S-locus encoding both S-RNases and F-box proteins as female and male determinants, respectively. S-RNase activity is essential for pollen rejection, and a minimum threshold value of S-RNases in the style is also required. Here we present biochemical evidence that eEF1A is a novel S-RNase-binding partner in vitro. We further show that the normal actin binding activity of eEF1A is enhanced by the presence of S-RNase. Lastly, we find that there is a co-localization of S-RNase and actin in the incompatible pollen tubes in structures reminiscent of the actin bundles formed by eEF1A. We propose that increased binding of eEF1A to actin in the presence of S-RNase could help explain the disruption of the actin cytoskeleton observed during SI reactions.Self-incompatibility (SI) is a genetic mechanism that allows flowering plants to identify and block fertilization by self-pollen. In the Solanaceae, SI is controlled by a multiallelic  S-locus S-locus are termed haplotypes, whereas variants of any of the individual genes in the S-locus are termed alleles Angiosperms represent the largest and most diverse group of land plants. In most species flowers have male (anther) and female (pistil) sexual organs in close proximity, increasing the risk of self-pollination. To prevent selfing and limit the deleterious effects of inbreeding, angiosperms have adopted several strategies to promote out-crossing. Among these strategies, self-incompatibility (SI) is one of the most widespread, reported in more than 100 families and estimated to be present in almost 40% of species, and is considered an important factor of the evolutionary success of angiosperms The female determinant is a polymorphic glycoprotein with ribonuclease activity called the S-RNase S-RNases Nicotiana alataPetunia hybrida pollen using a yeast-two-hybrid screen Petunia inflataNicotiana alata transmitting tract specific), NaPELPIII  and a 120 kDa glycoprotein have also been shown to interact with S-RNases immobilized on affinity resin Nicotiana. NaTrxh, was found to bind and reduce S-RNases in vitroPetunia hybrida ubiquitously expressed protein up-regulated in germinating pollen, PGPS/D3 Pyrus pirifoliaNicotiana alataFull manifestation of the SI response also requires several other proteins not encoded by the S-locus see , some ofSolanum chacoense, SSC has been used to estimate the threshold for the S12-RNase in a number of different genotypes The SI response also requires the presence of a minimum level of S-RNase in the style, an amount referred to as a threshold level. For example, transgenic plants must express more than this minimum level of S-RNase in order to show an SI phenotype, and several species show a phenomenon termed sporadic self-compatibility, or SSC   allows a range of glycoproteins to be purified from crude stylar extracts, of which the major component is the mono-glycosylated S11-RNase (11-RNase is more abundant than the S12-RNase 12-RNase which has four glycosylation sites In Solanaceae-type gametophytic self-incompatibility (SI), glycosylated stylar ribonucleases, termed S-RNases, are taken up indiscriminately by growing pollen tubes 11-RNase . The S1111-RNase only two proteins were present, eEF1A and actin. Thus, no other proteins appear to be involved in the interaction between these three components.The finding that a translation factor was an S-RNase binding partner was intriguing considering that the role of the S-RNase is to catalyze RNA degradation, thus blocking translation. To eliminate the possibility that a protein other than the S-RNase in the glycosylated protein fraction might be responsible for binding eEF1A, we prepared an even more highly purified S-RNase using ion exchange chromatography (IEX) to fractionate the conA-purified stylar extract prior to immobilization on a new ConA column . The entS. chacoense S11-RNase and eEF1A, we next immobilized eEF1A using a GST-tagged S. chacoense eEF1A expressed in bacteria. For this experiment, GST-eEF1A was purified using a glutathione agarose resin, then mixed with a total stylar protein extract and passed again through the affinity resin. Western blot analysis of the specific eluate showed that both S-RNase and actin were again retained on the immobilized eEF1A . Furthermore, this binding does not require actin although the amount of actin binding to eEF1A in our pull-down experiments is stimulated markedly by the presence of S-RNase  of 2, 1 mM MnCl2, 1 mM CaCl2, 0.02% 2-mercaptoethanol) by chromatography on PD-10 columns  and the extract incubated with 0.5 mL of Concanavalin A resin  at room temperature for 1 h. After washing the resin with ConA binding buffer, the bound proteins were washed with low-salt ConA binding buffer . The bound proteins were then either used directly for the pollen protein binding assay on the resin or were eluted using ConA elution buffer  for further purification. To further purify the S-RNase, the buffer in the ConA eluate was replaced with IEX binding buffer (50 mM MES pH 5.5) by PD-10 chromatography and the extract applied to a Resource S cation-exchange column using an AKTA FPLC system . Proteins were eluted with a linear gradient from 0 to 1 M NaCl in IEX binding buffer, and all fractions containing the S11-RNase, as verified by immunoblotting, were pooled. The IEX elution buffer was then replaced with ConA binding buffer by PD-10 chromatography and the purified S11-RNase extract immobilized on 0.5 mL of ConA resin. The resin was then washed with low-salt ConA binding buffer before use for the pollen protein-binding assay.Stylar proteins were extracted from 0.5 g of L25 styles ground in liquid nitrogen using ConA extraction buffer . After clarification by centrifugation, the extraction buffer was replaced with ConA binding buffer , whereas the entire eluate containing proteins binding the Resource S-purified S11-RNase was sequenced directly.Pollen proteins were extracted from 0.5 g of mature L25 pollen in ConA extraction buffer using a French press. After clarification by centrifugation, the extraction buffer was replaced with low-salt ConA binding buffer and the sample incubated twice with 0.5 mL of Concanavalin A resin to deplete Concanavalin A-binding proteins from the pollen extract. Unbound proteins were then incubated with the ConA-bound stylar proteins or ConA-bound purified SS. chacoense eEF1A coding sequence (GenBank Accession Number KF573426) was cloned and sequenced using sequence information derived from Illumina sequencing of pollen RNA from lines V22 and 2548 using BamHI-ScEF1a-F (5′ GGGGGGATCCGGTAAGGAAAAGATTCACAT 3′) as a forward primer and ScEF1a-BamHI-R (5′ GGGGGGATCCTCACTTTCCCTTCTTCTGGG 3′) as a reverse primer. The sequence was subcloned into the BamHI site of the pGEX-4T-2 protein expression vector  in frame with the N-terminal GST tag. The derived amino acid sequence of this pollen tube eEF1A contains all the amino acids determined by MS sequencing of the ConA column eluate. An eEF1A coding sequence from the dinoflagellate Lingulodinium polyedrum was also amplified using primer sequences derived from a sequence database generated by Illumina RNA-Seq (GenBank Accession number JO706501), forward primer BamHI-EF1a-F (5′ GGGGGGGGATCCTCTGATCAAAAAGAACATGTCTCTA 3′) and reverse primer Ef1a-NotI-R (5′ GGGGGGGCGGCCGCTTATTCAATCTTTGTAATTTTACCA 3′) and using L. polyedrum cDNA as template. This sequence was similarly subcloned in pGEX-4T-2 to create a N-terminal GST fusion protein.A full length E. coli  according to the manufacturer’s instructions. For protein expression, 100 mL of each culture at an OD of 0.5 were induced with 0.1 mM IPTG and incubated for 3 hours at 28°C. Cultures were lysed in extraction buffer  using a French press. The GST-tagged recombinant proteins were purified by incubating clarified crude bacterial extracts for 2 h with 200 µL GST resin . After washing the resin with the same buffer, bound proteins were eluted using elution buffer . The buffer in the eluted proteins was replaced with interaction buffer  by chromatography on PD-10 columns . The protein concentration of the purified extracts was determined using a Bio-Rad Protein Assay kit .The expression vectors were used to transform BL21(DE3) competent 11-RNase, 10 µg of a commercial bovine actin preparation  or both together. The resin was then washed and eluted as above.For pull-downs using only ConA purified S-RNase as in , stylar 11-RNase antibody Antibodies for western blots 11-RNase were performed as described using a 20 nm colloidal gold-labeled goat anti-rabbit secondary antibody Immunolocalization experiments with a 1/50 dilution of a rabbit anti-SFigure S1S11-RNase-enriched preparations by ConA chromatography do not contain actin. A partially purified S11-RNase fraction eluted from a ConA column contains the S11-RNase as the principal component. Stylar actin does not co-purify with the S11-RNase as shown by the lack of anti-actin antibody staining in the ConA eluate.(JPG)Click here for additional data file.Table S1Proteins interacting with a crude S-RNase preparation.(DOCX)Click here for additional data file.Table S2List of peptides sequenced from proteins interacting with an immobilized highly purified S-RNase.(DOCX)Click here for additional data file.


