Featured sets from: Evie Wilson, Kit Rayne, Bryony Doran and music from Sinead Campbell.
Open Mics from Beth Pearson, Alice Johns, Lauren Hart, Laura and Kate Garrett.
SheFest 2018: Wednesday 7th March 2018, 7.30pm-10pm, Theatre Delicatessen, 202 Eyre Street, S1 4QZ – In collaboration with SheFest Sheffield for International Women's Day.
What a festival it's been! Thank you SheFest for allowing us to be part of it once again! Thank you to all the performers for sharing your truth with us. Thank you for your vulnerability. Thank you for the love.
We heard featured sets from Fran Lock (London), Sarah Dixon (Huddersfield), Dan Webber (East Midlands), and Khadijah Ibrahiim (Leeds).
Camille McCawley, Carly Mountain, Evie Wilson, Zara Ade, Meg McGrath also joined us for open mic slots.
Rehearse The Verse 4th February 2018 at Theatre Deli Sheffield.
Anna May Fox (Spoken Word Artist, Norwich), ADENICO (musician, Huddersfield), Sarah Crutwell (Spoken Word Artist, Stockton-on-Tees) and Chrystine Moon (Perfomer, musician, Sheffield) helped bring 2018 in with a bang.
The anthology showcases the variety and richness of voices that often go unheard in mainstream poetry.
The Verse Matters anthology has been published by Valley Press, and all royalties will go to support ASSIST and The Archer Project, who support homeless people in Sheffield.
You can buy the anthology from Valley Press by going to their website .
We enjoyed fabulous featured performances from Cora Greenhill (The People's Priestess, poet, Sheffield) Tim Holmes (poet, Sheffield) Ros Ayres (poet and food blogger) and rap from Chanda Kay (rapper and producer from Huddersfield).
and Hive Young Writers: Amy King and Rebecca Watson.
Featured performances from Ralph Dartford & The Bleeding Obvious (A Firm of Poets), Sez Thomasin (poet, Sheffield), Rose Condo (poet) and Otis Mensah (hip hop artist).
Kate Fox Fox (poet), Salena Godden (poet), Mina Salama (Musician, Arts On The Run), and COR/PO Performance including Angelina Abel (Arts on the Run).
Anna Percy (poet, Stirred, Manchester), Carol Eades (poet, Gorilla Poetry, Sheffield), Louise Clines (poet, Broomhill Writers, Sheffield), Chella Quint (performer, Sheffield) and Emily Johnson (musician).
Plus open mic performances from: Aiyanna Lund (poet), Raluca Deso (poet), Louise Delamere (poet), Lily Karenza (poet) & Aaron Thierry (poet).
Featured artists: Malaika Kegode (Poet, Bristol), Kirsty Taylor (A Firm of Poets), Rhiannon Scutt (musician, Sheffield).
Open mic performances from: Jo Irwin (poet), Salomée Béranger (poet), Vertaa Lune (poet), Esme Brown (poet), L Worthy (poet), Dom Heslop (poet).
Open mic: Kate Garrett (poet), Umor Haque (poet), Dave Attrill (poet), Ros Arksey (poet), Gav Roberts (poet), Notuo (poet), Warda Yassin (poet), Lee Moore (v. short stories).
Open mic performances from Nick Allen (rapper/poet), Johnny Albrow (poet), Rex Davies (poet), Alex Carr-Malcolm (poet), Richard C Bower (poet) and Leanne Moden (poet).
Open mic performances from Pammie Grant (poet), Mimi (poet) – trans by Kate Marks (Conversation Club, ASSIST), 'Grapes in My Father's Yard' by poets from Material Stories of Migration (University of Sheffield).
Open mic performances include: Nicole May (Poet, Young Identity/ AndWhatTV), Steve Kay (Sheffield Authors), Susie Halksworth (Poet), Sez Thomasin (Poet), Vicky Morris (Poet), Anne Garage (short stories).
Open mic performances include: Ayun Abdi (Poet), Suzannah Evans (Bank St Arts, Poet), Marina Poppa (Firm of Poets), Katherine Henderson (Poet), Laura Church and Janine Smedley (Poet).
Open mic performances include Ingrid Hanson (Poet), Harry Man (Poet), Abigail Ward (Poet), Dave Attrill (Poet), Chris Rust (Musician), Johnny Albrow (Rapper/ Poet) and Norah Hamill (poet).
Open mic performances include: Louise Clines (poet), Liz Searle (Poet), Keira James (Poet/Singer), Kev Titterton (Poet), Jude Calvert-Toulmin (Poet), , Fran Critchlow (Poet).
Featured artists: Poet/Writer of Dramatic Monologues Shelley Roche-Jacques, Poet Sarah Thomasin, Musician Gemma Gray and Saxophonist Hervé Perez.
Open mic performances included: Amy Kinsman (Poet), Katie Wood (Fiction/ Poetry), Kworyl (Acoustic/ Poetry), Janet Murray (Poet) and Rachel Garner (Poet/ Singer).
Verse Matters was back in September with a fantastic line-up of talented artists. Everyone loved the featured artists: from Toria Garbutt's high-energy poems to Genevieve Carver's wit and Lou Richards' haunting melodies.
We were also very proud to host several poets who had never read their work in public before. Ross Walcott performed two powerful poems, Luke Worthy gave a moving reading of his work and Alice Pickersgill read her rousing poems to great applause.
and there were some fantastic open mics, including Katherine Henderson (poet), Sarah Sharp (musician), Cherry Barham (Meerly Words) and Clare Ibberson-John (Poet). Come back again next month for another fantastic night of poetry, music and solidarity!


