To assess the optimal time point of diffusion-weighted imaging (DWI) for early prognosis of breast cancer following tamoxifen therapy using a methylnitrosourea (MNU)-induced ER-positive breast-cancer model.Two groups of Sprague-Dawley rats  were used. All animals (50 days old) were intravenously injected with MNU (50 mg/kg body weight) to induce ER-positive mammary tumors. When tumors were approximately 2 cm in diameter, DWI was performed on days 0, 3, and 7, and intratumoral apparent diffusion coefficient (ADC) values were measured. Therapy started on day 0 with tamoxifen (10 mg/kg diet) and continued for 4 weeks for group 1, but only 1 week for group 2, while tumor volume was measured by caliper twice weekly. All animals of group 2 were euthanized on day 7 after imaging, and Ki-67, TUNEL, ERα, and ERβ staining were performed on tumor tissue.3D) was significantly correlated with tumor-volume change until day 11, but the significant correlation between ADC change for 7 days (ADC7D) and the tumor-volume change was observed until day 18. Similarly, for group 2, either ADC7D or ADC3D was significantly correlated with the tumor-volume change, but the higher significance was observed for ADC7D. Furthermore, ADC7D was significantly correlated with apoptotic (TUNEL stained), proliferative (Ki-67 stained), and ERβ-positive cell densities, but ADC3D was not significantly correlated with any of those.DW images of MNU-induced mammary tumors were successfully obtained with minimal motion artifact. For group 1, ADC change for 3 days after therapy initiation (ADC7D might be a more reliable surrogate imaging biomarker than ADC3D to assess effectiveness of tamoxifen therapy for ER-positive breast cancer, which may enable personalized treatment. The significant correlation between ADC7D and ERβ-positive cell density suggests that ERβ may play an important role as a therapeutic indicator of tamoxifen.ADC Estrogen stimulates cell growth via binding to the estrogen receptor (ER), and approximately 75% of breast cancers in the United States are ER positive. Thus, anti-ER drugs like tamoxifen, toremifene, and fulvestrant have been used for treatment of both early and advanced ER positive breast cancers Individualized optimal treatment, called personalized medicine, can be guided by molecular biomarkers obtained from biopsies, or by the use of imaging biomarkers. Although minimally invasive biopsy techniques are available The primary goal of this study was to estimate the optimal DWI time point, evaluating tamoxifen therapy in higher accuracy, using a methylnitrosourea (MNU)-induced ER-positive breast-cancer murine model. Standard cell-line driven xenograft models, routinely used for drug testing, poorly predict for human cancer response. Tumor cells in culture have selective pressure resulting in less differentiated but more homogeneous cells; therefore, the tumors implanted into animals no longer maintain original characteristics The secondary goal was to explore the potential of ERα and ERβ as prognostic biomarkers for tamoxifen therapy. Estrogen receptors are generally classified into two different kinds such as ERα and ERβ All reagents were from Fisher  unless otherwise specified. MNU was purchased from National Cancer Institute (NCI) Chemical Repository , and teklad mash diet was purchased from Harlan Teklad . Tamoxfien was purchased from Agvar Chemical, Inc .This study strictly followed the recommendations of National Institute of Health (NIH) for the Care and Use of Laboratory Animals. Animal experiments were reviewed and approved by the Committee on the Ethics of Animal Experiments of the University of Alabama at Birmingham (Permit Number: 120609080). All efforts were made for minimizing animal suffering. Two groups of Sprague-Dawley rats  were used. When the rats were 50 days old, they were injected with MNU (50 mg/kg body weight) through the left jugular vein to induce mammary tumors. Tumors formed in multiple places, and one tumor of about 2 cm in diameter was selected. T2-weighted MR imaging and DWI were performed for each tumor on day 0 (baseline), and days 3 and 7 after the initiation of tamoxifen therapy. Two plastic bars with wedges were used to lift up the tumor to separate it from respiratory motion, as illustrated in b values  were applied in the x direction with the following parameters: NEX = 1, TR = 3500 ms, TE = 32 ms, diffusion separation time = 16 ms, diffusion gradient duration = 6 ms, FOV = 30×30 mm, and matrix size = 128×128. A total of seven 1-mm thick slices were used to cover the central tumor region, and ADC value was averaged over all those slices. ADC value was calculated by finding the best fitting curve to the equation, S is the intensity of DW images, 0S is a constant, and D is ADC value. The tumor region of interest (ROI) was determined in T2W images; skin and connective tissue were manually excluded based on tumor anatomy, and then global thresholding technique was applied using ImageJ , while threshold value was manually determined for each image slice. The ROI obtained from T2W images was also used to determine the tumor region in ADC maps; minimal geometrical distortion was observed even in the high-b-value DW images. Tumor volume and ADC quantification was implemented with software developed with Labview 2010, version 10.0.1 . Dr. Zhai implemented all MR image analyses.Small-animal imaging was performed on a 9.4T MR imaging system  with a surface coil (Bruker BioSpin) as a receiver. An MR imaging compatible small-animal respiratory gating device  was used. Anatomic MR images were acquired with a T2-weighted (T2W) fast spin echo sequence (rapid acquisition with relaxation enhancement) with the following parameters: NEX = 1, TR = 3000 ms, TE = 34 ms, rare factor = 4, FOV = 30×30 mm, and matrix size = 128×128. Continuous 1-mm thick slices were used to cover the entire tumor region. Tumor volume was calculated by summing all voxels inside of the tumor boundary of the anatomical MR images. Diffusion weighted imaging was obtained with a diffusion weighted multi-slice 2-dimensional spin echo sequence. Four Ki67 and TUNEL  staining were performed for tumor tissues of group 2 with the same procedure as reported previously 2) instead. Uneven background intensity was corrected using “Rolling Ball” algorithm Two digital pictures (×200) were randomly taken by Dr. Umphrey, a board-certified pathologist, in a blinded manner for each tumor slice that had undergone TUNEL, Ki67, ERα, or ERβ staining using SPOT camera on a Nikon Optiphot-2 microscope , interfaced with personal computer and SPOT software. The apoptotic (TUNEL) cells were segmented by color difference between the target cells (brown) and non-target cells (blue) or background , while tumor cells (either apoptotic or non-apoptotic) were segmented by brightness and roundness differences, and then counted in all two pictures per tumor. Apoptotic cell density (%) was calculated by the ratio of the number of apoptotic cells to the total number of tumor cells. The proliferative (Ki67), ERα, and ERβ expressing cells were segmented by the signal-intensity difference, while the intensity threshold was manually determined; since the boundaries of the non-target cells were often vague, the cell densities were calculated by the number of target cells per unit area  was normalized to 100%; the therapy durations for 50% volume reduction of sensitive, intermediate, and resistant tumors were 5.9±0.3 days (n = 5), 12.3±1.5 days (n = 6), and more than 28 days (n = 4), respectively. −4 mm2/s) were normalized to 0%. The mean ADC changes of sensitive, intermediate, and resistant tumors for 3 days after therapy initiation were 15.5±2.7%, 5.5±4.2%, and 7.5±2.6%, respectively, while those for 7 days were 27.7±6.1%, 12.7±5.6%, and 4.7±1.8%, respectively. Asterisk and hash mark represent statistical difference from the resistant and intermediate groups, respectively. 3D) was significantly correlated with the tumor-volume change until day 11, but the significant correlation between the ADC change for 7 days (ADC7D) and the tumor-volume change was observed until day 18.Differential therapy effect was induced by medium dose of tamoxifen and significantly correlated with early ADC changes of tumor region. 3D of sensitive, intermediate, and resistant tumors were 11.6±3.2%, 2.4±6.0%, and −3.4±0.3%, while ADC7D of those groups were 26.7±7.8%, 9.5±5.5%, and −8.0±2.6%, respectively; similar to the tumor-volume change, statistical significance was detected only between sensitive and resistant groups (p = 0.0058). Also, ADC3D was significantly correlated with the tumor-volume change for either 3 or 7 days , but the higher correlation was observed between ADC7D and tumor-volume change for 7 days . The initial mean tumor volume and ADC value of group 2 were 4.16±0.49 cm3 and 9.34±0.18×10−4 mm2/s, respectively.The significant correlation between the changes of ADC value and tumor volume was observed for group 2 as well. For group 2, the tumor volumes of sensitive and intermediate sub-groups decreased 61.5±4.5% (n = 4) and 27.0±6.2% (n = 3), respectively, but that of resistant sub-group increased 24.4±16.8% (n = 3), during 7 days of therapy; statistical significance was detected between sensitive and resistant groups (p = 0.0138), but not among the others. ADC7D and the cell densities; ADC7D was significant correlated with apoptotic, proliferating, and ERβ-positive cell densities , but not with ERα-positive cell density . The tumor-volume change for 7 days was significantly correlated with proliferating and ERβ-positive cell densities , but not with apoptotic and ERα-positive cell densities (p>0.05). No correlation was observed between ADC3D and any of the cell densities (p>0.05). The tumor-volume change for 3 days was significantly correlated with only ERβ-positive cell density , but not with the others (p>0.05).The early ADC change was significantly correlated with apoptotic, proliferating, and ERβ-positive cell densities, but not with ERα-positive cell density. b values, and it can be also applied for orthotopic breast-cancer xenograft murine models easily. The plastic bars with wedges, however, may create pressure on the tumor region contacting the wedges altering tumor architecture and water diffusion. But, in this study, the plastic bars were applied parallel to the animal body as shown in To our knowledge, this is the first report of DWI application for an MNU-induced breast-cancer murine model. To date, DWI studies of tumors located in mammary fat pad have achieved little success due to severe motion artifact. In this study, however, a simple method to separate a tumor from the chest motion was introduced, enabling to repeatedly obtain DW images with minimal motion artifacts even at high 3D or ADC7D was significantly correlated with long-term tumor volume change, but ADC7D presented higher accuracy presumably because the extra-cellular water molecules were gradually increased during 7 days of therapy for sensitive tumors.The mechanism of apoptosis induced by tamoxifen has been well studied However, care should be taken to propose 7 days post therapy initiation as the optimal DWI time point to evaluate tamoxifen efficacy, because ADC value is dependent not only on the amount of extra-cellular water molecules, but also on interstitial fluid pressure (IFP); the higher IFP a tumor has, the more rapidly water molecules can be pushed out of the ROI. Tumor IFP is mainly determined by microvasculature et alMNU-induced mammary tumors expressed both ERα and ERβ, and the therapeutic efficacy was associated with the level of ERβ, not ERα. However, one study showed that ERβ level assessed by immunohistochemistry was significantly correlated with the improved survival of ERα-negative breast cancer patients during 2-years tamoxifen adjuvant therapy, but not with that of ERα-positive breast cancer patients The feasibility of DWI for early evaluation of tamoxifen therapy was demonstrated for ER-positive breast cancer using a human-like rat model; identification of tamoxifen-sensitive patients using DWI may enable personalized tamoxifen treatment. The significant correlation between early ADC change and ERβ-positive cell density suggests that ERβ may play an important role as a therapeutic indicator of tamoxifen.