Mucoepidermoid carcinoma (MEC) can be classified into low-, intermediate-, and high-grade tumors based on its histological features. MEC is mainly composed of three cell types , which correlates with the histological grade and reflects its clinical behavior. Most cancers exhibit reduced methylation of repetitive sequences such as Long INterspersed Element-1 (LINE-1) and Alu elements. However, to date very little information is available on the LINE-1 and Alu methylation status in MEC. The aim of this study was to investigate LINE-1 and Alu element methylation in MEC and compare if key differences in the methylation status exist between the three different cell types, and adjacent normal salivary gland cells, to see if this may reflect the histological grade.LINE-1 and Alu element methylation of 24 MEC, and 14 normal salivary gland tissues were compared using Combine Bisulfite Restriction Analysis (COBRA). Furthermore, the three different cell types from MEC samples were isolated for enrichment by laser capture microdissection (LCM), essentially to see if COBRA was likely to increase the predictive value of LINE-1 and Alu element methylation.p<0.001) between the cell types, and showed a stepwise decrease from the adjacent normal salivary gland to the intermediate, mucous and squamous cells. The reduced methylation levels of LINE-1 were correlated with a poorer histological grade. In addition, MEC tissue showed a significantly lower level of LINE-1 and Alu element methylation overall compared to normal salivary gland tissue (p<0.001).LINE-1 and Alu element methylation levels were significantly different (Our findings suggest that LINE-1 methylation differed among histological grade mucoepidermoid carcinoma. Hence, this epigenetic event may hold value for MEC diagnosis and prognostic prediction. Mucoepidermoid carcinoma (MEC) is a malignant neoplasm of salivary glands that occurs in both adults and children -4. MEC tHistologically, MECs are primarily composed of three morphological cell types, which include squamous or epidermoid, mucous and intermediate cells, and these can take the form of a solid nest or cystic structure. According to the WHO classification system, MECs are classified as low-, intermediate- or high-grade based on five histological features: the presence of a cystic component, neural invasion, necrosis, mitotic activity and anaplasia . HoweverH-ras gene at codon 12 and/or 13 (and none at codon 61), but however these were essentially detected in high-grade cases [To date, only a few genetic studies have proposed mechanisms for the etiology of MEC. Some MECs have been reported to have a t11:19)(q21:p13) translocation, and abnormality -16 that 1:19(q21:de cases .One of the most common epigenetic changes found in cancer is the genome-wide decrease in methylation (genome-wide hypomethylation) -23. LonguCuC) had a value that could significantly distinguish between normal peripheral blood mononuclear cells (PBMCs) and PBMCs from patients with cancers of the oral cavity, liver, colon, lung and the nasopharynx [Most methylation studies report only quantitative information about the methylation level. Recently, we reported that the methylation patterns of LINE-1s could provide more crucial information regarding carcinogenesis. For instance, the percentage of hypomethylation loci . Paraffin-embedded tissues from 24 salivary glands from MEC patients (diagnosed by histology) and 14 normal salivary glands from unrelated patients were obtained from the Department of Pathology, Faculty of Medicine, Chulalongkorn University. The limited clinical data available for each MEC patient was obtained from records, and this is shown in Table These specimens were cut into 3-μm-thick sections and mounted onto histological glass slides. After deparaffinization, and hydration, the sections underwent standard hematoxylin and eosin (H&E) staining. After, each slide underwent, histopathological evaluation by three independent pathologists , and those cases correctly identified as MECs were histologically graded according to the WHO diagnostic criteria . The MECMEC tissues underwent laser capture microdissection (LCM) using the method described in our previous study . Using oDNA was extracted from laser-captured microdissected tissue by proteinase K digestion and a standard phenol-chloroform extraction protocol . For whoTaqI and TasI in NEB buffer 3 , while the Alu amplicons (117 bp) were digested with TaqI in TaqI buffer . Both digestion reactions were incubated at 65°C overnight. The LINE-1 and Alu element digested products were then electrophoresed on an 8% non-denaturing polyacrylamide gel and stained with the SYBR green nucleic acid gel stain . Distilled water was used as negative control. All experiments were performed in duplicate.All DNA samples were treated with sodium bisulfite essentially following guidelines provided . For COBRALINE-1, the bisulfate-treated DNA was subjected to 40 PCR cycles with LINE-1-F (5’-CCGTAAGGGGTTAGGGAGTTTTT-3’) and LINE-1-R (5’-RTAAAACCCTCCRAACCAAATATAAA-3’) primers at an annealing temperature of 50°C. For COBRAAlu, the bisulfite-treated DNA was subjected to 40 cycles of PCR with two primers, Alu-F (5’-GGCGCGGTGGTTTACGTTTGTAA-3’) and Alu-R (5’-TTAATAAAAACGAAAT TTCACCATATTAACCAAAC-3’) at an annealing temperature of 53°C. After PCR amplification, the LINE-1 amplicons (160 bp) were digested with mCuC, 160 bp), hypomethylation , 1 methylated CpG  and 1 unmethylated CpG  /(C+A+A+B+D), percentage of mCuC = 100×(A)/(((C-D+B)/2)+A+D), percentage of uCmC = 100×(D-B)/(C-D+B)/2)+A+D, percentage of hypomethylated loci (uCuC) = 100×B/(((C-D+B)/2)+A+D) and percentage of hypermethylated loci (mCmC) = 100×((C-D+B)/2)/(((C-D+B)/2)+D+A). DNA samples isolated from HeLa, Jurkat and Daudi cell lines were used as positive controls in each experiment and for interassay variation normalization [The intensities of the COBRALINE-1 fragments on the polyacrylamide gel were quantified and analyzed using a Phosphoimager and the ImageQuant Software . COBRALINE-1 generated 4 products depending on the methylation state of the 2 CpG dinucleotides, as follows: partial methylation , partial methylation  and methylated loci  . Next, the frequency of each Alu element methylation pattern was calculated as follows: percentage of methylated loci (mC) = 100×(2C+2B)/(2A+2B+2C) = 100×(2D)/(2A+2D), percentage of hypermethylated loci (mCmC) = 100× C/(A+B+C), percentage of partially methylated loci (uCmC+mCuC) = 100×B/(A+B+C) and percentage of hypomethylated loci (uCuC) = 100×A/(A+B+C). DNA samples from HeLa, Jurkat and Daudi cell lines were used as positive controls in every experiment and to standardize interassay variation [The ImageQuant Software (Molecular-Dynamics) was used to quantify the intensities of COBRAAlu fragments on the polyacrylamide gel. COBRAAlu generated 3 bands based on the methylation status: hypomethylation uCuC, 17 bp, paariation .t-test to analyze among cell subtypes in paired samples. An independent sample t-test was performed to determine differences between LINE-1 and Alu element methylation patterns in total MEC tissue and normal tissue of the salivary gland. A receiver operating characteristic (ROC) analysis was performed to verify the ability of COBRALINE-1 and COBRAAlu to differentiate MEC lesions from normal salivary gland tissue. An area under the ROC curve (AUC) value of 1.0 indicated perfect accuracy, while an AUC value of 0.5 indicated an inability to distinguish between samples. The cut-off values were selected to determine the diagnostic value of this approach. All calculations were performed using the SPSS software for Windows, version 17.0  and the MedCalc statistical software. The results were considered statistically significant when the p-value was less than 0.05.Analysis of variance (ANOVA) was used to compare methylation patterns of LINE-1 and Alu elements among squamous, mucous, intermediate and adjacent normal salivary gland cells present in MEC lesions, as well as a paired mC) decreased from the adjacent normal salivary gland cells (N) to the intermediate cells (I), mucous cells (M) and squamous cells (S). The results showed significant differences between S:M, S:I, S:N and M:N (p<0.001). However, there was no significant difference between M:I and N:I   Figure A.uCuC) LINE-1s increased from N to M, I and S. Significant differences were found between S:M (p=0.048), S:I, S:N and M:N (p<0.001). However, no significant difference was found between M:I and N:I  . There was no significant difference between S:I (p=0.099), M:I (p=0.551), M:N (p=0.353), S:M and N:I (p=1.000).Additionally, the frequency of unmethylated  Figure B. A signp<0.001) of mC between N:M, N:S and M:S. Furthermore, the uCuC is also different when compared between N:M (p=0.013), N:S (p<0.001) and I:M (p=0.006). The detailed data of paired comparisons are shown in Additional file The paired comparisons among cell types displayed strongly significant difference p<0.00 of mC bemC) decreased from N to M, I and S. The results showed significant differences between S:M (p=0.001), S:I (p=0.002), S:N (p<0.001) and M:N (p=0.003). However, there was no significant difference between M:I and N:I (p=1.000) , S:I (p=0.002), S:N (p<0.001) and M:N (p=0.003). No significant difference was found between M:I and N:I (p=1.000)  and S:N (p=0.011). However, there was no significant difference between S:I (p=0.061), M:N, M:I and N:I (p=1.000). Moreover, the frequency of mCmC of Alu elements showed no significant difference between groups of microdissected cells.A significant difference in the percentage of mC and uCuC of Alu elements were observed as followed: N:M (p=0.032), N:S (p<0.001) and I:M (p=0.008). The detailed data of paired comparisons are shown in Additional file For the paired comparisons, significant differences in both :S p<0.00 and I:M mC) of microdissected cells in each cell type decreased from low-grade to intermediate-grade and high-grade MEC, p<0.001  in microdissected cells was not related to the histological grade of the MEC  (Table mC of LINE-1s in the low-grade MECs was higher than in intermediate-grade (p<0.001) and high-grade MECs (p<0.001), respectively  Table A and B. y Figure A and B.mC) in MEC tissue was also significantly lower than in normal salivary gland tissue (p=0.001). In agreement with these results, the frequency of uCuC of Alu elements in MEC tissue was significantly higher than in normal salivary gland tissue (p=0.001) (Table The frequency of each Alu element methylation pattern in whole MEC tissue is shown in Table 1) Table C and D. 1) Table C and D.mC and the mCmC patterns yielded ROC values indicative of diagnostic reliability. For the mC pattern of LINE-1, the area under the ROC curve (AUC) value was 0.974, while the cut-off value, sensitivity and specificity were 38.73%, 100% and 92.86%, respectively , essentially as we have previously shown that this is a very sensitive method for isolating a minimal number cells of interest from whole tissue sections and performing molecular analysis on the extracted nucleic acid. In this study, we procured ~1,500 microdissected cells, which provided sufficient DNA to allow the detection of LINE-1 and Alu methylation levels and pattern. However, LCM did not allow the sufficient isolation of all three cell types from most of the MEC cases, but with some exceptions, for example cases MEC23 Figure C and D, From our data we found that LINE-1 and Alu methylation levels were different among the three histological grades of MEC. We also observed that LINE-1 hypomethylation in adjacent normal salivary gland cells was dependent on the histological grade of the MEC Figure A. This aIn conclusion, our findings provide preliminary information of methylation levels between different cell components in MEC, which may be related to histological grading and prognosis of the neoplasm. The knowledge may be applied as a diagnostic tool or a prognostic marker for these tumors in addition to histological grading.MEC: Mucoepidermoid carcinoma; LINE-1: Long INterspersed Element-1; LCM: Laser capture microdissection; COBRA: Combine Bisulfite Restriction Analysis.The authors declare that they have no competing interests.PS carried out all experiments, performed the statistical analysis and drafted the manuscript. SK and KD participated in the patient enrollment. AM, KS and NK conceived the study, participated in its design and coordination, and revised the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6890/13/10/prepubPaired comparison of LINE-1 and Alu methylation patterns among MEC cell subtypes.Click here for file