Detection of serum biomarkers for early diagnosis of breast cancer remains an important goal. Changes in the structure of O-linked glycans occur in all breast cancers resulting in the expression of glycoproteins that are antigenically distinct. Indeed, the serum assay widely used for monitoring disease progression in breast cancer (CA15.3), detects a glycoprotein (MUC1), but elevated levels of the antigen cannot be detected in early stage patients. However, since the immune system acts to amplify the antigenic signal, antibodies can be detected in sera long before the antigen. We have exploited the change in O-glycosylation to measure autoantibody responses to cancer-associated glycoforms of MUC1 in sera from early stage breast cancer patients.
We used a microarray platform of 60mer MUC1 glycopeptides, to confirm the presence of autoantibodies to cancer associated glycoforms of MUC1 in a proportion of early breast cancer patients (54/198). Five positive sera were selected for detailed definition of the reactive epitopes using on chip glycosylation technology and a panel of glycopeptides based on a single MUC1 tandem repeat carrying specific glycans at specific sites. Based on these results, larger amounts of an extended repertoire of defined MUC1 glycopeptides were synthesised, printed on microarrays, and screened with sera from a large cohort of breast cancer patients (n = 395), patients with benign breast disease (n = 108) and healthy controls (n = 99). All sera were collected in the 1970s and 1980s and complete clinical follow-up of breast cancer patients is available.
The presence and level of autoantibodies was significantly higher in the sera from cancer patients compared with the controls, and a highly significant correlation with age was observed. High levels of a subset of autoantibodies to the core3MUC1 (GlcNAcβ1-3GalNAc-MUC1) and STnMUC1 (NeuAcα2,6GalNAc-MUC1) glycoforms were significantly associated with reduced incidence and increased time to metastasis.
Autoantibodies to specific cancer associated glycoforms of MUC1 are found more frequently and at higher levels in early stage breast cancer patients than in women with benign breast disease or healthy women. Association of strong antibody response with reduced rate and delay in metastases suggests that autoantibodies can affect disease progression.
Studies investigating the role of cancer associated antigens in the diagnosis of malignancy, and in cancer progression have focused on the MUC1 antigen, which is expressed in an aberrantly glycosylated form in most carcinomas including breast cancer [1–4]. Assays to detect the cancer-associated antigen in serum (for example, the CA15.3 assay) are widely used for monitoring disease progression and response to therapy in some late stage breast cancer patients, but this assay does not detect elevated levels of MUC1 in serum from patients with early stage disease . The detection of biomarkers in serum that could diagnose the presence of breast cancer, therefore, remains an important goal.
In contrast to antigens, autoantibodies can be detected in serum from early stage patients, the immune system serving to amplify the detection of antigen [6–10]. Most of the studies to date analyzing the profile of antibodies to MUC1 have used ELISA assays, generally detecting all Ig antibodies to the unglycosylated form of the mucin, or undefined glycoforms [1–3, 11, 12]. We have developed a novel O-glycopeptide array-based assay detecting IgG autoantibodies to specific glycoforms of MUC1 , which has low backgrounds and detects autoantibodies in sera from cancer patients. Our approach utilizes the added dimension of the changes in O-glycosylation that occur in cancer. This is in contrast to other methods used to identify specific targets for autoantibodies that do not allow for the detection of antibodies to epitopes resulting from cancer-associated post-translational modifications [1, 10, 14].
The pattern of O-glycosylation of membrane or secreted glycoproteins is consistently altered in virtually all cancers. In this type of glycosylation a family of polypeptide GalNAc-transferases add the sugar N-acetylgalactosamine (GalNAc) to serine and threonine residues in the core protein, and the glycan chain is extended by adding other sugars, individually and sequentially in the Golgi pathway. Chain termination occurs by adding specific sugars such as sialic acid or fucose . In breast cancer, glycan chains can be terminated early, in part due to changes in the expression of some glycosyltransferases , resulting in the expression of novel glycopeptide epitopes, which alter the immunogenicity of the glycoprotein [17, 18]. In the case of mucin glycoproteins, because of the presence of multiple tandem repeats which are rich in serine and threonine, the protein carries hundreds or even thousands of O-glycans, and changes in their composition can dramatically affect the immunogenicity of the cancer-associated glycoform.
MUC1 is the most widely expressed of the mucins, being found to some degree in epithelia lining glands or ducts. It is, however, upregulated in breast and other carcinomas as well as being differently glycosylated . The tandem repeat sequence in MUC1 is composed of 20 amino acids (VTSAPDTRPAPGSTAPPAHG) within which there are five potential O-linked glycosylation sites (serines and threonines), and the number of tandem repeats varies between 25 and 125 depending on the allele. The truncated glycans carried on breast cancer-associated MUC1 include the single sugar, αGalNAc (Tn), its sialylated derivative (STn), T (Galβ1,3GalNAcα) and sialylated T (see Figure 1). Our initial pilot studies used an array of 60mer MUC1 glycopeptides carrying various truncated glycans (enzymatically synthesized on the peptide in solution) for the detection of autoantibodies in a small number of breast cancer patients . While antibodies to both Tn and STn MUC1 glycopeptides were detected in some patients, a more frequent antibody response was seen to MUC1 carrying a truncated core3 glycan (GlcNAcβ1-3GalNAcα).
O-linked glycosylation in breast cancer and normal mammary gland and informative glycopeptides used. A, aberrant O-linked glycosylation results in the expression of Tn, STn, Core 1 and ST glycoforms, left of the dotted line. O-glycans in the normal mammary gland are mostly core2 based, right bottom. Note: core3 based glycans (top right) have not been demonstrated in the normal or malignant breast. B, Sequence of MUC1a and MUC1b showing sites of glycosylation and glycans carried.
We have recently demonstrated a novel method of on-chip synthesis of 20mer MUC1 glycoproteins to explore in detail the positions of the O-glycans attached to the MUC1 peptide in the glycopeptides eliciting an autoantibody response . Based on the initial analysis, and work presented here using on-chip synthesis, corresponding glycopeptides and additional glycoforms were selected for preparative scale synthesis in order to analyze antibody responses in a large cohort (395 serum samples) of breast cancer patients with Stage I or Stage II disease, comparing the autoantibody response to that in sera from patients with benign disease or age-matched healthy females. As with the pilot study, we find that the dominant antibody responses were to the MUC1 carrying the truncated core3 and STn glycans.
The analysis of autoantibodies to MUC1 glycoforms demonstrated an autoantibody response in early stage breast cancer patients that is highly significantly related to age, emphasizing the importance of using age matched controls in screening. Moreover, autoantibodies to specifc glycoforms of MUC1 were detected more frequently and at a higher level in breast cancer patients than in patients with benign breast disease or healthy females. Interestingly, autoantibody induction to the core3 based and STn glycoforms of MUC1 is significantly related to a lower incidence of metastases and increased time to metastasis, suggesting that autoantibodies to cancer-associated glycoforms of MUC1 may play a role in the development and progression of the disease.
Thirty-one Tn glycopeptides (each carrying one to five glycans) were chemically synthesized and extended on-chip as described (see Figure 2, ), using ST6GalNAcI for STn synthesis and the core3 β1-3 GlcNAc transferase for the synthesis of core3MUC1 glycopeptides. Five breast cancer sera selected as having antibodies to core3 MUC1 and/or STn MUC1, on the 60mer arrays, were screened on these arrays of MUC1 monomer glycopeptides.
Epitope mapping of autoantibodies in sera from breast cancer patients. Arrays were produced by on-slide glycosylation of the MUC1a glycopeptides carrying Tn at the sites indicated in red using core3 β3GlcNAc-T6 (middle panel) and ST6GalNAc-I (right panel). The arrays were stained with diluted sera (1:20) and Cy3 labelled anti-human-IgG. A, B, C show data obtained with sera from three individual patients.
The Tn-MUC1a, Tn-MUC1b, Core3-MUC1a and Core3-MUC1b glycopeptides were generously provided by GlycoZym Inc. (Beverly, MA, USA). and further elongated enzymatically in solution. Briefly, general enzymatic glycosylation reactions (2 to 5 μg enzyme/100 μg glycopeptide) were performed with one or several recombinant glycosyltransferases, T-synthase dC1GalT1 Drosophila melanogaster , β3Gal-TV , ST3GalI  and ST6GalNAcI  together with the corresponding donor sugar nucleotides (5 mM) in sodium cacodylate buffer solution (2.0 mL, 0.5 M, pH 7.2) containing manganese(II)chloride and incubated at room temperature for 24 hrs. All glycopeptides synthesised in solution, were individually diluted with water (1 mL), and slowly passed through a C-18 Isolute cartridge (0.5 g, previously washed with 5 mL of MeOH followed by 20 mL of water). After washing the column with water (10 mL), the desired material was eluted with a stepwise gradient (0 to 90% MeOH:water (0.5 mL aliquots). All fractions were analyzed by MALDI-TOF and fractions containing the desired product were pooled and lyophilized. Recombinant MUC1 based glycoproteins carrying ST and T were produced in CHOK1 cells as described by Bäckström et al.  and those without O-linked glycans or carrying the Tn glycan were produced in CHO ldlD cells. Glycopeptides and glycoproteins were used for printing of 48-well slides (for list of glycopeptides, see Table S1 in Additional file 1).
Printing was performed on Schott Nexterion® Slide H, Schott Nexterion®. Slide H MPX16 or MPX48 (Schott AG, Mainz, Germany). All salts for all the buffers, including TES, Triton-X-100 Tween 20, and ethanolamine were from Merck (Whitehouse Station, NJ, USA). Printing of the 48-well microarray slides was performed using a BioRobotics MicroGrid II spotter (Genomics Solution, Huntingdon, Cambridgeshire, UK) using Stealth 3B Micro Spotting Pins (Telechem International ArrayIt Division, Sunnyvale, CA, USA). The compounds were distributed into 384-well source plates (BD Falcon Microtest™ from BD Biosciences, Le Pont De Claix, France), 20 μL per well at 100 μM final concentration diluted in print buffer (PB) (150 mM phosphate, 0.005% CHAPS pH 8.5). The MPX16 well slides were printed in quadruplicates using an 8-pin (8 × 2) configuration within a 28 × 28 subgrid at a 0.20 mm pitch between each spot, and the MPX48 well slides were printed in three replicates using an 24-pin (6 × 4) configuration within a 12 × 12 subgrid at a 0.15 mm pitch between each spot. The pin dwell time in the wells was four seconds and the pins underwent three wash cycles in between source plate visits. The complete 8 × 2 or 6 × 4 array pattern was printed on a 16-well or 48-well slide in duplicate respectively. Scanning of the slides was performed on ProScanArray HT Microarray Scanner (PerkinElmer, Cambridge, Cambridgeshire, UK) followed by image analysis with ProScanArray Express 4.0 software (PerkinElmer). Human IgG was spotted (100 ug/mL in PB) for control of second antibody reactivity and for orientation of the scanned slides. Data were analyzed and plotted using Microsoft® Excel, GraphPad Prism software or MathWorks Matlab software (Microsoft®, Reading, Berkshire, UK). Quality control of printed glycopeptides were visualized by staining with glycoform-specific lectins and antibodies as described [13, 19] (see Figure S1 in Additional file 2).
The sera from cancer patients used in this study are part of a series of consecutive Stage I and Stage II breast cancer patients collected at Guy's Hospital, London between 1975 and 1980, known as the P-series. A total of 400 sera were randomly selected of which 5 did not fit the criteria and were, therefore, excluded leaving a cohort of 395. Complete clinical follow-up is available for these patients. The mean age of the cancer patients at the time of collection (one day pre-surgery) was 59.9 years, see Table 1 for clinical and treatment parameters of this cohort. Sera were taken with informed patient consent for the study of prognostic markers and were not taken as part of a particular clinical trial. At the time of taking these samples written informed consent was not required nor was ethnicity recorded. Ethical approval for use of the samples in the present study was obtained from the Outer South East London Research Ethics Committee, REC reference 07/H0809/51. Sera were also collected from 108 patients with benign breast disease who attended Guy's Hospital during the same period. Benign breast disease ranged from breast pain to benign tumours such as fibroadenomas. The mean age of the benign patients at the time of sera collection was 45 years. Sera from 99 healthy volunteer females living on the island of Guernsey formed the normal cohort (mean age 54.7) . Sera were taken from these women between 1977 and 1985. The women who donated these control sera were followed-up for between 25 and 30 years and at the time of sera selection (end of 2008) all except three were still alive and cancer free. However, none of the three that died had been diagnosed with cancer and one died in 2003 aged 102 and two in 2000 aged 93 and 95. Written informed consent had been obtained from each volunteer. This consent covered use of the serum for the investigations into the discovery of cancer biomarkers and access to the womens' medical records. Ethical approval to allow the access to patients' medical records of the volunteers who donated sera to the Guernsey bank has been obtained from the Guernsey and Alderney Committee.
