinaceae).Between 2009 and 2013, field investigations were conducted in southern part of China in order to look for eriophyoids on tea plants, leading to the discovery of a new genus and two new species. It is worth noting that the new genus and the new species were also found on the type host Acaphyllatheae (Watt), a major pest all over the world, inducing leaf russeting, and Calacaruscarinatus (Green), which causes bronzing and white cast skin streaks on both leaf surfaces. This paper describes the new genus and species, summarizes the main information on the eriophyoid mites found until now on tea plants, and provides a key to these mite species.Along with the current new records, thirteen species have been reported from tea plants worldwide Table . They inPlants were examined in field by the aid of hand-lens (30×) and eriophyoids, together with parts of their host plants, were placed in vials and stored in 75% ethanol. Each vial was marked with the collection data and herbaria were prepared for future identification of plant samples.Nanjing Agricultural University (NJAU), Jiangsu Province, China.In the laboratory, the liquid contents were poured into a Petri dish from the vials, mite specimens were picked up using a fine pin and slide-mounted using Keifer’s Booster and modified Berlese medium . SpecimePageBreakhttp://www.theplantlist.org/). Data on eriophyoid mites were extracted from the catalogue by Eriophyoidea ; a further record was added based on the searching made on the most common abstract indexes. When available, the following information was summarized for each listed eriophyoid species based on literature: a) previous genus name assignment and possible synonymies; b) information about the host plant species; c) documenting relationships between eriophyoid species and host plants based on literature; d) distribution within geographic realms according to Host plant names and their synonymies are in accordance with The Plant List  and by a wide furrow on the dorsal opisthosoma . The tarsal solenidion is located below the empodium similarly to Catachelamachaerii Keifer, 1969, Dechelaepelis Keifer, 1965 and Dechelaphoebe Wang, Han, Xue & Hong, 2014.The new genus is assigned to the Family Taxon classificationAnimaliaProstigmataEriophyidaehttp://zoobank.org/B7A8BE17-72C9-4524-BFB9-2E2C7CFBCE86Gnathosoma 19 (19–20), projecting obliquely downwards, pedipalp coxal setae (ep) 2 (2–3), dorsal pedipalp genual setae (d) 8 (7–8), cheliceral stylets 20 (19–20). Prodorsal shield 58 (57–58), 80 (80–82) wide, admedian lines complete curving mesally at their posterior ends; frontal shield lobe absent. Scapular tubercles ahead of rear shield margin, 25 (24–25) apart, scapular setae (sc) 2 (2–3), projecting upward. Coxigenital region with 5 (5–6) semiannuli between coxae and genitalia. Coxal plates with a few short lines, anterolateral setae on coxisternum I (1b) 7 (6–7), 15 (15–16) apart, proximal setae on coxisternum I (1a) 11 (10–11), 14 (14–15) apart, proximal setae on coxisternum II (2a) 12 (12–15), 33 (32–33) apart. Prosternal apodeme absent. Leg I 27 (26–27), femur 11 (10–11), basiventral femoral setae (bv) 5 (5–6); genu 3 (2–3), antaxial genual setae (l’’) 21 (20–21); tibia 3 (2–3), paraxial tibial setae (l’) 4 (4–5), located at 2/3 from the dorsal base; tarsus 7 (6–7), paraxial, fastigial, tarsal setae (ft’) 15 (15–16), antaxial, fastigial, tarsal setae (ft’’) 18 (18–20), paraxial, unguinal, tarsal setae (u’) 4 (4–5); tarsal empodium (em) 5 (4–5), simple, 5-rayed, tarsal solenidion (ω) 5 (5–6), slightly knobbed, located below empodium. Leg II 20 (20–21), femur 10 (10–11), basiventral femoral setae (bv) 6 (6–7); genu 3 (2–3), antaxial genual setae (l’’) absent; tibia 2 (2–3); tarsus 7 (6–7), paraxial, fastigial, tarsal setae (ft’) 3 (3–4), antaxial, fastigial, tarsal setae (ft’’) 18 (17–18), paraxial, unguinal, tarsal setae (u’) 4 (3–4); tarsal empodium (em) 5 (4–5), simple, 5-rayed, tarsal solenidion (ω) 7 (7–8), little knobbed, located below empodium. Opisthosoma dorsally with 26 (26–29) semiannuli, smooth, with a wide furrow, ventrally with 54 (52–54) semiannuli, with linear microtubercles. Setae c2 19 (18–19) on ventral semiannulus 6 (6–7), 85 (80–85) apart; setae d 35 (34–35) on ventral semiannulus 21 (20–21), 32 (30–32) apart; setae e absent, setae f 15 (15–16) on 5th–6th ventral semiannulus from rear, 20 (20–21) apart. Setae h1 absent, h2 50 (50–60). Genital coverflap 20 (18–20), 38 (38–39) wide, coverflap with 22–23 longitudinal ridges in one rank, some ridges not complete, setae 3a 6 (5–6), 24 (18–24) apart.FEMALE: . Body fusiform, white, 152 (152–153), 80 (80–85) wide. Gnathosoma 19 (18–19), projecting obliquely downwards, pedipalp coxal setae (ep) 2 (1–2), dorsal pedipalp genual setae (d) 6 (5–6), cheliceral stylets 17 (16–17). Prodorsal shield 53 (53–54), 65 (65–70) wide, admedian lines complete curving mesally at their posterior ends; frontal shield lobe absent. Scapular tubercles ahead of rear shield margin, 22 (22–24) apart, scapular setae (sc) 3 (2–3), projecting upward. Coxigenital region with 5 (5–6) semiannuli between coxae and genitalia. Coxal plates with a few short lines, anterolateral setae on coxisternum I (1b) 5 (5–6), 10 (10–12) apart, proximal setae on coxisternum I (1a) 7 (7–8), 14 (12–14) apart, proximal setae on coxisternum II (2a) 10 (10–11), 27 (27–28) apart. Prosternal apodeme absent. Leg I 20 (20–21), femur 12 (11–12), basiventral femoral setae (bv) 5 (5–6); genu 3 (2–3), antaxial genual setae (l’’) 17 (17–18); tibia 3 (2–3), paraxial tibial setae (l’) 4 (4–5), located at 2/3 from the dorsal base; tarsus 5 (4–5), paraxial, fastigial, tarsal setae (ft’) 13 (13–14), antaxial, fastigial, tarsal setae (ft’’) 17 (15–17), paraxial, unguinal, tarsal setae (u’) 4 (4–5); tarsal empodium (em) 5 (4–5), simple, 5-rayed, tarsal solenidion (ω) 5 (5–6), slightly knobbed, located below empodium. Leg II 18 (17–18), femur 9 (8–9), basiventral femoral setae (bv) 4 (4–6); genu 2 (2–3), antaxial genual setae (l’’) absent; tibia 3 (3–4); tarsus 5 (4–5), paraxial, fastigial, tarsal setae (ft’) 4 (4–5), antaxial, fastigial, tarsal setae (ft’’) 15 (14–15), paraxial, unguinal, tarsal setae (u’) 3 (3–4); tarsal empodium (em) 5 (4–5), simple, 5-rayed, tarsal solenidion (ω) 7 (7–8), slightly knobbed, located below empodium. Opisthosoma dorsally with 28 (26–28) semiannuli, smooth, with a wide furrow; ventrally with 51 (48–51) semiannuli, with linear microtubercles. Setae c2 12 (12–13) on ventral semiannulus 6 (5–6), 65 (64–69) apart; setae d 20 (18–22) on ventral semiannulus 16 (15–17), 27 (25–27) apart; setae e absent, setae f 14 (14–15) on 5th–6th ventral semiannulus from rear, 20 (18–20) apart. Setae h1 absent, h2 52 (51–52). Genitalia 20 (20–21) wide, setae 3a 5 (5–6), 15 (13–15) apart.MALE: . 142 (142–145), 69 (69–70) wide; white. Rapaneaneriifolia (Sieb. et Zucc.) Mez (Myrsinaceae)Camelliasinensis (L.) KuntzeVagrant. No damage to the host plant was observed.24°53'50"N, 113°01'18"E), elevation 1,408 m, 31 July 2012, coll. Qiong Wang, Hao-Sen Li and Jing-Feng Guo.Nanling National Forest Park, Guangdong Province , same collection data of the holotype.Holotype, single female on a microscope slide , from Camelliasinensis.10 females mounted on separate microscope slides (slide numbers NJAUAcariEriYN304B.1–NJAUAcariEriYN304B.10), from PageBreakrapaneae is the genitive case derived from the genus name of the type host plant, Rapanea.The specific designation MyrsinaceaeRapaneaneriifolia and also on Camelliasinensis. Slight morphological differences were observed between the populations found on the two host species: the population on Camelliasinensis is longer, thinner and with more ventral annuli (62–66) than the population on Rapaneaneriifolia which is provided with fewer ventral annuli (52–54). The population found on Camelliasinensis occurred with other eriophyoid species and no males were collected. Current data do not allow understanding if the mite species colonizes regularly and successfully Camelliasinensis and it needs further biological studies.This species is found on the Taxon classificationAnimaliaProstigmataEriophyidaehttp://zoobank.org/AC45840F-0982-40F3-8897-CDD76B3FD531Gnathosoma 35 (32–35), projecting obliquely downwards, pedipalp coxal setae (ep) 3 (2–3), dorsal pedipalp genual setae (d) 9 (8–9), cheliceral stylets 40 (38–40). Prodorsal shield 50 (50–52), 75 (70–75) wide, median line absent, admedian and submedian lines sinuous; front shield lobe present 12 (11–12). Scapular tubercles ahead of rear shield margin, 37 (35–37) apart, scapular setae (sc) 4 (3–4), projecting anteriorly. Coxigenital region with 8 (8–9) semiannuli between coxae and genitalia, smooth. Coxal plates smooth, anterolateral setae on coxisternum I (1b) 10 (10–11), 14–15 apart, anterolateral setae on coxisternum I (1a) 15 (13–15), 12 (11–12) apart, proximal setae on coxisternum II (2a) 40 (35–40), 30 (30–31) apart. Prosternal apodeme absent. Leg I 37 (37–40), femur 15 (14–15), basiventral femoral setae (bv) 15 (15–16); genu 5 (4–5), antaxial genual setae (l’’) 43 (42–43); tibia 8 (7–8), paraxial tibial setae (l’) 1, located at center; tarsus 8 (7–8), paraxial, fastigial, tarsal setae (ft’) 30 (29–30), antaxial, fastigial, tarsal setae (ft’’) 33 (33–34), paraxial, unguinal, tarsal setae (u’) 6 (5–6); tarsal empodium (em) 5 (5–6), divided, 5-rayed, tarsal solenidion (ω) 12 (11–12), slightly knobbed. Leg II 34 (32–34), femur 12 (12–14), basiventral femoral setae (bv) 11 (10–11); genu 5 (4–5), antaxial genual setae (l’’) absent; tibia 6 (5–6); tarsus 8 (7–8), paraxial, fastigial, tarsal setae (ft’) 9 (9–10), antaxial, fastigial, tarsal setae (ft’’) 26 (26–27), paraxial, unguinal, tarsal setae (u’) 5 (4–5); tarsal empodium (em) 5 (5–6), divided, 5-rayed, tarsal solenidion (ω) 11 (11–12), slightly knobbed. Opisthosoma dorsally with 38 (37–38) semiannuli, smooth, with three ridges, ventrally with 62 (62–69) semiannuli, with rounded microtubercles. Setae c2 35 (33–35) on ventral semiannulus 15 (12–15), 60 (57–60) apart; setae