Aconitum carmichaeli by ultra-high-pressure liquid chromatography coupled with high resolution LTQ-Orbitrap tandem mass spectrometry (UHPLC-LTQ-Orbitrap-MSn). According to accurate mass measurement and the characteristic neutral loss filtering strategy, a total of 42 diester-diterpenoid alkaloids (DDA) were rapidly detected and characterized or tentatively identified. Meanwhile, the proposed fragmentation pathways and the major diagnostic fragment ions of aconitine, mesaconitine and hypaconitine were investigated to trace DDA derivatives in crude plant extracts. 23 potential new compounds were successfully screened and characterized in Aconitum carmichaeli, including 16 short chain fatty acyls DDA, 4 N-dealkyl DDA and several isomers of aconitine, mesaconitine and hypaconitine.A rapid and effective method was developed for separation and identification of diester-diterpenoid alkaloids (DDA) in the roots of  Aconitum carmichaeli debx. (Fuzi), officially listed in the Chinese Pharmacopoeia, is one of the most widely used Chinese traditional medicinal herbs. It has been used for the treatment of rheumatism, neuralgia, and cardiac complaints for thousand years A. carmichaeli such as aconitine (AC), mesaconitine (MA) and hypaconitine (HA) have stimulated scientists’ strong interests in exploring their toxicity mechanisms and drug discovery 8 and C14 diester-substituted diterpenoid alkaloids. Therefore, development of new reliable strategy for rapid detection and identification of new or minor bio-active diterpenoid alkaloids and toxins in aconitum plants is of great significance.Previously, a number of analysis methods have been successfully applied to characterize diterpenoid alkaloids and their metabolites, including liquid chromatography (LC)-diode array detection (DAD) n method was developed to characterize the diester-diterpenoid alkaloids (DDA) in A. carmichaeli. The fragmentation pathways of different type DDA were studied by HR-MSn and the diagnostic fragmentation behaviors were summarized. With the strategy of neutral fragment monitoring and diagnostic fragmentation filtering, a total of 42 DDA including 16 new type of short chain lipo-DDA were rapidly detected and identified by UHPLC-LTQ/Orbitrap-MS, as well as N-deethyl type DDA and other potential new structural components of DDA were reported for the first time.In the present study, a simple and effective UHPLC-ESI-LTQ/Orbitrap MSA. carmichaeli were purchased from the GAP base in Jiangyou county, Sichuan province, China, and authenticated by Professor Zhihai Huang in our lab. Standards of aconitine (AC), mesaconitine (MA) and hypaconitine (HA) were obtained from National Institute for the Control of Pharmaceutical and Biological Products (NICPBP) .HPLC-grade acetonitrile, methanol and formic acid were purchased from Sigma Aldrich . Water (18.2 MΩ) was from a Milli-Q water system . The roots of µm membrane filter and directly analyzed by LC-MS.Powdered root (2 g) was refluxing extracted in 30 mL of methanol at 80°C for 1 h and then diluted 10 times with methanol. The diluted solution was filtered through a 0.22 18 column  at room temperature. The mobile phase consisted of acetonitrile (A) and water containing 0.2% formic acid (B) and the elution gradient was set as follows: 15% A (0 min), 40% A (15 min) and 85% A (20 min). The mobile phase flow rate was 200 µL/min and the injected volume was set at 1 µL.LC analyses were performed on a Thermo Accela UHPLC system  consisting of a quaternary pump, a diode-array detector (DAD), an autosampler, and a column compartment. Samples were separated on a Waters AQUITY UPLC CSH Cn experiments, a Thermo-fisher LTQ-Orbitrap XL hybrid mass spectrometer  was connected to the UHPLC instrument via an ESI interface. The conditions of ESI source were set as follows: Sheath and auxiliary gases flow, 25 and 3 arbitrary units, respectively; spray voltage, 4 KV; capillary temperature, 320°C and tube lens voltage: 120 V. Samples were analyzed in positive ion mode and the full scan mass range was set between m/z 350–1200 with the acquisition of centroided-type mass spectra. Accurate mass analyses were calibrated according to the manufacturer’s guidelines using a standard solution mix of sodium dodecyl sulphate, sodium taurocholate, the tetrapeptide MRFA acetate salt and Ultramark. In the full scan mode, resolution of the Orbitrap mass analyzer was set as 30,000 (FWHM as defined at m/z 400). In the MSn experiments, data-dependent MSn scanning was performed to minimize total analysis time as it can trigger fragmentation spectra of target ions and prevent repetition by dynamic exclusion settings. In the data-dependent scans, the FT resolution was 15, 000. The collision energy for collision-induced dissociation (CID) was adjusted to 40% of maximum, the isolation width of precursor ions was m/z 2.0 and default values were set for other CID parameters. A series of potential interference or contaminant ions were added to exclusion list to avoid triggering their MSn data acquisitions. A syringe pump was used for the direct infusion of standard solutions (about 10 µg/mL in methanol) in positive mode and flow rate was set at 10 µL/min. Capillary temperature was set at 275°C, sheath gas 5 arbitrary units and no auxiliary gas was needed for direct infusion. All other parameters were identical to those in LC-MSn experiments.For LC-ESI-MSm/z 60  and Xcalibur 2.1 (Thermo Fisher Scientific) was employed for data analysis. After the automated removal of noise and baseline signals, the theoretical extract chromatogram of main DDA was shown in the plot of neutral fragment for m/z 60 . Considen experiments, there are several options of fragmentation type provided by LTQ-Orbitrap analyzer . With the benefit of linear ion trap, MS5 CID fragmentation data are easily acquired when ion abundance is adequate. In this study, three different fragmentation measurements were conducted in independent LC-MSn experiments. In the first measurement the survey scan was performed in the Orbitrap analyzer  and was followed by MS2 scan in the Orbitrap . These high accurate tandem mass spectra can provide unequivocal evidences for identification of fragment and neutral loss. However, the high mass resolution and the scan rate were compromised in Orbitrap detection. In some cases, peaks were too narrow to produced sufficient data points for high resolution fragment data acquisition. To make up for it, other two separate measurements were performed, in which the CID MS2 and MS3 experiments were conducted by dynode detection, respectively. This faster acquisition of MSn spectra could provide more fragment ions and the order of fragmentation could be determined to show evidence of structural identification.For LC-MSn experiments of A. carmichaeli sample. In general, a low normalized collision energy of 30∼35% generated the minimal numbers of the fragments and the maximum intensity of fragment ions. In contrast, a 45% or higher energy made fragment ions abundances dropped significantly. As a result, compromised normalized collision energy of 40% was adopted in the LC-MSn experiments.The optimal CID collision energy was pre-tested by direct infusion experiments of reference compound hypaconitine (HA) and by LC-MSA. carmichaeli could be divided into three major groups, diester diterpenoid alkaloids (DDA), monoester diterpenoid alkaloids (MDA), and de-esterified diterpenoid alkaloids (DEDA) 8 and C14  and de-esterified diterpenoid alkaloids (DEDA). In the MS3, MS4 and MS5 high resolution mass spectra, consecutive neutral losses of MeOH denoted the successive losses of three methoxy groups at C1, C6, C16 or C18 sites from the skeleton. Chen et al.1, C6, C16 and C18 sites is: C16< C1< C6< C18. Thus the first neutral loss of CH4O in the MS2 spectrum referred to the methoxy group at C16 site. The most prominent ion of [M+H-AcOH-MeOH-CO]+ in MSn spectra, suggested that the most active elimination of MeOH occurred at C16 site and meanwhile could be a key fragmentation to determine the presence of 15-OH, where a neutral molecule of CO was eliminated when the enol form tautomerized into the ketone form 2O]+ in MS2 and MS3 spectra could be a diagnostic fragment to identify the hydroxyl group at C3 site, since the same fragment ion were not observed in MSn spectra of HA. This fragmentation pattern was partly consistent with previous work The high-resolution tandem mass spectrometric analyses of typical DDA (mesaconitine (MA), hypaconitine (HA), and aconitine (AC)) were exhaustively investigated by direct infusion. A. carmichaeli and related species. Basically, major DDA found in A. carmichaeli show 15-OH, 3-OMe and 16-OMe substitutions, while those with 10-OH and 3-OH substituted were identified as well 2O and their related neutral molecular. Lastly, the proposed fragmentation patterns were compared with reference compounds in the literatures A database was set to review all the DDA from A. carmichaeli. As for the introduction of UHPLC system and small particle-size (1.7 µm) column, the total analysis time for separation of A.carmichaeli was limited within 20 min with a high peak resolution, which was approximately four times faster than that of a conventional column packed with 5.0 µm particles. By profiling the acetyl-DDA with neutral fragment extracted chromatogram (NFC) and characteristic fragment ions searching, at least 42 DDA were detected and characterized, many of which were potential new DDA  were detected at tR 10.50 and 11.81 min, both of which showed the [M+H]+ ions at m/z 616.3127 with the elemental composition of C33H46O10N. The base ion at m/z 556.2891  in MS2 spectrum indicated that they were DDA derivatives. For peak 19, the characteristic [M+H-AcOH-MeOH-CO]+ ion at m/z 496.2682  observed in MS2 proved the presence of C15-OH substitution, while the [M+H-AcOH-MeOH-H2O]+ ion at m/z 506 wasn’t detected, suggesting that there was no hydroxyl group occupied at C3 site. The key fragmentation behaviors were contrary for peak 27. The presence of a hydroxyl group at C3 were resulted from the loss of [M+H-AcOH-MeOH-H2O]+ in MS2 and MS3 spectra to generate fragment ion at m/z 506.2530 , while the possibility of C15-OH substitution was excluded due to the absence of the neutral loss of MeOH+CO (60 Da) at m/z 496 in MSn. By reviewing the literatures 10 position. Thus, peak 19 and 27 were identified as 10-OH-13-deoxyhypaconitine and 15-deoxymesaconitine, respectively and their proposed fragmentation pattern were illustrated at Peaks of 31, 21 and 32 were unambiguously identified as hypaconitine (HA), mesaconitine (MA) and aconitine (AC) respectively, which