All blood samples were treated in an identical manner. Blood was allowed to clot, spun and the serum removed and aliquoted before storing at -20°C.
Microarray slides were blocked in 50 mM ethanolamine in sodium borate buffer pH 8.5 at room temperature for one hour with gentle agitation. Slides were washed three times with PBS (phosphate buffered saline; 0.5M Na2HPO4, 0.15M NaCl, 0.3M KCl, 0.5M KH2PO4, pH 7.4), rinsed in MilliQ water and dried by centrifugation (Labnet, Oakland, Rutland, UK), C1303-T-230V-UK, 4,800 rpm). 25 ul (MPX16 well slides) or 5 uL (MPX48 well slides) of serum diluted (1:25 in 1 × PLI-P Buffer, 0.0065M Na2HPO4, 0.5M NaCl, 0.003M KCl, 0.0015M KH2PO4, 1% BSA, 1% Triton-X-100, pH 7.4) was added to each well and the slides incubated at room temperature for one hour with gentle agitation. Slides were washed with PBS wash buffer (PBS containing 0.05% Tween-20) and then with PBS, and then probed with Cy3 labelled anti-human IgG secondary antibody (1:1,000 dilution, C2571 Sigma, Gillingham, Dorset, UK) at room temperature, for one hour, with gentle agitation. Slides were then washed again and scanned with a single laser power and detector gain setting. The images were quantified with ProScanArray Express software program (PerkinElmer). Spots were identified using automated spot finding or manual adjustments for occasional irregularities. The spot intensities were determined by subtracting the median compound for each sample. The operator was blinded as to which samples were from breast cancer patients or controls and all samples were screened in duplicate with the same positive control serum from a breast cancer patient being run on every slide. The reproducibility of the assay was assessed by screening a subset of samples at two sites (KCL and University of Copenhagen). A total of 57 samples from cancer patients were assessed as positive in Copenhagen and 55 of these were positive in the screen at KCL indicating 96% agreement.
Each glycoform of MUC1 was synthesised on two overlapping peptides known as MUC1a and MUC1b (see above and Figure 1). The combination of MUC1a and MUC1b carrying one type of glycan is defined as the feature. Sera were defined as being positive if they reacted with one or both of the MUC1a and MUC1b glycopeptides that carried the same glycan, taking the maximum of the two values. A positive value was defined as being two standard deviations (SDs) above the mean of the reactivity of sera from patients with benign breast disease or healthy female controls, on the corresponding feature. A strong positive was defined as being four SDs above the means. These cut offs were chosen as the data are close to a half-normal distribution and it would, therefore, be expected that at least 95% of the data to be below +2 SDs. After sample analysis and image processing a data set was constructed. Chi-squared tests were used to determine the P-values in Tables 2 and 3.
1Sera was defined as positive if it was two standard deviations or higher than the mean fluorescence for at least one feature.
1 Sera were defined as all positive if two standard deviations or higher than the mean fluorescence for at least one feature.
2 Sera was defined as strong positive if four standard deviations or higher than the mean fluorescence for at least one feature.
for a preselected threshold θ.
where w 1 ,...,w k are the weights for each feature which were set to 1 to reflect the fact that we did not have pre-disposition towards a particular feature. Also, we did not perform any model selection in order to fine-tune the best combination of weights: This was to avoid any data snooping inherent with such an approach.
Comparisons of the distribution of positive status with clinical parameters were made using the Chi-squared test of significance. Comparisons of metastasis-free survival by positive status were made using the Kaplan Meier Log Rank test and Cox Proportional Hazards models were used for the multivariate analysis. These tests were performed using the SPSS (v.16) statistical package (IBM, Feltham, Middlesex, UK).
After 5 minutes at 95°C, 40 cycles were performed: 15 seconds denaturation at 94°C, 30 seconds annealing at 60°C, 30 seconds extension at 72°C and fluorescence detection at 78°C. A melting curve fluorescence analysis was performed on each sample once the amplification cycles were completed to verify that a single product had been amplified. Each sample was normalized to the housekeeping gene of β-actin (ACTB). All qPCR reactions were performed in triplicate.
In our previous work screening for autoantibodies to MUC1 glycoforms, we used enzymatic solution synthesis of glycopeptides based on three tandem repeats of MUC1 and the sera were commercially obtained . Using these arrays we confirmed that autoantibodies to MUC1 carrying the truncated O-linked glycans core3, STn - and to a lesser extent Tn - were indeed present in a proportion of the sera (54/198) from the early stage breast cancer patients to be analysed here (data not shown).
To examine the specificity of these antibodies in more detail, and to test if glycopeptides based on a single tandem repeat could be used for antibody detection, we used solid-phase glycopeptide synthesis with on-chip microarray purification  to construct a micro-array of glycopeptides based on a single 20 amino acid tandem repeat of MUC1 (MUC1a, see Figure 1). Thirty-one Tn MUC1 glycopeptides, carrying each glycan at different sites were chemically synthesized and extended on-chip with glycosyltransferases to form core3 and STn MUC1 glycopeptides (see Figure 2,  and Materials and methods).
Analysis with these arrays of five of the sera from early breast cancer patients found to be positive on the 60mer arrays confirmed that IgG autoantibodies could be detected to MUC1 carrying Tn, core3 and STn when using only a single tandem repeat. Figure 2 shows three examples of the profile of autoantibodies documented in the patients' sera. For clarity in presentation the reactivity with only seven informative MUC1 glycopeptides is shown. Autoantibodies could be identified in the sera from an individual patient that recognised several glycoforms carried on MUC1a, that is, Tn, STn and core3 (patient A), or that reacted with one particular glycoform (sera from patients B and C). Moreover, even though the same glycoform could be recognised by sera from patients A and B (core3MUC1) or from patients A and C (STnMUC1), the site carrying the glycan recognised by the antibodies was different. Therefore, the on-chip glycosylation is a rapid approach to reveal detailed autoantibody signatures to glycopeptides of MUC1.
To produce sufficient quantities of glycopeptides required to screen large numbers of sera it was necessary to use a combination of chemical synthesis coupled with chain extension in solution with glycosyltransferases. To expedite the chemical synthesis of glycopeptides using Tn or core3 building blocks two MUC1 20mer glycopeptides (MUC1a and MUC1b) were designed with a 10mer overlap. MUC1a and MUC1b carrying Tn in three positions or core3 in two positions were chemically synthesized (see Figure 1B) and for technical reasons the glycopeptide carrying core3 at the PDTRP threonine position was synthesized separately. The TnMUC1 and core3MUC1 glycopeptides were enzymatically extended in solution to produce either the STn or ST glycoforms or the extended core3 structures respectively (see Table S1 in Additional file 1 for full list). These 20mer glycopeptides, and for comparison with the previous pilot study  a 60mer carrying core3, were printed onto slides and the arrays validated by the use of lectins and monoclonal antibodies (see Figure S1 in Additional file 2). The 60mer carrying core3 gave very similar results to the 20mer carrying core3 (see Figure S2 in Additional file 3). However, the use of the smaller glycopeptides allowed the synthesis of a wider selection of MUC1 glycoforms.
These arrays were used to screen 395 sera taken before surgery from breast cancer patients with Stage I or Stage II disease (P-series). Clinical parameters for these patients are listed in Table 1 and their average age was 59.9. The availability of extended clinical follow-up data allowed us to demonstrate that the cohort showed the expected correlations with the standard parameters for predicting prognosis (lymph node involvement, grade, tumour size and age at diagnosis, see Figure S3 in Additional file 4). Sera from 108 patients with benign breast disease who attended Guy's Hospital during a similar period were also analysed. In order to age-match the controls with the breast cancer patients, sera of age-ranged matched healthy females (n = 99, average age 54.7 years) who had not developed any form of cancer 25 to 30 years after donation were also included in the analysis.
The MUC1a and MUC1b glycopeptides giving informative results are listed in Figure 1B. Figure 3 shows the reactivity of the screened sera on the combined MUC1a and MUC1b individual glycan features both as a dot blot and a heat map, the latter allowing visualisation of the reactivity of each serum sample. The dot blot (Figure 3A) shows that the highest frequency of autoantibodies in the cancer patients' sera is to the core3 based and STn MUC1 glycopeptides and these also gave the strongest signals. Importantly, reactivity to the haptens core3 or STn linked to threonine, or to the unglycosylated MUC1 was weak and found in only a few patients thus demonstrating that the strongly binding autoantibodies were largely directed to epitopes determined by both the glycan and the core peptide.
Reactivity of auto-antibodies in sera with MUC1 glycoforms. A, dot blot showing the reactivity of auto-antibodies present in sera from breast cancer patients, red crosses; patients with benign breast disease, blue circles; and healthy females aged ranged matched to the cancer patients, green circles. Bars indicate the position for 2x the standard deviation of the mean of the benign serum values, (solid bar) or the aged matched normal values, (dotted bar). Glycopeptides are defined in figure 1B. Inserts show the reactivity of autoantibodies in the sera of breast cancer patients with MUC1a and b carrying STn (left insert) and MUC1a and b carrying core3 (right insert). Each red line represents an individual serum sample. B, heat maps of the reactivity of autoantibodies in sera to glycopeptides 1 to 10 defined in figure 1B. Breast Cancer Cont, breast cancer sera continued.
The autoantibodies in breast cancer sera that reacted with STnMUC1 reacted either with the STnMUC1a or the STnMUC1b but not with both (see insert in Figure 3A). In addition, the majority of sera containing antibodies to core3 MUC1 showed reactivity with core3 MUC1a, with only four sera showing reactivity with MUC1b (see right insert in Figure 3A). Fewer and weaker responses were seen to ST and Tn MUC1.
The core3 structure is synthesized by the enzyme core3 β3-N-acetylglucosamine transferase which is encoded by the β3GNT6 gene. This glycosyltransferase is not expressed in the normal mammary gland and it has not been found in breast carcinomas . We, therefore, looked by RT-qPCR at the expression of β3GNT6 in 58 Stage 1 and Stage 2 breast carcinomas (see Figure S4 in Additional file 5). As expected, normal colon showed reasonable levels of the transcript. However, in only 3 out of 58 breast carcinoma samples was a β3GNT6 transcript detectable and these were at least 100-fold lower than the glycosyltransferase, C1GALT1, that competes for the same sugar substrate to form core 1 (T). This observation together with published data, presents compelling evidence that core3 is not expressed by breast carcinomas suggesting that the MUC1-core3 glycan on the microarray may be acting as an epitope mimic.
Table 2 shows the number and percentage of positive (defined as two standard deviations above the means) and strongly positive (defined as four standard deviation above the means) with one or more of the glycopeptides described in Figure 1B for the breast cancer patients, patients with benign breast disease and healthy controls, using either the benign sera or the sera from healthy females to define the cut offs.
Thirty-one percent of the breast cancer patients had IgG autoantibodies to the glycopeptides whether the sera from patients with benign breast disease or sera from healthy women were used as controls. Both control cohorts showed a 7% rate of strongly positive sera, and using the cut off from data obtained from sera from patients with benign disease, 24% of the cancer patient sera were strongly positive. This was reduced to 20% when the sera from healthy females (who did not develop cancer) were used for establishing the cut off. We found a highly significant correlation of the presence of antibodies with age in the cancer patients, which was observed whether the cut-off used was from the benign or the more closely aged matched controls (see Table 3). Therefore this difference of 20% versus 24% may be due the difference in age of the control cohorts.
