The new nanocatalytic reaction of AgNP-NaCl-H2O2 was investigated by surface plasmon resonance (SPR) absorption, resonance Rayleigh scattering (RRS), surface-enhanced Raman scattering (SERS) and scanning electron microscope (SEM) techniques. The autocatalytic oxidization of Ag on AgNP surface by H2O2 was observed firstly and the AgNP/AgCl nanoparticles were characterized. The [Ag+] from AgNP is different to the Ag+ from AgNO3 that adsorb on the AgNP surface. An autocatalytic oxidization mechanism was proposed to explain experimental phenomena. The relationship between the SPR absorption peaks and the RRS peaks of AgNPB was studied, and three characteristic RRS peaks called as out-of-plane quadrupole, out-of-plane dipole and in-plane dipole RRS peaks were observed firstly. Using AgNP as nanoprobe, a simple, sensitive and selective RRS method was developed for assay of H2O2 in the range of 2.0 × 10−8-8.0 × 10−5 mol/L.The stable yellow nanosilver (AgNP) and blue nanosilver (AgNPB) sols were prepared by the NaBH It is rare report about nanosilver catalytic reaction. In synthesis of triangular nanosilver262O particle reaction of Fehling reagent-glucose, and it has been used to determine trace melamine by RRS technique2O2 analysis have not been reported.Nanoparticles have become research hot-spot in subjects such as physics, chemistry and materials due to their unique physical and chemical properties1234678101112131415171820212223242O2 is closely related to the photochemical reactions, oxidation-reduction reactions in natural water bodies. It is one of the important factors to affect the transfer, transformation, and ecological effect of chemical substances in water, and it is also one of the main reasons in the formation of acid rain2O2 content is also very important in biochemical reactions, clinical test, food safety, and other fields2021222324252627282930312O2 content are mainly including chemiluminescence, fluorescence, spectrophotometry, etc33342+, H2O2, HCl and so on36373839402843442O2 autonanocatalytic system yet. In this paper, the reaction mechanism of AgNP-NaCl-H2O2 was studied by SPR, RRS46472O2.The content of H4 reduction of AgNO3 in the presence of citrate. The SEM shows that they are spherical, with average size of 10 ± 2 nm  and H2SO4, the system had four RRS peaks at 290 nm, 360 nm, 455 nm and 500 nm  also produced HO· that oxidize AgNP to form [Ag+] and AgNP/[AgCl] particles. With H2O2 concentration increase its peak enhanced linearly due to more particles forming.In 2.0 × 10ery weak . With add 500 nm . Fe(II) 3 to the system of 2.0 × 10−3 mol/L NaCl-0.035% sodium citrate, AgCl particles were generated and exhibited strong scattering signal at 335 nm , the main reactions of AgNP-NaCl-H2O2 system are as follows,2O2 oxidize AgNP to form Agm-2k/[AgCl]2k complex particles become easily, as the result of the formation of AgCl particle with low solubility. The total reaction is as follow,According to the generation mechanism of HOAgNPBs exhibited special optical property that have one sharp out-of-plane quadrupole SPR absorption peak at 330 nm , one out5456I) of different H2O2 concentration was recorded, and the working curve between ΔI and H2O2 concentration was drawn. The linear range of AgNP-NaCl system was 2.0 × 10−8-8.0 × 10−5 mol/L, with a regression equation of ΔI = 71.4c-1.6, a correlation coefficient of 0.9852, and a detection limit of 8 × 10−9 mol/L. A 5.0 × 10−7 mol/L, 5.0 × 10−6 mol/L and 20 × 10−6 mol/L H2O2 were determined five times, and the related standard deviations (RSD) were 4.1%, 3.7% and 3.8% respectively, this showed that the RRS method has good accuracy. The linear range of AgNP-NaCl-H2SO4-FeSO4 system was 1.0 × 10−7-2.5 × 10−5 mol/L, with a detection limit of 2 × 10−8 mol/L. In the RRS analytical system, the AgNP-NaCl is most sensitive, simple, stabile and low blank  and Mg(II), 50 times of Cu2+, Mo6+, I−, triethanolamine, Co2+, NO2−, 10 times of Mn2+, Br−, citric acid, and 2.5 mg/L HSA did not interfere with the determination. This indicated that the method has good selectivity.Under the selected conditions , the RRSow blank , and it ex and emission wavelength λem (λex-λem = Δλ = 0) and the RRS intensity. A Model TU-1901 double beams spectrophotometer  was used to record the SPR spectra and the SPR intensity. A model JSM-6380LV scanning electron microscope , a model of JEM-2100F field emission transmission electron microscope , a model DXR smart Raman spectrometer , a moldel SK8200LH ultrasonic reactor , and a model magnetic stirrer  were used.A Model F-7000 fluorescence spectrophotometer  was used to record the RRS spectra by means of synchronous scanning excitation wavelength λ−3 mol/L AgNO3 solution, 1% (W/V) sodium citrate solution, 0.05 mol/L NaCl solution, and 1.0 × 10−3 mol/L FeSO4 solution were used. A 0.05% (W/V) NaBH4 was prepared freshly. A 0.100 mol/L H2O2 standard solution was prepared as follows: 1.02 mL H2O2 (30%) was diluted to 100 mL with water, it was standardized by KMnO4 procedure, and was diluted to 5.00 × 10−4 mol/L before use.A 1.0 × 10−4 mol/L AgNP was prepared as follows: 9.25 mL 1.0 × 10−3 mol/L AgNO3 and 3.5 mL 1% trisodium citrate were added into a conical flask with stirring and diluted to 40 mL with water, then 4 mL 0.05% NaBH4 was added slowly with about 15 min, the mixture was diluted to 50 mL, and it can be used after 24 h to make the NaBH4 decomposing completely. The preparation of AgNP sols was repeated five times, the SPR peak is at 395 nm with an average absorption value of 2.50 ± 0.10, and the sols were stabile within 20 days , and a certain amount of H2O2 solution were added into a 5 mL calibrated tube in turn, then diluted to 2 mL and mixed well. The RRS intensity at 330 nm (I) was recorded by a fluorescence spectrophotometer with synchronous scanning (λex-λem = Δλ = 0). A blank (I0) without H2O2 was recorded and the value of ΔI = I–I0 was calculated.A 1.0 mL 1.85 × 10G.Q.W. and Y.H.L. performed the experiment and measurement analysis. G.Q.W. and Z.L.J. prepared Fig.1–4. A.H.L. and Z.L.J. contributed to the discussion and measurement analysis. All authors contributed to the preparation of the manuscript and reviewed the manuscript.SUPPLEMENTARY INFO


The coordination sphere of the CdII atom is built of two symmetry-equivalent N atoms of one 2,9-dimethyl-1,10-phenanthroline (dmphen) ligand and two symmetry-equivalent I atoms, thus forming a distorted tetrahedral geometry. Inversion-related molecules interact along the c-axis direction by π–π stacking interactions between the phenanthroline ring systems, with centroid–centroid distances of 3.707 (9) and 3.597 (10) Å.In the title compound, [CdI DOI: 10.1107/S1600536811044667/pk2353Isup2.hkl            Structure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


Each CdII atom is seven-coordinated by a bidentate 5-nitro-1,10-phenanthroline (5-NO2-phen) ligand and two bidentate acetate anions, one of which also acts as a bridge linking the two Cd atoms. The crystal packing is stabilized by π–π interactions between the phen rings of neighboring molecules, with centroid–centroid distances of 3.491 (2) (intramolecular) and 3.598 (2) Å (intermolecular).In the binuclear title compound, [Cd DOI: 10.1107/S1600536811032259/bg2418Isup2.hkl            Structure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