were confirmed by comparing their A. carmichaeli. By means of diagnostic neutral loss screening and high-definition mass measurement, two isomers of aconitine were detected in A. carmichaeli in this study. Both peak 23 (tR: 11.20 min) and 25 (tR: 11.50 min) showed the identical [M+H]+ precursor ion at m/z 646.3227 (C34H48O11N). Comparing with AC, peak 23 exhibited very similar MSn spectra, except that the key neutral loss of [M+H-AcOH-MeOH-CO]+ for identification of C15-OH was not observed. The MS2 spectrum showed a base ion at m/z 586.2994 , as well as the [M+H-AcOH-MeOH]+ ion at m/z 554.2739  and [M+H-AcOH-MeOH-H2O]+ ion at m/z 536.2623 . We deduced that peak 23 was a 15-deoxy DDA and the hydroxyl group was substituted to C10 position. Thus peak 23 was tentatively identified as 10-OH-15-deoxyaconitine, which was a potential new aconitine-type C20-diterpenoid alkaloid 2 spectra of most DDA, the most abundant ions were [M+H-AcOH]+ fragments resulting from the elimination of a molecule of AcOH at C8 site. However the MS2 spectrum of peak 25 was so peculiar that the base ion was [M+H-AcOH-MeOH]+ at m/z 554.2737 , although the crucial fragment ion of [M+H-AcOH]+ at m/z 586.2994  was also found as an prominent ion in MS2 , suggesting they were MA isomers. Their MS2 spectra all gave m/z at 572 as base peak and the loss of 120 Da may consist of an AcOH, MeOH and CO groups as mentioned above. Therefore, peak 11 and 16 were characterized as 10-OH-hypaconitine isomers.Peak 11 and 16 both displayed a [M+H]m/z 614, there were three peaks (tR 11.85 (peak 28), 15.08 (peak 37) and 18.35 min (peak 42)). In the FT full scan spectra, peak 37 and 42 shared the identical elemental composition of C34H48O9N and further yielded the same fragment ion at m/z 554, suggesting these to be DDA derivatives. The key fragment ion of [M+H-AcOH-MeOH-CO]+ were detected only in the MSn spectrum of peak 42, which represented that their different substitution patterns at C15 site. There was no any sign of neutral elimination of MeOH+H2O for both peak 37 and 42. By investigating the reference data, peak 37 and 42 were identified as chasmaconitine and 3, 13-deoxyaconitine, the fragmentation pathways of which were reported previously In the extracted ion chromatogram (EIC) of + precursor ion at m/z 614.2953 (C33H44O10N) and gave value of the ring and double-bond equivalent (RDB) of 12.5, which was 1 more than that of all other DDA (RDB  = 11.5) detected in A. carmichaeli. It was preliminarily assumed that peak 28 was the dehydration product of MA (C33H46O11N). Its MSn spectra displayed the typical fragmentation behaviors of DDA, in which the neutral losses of acetic acid, MeOH and CO were observed, as well as the loss of BzOH. Neither the [M+H-AcOH-H2O]+ nor [M+H-AcOH-MeOH-H2O]+ product ion was detected in the MS2 spectrum, which allowed us to infer that the elimination of H2O could be assigned to C3 position to form 2,3-didehydro-hypaconitine Peak 28 showed the [M+H]m/z 630. They both exhibited [M+H]+ ion at m/z 630.3273 (C34H48O10N) and their MS2 spectra all gave ion at m/z 570.3051  as the base peak. On the basis of the characteristic fragment ion at m/z 510.2834 [M+H-AcOH-MeOH-CO]+, peak 33 and 38 could be easily discriminated as 13-deoxyaconitine (indaconitine) and 3-deoxyaconitine, respectively. Besides, a pair of isomers eluted at 13.54 and 13.88 min and they were tentatively identified as isodelphinine or delphinine A pair of deoxyaconitine isomers (peak 33 and 38) was detected in the EIC of 2 data and the literature reports The peaks at 8.80 min (peak 9) and 11.30 min (peak 24) had the similar fragmentation pathways with MA and AC, respectively. According to the MSA. carmichaeli and other species of Aconitum. However, no N-dealkyl-type DDA has been reported in A. carmichaeli so far. In this study, new N-dealkyl aconitine-type of DDA were plausibly characterized by means of high resolution mass spectrometry and key neutral loss patterns. For instance, peak 12 exhibited [M+H]+ ion at m/z 618.2914 , which showed less elemental composition of C2H4 than that of AC. Its MS2 spectrum gave a base fragment ion at m/z 558.2685 , consistent with it being a diester aconitine-type diterpenoid alkaloid. The successive losses of 32 to produce ions at m/z 526.2427, 494.2150 and 462.1888 suggested the methoxy groups linked to C1, C6 and C16 sites. In the MS3 spectrum, two expected characteristic ions at m/z 508 and 498, derived from neutral loss of 50 Da (MeOH+H2O) and 60 Da (MeOH+CO) respectively, were observed. Based on its fragmentation pattern . These three isomers could be deduced as the isomers of N-demethyl-hypaconitine by their MSn spectra. Based on the key neutral loss, they were tentatively characterized as N-demethyl-hypaconitine, N-demethyl-10-OH-13-deoxyhypaconitine and N-demethyl-15-deoxy-mesaconitine, respectively  all gave a [M+H]ectively .8 position is occupied by a long chain fatty acyl (14–25 carbon atoms) have been isolated and identified from A. carmichaelin spectra of the lipo-DDA were identical with that of acetylated counterparts at C8 position and the base ions in the MS2 spectra were corresponded to the neutral losses of fatty acids. Typical base ions in the MS2 were at m/z 586, 572, 556, 570 and 554, corresponding to the skeletons of benzoylaconine, benzoylmesaconine, benzoylhypaconine, benzoyldeoxyaconine and benzoyl-3,13-deoxyaconine, respectively n spectra with those of acetylated DDA and the type of fatty acid could be determined by acquiring high-resolution full scan mass spectra. For example, CID of m/z 704 at tR 10.16 min produced identical MS2 and MS3 spectra with that of AC to give characteristic fragment ions at m/z 586, 526, 536, 494 and 368 (4H6O4 were determined by high resolution mass of [M+H]+ at m/z 704.3311 (C36H50O13N), corresponding to the elimination of succinic acid. Therefore, the ion at m/z 704 corresponded to 8-scn-benzoylaconine (here scn denotes succinic acid).Lipo-alkaloids, in which the C and 368 , indicatm/z 690  , succinic acid (scn) and hydroxybutanoic acid (hbt) were confirmed by high resolution mass spectrometry. Thus peak 2, 5 and 8 were tentatively characterized as 8-mlc-benzoylhypaconine, 8-scn-benzoylmesaconine and 8-hbt-benzoylaconine, respectively. By using this method, the new finding of other twelve short chain lipo-DDA  in which C8 was occupied by a 4C, 5C or 6C fatty acid, hydroxyl fatty acid or diacid were demonstrated and they have not been reported previously in Aconitum genus.Three peaks gave the same protonated ion at .64 min)  and thein method has been successfully developed for separation and identification of diester-diterpenoid alkaloids in the roots of Aconitum carmichaeli. Based on the provided strategy, forty-two diester-diterpenoid alkaloids were extracted and characterized. A number of new diester-diterpenoid alkaloids including sixteen short chain fatty acid esters of DDA and four N-dealkyl-type DDA were detected. The UHPLC-LTQ-Orbitrap-MSn method presented here provided essential data for further pharmacological and toxicological studies of Aconitum plants. Moreover, LC-LTQ-Orbitrap mass spectrometry has been demonstrated to be an effective tool for the analysis of the components and searching for some novel compounds in a complex plant extract.In this study, a simple and rapid UHPLC-LTQ-Orbitrap-MS


ABCE1 in human lung adenocarcinoma tissues and metastatic lymph nodes, which was also correlated with clinical stages. Moreover, human lung adenocarcinoma A549 cells were infected with lentiviral vectors containing ABCE1-specific shRNA, and resulted in significant inhibition of cell growth. Using microarray assay, a number of differentially expressed genes were found after ABCE1 suppression. Our results demonstrated the potential role of ABCE1 in human lung adenocarcinoma, which may provide some molecular basis for the mechanisms of development and progress of human lung adenocarcinoma, and help to find new pharmacological targets.ATP-binding cassette transporter E1 (ABCE1), also known as RLI (RNase L inhibitor), is a new type of endoribonuclease inhibitor, which can specifically bind to RNase L and abolish its effect. ABCE1 binds to eIF2α and eIF5 to form a pre-translation initiation complex, suggesting its crucial role in cell growth, development and certain pathological processes. To probe the role of ABCE1 in the development and progress of human lung adenocarcinoma, we first detected the changes of its mRNA and protein expression in tissues, and found a high expression level of  Lung cancer is the maligant tumor with the highest incidence world-wide, and adenocarcinoma is one of the major pathological types. Due to its unknown developmental mechanism, early metastasis and insensitivity to radiation and chemo-treatment, 5-year survival rate is very low. Therefore, to probe the newly found related genes is an important task to improve the prognosis of lung cancer.ABCE1) gene is located at 4q31 and encodes a 68-kDa protein. The protein is composed of 599 amino acids, which is classified as an ABC transporter based on the sequence and organization of its ATP-binding domain, also known as nucleotide-binding folds (NBFs). The NBFs contain characteristic motifs, Walker A and B, signature C, Q-loop and H-loop. However, due to its lack of transmembrane (TM) domains, ABCE1 can not transport substrates across the membrane as other members of the family. ABCE1, also known as RLI (RNase L inhibitor), is a new type of endoribonuclease inhibitor, which can specifically bind to RNase L and abolish its effect  were obtained from the Liaoning Province Tumor Hospital. All human tissue samples were collected with informed consent of the patients. In all the patients lung adenocarcinoma was confirmed by pathological diagnosis. Human lung adenocarcinoma A549 cells  were maintained in RPMI-1640 medium  at 37°C under 5% COABCE1 and β-actin were: ABCE1 5′-TTGGTTGTGGGAAGTCGT-3′ (sense) and 5′-GCTTATGTAGTTAATGGGAGGT-3′ (antisense); β-actin, 5′-CTTCCTGGGCATGGAGTC-3′ (sense) and 5′-GCCGATCCACACGGAGTA-3′ (antisense). PCRs were carried out under optimized conditions. Agarose electrophoresis (2%) was used for detection. The integrated density values (IDV) were calculated with β-actin as an internal control.Total RNA was isolated from different tissues or A549 cells using TRIzol reagent , and cDNA was synthesized with the cDNA Synthesis Kit . The primers for Cell lysis from tissues or A549 cells were extracted as usual, separated by SDS-polyacrylamide gel and transferred to polyvinylidene difluoride membranes . The membranes were then incubated with an anti-ABCE1 or β-actin monoclonal antibody (Sigma-Aldrich) diluted by 1:200, followed by incubation with a rabbit anti-mouse horseradish peroxidase conjugated IgG. The ECL Western blot analysis kit  was used to observe the results.ABCE1 shRNA was cloned into lentiviral pSC-GFP vectors encoding the green fluorescent protein (GFP). Then 293T cells were transfected using calcium phosphate with the VIRPAC packaging construct pCMV-dR8.74 and pMD2G. Supernatant containing lentiviral particles were collected every 72 h after transfection and collected by centrifugation. Titers (IU/ml) were the cells with fluorescent signals (percent)/5×1.5×105×103. Subsequently, lentiviral particles with MOI of 1×105 IU/ml were directly added to 5×105 A549 cells and cells were cultured for 72 h. Fluorescent intensity was detected under a microscope.ABCE1 shRNA was synthesized and annealed using the following oligonucleotides: 5′-GATCCGCTACAGCGAGTACGTTTACCTGTGAAGCC ACAGATGGGGTAAACGTACTCGCTGTAGCTTTTTTG-3′ and 