ROC curves, shown in Figure 4, were constructed for each of the nine features (see 'Materials and methods') using either the benign serum values to provide the cut off or the values from the age matched healthy individuals. Giving an equal weight to all features (shown in pale grey lines) we formed a combination (generalised ROC curve) to give a single curve (red line). Figure 4 shows that this gave an area under the curve of 0.777 and 0.730 when using the benign sera or sera from healthy females to define cut-offs respectively. Using sera from benign patients to define cut-offs, the generalised ROC curve showed an improvement over the curves of the individual features. When looking at the dot plots in Figure 3A it is obvious that some glycoforms contribute more information and the STn and core3 glycoforms give both high positive breast cancers along with a low positive benign. Using these criteria, we constructed a ROC curve of this subset of glycoforms (combinational subset defined in Figure 1B) and this is shown by the solid black line in Figure 4. The area under this ROC curve is close to the ROC curve area for the combination of all features. When the generalised and combinational ROC curves were applied to the samples using sera from the healthy women as the controls these curves were not as informative as some of the individual features (see Figure 4B) but in these latter cases, only a small number of sera were positive (see Figure 3A).
Receiver operating characteristic analysis of individual and combinations of features. A, sera from patients with benign breast disease defines cut-offs; B, sera from healthy females defines cut-offs. Pale dotted grey lines represent individual features, solid red line represent the combination of all the features and solid black line represents the combinational subset defined in Table by * and Figure 1B by *. The table shows ROC curve area and sensitivity/specificity data for breast cancer sera on each MUC1 glycan, combination of all MUC1 glycans and a combination subset.
To investigate correlations with clinical parameters we used the healthy female control sera to define the cut-offs as they were more closely age matched. We used two standard deviations above the mean for each glycopeptide to define as all positives and four standard deviations above the mean to define strong positives. The presence of IgG autoantibodies was then compared to a series of clinical parameters including occurrence of metastases within 15 years, grade, lymph node involvement, tumour size, ER positivity, parity and age. When IgG autoantibodies to all the glycoforms screened were analysed there was a correlation with tumour grade but no other parameters (data not shown). However, using the subset defined in Figure 1B and Figure 4, Table 4 shows that the highly significant correlation with age remained (P = 0.001) and a significant correlation was observed with tumour grade when the strong positives were analysed (P = 0.016). Moreover, autoantibodies to this combinational subset were associated with a lower incidence of metastasis with a follow-up of 15 years (see Table 4).
B Total number in that group for the variable.
C Using Chi-squared test for equality of distributionsBold text indicates a significant correlation.
The metastasis-free survival was plotted for the sera containing IgG autoantibodies to glycopeptides in the combinational subset compared to negative sera. Figure 5 shows that there was a separation of the curves and Log Rank testing gave a P-value of 0.081 for all positives and 0.032 for the strong positives. However, when using Cox regression analysis adjusted for age, nodal status, grade and tumour size, this association was lost (P = 0.834, data not shown). This suggests that the presence of autoantibodies is not an independent variable but rather reflects the clinical condition of a patient with early disease. There was also no significant association of strongly positive sera with overall survival to 15 years in either univariate, see Figure 5C, or multivariate analysis (P = 0.601, data not shown).
Breast cancer patients with autoantibodies to MUC1 glycopeptide combinational subset show increased time to metastasis. A, metastasis free survival with patients with autoantibodies versus no auto-antibodies (P = 0.08). B, metastasis free survival of patients with strong autoantibodies versus no antibodies (P = 0.032). C, overall survival of patients with strong autoantibodies versus no antibodies. Thick lines indicate no auto-antibodies in sera. Dotted lines indicate autoantibodies. Vertical dashes indicate censored events.
The efforts to diagnose cancer by the use of serum assays have met with limited success, partly because the disease is heterogeneous at both causative and host response levels. The changes in O-linked glycosylation that occur in cancer [16, 23] result in the production of cancer-associated glycoforms of cancer-associated glycoproteins, which are antigenically distinct and are able to induce IgG autoantibodies [13, 24, 25]. By taking into account these modifications, the specificity of diagnostic markers might be expected to be improved, as they reflect both the patho-physiology of the disease and the hosts' response. Using novel micro-arrays carrying different glycoforms of MUC1 we have demonstrated the presence of autoantibodies in the sera of a proportion of breast cancer patients that recognise MUC1 carrying specific O-linked glycans. The difference in the percentage of sera strongly positive for the antibodies in cancer patients' sera versus sera from patients with benign disease or from healthy individuals who never developed cancer is highly significant.
Unglycosylated MUC1, or undefined glycoforms of MUC1, has been used in other ELISA based assays for detecting antibodies in sera from cancer patients [1–3, 12]. However, using the microarray assay described here, the antibody response to un-glycosylated MUC1 was very weak and only seen in a very small number of patients (see Figure 3A). In contrast, IgG autoantibodies to defined MUC1 glycoforms were found in 31% of Stage I and II breast cancer patients. Importantly, elevated levels of circulating MUC1 antigen (CA15-3) was not detected in these serum samples from early stage patients. In contrast, in late stage patients, where MUC1 antigen can be detected in sera, IgG autoantibodies were not detected (data not shown). This emphasises the importance of the autoantibody assay in early breast cancer when the antigen itself cannot be detected and the induction of autoantibodies represents an amplification of the antigenic signal.
Autoantibodies were detected to MUC1 carrying core3 based glycans and STn, with fewer responses seen to Tn and ST glycoforms. The STn glycan is relatively tumour specific, but expression of this glycan is seen in only 25 to 30% of breast cancers, which could explain the lower percentage of sera showing IgG autoantibodies to STnMUC1 compared to other cancers such as colo-rectal [13, 19]. Although the ST glycoform is the main MUC1 glycoform found in sera of advanced breast cancer patients , fewer and weaker responses were seen to STMUC1. ST is also found on other serum proteins, therefore, tolerance might be expected to be operative.
The dominance of antibodies to core3MUC1 in the breast cancer sera is surprising. The enzyme catalysing the addition of GlcNAc to GalNAc to produce core3 (β3GNT6) is generally only expressed in the cells of the gastrointestinal (GI) tract and it is dramatically downregulated in colon cancer [27, 28]. We found that the β3GNT6 transcript could only be detected in 3 of 58 breast carcinomas, and even then the level was at least 100-fold lower than the competing glycosyltransferase, C1GALT1. Moreover, core3 structures have not been found in normal breast or breast carcinomas . While clearly the autoantibodies recognise core-3 structures on MUC1 (but not the core3-threonine), it is possible that the antibodies were induced to a structure that is mimicked by MUC1 carrying core-3.
Our data (Table 3) show that the presence of autoantibodies in the breast cancer patients is highly correlated with age, emphasising the importance of using aged-matched controls for any autoantibody study. The increase of autoantibodies with age has been reported for both mice and man . Also, in a study by Pinheiro et al., measuring antibodies to unglycosylated MUC1 by ELISA in sera collected in the Nurses Health Studies, an association with age was noted in women with antibodies who went on to develop ovarian cancer . The increased specificity and sensitivity of the assay when using sera from patients with benign breast disease to determine cut-offs almost certainly reflects the younger age of the patients with benign disease. However, the correlation with age that we observed was maintained when the data were analysed using sera from the age range matched healthy females.
The availability of ideal control sera for use in retrospective studies can be a problem. The sera from patients with benign breast disease were collected at the same site and at around the same time as the cancer patients, thus reflecting the same population for both cohorts. However, the age demographics of benign breast disease results in the mean age of these patients being significantly younger than the breast cancer patients. The healthy control sera used were taken in the 1970s from healthy females living on the island of Guernsey  and allowed not only closer age matching, but extensive follow-up, although the ethnicity of the Guernsey and P-series populations may not be identical. Nevertheless, the availability of extensive follow-up allowed for the selection of sera from Guernsey women who had not developed any form of cancer for 25 to 30 years after blood donation. It should be noted that sera from all three cohorts were stored under identical conditions and for similar periods of time. Using sera from healthy women to determine cut-off points we found a significant association of the presence of autoantibodies to the combinational subset with increased time to metastasis (see Figure 5). Although this was not an independent variable, it should be noted that when the presence of autoantibodies to any of the other glycopeptides screened were analysed, no such significant association was observed (data not shown). This suggests that autoantibodies to the combinational subset could play a role in disease progression.
An important question which should be addressed is how long before clinical symptoms develop can IgG autoantibodies to aberrant glycoforms of MUC1 be detected? We have anecdotal evidence that the antibodies may appear early since one of the patients who had benign disease and high levels of IgG autoantibodies to MUC1 glycopeptides, developed breast cancer five years later. However, antibodies to the subset of MUC1 glycopeptides were found in a number of the control sera taken from the cohort of women on Guernsey where no cancer developed during 25 to 30 years of follow-up. The possibility arises that these antibodies may be functional in suppressing tumour development, as well as progression. This may be particularly true for the anti-STn-MUC antibodies (found in 2% of controls) as this glycoform is truly cancer specific and therefore the presence of antibodies should indicate the presence of tumour, albeit on a micro scale. However, as MUC1 and core3 are expressed in the colon there is the possibility that antibodies to MUC1core3 may be induced during an inflammatory response occurring in the colon resulting in expression of shorter glycans . However, following up individuals who have antibodies would not be an overwhelming procedure as it merely involves taking blood and screening with the arrays for antibody levels- possibly in parallel with mammography screening.
In searching for a simple, cost effective and non-invasive screening assay that could pick up breast malignancy in its early stages it should be noted that the MUC1 mucin is expressed by other carcinomas such as ovarian and lung, and IgG autoantibodies to MUC1 carrying core3 or STn glycans have been detected in sera from patients with these cancers . Thus, an assay based solely on aberrant glycoforms of MUC1 applied to sera from individuals without clinical symptoms would not necessarily distinguish between the different carcinomas. To extend this work we are using our on-chip technology and other glycopeptide platforms for high-throughput screening and discovery of disease-associated autoantibody glycopeptide epitopes in other proteins with more organ specific expression patterns.
Whereas the serum assay (CA15.3) used for detecting the cancer associated MUC1 antigen in breast cancer patient serum does not detect elevated antigen in early stage breast cancer, autoantibodies to specific tumour-associated glycoforms of MUC1 can be detected in sera from Stage I and Stage II breast cancer patients. The difference in the percentage of sera strongly positive for the antibodies in cancer patient sera versus sera from patients with benign disease or from healthy individuals who never developed cancer is highly significant. Follow-up of patients with benign breast disease with sera with high levels of autoantibodies may be appropriate. The lower incidence of metastases and delay in development of metastases in patients with high levels of antibodies to a subset of the glycoforms, suggests that the antibodies may play a role in inhibiting the progression of disease. Administration of antibodies with these specificities is, therefore, a possible therapeutic strategy. This would be particularly appropriate for breast cancer patients with STn positive cancers (25 to 30% of breast cancer patients) as the STnMUC1 glycoform is truly tumour specific.
This work was supported by grants from the European Union under the FP7 framework grant agreement number 201381, the NIH (PO1 CA052477 1U01CA128437-01), The Benzon Foundation, The Carlsberg Foundation, the Danish Research Council, the Danish Agency for Science, Technology and Innovation (FTP) and a centre of Excellence program from University of Copenhagen. The authors also acknowledge financial support from the Department of Health via the National Institute for Health Research (NIHR) comprehensive Biomedical Research Centre award to Guy's & St Thomas' NHS Foundation Trust in partnership with King's College London and King's College Hospital NHS Foundation Trust.
OB has shares in GlycoZym, Inc. and is a consultant for GlycoZym, Inc. The other authors declare that they have no competing interests.