OMe and LNA modifications were used to study the mechanism of their impact on translation. It turned out that the remaining RNase H activity of the lysate contributed to modulation of protein synthesis efficiency which was observed in the presence of antisense oligomers. A possibility of changing the ratio of the newly synthetized p53 and ΔNp53 in a controlled manner was revealed which is potentially very attractive considering the relationship between the functioning of these two proteins. Selected antisense oligonucleotides which were designed based on accessibility mapping of the 5′-terminal region of p53 mRNA were able to significantly reduce the level of p53 protein in MCF-7 cells. One of these oligomers might be used in the future as a support treatment in anticancer therapy.The p53 protein is a key player in cell response to stress events and cancer prevention. However, up-regulation of p53 that occurs during radiotherapy of some tumours results in radio-resistance of targeted cells. Recently, antisense oligonucleotides have been used to reduce the p53 level in tumour cells which facilitates their radiation-induced apoptosis. Here we describe the rational design of antisense oligomers directed against the 5′-terminal region of p53 mRNA aimed to inhibit the synthesis of p53 protein and its ΔNp53 isoform. A comprehensive analysis of the sites accessible to oligomer hybridization in this mRNA region was performed. Subsequently, translation efficiency from the initiation codons for both proteins in the presence of selected oligomers was determined in rabbit reticulocyte lysate and in MCF-7 cells. The antisense oligomers with 2′- However, cell radioresistance is observed in some malignant tumours via RNase H-dependent and independent mechanisms The antisense oligonucleotide strategy has been successfully used for decades to inhibit gene expression In our study the rational design of antisense oligonucleotides targeting the 5′-terminal region of p53 mRNA was used. Based on the comprehensive analysis of the sites accessible to oligomer hybridization in a model mRNA transcript several antisense compounds were designed. Modulation of protein translation by these antisense oligonucleotides was tested in rabbit reticulocyte lysate. To gain more details about the mechanisms which are responsible for changes in the synthesis of the full-length p53 and ΔNp53 isoform antisense oligonucleotides bearing different chemical modifications were also used. Subsequently, selected oligomers were applied in MCF-7 cells to verify their potential as regulators of p53 protein expression.5′-CTAGAGCCACCGTCCAGGGAGC-3′ complementary to exon 1, and 5′-TCCATTGCTTGGGACGGCAAGG-3′ complementary to exon 4. The restriction sites were then added using forward primer FXba 5′-AAGTCTAGAGCCACCGTCCAGG-3′ bearing restriction site for XbaI and reverse primer RCsp 5′-TCCATTGCTTGGGACGGCAAGG-3′ with Csp45I restriction site. The PCR reaction mixture contained: 0.5–20 ng of cDNA (or dsDNA) template, 0.5 µM of each forward and reverse primers, 75 mM Tris-HCl pH 8.8, 20 mM (NH4)2SO4, 0.01% Tween 20, 200 µM each dNTP, 1.5 mM MgCl2 and 30 U/ml Taq polymerase (Fermentas). The reaction products were purified by phenol/chloroform  extraction and precipitated with ethanol or purified using DNA Purification System (Promega). The obtained ΔNp53utr DNA insert was cloned into the pRL-CMV vector (Promega) between T7 promoter and Renilla luciferase coding sequence. The sequence of the construct was confirmed by sequencing.The DNA template for the 5′-terminal region of p53 mRNA, ΔNp53utr, was amplified from human liver cDNA (Ambion) using the following forward (F) and reverse (R) primers: OMe oligomer no. 1 and control oligomer were synthesized by Future Synthesis, Poznan. 2′-OMe oligomer no. 7b was a gift from prof. R. Kierzek. All GAP oligomers were synthesized by Exiqon.Primers and unmodified antisense oligonucleotides were synthesized by Oligo Service IBB PAS, Warsaw. 2′-in vitro transcription the plasmid construct was linearized using NotI restriction enzyme. In vitro transcription reaction with AmpliScribe T7, T3 and SP6 High Yield Transcription Kit (Epicentre Biotechnologies) or RiboMax Large Scale RNA Production System-T7 (Promega) was performed as recommended by the manufacturers. The 5′-capped ΔNp53utr-Luc RNA was synthesized in the presence of 3 mM Anti-Reverse Cap Analogue , 1.5 mM GTP and 7.5 mM of the remaining nucleotide triphosphates. After transcription reaction 1 unit of DNase I was added and the reaction was incubated for 15 min at 37°C. The RNA was purified using RNeasy MinElute Cleanup kit (Qiagen). The size and integrity of RNA transcript was verified using agarose gel electrophoresis and ethidium bromide staining.For E. coli RNase H (Fermentas) the 0.5 pmol of ΔNp53utr-Luc RNA was renatured in buffer containing: 40 mM Tris-HCl pH 8.0, 40 mM KCl, 10 mM MgCl2, 1 mM DTT, 0.1 mM EDTA by heating for 2 min at 65°C and slow cooling to 37°C. RNase H was added to the final concentration of 250 units/ml. The cleavage reactions were induced by adding separately four DNA 6-mer libraries  to four RNA samples . The reaction mixtures were incubated for 10 and 30 min at 37°C. The reactions were stopped by adding an equal volume of 20 mM EDTA. The reaction products were purified by phenol/chloroform  extraction and precipitated with 0.3 M sodium acetate pH 5.2, 1 µl of glycogen (20 mg/ml) and 3 volumes of ethanol at −20°C for one hour.Mapping of accessible sites in RNA transcripts was performed as described previously 2 to adjust to 0.5 mM final concentration of Mg2+ ions and the mixture was incubated for 10 min at 37°C. Subsequently, the RNA solution was added to 17.5 µl of RRL to a final volume of 23.75 µl and the mixture was incubated for another 10 min at 30°C. Finally, 1.25 µl of 5% DMS in ethanol  was added except the control reaction. All reactions were incubated for 2 min at 30°C. Modification with DMS was stopped by 10% ice-cold ß-mercaptoethanol. Immediately, the RNA was isolated using 300 µl of TriReagent  according to the manufacturer protocol.Ten pmol of ΔNp53utr-Luc RNA in 100 mM KCl was renatured for 3 min at 90°C and slowly cooled (0.1°C/sec) to 4°C. The RNA solution was supplemented with MgCl32P]-labelled primer 5′-TAATAAATGAATCAAGAACATCC-3′ which was complementary to nucleotides 81–103 in Renilla luciferase coding sequence. The reaction mixtures containing: RNA (0.5–1 pmol), DNA primer (2 pmol) in a final volume of 12 µl were incubated at 90°C for 1 min and placed on ice for 10 min. Subsequently, 8 µl of reverse transcription mix was added and the final reaction conditions were as follows: 50 mM Tris-HCl pH 8.3, 50 mM KCl, 4 mM MgCl2, 10 mM DTT, 500 µM each dNTP and 100 units of RevertAid™ M-MuLV reverse transcriptase (Fermentas). The reaction was incubated at 42°C for 40 min. The sequencing ladders were generated in the same way and the final concentration of each ddNTP was 0.2 mM. The obtained cDNAs were treated with 1 µl of 4 M NaOH, heated at 95°C for 5 min, placed on ice and mixed with 160 mM Trizma. Then cDNAs were precipitated with 0.3 M sodium acetate pH 5.2, 1 µl of glycogen (20 mg/ml) and 3 volumes of ethanol. The samples were heated at 95°C for 2 min in 8 M urea/dyes/20 mM EDTA solution and loaded onto 8% acrylamide, 0.75% bis-acrylamide and 7 M urea gels. The separated products were visualized using FLA 5100 image analyzer (Fuji).To determine RNase H cleavage sites primer extension reaction was performed with 5′-end--methionine (1000 Ci/mol)  and 20 units of recombinant ribonuclease inhibitor (Promega). The reaction mixture was incubated for 2 min at 30°C. Subsequently, antisense oligonucleotides were added to the final concentration of 0.5 or 2 µM and the reactions proceeded at 30°C for 90 min. A control reaction was performed without oligonucleotides. After incubation, RNase A was added to a final concentration of 0.2 mg/ml and the samples were incubated for 5 min at room temperature. Translation reaction products were resolved on 15% SDS-polyacrylamide gels, followed by radioisotope imaging with FLA 5100 image analyzer (Fuji). Band intensities were analyzed using MultiGauge software (Fuji).Translation reactions were performed in nuclease-treated rabbit reticulocyte lysate (RRL) as described by the manufacturer (Promega). 