d 70 (67–70) on ventral semiannulus 26 (25–27), 45 (43–45) apart; setae e 50 (50–52) on ventral semiannulus 41 (41–44), 20 (19–20) apart, setae f 23 (23–25) on 6th–7th ventral semiannulus from rear, 15 (15–16) apart. Setae h1 absent, h2 55 (52–55). Genital coverflap 15 (15–16), 30 (29–30) wide, coverflap with 23 (18–23) longitudinal ridges and dense short lines at base, setae 3a 14 (13–14), 20 (20–21) apart.FEMALE: . Body fusiform, 200 (190–200), 80 (75–80) wide; white. PageBreakPageBreakMALE: Unknown.Camelliasinensis (L.) KuntzeVagrant. No damage to the host plant was observed.26°33'05"N, 98°55'08"E), elevation 2,122 m, 26 June 2013, coll. Xiao Han, Qiong Wang and Jing-Feng Guo.Pihe Village, Fugong County, Nujiang Lisu autonomous prefecture, Yunnan Province .Holotype, single female on a microscope slide . theacea is derived from the family name of the host plant; feminine in gender.The specific designation Paracaphyllisaadinandrae Kuang & Luo, 2005, but can be differentiated from the latter by the design of prodorsal shield which is provided with admedian and submedian lines , smooth coxal plates  and coverflap with 23 (18–23) longitudinal ridges and dense short lines at its base (coverflap is smooth in Paracaphyllisaadinandrae).This new species is similar to The new species is surrounded with white hairs around the body.Taxon classificationAnimaliaProstigmataEriophyidaeAcaphyllaindiae ; Acaphyllisaindiae ; Camelliasinensis (L.) Kuntze.Vagrant, causing leaf rusting.Indomalayan region.Taxon classificationAnimaliaProstigmataEriophyidaeKeifer, 1978Acaphyllisaparindiae ; Camelliasinensis (L.) Kuntze.Vagrant, causing leaf rusting.Indomalayan region.PageBreakTaxon classificationAnimaliaProstigmataEriophyidaePhytoptustheae Watt, 1898Eriophyestheae ; AcaphyllatheaeAcaphyllasteinwedeni Keifer, 1943). ; Acaphyllasteinwedeni ; Acaphyllasteinwedeni ; Acaphyllasteinwedeni ; Acaphyllasteinwedeni ; Acaphyllasteinwedeni ; Camelliajaponica L., Camelliaoleifera Abel, Camelliareticulata Lindl., Camelliasasanqua Thunb., Camelliasinensis (L.) Kuntze, Camelliasinensis var. assamica (J.W. Mast.) Kitam.Calacaruscarinatus (Green) ). Acaphyllasteinwedeni does not leave as much debris on the leaves as Calacaruscarinatus, but it may actually be more important as a rust mite. Both mite species overwinter on the leaves and show no deuterogyny.Vagrant. This species occurs on the undersurface of the leaves and is often associated with Australian, Nearctic, Indomalayan and Palaearctic regions.Phytoptustheae was reported infesting Camelliasinensis by Phytoptustheae Watt & Mann, 1903, based on the more available publication. Improperly, Phytoptustheae as a nude name. Acaphyllasteinwedeni from specimens provided by Dr. Das and which were referred to have been the object of the name Phytoptustheae. Acaphyllasteinwedeni as junior synonym of Acaphyllatheae, making this last one as the genotype of the genus Acaphylla. Finally, Acaphyllatheae as possible synonym (they marked the synonymy with a question mark) of Acaphyllasteinwedeni without any further indication about it and the use of the names.Taxon classificationAnimaliaProstigmataEriophyidaeKadono, 1992Acaphyllatheavagrans ; Camelliasinensis (L.) Kuntze.Vagrant, causing rust.Indomalayan region.Taxon classificationAnimaliaProstigmataEriophyidaeTyphlodromuscarinatus Green, 1890: 35.PageBreakEriophyescarinatus ; Epitrimerusadornatus ; Calacaruscarinatus ; Camelliacaudata Wallich, Camelliasinensis (L.) Kuntze, Camelliakissi Wallich, Camelliajaponica L., Camelliasasanqua Thunb., Capsicumannuum L. (Solanaceae), Viburnumopulus L. (Adoxaceae).Vagrant, causing bronzing and leaving white cast skin streaks. Wax is produced on the five ridges and prodorsal shield.Africotropical, Australian, Indomalayan, Nearctic, Palaearctic regions.Acaphyllasteinwedeni. They both overwinter on the leaves and show no deuterogyny.This free-living species leaves much debris on the host leaves and occurs on the leaves associated with Taxon classificationAnimaliaProstigmataEriophyidaeAceriacamelliae ; Cosetacuscamelliae ; Cosetacuscamelliae ; Camelliajaponica L., Camellia sp.Vagrant. The mite lives under leaf and flower buds, probably causing premature flower drop.Australian, Nearctic, Neotropic, Palaearctic regions.Taxon classificationAnimaliaProstigmataDiptilomiopidaeWang & Chen, 2013Diptilomiopuscamelliae ; Camelliacaudata Wallich.Vagrant. No damage to the host was observed.On the border between Palaearctic and Indomalayan regions.Taxon classificationAnimaliaProstigmataEriophyidaeKuang & Huang, 1994Paracolopodacuscamelliae ; Camelliaoleifera Abel.Vagrant.On the border between Palaearctic and Indomalayan regions.PageBreakTaxon classificationAnimaliaProstigmataEriophyidaeKuang & Lin, 2002Phyllocoptacuscamelliae ; Phyllocoptacuscamelliae ; Camelliasinensis (L.) Kuntze.Vagrant.On the border between Palaearctic and Indomalayan regions.Taxon classificationAnimaliaProstigmataEriophyidaeSong, Xue & Hong, 2008Shevtchenkellacamelliae ; Camelliasinensis (L.) Kuntze.Vagrant, causing no apparent damage to the host plant.On the border between Palaearctic and Indomalayan regions.Taxon classificationAnimaliaProstigmataEriophyidaeWei, Feng & Huang, 1999Tergilatuscamelliae ; Camelliasinensis (L.) Kuntze.Not stated.On the border between Palaearctic and Indomalayan regions.