text:
KEYWORD "Bank of Mum and Dad" - 13 RESULTS
West One enhances second charge products for BoMaD
West One has launched an initiative to support the Bank of Mum and Dad (BoMaD) via its second charge mortgage range. The...
Borrowers can still benefit from the Bank of Mum and Dad - claim
As the industry starts to tighten its criteria, Saffron Building Society is affirming that there is still hope for borrowers as...
Hinckley slashes rates for first-time buyer-friendly mortgages
Hinckley & Rugby Building Society has cut the interest rates on three mortgages particularly attractive to first-time buyers. The fee-free mortgages are...
Knight Frank Finance first property firm to launch later life lending
Knight Frank Finance has launched a later life finance team, making it the first major player in the property sector to...
Average Bank of Mum and Dad mortgage contribution rises by £6,000
The Bank of Mum and Dad (BoMaD) is currently spending an average of £6,000 more than in 2018 to help loved...
The Bank of Mum and Dad to initiate tougher lending rules
Tougher lending rules are surfacing after the expansion of the Bank of Mum and Dad (BOMAD) into one of the UK's...
Bank of Mum and Dad seeking financial and legal advice
The Bank of Mum and Dad is now seeking financial advice and guidance, according to new research from Key. The independent equity...
Parents provide financial aid to one in four new buyers, research shows
The Bank of Mum and Dad is very much prevalent, with recent research showing that UK parents will help 316,000 young...
Research reveals the scope of the Bank of Mum and Dad
Six in ten parents can afford to financially support their children to buy their first home, while only 5% can pay...
Bank of Mum and Dad to lend 23% more in 2017
The so-called Bank of Mum and Dad will lend 23% more funds in 2017, putting it on a par with the...
Remortgage activity continues to increase, research reveals
Homeowners remortgaged for an average of £15,000 more in 2016 than they did two years ago, with 57% of remortgage cases...
Furness unveils family first-time buyer deal
Furness Building Society has launched a new first-time buyer mortgage charging 3.50% to 95% LTV. There is no product fee on the two-year...
'Bank of mum and dad' to pump £5bn into property market
UK parents will lend or give more than £5billion over the course of 2016 to help their children get onto or move...