2.5 pmol of 5′-capped ΔNp53utr-Luc RNA in a volume of 5.5 µl was denatured at 65°C for 3 min and immediately placed on ice for 5 min. The RNA solution was added to 19.5 µl of the translation mixture containing: 17.5 µl of RRL, 20 µM amino acid mix without methionine, 1 µl of [32P]-labelled ΔNp53utr-Luc RNA was heated at 65°C for 2 min and chilled on ice for 5 min. The RNA was combined with 17.5 µl of RRL and the mixture was incubated for 5 min at 30°C. Then, the antisense oligonucleotide no. 1 or no. 7b was added to the final concentration of 2 µM. After 30 min incubation at 30°C all reactions were immediately placed on ice. RNAs were isolated using TriReagent as described previously.2.5 pmol of 5′-end-[in vivo experiments) to confirm that the oligomers enter to MCF-7 cells (data not shown).MCF-7 cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS), non-essential amino acids (Gibco-BRL), 100 U/ml of penicillin G, 0.1 mg/ml streptomycin sulphate and 0.25 µg/ml amphotericin B (Antibiotic Antimycotic – Sigma) at 37°C in 5% carbon dioxide atmosphere. Cells between 4 and 15 passages were used for transfection. Transfection was performed when cell confluence reached 50–80%. Antisense oligonucleotides with the final concentration of 1 µM  were transfected into cells using Lipofectamine 2000 or Lipofectamine RNAiMax according to a transfection protocol (Invitrogen). Four hours post transfection cells were washed in PBS and fresh medium was supplied. Then cells were washed in PBS and lysed at each time point, respectively. Prior to antisense oligonucleotide transfection experiments the transfection assay was performed using 5′-Fam-conjugated DNA oligomer  and GAPDH (Santa Cruz Biotechnology). Primary antibody was detected by Goat Anti-Mouse-HRP (Thermo Scientific Pierce) and visualization was performed by using enhanced chemiluminescent visualization (ECL) system (Thermo Scientific Pierce).18 primer and 100 units of SuperScript™ III reverse transcriptase (Invitrogen). cDNA was treated with 250 units/ml of RNase H (Fermentas) for 20 min at 37°C. Equal volumes of cDNA were used to amplify p53 and β-actin using following primers: p53 F, 5′-CTAGAGCCACCGTCCAGGGGAGC-3′, p53 R, 5′-GTCTTGGCCAGTTGGCAAAACATC-3′, β-actin F, 5′-AGAGCAAGAGAGGCATCCTG-3′, β-actin R, 5′-CGACGTAGCACAGCTTCTCC-3′. PCR reactions for p53 and β-actin were performed in the following conditions: 30 cycles of denaturing at 95°C for 30 s, annealing at 62°C (for p53) and 60°C (for β-actin) for 30 s and extending 72°C for 30 s. The obtained products were resolved on 1% agarose gels.For RT-PCR after cells transfection with antisense oligonucleotides total RNA was isolated from MCF-7 cells using TriReagent  according to the manufacturer protocol. First strand cDNA was synthesized from 300 ng of RNA using 100 ng of oligo(dT)Renilla luciferase , which hybridize to the functionally important elements of the 5′-terminal region of p53 mRNA, induced significant changes in protein synthesis we decided to design a few more antisense oligonucleotides. They covered the surroundings of the binding sites of oligomers no. 1 and no. 7 .O-methyl residues (2′-OMe) and LNA/phosphorothioates – gapmers (GAP) were designed . The RT-PCR revealed no changes in the amount of p53 mRNA in the presence of 2′-OMe oligomers no. 1 or no. 7b . Oligomers no. 1 and no. 7b exhibited a strong impact on the translation occurring from both the initiation codons AUG1 and AUG2. Interestingly, in the presence of oligomer no. 7b the amount of protein synthetized from AUG2 was highly increased . It has increased radiation-induced apoptosis of tumour cells via regulation of p53 expression in some tumours.In our report, the potential applications of antisense oligonucleotides in the modulation of translation of p53 and ΔNp53 proteins were shown. The approaches based on antisense oligomers would shed more light on the way how the structural elements of the 5′-terminal region of p53 mRNA together with cellular proteins orchestrate to govern the translation initiation process. This is particularly attractive for studies performed in cells under stress conditions when a high increase of activity of p53 IRES is observed. Moreover, the antisense strategy can be potentially useful for the purpose of establishing an alternative medical treatment Figure S1Structure probing of ΔNp53utr RNA. (A) Autoradiograms show the products of Pb2+-induced cleavage analyzed by primer extension on 8% polyacrylamide gels in denaturing conditions. Guanosine residues are labelled on the left and the short and long run of the gel is shown. Lanes: (−) – control reaction in the absence of Pb2+ ions; A, T, C, G – sequencing reaction with adenosine, thymidine, cytosine and guanosine dideoxy terminating nucleotides, respectively. The cleavages induced in the presence of Pb2+ ions are displayed in panel D as black triangles. (B) Histogram of integrated and normalized DMS reactivity as a function of nucleotide position. Sites of modification were identified using the fluorescent-labelled DNA primer which anneals to a reporter protein coding sequence. The cDNA fragments were resolved by capillary electrophoresis. In order to create quantitative normalized SHAPE reactivity the raw data from capillary electrophoresis were analyzed using ShapeFinder software. Bars are coloured using the scale shown in panel D. (C) Normalized SHAPE reactivity as a function of nucleotide position. The nucleotides modified by DMS were determined by primer extension with a fluorescent-labelled DNA primer on a single capillary electrophoresis. Bars are coloured according to the scale shown in panel D. (D) The secondary structure model of ΔNp53utr RNA generated by RNAStructure 5.2 program by incorporation of the SHAPE reactivity as an energy function into the SHAPE-constrained algorithm. Nucleotide symbols are coloured according to their SHAPE reactivity. Nucleotide positions modified by DMS are denoted by circles and the cleavages induced by Pb2+ ions are displayed as black triangles.(TIF)Click here for additional data file.Figure S2RNase H assay in the presence of antisense oligomer no. 1. The RNase H assay on ΔNp53utr-Luc RNA with unmodified and GAP-modified oligomer no. 1 was performed. After 10 and 30 min incubation with RNase H from E. coli, RNA was phenol/chloroform purified and resolved on 8% polyacrylamide gel in denaturing conditions. Lanes: (−) – control reaction without antisense oligonucleotide, T1– limited hydrolysis by RNase T1. Selected guanosine residues are labelled on the left side of the autoradiogram.(TIF)Click here for additional data file.Materials and Methods S12+-induced cleavage, DMS modification, RNase H assay.SHAPE Analysis, Pb(DOC)Click here for additional data file.


Bacillus licheniformis P003 in basal medium containing glucose, peptone, K2HPO4, MgSO4 and Na2CO3 at pH 10. The effect of culture conditions and medium components for maximum production of alkaline protease was investigated using one factor constant at a time method along with its characterization. Maximum level of enzyme production was obtained after 48h of incubation with 2% inoculum size at 42°C, under continuous agitation at 150 rpm, in growth medium of pH 9. Highest enzyme production was obtained using 1% rice flour as carbon source and 0.8% beef extract as organic nitrogen source. Results indicated that single organic nitrogen source alone was more suitable than using in combinations and there was no significant positive effect of adding inorganic nitrogen sources in basal medium. After optimization of the parameters, enzyme production was increased about 20 fold than that of in basal medium. The crude enzyme was highly active at pH 10 and stable from pH 7–11. The enzyme showed highest activity (100%) at 50°C, and retained 78% relative activity at 70°C. Stability studies showed that the enzyme retained 75% of its initial activity after heating at 60°C for 1h. The enzyme retained about 66% and 46% of its initial activity after 28 days of storage at 4°C and room temperature (25°C) respectively. Mn2+ and Mg2+ increased the residual activity of the enzyme, whereas Fe2+ moderately inhibited its residual activity. When pre-incubated with Tween-20, Tween-80, SDS and H2O2, each at 0.5% concentration, the enzyme showed increased residual activity. These characteristics may make the enzyme suitable for several industrial applications, especially in leather industries.Proteolytic enzymes have occupied a pivotal position for their practical applications. The present study was carried out under shake flask conditions for the production of alkaline protease from  Bacillus dominate in the industry , but the enzyme production was found to be highest (89.30 U/ml) at pH 9.0. The enzyme production was drastically reduced at pH 12.0  after 48h of cultivation period  was found when (NH4)2SO4 was used as sole inorganic nitrogen source 2SO4. However, Bhunia et al. (Low activity (4.81 U/ml and 9.62 U/ml) was found in case of NHa et al.  found maa et al. .Figure 2Alkaline protease production was significantly increased about 20 fold (930.02 U/ml) using the simulated media after optimization of different parameters in comparison to the basal medium (46.10 U/ml).The activity of the protease towards casein at different pH levels was determined. It can be observed from the figure Figure  that proB. licheniformis MH31 was stable (75%) up to 60°C after 1 h of incubation, whereas Deng et al. . The inoculated flasks were placed in a thermostated orbital shaker for 48 hours, at 37°C and 150 rpm. Samples were withdrawn at regular intervals and centrifuged in a refrigerated centrifuge machine at 10000 rpm for 15 minutes at 4°C. The cell free supernatant was preserved at 4°C and used for enzyme assay and protein estimation.The seed culture (1 ml) was transferred to 100 ml of basal medium in a 250-ml Erlenmeyer flask. Basal medium contained Glucose 10.0 g/l, Peptone 10.0 g/l, KExtracellular soluble protein in culture filtrate was estimated by Lowry’s method using bovine serum albumin (BSA) used as Standard  equivalent to 1 μmol of tyrosine, in the presence of the Folin-Ciocalteau reagent by using a tyrosine standard curve , temperature (32-45°C), incubation time (24–120 h), inoculums concentration (0.5-3.5%) and medium composition  were evaluated. All the experiments were conducted in triplicate and then the mean values were considered.The fermentation condition for protease production by Data analysis was performed using SPSS software version 10 . The results were presented as mean ± SE.