We therefore examined to which extent T cell (co)stimulation and/or stereotactic body radiation therapy (SBRT) could further enhance the therapeutic efficacy of T cell checkpoint blockade in a genetically engineered mouse melanoma model that is driven by PTEN-deficiency, and BRAFV600 mutation, as in human, but lacks the sporadic UV-induced mutations. Tumor-bearing mice were treated with different combinations of immunomodulatory antibodies  or interleukin-2 (IL-2) alone or in combination with SBRT. None of our immunotherapeutic approaches  had any anti-tumor efficacy, while SBRT alone delayed melanoma outgrowth. However, α-CD137 combined with α-PD-1 antibodies significantly enhanced the anti-tumor effect of SBRT, while the anti-tumor effect of SBRT was not enhanced by interleukin-2, or the combination of α-CTLA-4 and α-PD-1. We conclude that α-CD137 and α-PD-1 antibodies were most effective in enhancing SBRT-induced tumor growth delay in this mouse melanoma model, outperforming the ability of IL-2, or the combination of α-CTLA-4 and α-PD-1 to synergize with SBRT. Given the high mutational load and increased immunogenicity of human melanoma with the same genotype, our findings encourage testing α-CD137 and α-PD-1 alone or in combination with SBRT clinically, particularly in patients refractory to α-CTLA-4 and/or α-PD-1 therapy. T cell checkpoint blockade with antibodies targeting programmed cell death (ligand)-1 (PD-1/PD-L1) and/or cytotoxic T lymphocyte-antigen 4 (CTLA-4) has improved therapy outcome in melanoma patients. However, a considerable proportion of patients does not benefit The online version of this article (doi:10.1007/s00262-016-1843-4) contains supplementary material, which is available to authorized users. Cancer immunotherapy, aimed at stimulating tumor-reactive T cells to eliminate (disseminated) tumors, is a promising new treatment approach in various cancers, including melanoma, a cancer with a high (UV-induced) mutational load and potential immunogenicity . InterleA possible reason for failure to respond to checkpoint inhibitors is the insufficient presence, recruitment and/or functionality of T cells into the tumor microenvironment . TherefoOne way to enhance tumor-specific T cell responses is to use agonistic antibodies that engage T cell costimulatory receptors, including cluster of differentiation proteins (CD)137 (4-1BB), CD27 and CD134 (OX40). These antibodies have been shown to improve anti-tumor T cell responses in preclinical and early clinical studies (reviewed in ) and can+ T cells . Severas  could enhance the anti-tumor effect of SBRT in an inducible mouse model of human BRAFV600-mutant and PTEN-deficient melanoma , 26. ThiTyr::CreERT2;PtenloxP/loxP;BrafCA/+ mice as previously described [Tumors were induced on the skin of C57Bl/6J escribed , 26, 28.Rat α-mouse CD137 mAb  , derived2. Radiotherapy of melanomas was conducted as described using the XRAD225-Cx system  commenced when tumors reached ~20 mm5 IU) twice daily for three consecutive days starting 3 days following radiotherapy. This dosing schedule was chosen to mimic the dosing schedule used in clinical trials as closely as possible [2. A tumor size of 100 mm2 was set as designated end point.Immunomodulatory α-PD-1, α-CD137, α-CTLA-4 or Control 2A3 mAbs diluted in PBS were administered at 100 μg/mouse intraperitoneally twice weekly for 2 weeks with the first dose delivered immediately after radiotherapy. IL-2 (in PBS) was administered as a high dose  tumor sizes taken of all analyzable mice in each group. In case the tumor reached >100 mm−) and immune (CD45+) cells. NK cells, CD4 and CD8 T cells were identified using α-TCRβ-PE-Cy5 (H57-597), α-CD8-PerCpCy5.5 , α-CD4-FITC , α-NK1.1-allophycocyanin (APC)-Cy7 . On each of these immune cell populations, CD25 (PC61), CTLA-4 (UC10-4F10-11) and PD-1  were detected using indicated PE-conjugated antibodies and CD137 was detected using a biotinylated antibody  followed by APC-conjugated streptavidin. This staining strategy allowed us to examine co-expression of CD137 and any of CD25, PD-1 and CTLA-4 in any of the CD4 T cell, CD8 T cell and NK cell subsets. Biotinylated or PE-conjugated isotype Controls were included for stainings to CD137, PD-1, CTLA-4 and CD25. The frequency of positive cells was determined by subtracting the % positive fraction in isotype Control staining from the % positive fraction in the staining specific for CD25, PD-1, CTLA-4 or CD137. This number is indicated in the graphs and was used for statistical analyses. CD137 staining was analyzed in triplicate in each sample and was averaged for statistical analysis. Numbers in text represent mean ± SEM. Samples were analyzed on a BD Fortessa. Examples of gating