redpajama_set_name: RedPajamaCommonCrawl


1st & 3rd Monday, 7:00 p.m.
The Lions Club of Perth was chartered on November 11th 1953 with a total of 60 members; sponsored by the Smiths Falls (ON) Lions Club.
Our club is honoured to recognize Past International Director Lion H.H. Ernie Moore who was the first Perth Lions Club Secretary. Lion Ernie was recognized by the Lions Clubs International and rose to the position of International Director for Canada for the period 1985 - 1987.
Since 1953 our club has contrinuted significantly not only to the community in which we live but to worthy causes throughout the world through contributions to Lions Clubs International. We have also contribute to numerous charities and persons who are in need of assistance when requested to do so in and around the community of Perth.
The Salvation Army, The Perth Food Bank, The Great War Memorial Hospital (now in excess of $125,000.00), The Heart Institute, The Children Hospital of Eastern Ontario, The Canadian Diabetes Association, The Canadian Cancer Society, The Children Breakfast program at a local elementary school, bursaries to two high schools,four bursaries to students enrolled in Algonquin College, money for unfortunate families who have been burn out victims or have another serious need, eye glasses for very specialized need by children, collect eye glasses for third world countries, The Canadian National Institute of the Blind, Camp Dorset for persons requiring dialyses treatment and sponsored children to attend this specialized camp as well, we have also sponsor other children to attending other camps, and have purchased a dog for a person who is visually impaired.
In addition, our club donated the Millennium Gates to the Town of Perth located in Stewart Park, a soccer field at Conlon Farm and contributed monies towards pillars in conjunction with the Perth Lioness Club to the entrance of the Glass Palace at the entrance to the Perth Basin.
To date, the Lions Club of Perth remains an active club with a current membership of 41 Lions and we remain proud of our accomplishments.
The International Association of Lions Clubs is the largest service organization in the world with a membership of over 1.4 million members in 207 countries and geographical areas spanning the globe and continues to grow.


text:
DeMatteo
CREATOR / PERFORMER
© 2018 by Nick DeMatteo
Nikmusik@gmail.com
The Thursday Throwback Track - Episode 184: Agent Orange & "In Your Dreams Tonight"
Welcome to WEEK 184 of 4T! (Video Episode #10)
Agent Orange – This is the Voice (1986)
Ya Gotta Understand
https://en.wikipedia.org/wiki/This_Is_the_Voice
When you're in high school, you're looking for two wildly divergent things. First, you're looking to belong. That'...
The Thursday Throwback Track - Episode 183: Chicago & "One Minute Shy of Forever"
Welcome to WEEK 183 of 4T! (Video Episode #9)
Chicago – (1967-present)
Why? – Devolution!
https://en.wikipedia.org/wiki/Chicago_(band)
How is it that a band mostly known for its awesome but highly cheesy 1980s power ballads and launching the solo career of Peter Cetera h...
Pac-Man Fever & "Korean Pop Song"
Buckner & Garcia – Pac-Man Fever (1982)
The Future Was Then
https://en.wikipedia.org/wiki/Pac-Man_Fever_(album)
If you didn't live it, you can't feel it. The title track of this album was 100% accurate. While video games ha...
Wham! & "Up All Day" - Happy Holidays 2019!
Wham! – Music from the Edge of Heaven (1986)
There Are Always New Classics
https://en.wikipedia.org/wiki/Music_from_the_Edge_of_Heaven
Music & nostalgia are SO closely linked. You can hear a song and it immediately takes you...
New Music for 2020!
The Thursday Throwback Track - Episode 180: Run-D.M.C. & "The Power of Repetition (Everlasting)"
The Thursday Throwback Track - Episode 179: Talking Heads & "Make Me Mic My Mouth"
The Thursday Throwback Track - Episode 178: Royal Crescent Mob & "Some Things Happen"
The Thursday Throwback Track - Episode 177 - Video #3 - The Dead Milkmen & Me
The Cure for My Music


text:
package com.opengamma.financial.convention.rest;

import java.net.URI;
import java.util.List;

import javax.ws.rs.GET;
import javax.ws.rs.Path;
import javax.ws.rs.PathParam;
import javax.ws.rs.QueryParam;
import javax.ws.rs.core.Response;
import javax.ws.rs.core.UriBuilder;

import org.fudgemsg.FudgeMsg;
import org.fudgemsg.mapping.FudgeSerializer;

import com.opengamma.financial.convention.ConventionBundle;
import com.opengamma.financial.convention.ConventionBundleSource;
import com.opengamma.id.ExternalId;
import com.opengamma.id.ExternalIdBundle;
import com.opengamma.id.UniqueId;
import com.opengamma.util.ArgumentChecker;
import com.opengamma.util.fudgemsg.OpenGammaFudgeContext;
import com.opengamma.util.rest.AbstractDataResource;

/**
 * RESTful resource exposing a {@link ConventionBundleSource} to remote clients.
 * <p>
 * This is the server to {@link RemoteConventionBundleSource}.
 */
public class DataConventionBundleSourceResource extends AbstractDataResource {

  private final ConventionBundleSource _underlying;

  public DataConventionBundleSourceResource(final ConventionBundleSource underlying) {
    ArgumentChecker.notNull(underlying, "underlying");
    _underlying = underlying;
  }

  protected ConventionBundleSource getUnderlying() {
    return _underlying;
  }

  private FudgeMsg encodeBundle(final ConventionBundle bundle) {
    if (bundle == null) {
      return null;
    }
    return new FudgeSerializer(OpenGammaFudgeContext.getInstance()).objectToFudgeMsg(bundle);
  }

  @GET
  @Path("identifier/{id}")
  public Response getByIdentifier(@PathParam("id") final String idStr) {
    final ExternalId id = ExternalId.parse(idStr);
    return responseOkObject(encodeBundle(getUnderlying().getConventionBundle(id)));
  }

  public static URI uriGetByIdentifier(final URI baseUri, final ExternalId id) {
    final UriBuilder bld = UriBuilder.fromUri(baseUri).path("identifier/{id}");
    return bld.buildFromEncoded(id.toString().replace("/", "%2F"));
  }

  @GET
  @Path("bundle")
  public Response getByBundle(@QueryParam("id") final List<String> idStrs) {
    final ExternalIdBundle ids = ExternalIdBundle.parse(idStrs);
    return responseOkObject(encodeBundle(getUnderlying().getConventionBundle(ids)));
  }

  public static URI uriGetByBundle(final URI baseUri, final ExternalIdBundle ids) {
    final UriBuilder bld = UriBuilder.fromUri(baseUri).path("bundle");
    bld.queryParam("id", ids.toStringList().toArray());
    return bld.build();
  }

  @GET
  @Path("unique/{id}")
  public Response getByUniqueId(@PathParam("id") final String idStr) {
    final UniqueId id = UniqueId.parse(idStr);
    return responseOkObject(encodeBundle(getUnderlying().getConventionBundle(id)));
  }

  public static URI uriGetByUniqueId(final URI baseUri, final UniqueId id) {
    final UriBuilder bld = UriBuilder.fromUri(baseUri).path("unique/{id}");
    return bld.buildFromEncoded(id.toString().replace("/", "%2F"));
  }

}

redpajama_set_name: RedPajamaGithub


text:
The Day of Double Length
November 5, 2009 /
I was just thinking about how little I'd done, then I remembered that the lecture was today, not yesterday, so I must have done a fair amount. It's weird when you have those days that just seem to go on and on.
I had to add a few items to my To Do list, which is very depressing, because I couldn't get everything done, though I did work on all of it. So let's review, shall we?
Get a production assistant. – Done… probably.
Write a proposal for a writing workshop. – Done.
Go to the lecture I'm completely unprepared for – Done, obviously
Go to meeting to decide on play for Am Dram society – Done
Addition: Get permission to film at Tesco and Oddbins – Done and failed
That's not really so bad!
Luckily, Radim managed to cajole Rebecca and Ralph from my class into helping us out on Saturday and Sunday respectively. Ralph is a definite, at least. Rebecca still hasn't gotten back to me with final words, so I might have to get a backup.
Spent most of the day between 1.30 PM and 6 PM trying to get permission to film in various shops and buses. The dickhead branch manager at one major grocery store blankly told me no, but when I rang head office, they told me they have a whole system just for dealing for those kinds of requests. I submitted a request but kind of doubt they'll get back to me in a single day. So that's out then. Oddbins basically said it'd take weeks to get them to respond and Lothian Buses told me I'd have to speak to the marketing manager, Ian Cooper, but that she couldn't give out e-mail addresses. Are you joking me?! He's your marketing manager! Anyway… We'll just pay the fare and start filming, fingers crossed we won't be tossed out, but if we are, we'll get a new one.
The drama society I'm in was having trouble with the play we wanted to put on for easter, Twelve Angry Jurors (a less sexist version of Twelve Angry Men), which we couldn't get the rights to put on. However, there's a loophole for adaptations, so we'll adapt it to Edinburgh in slightly more modern times and we're golden!
Then, just before starting this blog post, I wrote a proposal for a writing workshop that I would be holding for school kids aged 16-18 on behalf of Ren Theatre, the theatre company I'm a co-founder of which will be putting on my original play "Pieces" next year. I've never had a workshop like this in my life before and I still have a lot to learn myself about writing, but I think it could be a lot of fun to do this. I've enjoyed film editing workshops I've done in the past, so this could be a lot of fun!
Oh, I also diced up the pumpkin I intended to carve for Halloween. Instead of a moldy Jack-o-Lantern, I now have 3 kgs of pumpkin flesh!
Time for my list for tomorrow!
Start compiling research and thinking of questions for documentary.
Pick up equipment and photograph docu subject's flat.
Try yet again to get photographs off other subject.
Get final word from production assistants