H2) immune responses, with IL-13 production being essential for Nippostrongylus brasiliensis expulsion. This TH2 response results in intestinal inflammation associated with local infiltration by T cells and macrophages. The resulting increased IL-4/IL-13 intestinal milieu drives goblet cell hyperplasia, alternative macrophage activation and smooth muscle cell hypercontraction. In this study we investigated how IL-4-promoted T cells contributed to the parasite induced effects in the intestine. This was achieved using pan T cell-specific IL-4 receptor alpha-deficient mice (iLckcreIL-4Rα−/lox) and IL-4Rα-responsive control mice. Global IL-4Rα−/− mice showed, as expected, impaired type 2 immunity to N. brasiliensis. Infected T cell-specific IL-4Rα-deficient mice showed comparable worm expulsion, goblet cell hyperplasia and IgE responses to control mice. However, impaired IL-4-promoted TH2 cells in T cell-specific IL-4Rα deficient mice led to strikingly reduced IL-4 production by mesenteric lymph node CD4+ T cells and reduced intestinal IL-4 and IL-13 levels, compared to control mice. This reduced IL-4/IL-13 response was associated with an impaired IL-4/IL-13-mediated smooth muscle cell hypercontractility, similar to that seen in global IL-4Rα−/− mice. These results demonstrate that IL-4-promoted T cell responses are not required for the resolution of a primary N. brasiliensis infection. However, they do contribute significantly to an important physiological manifestation of helminth infection; namely intestinal smooth muscle cell-driven hypercontractility.Gut-dwelling helminthes induce potent IL-4 and IL-13 dominated type 2 T helper cell (T H2 differentiation and B cell isotype switching is dependent on IL-4 signalling via the type 1 IL-4Rα H2 program in polarized CD4+ T cells IL-4 and IL-13 share a common signalling pathway through the IL-4 receptor alpha (IL-4Rα) chain. A functional IL-4R (type I) requires assembly of IL-4Rα with a gamma c chain, while interaction of IL-4Rα with an IL-13Rα1 subunit leads to formation of a functional IL-13 receptor (type II). IL-4Rα–deficient mice lack responsiveness to IL-4 and IL-13. Expression of IL-4Rα reflects the pleiotropic nature of IL-4/IL-13 biology, as this receptor subunit is expressed upon a wide range of cells H2 cells stimulate the production of IL-4 and IL-13 which enhances epithelial cell permeability N. brasiliensis expulsion occurs in mice deficient in STAT-6 In the gastrointestinal tract activated TN. brasiliensis infection studies in experimental murine models are analogous to human hookworm infections N. brasiliensis infection resulted in impaired TH2 responses in global IL-4Rα and smooth muscle cell-specific IL-4Rα deficient BALB/c mice and accompanied by delayed goblet cell hyperplasia in these mice H2 response may contribute to smooth muscle cell contraction. In contrast, macrophage/neutrophil-specific IL-4Rα deficient mice, which have impaired IL-4Rα-activated alternative macrophages N. brasiliensis infection accompanied by goblet cell hyperplasia.+ T cells and macrophage/neutrophils is not required for N. brasiliensis expulsion +, CD8+, NK T and γδ) T cell-specific IL-4Rα (iLckcreIL-4Rα−/lox) deficient mice Our previous studies have shown that the expression of IL-4Rα specifically on CD4All experiments were approved by the University of Cape Town Animal Ethics Committee  and all efforts were made to minimize suffering.creIL-4Rα−/lox) IL-4Rα deficient mice were generated as previously described −/lox mice (littermate control mice) and homozygous IL-4Rα−/− mice (IL-4Rα KO mice) were used as controls. iLckcreIL-4Rα−/lox mice are described as tm1Fbb/Il4ratm2FbbTg(Lck-cre)C.Cg-Il4ra, and IL-4Rα−/− are tm1Fbb/Il4ratm1FbbIl4ra. All mice used were on a BALB/c background. In addition, BALB/c mice were compared to hemizygous IL-4Rα−/lox mice and improved iLckcreIL-4Rα−/lox compared with LckcreIL-4Rα−/lox mice Eight- to 12-week-old mice were obtained from the University of Cape Town specific-pathogen-free animal facility and kept in individually ventilated cages. T cell-  for quantification of intestinal goblet cell hyperplasia, which was carried out as previously described + T-cells isolated by negative selection using Biomag beads (Qiagen) with a purity of >90% [as previously described 20] were restimulated for 48 h with 20 µg/ml anti-CD3 antibody 145-2C11. Supernatants were then collected and stored at −80°C until they were analyzed. Cytokine levels in supernatants and total IgE antibody levels in serum of individual infected animals were determined as previously described 3PO3. The absorbance at 450 nm was determined with a Versamax microplate spectrophotometer . Total IgE >0.002 ug/ml and IL-4, IL-13 or IFN-g >0.412 ng/ml were detected.CD4For intestinal cytokine detection the jejunum was removed from naive and infected mice and homogenized in lysis buffer containing protease inhibitors (Sigma). The homogenates were centrifuged at 14000 rpm for 20 min and the protein concentration in the supernatant was determined using the BCA assay . Protein concentration for all samples were equalised to 3 mg/ml and the levels of the cytokines IL-4 and IL-13 were determined using ELISA (see above).Whole tissue sections, 1 cm long were dissected from the jejunum region of the small intestine and suspended in a four chamber automatic organ bath system in oxygenated Krebs buffer at a resting tension of 0.5 g as previously described Values are expressed below as means ± standard deviations or means ± standard errors of the means, and significant differences were determined using the Mann-Whitney U test, an unpaired two-tailed Student t test or a One-Way ANOVA (GraphPad Prism4).N. brasiliensis infection, IL-4Rα−/−, pan T cell-specific IL-4Rα deficient (iLckcreIL-4Rα−/lox) and heterozygous IL-4Rα−/lox littermate control mice were infected by subcutaneous injection of 750 L3 N. brasiliensis larvae. Parasite egg production was determined on days 6–14 PI (−/lox control and iLckcreIL-4Rα−/lox mice showed similar egg production throughout the infection with egg counts peaking at day 7 and clearing by day 9 post infection (PI). Intestinal worm burdens in both mice strains were similar at day 7 PI and absent by day 10 PI. As previously demonstrated, IL-4Rα−/− mice did not clear infection efficiently showing a maintained egg production at day 11 PI and the presence of adult worms detected at day 10 PI + T cell-specific IL-4Rα deficient mice (LckcreIL-4Rα−/lox) To investigate the role of IL-4Rα-responsive T cells in the control of  6–14 PI  A and in+ T cells are also known to play a key role in N. brasiliensis clearance, such as γδ T cells which initiate rapid expulsion of adult worms from the intestine and limit egg production +, γδ and NK T cells plays a role in N. brasiliensis expulsion we compared pan T cell-specific IL-4Rα deficient mice (iLckcreIL-4Rα−/lox) described in this paper with the previously described CD4+ T cell-specific IL-4Rα deficient (LckcreIL-4Rα−/lox) mice which have partial or normal IL-4Rα surface expression on CD8+, γδ and NK T cells . Quantification of PAS-stained mucus-containing goblet cells in the small intestine resulted in similar number per villi between control and iLckcreIL-4Rα−/lox mice  . Whereas total IgE antibody concentration was below detection limit in the sera of global IL-4Rα−/− mice, IgE antibodies were present in naive iLckcreIL-4Rα−/lox mice and increased during infection, though to a lesser extent than infected control mice  with day 7 and 10 post infection  (+ T cells derived from iLckcreIL-4Rα−/lox mice showed a similar reduction of IL-4 as CD4+ T cells derived from IL-4Rα−/− mice). However, IL-13 and IL-17 secretion by iLckcreIL-4Rα−/lox mice was not significantly different to control mice  . Interesrol mice . TogetheN. brasiliensis infection induced the TH2 cytokines IL-4 and IL-13 in the jejunum of IL-4Rα−/lox control mice  maintained their ability to produce IL-13 in contrast to the CD4+ T cells isolated from digested lung + T cells and impaired IL-4 and IL-13 levels in the jejunum of N. brasiliensis-infected T cell-specific IL-4Rα deficient mice.To determine levels of IL-4 and IL-13 in the jejunum, soluble homogenates of tissue were analysed by ELISA. As expected, rol mice . In cont−/− mice and reduced in smooth muscle cell-specific IL-4Rα−/− mice ex vivo contractile ability of jejunum from infected iLckcreIL-4Rα−/lox mice was compared to control IL-4Rα−/lox and global IL-4Rα−/− mice after 7 or 10 days PI. Jejunum weight was equivalent between all strains under naive conditions and at 7 days PI, while at day 10 PI the tissue weight was increased in the global IL-4Rα−/− but not in iLckcreIL-4Rα−/lox mice compared to controls (data not shown). Jejunum contractile responses to stimulation with potassium chloride and acetylcholine in naïve mice were similar in all groups Click here for additional data file.Figure S2N. brasiliensiscreIL-4Rα−/lox and LckcreIL-4Rα−/lox mice. infection is comparable between iLck iLckcreIL-4Rα−/lox, LckcreIL-4Rα−/lox and control mice were infected with 750 N. brasiliensis L3 larvae. Faeces were collected from day 5 to 10 post infection (PI) and egg production was calculated using the modified McMaster technique (A). At days 7 and 10 PI the worm burden in the small intestine was assessed (B). Intestinal goblet cell hyperplasia was assessed by determining the total number of PAS-positive goblet cells per 5 villi in histological sections of the small intestine at day 7 and 10 PI (C). Total IgE production in the serum was measured by ELISA at day 7 and 10 PI (D). The data are representative of the results of two independent experiments with mean values+SEM and n = 4 or 5 mice per group. ND, not detected, ns = not significant. One-Way-ANOVA, *P<.05, **P<.01.(TIF)Click here for additional data file.Figure S3N. brasiliensis−/lox mice. infection is comparable between BALB/c and IL-4Rα Five mice per group were infected with 750 N. brasiliensis L3 larvae. Faeces were collected from day 5 to 10 post infection (PI) and egg production was calculated using the modified McMaster technique (A). At days 7 and 10 PI the worm burden in the small intestine was assessed (B). Intestinal goblet cell hyperplasia was assessed by determining the total number of PAS-positive goblet cells per 5 villi in histological sections of the small intestine at day 7 and 10 PI (C). Total IgE production in the serum was measured by ELISA at day 7 and 10 PI (D). Comparison of the response of infected BALB/c and IL-4Rα−/lox mice to acetylcholine is also shown for day 7 p.i. The data represents one (A-D) and two (E) independent experiment with n = 5 per group and mean values + SEM. ND, not detected. Unpaired two-tailed Student t test, ns = not significant.(TIF)Click here for additional data file.Table S1Summary of IL-4Rα surface expression on T cell subpopulations.+, CD8+, γδ T cells and NK T cells.(DOC)Click here for additional data file.


H-1,5-benzodiazepin-2(3H)-one reacts in the presence of a concentrated aqueous solution of sodium hydroxide and a quaternary ammonium salt  in benzene  with propargyl bromide, affording the title benzodiazepine derivative, C18H14N2O. In the molecule, the mean plane of the propargyl substituent is almost perpendicular with that of the amide group [dihedral angle = 87.81 (8)°]. In the crystal, the molecules are linked by C—H⋯O and C—H⋯N interactions.4-Phenyl-1 DOI: 10.1107/S1600536811027371/tk2762Isup2.hkl            Structure factors: contains datablock(s) I. DOI: 10.1107/S1600536811027371/tk2762Isup3.cml            Supplementary material file. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


Interesting special cases of theorems are alsodiscussed. Further, the results proved in this paper may find certainapplications of q-Sumudu transforms to the solutions of the q-integrodifferential equations involving q-Bessel functions. The resultsmay help to extend the q-theory of orthogonal functions.The main purpose of this paper is to evaluate  Purohit and Kalla [q-Laplace transforms of a product of basic analogues of the Bessel functions. They gave several useful special cases as application. Recently, Albayrak et al. [q-Sumudu transforms and established several theorems related to q-images of some elementary functions. Subsequently, the same authors evaluated the q-Sumudu images of a number of q-polynomials and q-hypergeometric functions .q-Sumudu images of a product of q-Bessel functions. q-Bessel functions were introduced by Jackson [q-Bessel functions. Some q-analogues of the Bessel functions are given byIn this section, we purpose to add one more dimension to this study by giving some theorems which give rise to  Jackson  and are q-Bessel functions are related by the following equality:q-analogues have been studied extensively by Ismail in [q-analogue of the Bessel function is given byq-Bessel function is also defined by Jackson and sometimes it is called Hahn-Exton q-Bessel function. The notation 1Φ1 in , isHahn  showed tsmail in , 12. Thegiven byJν(3)lim∈ℝ).See  for all fined by (9)Φrs[aq-analogues of the Sumudu transform by means of the following q-integrals:q-analogues of the classical exponential functions are defined byq-integrals as followsq-Sumudu transforms can be expressed asq-gamma function are defined byK is the following remarkable function [q-gamma and K function have the following properties [q-gamma function can be expressed asAlbayrak et al.  have deffined byeq(t)=∑n= follows∫0xf(t)dq follows∫0xf(t)dq follows∫0xf(t)dq follows∫0xf(t)dqq-Sumudu transforms of a product of q-Bessel functions.In this section we will evaluate j = 1,2,…, n, be n different q-Bessel functions andq-Sumudu transform of f(t) ism = m1 + m2 + ⋯+mn and M = μ1 + ⋯+μn, Re(s) > 0 and Re(υ + M) > 0.(a) Let j = 1,2,…, n, be n different q-Bessel functions andq-Sumudu transform of f(t) ism = m1 + m2 + ⋯+mn and M = μ1 + ⋯+μn, Re(s) > 0 and Re(υ + M) > 0.(b) Let j = 1,2,…, n, be n different q-Bessel functions andq-Sumudu transform of f(t) ism = m1 + m2 + ⋯+mn and M = μ1 + ⋯+μn, Re(s) > 0 and Re(υ + M) > 0.(c) Let m = m1 + m2 + ⋯+mn and M = μ1 + ⋯+μn. By using the property of =tυ−1perty of  and thenj = 1,2,…, n, be n different q-Bessel functions andq-Sumudu transform of f(t) ism = m1 + m2 + ⋯+mn and M = μ1 + ⋯+μn, Re(s) > 0 and Re(υ + M) > 0.(a) Let j = 1,2,…, n, be n different q-Bessel functions andq-Sumudu transform of f(t) is given bym = m1 + m2 + ⋯+mn and M = μ1 + ⋯+μn, Re(s) > 0 and Re(υ + M) > 0.(b) Let A = s and α = υ + M + m, where M = μ1 + ⋯+μn and m = m1 + ⋯+mn, we getWe will only give the proof of , becausefinition  and makefinition ; then weation of , if we wrties of  and 22)(39)f(t)=q-Sumudu transforms involving the q-Bessel functions as applications of our main results.In this section we evaluate the n = 1, μ1 = v, v = μ, and a1 = a in above theorems, respectively, one hasRe(s) > 0 and Re(μ + υ) > 0.If one takes v/2) + 1 and (υ/2) instead of μ and υ, respectively, one obtainsRe(s) > 0 and Re(υ + 1) > 0.In v = 1, respectively, one getsRe(s) > 0.In v = 0 in Re(s) > 0.Similarly, if one chooses υ = 0 and then a = 0, one findsRe(s) > 0 and Re(μ) > 0.In The results in q-Sumudu transforms of many other q-Bessel functions can be evaluated in this manner by applying the above theorems and their various corollaries and consequences considered here. Also, the results obtained here will be used in the forthcoming paper where some q-difference equations are solved.We conclude this investigation by remarking that