5′-AATTCAAAAAAGCTACAGCGAGTACGTTTACC CCATCTGTGGCTTCACAGGTAAACGTACCTGCTGTAG CG-3′. And the double-strand 6) were placed in 96-well plates with RPMI-1640 in a final volume of 500 μl. The following day, cells were transfected with pSC-ABCE1. Then cell proliferation was assessed by MTT assay after 24, 48, 72, 96 or 120-h culture. Following incubation at 37°C for 3 h, the reaction was stopped by the addition of 150 μl DMSO. After the crystal dissolved, the absorbency of the samples was determined at 492 nm.Cell viability was examined by routine 3--2,5-diphenyltetrazolium bromide assay. A549 cells . RNA quantity was measured by A260/A280 ratio. RNA quality was assessed using agarose electrophoresis. Samples with high-quality RNA were hybridized to human WG-6V3 Beadchip KIT expression microarray  in accordance with the Illumina standard protocol, and the data were analyzed using Illumina BeadArray Reader.Experiments were performed a minimum of three times. Results represent the mean ± SD from three experiments. Representative results are depicted. We compared data using one-way ANOVA or t-test as appropriate, and defined statistical significance at p<0.05.ABCE1 gene in human lung adenocarcinoma, we first detected its expression in human lung adenocarcinoma tissues. The results of RT-PCR and Western blot analysis showed that ABCE1 mRNA and protein were expressed in lung adenocarcinoma tissues, normal lung tissues and metastatic lymph nodes  and lower than in metastatic lymph nodes (p<0.05) (To probe the role of ph nodes . By comp(p<0.05)  and II.ABCE1 expression of the 18 cases of lung adenocarcinoma. As shown in ABCE1 mRNA and protein were differentially expressed in lung cancers of different clinical stages. The expression in cases of stage III was significantly higher than cases of stage I and II. And the expression in group of N1+2 was significantly higher than cases of N0.Subsequently, we analyzed the association between the clinical stages (classified according to the 1997 TNM classification of UICC) and ABCE1 shRNA fragment was cloned into the lentiviral pSC-GFP plasmid and confirmed by sequencing (termed pSC-ABCE1). Then pSC-ABCE1, pCMV-dR8.74 and pMD2G were co-transfected into incasing 293T cells. Twenty-four hours later, green fluorescence was detected under a fluorescent microscope , including 405 known protein-encoding genes, which could be divided into the following categories: inflammatory response factors, signal transduction proteins, metabolism-related proteins, cell proliferation, development, and differentiation-related factors, coagulation factors, gene transcription, translation and modification factors and cell adhesion and apoptosis-related molecules , including 405 known protein-encoding genes, which could be divided into categories such as inflammatory response factors, signal transduction proteins, metabolism-related proteins, and cell proliferation-related factors, indicating the wide role of ABCE1 in the physiological and pathological processes. For example, the expression of GADD45 (growth arrest and DNA damage 45) was increased after ABCE1 silence. As a cell cycle-dependent protein, the expression of GADD45 is changed following the progress of cell cycle, at the highest in G1 phase and lower in S phase –11. It pABCE1 gene in human lung adenocarcinoma and its effect in the proliferation of lung adenocarcinoma cells. A number of differentially expressed genes were found after ABCE1 silencing. The present study may provide some molecular basis for the mechanisms of development and progress of human lung adenocarcinoma, and help to find new treatment targets.Collectively, our results demonstrated abnormal expression of


USH2A gene were identified. Biparental mutations in USH2A were revealed in 2 families with pseudo-dominant inheritance. A proband was found to have triple mutations, two of them were supposed to locate in the same chromosome. In conclusion, this study revealed the genetic defects in the USH2A gene and demonstrated the robustness of targeted exome sequencing to precisely and rapidly determine genetic defects. The methodology provides a reliable strategy for routine gene diagnosis of USH.Usher syndrome (USH) is a leading cause of deaf-blindness in autosomal recessive trait. Phenotypic and genetic heterogeneities in USH make molecular diagnosis much difficult. This is a pilot study aiming to develop an approach based on next-generation sequencing to determine the genetic defects in patients with USH or allied diseases precisely and effectively. Eight affected patients and twelve unaffected relatives from five unrelated Chinese USH families, including 2 pseudo-dominant ones, were recruited. A total of 144 known genes of inherited retinal diseases were selected for deep exome resequencing. Through systematic data analysis using established bioinformatics pipeline and segregation analysis, a number of genetic variants were released. Eleven mutations, eight of them were novel, in the  Usher syndrome (USH) is an autosomal recessive disorder characterized by visual loss due to retinitis pigmentosa (RP), sensorineural hearing impairment and variable vestibular dysfunction, with remarkable clinical and genetic heterogeneity This study adhered to the tenets of the Declaration of Helsinki. The protocol was approved by the ethics committee of The Eye Hospital of Wenzhou Medical College and written informed consent has been obtained from all study subjects. Comprehensive ophthalmic examination, including routine eye tests, perimetry, electroretinography (ERG), and optical coherence tomography (OCT), were carried out in each patient. Detailed family history was obtained through personal interviews with patients and their relatives . PeripheGenomic DNA was extracted from whole blood using a DNA Extraction kit  using the manufacturer's instructions. The DNA was quantified with Nanodrop 2000 . A minimum of 3 μg DNA was used for the indexed Illumina libraries according to manufacturer's protocol. The final library size 350 bp–450 bp including adapter sequences was selected.A total of 144 disease genes  were selhttp://code.google.com/p/cutadapt/), respectively. SOAPaligner program http://122.228.158.106/exomeassistant). MagicViewer After Solexa HiSeq 2000 sequencing, high-quality reads were retrieved from raw reads by filtering out the low quality reads and adaptor sequences using the Solexa QA package DNA samples of all the family members  were takTypical RP signs in the fundi, including bone-spicule hyperpigmentation and attenuated arteries, were observed in all patients. ERG showed extinguished response of the rods and OCT clearly displayed thinner retinal thickness and disorganized inner or outer segment structure . Three pWe performed targeted exome resequencing of 144 genes implicated in inherited retinal degeneration. For the samples subjected to TES, the average sequencing depths on the targeted regions were yielded from 104.38 to 140.13 . Each sade novo dominant mutation, which was afterward transmitted to F1-III-2. Family F3 also showed a genetic continuity which was considered as a dominant pedigree. However, the TES results of the two probands were probably indicated to be the USH2A gene defects. To validate the TES results from proband, we extended the same procedure to the parents and a sibling in the F1 pedigree. Interestingly, expanded familial validation successfully revealed two compound heterozygous USH2A mutations (C3416G and R3484X) in the affected father (F1-II-4), a heterozygous USH2A mutation (Q3157X) in the unaffected mother (F1-II-5) and a heterozygous USH2A mutation (R3484X) in the unaffected sibling (F1-III-3), while the proband (F1-III-2) harbored two compound heterozygous USH2A mutations (Q3157X and R3484X)  were inherited from the paternal grandfather and maternal grandfather respectively. Furthermore, the affected father (F1-II-4) harbored another mutation in USH2A leading to the disease. Co-segregation analysis was performed to confirm the extracted mutations in the other 4 pedigrees. As a result, all mutations were confirmed to co-segregate well with the disease in these families. In the pseudo-dominant family F3, both the proband (F3-III-15) and the affected sister (F3-III-13) were revealed to have two compound heterozygous mutations, c.538T>C (p.S180P) and IVS48+1G>A. The splice site mutation was transmitted from proband to her unaffected son (F3-IV-2). It was suspected that the deceased affected mother (F3-II-6) carried another unknown mutation in the USH2A gene. In family F2, the proband (F2-III-2) was found to carry three mutations. This rare condition was further confirmed by intra-familial validation. Two heterozygous mutations, c.15427C>T (p.R5143C) and c.5581G>T (p.G1861S), were confirmed in the elder daughter (F2-IV-1) who was unaffected, which indicates the bi-mutation (c.5581G>A and c.15427C>T) in the maternal allele; while another frameshift mutation, c.8602delA, was identified in the unaffected younger daughter (F2-IV-2). In the pedigree F4, two affected siblings (F4-III-2 and F4-III-4) were revealed with the same heterozygous mutations, c.8212G>A (p.D2738N) and c.5528C>T (p.P1843L). In the last pedigree F5, a novel homozygous frameshift mutation (c.4383delT) was identified in the proband (F5-II-4). The unaffected son (F5-III-3) was proved to be a carrier. Taken together, causative mutations  . Sanger utations  were sucUSH2A gene.In this study a total of 11 identified mutations were presumed to be pathological. Both the nonsense and frameshift mutations were predicted to create a premature stop codon, strongly indicating the protein dysfunction. The splicing site mutation, IVS48+1G>A, is predicted to be damaging in silico. In addition, all missense mutations were located in highly conserved  and funcUSH2A mutations USH2A in the population . In family F2, we identified three distinct USH2A mutations in the proband (F2-III-1). In these cases, targeted exome sequencing allows excavating complete information of genetic defects which may be undetectable by traditional methods.It is notable that in our strategy revealed genetic mutations in several extraordinary cases in this study. USH is well known as an autosomal recessive disease, however, among our families, pedigree F1 showed a successive transmission in a pseudo-dominant trait. This unusual case raised a question whether the patient F1-II-4 carried a In the present study, we have successfully performed genetic diagnosis of Usher syndrome by utilization of NGS and have proved that it can serve as a rapid, high-throughput and efficient screening strategy. In brief, targeted exome sequencing of the 144 known genes is sufficient and clinically utilizable to comprehensively reveal genetic defects in patients with genetic retinal disease.Figure S1Identified mutations confirmed by Sanger sequencing. Corresponding chromatograms showing mutant and wild-type alleles are as indicated.(TIF)Click here for additional data file.Figure S2Conserved amino acid sequence. Conservation of amino acid residue across species is highlighted.(TIF)Click here for additional data file.Table S1Known causative genes responsible for Usher syndrome.(DOC)Click here for additional data file.Table S2List of the genes captured in the present study.(DOC)Click here for additional data file.Table S3List of all probes used to enrich for the target genes.(XLSX)Click here for additional data file.Table S4Data summary of the targeted exome resequencing. Asterisk indicates proband patient.(DOC)Click here for additional data file.Table S5Expanded familial validation.(DOC)Click here for additional data file.