text:
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Halo Wars: Definitive Edition review
by Roman
As a strategy this game doesn't offer anything interesting, it's not deep and it's possible to beat the entire campaign by one tactic. Plot is dull, despite the high quality CGI video between missions. Still this game is quite fun in co-op mode for a few evenings, so if you have a friend it's not a waste of time.
«Better with friends»
RomanJan 29, 2018
2 of 3 users found this helpful
Other reviews4
Microsoft from Deutsch
Graphic is quite ok. Controlling the Units is very annoying as you can't do failed Micromanagement. The Units block the one, or if you command them away from the Enemy, they run back. Getting Damaged Units out of the Battlefield is very tedious. The Tutorial Crashes so you have to restart your PC. The Buildings can only be built in predefined Places and still buried. I wouldn't Give it to myself.
Steam, Apr 19, 2019
First Of all: I myself was and am a big Fan of the Halo series (this Part in particular did it to me). As a result, my little Review could be More partisan than I write it from a neutral standpoint. As a small hit, I got an Xbox 360. With this Game. I was there and away. Great campaign, great graphics, good soundtrack, and a great bot-mode. Later I learned that there was also a PvP mode. But quickly the disillusionment: Lags, Leaver, Rager. And lo and behold, it's on Steam today. And it's 1 in 1 the same game. 10/10 nostalgia (All of The above points are to be transferred to the Steam version)
TODESSTRAHLEN
Finally, it's also available from Steam! For a long Time I've been waiting for the RTS "Lone Star" from the Halo Universe and now it's here. With a Building System that compares to that of Battle for Middle Earth 1 and settled in the Halo Universe, Halo Wars offers the full Load of Halo. The Story is quite ok, the Implementation is pretty good and the Cutscenes I find very good! I find It particularly problematic that Halo Newcomers are almost not introduced into the Universe ... Who is the Alliance? Who are the Brothers or Prophets? What are the Flood? What is the UNSC or a Spartan? There, the Game really has its biggest Weak points: Namely, in its Introduction to the Game world and the overall depth of The Story. The Halo Universe is very extensive in terms of history and has many interesting historical Contents to offer, so a comprehensive History book or the like would provide more Depth in the Game. This Was implemented with a Timeline, but very suboptimal. The Campaign Was very short in terms of Genre technology, which annoys me a lot with €20! And all the Content available. Comparable Genre mates offer 10-15h (mostly more) Campaign at normal Difficulty. Here I was through in about 7 or 8h with the Campaign, although I always fulfilled as many side Missions as possible etc. Pretty disappointing. Only the UNSC (People) is playable in the CAMPAIGN, while we are constantly competing against the Alliance and later Flood. Alliance Campaign or even one with the Flood? Bad Ad. I was a bit disappointed because you could have done a lot with you. Multiplayer mode offers a normal Battle mode (PvE and PvP). In addition, the Campaign can also be played in Co-op Mode. Exactly What this looks like, I don't know, as I only played through it in Single Player. Despite everything, I can really recommend the Game, but at the earliest in the Sale!
Indrik Boreale
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Magento is a great open source ecommerce platform but every now and then I'll come across a feature that seems to be missing. Recently, when developing a website that intended on using Magento's bundled products to create product kits as single items, I realized that the price was nowhere to be found on the bundled product page. Unfortunately, this is on purpose because the user is meant to select each individual item or product they wish to include in the bundle.


The Claude Moore Recreation and Community Center offers rooms to accommodate up to 100 people, with a variety of setups.
Please call 571-258-3600 for more details and availability. To request a room, complete the Room Rental Request Form (PDF) Opens a New Window. .


text:
DLM, sigle composé des trois lettres D, L et M, peut faire référence à :
 division légère mécanique, par exemple, la  légère mécanique ;
 dose létale minimale, la plus petite dose d'une substance nécessaire pour provoquer la mort chez une espèce animale particulière ;
 .

redpajama_set_name: RedPajamaWikipedia


The river wharfe near Bolton Abbey in Yorkshire is a beautiful picnic spot and is great for a peaceful evening walk but there is only one problem. There is a dark and sinister side to the area where a narrow stretch of water called the Bolton Strid is a death corridor. Falling in means CERTAIN DEATH.
Called the Bolton Strid near Bolton Abbey or the stream that swallows people, it is a narrow stretch of treacherous underwater rocks and deadly fast currents. Although there are foolhardy people who try to jump across, if you miss your step and fall in, it's certain death.
Located between Barden Tower and Bolton Abbey in Yorkshire, England is nature's deadliest booby trap. A small inconspicuous looking stream that many may define as quaint or pretty is a demon in disguise ready to ensnare you to your death. Below the surface is a deep chasm with extremely strong currents that can just about pull anyone falling into it. It is widely believed that not a single person who has fallen into the Strid has made it out alive. Even their bodies weren't found.
2 Why is the strid so dangerous?
How does a small little mountain brook become so dangerous? It is just 6 feet wide. If you walk along the stream for 100 yards, it opens out into a 30 feet wide river wharfe running through Yorkshire. In the area near Bolton Abbey, it is in the form of what is known as the Bolton Strid, a small narrow gap which results in the water gaining even more power, speed and depth.
What most don't know is that there is a network of caverns and tunnels hidden beneath the surface and the narrow gap is just a natural illusion making it a dangerous and evil booby trap. The image below is of the wharfe with Bolton Abbey in the background.