The aim of our work was to analyze Vγ9Vδ2 T-cell functionality during chronic HCV infection, studying the role of IFN-α on their function capability. IFN-γ production by Vγ9Vδ2 T-cells was analyzed in vitro in 24 HCV-infected patients and 35 healthy donors (HD) after PhAg stimulation with or without IFN-α. The effect of in vivo PhAg/IFN-α administration on plasma IFN-γ levels was analyzed in M. fascicularis monkeys. A quantitative analysis of IFN-γ mRNA level and stability in Vγ9Vδ2 T-cells was also evaluated. During chronic HCV infection, Vγ9Vδ2 T-cells showed an effector/activated phenotype and were significantly impaired in IFN-γ production. Interestingly, IFN-α was able to improve their IFN-γ response to PhAg both in vitro in HD and HCV-infected patients, and in vivo in Macaca fascicularis primates. Finally, IFN-α increased IFN-γ-mRNA transcription and stability in PhAg-activated Vγ9Vδ2 T-cells. Altogether our results show a functional impairment of Vγ9Vδ2 T-cells during chronic HCV infection that can be partially restored by using IFN-α. A study aimed to evaluate the antiviral impact of PhAg/IFN-α combination may provide new insight in designing possible combined strategies to improve HCV infection treatment outcome.In chronic HCV infection, treatment failure and defective host immune response highly demand improved therapy strategies. Vγ9Vδ2 T-cells may inhibit HCV replication  Most Hepatitis C virus (HCV) infections evolve in persistent infection, which may progress to fibrosis, cirrhosis, liver failure or even hepatocellular carcinoma HCV persistence is mainly due to the failure of the host’s immune system to effectively and definitively clear the infection and generate protective cellular immunity. Indeed, marked quantitative and qualitative defects of HCV-specific CD8 T-cells have been described in HCV patients, correlated with innate immune cell impairment such as dendritic cell (DC) in vitro and in vivo by using phosphoantigens (PhAgs) Among innate immune cells, Vγ9Vδ2 T-cells represent a good target for immunotherapy in infectious diseases Vγ9Vδ2 T-cells are involved in host response to many chronic viral infections, including HCV in vivo in a non-human primate model.In this work, phenotype and function of Vγ9Vδ2 T-cells were analyzed during chronic HCV infection, evaluating possible strategies aimed to improve their effector response. This approach was validated This study was approved by the Ethics Committee of the National Institute for Infectious Diseases “L.Spallanzani”, and all enrolled individuals provided written informed consent.All experiments on monkeys were performed in accordance with the recommendations of the Weatherall report, and were previously approved by the regional ethical committee  .24 HCV-infected patients  naïve to treatment, and 35 healthy age-matched individuals , were recruited at the INMI L. Spallanzani. Patients clinical features are described in Plasma HCV-RNA levels were assayed by Abbott RealTimeHCV assay . Moreover, HCV genotype was determined by Abbott RealTime HCV Genotype II Amplification Reagent kit.Peripheral blood mononuclear cells (PBMC) were isolated by Lympholyte . In selected experiments, γδ T-cells were purified from PBMC by immunomagnetic separation using anti-γδ-conjugated magnetic microbeads . The purity of cells fraction was >95% in all experiments, as measured by flow cytometry analysis (data not shown).6 cells/ml) were incubated with mAbs cocktail for 15 min a 4°C, washed twice with wash buffer  and fixed with 1% paraformaldehyde . Samples acquisition and data analysis were performed by a FACS Canto II flow cytometer (Becton Dickinson) by using Diva software.Phenotypic analysis of Vδ2 T-cells from HCV and from HD was performed by flow cytometry. Specifically, Vδ2 T-cell subsets were analyzed by using the following monoclonal antibodies: anti- Vδ2 FITC (clone IMMU389), anti-CD3 PerCP-PC5 (clone UCHT-1), from Beckman Coulter ; anti-CD27 APC (clone L128), anti-CD45RA CY-Chrome (clone HI100), anti-CD69 APC-Cy7 (clone FN50), anti-CD25 APC (clone M-A251) from BD Biosciences . Briefly, thawed PBMC  able to specifically activate only Vγ9Vδ2 T-cells 3, 1% BSA, 0.5% saponin). After washing , cells were acquired by flow cytometer (FACS Canto II flow cytometer) and data were analyzed by using Diva software. The frequency of IFN-γ-producing Vγ9Vδ2 T-cells and the IFN-γ MFI (Median Fluorescence Intensity) were compared between HD and HCV-infected patients.Moreover, in selected HCV and HD, the frequency of IFN-γ-producing Vγ9Vδ2 T-cells was monitored. Briefly, PBMC were stimulated for 18 h with PhAg or PhAg/IFN-α in the presence of Brefeldin A (10 µg/ml)  to block cytokine secretion. Intracellular staining was performed by staining cells for 15 minutes at 4°C with anti-Vδ2 FITC antibody; after washing, cells were fixed with 1% PFA  and stained at room temperature with an APC-labeled IFN-γ specific antibody (clone B27), in permeabilizing solution  were included in the study: 6 animals were purchased from Noveprim  and 2 from CDP . Animal welfare conditions conformed to the European requirements, comprising monitored temperature, humidity, air change, and lighting cycle. All experiments were previously approved by the regional ethical committee  . At the beginning of the study, body weights ranged from 2.2 to 5.8 kg. In order to avoid suffering, animals were anaesthetized with Ketamine 1000 ND (10 mg/kg IM) before any procedure. Group 1  was injected s.c. with 3 mg/Kg of IPH1201 (C-HDMAPP) a second generation synthetic PhAg able to specifically activate Vγ9Vδ2 T-cells  . Group 2  was injected subcutaneously (s.c.) with 3 mg/Kg of IPH1201 and with 27 µg/animal s.c. of Interferon α-2a pegylated, Pegasys® (Roche). The dose of Pegasys is the same as that used in the clinical care of HCV patients. Blood samples were collected before and after 4, 8, 12, 16, 20, 24, 28 hours after treatment, and sera were stored for further analysis. IFN-γ plasma levels were analyzed by ELISA (Biosource).8 naïve cynomologus macaques . One µg total RNA was reverse transcribed by TaqMan Reverse Transcription Reagent kit  according to manufacturer’s instructions. IFN-γ-mRNA level was quantified by qPCR performed using Taqman 2× PCR Master mix  and a 7900 HT Fast Real-Time PCR system machine by using primers and probe sets for IFN-γ-mRNA and β-actin as described in mRNA stability was evaluated by adding 10 µg/ml actinomycin D (ActD) after 18 hours of PhAg (IPH1101: 3 µM) or IFN-α (100 IU/ml) stimulation. IFN-γ-mRNA levels were evaluated by qRT-PCR just before Actinomycin addiction (t0), and after 30 and 120 minutes of culture, and expressed as normalized to β-actin. mRNA stability was evaluated by calculating half-life times of IFN-γ-mRNA by linear regression (GraphPad Prism).Statistical significance was determined by GraphPad Prism software (GraphPad). Differences between groups were evaluated by non parametric Mann-Whitney test; Wilcoxon test was used when comparing different culture conditions of the same cells. Tests were considered significant when p<0.05. IFN-γ-mRNA half life was evaluated by linear regression.Vγ9Vδ2 T-cell subsets were analyzed in 24 HCV-infected patients (HCV), naïve to treatment, and compared with 35 healthy donors (HD). In chronic HCV patients a slight but significant decrease in circulating Vγ9Vδ2 T-cell frequency was observed [HCV: median 1.140% (IQR: 0.49–2.16) vs. HD: 1.770% (1.080–2.290), p = 0.0362, μM). The amount of IFN-γ released in supernatants was measured by ELISA .In order to evaluate whether IFN-α could improve Vγ9Vδ2 T-cell responsiveness to PhAg, purified Vγ9Vδ2 T-cells were stimulated with single PhAg (3 µM), single IFN-α (100 IU/ml) and combined (PhAg/IFNα) for 18 hours. At the end of incubation IFN-γ released by Vγ9Vδ2 T-cells was evaluated by ELISA test . As showin vivo Vγ9Vδ2 T-cell responsiveness to PhAg stimulation, non-human primates (M. Fascicularis) were injected with 3 mg/Kg s.c. of PhAg  or with 3 mg/Kg of PhAg s.c. and 27 µg/animal s.c. of PEG-IFN-α . Plasma IFN-γ levels were analyzed before administration, and after 4, 8, 12, 16, 20, 24, 28 hours. As shown in max PhAg: 1,370 pg/ml vs. Cmax PhAg/IFN-α: 2,155 pg/ml), showing that PhAg/IFN-α combination is able to boost in vivo IFN-γ production.To verify if IFN-α could also improve in vitro stimulations was evaluated on PBMC from 24 HCV-infected patients and 35 HD. Since IFN-α was able to improve Vγ9Vδ2 T-cell responsiveness in HD, we wondered whether it can restore the impaired functional activities of Vγ9Vδ2 T-cells during chronic HCV infection. To this aim, IFN-γ production  after 24A quantitative analysis of IFN-γ-mRNA after PhAg and PhAg/IFN-α stimulations of purified Vγ9Vδ2 T-cells was performed by qRT-PCR . As expeIFN-γ-mRNA persistence was studied by blocking transcription with actinomycin D after 18 hours of stimulations . We defiMain aim of our work was to study the effects of chronic HCV infection on Vγ9Vδ2 T-cell phenotype and function, and on possible strategies aimed to improve their effector activity.Chronic HCV infection induced a slight but significant decrease in the frequency of Vγ9Vδ2 T-cells. An increased liver tissue compartmentalization of these cells may represent an additional factor Vγ9Vδ2 T-cells play a pivotal role in viral infections, for their ability to mediate broad antiviral and immunomodulating activities et al., showing an increase of CD69 after IFN-α treatment in vivo by combining standard IFN-α treatment and PhAg stimulation may have a positive impact on HCV inhibition. Indeed several reports show that IFN-α and IFN-γ may synergistically inhibit HCV replication in vitroin vivo in pre-clinical trials on non-human primates, inducing an increase in IFN-γ amount in animals sera. A time-course study of in vivo IFN-α treatment on Vγ9Vδ2 T-cell responsiveness to PhAg in HCV-infected patients is currently in progress.The main result of our work is the demonstration that Vγ9Vδ2 T-cell function may be improved by IFN-α both in HD and in HCV-infected patients, resulting in a higher IFN-γ production. A first demonstration that type-I IFN may be sensed by Vγ9Vδ2 T-cells was reported by Kunzmann About possible mechanisms mediating this improvement, we found that IFN-α acts by increasing IFN-γ-mRNA persistence, that may result in increased IFN-γ translation levels. Similar observations were reported on NK cells, as IFN-γ production after IL-12 and IL-18 stimulation was regulated by mechanisms involving IFN-γ-mRNA stabilization in vivo by PhAgs in vivo studies on HCV-infected non-human primates aimed to define the antiviral capability of the combined treatment are necessary both to assess safety and antiviral effectiveness of this combined approach, and to disclose the cellular/molecular mechanisms involved.In recent years, a new attention on new direct antiviral drugs for chronic HCV infection is growing. Nevertheless, a combination of these new treatments with IFN-α/Ribavirin seem necessary to avoid the emergence of drug resistance