The gold discs used for surface plasmon resonance (SPR) measurements were modified with thioaliphatic acid. The surface functionalized with carboxylic groups was used for covalent attaching of Aβ40 probe by creation of amide bonds in the presence of EDC/NHS. The modified SPR gold discs were used for exploring the Aβ40 aggregation process in the presence of selected alkaloids: arecoline hydrobromide, pseudopelletierine hydrochloride, trigonelline hydrochloride and α-lobeline hydrochloride. The obtained results were discussed with other parameters which govern the phenomenon studied such as lipophilicity/hydrophilicy and Aβ40-alkaloid association constants.The main objective of the presented study was the development of a simple analytical tool for exploring the influence of naturally occurring compounds on the aggregation of amyloid-β peptide (Aβ Soluble α-helical or random-coil structures can convert into β-sheet protofibrilar intermediates which act as a seed for amyloid formation and plaque precipitation [Abnormal folding and aggregation of proteins characterizes many, if not all, neurodegenerative disorders such as Alzheimer’s (AD), Parkinson’s, Creutzfeldt-Jakob disease and large group of poliglutamine disorders including Huntington’s disease . Aggregapitation . Formatipitation . Thus tain vitro and in vivo but special attention has been reserved for the alkaloid nicotine and its derivatives, as they are well known to exert an influence on cognitive functions [in vitro studies suggesting that this alkaloid can alter Aβ amyloidogenesis directly [40 with nicotine and cotinine as well as the other pyridine- and piperdine-derived alkaloids were determined electrochemically [Numerous plant-derived molecules have been tested unctions . While iunctions –10, therdirectly –14. The emically ,16. Sincin vitro anti-amyloidogenic activity of metal ions chelators, short peptides, antibody fragments and flavonoids has been tested utilizing SPR-based methods [Surface plasmon resonance (SPR) spectroscopy has been successfully employed in studies concerning molecular interactions associated with different amyloidoses . Various methods –23. In m methods –24.40 peptide probe to the carboxyl-terminated surface was performed via amide bond formation.In the present study we proposed a simple in preparation SPR biosensor based on a thioaliphatic acid self-assembled monolayer (SAM) formed on gold SPR disc. The attachment of Aβin situ monitoring of Aβ40 early aggregation stages (oligomerization) in the presence of potential aggregation inhibitors. The influence of arecoline, α-lobeline, trigonelline and pseudopelletierine (40 aggregation was examined. These compounds derived from domestic plants are either commonly used as psychoactive substances (arecoline and α-lobeline) or are consumed as a dietary component (trigonelline and pseudopelletierine). Plants and products containing the considered alkaloids are easily available, which creates the possibility of using them (or their derivatives) as a diet supplementation in AD prevention and treatment. For comparison, one pharmacologically unrelated compound, pyridoxine (vitamin B6) was tested too.The proposed biosensor was applied for etierine  on Aβ40 2.2.1.40 was purchased from Bachem, Switzerland (lot 1020370). Ammonium hydroxide, 11-mercaptoundecanoic acid (11-MUA), trifluoroacetic acid, 2-morpholinoethanesulfonic acid (MES), N-hydroxysuccinimide (NHS), N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC), ethanolamine, pyridoxine, arecoline hydrobromide, pseudopelletierine hydrochloride, trigonelline hydrochloride, α-lobeline hydrochloride and PBS components: sodium chloride, potassium chloride, sodium phosphate dibasic, potassium phosphate monobasic were supplied by Sigma-Aldrich, Poznań, Poland. Ethanol absolute, hydrochloric acid and sodium hydroxide were obtained from POCH, Poland. Reagents and solvents were of analytical grade and used without any purification step. All aqueous solutions were prepared using autoclaved Milli-Q water, resistivity 18.2 MΩ·cm . All buffers used for SPR experiments were rigorously filtrated through 0.22 μm sterile PVDF filters .Amyloid β protein (1–40), trifluoroacetate salt Aβ2.2.40 probe immobilization and Aβ40 aggregation. The experiments were carried out continuously and the immobilization step was followed by aggregation procedure, as described below.An Autolab Springle SPR system  equipped with a thermostatic water bath  was used. Springle SPR was employed for monitoring both Aβ2.3.40 peptides were dissolved in 0.02% ammonia solution at the concentration of 300 μM by brief vortexing. Then the solution was sonicated for 2 min in a water bath at 4 °C. The Aβ40 aliquots were stored in at −85 °C.The lyophilized synthetic Aβ2.4.et al. [Gold-coated SPR discs were supplied by Eco Chemie, The Netherlands. Prior to modification, discs were rinsed with Milli-Q water then with ethanol and dried thoroughly in a stream of nitrogen. Gold discs were treated with UV-ozone chamber  for 20 min in order to remove any organic contaminations. Clean gold SPR discs were immediately immersed for 30 min in 1.0 mM solution of 11-MUA in ethanol containing 2% of trifluoroacetic acid. Afterwards discs were rinsed with ethanol, 10% solution of ammonia in ethanol, again with ethanol and gently dried in a stream of nitrogen before placing in SPR chamber. Formation of 11-MUA SAM was carried out according to modified method described by Wang et al. .2.5.40 aliquots were diluted with 50 mM MES (pH 6.5) to 30μM, briefly vortexed and injected on the activated disc surface. After 20-min incubation peptide solution was rinsed off with 2 mL of running buffer and followed by quenching the remaining NHS-activated sites with 1.0 M ethanolamine in 50 mM MES (pH 8.5) for 10 min. Finally, discs were rinsed with 4 mL of the running buffer and the value of SPR angle shift was recorded 10 min after the end wash.Probe immobilization was performed at 22 °C using 50 mM MES (pH 5.5) as running buffer. Freshly 11-MUA-modified discs were placed in the SPR chamber and conditioned in running buffer for 2 h. Every 30 min discs were washed with 4 mL of running buffer. After conditioning, when stable readout was achieved, the carboxyl groups of 11-MUA SAM were activated by treating with a mixture of 100 mM EDC and 50 mM NHS in 50 mM MES (pH 5.5) for 15 min. Subsequently discs were washed with 2 mL of running buffer and the value of SPR angle shift was recorded after 3 min. Aβ2.6.40 aliquots were diluted within PBS to 30 μM. 1.0 mM alkaloids: arecoline, pseudopelletierine, trigonelline and α-lobeline as well as pyridoxine were dissolved in PBS. Final pH was adjusted to 7.4 ± 0.05. Then discs were incubated in a solution of Aβ40 with a supplementation of selected alkaloid for 2 h followed by washing with 4 mL of running buffer. The same experiment, but without tested compounds has been preformed too (control aggregation). Values of SPR shift angle were recorded 20 min after the end wash.Aggregation experiments were performed at 37 °C using 10 mM PBS (pH 7.4) as running buffer. Prior to the experiment, discs with immobilized probe were washed with 4 mL of running buffer and conditioned until the stable readout was gained. SPR angle shift values were recorded after 1 h of conditioning. Aβ2.7.40 peptide, a solution containing 50 mM MES (pH 5.5) with 0.002% ammonia only was injected, incubated for 20-min and rinsed off with 2 mL of running buffer. This was followed by quenching the remaining NHS-activated sites with 1.0 M ethanolamine in 50 mM MES (pH 8.5) for 10 min. Finally, discs were rinsed with 4 ml of the running buffer and the value of SPR angle shift was recorded 10 min after the end wash. Aggregation was performed at 37 °C using 10 mM PBS (pH 7.4) as running buffer. Prior to the experiment, discs were washed with 4 mL of running buffer and conditioned until the stable readout was gained. SPR angle shift values were recorded after 1 h of conditioning. Then discs were incubated in a solution of 30 μM Aβ40 in PBS for 2 h followed by washing with 4 mL of running buffer. Values of SPR shift angle were recorded 20 min after the end wash.Freshly 11-MUA-modified discs were placed in SPR chamber and conditioned in 50 mM MES (pH 5.5) running buffer for 2 h. Every 30 min discs were washed with 4 mL of running buffer. After conditioning, when stable readout was achieved, the carboxyl groups of 11-MUA SAM were activated by treating with a mixture of 100 mM EDC and 50 mM NHS in 50 mM MES (pH 5.5) for 15 min. Subsequently discs were washed with 2 mL of running buffer and the value of SPR angle shift was recorded after 3 min. Instead of diluted Aβ2.8.Experiments were performed at 37 °C using 10 mM PBS (pH 7.4) as running buffer. Prior to the experiment, discs with immobilized probe were washed with 4 mL of running buffer and conditioned until the stable readout was gained. SPR angle shift values were recorded after 1 h of conditioning. 1.0 mM alkaloids: arecoline, pseudopelletierine, trigonelline and α-lobeline as well as pyridoxine were dissolved in PBS containing 0.002% ammonia solution. Final pH was adjusted to 7.4 ± 0.05. Then discs were incubated in alkaloid solution for 2 h followed by washing with 4 mL of running buffer. Values of SPR shift angle were recorded 20 min after the end wash.2.9.Aggregation assay results were presented as means ± standard deviation for at least five replicates for each experimental group. Results were submitted to the t-test  and values of p < 0.05 were considered as statistically significant.3.40 peptide probe. The immobilization procedure scheme is shown in 40 peptide probe, performed via EDC/NHS coupling chemistry [40 peptide attached per 1 mm2 of disc surface.The 11-MUA-modified SPR gold discs were utilized for the covalent binding of Aβhemistry , was mon40 solution was injected on the surface of Aβ40 functionalized disc (control aggregation), and in second, the same solution was injected on the disk surface modified only with 11-MUA SAM, which was treated with ethanolamine after activation with EDC/NHS (background aggregation). The value of SPR angle shifts for control aggregation was 125.7 ± 15.6 m° and for background only 60.1 ± 3.6 m°  was reported in the literature [3-SAM has a lower energy barrier for Aβ monomer adsorption than OH-SAM [2-SAM bearing relatively large charge in the head groups, electrostatic interactions between Aβ and SAMs provide the additional driving force for Aβ adsorption [40-functionalized SPR gold disc were checked. Incubations of probe-modified discs with the tested alkaloids injected alone onto disc surface revealed that SPR angle shifts were negligible . Thus, the proposed assay was deemed selective and suitable for performing the Aβ40 aggregation processes in the presence of selected compounds.In order to check if the proposed biosensor is able to detect the early steps of the aggregation process, two series of control aggregations were carried out. In first, the Aβ± 3.6 m° . This interature . The autn OH-SAM . For thesorption . The bacsorption . Prior t40-probe-functionalized gold discs with Aβ40 solution containing 1.0 mM of tested alkaloid. For comparison the representative binding curve for incubation of Aβ40-probe-functionalized disc with Aβ40 solution without any additives (control aggregation) was included.40 aggregation. Values of SPR angle shifts decreased from 125.7 ± 15.6 m° for control incubation  to 83.1 ± 16.5 m° for arecoline (40 peptide deposition and SPR angle shift resulted in 121.9 ± 25.6 m° (6), did not influence Aβ40 deposition  decreased Aβrecoline  and 74.8recoline . On the recoline . Trigone 25.6 m° . Aggrega40 deposition obtained for incubations with tested compounds was calculated relative to the aggregation control (100%)  .40 peptide and selected alkaloids were established electrochemically [40 was determined for arecoline: 2.30 × 108 M−1. The association constant for α-lobeline was equal to 0.90 × 108 M−1, while binding of pseudopelletierine (0.57 × 108 M−1) and trigonelline (0.28 × 108 M−1) were relatively weaker [40 aggregation and their therapeutic and/or prophylactic significance can be suggested.In our previous study, association constants for Aβemically . The higy weaker . In the 40 peptide interactions. The second step of considered phenomenon refers to the direct interaction between the drug-and receptor site in the peptide. These two factors, ability to cross the interface and binding to the specific fragment of the peptide can be crucial for determining the effects of alkaloid influence on Aβ40 aggregation observed herein.The phenomenon of interaction between examined compounds and Aβ peptide, resulting in altered aggregation, can be considered as a process composed of two steps. At the first stage alkaloid passes through the water-peptide interface, moving from aqueous phase to the relatively lipophilic environment consisted of Aβ molecules, either deposited on gold disc surface or remaining in the solution. In that case the permeability of substance is