While the role of adenosine receptor-subtypes has been well defined in the setting of ischemia-reperfusion, far less is known regarding their roles in protecting the heart during other forms of cardiac stress. Because of its ability to increase cardiac contractility and heart rate, we hypothesized that enhanced signaling through A2A-R would protect the heart during the stress of transverse aortic constriction (TAC). Using a cardiac-specific and inducible promoter, we selectively over-expressed A2A-R in FVB mice. Echocardiograms were obtained at baseline, 2, 4, 8, 12, 14 weeks and hearts were harvested at 14 weeks, when WT mice developed a significant decrease in cardiac function, an increase in end systolic and diastolic dimensions, a higher heart weight to body weight ratio (HW/BW), and marked fibrosis when compared with sham-operated WT. More importantly, these changes were significantly attenuated by over expression of the A2A-R. Furthermore, WT mice also demonstrated marked increases in the hypertrophic genes β-myosin heavy chain (β-MHC), and atrial natriuretic factor (ANF) – changes that are mediated by activation of the transcription factor GATA-4. Levels of the mRNAs encoding β-MHC, ANP, and GATA-4 were significantly lower in myocardium from A2A-R TG mice after TAC when compared with WT and sham-operated controls. In addition, three inflammatory factors genes encoding cysteine dioxygenase, complement component 3, and serine peptidase inhibitor, member 3N, were enhanced in WT TAC mice, but their expression was suppressed in A2A-R TG mice. A2A-R over-expression is protective against pressure-induced heart failure secondary to TAC. These cardioprotective effects are associated with attenuation of GATA-4 expression and inflammatory factors. The A2A-R may provide a novel new target for pharmacologic therapy in patients with cardiovascular disease.Adenosine binds to three G protein-coupled receptors (R) located on the cardiomyocyte (A Activation of A2A -Rs results in coupling to Gs proteins and activation of adenylyl cyclase 1- and A3-Rs inhibits adenylyl cyclase and modulates other signaling pathways regulated by Gi/o. Studies using murine models in which the A1- and A3-Rs have been genetically manipulated demonstrate a critical role for these receptors in cardiac protection during ischemia and reperfusion. 2A-Rs have been shown to promote post ischemic protection through inhibition of inflammatory responses. Adenosine is an endogenous purine nucleoside that plays an important role in protecting the heart during ischemia. The cardiovascular effects of adenosine (A) are mediated by 4 G-protein–coupled receptors (A1-R effectively attenuated the development of cardiac hypertrophy and prevented heart failure in mice that underwent transverse aortic constriction (TAC) 1-R gene-deficient had a higher mortality when compared with wild-type controls but did not demonstrate altered ventricular hypertrophy or increased cardiac dysfunction. 3-R had been knocked out demonstrated an improved survival, decreased fibrosis and hypertrophy and a more robust left ventricular function after TAC when compared with wild-type controls. The role of the A2-R in cardiac remodeling has not been defined.Owing at least in part to its pharmacological effects on neurohormone and cytokine activation, 2A -R induced a hyper-contractile phenotype with enhanced calcium handling that prevented heart failure in a transgenic model 2A -R might also have salutary effects on cardiac remodeling. To test this hypothesis we assessed the effects of TAC on cardiac morphology, function and gene expression in wild type mice and in mice with cardiac specific and controlled (adult) over-expression of the A2a-R.Previously, we demonstrated that constitutive and cardiac specific over-expression of the A1-R Sustained myocardial hypertrophy secondary to pressure overload is a leading cause in the development of heart failure and sudden death in humans 2A-AR. The human A2A-AR cDNA was cloned into a cardiac-specific and inducible controlled vector (TREMHC) composed of a modified mouse α-myosin heavy chain (α-MHC) minimal promoter fused with nucleotide binding sites for tetracycline transactivating factor (tTA) (2A-AR transgenic (TG) mice were engineered on an FVB background  and crossed with mice that expressed tTA in the heart  Fig. 1A.2A-R TG mice demonstrated a significant increase in fractional shortening by 15–20% compared with non-transgenic littermates  after TAC, but not in A2A-R TG mice , as shown in To assess the effects of pressure overload on gene expression in A2A-R are responsible for its anti-inflammatory effects 2A-R TG mice. Interestingly, toll-like receptor (TLr 7), which synergize with A2A-R agonists and adenosine to up-regulate VEGF, while simultaneously strongly down-regulating TNFα expression 2A-R TG mice even without TAC  is cardiac specific, thereby obviating effects of adenosine receptor signaling in the peripheral vasculature or in the central nervous system; (2) can be “controlled” in order to preclude the known effects of adenosine receptor signaling on cardiac and neural development; and (3) avoids the potentially confounding effects of using non-selective or partially selective adenosine receptor pharmacologic agonists and antagonists.The present study demonstrates for the first time that activation of the A2A-R TG mice after TAC.In concentric hypertrophy induced by pressure overload, it has been suggested that myocytes grow in width to increase wall thickness in order to regulate the pressure induced by increased wall stress 2A-R signaling were associated with a marked attenuation in the expression of the hypertrophy-associated genes β-MYC and ANF and the transcription regulatory protein GATA-4. β-MHC is characterized by low adenosine triphosphate activity and low filament sliding velocity but can generate cross-bridge force with higher economy of energy consumption 2A-R TG mice is associated with a decrease in GATA-4 expression may imply a link between A2A-R signaling and the expression of hypertrophy genes. GATA-4 expression level varies between tissues, between developmental stages, and in disease states. Although it is often used as a marker of cardiomyocytes, in fetal heart GATA-4 expression is the highest in the proepicardium followed by endocardial cushions and then cardiomyocytes. GATA-4 expression in the adult heart had been reported to increase by approximately twofold in heart disease 2A-R, another Gs protein-coupled receptors that also signals through activation of adenylate cyclase, appears to diminshe GATA-4 expression. However, by contrast with the β1-AR, the A2A-R can also mediate activation of MAPKs and PKC 2A-R signaling. However, further studies will be required to test this hypothesis.These salutary affects of enhanced A1- and A3-adenosine receptors in protecting the heart from the stress of pressure overload remains less clear. Using the selective A1-adeonsine agonist N6-cyclopentyladeonsine (CPA), Liao et al found that A1-R signaling attenuated TAC-induced changes in left ventricular fractional shortening and heart to body-weight ratios in C57B6 mice. However, when the A1-R was genetically deleted, TAC had identical effects on ventricular hypertrophy and dysfunction. 3-R attenuated TAC-induced left ventricular hypertrophy, fibrosis and dysfunction, suggesting that over-expression of the A3-R would have a deleterious effect. Since A1- and A3-R signaling inhibit adenylyl cyclase, slow heart rate, and inhibit cardiac contractility while A2A-R signaling increases adenylyl cyclase activity and enhances cardiac contractility, it is not surprising that these different adenosine receptor-subtypes have disparate effects in the context of pressure-induced stress. Earlier studies have suggested a role for adenosine in cardioprotection during pressure-induced stress. For example, treatment with dipyridamole, an adenosine uptake blocker that increases myocardial adenosine levels, attenuated chamber remodeling in rats with pressure overload hypertrophy. 2A-R agonist displays rapid anti-inflammatory properties in a variety of in vitro and in vivo models 2A-R, which might be attributable to its salutary effects on cardiac remodeling. Future studies will be required to determine why the enhanced cardiac specific A2A-R signaling suppresses myocardial inflammation and what the molecular relationship is between mycoytes, inflammatory cells, and fibroblast during enhanced A2A-R signaling.A2A-R over-expression is protective against pressure-induced heart failure secondary to TAC. These cardioprotective effects are associated with inhibition of GATA-4 expression and attenuation of the up-regulation of hypertrophy gene program that characterizes the pressure overloaded heart. Taken together, these results suggest that the A2A-R may be a therapeutic target in the treatment of patients with hypertension or hypertrophic heart disease.In summary our study demonstrates that A2A-R TG as previously described 2A-R TG in FVB mice after removal of doxycycline (DOX) from their diet at 3wks. Animal studies were approved by the Institutional Animal Care and Use Committee of Thomas Jefferson University.Experiments were carried out in transgenic mice with controlled cardiac restricted over expression of the human A2A-R TG and WT mice were exposed to 0.67 µM of fura-2 AM for 15 minutes at 37°C. Fura-2-loaded myocytes were field-stimulated to contract  in medium 199 containing 1.8 mM [Ca2+]o. Fura-2-loaded myocytes mounted on [Ca2+]i transient measurements using a Dvorak-Stotler chamber situated in a temperature-controlled stage (37°C) of a Zeiss IM 35 inverted microscope  were performed as previously described Myocytes from A2A -R littermates (N = 10) underwent transverse aortic banding (TAC) as previously described Eight-week-old male wild-type FVB mice (N = 17) and ALeft ventricular (LV) function was evaluated with transthoracic echocardiography at baseline, 2, 4, 8, 12, and 14Wks. A Visual Sonic Vevo 770 imaging system was used . Mice were lightly sedated with isoflurane. A parasternal short-axis view was obtained for LV M-mode imaging at the papillary muscle level. Three independent M-mode images were used for measurements of LV end-diastolic internal diameter (LVEDD) and LV end-systolic internal diameter (LVESD) in two consecutive beats according to the American Society of Echocardiography leading edge method. Fractional shortening (FS) was calculated as FS%  = [(LVEDD – LVESD)/LVEDD]×100. Anterior (AWT) and Posterior Wall thickness (PWT) were also measured. Hearts were harvested at 14 weeks.2-AR, atrial natriuretic peptide (ANP), GATA-4, and β-MHC. cDNA was reverse transcribed from 1µg of total RNA extracted from the left ventricular myocardium of male mice (n = 6 for each group) with the primers as shown in Reverse-transcribed cDNA from myocardial mRNA was used to determine the expression of APicrosirius red staining for assessment of fibrosis was performed by the Research Animal Diagnostic Laboratory (University of Missouri). To determine fibrosis, 5 independent high-power fields of stained images from each animal were analyzed by a blinded observer with Image-Pro Plus software .p<0.05 was taken to be statistically significant.All results are expressed as means ± SE. Two-way analysis of variance was used to analyze the calcium transient. Repeated measurement ANOVA was used to analyze the contractile function after TAC. Commercial software package were used for all statistical analysis  two group comparisons were made with the unpaired student t-test. In all analyses,


Rilpivirine (RPV) is a second-generation non-nucleoside reverse transcriptase inhibitor (NNRTI) that was recently approved for the treatment of antiretroviral-naïve individuals with HIV-1 viral load of <100,000 copies/ml. As transmission of the drug resistance mutations to this NNRTI may affect treatment outcomes, the frequency of primary, RPV-associated drug resistance mutations was assessed in this study.in vitro and in vivo data.For the study, 244 viral genome sequences from antiretroviral-naïve individuals were obtained by bulk sequencing. RPV-associated mutations were divided into RPV resistance mutations  according to the International AIDS Society-USA (IAS-USA) mutation list and variants potentially affecting RPV susceptibility  based on the n=2, 2.5%) and subtype B  clades. Clustering of RPV mutations was infrequent.IAS-USA RPV drug resistance mutations were found in 5.3% sequences, with E138A and E138G being the most common , followed by K101E (0.4%) and Y181C (0.4%), with no significant differences in the frequency between subtype B and non-B clades. Mutations potentially reducing RPV susceptibility were found in 2.5% of sequences, and they included V179D (1.6%) and G190A (0.8%), with equal distribution among non-B (Prevalence of RPV-associated drug resistance mutations was low in the analysed sample and did not vary across the subtypes. The frequency of variants with potential influence on RPV susceptibility was similar among non-B variants if compared to B clades. Transmitted drug resistance to RPV is uncommon, which makes this a good option for the treatment of ARV-naïve patients; however, genotype resistance testing should remain compulsory before starting an RPV-based regimen. Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are commonly affected by transmitted, primary drug resistance . In Euroin vivo and in vitro studies, other candidate drug resistance mutations have been identified, including V90I, L100I, K101H/T, V106L, E138S,V179F/D/G/I/T, V189, G190A/E/S, F227L and M230V . The total frequency of the RPV drug resistance mutations and variants potentially affecting RPV susceptibility was 7.8%, being similar among the non-B clades (8.6%) compared to the B sequences (7.4%)   vs. 4 (2.5%), respectively, odds ratio (OR) 0.99 : 0.15–7.99), , p=n.s] .According to the Stanford HIVDB, in five (2.1%) cases resistance to RPV was assigned .In phylogenetic analysis of the subtype B sequences, no clusters with key drug resistance mutations were noted . Among sThere were no trends over time in the frequency of the RPV-associated mutations, nor were there differences for gender, age or year of infection.RPV is a first-line choice for the treatment of antiretroviral treatment-naïve patients according to the current guidelines, and it is often selected because of its favourable safety and resistance profile . In thisFrequency of the transmission of NNRI drug resistance has been stable since 2003, being observed in <5% of newly diagnosed patients . It mustet al. [et al., frequencies of the RPV drug resistance mutations from the German cohort of the Frankfurt Resistance Database were lower – E138K was found in 0.4% of sequences, Y181I/V in 0.9% and K101E in 2.4%. Frequencies of the RPV drug resistance mutations and polymorphic variants were in accordance with the previous data reported not only by Lambert-Niclot et al. but also in the pooled ECHO and THRIVE data reported by Vingerhoets et al. [Observed prevalence of RPV-associated key drug resistance mutations was similar to that found in the studies performed by Lambert-Niclot et al. . In our s et al. ,19. It ss et al. .in vitro study among mutants harbouring Y181C+V179D (a six-fold reduction), V179I+Y181C (3.7-fold), L100I+V179I+Y181C (15.2-fold) and K103N+V179I+Y181C (10.5-fold) [The V179I and V179D variants are not associated with a significant reduction in the RPV fold change susceptibility values. None of the potential RPV mutations observed in our data set alone, if not co-emergent with IAS-USA-listed RPV drug resistance mutations, have been associated with virological failure to RPV . Decreas.5-fold) .et al., but generally in line with the results on the frequency of NNRTI DRM transmission [Among double mutants found in our data set, two (K101EK+G190AG and V179D+Y181C) were assigned an intermediate resistance level and one (E138G+V179D) a low level of resistance by the Stanford HIVDB. According to this algorithm, 2% of samples were RPV resistant, which is lower than the previous result of Lambert-Niclot To conclude, RPV-associated drug resistance mutations associated with virologic failure are infrequent (94.7% of patients with complete RPV susceptibility) among antiretroviral treatment-naïve patients from northern Poland. If all analysed RPV mutations are considered, both potential and IAS-USA defined, the prevalence of resistance-free clades is 92.2%, being similar across HIV subtypes and recombinant clades (92.6% for subtype B versus 91.4% for non-B clades). The clinical significance of potential RPV mutations requires further investigation; however, resistance testing in antiretroviral drug-naïve populations should remain compulsory prior to the initiation of RPV-based treatment, as outlined in the resistance testing and treatment guidelines ,6.