A physiological role played by PAT2 in amino acid reabsorption in the renal proximal tubule is demonstrated by mutations in SLC36A2 that lead to an iminoglycinuric phenotype (imino acid and glycine uria) in humans. A number of proline, GABA and tryptophan derivatives were examined to determine if they function either as transported substrates or non-transported inhibitors of PAT2. The compounds were investigated following heterologous expression of rat PAT2 in Xenopus laevis oocytes. PAT2 function was characterised by: radiotracer uptake and competition (cis-inhibition) studies; radiotracer efflux and trans-stimulation; and measurement of substrate-induced positive inward current by two-electrode voltage-clamp. In general, the proline derivatives appeared to be transported substrates and the relative ability to induce current flow was closely related to the inhibitory effects on PAT2-mediated l-[3H]proline uptake. In contrast, certain heterocyclic GABA derivatives (e.g. l-pipecolic acid) were translocated only slowly. Finally, the tryptophan derivatives inhibited PAT2 function but did not undergo transport. l-Proline uptake was inhibited by 5-hydroxy-l-tryptophan (IC50 1.6 ± 0.4 mM), α-methyl-d,l-tryptophan (3.5 ± 1.5 mM), l-tryptophan, 1-methyl-l-tryptophan and indole-3-propionic acid. Although neither 5-hydroxy-l-tryptophan nor α-methyl-d,l-tryptophan were able to elicit inward current in PAT2-expressing oocytes both reduced the current evoked by l-proline. 5-Hydroxy-l-tryptophan and α-methyl-d,l-tryptophan were unable to trans-stimulate l-proline efflux from PAT2-expressing oocytes, confirming that the two compounds act as non-transported blockers of PAT2. These two tryptophan derivatives should prove valuable experimental tools in future investigations of the physiological roles of PAT2.The H d,l-tryptophan) are non-transported inhibitors.►PAT2 (SLC36A2) transports proline, glycine, alanine and hydroxyproline. ►Heterocyclic proline derivatives are transported substrates. ►Heterocyclic GABA derivatives are translocated slowly. ►Tryptophan derivatives (e.g. α-methyl- In five pedigrees a single amino acid substitution (G87V) in PAT2 was identified Xenopus oocytes produced a transporter with reduced activity compared to wild-type PAT2 because of decreased affinity for proline and glycine SLC36A2 was identified. This mutation produced a truncated protein with no function In contrast to PAT1, much less is known about the other members of the SLC36 family. SLC36A3/PAT3 and SLC36A4/PAT4 remain orphan transporters with no known function. Based upon homology to PAT1  share 72% identity in amino acid sequence), PAT2 was isolated from mouse +-coupled amino acid transporter PAT2. A previous study Xenopus laevis oocytes and transport function characterised using a combination of experimental approaches including radiotracer uptake and competition (cis-inhibition) studies, radiotracer efflux and trans-stimulation, and measurement of substrate-induced positive inward current using the two-electrode voltage-clamp (TEVC) technique.Although major advances have been made over recent years in our understanding of membrane transporter function in mammalian tissues it is also likely that many unidentified transport systems will play unique physiological roles. These roles can be difficult to define because the function of an individual transporter may be masked, for example, due to coexpression with other transport systems, low level of expression, or limited expression in a sub-population of cells within a mixed tissue. In the case of amino acid transporters, a major difficulty in attributing transport of any individual compound to any single transport system is due to the overlapping substrate specificities and affinities observed between many transporters. The development of a panel of selective substrates or, preferably, non-transported inhibitors will allow discrimination between movements through distinct transport systems. Thus, the objective of this investigation was to identify non-transported inhibitors of the H22.1d,l-tryptophan, d- and l-pipecolic acids ; and serotonin . Radiolabelled amino acids used were: β-[3-3H]alanine (specific activity 50 Ci mmol− 1) and l-pipecolic acid (20 Ci mmol− 1) ; [2-3H]glycine (21 Ci mmol− 1) ; and l-proline (75–85 Ci mmol− 1) .All amino acids, analogues and derivatives were purchased from Sigma-Aldrich  except: guvacine and isoguvacine ; α-methyl-2.2Xenopus laevis frogs were killed following Schedule 1 methods prior to surgical removal of the ovaries. Oocytes were prepared as described previously − 1) synthesised by in vitro transcription of pCRII containing mouse PAT1 Female 2.3+-free solution . Radiolabel uptake experiments were initiated by replacement of wash solution with 200 μl Na+-free solution (pH 5.5 unless stated otherwise) supplemented with [3H]amino acid . For cis-inhibition experiments, uptake of [3H]amino acid was measured in the absence (control) and presence of competitor compound (0.01–30 mM). Unless stated otherwise, amino acids and derivatives are used routinely in their l-forms. Uptakes were measured over 40 min at 22 °C after which time the oocytes were washed three times with ice-cold Na+-free solution and lysed in 10% SDS before addition of scintillation cocktail (PerkinElmer). For trans-stimulation experiments, oocytes were pre-loaded with proline by microinjection of 50 nl l-[3H]proline . Assuming an effective oocyte volume of 250 nl +-free solution (pH 5.5) and incubated  in 100 μl Na+-free solution (pH 5.5) in the presence or absence of various compounds . After the 10 min efflux period, the incubation solution was removed for scintillation counting and oocytes washed three times with ice-cold Na+-free solution. Oocytes were lysed in 10% SDS before addition of scintillation cocktail. Sample radioactivity was determined using an LS6500 liquid scintillation counter .Oocytes were washed with Na2.4+-free solution (composition as per radiolabel flux experiments) whilst clamped at a membrane potential of − 60 mV. Current changes evoked by exposure (60–120 s) to experimental compounds (0.2–30 mM) were recorded using pClamp software connected to a Geneclamp 500 amplifier and Digidata 1200  and analysed using Clampfit 8.2 (Axon Instruments). Compound-induced current changes were calculated by subtraction of baseline (average current recorded over 15 s before addition of compound) from the average current recorded over the final 15 s of the exposure. Results are presented as cRNA-specific current  and representative trace recordings.Two-electrode voltage-clamp (TEVC) experiments were performed as described previously 2.5Data are mean ± SEM (n = number of oocytes), except where indicated in the text. Unless stated otherwise, results are expressed as the cRNA-specific response . Statistical significance was determined by unpaired or paired, two-tailed Student's t-test or one-way analysis of variance (ANOVA) with Tukey–Kramer multiple comparisons post-testing as appropriate. p < 0.05 was deemed statistically significant. Kinetic parameters were determined by fitting data to Michaelis–Menten kinetics. Statistical and kinetic analyses were performed using GraphPad Prism v.4 .33.1l-alanine and l-proline are transported substrates for PAT2 with the five-membered ring heterocyclic l-proline (m 0.1–0.2 mM) l-enantiomers of amino acids although this preference is weaker than that observed in most other mammalian amino acid carriers d-proline and trans-4-hydroxy-l-proline have similar affinities for PAT2 to that observed with l-proline l-proline transport via PAT2 is less than that observed with glycine The substrate specificity of PAT2 has been characterised to some degree -proline  having tl-[3H]Proline uptake (over 40 min) under optimal transport conditions  via PAT2 was increased 17-fold above that in water-injected oocytes. PAT2-mediated l-[3H]proline uptake was reduced by the proline derivatives  l-thiaproline , trans-4-hydroxy-l-proline (T4HP), 3,4-dehydro-d,l-proline (DHP), cis-4-hydroxy-d-proline (CHDP), trans-3-hydroxy-l-proline (T3HP) and cis-4-hydroxy-l-proline (CHLP)  was well tolerated although the position and orientation of the hydroxyl group  affected the relative affinity of the compounds for PAT2  was demonstrated previously The lower turnover rate of PAT2 observed at high (mM) concentrations of heterocyclic prolines e (CHLP) A. Modifiyl group  in the pfor PAT2 A–B. Tran3.2d-cycloserine and l-2-azetidine carboxylate are excellent PAT2 substrates. Thus, to try and identify non-transported inhibitors, we focussed on amino acid analogues containing bulkier ring structures. The six-membered ring cis-3-aminocyclohexanecarboxylic acid is used to selectively discriminate between GABA transport by the four GAT transporters . Cis-3-aminocyclohexanecarboxylic acid has a higher affinity for GAT1 whereas β-alanine has a preference for GAT3 and 4 l-[3H]proline uptake  l-[3H]proline uptake (Other heterocyclic GABA-related compounds are used to selectively inhibit GAT transporters (such as guvacine) or as agonists for GABAe uptake A and thee uptake B–C suggeA receptor agonist with a similar structure to isoguvacine) differ from each other by the distance between the amino and carboxyl groups . However, all four compounds were relatively poor at inducing inward currents via PAT2 The six-membered heterocyclic ring (saturated) piperidine carboxylates pipecolic acid, nipecotic acid (a GAT transport inhibitor with a similar structure to guvacine) and isonipecotic acid (a GABAl groups . Our prem for glycine 0.5–0.7 mM) l-pipecolic acid (l-Pip) (10 mM l-pipecolic acid plus 0.3 mM glycine) a decrease in the amino acid-induced current is observed  than that observed in the presence of 10 mM glycine (d-pipecolic (d-Pip), nipecotic (Nip) and isonipecotic (Isonip) acids  than that observed following exposure to 3 mM glycine. In addition, the current induced by the combination of saturating concentrations of l-pipecolic acid and glycine  is significantly less (p < 0.05) than that induced by 3 mM glycine alone  to a saturating (10 mM) concentration (Kobserved A and C.  glycine A. Similap) acids B–C. The ne alone D–E. In ttic acid , improvetic acid B–C.l-pipecolic acid is due to a dissociation of amino acid and proton transport via PAT2 such that l-pipecolic acid is transported in a partially uncoupled mode. However, that does not appear to be the case as PAT2-mediated l-[3H]pipecolic acid uptake is much reduced (p < 0.001) compared to [3H]glycine due to a reduction in Vmax from 934 ± 22 to 224 ± 19 pmol oocyte− 1 (40 min)− 1  investigated − 1 F. Thus, stigated G. The we3.40,+ (SLC6A14), respectively l-[3H]Proline uptake was inhibited significantly (p < 0.001) by 5-hydroxy-l-tryptophan (OH-Trp), α-methyl-d,l-tryptophan (Me-Trp), l-tryptophan (l-Trp), 1-methyl-l-tryptophan (1-Me-l-Trp) and indole-3-propionic acid (3-IPA)  whereas the d-enantiomers d-tryptophan (d-Trp) and 1-methyl-d-tryptophan (1-Me-d-Trp) were without effect (p > 0.05)  or nitrogen within the indole ring structure (1-methyl-l-tryptophan)  , have be > 0.05) A. The pre uptake A whereasptophan)  was tolel-tryptophan and α-methyl-d,l-tryptophan and their interaction with PAT2 was investigated further. 5-Hydroxy-l-tryptophan and α-methyl-d,l-tryptophan inhibited l-[3H]proline uptake in a concentration-dependent manner with IC50 values of 1.6 ± 0.4 and 3.5 ± 1.5 mM, respectively  alone were able to elicit significant (p > 0.05 versus water-injected oocytes) current in PAT2-expressing oocytes  caused significant (p < 0.001) reductions in current evoked by 0.2 mM l-proline  inhibit PAT2 with similar efficacy whereas 5-hydroxy-l-tryptophan causes a significantly greater (p < 0.001) inhibition of amino acid uptake via PAT1 than α-methyl-d,l-tryptophan  difference in l-[3H]proline efflux from PAT2-expressing (22 ± 3 (n = 50) pmol oocyte− 1 (10 min)− 1) or water-injected (21 ± 2 (n = 50) pmol oocyte− 1 (10 min)− 1) oocytes (l-[3H]proline efflux was observed from PAT2 (272 ± 10 (n = 50) pmol oocyte− 1 (10 min)− 1) but not water-injected (19 ± 2 (n = 50) pmol oocyte− 1 (10 min)− 1) oocytes, which represents a 14-fold increase in efflux. An example is shown in l-[3H]proline efflux whereas 5-hydroxy-l-tryptophan and α-methyl-d,l-tryptophan (both 10 mM) have no effect (p > 0.05 versus water-injected oocytes). These data are consistent with the inhibitory effects of 5-hydroxy-l-tryptophan and α-methyl-d,l-tryptophan on amino acid-induced current via PAT2 , l-alanine (Ala) and sarcosine (Sar) all trans-stimulate l-[3H]proline efflux (l-pipecolic acid (l-Pip). A direct relationship between the relative ability of compounds to cis-inhibit PAT2-mediated amino acid uptake and to trans-stimulate PAT2-mediated amino acid efflux can be observed in l-tryptophan and α-methyl-d,l-tryptophan, provides evidence for the inhibitory effects being due to the compounds acting as transport blockers rather than transported substrates.As suggested above, a potential limitation of measurement of substrate-induced currents by the TEVC technique is that electroneutral movement of compounds through the carrier cannot be detected. This has been demonstrated previously where H oocytes A. Howevevia PAT2  being due efflux B wherease efflux B. The we4+-coupled amino acid transporter PAT2. In general, the proline analogues were transported substrates with varying affinity for the transporter whereas the GABA analogues, in particular l-pipecolic acid, appear to be translocated relatively slowly. In contrast, both 5-hydroxy-l-tryptophan and α-methyl-d,l-tryptophan function as non-transported inhibitors of PAT2. Although the affinity of both compounds is relatively low, compared to the high affinity (IC50 approximately 250 μM) observed for the effect of α-methyl-d,l-tryptophan on amino acid transport via ATB0,+SLC36A2 gene is highly upregulated in uterine tissues, perhaps reflecting the increased nutrient demands of the developing conceptus A number of proline, GABA and tryptophan derivatives were examined to determine if they function either as transported substrates or non-transported inhibitors of the H