determined by its lipophilicity. This attribute also governs drug bioavailability and cannot be neglected when potential therapeutic or prophylactic benefits of compound administration are considered. Thus, the lipophilicity of drug candidate is an important factor which enables any subsequent ligand-Aβ40 aggregation.In light of those assumptions, the lipophilicity of tested compounds was taken into account. Log D (pH 7.4) values of the examined alkaloids and pyridoxine were found in ChemSpider online database  and was 40 peptide as was recorded for trigonelline or α-lobeline [40 aggregation process. The specific non-flat geometry of pseudopelletierine molecule  which is known as a neutral compound concerning Aβ40 peptide aggregation.The log D values of pseudopelletierine and trigonelline are −0.96 and −3.31, respectively, showing the predominantly hydrophilic character of these compounds under physiological pH. Pseudopelletierine demonstrated similar affinity towards Aβlobeline , but at molecule  could beProtein aggregation processes have been widely explored by fluorescence assays ,21,23,3240-peptide probe via amide bond formation. It is worth underlining that the surface of 11-MUA SAM displayed good selectivity. The unspecific aggregation of Aβ40 was significantly lower in the comparison to the Aβ40 aggregation performed on the surface with Aβ40 probe covalently bound via amide bonds , which is known as a neutral compound for Aβ40-peptide aggregation, displayed no influence on this phenomenon when studied by the analytical tool proposed. The obtained results showed that developed SPR-based biosensors could be applied for monitoring of Aβ40 aggregation phenomena in real-time, without using any chemical markers influencing the process.The proposed SPR biosensor based on 11-mercaptoundecanoic acid (11-MUA) was useful for study of the influence of several alkaloids—arecoline hydrobromide, pseudopelletierine hydrochloride, trigonelline hydrochloride, α-lobeline hydrochloride—on the aggregation process of Aβ


This method facilitated optimization of a biochemical procedure to fractionate autophagosomes. A combination of LC-MS/MS with subsequent statistical analyses revealed a list of autophagosome cargo proteins; some of these are selectively enclosed in autophagosomes and delivered to the vacuole in an Atg11-independent manner. The methods we describe will be useful for analyzing the mechanisms and physiological significance of Atg11-independent selective autophagy.Macroautophagy (autophagy) is a bulk protein-degradation system ubiquitously conserved in eukaryotic cells. During autophagy, cytoplasmic components are enclosed in a membrane compartment, called an autophagosome. The autophagosome fuses with the vacuole/lysosome and is degraded together with its cargo. Because autophagy is important for the maintenance of cellular homeostasis by degrading unwanted proteins and organelles, identification of autophagosome cargo proteins  will aid in understanding the physiological roles of autophagy. In this study, we developed a method for monitoring intact autophagosomes  Macroautophagy, hereafter referred to as autophagy, is a system for bulk degradation of cytoplasmic materials. It is mediated by a double-membrane-bound organelle, the autophagosome. Bulk autophagy serves to maintain the intracellular pool of free amino acids that is necessary for protein synthesis under nutrient-starvation conditions autophagy-related 11) and the other independent of this protein cytoplasm-to-vacuole targeting (Cvt) pathway, transports the Cvt complex to the vacuole Autophagy is predominantly a non-selective degradation system, but several selective cargoes have recently been reported. Two modes of selective autophagy have been proposed, one dependent on Atg11  equipped with a UPlanSApo100×Oil (NA: 1.40) and a CoolSNAP HQ CCD camera , as described For R18 staining, samples were stained with 10 µg/ml of R18 (1 mg/ml stock dissolved in dimethyl sulfoxide) for 10 minutes at room temperature. The samples were observed after they were washed twice with fresh buffer.Electron microscopy was performed by Tokai-EMA, Inc. (Japan) as described 600 = ∼1.5 in 50 ml of YEPD medium were starved in 50 ml of SD(-N) medium. Collected cells were converted to spheroplasts with Zymolyase 100T, and then mechanically disrupted with 3.0 µm–pore polycarbonate filters (Whatman) in lysis buffer . After cell debris was removed, lysates were divided into aliquots and treated with or without 1% Triton X-100 , 1 mg/ml proteinase K (Roche), or both for 30 minutes on ice. The reactions were terminated by addition of 20% trichloroacetic acid (TCA). Precipitates were washed with acetone and dissolved in SDS-PAGE sample buffer containing 4 mM Pefabloc SC (Roche). Equivalent quantities of protein were subjected to immunoblot analysis.Cells grown to ODg for 15 minutes at 4°C. The pellets were resuspended in 500 µl HES1.0 buffer, and then layered onto 10 ml of 0–30% continuous iodixanol gradients using Optiprep (Axis-Shield), which is a solution of 60% iodixanol in water. The gradients were constructed in 13 PA tubes  using a Gradient Master gradient maker (Biocomp Instruments). Loaded gradients were ultracentrifuged at 100,000×g for 20 hour at 4°C in a P40ST rotor on a CP-80β ultracentrifuge . Twenty-four fractions (approximately 440 µl each) were taken from each tube, starting from the top of the gradient. Each fraction was mixed with 70 µl of 100% TCA, and then incubated for 15 minutes on ice. The TCA-precipitated fractions were centrifuged at 15,000×g for 10 minutes at 4°C in a T15AP31 rotor on a CF15RX centrifuge . The pellets were washed once with 150 µl of ice-cold acetone using a bath sonicator . After the pellets were centrifuged at 15,000×g for 5 minutes at 4°C, the acetone was discarded and the samples were dried using a VC-15SP centrifugal concentration apparatus . The pellets were dissolved in 70 µl of SDS-PAGE sample buffer, and 10-µl aliquots were subjected to immunoblot analysis.Cell lysates were prepared as described above and centrifuged at 15,000×Primary antibodies used were anti-Ape1, anti-Pgk1 , anti-Rpl17  600 = ∼1.5 in 1 L YEPD medium at 30°C, washed once with distilled water, and starved in 300 ml of SD(-N) medium for 3 hours. Collected cells were suspended in 40 ml of pre-spheroplasting buffer  and incubated for 10 minutes at 30°C. The cells were collected by centrifugation at 2,000×g for 2 minutes in a TS-7LB rotor on a LX-120 centrifuge . The pelleted cells were suspended in 8 ml of spheroplasting buffer  containing 1 mg/ml Zymolyase 100T , and the resultant suspension was diluted with 32 ml of spheroplasting buffer . The cells were converted to spheroplasts by incubation for 25 minutes at 30°C with gentle shaking. Spheroplasts were collected by centrifugation at 1,000×g at 4°C, washed twice with 40 ml of 1.4 M sorbitol, resuspended in 40 ml of HES1.0 buffer, and then mechanically disrupted with 3.0 µm–pore polycarbonate filters. After cell debris was removed by centrifugation at 300×g for 1 minute at 4°C, cell lysates were passed through 2.0 µm–pore polycarbonate filters (Whatman). The lysates were again centrifuged at 500×g for 1 minutes at 4°C and the cleared lysates were centrifuged at 15,000×g for 15 minutes at 4°C. The pellets were suspended in 900 µl of HES1.0 buffer, and then 100 µl of 10 mg/ml proteinase K dissolved in HES1.0 buffer was added. This mixture was incubated at 37°C for 30 minutes; reactions were terminated by addition of 10 µl of 400 mM Pefabloc SC dissolved in HES1.0 buffer, and then filtered through 0.8 µm–pore polycarbonate filters (Whatman). Samples were layered onto discontinuous iodixanol gradients , which were constructed in 13.2-ml polyallomer centrifuge tubes . Loaded gradients were ultracentrifuged at 100,000×g for 60 minutes at 4°C in a SW41Ti rotor on an Optima L-80 XP ultracentrifuge (Beckman Coulter). GFP fluorescence was visualized using a blue LED light with a band-pass filter . Bands at the 10–20% interface were taken with Pasteur pipettes (500–1000 µl each) and precipitated with 1/40 volume of 20% Triton X-100  and 1/5 volume of 100% TCA . The pellets were washed twice with 600 µl of ice-cold acetone and dissolved in 50 µl of phosphate-buffered saline containing 8 M urea (2–5 mg protein/ml). Protein concentrations were measured using the BCA Protein Assay reagent (Thermo Scientific).Cells were grown to ODMethods for LC-MS/MS spectrometry are described in ypt7Δ autophagosome fractions were selected from 673 proteins  and for cytosol  prepared from ypt7Δ cells (gray-labeled parameters in Saccharomyces Genome Database (http://www.yeastgenome.org/)  . Finallyypt7Δ and ypt7Δatg1Δ autophagosome fractions  fraction from nitrogen-starved ypt7Δ cells and subjected the fraction to immunoelectron microscopy using an anti-Ape1 antiserum. Although numerous membrane structures were non-specifically labeled, we could not identify autophagosomes (data not shown). We overexpressed GFP-prApe1 in ypt7Δ cells to increase the signal in fluorescence and immunoelectron microscopy. We then monitored fluorescence of GFP-prApe1 by fluorescence microscopy. Under nitrogen-starvation conditions, only one bright dot was seen in ypt7Δatg1Δ cells (data not shown), suggesting that this dot corresponded to the Ape1 complex localized outside autophagosomes ypt7Δ cells, several weaker dots were seen in addition to the bright dot  fraction  fraction’.The band from with R18 , implyinwith R18 , indicatwith R18 . Hereaftypt7Δ cells by LC-MS/MS. To quantify the relative abundance of each protein, we used the spectral-counting method, which is based on the fact that the number of spectral counts detected by LC-MS/MS reflects the amount of each protein ypt7Δ cells  prepared from ypt7Δ cells, in three independent experiments  mutants In addition to the ribosomal and ribosome-associated proteins, several cytoplasmic proteins were plotted along the regression line with negative slope; for example, Hsc82, Fas1/2 (subunits of fatty-acid synthetase), Pfk1/2 (subunits of phosphofructokinase), Gdh1 (NADP(+)-dependent glutamate dehydrogenase), and Met6 (methionine biosynthesis) were concentrated in the fraction , suggestpep4Δ cells upon starvation pep4Δ and pep4Δatg7Δ cells into S15 (cytoplasm) and P15 (vacuole) fractions, and subjected the resulting fractions to immunoblot analysis Click here for additional data file.Figure S2Principal component analysis of AP fractions from ypt7Δ, ypt7Δatg1Δ, and ypt7Δatg11Δ cells. The first and second principal components (PC1 and PC2) are plotted. Green diamonds indicate prApe1 and Ald6, both of which are previously reported selective cargoes of autophagosomes. Black diamonds indicate nonselective cargoes . Blue diamonds indicate other cytoplasmic proteins.(TIF)Click here for additional data file.Figure S3Immunoblot analysis of cytoplasmic and vacuole fractions. Cells grown in YEPD medium were starved in SD(-N) medium for 4.5 hours. Cells were converted to spheroplasts, and then physically disrupted with 3.0 µm-pore polycarbonate filters. After debris was removed, cell lysates were centrifuged at 15,000 x g for 15 minutes at 4°C and separated into supernatant (S15) and pellet (P15) fractions. The P15 fractions were washed once with lysis buffer. As a representative of cytoplasm, the S15 fraction of pep4Δatg7Δ cells was serially diluted (lanes 1–3). To detect Adh, one-eighth volume of the S15 fraction was loaded for each lane (lanes 1–3). Serially diluted P15 fractions were loaded as follows: pep4Δ cells: lanes 4, 6, and 8; pep4Δatg7Δ cells: lanes 5, 7, and 9. Intensities of chemiluminescent signals were quantified using Image Gauge software . To calculate Pfk/Adh ratios in cytoplasm (lanes 1–3), signals for Pfk1/2 and Adh were quantified by subtracting the corresponding background intensities from the band intensities. To calculate the ratios in autophagic bodies, Pfk and Adh signals of the pep4Δ P15 fractions were adjusted by subtracting the corresponding signals of pep4Δatg7Δ P15 fractions. Calculated ratios were normalized to the ratio of lane 1.(TIF)Click here for additional data file.Table S1Complete data obtained from mass spectrometric analysis (673 proteins).(XLSX)Click here for additional data file.Table S2Data used for principal component analyses Click here for additional data file.Table S3Principal component analysis for  (378 proteins).(XLSX)Click here for additional data file.Table S4Protein localization manually annotated (for ).(XLSX)Click here for additional data file.Table S5Principal component analysis for (XLSX)Click here for additional data file.Table S6Principal component analysis for  .(XLSX)Click here for additional data file.Table S7Top 40 proteins identified in the AP fraction of ypt7Δ cells.(XLSX)Click here for additional data file.Table S8Top 40 proteins identified in the AP fraction of ypt7Δatg1Δ cells.(XLSX)Click here for additional data file.Appendix S1Supplemental Materials and Methods for LC-MS/MS spectrometry.(DOC)Click here for additional data file.