Corvus frugilegus) represent good indicators for the potential circulation of antibiotics in their native habitat, two populations with different migratory behavior were tested for the presence of beta-lactamase producing Enterobacteriaceae and methicillin-resistant Staphylococcus aureus (MRSA). In all, 54 and 102 samples of fresh feces of a migratory and a resident population were investigated. A total of 24 and 3 cefotaxime-resistant enterobacterial isolates were obtained from the migratory and resident population, respectively. In these isolates CTX-M-1 (n = 15), CTX-M-3 (n = 3), and CTX-M-15 (n = 3) genes were detected. TEM-1 and OXA-1 were associated with CTX-M in 3 and 2 isolates, respectively. In two E. coli isolates CMY-2 could be detected, where from one isolate displayed an overexpression of chromosomal AmpC as well. Among E. coli isolates the most common phylogenetic group was A (n = 11) and ST1683 (n = 5). In one E. coli of B2-ST131 the rfbO25b locus was detected. Three Enterobacter isolates were stably derepressed AmpC-producers. In five samples of the migratory population, PVL positive MRSA could be isolated. Two isolates were typed SCCmec IVa, spa type t127, and ST1. Three isolates carried a SCCmec type IVc, with spa type t852 and ST22. The highly significant difference of the occurrence of antibiotic resistance between the migratory population from eastern Europe compared to resident population in our study indicates that rooks may be good indicator species for the evaluation of environmental contamination with antibiotic resistant bacteria, especially due to their ecology, foraging behavior and differing migratory behavior.In order to test whether rooks ( Enterobacteriaceae. Emergence and dissemination of resistance among Enterobacteriaceae represent a serious threat to public health Enterobacteriaceae isolates in wildlife. Studies on the presence of Escherichia coli (E. coli), especially extended-spectrum β-lactamase producing E. coli (ESBL) are performed to detect antibiotic resistance in populations of wild animals E. coli is a common inhabitant of the gastrointestinal tract of birds and mammals. It is also found in the environment and can therefore be used as indicator of antimicrobial resistance change Enterobacteriaceae from wildlife in Austria. Nevertheless, in the last years a few sources regarding wildlife resistant enterobacterial isolates from neighboring countries are reported. β-lactamase producing E. coli isolates were found in various mammalian and avian hosts in central Europe Enterobacteriaceae other than E. coli in wildlife are scarce Staphylococcus aureus (MRSA) in wildlife Corvus frugilegus) is considered a good indicator for the potential circulation of antibiotics in their habitat. The biggest coherent population, comprising 5 to 10 million breeding pairs, is located in Russia Enterobacteriaceae and b) MRSA isolated from feces of rooks (Corvus frugilegus) collected from a small resident Austrian population in Burgenland as well as an eastern European migratory population with wintering grounds in the city of Vienna.Antibiotic resistance in bacteria of animal origin and its zoonotic potential has elucidated considerable attention worldwide. Microorganisms responsible for increased resistance are often those from the group of gram-negative rods, mostly beta (β)-lactamase producing members of the family No specific permission was required to non-invasively collect fresh fecal samples from the ground on public land. While the rook is protected in the EU birds directive (79/409/EWG) and in the respective national legislation in Austria this study did not involve a protected species. This field study did not involve any contact or disturbance of actual birds as fresh fecal samples were collected from the ground and tree leaves in public areas used by rooks on a daily basis. No permissions are required for this type of activity.No animals were used, disturbed or otherwise impacted by this study and therefore no Institutional Animal Care and Use Committee (IACUC) permission was required.Enterobacteriaceae, 200 µl of suspended feces was precultured at 37°C overnight in buffered peptone water (BPW)  supplemented with cefotaxime (1 mg/L) and then cultivated at 37°C overnight on MacConkey agar (MCA)  supplemented with cefotaxime (1 mg/L), which select for broad-spectrum-cephalosporin-resistant isolates. For MRSA isolation, another 200 µl suspended feces was incubated at 37°C overnight in Trypticase soy broth (TSB)  with 6.5% NaCl and then streaked in a BBL™ CHROMagar™ MRSA II  and on Columbia CNA Agar with 5% Sheep Blood, Improved II  and incubated for at 35°C for 24 h.In March 2013, 54 (1 k–54 k) samples of fresh feces of a migratory population of rooks in the Lobau  and 102 (55 k–106 k) samples of fresh feces of a resident, non-migratory population in Wulkaprodersdorf  were collected in sterile tubes and transported directly to the laboratory. Each fecal sample was suspended in 1.5 ml of 0,9% sterile saline. For isolation of Enterobacteriaceae standard bacteriological techniques were used. Isolates that were oxidase negative produced acid from glucose by oxidation as well as by fermentation were screened for ESBL production by combination disk tests using cefotaxime and ceftazidime with and without clavulanic acid  according to Clinical and Laboratory Standards Institute (CLSI) After incubation on MCA one colony representing each a distinct colonial morphotype was regrown on the same plate and on Müller Hinton Agar II . For detection of members of E. coli-like colonial morphotypes were tested using species specific PCR http://eztaxon-e.ezbiocloud.net/. Accessed 2013 September 15) Antimicrobial-susceptibility testing of enterobacterial isolates was performed by the agar disk diffusion method according to CLSI for the following antibiotics: ampicillin, piperacillin, aztreonam, doripenem, ertapenem, imipenem, meropenem, gentamicin, tobramycin, amikacin, tetracyclin, ciprofloxacin, trimethoprim-sulfamethoxazole, chloramphenicol, fosfomycin . Additionally minimal inhibitory concentration (MIC) was performed by Etest  for cefoxitin (AmpC-hyperproducing isolates (stably derepressed only) and ceftazidime, and M.I.C. Evaluator test  for ciprofloxacin, gentamicin, meropenem and cefotaxime. Escherichia coli ATCC 25922 was used as quality control strains for MIC tests. AmpC-hyperproducing isolates (stably derepressed) of Enterobacter sp. were defined as those with a cefotaxime and ceftazidfime MIC of ≥32 mg/L, without ESBL production and a negative cefoxitin-cefotaxime disk antagonist test blaOXA, blaTEM, blaSHV and blaCTX-M genes in the ESBL-positive isolates was performed by multiplex-PCRs as described earlier http://www.lahey.org/studies/ - accessed August 2013). The E. coli isolates displaying AmpC phenotype were also tested for mutations in the chromosomal ampC promoter/attenuator region as described previously tet(A), tet(B), tet(C), tet(D), tet(E), tet(G), cat, cmlA, floR, sul1, sul2, sul3, strA, int1, int2, variable regions of class 1 and class 2 integrons, and gene cassettes dhfr1, dhfr12, dhfr17, aadA1, aadA2, aadA5, estX, and sat1 and sat2 were tested with primers and under conditions as described elsewhere qepA, qnrA, qnrB, qnrC, qnrD, qnrS, and aac(6′)-Ib-cr by PCR Preliminary screening for the presence of blaCTX-M genes, such as upstream regions ISEcp1 and IS26 was investigated by PCR and sequencing as previously described Ecp1 region was searched using ISEcp1 5′ primer binding in the 5′ region of ISEcp1 and using ISEcp1 UP primer binding to the transposase gene tnpA of ISEcp1, located in the immediate blaCTX-M upstream region. The presence of IS26 linked to blaCTX-M was tested using tnpA IS26 primer recognizing the transposase gene of the IS26 element.The genetic environment of bla genes was carried out by broth mating using a sodium azide-resistant E. coli J53 and sodium azide- and rifampicin resistant E. coli MT 102 as recipients. The strains were grown to the exponential phase and then mixed (1∶2), and the donor and recipient mixture was incubated in Müller-Hintorn (MH) broth overnight at 37°C. Transconjugants were selected on MH agar supplemented with sodium azide (200 mg/L), cefotaxime (1 mg/L), and MH supplemented with sodium azide (150 mg/L), rifampicin (50 mg/L), and cefotaxime (1 mg/L), respectively. Selected transconjugants were further characterized for their antimicrobial susceptibility, presence of the relevant bla genes, and co-transfer of other resistance genes as describe above for the wild isolates.The transferability of Plasmid DNAs were isolated using the GenElute™ Plasmid Miniprep Kit  according to the manufacturer's instructions. The presence of replicons was tested by PCR-based method as described previously E. coli isolate was determined by recently described revisited Clermont method E. coli isolates was performed as described previously E. coli MLST website (http://mlst.ucc.ie/mlst/dbs/Ecoli. Accessed 2013 September 15.) scheme. In case of E. coli ST131, allele-specific PCR was performed to identify the O25 clone of E. coliThe phylogenetic group of each mecA PCR and further investigated by the PCR mec (SCCmec) typing using multiplex PCR methods with subsequent subtyping, staphylococcal Protein A (spa) typing, and multiple-locus variable-number tandem repeat analyses (MLVA) using multiplex-PCR arcC, aroE, glpF, gmk, pta, tpi, yqiL) as previously described http://saureus.mlst.net/. Accessed 2013 September 15.).MRSA isolates were confirmed by All statistical analyses were conducted using the statistical software R Enterobactericeae isolates were obtained from the migratory population, and 3 cefotaxime-resistant enterobacterial isolates were obtained from the resident population. 22 isolates displayed ESBL phenotype, and 5 isolates displayed AmpC phenotype, wherefrom three Enterobacter isolates were stably derepressed AmpC-producer. Apart from resistance to amipicillin and piperacillin antibiotics, resistance to tetracycline (85% of the isolates), trimethoprim-sulfamethoxazole (62%) and chloramphenicol (48%) was the most prevalent among isolates obtained from migratory population (E. coli. The highest 16S rDNA gene sequence similarity observed for the isolate 4.2 k (KF022262) was 99.65% with the type strain of Klebsiella pneumoniae subsp. ozaenae ATCC 11296T (Y17654). Isolates isolate 24 k (KF022261) (99.5%) and 44.1 k (KF022259) (99.1%) showed highest similarity scores with type strain of Enterobacter hormaechei ATCC 49162T (AFHR01000079). Isolate 22.1 k (KF022260) was most closely related with members of Citrobacter youngae group (Citrobacter youngae ATCC 29935T (M59291)).Out of 54 samples, a total of 24 cefotaxime-resistant pulation . Three cpulation . Using sblaCTX-M group. In all but one isolate from both rook populations blaCTX-M was detected. The genes blaCTX-M-1, blaCTX-M-3, or blaCTX-M-15 were detected solely in 15, 2, and 1 isolates, respectively. The gene blaCTX-M-1 was detected in combination with blaTEM-1 in 2 isolates, blaCTX-M-15 was detected in combination with blaOXA-1 in one isolate from the resident and in one from the migratory population. The gene blaCTX-M-3 was detected in a Citrobacter isolate as well as in a Klebsiella isolate in combination with blaTEM-1. blaTEM-15 was detected in one E. coli isolate. In one E. coli (47.2 k) isolate displaying AmpC phenotyp the combination of plasmidic AmpC (CMY-2) and overexpression of chromosomal AmpC was detected. Mutations in the promoter and attenuator region of the chromosomal ampC gene from E. coli were found at positions −18 (G→A), −1 (C→T), and +58 (C→T) of the ampC gene of E. coli K12. In one E. coli (33 k) isolate CMY-2 was solely detected. The non-β-lactamase genes strA (n = 14), sul2 (n = 13), and tetB (n = 10) were predominant . 