Natural microtubules typically include one A-lattice seam within an otherwise helically symmetric B-lattice tube. It is currently unclear how A-lattice seams influence microtubule dynamic instability. Here we find that including extra A-lattice seams in GMPCPP microtubules, structural analogues of the GTP caps of dynamic microtubules, destabilizes them, enhancing their median shrinkage rate by >20-fold. Dynamic microtubules nucleated by seeds containing extra A-lattice seams have growth rates similar to microtubules nucleated by B-lattice seeds, yet have increased catastrophe frequencies at both ends. Furthermore, binding B-lattice GDP microtubules to a rigor kinesin surface stabilizes them against shrinkage, whereas microtubules with extra A-lattice seams are stabilized only slightly. Our data suggest that introducing extra A-lattice seams into dynamic microtubules destabilizes them by destabilizing their GTP caps. On this basis, we propose that the single A-lattice seam of natural B-lattice MTs may act as a trigger point, and potentially a regulation point, for catastrophe. Microtubules are tubes in which helical symmetry is broken at a single ‘A-lattice’ seam. Katsuki et al. show that microtubules containing additional A-lattice seams exhibit decreased stability, and propose that such seams may act as trigger points for microtubule catastrophe. Microtubules (MTs) play a central role in the self-organization of eukaryotic cells, driving directional transport of cellular components either by using their own dynamics, or by serving as rails for cargo-carrying motor proteins. MTs self-assemble from α–β tubulin heterodimers to form hollow tubes of ∼25 nm diameterCatastrophe, the process of conversion from sustained growth to rapid shrinkage, ultimately results in the breaking of lateral bonds between protofilaments (PFs), leading to rapid shrinkage of the MT with coupled unpeeling of outwardly curved GDP–tubulin PFs58111213in vitroin vivo252627in vitroin vivo. Instead, there appears to be a biological drive to build B-lattice MTs that break symmetry by including an A-lattice seam. Why is this? One possibility is that A-lattice seams have a stability different from that of the B-latticein vivo have a flared structure, suggesting no preferential formation of any particular lateral contacts and therefore little difference in the stability of seam and sheet contactsMTs in mammalian cells typically contain 13 straight PFs arranged in the B-lattice2122in vitro assembly of MTs with either no A-lattice seamsS. pombe EB protein Mal3, when added in high concentration during nucleation can drive the assembly of A-lattice-enriched 13 PF MTs, with a population mean of ∼50% A-lattice contentA direct experimental test of the influence of A-lattice seams on MT catastrophe has hitherto been lacking, because it has not previously been possible to vary the A-lattice content of MTs. Spontaneous To examine the properties of MTs with extra A-lattice content we developed a kinesin motor surface clamp-release assay. We found that MT shrinkage can be inhibited by clamping a MT to a kinesin-coated coverslip . We usedS. pombe and pig brain tubulin−1 . B-lattice single-seam seeds containing Alexa-480 fluorescent label  versus 675±9 nm s−1 (229) mean±s.e.m. (n); P=0.0043, t-test with Welch’s correction for unequal variances)  , indicatS. pombe MTs nucleated by B-lattice single-seam pig tubulin seedsS. pombe tubulin and GTP were then flowed in. The added tubulin polymerizes from the seeds to form dynamic MTs, which are only anchored to the surface through the antibody-bound seed ), ∼60 times slower than the plus-end shrinkage of MTs not tethered by kinesin (−1 (5) (mean±s.e.m. (n)) (−1 (5) and minus-end shrinkage at 2.78±0.13 nm s−1 (5) (mean±s.e.m. (n)). Thus, the GDP core of dynamic A-lattice-enriched MTs is much less stable than dynamic B-lattice single-seam MTs ) , Table 3.m. (n)) , Table 1.m. (n)) , even wiThe single-seam of 13-3 B-lattice MTs creates a line of A-lattice PF contacts2924We found that shrinkage rates varied along the length of A-lattice-enriched GMPCPP MTs co-assembled with Mal3–N143. We speculate that this indicates a variation of A-lattice content along the length of our GMPCPP MTs, with sharp transitions between different zones. Localized zones of additional A-lattice can only be created by a shift in the lateral registration of one PF with its neighbours, creating a defect (a hole) in the MT lattice. Such defects have been observed for B-lattice MTs with variable PF numbers24We find that increasing the A-lattice content of seeds used to nucleate the growth of dynamic MTs enhances catastrophe in the resulting MTs at both their plus and minus ends. Usually, a reduction in MT growth rate correlates with an increase in catastrophe frequencyIncreased A-lattice content causes a dramatic decrease in GMPCPP MT lattice stability yet our observations suggest only a modest effect on catastrophe frequencies in dynamic MTs. Several factors may have led us to underestimate the influence of A-lattice seams on dynamics. Our structural studyIt is also unclear how efficiently the A-lattice content of the nucleating seeds is propagated into the dynamic MTs as they extend from the seeds. We observed rapid short fluctuating MT growths from seeds that we termed MT dithering. MT dithering increased dramatically on MT seeds with increased A-lattice content. We speculate that MT dithering arises when the growing MT contains a high fraction of A-lattice, such that catastrophe occurs very shortly after growth has initiated. Consistent with this idea, time spent dithering increases with increasing Mal3FL concentrations during seed assembly , Table 1Lastly, we observed that for dynamic MTs that were only attached to the surface via their seeds, A-lattice enrichment accelerated post-catastrophe shrinkage of the GDP core of the MT at the minus ends but not at the plus ends. We examined this further using our kinesin-clamp assay. We found that B-lattice GDP MTs with a single-seam were stabilized by binding to a rigor kinesin surface. In contrast, for MTs with extra A-lattice seams, the stabilizing effect of the rigor kinesin clamps was reduced so that the GDP lattice shrank even before the addition of ATP, with the plus end shrinking faster than the minus end. Thus, while a difference in the plus- and minus-end shrinkage rates is detectable when shrinkage is slowed by the kinesin clamps, in dynamic MTs not directly attached to the surface, the plus-end shrinkage rate is saturated and insensitive to changes in the lattice arrangement. Our overall conclusion is that like the GTP cap, the GDP core is also destabilized by A-lattice enrichment.in vivo and in vitro have produced apparently contradictory observations , PIPES from Melford (UK) and other reagents from Sigma (UK) except where stated.2, 2 mM EGTA, 0.1 mM EDTA, 1 mM Mg.ATP, 0.1 mM GTP, 1 mM DTT, 4 μM DCI, 10 μg ml−1 Leupeptin, 10 μg ml−1 Pepstatin, 1 μg ml−1 Aprotinin) and centrifuged at 35,800 g in an SLA1000 (Thermo) rotor for 50 min at 4 °C. The supernatant was adjusted to 33% (v/v) glycerol, 5 mM MgCl2, 1 mM GTP and incubated at 37 °C for 50 min, then centrifuged in an SLA1000 rotor at 35,800 g for 2.5 h at 35 °C. The pellet was resuspended in homogenization buffer at 4 °C, pelleted in a T865 (Thermo) rotor at 163,000 g for 1 h at 4 °C. The supernatant was adjusted to 33% (v/v) glycerol, 5 mM MgCl2, 1 mM GTP and incubated at 37 °C for 40 min, then centrifuged in a T865 (Thermo) rotor at 163 000 g for 1 h at 35 °C. The pellet was resuspended in 50 mM K-PIPES (pH 6.9), 1 mM EGTA, 0.2 mM MgCl2, 0.1 mM GTP, clarified by centrifugation in a TLA 100.3 rotor (Beckman) at 135,000 g for 30 min at 4 °C and loaded on a P11 phosphocellulose column (Whatman). Tubulin was eluted using 50 mM K-PIPES (pH 6.9), 1 mM EGTA, 0.2 mM MgCl2 and 10 μM GTP, then desalted into 100 mM K-PIPES (pH 6.9), 1 mM MgSO4, 2 mM EGTA and 20 μM GDP using a HIPREP 26/10 desalting column . The tubulin was then aliquoted, flash-frozen and stored in liquid nitrogen. Pig tubulin concentration was determined using E280=105,838 M−1 cm−1.Pig brain tubulin−1 in 0.1 M HEPES (pH 8.6), 1 mM MgCl2, 1 mM EGTA, 1 mM GTP, 40% (v/v) glycerol at 37 °C. Alexa Carboxylic acid succinimidyl ester dyes (40 mM) of Alexa-488 or Alexa-680 in DMSO were added to 4 mM final concentration, vortexed and incubated for 10 min at 37 °C, with vortexing every 2 min. Two volumes of 0.16 M MES (pH 6.8), 1 mM MgCl2, 1 mM EGTA, 1 mM GTP, 0.1 M K-glutamate, 40% (v/v) glycerol at 37 °C were added and vortexed. The MTs were pelleted through a 0.4 ml cushion of 80 mM MES (pH 6.8), 1 mM MgCl2, 1 mM EGTA, 1 mM GTP, 60% (v/v) glycerol in a TLA 120.2 rotor (Beckman) at 174,000g for 30 min at 35 °C. The pellet was washed with 80 mM MES (pH 6.8), 1 mM MgCl2, 1 mM EGTA and 1 mM GTP, at 37 °C then resuspended in the same buffer at 4 °C for 10 min and centrifuged at 170,000 g in a TLA120.2 rotor (Beckman) for 15 min at 4 °C. Glycerol (0.5 volume) was added to the supernatant, which was then incubated at 37 °C for 30 min. The MTs were again pelleted through the glycerol cushion, washed and resuspended in 80 mM MES (pH 6.8), 1 mM MgCl2, 1 mM EGTA and 1 mM GTP, at 4 °C, clarified by centrifugation at 170,000 g in a TLA120.2 rotor (Beckman) for 15 min at 4 °C, aliquoted, flash-frozen and stored in liquid nitrogen. Labelled tubulin concentrations  were calculated using a corrected OD280=OD280measured−(OD495 × 0.11) for Alexa-488 and corrected OD280=OD280measured−(OD679 × 0.05) for Alexa-680-labelled tubulin. The OD280corrected was used to determine the pig tubulin concentration using E280=105,838 M−1 cm−1.Pig brain tubulins labelled with Alexa-488 and Alexa-680 fluorophores (Invitrogen) were preparedS. pombe single-isoform tubulin29S. pombe cell pellets in 1.8 l of 100 mM K-PIPES (pH 6.9), 10 mM MgSO4, 2 mM EGTA, 50 mM NaCl, 1 mM GTP, 5 mM DTT, 1 mM TAME, 1 μg ml−1 aprotinin, 1 μg ml−1 AEBSF, 1 μg ml−1 E-64, 1 μg ml−1 Pepstatin A, 1 μg ml−1 Chymostatin, 1 μg ml−1 Antipain, 2 μg ml−1 Leupeptin in a Dyno-Mill (Willy A. Bachofen) using 500 μm glass beads at 4 °C. After centrifugation at 24,400g in an SLA-3000 rotor (Thermo) for 30 min at 4 °C the supernatent was batch bound with gentle stirring to 180 g of equilibrated DE-52 resin (Whatman) at 4 °C for 30 min. After settling for 30 min the supernatant was discarded and the resin washed with 450 ml of DE-52 low-salt buffer (100 mM K-PIPES (pH 6.9), 10 mM MgSO4, 2 mM EGTA, 50 mM NaCl, 10% (v/v) glycerol, 0.1 mM GTP, 5 mM DTT, 1 μg ml−1 aprotinin, 1 μg ml−1 AEBSF, 1 μg ml−1 E-64, 1 μg ml−1 Pepstatin A, 1 μg ml−1 Chymostatin, 1 μg ml−1 Antipain, 2 μg ml−1 Leupeptin) and allowed to settle for 50 min. The supernatant was removed and the resin packed in an X50/30 column . Using an Akta purifier  the resin was washed with 3–4 column volumes of DE-52 low-salt buffer. The tubulin was eluted using a 0–60% gradient of high salt DE-52 buffer (100 mM K-PIPES (pH 6.9), 10 mM MgSO4, 2 mM EGTA, 1 M NaCl, 10% (v/v) glycerol, 0.1 mM GTP, 5 mM DTT, 1 μg