Previous studies implicated that IL23R and IL17 genes play an important role in autoimmune inflammation. Genome-wide association studies have also identified multiple single nucleotide polymorphisms (SNPs) in the IL23R gene region associated with inflammatory bowel diseases. This study examined the association of IL23R and IL17A gene SNPs with ulcerative colitis susceptibility in a population in China.A total of 270 ulcerative colitis and 268 healthy controls were recruited for the analyses of 23 SNPs in the IL23R and IL17A regions. Genomic DNA was extracted and analysis of these 23 SNPs using ligase detection reaction allelic (LDR) technology. Genotype and allele associations were calculated using SPSS 13.0 software package.vs 3.3%, P = 0.002; 60.4% vs 53.2%, P = 0.0017, respectively). The linkage disequilibrium (LD) patterns of these SNPs were measured and three LD blocks from the SNPs of IL23R and one block from those of IL17A were identified. A novel association with ulcerative colitis susceptibility occurred in haplotypes of IL23R  and IL17A (H4 P = 0.034). Pair-wise analyses showed an interaction between the risk haplotypes in IL23R and IL17A (P = 0.014).Compared to the healthy controls, the variant alleles IL23R rs7530511, and rs11805303 showed a statistically significant difference for ulcerative colitis susceptibility (0.7% Our study demonstrated that rs7530511, and rs11805303 of IL23R were significantly associated with ulcerative colitis susceptibility in the Chinese population. The most noticeable finding was the linkage of IL23R and IL17A gene region to ulcerative colitis risk due to the gene-gene interaction. Ulcerative colitis (UC) is characterized by chronic and relapsing inflammation in the gastrointestinal tract, and mostly affects the colorectal region of the gastrointestinal tract et al. et al. Recently, it is shown that the IL-23/IL-17 axis plays an important role in IBD development Thus, in this study, we examined the distribution of 16 SNPs in the IL23R gene and 7 SNPs in the IL17A gene for analysis of the allele/genotype–phenotype association and gene-gene interaction, which could provide insightful information for their role in UC development in a Chinese Han population.In this study, we recruited 270 cases of UC and 268 healthy controls from four different hospitals  between 2008 and 2011. The diagnosis of UC was based on standard clinical, endoscopic, radiological, and histological criteria This study was approved by the ethics committees of all four hospitals  involved in this study, and all patients signed informed consent forms for participation in this study. Clinical data were collected from the patients’ medical history and from questionnaires.2, 0.3 µl dNTP (10 mM), 0.5 µl of each primer and H2O for an initial DNA denature at 94°C for 2 min and then 35 cycles of 94°C for 20 s, 56°C for 20 s, and 72°C for 40 s and a final extension at 72°C for 3 min. Then, 3 µl of the PCR product was mixed with 1 µl of 10× Taq DNA ligase buffer, 0.125 µl of 40 U/µl Taq DNA ligase, 0.01 µl of probe (10 p)/Article and then PCR amplified for 30 cycles of 94°C for 30 s and 56°C for 3 min. After that, 1 µl of PCR product was added to 10 µl of sample loading buffer containing a molecular weight marker, and then denatured at 95°C for 3 min and immediately put into an ice bath for DNA sequence analysis using an ABI 3730 system.Approximately 2 ml of blood from the cases and controls was collected and stored in tubes containing EDTA. Blood cells and serum were separated in conditions of 4°C at 3000 rpm/min for 10 min within 4 h of blood collection. Genomic DNA was then extracted from peripheral blood lymphocytes using the RelaxGene Blood DNA System . The single nucleotide polymorphisms were genotyped by a predesigned ligase detection reaction allelic (LDR) technology . The PCR amplification was carried out in a volume of 15 µl, including 1 µl of genomic DNA, 1.5 µl 10× buffer, 1.5 µl MgCl2) test, Fisher’s exact test or Student’s t-test, when each was appropriate. Odds radios (OR) and 95% confidence intervals (95% CIs) were applied for 2×2 tables. Bonferroni correction was used for multiple comparisons. Bivariate correlate analysis and binary classification Logistic regression analysis were used to search risk factors associated with SNPs loci mutations. Subgroups were based on clinical and endoscopic severity, disease location, extra-intestinal manifestations, and tobacco smoke history. Subgroups dependent on the relative risk factors were used to find the different distribution of alleles and genotypes.Allele frequencies were first calculated for the Hardy-Weinberg balance test. Genotype, haplotype, and allele frequencies were compared between cases and controls using the chi-square (χhttp://www.broadinstitute.org//haploview/haploview-downloads). SNPs with minor allele frequencies (MAF) below 5% were excluded. Tagger SNPs were picked up as r2>0.8, of which adjacent SNPs with 95% CI = (0.70–0.98) of D’ value were considered the haplotype. Mantel-Haenszel and logistic regression methods were used to determine the interaction between haplotypes in different genes. For all the analyses, differences were considered statistically significant when the P value was less than or equal to 0.05.Haplotype frequencies and linkage disequilibrium (LD) were estimated and visualized by Haploview 4.2 . The data showed that one disease risk and two protective mutations in the IL23R polymorphisms were found . Compared to the controls, the mutant allele T for IL23R rs11805303 was significantly higher in UC patients . Only two (rs7530511 and rs11805303) genome-wide significant SNPs passed the Bonferroni correction threshold (0.05/16 = 0.0031). Our study only showed wild type alleles in IL23R rs11209026 and rs11465804, and heterozygous alleles in IL23R rs11465788 of all the participants. However, we did not find any association between IL17A variant alleles and UC risk.Allele frequency data are shown in re found . The frevs 32.1%, P<0.001, OR = 0.49, 95%CI = 0.33–0.76, P = 0.024, OR = 0.45, 95% CI = 0.21–0.91, P<0.001) and IL7A rs2275913 in the severe patients (P = 0.001). From P = 0.028), while the IL17A rs2275913 was negatively correlated to UC disease severity (AA+AG: P = 0.035). The IL23R rs6677188 genotype distribution between UC endoscopic remission and activity was significantly different (P = 0.010). The heterozygous type AT frequency distribution was significantly higher in the endoscopic inactivity group than that in the endoscopic activity group . The rare haplotype (GGTT) of IL17A, formed with four SNPs , was also a risk haplotype with a frequency of 3.3% in UC patients and 0.7% in controls (P = 0.034). Another two haplotypes of IL23R were both protective factors, one was tagged by the rs10489629-A and rs2201841-T alleles, and the frequency of this haplotype was 0.7% in UC patients and 3.7% in controls (P = 0.019). Another rare haplotype formed with three wild type alleles (GTG) of rs11209032, rs6677188 and rs1495965, the frequency of which was 1.1% in UC patients and 4.1% in controls (P = 0.029).Then, we measured the linkage disequilibrium (LD) patterns of the SNPs and identified three LD blocks from the SNPs of IL23R and one LD block in the SNPs of IL17A . To deteP = 0.004 by Mantel-Haenszel test, P = 0.014). Furthermore, the “risk” haplotypes of IL23R and IL17A synergistically increased UC risk Click here for additional data file.Table S2The association between IL23R rs17375018/IL17A rs2275913 and Clinical severity of UC (Student’s t-test).(DOC)Click here for additional data file.