strategies for lymph node and tumor are presented in Supplemental Figs. Mice bearing 2–3 established melanomas were subjected to 14 Gy localized radiotherapy to one of the tumors. One week later, single-cell suspensions were prepared from non-irradiated tumors, irradiated tumors or inguinal lymph nodes, stained with the fluorochrome-conjugated mAbs (from BD Pharmingen unless otherwise specified) indicated below and analyzed by flow cytometry according to the following gating strategy: Single-cell suspensions were gated on live (DAPI negative) cells. α-CD45-PE-Cy7 (104) was used to discriminate tumor . Substrate was developed with either 3-amino-9-ethylcarbazole (AEC) or diaminobenzidine (DAB) (DAKO). Primary antibodies were α-CD3 , α-CD4 (cat. 14-9766 eBioscience), α-FoxP3 (cat. 14-5773 eBioscience).For immunohistochemical analysis, tumors (three mice per group) were fixed for 24 h in ethanol (50 %), acetic acid (5 %), formalin (3.7 %), embedded in paraffin, randomly sectioned at 4 µm. Staining was performed as previously described . BrieflyCD8 staining was performed on optimal cutting temperature compound (OCT) embedded, cryopreserved tumor pieces using standard procedures. Briefly, tumor pieces were thawed to room temperature, rehydrated in PBS and blocked for avidin and biotin (Vector SP-2001). After sections were blocked in 5 % normal goat serum and 2.5 % BSA, sections were incubated for 1 h with primary α-CD8 antibody (clone 2.43). After washing, sections were incubated with biotinylated secondary antibodies, followed by incubation with HRP-conjugated streptavidin–biotin complex and substrate was developed with DAB.Slides were counterstained with hematoxylin and slides scanned using the Aperio ScanScope (Leika) (20× objective). ImageJ software was used to quantify # positive cells  or % positive area (CD8) from 3 to 5 random fields of view (FOV) per slide.U test using GraphPad Prism (GraphPad Software) and considered significant when p < 0.05.Statistical differences between groups were analyzed with the Mann–Whitney + and CD8+ T cells and natural killer (NK) cells were examined for expression of CD25 (IL-2 receptor α-chain), CTLA-4, PD-1 and CD137. Examination of CD25 was chosen because of potent combined effects of SBRT and IL-2 in the clinic [First, we investigated whether relevant cell surface receptors were available for targeting on melanoma tumor-infiltrating lymphocytes (TILs) before and after radiotherapy. For this purpose, CD4e clinic ; CTLA-4 e clinic  and CD13e clinic , 22.In mice bearing 2–3 melanomas, one of these tumors was subjected to 14 Gy SBRT. Pilot experiments revealed that this radiotherapy dose induced tumor growth delay of irradiated tumors without inducing complete tumor regression. Therefore, this dose provided a window to read out the combined effect of immune-modulatory agents on radiotherapy-induced tumor growth delay.One week after radiotherapy, single-cell suspensions were prepared from irradiated and non-irradiated tumors of the same mice, as well as from their  lymph nodes and flow cytometric detection of receptors was performed .+ T cells (60.8 ± 2.6 %), on small populations of CD8+ T cells (6.0 ± 2.7 %) and NK cells (6.1 ± 5.6 %). Radiotherapy did not significantly alter this expression. In lymph nodes, CD25 was expressed on a subset of CD4+ T cells (17.9 ± 2.4 %) and on a small proportion of NK cells (4.8 ± 3.7 %) and was hardly found on CD8+ T cells  on the majority of CD4 cells 6. ± 5.6 %.+ T cells (7.4 ± %1.5 %), a subset of NK cells (4.2 ± 3.1 %), but not on CD8+ T cells. In irradiated tumors, CTLA-4 was occasionally detected on CD8+ T cells (2.5 ± 4.9 %). In lymph nodes, CTLA-4 was only expressed on a small percentage (1.2 ± 0.6 %) of CD4+ T cells  and on NK cells (25.6 ± 7.3 %). Radiotherapy did not significantly alter PD-1 expression. Expression of PD-1 in lymph nodes was found to a lesser extent as compared to TILs , a small fraction of CD8+ T cells (1.5 ± 0.8 %) and a sizable fraction of NK cells (26.5 ± 2.5 %). Radiotherapy slightly increased the frequency of CD137-expressing CD4+ and CD8+ T cells, but this did not reach statistical significance. In lymph nodes, CD137 expression was detected on a fraction of NK cells (5.9 ± 1.4 %), but expression on CD4+ and CD8+ T cells was negligible . Due to the small number of T cells recovered from these tumors, the variability within the groups is relatively large, leaving us unable to draw strong conclusions. However, we again observed no significant differences between TILs in irradiated versus non-irradiated tumors.