ml−1 aprotinin, 1 μg ml−1 AEBSF, 1 μg ml−1 E-64, 1 μg ml−1 Pepstatin A, 1 μg ml−1 Chymostatin, 1 μg ml−1 Antipain, 2 μg ml−1 Leupeptin). Tubulin containing fractions were precipitated by adding 375 mg of ammonium sulphate per ml and incubating overnight at 4 °C. The precipitate was pelleted at 10,000 g in an SS34 rotor (Thermo) for 30 min at 4 °C then resuspended in 12 ml of resuspension buffer (100 mM K-PIPES (pH 6.9), 1 mM MgSO4, 2 mM EGTA, 0.1 mM GTP, 1 μg ml−1 aprotinin, 1 μg ml−1 AEBSF, 1 μg ml−1 E-64, 1 μg ml−1 Pepstatin A, 1 μg ml−1 Chymostatin, 1 μg ml−1 Antipain, 2 μg ml−1 Leupeptin) at 4 °C for 45 min. After centrifugation at 18,000 g in a microcentrifuge for 2 min at 4 °C the supernatant was desalted into resuspension buffer using a HiPrep 26/10 desalting column . Tubulin fractions were loaded onto a 5 ml HiTrap Q HP column  and washed with Q low-salt buffer (100 mM K-PIPES (pH 6.9), 1 mM MgSO4, 2 mM EGTA, 50 mM KCl, 50 μM GDP) then eluted using a 0–70% gradient of Q high-salt buffer (100 mM K-PIPES (pH 6.9), 1 mM MgSO4, 2 mM EGTA, 1 M KCl, 50 μM GDP). Tubulin-containing fractions were polymerized by adding GMPCPP to 0.6 mM, 1/10 vol of DMSO and 1/10 vol of glycerol then incubating for 1 h at 32 °C. MTs were pelleted at 124,000 g in a TLA100.3 rotor (Beckman) for 10 min at 32 °C and the pellet washed with 100 mM K-PIPES (pH 6.9), 1 mM MgSO4, 2 mM EGTA, 20 μM GMPCPP at 32 °C. Pellets were resuspended in 5 ml of 20 mM Na-PIPES (pH 6.9), 1 mM MgSO4, 5 mM CaCl2, 1 mM GDP at 32 °C for 10 min then incubated at 4 °C for 1 h with mixing. After centrifugation at 124,000 g in a TLA 100.3 rotor (Beckman) for 10 min at 4 °C the supernatant was loaded on a Superdex 200 XK16/60 gel filtration column  equilibrated with 100 mM K-PIPES (pH 6.9), 1 mM MgSO4, 2 mM EGTA, 150 mM NaCl, 50 μM GTP and eluted in the same buffer. The tubulin was then desalted into 100 mM K-PIPES (pH 6.9), 1 mM MgSO4, 2 mM EGTA, 20 μM GDP using a HiPrep 26/10 desalting column . The tubulin was then concentrated to 30–40 μM using a Vivaspin 15R 10,000 mwco Spin Concentrator (Viva products) at 2,675 g in a swing out rotor (Centra MP4R centrifuge) at 4 °C. The tubulin was aliquoted, flash-frozen and stored in liquid nitrogen. Tubulin concentration was measured using E280=108,390 M−1 cm−1.E. coli containing the pET vector expression constructs were grown to OD600=0.6 in LB, 100 μg ml−1 ampicillin at 37 °C, 250 r.p.m. Temperature was reduced to 20 °C, expression induced by adding IPTG to 0.5 mM and incubation continued at 20 °C, 250 r.p.m. for 6 h. Cells were pelleted at 1,500 × g in an SLA-3000 rotor (Thermo) at 4 °C for 15 min, then 12 g of cell pellet was resuspended in 60 ml of 15 mM Bicine (pH 8.0), 2 mM Mg Acetate, 0.4 M NaCl, 1 mM PMSF containing protease inhibitors . Cells were lysed by sonication (4 × 15 s at 4 °C) and the homogenate centrifuged in a T865 rotor (Thermo) at 206,000 g for 20 min at 4 °C. The supernatant was batch bound to 4 ml of Nickel-NTA resin (Qiagen) then packed in an XK 16/20 column  and washed in 15 mM Bicine (pH 8.0), 2 mM MgAcetate, 0.4 M NaCl, 25 mM imadazole using an Akta purifier . Mal3 was eluted using 15 mM Bicine pH 8.0, 2 mM MgAcetate, 0.4 M NaCl, 100 mM imadazole then loaded on a Superdex 200 16/60 gel filtration column  equilibrated with 100 mM K-PEM (pH 6.9), 1 mM MgSO4, 2 mM EGTA, 100 mM NaCl and eluted in the same buffer. Aliquots were flash-frozen and stored in liquid nitrogen. Mal3-308 concentration was determined using E280=37,025 M−1 cm−1 and Mal3-143 using E280=32,555 M−1 cm−1.Mal3-308 full-length and Mal3-143 encoding the N-terminal 143 amino acids of Mal3 were expressed, purified and quantifiedv/v) mixture of methanol and HCl for 30 min, rinsed by sonication in ultrapure water  for 4 × 5 min, sonicated in 0.2 M KOH for 60 min, then washed by sonication in ultrapure water 5 × 5 min. Coverslips were spun  and then dried in an oven at 45 °C before silanization in 0.05% (v/v) dichlorodimethylsilane (DDS) in trichloroethylene (TCE) for 1 h. Coverslips were rinsed in methanol, then sonicated 6 × 5 min in methanol before being spun dry and stored in a desicator (Secador). Slides (Matsunami) were used without further processing. Flow cells were assembled by attaching the silanized coverslip to the slide using double-sided sticky tape .Coverslips were cleaned, silanized and flow cells assembled4, 2 mM EGTA (Fisher) adjusted to pH 6.9 with KOHv/v) Glycerol in K-PEM at 90,000 g in a TLA100 rotor at 35 °C for 5 min using a Beckman Optima ultracentrifuge. The supernatant was discarded and the seeds resuspended in K-PEM buffer at 25 °C.Dual-labelled B-lattice single-seam MT seeds were assembled using 0.68 μM pig brain tubulin, 0.32 μM Alexa-680-labelled pig brain tubulin, 0.1 μM Alexa-488-labelled pig brain tubulin, 0.5 mM GMPCPP in K-PEM buffer , 1% (v/v) 2-mercaptoethanol in K-PEM.A flow cell was directly coated with rat Kinesin-1 rK430GSTS. pombe MTs were assembled by incubating 10 μM S. pombe tubulin, 1 mM GTP in K-PEM at 37 °C for 30 min. A-lattice MTs were assembled by incubating 5 μM S. pombe tubulin, 5 μM Mal3FL, 1 mM GTP in K-PEM at 37 °C for 30 min. MTs were then flushed into a flow cell coated with rKin430GST. After attachment for 10 min the flow cell was flushed with K-PEM buffer to remove unattached MTs and Mal3. B-lattice motility was examined by flushing in 1 μM Mg.ATP, GCO oxygen scavenger  in K-PEM.B-lattice single-seam GDP 4, 2 mM EGTA) at 4 °C for 20 min then 37 °C for 60 min. An extension mix of 1 μM pig brain tubulin. containing 10% Alexa-488-labelled subunits in 50 μM Mal3FL, 0.5 mM GMPCPP, K-PEM was incubated at 4 °C for 20 min then pre-warmed at 30 °C for 10 min. Alexa-680 seeds (1/10 volume) were added to the extension mix and incubated at 30 °C for 15 min and then 37 °C for 45 min. The MTs were pelleted at 90,000 g in a TLA100 rotor (Beckman) for 5 min at 35 °C then resuspended in K-PEM. The MTs were incubated at room temperature before use to allow them to anneal to create the ‘lemur tail’ pattern.A-lattice GMPCPP MT seeds with alternating segments of fluorescent labelling (Lemur tail patterned)S. pombe single-isoform tubulin, 1 mM GTP and GCO oxygen scavenger in K-PEM.A flow cell was coated with Anti-Alexa-488 antibodies (Molecular Probes) diluted 10-fold with K-PEM, then GMPCPP Alexa-488-labelled B-lattice single-seam or A-lattice-enriched pig tubulin MT seeds were added. Flow cells were flushed with K-PEM buffer to remove unbound seeds and, for A-lattice-enriched seeds, any residual Mal3 used for co-assembly. Dynamic MTs were assembled from the seeds using unlabelled 5.5 μM EM+DU-897E, Andor). Epifluorescence illumination used a stabilized mercury lamp  coupled to the microscope via a light pipe. For dark-field illumination a 100-W mercury lamp was connected via a fibre optic light scrambler , cold mirror and GIF 500–568 nm band-pass filter (Nikon). Fluorescence excitation and emission filters (Chroma) E460SPUVv2 and D525/20m for Alexa-488 together with S654/24x and S710/60m for Alexa-680 plus ND filters were mounted in motorized filter wheels (LEP MAC5000). An FF502/670-Di01 dichroic mirror (Semrock) permitted fluorescence and dark-field imaging. Electronic shutters were used to rapidly switch between fluorescence and epifluorescence modes. Metamorph software (Molecular Devices) was used to control the microscope and camera. Images for GTP dynamics were captured at 0.5 s intervals using 100 ms exposures at a spatial resolution of 0.128 μm pixel−1. GMPCPP kinesin-clamp assays used 1 s intervals and 0.128 μm pixel−1.A Nikon E800 fluorescence microscope with a customized dark-field illumination system was used for image capturehttp://imagej.nih.gov/ij/, 1997–2012) to automatically digitize MT end locations from the KymographsMetamorph software was used to generate kymographs of dynamic MTs. A custom macro GetEdge was used in ImageJ  or Minitab software.M.K. and D.R.D. carried out the experiments. M.K., D.R.D. and R.A.C. devised and analysed experiments and prepared the manuscript.How to cite this article: Katsuki, M. et al. Ectopic A-lattice seams destabilize microtubules. Nat. Commun. 5:3094 doi: 10.1038/ncomms4094 (2014).Supplementary Table 1 and Supplementary ReferencesB-lattice MTs (right hand panel B) assembled from pig brain tubulin labelled with Alexa-480 fluorescent dye and GMPCPP, and A-lattice enriched MTs (left hand panel A-rich) co-assembled from pig brain tubulin labelled with Alexa-488, Mal3-N143 (CH domain) and GMPCPP were added to flow cells containing rat kinesin-1 motor protein dimers. Flow cells were flushed with buffer to remove Mal3 and unbound MTs. Buffer containing ATP was added and MT motility observed by timelapse epi-fluorescence microscopy. A-lattice enriched MTs shrink faster than B-lattice MTs. Time is in minutes: seconds. Scale bar is 10 μm.B-lattice MTs (red) were assembled using pig brain tubulin labelled with Alexa-680 and Alexa-480 fluorescent dyes and GMPCPP, and A-lattice enriched MTs (green) co-assembled with pig brain tubulin labelled with Alexa-488, Mal3FL and GMPCPP. After separate assembly reactions the B and A-lattice enriched MTs were mixed and added to a flow cell coated with rat kinesin-1 motor protein dimers. The flow cell was flushed with buffer to remove Mal3 and unbound MTs. Buffer containing ATP was added and MT motility observed by timelapse epi-fluorescence microscopy. A-lattice enriched MTs shrink faster than B-lattice MTs. Time is in minutes: seconds:milliseconds. Scale bar is 15 μm.pombe single isoform tubulin and GTP was added and MT dynamics observed using timelapse epifluorescence (for seeds) and darkfield (for dynamic unlabelled S. pombe MTs) microscopy. The MTs grown from the A-lattice enriched seeds had a higher catastrophe frequency and the minus ends a faster shrinkage rate. Green, Fluorescent MT seed; Grey, unlabelled dynamic MT; Yellow triangle, faster growing MT plus end. Time is in minutes: seconds. Scale bar is 3 μm.B-lattice MT seeds (lower panel red outline) were assembled from pig brain tubulin labelled with Alex-488 fluorescent dye and GMPCPP. A-lattice enriched MT seeds (upper panel green outline) were co-assembled from pig brain tubulin labelled with Alexa-488, Mal3FL and GMPCPP. Seeds were attached to flow cells coated with anti-Alexa488 antibodies. Flow cells were flushed with buffer to remove Mal3 and unbound MTs. Buffer containing unlabelled S. pombe single isoform tubulin (grey) was added and MT dynamics observed using timelapse epifluorescence (for seeds) and darkfield (for dynamic unlabelled S. pombe MTs) microscopy. MTs grown from the A-lattice enriched seeds had frequent episodes of transient short MT growth, which we have named piku-piku or MT dithering (blue label). Time is in minutes. Scale bar is 5 μm.A-lattice enriched MT seeds (green) were co-assembled from pig brain tubulin labelled with Alexa-488, Mal3FL and GMPCPP. Seeds were attached to flow cells coated with anti-Alexa488 antibodies. Flow cells were flushed with buffer to remove Mal3 and unbound MTs. Buffer containing GTP and unlabelled S.