Upon lipopolysaccharide (LPS) stimulation, activation of both the Toll-like receptor 4 (TLR4) and phosphoinositide 3-kinase (PI3K) pathways serves to balance proinflammatory and anti-inflammatory responses. Although the antagonist to TLR4 represents an emerging promising target for the treatment of sepsis; however, the role of the PI3K pathway under TLR4-null conditions is not well understood. This goal of this study was to investigate the effect of inhibition of PI3K on innate resistance to LPS toxicity in a murine model.Escherichia coli serotype 026:B6 after 7 d was 100%, 10%, and 10%, respectively. In contrast, no mortality was noted after 500-μg LPS injection in Tlr4-/- mice. When the PI3K inhibitor LY294002 was injected (1 mg/25 g body weight) 1 h prior to the administration of LPS, the overall survival of the Tlr4-/- mice was 30%. In the Tlr4-/- mice, the LPS injection induced no NF-κB activation but an increased Akt phosphorylation in the lung and liver, when compared to that of the C57BL/6 mice. Injection of 500 μg LPS led to a significant induction in O2- detected by electron paramagnetic resonance (EPR) spin trapping spectroscopy in the lung and liver at 3 and 6 h in C57BL/6 but not Tlr4-/- mice. Addition of LY294002 only significantly increased the O2- level in the lung and liver of the Tlr4-/- mice but not in the C57BL/6 mice following 500-μg LPS injection. In addition, the serum IL-1β and IL-2 levels were more elevated in C57BL/6 mice than in Tlr4-/- mice. Notably, IL-1β and IL-2 were significantly increased in Tlr4-/- mice but not in the C57BL/6 mice when the PI3K pathway was inhibited by LY294002 prior to LPS injection.The overall survival of the cohorts receiving intraperitoneal injections of 100, 500, or 1000 μg LPS from Tlr4-/- mice is impaired by inhibition of the PI3K pathway, with a corresponding increase in mortality and production of tissue O2- and inflammatory cytokines.In this study, we demonstrate that innate resistance to LPS toxicity in  TLRs sid in vivo. LPS-indd in vivo, and cand in vivo.The PI3K/Akt signaling pathway is one of the most critical pathways involved in regulation of cell survival  and inflTlr4 gene, and are thus refractory to LPS effects [Tlr4-/- mice is reduced by the inhibition of the PI3K pathway, with a corresponding increase in mortality and production of tissue superoxide and inflammatory cytokines.Sepsis mice induced by the administration of LPS were employed as a redox disruption model because sepsis is well known to be a systemic inflammatory response syndrome related to the generation of reactive oxygen species (ROS) . LPS has effects ,26. TLR4 effects ,28. The  effects ; AlthougTlr4-/- (C57BL/10ScNJ) mice were purchased from Jackson Laboratory . C57BL/6 mice were purchased from the National Laboratory Animal Center, Taiwan. The murine strain C57BL/10ScNJ mice have a deletion of the Tlr4 gene that results in absence of both mRNA and protein and thus in defective response to LPS stimulation. C57BL/6 was used as a control. All housing conditions and surgical procedures, analgesia, and assessments were in accordance with national and institutional guidelines, and an Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)–accredited SPF facility was used. The animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Kaohsiung Chang Gung Memorial Hospital.Eight to twelve week old male mice weighing 25–30 g were used in the study. Escherichia coli serotype 026:B6  was purchased from Sigma-Aldrich . To profile the LPS toxicity, C57BL/6 mice were injected intraperitoneally (i.p.) with 100, 500, 1000 μg of LPS reconstituted in 100 μL of phosphate-buffered saline (PBS) (n = 10 in each group) for survival studies. The mice were returned to their cages after LPS injection and closely monitored for up to 7 d. Mice were given ad libitum access to food and water at all times. Additional groups of Tlr4-/- mice received i.p. injections of 500 μg LPS in the presence or absence of i.p. PI3K inhibitor LY294002  and were observed for survival studies (n = 10 in each group). LY294002 was injected 1 h prior to the administration of LPS.LPS from Tlr4-/- mice were injected i.p. with 500 μg LPS and sacrificed at 3- or 6-h post-injection. The control group was injected with 100 μL PBS. Additional groups of Tlr4-/- mice received i.p. injections of (1 mg/25 g body weight) LY294002 1 h prior to the injection of 500 μg LPS (n = 6 in each group). Whole blood was drawn from the mice, and tissues, including the lungs, liver, and spleen, were harvested for western blot analysis of Akt phosphorylation and nuclear translocation of p65 as well as the measurement of superoxide (O2-) production. Whole blood samples (1 mL per mouse) were collected into tubes containing an anticoagulant and incubated at room temperature for 15 min prior to centrifugation at 3,000 × g for 10 min. White blood cells (WBCs) were slowly removed from the corresponding layers and the serum was extracted and stored at -80°C before processing for cytokine assays.C57BL/6 and Tlr4-/- mice injected with 500 μg LPS in the presence or absence of LY294002 were harvested for detection of pAkt/Akt and nuclear p65/lamin B1 proteins. We extracted the cytoplasmic and nuclear protein fractions using NE-PER extraction reagents according to the manufacturer’s protocol . Cytoplasmic and nuclear protein extracts were used for Western blot analysis. The protein samples (30 μg) were resolved on a 10% SDS-polyacrylamide gel and transferred to polyvinylidenedifluoride membranes. The membranes were then blocked with 5% skim milk in Tween-20/ PBS and incubated with various primary antibodies, including rabbit anti–phospho-Akt (Ser473), anti-Akt , anti-p65, and anti-lamin B1  at 4°C overnight. The blots were then incubated with horseradish peroxidase–conjugated secondary antibodies at room temperature for 60 min, and developed using the ECL™ Western Blotting System . The protein bands were quantified with the FluorChem 8900 imaging system and the AlphaEaseFC software .The harvested lung, liver, and spleen tissues from wild type C57BL/6 mice and Superoxide formation was measured using electron paramagnetic resonance (EPR) spin trapping spectroscopy . BrieflyThe concentrations of IL-1β and IL-2 in the serum samples were determined using enzyme-linked immunosorbent assay (ELISA) kits for IL-1β and IL-2 , as per the manufacturer’s protocol. Sample concentrations were then calculated from a standard curve. Results are expressed as picograms per milligram (pg/mg) of protein.All results are presented as the mean ± standard error. An overall analysis of the differences between the group means was calculated by a one way analysis of variance (ANOVA) and an appropriate post hoc test using SPSS statistical software . A value of P < 0.05 was considered to indicate statistical significance.Tlr4-/- mice, no mortality was noted throughout the experiment. In addition, the symptoms observed following LPS injection were less pronounced during the entire observation period and the mice regained full mobility quickly. When 1 mg/25 g body weight LY294002 was injected 1 h prior to the administration of LPS, 30% and 70% of the Tlr4-/- mice died within 48 and 72 h, respectively  synthesis [-/- mice [In this study, we demonstrate that the innate resistance to LPS toxicity in  pathway . However pathway -20. In tynthesis ,32. In c-/- mice . However-/- animals or targeted gene silencing of Tlr4 have shown that both inflammation and injury responses like those in ischaemia/reperfusion (I/R) are partially TLR4-dependent [TLR4 activates immune responses by sensing not only microbial structures such as bacterial LPS but also some endogenous “danger” molecules released by damaged host cells ,35. Inflependent ,38-40. Sependent . The abiependent . Pharmacependent .Tlr4-/- mice 3 and 6 h after injection of 500 μg LPS. These data are consistent with a report that inhibition of PI3K in vivo resulted in significant increases in circulating IL-1β, IL-2, IL-6, IL-10, IL-12, and TNF-α during polymicrobial sepsis [in vitro studies [Tlr4-/- mice, the increase in serum IL-1β and IL-2 levels 3 h after LPS treatment and a concurrent inhibition of the PI3K pathway was still not as high as the corresponding increase in serum cytokine levels in C57BL/6 mice. In addition, at 6 h, the level of IL-2 in Tlr4-/- mice receiving LY294002 was significantly higher than that in the serum of the C57BL/6 mice after LPS treatment. The significance and mechanism of this differential regulation of in vivo cytokine expression after PI3K inhibition in the Tlr4-/- mice is not clear. However, it should also be noted that the precise role of circulating cytokines in the pathophysiology of sepsis/septic shock is still controversial [In a study of time courses expression of inflammatory mediators, including IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-12(p40), IL-13, Eotaxin (CCL11), G-CSF, GM-CSF, IFN-γ, KC (CXCL1), MCP-1 (CCL2), MIP-1α, (CCL3), MIP-1β (CCL4), RANTES (CCL5) and TNF-α, of C57BL/6 mice receiving intraperitoneal injection of LPS , signifil sepsis  as well  studies . Our resoversial , and theoversial .Tlr4-/- mice is impaired by inhibition of the PI3K pathway, with a corresponding increase in mortality and production of tissue O2- and inflammatory cytokines.In this study, we demonstrate that innate resistance to LPS toxicity in The authors declare that they have no competing interests.JCY, SCW, and CSR contributed to analysis and acquisition of all data and the writing of the manuscript. THL and SLT participated in the animal surgery and acquisition of the study specimens. YCC and CJW participated in Western blotting experiment and superoxide measurement. YCW contributed to ELISA study. MWL participated in analysis of all data. CHH contributed to the design of animal study, interpretation of the analyzed results and the writing of the manuscript. All authors read and approved the final manuscript.