+ T cells. Their frequency was not significantly altered by radiotherapy . In addition, radiotherapy did not alter the frequency of CD4+ T cells, CD8+ T cells and NK cells . CTLA-4, CD25 and CD137 were expressed on a higher frequency of regulatory (Foxp3+) CD4 TILs cells than on ‘non-regulatory’ (Foxp3−) CD4 TILs cells, whereas PD-1 expression was similar between the two subsets. This was not significantly affected by radiotherapy .Foxp3, the hallmark protein of regulatory T cells (Tregs) was detected in 30–75 % of the intratumoral CD4The presence of CD25, CTLA-4, PD-1 and/or CD137 on T cells and NK cells indicates that IL-2 or antibodies targeting these receptors may have an immune modulatory and potentially therapeutic effect. We therefore tested the effectiveness of IL-2 or mAbs targeting CTLA-4, PD-1 and CD137 alone and in combination with SBRT.2) tumors were divided into the following treatment groups that consisted of immunotherapy alone or in combination with 14 Gray (Gy) SBRT: (1) Control Ig (Ctr), (2) IL-2, (3) α-PD-1 and α-CTLA-4, (4) α-PD-1, (5) α-CD137, (6) α-PD-1 and α-CD137, (7) SBRT, (8) SBRT + IL-2, (9) SBRT + α-PD-1 and α-CTLA-4, (10) SBRT + α-PD-1 and α-CD137, (11) SBRT + α-PD-1 and (12) SBRT + α-CD137.Mice bearing established (>20 mmp < 0.0001). IL-2 or α-PD-1/α-CTLA-4 treatment did not enhance the anti-tumor effect of SBRT (mean TDT of 40.7 (p = 0.71) and 36.8 days (p = 0.60), respectively). However, concomitant targeting of PD-1 and CD137 significantly enhanced the therapeutic effect of SBRT ). α-PD-1 or α-CD137 mAbs alone did not significantly enhance the anti-tumor effect of SBRT .Treatment in all groups was well tolerated, and adverse events such as weight loss or diarrhea were not observed. None of the immunotherapeutic approaches exhibited any single-agent activity Fig. . Tumor gAnalysis of tumor sizes at different time points after initiation of therapy  revealed an immediate effect of SBRT on tumor growth delay: At day 9, tumor sizes of all groups that received SBRT were significantly smaller compared to mock-treated mice, while immunotherapy again did not reveal any single-agent activity. At day 35, when most of tumors of non-irradiated mice had grown out, there was a statistically significant difference in tumor size between mice that received SBRT and mice that received SBRT + IL-2, SBRT + α-CTLA-4/α-CD137, SBRT + α-PD-1/α-CD137 or SBRT + α-CD137. At day 55, a time point at which most tumors treated with SBRT had started to grow out; significant differences in tumor sizes were still observed between mice treated with SBRT and mice treated with SBRT + α-PD-1/α-CD137 or SBRT + α-CD137 . Concomitant targeting of PD-1 and CD137 significantly extended survival of mice that were treated with radiotherapy only . In this analysis, α-CD137 treatment also slightly extended survival compared to mice that were treated with radiotherapy only , while α-PD-1 treatment had no such effect . In addition, despite not reaching statistical significance, the TDT of CD4+ and CD8+ T cell-depleted mice treated with radio-immunotherapy (32.3 days) was shorter compared to mock-depleted mice .To test this possibility, we depleted CD4apy Fig. a. Combin+ and CD8+ T cells, which are in part required for melanoma Control.Collectively, our data suggest that concomitant triggering of CD137 and blocking of PD-1 signalling within irradiated melanomas enhance the intratumoral presence of both CD4T cell checkpoint inhibitors like α-CTLA-4 and α-PD-1/PD-L1 mAbs have revolutionized treatment of melanoma . HoweverWe demonstrate that combined targeting of the T cell costimulatory receptor CD137 and coinhibitory receptor PD-1 enhances the therapeutic efficacy of SBRT in a mouse model of human BRAFV600-driven melanoma. While none of our immunotherapy approaches  possessed any anti-tumor efficacy themselves, only α-CD137/α-PD-1 enhanced the anti-tumor effect of SBRT. Therefore, α-CD137/α-PD-1 therapy outperformed the capacity α-CTLA-4/α-PD-1 or IL-2 treatment to synergize with SBRT in this mouse model, even though IL-2 in combination with SBRT-induced anti-tumor responses in human melanoma patients . Our dat+ and CD8+ T cells with an effector phenotype, which contributed to the therapeutic effect of this radio-immunotherapy approach.The enhanced anti-tumor effect of α-CD137/α-PD-1 mAbs when combined with radiotherapy was associated with accumulation (>60-fold) of intratumoral CD4However, depletion of CD4 and CD8 T cells did not completely abrogate the therapeutic effect. Taking into consideration that α-CTLA-4 or α-PD-1 mAbs did not enhance the therapeutic effect of radiotherapy, these findings indicate that CD137 triggering may also mobilize other effector mechanisms of cell types other than T cells and NK cells, including dendritic cells, monocytes, B cells, neutrophils and mast cells (reviewed in ). ActivaPten and gain of mutant Braf. As a result, the tumors induced in this model are probably less immunogenic than tumors arising in melanoma patients, likely explaining the absence of responses upon treatment with CTLA-4, PD-1 mAbs, IL-2 alone Below is the link to the electronic supplementary material.