Artemia franciscana by repeated lipid extraction, alkaline methanolysis, acid treatment, successive column chromatography, and post-source decay measurements by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Structures of the glycosphingolipids were elucidated by conventional structural characterization and mass spectrometry, and the compounds were identified as GlcNAcβ1-3GalNAcβ1-4(GlcNAcα1-2Fucα1-3)GlcNAcβ1-3Manβ1-4Glcβ1-Cer, GalNAcβ1-4(Fucα1-3)GlcNAcβ1-3GalNAcβ1-4(GlcNAcα1-2Fucα1-3)GlcNAcβ1-3Manβ1-4Glcβ1-Cer, and GalNAcβ1-4(GlcNAcα1-2Fucα1-3)GlcNAcβ1-3GalNAcβ1-4(GlcNAcα1-2Fucα1-3)GlcNAcβ1-3Manβ1-4Glcβ1-Cer. These compounds also contained a branching, non-arthro-series disaccharide with an α-GlcNAc terminus, similar to that found in a previously reported ceramide hexasaccharide (III3(GlcNAcα2Fucα)-At4Cer). The glycans within these complex GSLs are longer than reported glycans of the animal kingdom containing α-GlcNAc terminus. These complex GSLs as well as the longest GSL with ten sugar residues, ceramide decasaccharide (CDeS), contain the fucosylated LacdiNAc sequence reported to associate with parasitism/immunosuppression and the α-GlcNAc terminus reported to show a certain antibacterial effect in other reports. CDeS, the longest GSL of this species, was found in the highest amount, which indicates that CDeS may be functionally important.Brine shrimp are primitive crustacean arthropodal model organisms, second to daphnia, which can survive in high-salinity environments. Their oviposited cysts, cuticle-covered diapausing eggs, are highly resistant to dryness. To elucidate specialties of brine shrimp, this study characterized glycosphingolipids, which are signal transduction-associated material. A group of novel and complex fucosyl glycosphingolipids were separated and identified from cysts of the brine shrimp The online version of this article (doi:10.1007/s10719-012-9436-8) contains supplementary material, which is available to authorized users. Glycosphingolipid (GSL) is an amphipathic compound consisting of a sugar chain and a ceramide composed of a fatty acid and sphingoid base. GSLs are ubiquitous on the outer surface of the plasma membrane in animal cells, aggregated into patches called microdomains, and play an essential role in intercellular interaction and recognition –3. Howev3Cer) has been characterized. Functional analyses of GSLs by knockout experiments of egghead and brainiac have shown that At3Cer is essential for insect development 1H-NMR as previously reported +), 1495 ([M−2HexNAc+Na]+), 1348 ([M−2HexNAc−Fuc+Na]+), 1144 ([M−3HexNAc−Fuc+Na]+), 941 ([M−4HexNAc−Fuc+Na]+), and 779 ([M−4HexNAc−Fuc−Hex+Na]+), corresponding to CMS with d16:1/C22:0 , 1551 ([M−HexNAc−Fuc+Na]+), 1496 ([M−2HexNAc+Na]+), 1349 ([M−2HexNAc−Fuc+Na]+), 1292 ([M−3HexNAc+Na]+), 1145 ([M−3HexNAc−Fuc+Na]+), 942 ([M−4HexNAc−Fuc+Na]+), and 779 ([M−4HexNAc−Fuc−Hex+Na]+), corresponding to CMS with d16:1/C22:0 , 1697 ([M−2HexNAc+Na]+), 1494 ([M−3HexNAc+Na]+), 1348 ([M−3HexNAc−Fuc+Na]+), 1145 ([M−4HexNAc−Fuc+Na]+), 941 ([M−5HexNAc−Fuc+Na]+), and 779 ([M−5HexNAc−Fuc−Hex+Na]+), corresponding to CMS with d16:1/C22:0 , 1843 ([M−HexNAc+Na]+), 1754 ([M−2Fuc+Na] +), 1697 ([M−HexNAc−Fuc+Na]+), 1552 ([M−HexNAc−2Fuc+Na]+), 1494 ([M−2HexNAc−Fuc+Na]+), 1348 ([M−2HexNAc−2Fuc+Na]+), 1290 ([M−3HexNAc−Fuc+Na]+), 1145 ([M−3HexNAc−2Fuc+Na]+), 941 ([M−4HexNAc−2Fuc+Na]+), and 779 ([M−4HexNAc−2Fuc−Hex+Na]+), corresponding to CMS with d16:1/C22:0 , 2047 ([M−HexNAc+Na]+), 1901 ([M−HexNAc−Fuc+Na]+), 1698 ([M−2HexNAc−Fuc+Na]+), 1494 ([M−3HexNAc−Fuc+Na]+), 1348 ([M−3HexNAc−2Fuc+Na]+), 1291 ([M−4HexNAc−Fuc+Na]+), 1144 ([M−4HexNAc−2Fuc+Na]+), 941 ([M−5HexNAc−2Fuc+Na]+), and 779 ([M−5HexNAc−2Fuc−Hex+Na]+), corresponding to CMS with d16:1/C22:0 , 2104 ([M−HexNAc−Fuc+Na]+), 2046 ([M−2HexNAc+Na]+), 1900 ([M−2HexNAc−Fuc+Na]+), 1697 ([M−3HexNAc−Fuc+Na]+), 1494 ([M−4HexNAc−Fuc+Na]+), 1348 ([M−4HexNAc−2Fuc+Na]+), 1290 ([M−5HexNAc−Fuc+Na]+), 1144 ([M−5HexNAc−2Fuc+Na]+), 940 ([M−6HexNAc−2Fuc+Na]+), and 778 ([M−6HexNAc−2Fuc−Hex+Na]+), corresponding to CMS with d16:1/C22:0  for β-Glc, at 4.52 ppm  for β-Man, at 4.60 and 4.63 ppm  for β-GlcNAc, at 4.29 ppm  for β-GalNAc, and at 4.99 and 4.98 ppm  for overlapped α-Fuc and α-GlcNAc ; at 4.15 ppm  for β-Glc, at 4.51 ppm  for β-Man, at 4.58 and 4.65 ppm  for β-GlcNAc, at 4.35 ppm  for β-GalNAc, and at 5.01 and 5.02 ppm  for overlapped α-Fuc and α-GlcNAc ; at 4.15 or 4.16 ppm  for β-Glc, at 4.52 ppm  for β-Man, at 4.63 and 4.64 ppm  for β-GlcNAc, at 4.32 and 4.34 ppm  for β-GalNAc, at 5.01 ppm  for overlapped α-Fuc and α-GlcNAc, and 4.84 ppm  for terminal α-Fuc  for β-Glc, at 4.51 ppm  for β-Man, at 4.64 and 4.68 ppm  for β-GlcNAc, at 4.34 and 4.37 ppm  for β-GalNAc, and at 5.02 ppm  for overlapped α-Fuc and α-GlcNAc -III3-(GlcNAcα2Fucα)-At4Cer (CHpS2), IV3-(fucosyl-LacdiNAc)-III3-(GlcNAcα2Fucα)-At4Cer (CNS), and IV3-(2-α-N-acetylglucosaminylfucosyl-LacdiNAc)-III3-(GlcNAcα2Fucα)-At4Cer (CDeS). The following complex GSL structures were also analyzed: IV3,4--IV3,4-(GlcNAcα2Fucα)-At4Cer (CHpS1), GalNAc1-3,4GlcNAc1-2Fuc1-3GalNAc1-4GlcNAc1-3Man1-4Glc1-Cer (COS1), and IV3- (fucosyl-LacdiNAc)-III3-(Fucα)-At4Cer (COS2). Although “1,3,4GlcNAc” was observed as a branching GlcNAc in both CHpS1 and COS1, it was impossible to confirm sugar substitution at the C3/C4 position without hydrolysis analysis, which requires a large amount of CHpS1 and COS1.The following complex GSL structures were speculated by means of the conventional analyses plus MALDI PSD measurements: IV1H-NMR spectroscopy to identify anomeric configurations by using only 1-mg samples of each complex GSL separated from A. franciscana. Although PSD spectra of complex GSLs were confusing, sugar sequence analysis using sequences speculated from fragments, with the ceramide moiety observed in the high mass region, were consistent with sequences speculated from mere glycan fragments observed in the low mass region.Structural characterization of complex GSLs can be a painstaking process because the results obtained by different analyses must converge on a single structure. For example, characterization by liquid chromatography-mass spectrometry (LC-MS) and tandem MS (MS-MS) provides accurate mass values of ceramide moieties, fatty acids, and sphingoids, but not of sugar species. It is also possible that liquid chromatographic separation would not be able to fully separate GSLs with different sugar chains of identical molecular weight. The conventional analyses, which consist of GC and MS, can be used to determine sugar species and molar ratios without overlooking the possibility of GSLs with different sugar chains of identical molecular weight. Another benefit of the conventional analysis is the ability to determine branching structures by hydrolysis. No matter the analysis, it is difficult to characterize complex GSLs because each result is also complex. A few decades ago, characterization of a complex GSL required 10 mg of isolated GLS for chemical degradation, which liberated shorter GSLs, followed by GC analysis to determine sugar sequence and exoglycosidase treatment or chromic acid oxidation to determine anomeric configurations. In this study, we conducted MALDI PSD analysis to determine sugar sequence and A. franciscana. Of them, a fucosylated LacdiNAc trisaccharide structure  was found in CHpS2, COS2, CNS, and CDeS, similar to a previous report on CHS [S. mansoni [This study examined the structures of complex GSLs separated from the brine shrimp t on CHS . The tri mansoni  and in t mansoni .2, CNS, and CDeS, as described in previous reports on nAtCTeS and CHS. The α-anomeric GlcNAc residue is rare, and there have been few reports of this residue among the carbohydrate portions of GSLs and glycoproteins. A human gastric mucin with an α-anomeric GlcNAc residue could arrest the proliferation of Helicobacter pylori [The arthro-series core structure and a branching non-arthro-series disaccharide were also found in CHpSr pylori .2 is elongated with an additional Fucα group, forming COS, and that COS is further elongated with branching GalNAcβ to form CNS. Although we could not separate COS as a direct precursor of CNS, we believe it exists in the biosynthetic pathway  residue in the rainbow trout Salmo gairdneri [We speculate that CHpSway Fig. . CNS is cing end , 28, 29.airdneri .Fig. 6Su3Cer, II3Fucα-MacCer (non-arthro-CTS), At4Cer, II3(GlcNAcα2Fucα)-MacCer (non-arthro-CTeS), III3Fucα-At4Cer (CPS), III3(GlcNAcα2Fucα)-At4Cer (CHS), CHpS1, CHpS2, COS1, COS2, CNS, and CDeS as GSLs of A. franciscana. The complex GSLs reported this time are as the novel fucomannolipids. However, we confirmed that gangliosides and their functional alternative materials (other acidic GSLs) were below the limit of detection. In mammals, acidic GSLs such as gangliosides play important roles in the formation of the plasma membrane surface environment for signal transduction. It is interesting that A. franciscana can survive without significant amounts of acidic GSLs.A series of structural analyses, including this study, has established the existence of sphingomyelin as a sphingophospholipid and GlcCer, MacCer, AtA. franciscana cysts, which are developmentally diapaused gastrulae, we established a GSL profile during embryonic development. Similar GSL analyses have been carried out in other species, including the frog Xenopus laevis [Gallus gallus domesticus [Mus musculus [A. franciscana is a very suitable organism for studying GSL profiles during early embryogenesis, as they require 5 days from fertilization to the gastrula stage.Performing structural analyses of complex GSLs in s laevis , the chimesticus , and themusculus . HoweverA. franciscana, there are two reproduction patterns: oviparity, which generates diapausing eggs (cysts), and ovoviviparity, which generates nauplii. Oviparous eggs are resistant to dryness due to the existence of the heat shock protein p26 [et al. [A. franciscana, might be of major significance in studies of embryogenesis and signal transduction.In the brine shrimp tein p26 , which s [et al.  conducteA. franciscana contains complex GSLs such as CDeS, CHpSes, and COSes. Other invertebrates containing complex GSLs with more than five sugar residues are [L. caesar, C. vicina, Drosophila melanogaster (Insecta), the parasitic nematode Ascaris suum (Nematoda), the liver fluke Fasciola hepatica, the blood fluke S. mansoni (Platyhelminthes: Trematoda), the pseudophyllidean tapeworm Spirometra erinacei, the tapeworm Diphyllobothrium hottai (Platyhelminthes: Cestoda), the earthworm Pheretima hilgendorfi (Annelida: Oligochaeta), the annelidan worm Pseudopotamilla occelata (Annelida: Polychaeta), the bivalves H. schlegeli, C. sandai, M. lusoria, the oyster O. gigas (Mollusca), the abalone Haliotis japonica (Mollusca: Gatropoda), the lamp shell Lingula unguis (Brachiopoda), and the sea urchin Hemicentrotus pulcherrimus (Echinodermata). Of these, only half contain GSLs with the same number of sugar residues and a different sugar chain sequence. As in vertebrates, GSLs with multiple sugar chain sequences may exhibit tissue specificity. These GSLs can be roughly divided into two groups: one with straight complex GSLs, such as those found in flies, the nematode, the blood fluke, and the lamp shell, and those with branching complex GSLs. Complex GSLs with more than seven sugar residues have been reported as follows: GlcNAcβ1-3Galβ1-3GalNAcα1-4GalNAcβ1-4GlcNAcβ1-3Manβ1-4Glcβ1-Cer (At7Cer) from the fly C. vicina [7Cer, VII3-β-GalNAc-At7Cer (At8Cer), and VIII3-β-Gal-At8Cer (At9Cer) from the fly L. caesar [C. sandai [H. schlegeli and M. lusoria [O-methyl sugar residue. Organisms can also be divided into two groups according to the presence or absence of acidic or polar GSLs. The former group includes the flies and the bivalves. The latter group contains A. franciscana, the blood fluke, and the oyster. Furthermore, a fucosylated LacdiNAc structure and a GlcNAcβ1-3GlcNAc disaccharide are common to both A. franciscana and the blood fluke. Shorter GSLs seem to be conserved within the same phyla while longer GSLs are more commonly unique to a given species.dues are  the flie. vicina ; At7Cer,. caesar ; Fucα3Ga. caesar –14; Gal4. sandai ; Fuc3Meα lusoria , 17; and lusoria . All of A. franciscana showed that the ceramide composition is common among nearly all neutral GSLs, irrespective of the length of the sugar chain, with the exception of CMS, which contains hydroxy fatty acid, similar to millipedes [L. caesar and C. vicina [A. franciscana; d17:1 and smaller amounts of branched d18:1 as sphingoid; C22:0 and smaller amounts of C23:0/C24:0 as fatty acid in the millipede; d14:1 and smaller amounts of d16:1 as sphingoid; C20:0 and smaller amounts of C18:0/C22:0 as fatty acid in the flies and in the High Five insect cell line derived from the cabbage looper Trichoplusia ni [A. franciscana and the millipede, especially odd-numbered sphingoids, with the highest amount being C22:0. In addition, all of the neutral GSLs isolated from flies were straight, while the millipede contained a branching neutral GSL [A. franciscana contained branching neutral GSLs and sphingomyelin instead of a sphingolipid with a phosphoryl-ethanolamine group [A. franciscana is more closely related to the millipede than to insects.This study of the entire GSL structure in llipedes , but not. vicina , 11, 36.lusia ni . As menttral GSL  and a cetral GSL  instead tral GSL . A. franne group . This coD. melanogaster and other species, which might aid in the elucidation of a relationship between morphogenesis and the functions of GSLs in Crustacea.Currently, it is not possible to use molecular biological approaches in brine shrimp because its genome has not been sequenced. However, there has been a report detailing the RNAi knockdown of a homeotic gene using microinjection . It has Fig. S1a) CHpS2; (b) COS1; (c) COS2; and (d) CDeS. (PDF 115 kb)Positive-ion PSD spectra in MALDI-TOF MS of the separated GSLs. (Table S1A. franciscana. Symbols A and B, denoted in the left column, show predominant peaks mentioned in Fig. Summary of MALDI-TOF MS spectra obtained for complex GSLs purified from Below is the link to the electronic supplementary material.