text:
Colts at Titans, 8:20 p.m. Sunday, NBC
INDIANAPOLIS – T.Y. Hilton is heading slowly to midfield. Walking, you might call it, though at the moment he seems to be dragging the injured ankle that caused him to miss all but one practice this week. Hilton played anyway on Sunday, another monster performance that continued his one-man rampage since mid-November and helped the Indianapolis Colts rally to a season-saving, 28-27 victory against the New York Giants.
But now the game is over and the endorphins are gone and the pain is here. And he's walking – sorry, dragging – to midfield.
Now comes the real dilemma. Hilton needs to take off his cleats, but it's not easy with one leg hurting this badly. Slowly he lowers himself, slides off one cleat, and slowly – slowly – slides off the other. He adds his to the small pile of shoes at midfield for Colts safety Matthias Farley, who is raising money and donating shoes to Samaritan's Feet, a non-profit organization from his hometown of Charlotte, N.C., that has donated more than 7.5 million shoes since 2003.
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More:Colts' Chester Rogers knew Andrew Luck was coming his way on game-winning play
Hilton hobbles to the locker room in his socks, with one arm around a trainer. He'll get there eventually, and when he does he's in a great mood. Right away reporters are surrounding the nearby locker of receiver Chester Rogers, whose 1-yard touchdown reception from Andrew Luck with 55 seconds left provided the Colts their only lead of the game, the only one that matters.
"Y'all want to talk to Chester 'D-Wade' Rogers!" Hilton is shouting to everyone and nobody, happy as he can be that one of his good buddies is getting some deserved attention. But the media finishes with Rogers soon, and now we're congregating around Hilton's locker, and he's grumbling good-naturedly at Rogers.
"Hey Chester, you did a bad job," T.Y. is saying. "They're supposed to still be talking to you!"
Chester smiles.
"Nope," he says. "They want to talk to you."
And we did. We do. Hilton is playing the best football of his career, that's a statistical fact, and when someone who has put up borderline Hall of Fame numbers since 2012 reels off the best seven-game stretch of that career? We want to talk to him. Before we get to what Hilton said, here's what he has done in these past seven games: 48 catches for 856 yards, the most yards in the NFL since Week 10, and the most of any seven-game stretch of a career that has seen Hilton reach 500 catches and 8,000 yards (he passed both on Sunday, and now has 505 career catches for 8,036 yards) at age 29.
Hilton had seven catches for 138 yards on Sunday, his fourth 100-yard game in the last seven – he had 85, 77 and 77 in the other three – and made the biggest play that led to each of the Colts' four touchdowns as they rallied from a 14-0 deficit against the Giants.
That's what he did. Here's what he said, when his news conference with reporters was over and someone asked him about a game later Sunday, one with postseason implications for the Colts: "So," a reporter wanted to know as he was walking away, "are you going to watch Pittsburgh and New Orleans?"
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Hilton started chuckling.
"Pittsburgh? Noooo," he said. "I'm fixing to go home and rest. Get this leg right."
His locker cleared out. I stayed. This, the ankle, was the topic I wanted to ask him about. Alone with Hilton, I asked:
How is that leg?
"Ho ho!" he said. "Terrible. It's terrible!"
T.Y. is laughing, so I'm laughing. I mean, he just caught seven passes for 138 yards. He made the biggest play – gains of 12, 25 and 55 yards – to set up touchdowns on three different drives, and on a fourth drive he beat Giants cornerback Janoris Jenkins so badly on a route that Jenkins had to grab him, getting flagged for a penalty that negated a strip-sack of Luck. It's funny, me asking Hilton about his leg after a game like that.
But then I remember the way he dragged his leg to midfield. I remember the shoes. And so I ask:
That's not sarcasm, is it?
"No," he says softly. "No sarcasm."
This is not the T.Y. Hilton that we've known around here, a player whose toughness has been questioned over the years – rightly or wrongly, lots of us have questioned it – but a player who has put that talk to rest this season. In the last three weeks, Hilton has been able to practice just twice. He couldn't practice all week before the game in Houston on Dec. 9, limited by a shoulder injury, but then caught nine passes for 199 yards in a 24-21 victory against the Texans.
Hilton injured his ankle on a 60-yard reception that day, finishing the game but practicing just once the next week before the Dallas game. And then Hilton, saying he was motivated by a young cardiac patient named Tyler Beikes, had five catches for 85 yards in the Colts' 23-0 shutout of the Cowboys.
Now this. Hilton doesn't practice until Friday this week, then catches seven passes for 138 yards to spark a 28-27 comeback win.
So I'm asking T.Y., a player whose toughness lots of us have questioned in the past – yes, me included – how he is playing so well on an ankle so obviously injured.
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"I'm just giving it all to my team, man," he says. "I'll get all offseason to rest, whenever that comes. I'll be sick if I miss a game and we lose. I'll be sick. I'll continue giving everything to this team, man. I love those guys, and I'm showing them out there."
Since we're alone, I ask Hilton something else, something I've been wanting to ask him for weeks. About a month ago, Colts coach Frank Reich told the media about Hilton's "unorthodox" route-running, and relayed a preseason conversation he'd had with Andrew Luck.
"Frank," Luck told Reich, "(Hilton) does some unorthodox things in his route-running, but I'm just telling you, you're going to learn to trust him like I've learned to trust him. He has this unique ability to understand leverage and spacing."
Reich continued.
"This was Andrew speaking: 'I've just learned to trust him, I have a feel for him and you are going to see it,'" Reich said. "And he was exactly right."
Luck has described Hilton's route-running as "instinctive," while offensive coordinator Nick Sirianni has called it "unique" and "not conventional." So now I'm asking Hilton what on earth does he do that's so unusual? Does he even know?
"I don't know!" he says, barking those three words happily. "But it's our seven years together. (Andrew) knows how I run the routes, and I know where he wants me at. As long as I'm in the spot, we'll be all right."
All right? Ho ho! For nearly two months, Hilton and the Colts have been better than "all right." Almost indefensible. Almost unbeatable. Barreling down on a playoff spot after a 1-5 start.
And no, I'm saying softly. That's not sarcasm.
Find IndyStar columnist Gregg Doyel on Twitter: @GreggDoyelStar or at facebook.com/gregg.doyel.


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Q: How do I order a string array ascending and descending alphabetically? C# .NET or Java Well, I have a string array:
String array[] = new String[5];

array[0]="abc";
array[1]="aab";
array[2]="aaacb   sf";
array[3]="  ab";
array[4]="hello";

How do I order ascending and descending?

A: Here is a sample: The "Array.Sort" function does this for you:
char[] array = { 'z', 'a', 'b' }; // Input array.
Array.Sort<char>(array); // Sort array.
foreach (var c in array)
    Console.WriteLine(c);

Look at dotnetPearls.
The LINQ functions ordering the array ascending and descending is also very well described with an sample.

A: Using LINQ, you could do:
array = array.OrderBy(d => d).ToArray();

to sort ascending
And
array = array.OrderByDescending(d => d).ToArray();

to sort descending
Remember to add using System.Linq;.

A: You can use ORDERBY from LINQ to sort any collection (in .NET).

redpajama_set_name: RedPajamaStackExchange


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Bartter syndrome is a genetically heterogeneous kidney disorder with defective sodium, potassium and chloride reabsorbtion in the thick ascending loop of Henle. Bartter syndrome is divided into two main forms; antenatal and classic Bartter syndrome. The classic form is typically less severe than the antenatal form with the age of onset in early childhood. Depending on the age of onset, severity of the disorder and the gene involved, the clinical findings may consist of polyhydramnios, premature birth, polyuria, polydipsia, muscle weakness, nephrocalcinosis, osteopenia, chondrocalcinosis, and deafness.
The Bartter syndrome and related disorders NGS panel consists of nine genes: BSND, CASR, CLCNKA, CLCNKB, GNA11, KCNJ1, MAGED2, SLC12A1 and SLC12A3.
Copy number variation (CNV) analysis of the Bartter syndrome and related disorders genes is also offered as a panel. Additionally, CTGT offers a comprehensive test (both NGS and CNV panels) for these genes. Panel genes are also offered as individual sequencing and deletion/duplication tests, unless otherwise indicated.


text:
Venues to whip up FIFA U-17 World Cup fever
Taking full advantage of the football fever gripping Chile thanks to the ongoing Copa America, on Saturday 20 June the Local Organising Committee (LOC) of the FIFA U-17 World Cup Chile 2015 began the Dome Tour, a promotional campaign set to take in all but one of the host venues of the global youth showpiece.
Puerto Montt was chosen as the first stop on this tour, whose objective is to educate and inform the whole community on the progress made by and the benefits to those cities which will host matches at the competition. The tour will take 22 days and comes to a close on 11 July in Vina del Mar.
Each of these events, at which some of the tournament's sporting ambassadors will be present, will endeavour to appeal to all football fans through games, presentations and information sheets. The background goal is to engage as many families as possible with the U-17 World Cup, while always staying in tune with the aims of sport promotion and the decentralisation of major sporting events.
"We're aiming for the whole community, both in the host cities and their surrounding areas, to have access to the widest array of information possible, while at the same time having an entertaining and pleasant time taking part in activities in this huge dome," said LOC Executive Director Carlos Honorato. "Everybody needs to be aware that there's going to be a tremendous World Cup experience taking place in their city or region."
The dome will be located in the busiest areas of each host city, and it is hoped that the collective enthusiasm of adults, young people and children will help each city thoroughly buy into the idea of "Una fiesta en nuestra cancha" (A party on our pitch), the tournament motto.
"This family activity helps to transmit the values associated with sport and the World Cup," was the view of Gervoy Paredes, the Mayor of Puerto Montt. "What's more, it helps prove to the whole world that we're ready to welcome sport and football lovers, and to show them the best that Puerto Montt and the attractive Lake District have to offer."
The only host venue where the tour will not stop is the capital Santiago de Chile, where there will be a launch event for the tournament's Official Mascot and Official Song on 9 July.
Dome Tour schedule:
Puerto Montt: 20 and 21 June
Chillan: 24 and 25 June
Concepcion: 27 and 28 June
Talca: 30 June and 1 July
La Serena: 4 and 5 July
Coquimbo: 6 and 7 July
Vina del Mar: 10 and 11 July
Chile 2015: Match Calendar


CHARLOTTE - The N.C. Department of Transportation today is preparing Charlotte area roads for a weather event predicted to create hazardous driving conditions Wednesday morning. A mix of rain and snow flurries is forecasted to arrive this afternoon, bringing gusty winds and plunging temperatures.
NCDOT crews in Charlotte are using 10 trucks to spray a brine mixture on Mecklenburg County roadways, planning to finish by 4 p.m.
At 5 p.m., the department will have 15 contractor trucks staged along interstates to place materials on the road before temperatures dip below freezing. Should any rain or snow fall above freezing, crews will wait for the precipitation to end before treating roads, bridges and overpasses. Engineers will continue to monitor conditions overnight and will respond to treat any trouble spots.