16 isolates contained the transposase gene of the insertion sequence IS26 as analyzed by PCR. As confirmed with sequencing all these isolates showed the presence of IS26 disrupted by ISEcp1. All CTX-M-1-positive isolates had a 48 bp region (W sequence) upstream of the blaCTX-M-1 gene and an additional 32 bp X sequence, as observed in a previous sequence reported in the GenBank database (accession no. AM003904). The CTX-M-15-positive isolates had only the 48 bp W sequence, as previously described (AM040707).PCRs applied different primer combination identified the insertion sequence ISisolates .. PCR usE. coli isolates belonged to phylogenetic group A, 4 to B1, 2 to B2, 1 to C, 2 to D and one to E. E. coli MLST typing identified 11 different types among the tested isolates , ST744 (n = 3), ST34 (n = 2), ST58 (n = 2), ST69 (n = 2), and ST162 (n = 2), whereas isolates belonging to the ST23, ST90, and ST131 genotypes were also identified. The CMY-2 isolate belonged to ST224. One CMY-2 isolate could not be assigned. Allele-specific PCR confirmed the presence of the rfbO25b locus in E. coli of ST131 (phylogenetic group B2) and, showing that it belonged to the O25b type. ERIC-PCR analysis was used to analyze the molecular epidemiology of all enterobacterial isolates and showed that two ST162 (B1) isolates , two ST1683 (A) , two ST34 (A) (25 k and 39 k), two ST58 (B1) , two ST69 (D) isolates from resident population (69 k and 100 k) as well as two derepressed AmpC Enterobacter sp. were clonally related due to identical ERIC fingerprint (data not shown).Eleven isolates . Among EmecA and PVL positive as determined by PCR. Two isolates (3mrsak and 5mrsak) carried a SCCmec type IVa cassette, belonged to spa type t127, and shared the same MLVA genotype. These two isolates carried tet(K) resistance genes as well as seh toxin genes. MLST revealed that these two isolates belonged to sequence type (ST) 1. Other three isolates  carried a SCCmec type IVc, with spa type t852 and were indistinguishable after multiplex-PCR MLVA, carried aacA-aphD and msrA resistance genes and sed toxin genes. These isolates were typed by MLST as ST22. Isolates 3mrsak and 5mrsak were resistant to β-lactams and tetracycline, whereas 39mrsak, 41mrsak and 53mrsak were resistant to β-lactams and ciprofloxacin. The MICs of oxacillin for all isolates ranged from 32 to >256 mg/L, the MIC of cefoxitin from 24 to 48 mg/L  collected in the migrating population showed at least resistance to one of the tested antibiotics compared to 2.9% (3 of 102) in the resident population. As shown in Enterobacteriaceae as well as methicillin resistant Staphylococcus aureus isolates from two different rooks populations were investigated. The isolation rates (44.4%) of cefotaxime resistant enterobacterial isolates was significantly higher in migratory population  than in the resident population.In the present study, the phenotypic and genotypic characteristics of β-lactamase producing E. coli harbored blaCTX-M-1 genes and belonged to six different STs: ST34 (phylogenetic group A), ST58 (B1), ST69 (D), ST162 (B1), ST744 (A and C), and ST1683 (A). CTX-M-1, a major ESBL type in livestock in Europe E. coli harboring blaCTX-M-1 and blaTEM-1 belonged to ST744 (A) were isolated from different birds of prey  in Germany E. coli CTX-M-1 clonally related isolates also harbored blaTEM-1 and belonged to ST58. ST58 isolates harboring the same genes were also isolated from seagulls in Portugal E. coli isolates retrieved from the resident rook's population belonged to the phylogenetic group D and ST69. This is especially interesting because previous studies have reported the association of E. coli isolates of group D with extra-intestinal infections in humans E. coli isolates from seagulls E. coli and two isolates, identified based on their 16S rDNA similarity as a members of genera Citrobacter and Klebisiella from migratory population carried blaCTX-M-3 genes, The Klebsiella sp. isolate harbored also the blaTEM-1 gene. To the authors knowledge, there are no reports describing isolation of E. coli, Citrobacter sp. and/or Klebsiella sp. harboring CTX-M-3 type alone or in combinations with other β-lactamases from rooks. Two E. coli (32 k and 38 k) isolates from the migratory as well as one (58 k) from the resident population harbored the blaCTX-M-15 gene, which is the dominant ESBL gene in strains isolated from humans and is rarely found in animals blaOXA-1 gene as well. CTX-M-15 producing ST90 (A) E. coli variant has been detected previously from yellow legged gulls in the South of France E. coli clone B2-O25b-ST131population was isolated. The B2-O25b-ST131 clone presents an anthropogenic zoonotic pathogen E. coli harbouring the CTX-M-9 type in a feral urban brown rat (Rattus norvegicus) 26 and blaCTX-M-15 in isolate 32 k has been observed previously in the B2-O25b-ST131 clone E. coli isolate (8 k) harboring the blaTEM-15 gene belonged to ST23 (A). This type of E. coli has not been detected yet in rooks.Genotypic characterization of the isolates revealed that the majority of ESBL-Enterobacteriaceae were isolated, plasmid mediated AmpC (pAmpC) genes in E. coli isolates, combination of pAmpC and overexpression of chromosomal AmpC, and derepressed AmpC in three Enterobacter sp. isolates. The presence of pAmpC β-lactamases have been found worldwide but are less common than ESBLs E. coli isolates harbored pAmpC. Among the AmpC β-lactamases, blaCMY-2 is by far the most common worldwide, which was detected in isolates from human, livestock and companion animals Enterobacteriacea in wildlife is scarce. Recently, E. coli harboring CMY-2 belonged to different phylogenetic groups and STs were isolated from wild coastline birds in Miami Beach, Florida, USA E. coli isolate belonged to B1-ST224. E. coli isolate 47.2 k analyzed in this study belonged also to the same phylogenetic group and shared the same ST with those from USA. Based on the results obtained, three Enterobacter isolates most closely related to type strain of Enterobacter hormaechei, were detected as a derepressed AmpC mutants. Derepressed production of AmpC β-lactamase has been documented to be a predominant mechanism of extended-spectrum cephalosporin resistance in clinical isolates of Enterobacter spp. Enterobacter isolates from wildlife. In the present study, for all E. coli isolates, the bla genes could be transferred by conjugation to both E. coli recipient strains. Additionally, the majority of non-β-lactamase genes were co-transferred. Our results indicate that the horizontal dissemination of bla genes is due to multiple plasmids, because the majority of replicon types were detected both on conjugative plasmids as well as in the wild isolates. Association of ISEcp1 and blaCTX-M was observed in all but one blaCTX-M isolates. The presence of ISEcp1 element upstream of blaCTX-M in E. coli isolates originated from wildlife has been observed previously bla genes, in case of E. coli belonged to the same phylogenetic group and ST type, replicon combination, resistance pattern) indicating that resistances to antibiotics spread, not only due to dissemination of different β-lactamases, but also due to clonal transmission. This is probable, as they originated from rooks sharing the same habitat.During this study three kind of AmpC-producing E. coli clone B2-O25b-ST131, the isolation of MRSA isolates, even in a low prevalence , is an important finding as there are only few reports regarding this issue. In 2012, three MRSA isolates from lesser yellowlegs (Tringa flavipes) and eastern cottontails (Sylvilagus floridanus) in Central Iowa, USA have been isolated spa typed as t002 and t008, wherefrom t008 was PVL positive. MRSA was also detected in brown rats (Rattus norvegicus), chaffinch (Fringilla coelebs), common seals (Phoca vitulina) mecC gene. Recently, our working group also detected mecC positive MRSA isolates in wildlife, particularly in European brown hare (Lepus europaeus), European otter (Lutra lutra) and a European hedgehog (Erinaceus europaeus) mec IVa, belonged to spa type t127 and ST1, were PVL positive. MRSAs belonging to SCCmec IVa, t127, ST1 are commonly isolated from humans as well as from animals mec IVa, t127, ST1 has been reported. Three SCCmec IVa, t127, ST1 MRSA isolates originated from a veterinary hospital,  and caused infections in horses. Two isolates originated from horse-associated personnel. All five isolates were PVL negative spa type t852 and ST22 and carried SCCmec type IVc are a variant of epidemic methicillin-resistant Staphylococcus aureus (EMRSA-15) http://spaserver.ridom.de/, Accessed 2013 August 26.). To the authors' knowledge there are no reports on presence of these MRSA variants in rooks.Together with the isolation of pandemic CTX-M-15-producing Based on the significant differences recorded between the migratory and non-migratory populations, it is the authors' opinion that rooks may be good indicator species for the evaluation of environmental contamination with antibiotic resistant bacteria. Populations originating from North and Central-Russia to the Ural, travel the longest distance by migrating along the Baltic to Western Europe . Rook-flocks wintering in Vienna mostly originate from Russia, Belarus, Poland and the region North-East of the Carpathian mountains and arrive regularly every winter with the first small flocks of rooks usually being observed in the beginning of October followed by a peak in the middle of the month Insufficient degradation of antibiotics in wastewater treatment plants results in antibiotic residues entering the environment in moderate concentrations, and over time, selection pressure enhances survival of organisms bearing resistance traits Enterobacteriaceae as well as MRSA can be found in wildlife. The presence of pandemic isolates in wildlife emphasizes the complexity in the dissemination of antimicrobial drug resistance. Moreover, the present study confirmed that rooks might represent an important vector not only of β-lactamase producing enterobacteria including pandemic zoonotic isolates but also as a vector of MRSA.In conclusion, our study contributes to the growing evidence that multidrug resistant members of