The title compound diethyl 2-acetoxy-2-[3-(4-nitrophenyl)-3-oxo-1-phenylpropyl]malonate possesses a three-dimensional supramolecular structure formed through weak C—H⋯O and C—H⋯π hydrogen bonds. 24H25NO9, the dihedral angle between the planes of the two benzene-ring systems is 80.16 (6)°, while the side-chain conformation is stabilized by a methylene–carboxyl C—H⋯O hydrogen bond. Weak intermolecular C—H⋯O hydrogen bonds form inversion dimers [graph set R22(16)] which are linked into chains extending along a. Further C—H⋯O hydrogen bonding extends the structure along b through cyclic R22(10) motifs. Although no π–π aromatic ring interactions are present in the structure, C—H⋯π ring interactions across c generate an overall three-dimensional supramolecular structure.In the racemic title compound, C Despiteg) Fig. 4, resultiviz. Cambridge Structural Database -4-methyl-4-nitro-1,3-diphenylpentanone; Bakó et al., 1997et al., 2012P212121 and Pca21 space groups, respectively, and LAPKEU crystallized as a racemic mixture in the centrosymmetric P21/c space group. Comparing the dihedral angles between the planes of the two benzene rings, the steric effect of the bulky substituents on atom C2 can be seen. This value is 62.5° for the methyl derivative (DULJOK) and 68.4° for the nitrile (LAPKEU), but significantly higher for the bulky methyl–nitro derivative  or the title compound (80.2°).The structures of different derivatives of 1,2-diphenylpentan-1-one, carrying methyl or nitrile substituents on the chiral C atom, have been reported, E)-3-(4-nitrophenyl)-1-phenylprop-2-en-1-one] with diethyl 2-acetoxymalonate. The reaction was carried out in a solid/liquid two-phase system [Na2CO3/tetrahydrofuran (THF)] in the presence of a glucopyranoside-based crown ether catalyst. The compound was isolated by preparative thin-layer chromatography (TLC) (silica gel) in good yield. The structure of the compound was confirmed by 1H and 13C NMR and mass spectroscopy (m.p. 366–369 K). The details of the synthesis are presented in Rapi et al.  = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989016001432/zs2354sup1.cifCrystal structure: contains datablock(s) I, header. DOI: 10.1107/S2056989016001432/zs2354Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989016001432/zs2354Isup3.cmlSupporting information file. DOI: 1449223CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The structures of three disaccharide molecules, precursors to novel therapeutics, as determined from weakly diffracting crystals are presented. The crystal packing depends mainly on weak C—H⋯O hydrogen-bond interactions, augmented by C—H⋯π contacts in the best-defined structure. O-[6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-4-O-(9-fluorenylmethyloxycarbonyl)-α-d-glucopyranosyl]-2-O-benzoyl-3-O-benzyl-6-O-chloroacetyl-α-l-idopyranoside, C59H56ClN3O16, (I), the ido-relative of a reported gluco-disaccharide  and 4-methoxyphenyl 4-O-[6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-4-O-(9-fluorenylmethyloxycarbonyl)-α-d-glucopyranosyl]-2-O-benzoyl-3-O-benzyl-6-O-methoxyacetyl-α-l-idopyranoside, C60H59N3O17, (II). Both exhibit similar conformational disorder of pendant groups. The third compound 4-methoxyphenyl 4-O--2-O-benzoyl-3-O-benzyl-6-O-methoxyoacetyl-β-d-glucopyranoside, C52H55N3O15, (III), illustrates that a slightly larger set of weak intermolecular interactions can result in a less disordered molecular arrangement. The molecules are bound by weak C—H⋯O(ether) hydrogen bonds in (I) and (II), augmented by C—H⋯π interactions in (III). The absolute configurations were determined, although at varying levels of significance from the limited observed data.The three title compounds form part of a set of important precursor dissacharides which lead to novel therapeutics, in particular for Alzheimer’s disease. All three crystallize as poorly diffracting crystals with one independent molecule in the asymmetric unit. Two of them are isostructural: 4-methoxyphenyl 4- HS is involved in the regulation of many important biological processes  is a linear polysaccharide with a disaccharide repeating unit of et al., 2013In our recent paper -α-d-glucopyranosyl]-2-O-benzoyl-3-O-benzyl-6-O-chloroacetyl-β-d-glucopyranoside, hereafter RSTE -α-O-benzoyl-3-O-benzyl-6-Ol-idopyranoside, (I) (hereafter OZTF)-chloroacetyl-α-S), C2(R), C3(S), C4(S), C5(S), C30(S), C31(R), C32(S), C33(R), C34(R), C47(R). Conformational two-site disorder models were required for the pendant 6-O-chloroacetyl and methyl of the 6-O-acetyl groups.The asymmetric unit contains one independent molecule of the title compound Fig. 1 with the(I4-Methoxyphenyl 4-O-[6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-4-OD-glucopyranosyl]-2--(9-fluorenylmethyloxycarbonyl)-α-O-benzoyl-3-O-benzyl-6-Ol-idopyranoside, (II) (hereafter RNSB)-methoxyoacetyl-α-This molecule Fig. 2 crystall4-Methoxyphenyl 4-O--2--benzyl-2-deoxy-α-O-benzoyl-3-O-benzyl-6-Od-glucopyranoside, (III) (hereafter RSTN)-methoxyacetyl-β-Compound (III) Fig. 4 crystallThe conformational data given in Tables 1C(8), C(12) and C(20)  interaction  is provided by weak C—H⋯O(ether), C—H⋯O (carbonyl) hydrogen bonds and one C—H⋯π interaction Table 3. These in Table 4; this isb axis in which C(n)  motifs , the five C—H⋯O(ether and ketone) interactions are augmented by five C—H⋯π interactions Table 5. These