FCGR polymorphisms and cryptococcal meningitis.As important regulators of the immune system, the human Fcγ receptors (FcγRs) have been demonstrated to play important roles in the pathogenesis of various infectious diseases. The aim of the present study was to identify the association between FCGR2A 131H/R, FCGR3A 158F/V, FCGR3B NA1/NA2, and FCGR2B 232I/T, in 117 patients with cryptococcal meningitis and 190 healthy controls by multiplex SNaPshot technology. Among the 117 patients with cryptococcal meningitis, 59 had predisposing factors. In patients with cryptococcal meningitis, the FCGR2B 232I/I genotype was over-presented  and the FCGR2B 232I/T genotype was under-presented  in comparison with control group. In cryptococcal meningitis patients without predisposing factors, FCGR2B 232I/I genotype was also more frequently detected , and the FCGR2B 232I/T genotype was also less frequently detected  than in controls. No significant difference was found among FCGR2A 131H/R, FCGR3A 158F/V, and FCGR3B NA1/NA2 genotype frequencies between patients and controls.In this case control genetic association study, we genotyped four functional polymorphisms in low-affinity FcγRs, including FCGR2B 232I/T genotypes, which suggested that FcγRIIB might play an important role in the central nervous system infection by Cryptococcus in HIV-uninfected individuals.We found for the first time associations between cryptococcal meningitis and  Cryptococcal meningitis is the most common opportunistic fungal infection of the central nervous system in AIDS patients. Among HIV-uninfected patients, several predisposing factors for cryptococcal meningitis such as corticosteroid medication, solid organ transplantation and malignancy, etc, have been indentified. Yet cryptococcal infections in apparently healthy individuals are also increasingly being reported, especially from Asian data FCGR2A, FCGR2B, FCGR2C, FCGR3A, and FCGR3B genes, respectively.Fc gamma receptors (FcγRs) mediate a variety of immune responses after binding to IgG-opsonized pathogens or immune complexes, and therefore act as immune regulators in both autoimmune and infectious diseases FCGR polymorphisms had been associated with the susceptibility and severity of various infections. FCGR2A 131R/R had been reported to attribute to the susceptibility of meningococcal infection, community-acquired pneumonia (CAP) caused by Haemophilus influenza, and the development of severe malaria FCGR2A 131H/H was reported to contribute to higher risk of bacteremia in pneumococcal CAP patients FCGR2A 131R/R genotype progressed to a low CD4+ cell count at a faster rate, but conversely in individuals carried FCGR2A 131H/H there was an increased risk of Pneumocystis jiroveci pneumonia FCGR3A 158F/V gene polymorphism was not associated with progression of HIV infection, but predicted the risk of Kaposi’s sarcoma FCGR2A 131H/H was associated with a decreased risk of pneumonia, FCGR3B NA1/NA1 associated with infections, and FCGR3A polymorphisms not associated with infections FCGR3A 158V/F and FCGR2A 131H/R polymorphisms on susceptibility to pulmonary tuberculosis in the Moroccan population but no association was found FCGR2B 232T/T genotype provided protectiveness for children against severe malaria FCGR polymorphisms in patients with cryptococcosis, and found that FCGR2A 131R/R and FCGR3A 158V/V were over-presented, and FCGR3B NA2/NA2 was under-presented in patients with cryptococcosis FCGR polymorphisms in our series of patients to further verify the association between FCGR and cryptococcal meningitis.A previous study by Meletiadis et al. investigated A total of 117 HIV-uninfected patients with cryptococcal meningitis were included. Subjects from both the patient and control groups were of Chinese Han ethnicity. Clinical information and predisposing factors of the patients are summarized in FCGR3A and 2 samples failed in genotyping of FCGR2B. Allele distributions of the tested FCGR genes in the control group were in Hardy-Weinberg equilibrium. The frequencies of FCGR2A, FCGR3A, FCGR3B and FCGR2B genotypes were shown in FCGR2B 232I/T genotypes and cryptococcal meningitis based on dominant and over-dominant model. The FCGR2B 232I/I genotype was over-presented  and the FCGR2B 232I/T genotype was under-presented  in patients with cryptococcal meningitis in comparison with controls. No significant difference was found in the distribution of FCGR2A 131H/R, FCGR3A 158 F/V and FCGR3B NA1/NA2 genotypes.Two samples failed genotyping of FCGR2A, FCGR3A, FCGR3B and FCGR2B between the 58 patients without predisposing condition and controls. Similar to results from the overall patient group, associations were also found between FCGR2B 232I/T genotypes and cryptococcal meningitis based on dominant and over-dominant model. Specifically, FCGR2B 232I/I genotype was also more frequently detected , and FCGR2B 232I/T genotype was also less frequently detected  in patients without predisposing factor than in controls. For the genotype distribution of other polymorphisms , there was also no significant difference between patients and controls.We further compared the genotype distribution of FCGR polymorphisms has been reported to exhibit substantial inter-ethnic variation. According to our population, frequencies of FCGR2A 131R/R, FCGR3B NA2/NA2, and FCGR2B 232T/T in all subjects were 16%, 11%, and 7% respectively, similar to those reported among other Asian populations  FCGR3A 158F/V genotypes between the Asian and Caucasian populations The distribution of FCGR2A, the G>A SNP at amino acid position 131 results in a histidine (H) to arginine (R) change (FCGR2A 131H/R), resulting in reduced affinity of the correspondent receptor to IgG2 FCGR3A causes a phenylalanine (F) to valine (V) substitution (FCGR23A 131F/V) and FCGR3A 158V/V encoded receptors show higher affinity to IgG1 and IgG3 FCGR3B gene, five nucleotides  are combined to form two main haplotypes termed FCGR3B NA1 and FCGR3B NA2, and receptor encoded by FCG3B NA1 haplotype binds to IgG1 and IgG3 more easily FCGR2B 232I/T causes an isoleucine (I) to threonine (T) substitution in the transmembrane domain FCGR2B 232T are unable to interact with activating receptors The four polymorphisms of low-affinity receptors genotyped in our study have each been demonstrated to affect functions of their encoded receptors. In FCGR polymorphisms have been demonstrated to be associated with susceptibility and severity of numerous infections, there has only been one previous genetic association study on the relationship of FCGR genotypes and cryptococcosis FCGR2A 131H/R, FCGR3A 158F/V and FCGR3B NA1/NA2 in 103 cryptococcosis patients and 395 healthy controls. They found that in patients with cryptococcosis FCGR2A 131R/R and FCGR3A 158V/V were over-presented , while FCGR3B NA2/NA2 was under-presented .Although FCGR2B 232I/T genotypes, which was not reported in Metediatis’ study. As the only known inhibitory FcγR, FcγRIIB has an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain, and thus it plays an important role in regulating the immune system FCGR2B 232I/T is located in the transmembrane domain, and the FcγRIIB receptors encoded by FCGR2B 232T are unable to interact with activating receptors and exert inhibitory activity FCGR2B 232T/T to be a susceptible genotype for systemic lupus erythematosus C. neoformans, glucuronoxylomannan (GXM). In a previous study by Monari et al., the immunosuppressive effect of GXM was demonstrated to be dependent on FcRγIIB, based on the evidences that FcγRIIB blockade inhibits GXM-induced IL-10 production and induces TNF-α secretion, and that the addition of monoclonal antibody to GXM reverses GXM-induced immunosuppression by shifting recognition from FcγRIIB to FcγRIIA FCGR2B 232I/T heterozygote instead of the minor homozygote 232T/T that is under-presented in patient group. One study on children with idiopathic thrombocytopenia (ITP) also showed a similar pattern, that the FCGR2B 232I/T was less frequently detected in acute ITP in comparison with chronic ITP In our study, we found for the first time that cryptococcal meningitis was associated with the FCGR2A 131H/R, FCGR3A 158F/V, FCGR3B NA2/NA2 and cryptococcal meningitis was found in our study. The cause for discrepant results may be multifactorial. One was the ethnic differences between the two studies. Subjects in our study were of Chinese Han ethnicity, while the majority of subjects in Meletiadis’ study were Caucasians (60%). As a matter of fact, the FCGR3A 158V allele was significantly increased only in patients who were Caucasian in Meletiadis’ study. Secondly, all the cases in our study were diagnosed with cryptococcal meningitis, while some patients from Meletiadis’ study were cryptococcosis without central nervous system involvement. Furthermore, both studies had relatively small sample sizes, which could be underpowered to generate positive results.Unlike results from Meletiadis’ study, no association among In conclusion, our study suggested that FcγRIIB genetic polymorphism may contribute to the susceptibility of cryptococcal meningitis. The overall association is relatively weak, which warrants validation in larger population.This study was reviewed and approved by the Ethic Committee/Institutional Review Board (HIRB) of Huashan Hospital, Fudan University, and informed written consent was obtained from each participant.C. neoformans from cerebrospinal fluid (CSF) by culture or positive India ink smear, and (2) compatible histopathological findings, which are 5–10 µm encapsulated yeasts observed in brain tissue. Patients who had no microbiological or pathological documentation but present with positive cryptococcal antigen titer (≥1∶10) in CSF and met at least one of the following criteria were regarded as probable cryptococcal meningitis: (1) abnormal laboratory tests or an increased open pressure (≥200 mmH2O) of CSF, (2) abnormalities of cranial imaging (Computerized Tomography or Magnetic Resonance Imaging) which could not be explained by other factors, and (3) comorbidities that compromise the host immune system. Cryptococcal antigen was determined using diluted CSF with the Latex-Cryptococcus antigen detection system (Immuno-Mycologics). Patients and volunteers were assessed for predisposing factors as follow, immunocompromising diseases  + T lymphocytopenia (unexplained CD4+ T lymphocytopenia with CD4+ T lymphocyte count <300 cells/mm3) A total of 200 volunteers and 117 unrelated patients with proven or probably diagnosed cryptococcal meningitis who were referred to Huashan Hospital, Fudan University, China, from 2001 through 2011 were recruited for the present study. Patients who met at least one of the following criteria were considered as proven cryptococcal meningitis: (1) Isolation of FCGR polymorphisms including FCGR2A 131H/R, FCGR3A 158F/V, FCGR3B NA1/NA2, and FCGR2B 232I/T were selected for genotyping after literature review of previous studies on association between FCGR polymorphisms and infectious diseases Four functional FCGRs  according to manufacturer’s instructions. Genotyping of 8 SNPs in FCGRs  was perfhttp://frodo.wi.mit.edu/primer3/) according to the reference sequences from dbSNP (http://www.ncbi.nlm.nih.gov/SNP). There were homologous sequences between FCGRs, the specificity sequences were checked with the sequence databases using BLAST (http://www.ncbi.nlm.nih.gov/blast/blast.cgi). These sequences were also verified by SNPmasker1.1 (http://bioinfo.ebc.ee/snpmasker) to make sure that the different bases were caused by SNP http://www.cstl.nist.gov/biotech/strbase/AutoDimerHomepage/AutoDimerProgramHomepage.htm). The primers used in this study were listed in Four pairs of primers for PCR amplification including 5 fragments of 587–2394 bp and 8 primers for SNaPshot extension reactions were designed by Primer3 online software (v.0.4.0) . The cycling conditions for FCGR2A and FCGR3A were 95°C for 2 min, 35 cycles using 96°C for 20 s, 62°C for 2 min, and 72°C for 3 min, then 72°C for 10 min, and finally kept at 4°C. The cycling conditions for FCGR2B and FCGR3B were 95°C for 2 min, 7 cycles using 96°C for 20 s, 55°C for 2 min, and 72°C for 3 min, then 72°C for 10 min, and finally kept at 4°C. PCR products were then purified .The PCR reactions were performed with 1 µL of DNA and 1 µL multiple PCR primers (the concentration was 1 µM) in a total volume of 20 µL containing 1× HotStarTaq buffer, 2.0 mM MgThe extension reaction to identify single nucleotide polymorphisms in the PCR products was performed in a total volume of 10 µL containing 2 µL purified PCR product, 1 µL primer (the concentration was 0.8 µM), 5 µL SNaPshot Multiplex Kit , and 2 µL ultrapure water. The cycling conditions for extension were 96°C for 1 min, 28 cycles of 96°C for 10 s, 52°C for 5 s, and 60°C for 30 s, and kept at 4°C. Then each extended product was added to 1 U shrimp alkaline phosphatase, incubated at 37°C for 1 hour, and the enzyme inactivated at 75°C for 15 min. Then, 0.5 µL was added to 0.5 µL Liz120 SIZE STANDARD , 9 µL Hi-Di , and sequenced by ABI3130XL Sequencer . Finally, the primary data was analyzed by GeneMapper 4.0 . Genotypes were determined by the type of nucleotide presented at SNP site, which was visualized by one or two different color peaks on the figures.http://www.ncbi.nlm.nih.gov/projects/SNP), and the data were matched well. Genotyping was performed blind to group status.For quality control, a random sample of 5% of the cases and controls was genotyped twice by different researchers, with a reproducibility of 100%. The minor allele counts were compared with database  and 95% confidence intervals (CIs) were obtained by SPSS 16.0 for Windows . P-values <0.05 were considered statistically significant.Dominant, over-dominant, recessive and allelic models were applied for the analysis of genotype distribution. Hardy-Weinberg equilibrium, differences in gene polymorphism distributions between patients and controls were analyzed with 2×2 χ