IV atom in the title carboxylate-bridged polymer, [Sn(C7H7)3(C9H7O2)]n, exists in a trans-C3SnO2 trigonal–bipyramidal geometry . The polymer propagates as a helical chain along the b axis with a repeat distance that is half the b-axial length. There are four independent formula units in the asymmetric unit; two are disposed about a false center of inversion with respect to the other two so that the space group emulates a centric space group.The Sn For the al. 1995. For the al. 2008. For the al. 2008; Spek 3(C9H7O2)] = 0.042                           wR(F                           2) = 0.087                           S = 0.97                           22492 reflections1189 parameters1 restraintH-atom parameters constrainedmax = 0.83 e Å−3                        Δρmin = −0.79 e Å−3                        ΔρAbsolute structure: Flack 1983, 10328 FFlack parameter: 0.00 (2)                                 APEX2  used to solve structure: SHELXS97 (Sheldrick, 2008SHELXL97 (Sheldrick, 2008X-SEED (Barbour, 2001publCIF (Westrip, 2010Data collection: 10.1107/S1600536811017247/si2355sup1.cif            Crystal structure: contains datablocks global, I. DOI: 10.1107/S1600536811017247/si2355Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


LPS and LTA increased intracellular Ca2+ concentrations while Ca2+ chelation by BAPTA decreased LPS- and LTA-mediated cytokine responses. BAPTA blocked the effects of low pH stress on most of LPS-stimulated cytokines but not of LTA-stimulated responses. In vivo mouse model demonstrates suppressed E. coli and S. pneumoniae clearance following acid aspiration. In conclusion, low pH stress inhibits antibacterial cytokine response of aMØs due to impaired TLR2 (MyD88 pathway) and TLR4 signaling (MyD88 and TRIF pathways). The role of Ca2+ in low pH stress-induced signaling is complex but appears to be distinct between LPS- and LTA-mediated responses.Gastric aspiration increases the risks for developing secondary bacterial pneumonia. Cytokine elaboration through pathogen recognition receptors (PRRs) is an important mechanism in initiating innate immune host response. Effects of low pH stress, a critical component of aspiration pathogenesis, on the PRR pathways were examined, specifically toll-like receptor-2 (TLR2) and TLR4, using isolated rat alveolar macrophages (aMØs). We assessed the ability of aMØs after brief exposure to acidified saline to elaborate proinflammatory cytokines in response to lipopolysaccharide (LPS) and lipoteichoic acid (LTA) stimulation, known ligands of TLR4 and TLR2, respectively. Low pH stress reduced LPS- and LTA-mediated cytokine release (CINC-1, MIP-2, TNF- Gastric aspiration pneumonitis is a major cause of death and disability. An important complication of this acute lung injury is the development of a secondary pulmonary bacterial pneumonia from inhalation of oropharyngeal or gastric bacterial flora. Patients that are specifically susceptible to gastric aspiration pneumonitis are those with impaired airway defense reflexes such as anesthetized patients or patients with altered mental status. Consequences of low pH gastric aspiration can range from benign aseptic pneumonitis to acute respiratory distress syndrome (ARDS) with or without a fulminant secondary bacterial infection. It is estimated that complications from gastric aspirations can be seen in 1 of every 2,000–4,000 anesthetic cases every year and possibly 10–15% of all community acquired pneumonia i) is an important regulator of macrophage function [2+]i in mediating the effects of low pH stress on LPS-mediated responses, changes in [Ca2+]i were determined using the Ca2+-sensitive fluorescent dye, Fura-2 AM (see R340/380) were measured at different time intervals within this 24 hr period to identify variations in the temporal Ca2+ levels: immediate postexposure to LPS, after 30 min, 1 hr, 4 hrs, and 24 hrs of LPS exposure. Intracellular Cafunction –18. To a2 AM see . AMØs we2+]i due to low pH stress alone were assessed. Low pH stress increased R340/380, correlating with increased [Ca2+]i, in a biphasic pattern . This was followed by another rise in R340/380 at 4 hrs and 24 hrs after exposure . First, changes in i. Changes in [Ca2+]i were assessed at increasing duration of LPS incubation as described earlier. R340/380 increased immediately following LPS stimulation from control levels of 0.77 ± 0.01 to 1.04 ± 0.06 (P = 0.02)  . The ele2+]i profile was then investigated. AMØs exposed to a transient low pH stress prior to LPS stimulation resulted in increased [Ca2+]i at 30 min after LPS exposure (P < 0.001). The increased R340/380 persisted at the 1 hr (P < 0.001), 4 hrs (P < 0.001), and 24 hrs LPS exposure times (P < 0.001).The effect of low pH stress on the LPS-mediated i in mediating the effects of low pH stress on LTA-mediated responses, we identified changes in [Ca2+]i in response to LTA alone and following combined low pH stress and LTA stimulation (R340/380 from baseline levels of 0.40 ± 0.00 to 1.50 ± 0.06 (P < 0.001). This elevation persisted at 30 min and returned to baseline levels at 1 hr . After an initial return to baseline levels, the R340/380 ratio developed a second wave of increased [Ca2+]i at 4 hrs and 24 hrs, 0.62 ± 0.02 (P < 0.001 versus baseline) and 0.58 ± 0.03 (P < 0.001), respectively. To examine the role of [Camulation . In Fura2+ profile was then investigated. LTA-stimulated aMØs were exposed to low pH conditions and changes in R340/380 monitored. There was immediate suppression by low pH stress on the LTA-induced [Ca2+]i increase, 1.50 ± 0.06 (LTA) versus 1.15 ± 0.07 (LTA/Low pH), P < 0.01, and the [Ca2+]i suppression persisted at 30 min after LTA exposure. At 1 hr and 4 hrs, there was no difference in the LTA-stimulated response, with or without low pH stress. At 24 hrs, low pH stress resulted in a robust increase in R340/380, 0.58  ±  0.03 (LTA) versus 1.45  ±  0.25 (LTA/Low pH), P < 0.001. The effect of low pH stress on LTA-induced Ca2+]i are induced by LPS and LTA, and the effect of low pH stress on [Ca2+]i appears unique to the specific ligand/TLR interaction. In LPS-stimulated cells, low pH stress resulted in low level augmentation of [Ca2+]i while, in LTA-stimulated cells, low pH stress initially suppressed the [Ca2+]i increase but was followed by a robust augmentation in [Ca2+]i.These findings indicate that complex changes in i [2+]i is necessary as an intermediate signal for LPS responses, BAPTA-AM will effectively inhibit these responses. AMØs were stimulated with LPS in the presence or absence of BAPTA-AM and cytokine production quantified. BAPTA-AM alone (control) did not result in any significant change in measured cytokines (P < 0.001), MIP-2 by 67% (P < 0.001), MCP-1 by 79% (P < 0.001), and IFN-β (P < 0.001) from their LPS treatment levels. TNF-α levels trended down but were not statistically significant (P = 0.07).To further investigate the role of Ca [Ca2+]i . The genytokines . In LPS 2+-chelated, LPS-stimulated aMØs (β production in LPS/BAPTA-AM-treated macrophages but significantly reduced TNF-α production by 82% (P < 0.001). It was subsequently tested whether low pH stress can further modulate cytokine production in Cated aMØs . Low pH P < 0.001), MIP-2 by 53% (P < 0.01), TNF-α by 65% (P < 0.05), and MCP-1 by 51% (P < 0.05), when compared with LTA treatment in the absence of BAPTA-AM. It was subsequently investigated whether low pH stress would further modulate cytokine production in Ca2+-chelated, LTA-stimulated aMØs (P < 0.001), MIP-2 by 46% (P < 0.01), and TNF-α by 82% (P < 0.001) in LTA/BAPTA-AM-treated cells. Low pH stress tended to suppress MCP-1 (P = 0.06). A set of experiments were performed using LTA-stimulated aMØs with BAPTA-AM. BAPTA-AM, in LTA-stimulated macrophages , reducedted aMØs . Low pH in vivo, mice were exposed to an acidic pulmonary aspiration injury followed by intratracheal instillation of either E. coli or S. pneumoniae  . Twenty-four hours after intratracheal E. coli challenge, resulting lung bacterial loads in the absence of low pH injury were 5.3 × 103 ± 4.4 × 103 cfu (n = 13) versus 1.1 × 105 ± 0.3 × 105 cfu (n = 17) in the presence of low pH injury, demonstrating an increased E. coli load after low pH injury. Estimated E. coli clearance  was 3.7 ± 0.2 versus 1.9 ± 0.1, respectively (P < 0.001). In animals inoculated with S. pneumoniae, starting inoculum was 4.5 × 105 ± 0 (n = 5). Twenty-four hours after intratracheal S. pneumoniae challenge, resulting lung bacterial loads in the absence of low pH injury were 5.8 × 103 ± 2.9 × 103 cfu versus 8.4 × 107 ± 3.1 × 107 cfu in the presence of low pH injury, demonstrating a profound increase in total S. pneumoniae load, in excess of the starting inoculum. Estimated S. pneumoniae clearance was 2.4 ± 0.4 versus −2.19 ± 0.2, respectively (P < 0.001). To determine the effects of low pH stress on bacterial clearance sp.) see . In animIn vivo animal studies have demonstrated that gastric acid aspiration results in a severe heterogeneous lung injury i are also required for optimal TLR4 trafficking to the endosomes and the phosphorylation/nuclear localization of IRF3 i increases in response to LPS and LTA , low pH stress failed to modify LPS-mediated production of proinflammatory cytokines (2+-dependent mechanisms, while that of TLR2 may involve Ca2+-independent mechanisms.To further investigate the role of Cauestrant , 48–50. ter Ca2+ , 48–50. ytokines  while coytokines . This di2+ is an essential component for efficient elaboration of LPS- and LTA-mediated cytokine response but plays a differential role in mediating the suppressive effects of low pH stress. Changes in [Ca2+]i appear to be required for LPS-stimulated responses but are less important for the LTA-mediated response. The mechanism for this differential role of cytosolic Ca2+ signaling needs further investigation, but the data presented in this study suggest that the mechanistic effect of low pH stress cannot be generalized across the various TLRs signaling cascades. These findings further our understanding of aMØs dysfunction following acute gastric aspiration and the role of this resident sentinel lung cell in the increased susceptibility to bacterial infection. In summary, our findings demonstrate a strong suppressive direct effect of low pH stress on the ability of aMØs to mount an adequate antibacterial proinflammatory response following TLR2 and TLR4 activation. The site of action of the low pH stress appears to involve both the MyD88 and TRIF pathways. Ca


The LuIII atom in the cation is coordinated by eight O atoms from two nitrate groups and four hmp ligands in a distorted square-antiprismatic geometry. Together with the two nitrate groups, 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 161.66 (2) and 153.503 (12)°, respectively. The title complex is isotypic with the Y, Yb, Eu, Nd, La, Dy and Sm analogues.In the title compound, {[Lu(NO For the al. 2007; Zhang 2(C6H18N3OP)4][AgWS4] = 0.043wR(F2) = 0.089S = 1.099613 reflections532 parametersH-atom parameters constrainedmax = 1.35 e Å−3Δρmin = −1.46 e Å−3ΔρCrystalClear  used to solve structure: SHELXTL  I, global. DOI: 10.1107/S1600536812020983/rz2754Isup2.hklStructure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:


There was an error in affiliation 1 for author Pascual López-López. Affiliation 1 should be: Vertebrates Zoology Research Group, CIBIO, University of Alicante, Alicante, Spain


One SmIII ion is eight-coordinated in a bicapped trigonal–prismatic coordination 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 two SmIII ions are nine-coordinated in a tricapped trigonal–prismatic coordination geometry; one of these SmIII 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 SmIII 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. The crystal structure is further stabilized by N—H⋯O, O—H⋯O, and C—H⋯O hydrogen-bonding interactions.In the title three-dimensional compound, [CoSm DOI: 10.1107/S1600536811030169/om2450Isup2.hkl            Structure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


Three-dimensional high-resolution PSIR LGE , flow-in3, FOV=270×270×30 mm3) where one heartbeat (interleaf 1) was acquired with T2prep prepulse, and the subsequent one (interleaf 2) without [The right coronary artery (RCA) of 7 subjects (29.6±12.6 yrs) were imaged on a 1.5T Philips Achieva magnet with a targeted i-T2prep sequence using an interleaved SSFP acquisition (TR/TE/α=4.3/2.1 ms/90°, resolution=1×1×3 mmFigure We have investigated two navigating approaches for high-resolution interleaved sequences. Examples and quantitative data show that while the asynchronous scheme allows a significantly shorter scan time, independently accepting data from interleaves results in loss of image quality and sharpness, even though a 5 mm gating window is utilized. Techniques that require voxel-by-voxel processing of images from multiple interleaves may require motion correction within the gating window if a synchronous scheme cannot be utilized due to a higher number of interleaves.NIH:K99HL111410-01;R01EB008743-01A2; BHF: RG/12/1/29262