iet al., 2013et al., 2004et al., 1997There are only a few reported 2-azido pyranose-based disaccharide structures in the Cambridge Structural Database  = 1.5Ueq(C), but were allowed to rotate freely about the adjacent C—C bonds. All other H atoms were placed in geometrically idealized positions and constrained to ride on their parent atoms with C—H distances of 0.95 (aromatic), 0.99 (methylene) or 1.00 (tertiary) Å with Uiso(H) = 1.2Ueq(C) or 1.5Ueq(C) (for methyl C) of their parent atom. Specific variations were:Crystal data, data collection and structure refinement details are summarized in Table 6(I) Data at resolution less than 1.12 Å was not significantly above the noise level and was excluded from the refinement. One other reflection  was OMITted as an outlier. Data analysis shows that there are many data in the resolution range 1.40–1.12 Å that are in poor agreement reflecting crystal quality.A and B) groups. Because of proximity, and poor data quality, these atoms were unable to be refined with anisotropic thermal parameters. It proved advisable to add additional restraints to retain known geometries based on published structures for these groups. So (SHELXL DFIX) C28–C29 pairs were held to 1.50 (3) Å; C28—O9 to 1.20 Å and same-distance constraints  were applied to C29–Cl1, C36–C37 and C36–O11. Thermal parameters were also linked using SIMU for ring atoms C6–C11 and atom pairs C53 and C54, O12A and O12B, C37A and C37B, and C36A and C36B. Finally, rings C6–C11 and C14–C19 were constrained to hexagonal geometry with C—C = 1.390 Å. Final A:B occupancies for the chloroacetoxy group were 0.509 (17):0.491 (17) and for the methoxycarbonyloxy, 0.44 (4):0.56 (4).There was conformational disorder in the chloroacetoxy  and the methoxycarbonyloxy  groups which was modelled as two . Atoms C13, C33, C34, C30, C361 and C36B were restrained to isotropic-like behaviour (using ISOR) and the two-model disordered atoms  were given the same anisotropic thermal parameters. Distance constraints  were applied to the C36A—O12A and C36B—O12B bonds. Final A:B occupancies for the methoxyacetyl atoms were 0.797 (16):0.203 (16).(II) Data at resolution less than 0.81 Å was not significantly above the noise level and was excluded from the refinement. Two reflections (Fo<<Fc). The molecule showed two major orientations for the benzyl group (atoms C13–C19) refined by two refining set occupancies [A:B 0.793 (6):0.207 (6)] coupled with equivalent U values (SIMU for each ring set) and with each ring restrained to a regular hexagon (C—C 1.39 Å). In a similar manner, two orientations of atoms C29, O52 and C52 were refined as two conformations: final A:B ratio 0.687 (8):0.313 (8).(III) One reflection was removed as an outlier as well as nine low angle reflections affected by the beamstop  global, OZTF, RNSB, RSTN. DOI: 10.1107/S2056989015008518/sj5456OZTFsup2.hklStructure factors: contains datablock(s) OZTF. DOI: 10.1107/S2056989015008518/sj5456RNSBsup3.hklStructure factors: contains datablock(s) RNSB. DOI: 10.1107/S2056989015008518/sj5456RSTNsup4.hklStructure factors: contains datablock(s) RSTN. DOI: Click here for additional data file.10.1107/S2056989015008518/sj5456RSTNsup5.cmlSupporting information file. DOI: 1062536, 1062535, 1062534CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In the title compound, the two indole ring systems are approximately perpendicular to one another, with a dihedral angle between their planes of 84.0 (5)°. 27H21FN2O4, the mean planes of the indole ring systems (r.m.s. deviations = 0.0263 and 0.0160 Å) are approximately perpendicular to one another, making a dihedral angle of 84.0 (5)°; the fluorobenzene ring is twisted with respect to the mean planes of the two indole ring systems at 89.5 (5) and 84.6 (3)°. In the crystal, pairs of N—H⋯O hydrogen bonds link the molecules into inversion dimers, which are further linked by N—H⋯O hydrogen bonds into supramolecular chains propagated along the b-axis direction. Weak C—H⋯π interactions are observed between neighbouring chains.In the title compound, C Yield was 90%. Single crystals of the title compound suitable for X-ray analysis were obtained by slow evaporation of a methanol solution. 1H NMR  δ 11.81 , 7.59–7.36 , 7.13 , 6.71 , 6.60 , 3.77 .Methyl indole-2-carboxylate  was dissolved in 200 ml methanol; commercially available 4-fluorobenzaldehyde  was added and the mixture was heated to reflux temperature. Concentrated HCl (3.7 ml) was added and the reaction was left for 1 h. After cooling the white product was filtered off and washed thoroughly with methanol. The reaction was monitored by TLC (CHClUiso(H) = xUeq, where x = 1.5 for methyl H atoms and 1.2 for the others.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S205698901501628X/xu5870sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S205698901501628X/xu5870Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S205698901501628X/xu5870Isup3.cmlSupporting information file. DOI: 1421585CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: