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International Journal of Cell Biology
Volume 2013 (2013), Article ID 839329, 10 pages
Identification of Misfolded Proteins in Body Fluids for the Diagnosis of Prion Diseases
Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy
Received 21 May 2013; Revised 10 July 2013; Accepted 11 July 2013
Academic Editor: Alessio Cardinale
Copyright © 2013 Francesca Properzi and Maurizio Pocchiari. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Transmissible spongiform encephalopathy (TSE) or prion diseases are fatal rare neurodegenerative disorders affecting man and animals and caused by a transmissible infectious agent. TSE diseases are characterized by spongiform brain lesions with neuronal loss and the abnormal deposition in the CNS, and to less extent in other tissues, of an insoluble and protease resistant form of the cellular prion protein (), named . In man, TSE diseases affect usually people over 60 years of age with no evident disease-associated risk factors. In some cases, however, TSE diseases are unequivocally linked to infectious episodes related to the use of prion-contaminated medicines, medical devices, or meat products as in the variant Creutzfeldt-Jakob disease (CJD). Clinical signs occur months or years after infection, and during this silent period , the only reliable marker of infection, is not easily measurable in blood or other accessible tissues or body fluids causing public health concerns. To overcome the limit of detection, several highly sensitive assays have been developed, but attempts to apply these techniques to blood of infected hosts have been unsuccessful or not yet validated. An update on the latest advances for the detection of misfolded prion protein in body fluids is provided.
1. Introduction
There are several forms of Transmissible Spongiform Encephalopathy (TSE) diseases or prion diseases affecting humans and different species of farm and wild animals (i.e., sheep, cattle, and cervids). Some of them have an apparently spontaneous occurrence (i.e., sporadic and genetic TSEs; some forms of atypical bovine spongiform encephalopathy or scrapie), while others are linked to the consumption of prion-contaminated food as in the variant Creutzfeldt-Jakob disease (CJD) [1], feedstuff in bovine spongiform encephalopathy (BSE) [2], or medical and surgical devices in iatrogenic CJD [3]. Transmission of variant CJD via blood transfusion and possibly plasma-derived factor VIII from asymptomatic donors [4] indicates that prion infectivity is present in blood months or years before clinical onset. Thus, the occurrence of epidemics in farm animals and episodes of human cases linked to prion infection pose serious public health issues that are often difficult to solve [5]. An eloquent example is given by the yet unexplained discrepancy between mortality (176 death from 1995 to June 2013) [6] and estimated prevalence data (1 in 4,000 to 1 in 10,000 people) of variant CJD in the British population [7]. This incongruity is causing great concerns because healthy and infected donors who are not promptly identified might transmit disease by blood transfusion, surgical instruments, or plasma-derived products.
The only validated surrogate marker of infection is the abnormally misfolded isoform of the cellular prion protein (PrPC) despite intensive but disappointing search for the identification of other disease-specific biomarkers in easily accessible tissues or body fluids [8, 9]. Misfolded PrP (PrPTSE) accumulates in the CNS and other tissues of infected hosts assuming different conformations that are related to the strain of prions [10]. PrPTSE is easily detected by western blot or immunohistochemistry methods after removing the cellular isoform (PrPC). Most anti-PrP antibodies, in fact, do not distinguish between PrPTSE and PrPC requiring the removal of the cellular isoform for achieving disease-specific signals. This is usually realised by pretreating samples with proteases (usually proteinase K) that partially digest PrPTSE but completely remove PrPC. The use of proteinase K (PK), however, removes fractions of poorly aggregated misfolded PrPTSE that is usually present in blood [11] and likely other body fluids decreasing the chance of detection. Finally, it is still debated whether PrPTSE is unequivocally associated with prion infectivity as there are occasions in which PrPTSE is either not associated with infectivity [12] or absent in infected hosts [13]. Despite these limits, PrPTSE remains the best available choice for confirming the diagnosis of prion diseases and for the identification of prion-associated infectivity in tissues and body fluids. Moreover, the profile that assumes PrPTSE in western blot, reflecting different pathological conformations, is of great help for making a correct molecular diagnosis of sporadic CJD and for differentiating sporadic from variant CJD [14].
In the last 15 years several methods have been developed for increasing the sensitivity of PrPTSE detection with the aim of finding a reliable assay for an early diagnosis of prion diseases in easily accessible tissues or body fluids. An overview of these developments is the objective of this work.
2. Protein-Misfolding Cyclic Amplification (PMCA)
In 2001, Saborio and colleagues [15] developed a novel protocol for the in vitro amplification of the misfolded prion protein based on the principle that disaggregated PrPTSE incubated in the presence of a large excess of PrPC produces novel PrPTSE. Disaggregation of fibrils requires a sonication step, which can be repeated several times, in a cyclic process, to allow sensitive detection of the misfolded PrP of the original seed. The protein-misfolding cyclic amplification (PMCA) was originally developed using hamster brain homogenate and has since been shown to be an efficient method for the amplification of brain PrPTSE of other species including mouse, sheep, cattle, bank voles, cervids, and humans [16–23]. In human samples, the amplification of PrPTSE is strongly influenced by the correct matching of methionine/valine in the 129 residue of PrP, suggesting that this polymorphic site of the protein is important for the amplification of PrP misfolding by the PMCA assay [24–26].
Ten cycles of sonication are sufficient to increase the sensitivity of standard western blots from 6–12 picograms to 0.3–0.5 picograms of brain PrPTSE and, with an improved automated protocol which enables a substantial increase in the number of amplification cycles, up to femtogram levels [27]. PMCA is therefore a promising platform for prion diagnosis in body fluids (blood, urine, and CSF) where the level of PrPTSE is estimated in the range of picograms per mL.
The group led by Soto reported the first successful identification of PrPTSE in blood (buffy coat) of scrapie affected hamsters with 89% sensitivity and 100% specificity [27] and positive signals in 50% of samples taken in the preclinical stage of disease as early as 20 days after intraperitoneal 263K scrapie injection [28]. The detection of PrPTSE in blood of preclinical scrapie-infected hamsters is consistent with data on infectivity detection in blood [8].
Since then, automated PMCA revealed the presence of PrPTSE in plasma fractions [29], urine [29–31], and cerebral spinal fluid (CSF) [32] of scrapie-diseased hamsters with sensitivity ranging from 50 (plasma) to 100 percent (CSF) (Table 1). In the CSF samples from scrapie-infected hamsters, PMCA was performed by using a further improved protocol (rPrP-PMCA) in which PrPC was replaced by recombinant PrP (recPrP), allowing a sensitivity greater than that observed with previous PMCA protocols [32].
Other than in hamster models, PrPTSE was amplified from blood leukocytes of both naturally [20, 34] and experimentally scrapie-infected sheep [33] where PrPTSE bands were detected as early as 90 days postinoculation and correlated with infectivity titres [33]. On leukocytes of naturally scrapie-infected sheep, PrPTSE was detected in all tested animals with 100% specificity by using an enhanced (i.e., addition of poly-A PMCA) protocol [20].
Attempts to detect PrPTSE in blood of other species such as cattle with BSE and cervids with CWD produced negative or controversial results [34, 37, 58]. In patients with various forms of prion diseases, the detection of PrPTSE by PMCA was not attempted (or results were not published) in sample of blood, blood derivatives, plasma, urine, or CSF despite amplification of PrPTSE was successfully reported in human brain samples taken from both sporadic and variant CJDs [16, 24–26].
Finally, PMCA amplification of PrPTSE in samples from body fluids, other than blood, taken from prion-infected hosts was successfully achieved in a variety of species and included saliva and urine in sheep with scrapie [36, 36]; saliva, urine, and CSF in cervids with CWD [38, 58]; and CSF and saliva in cattle with BSE [37]. A list of prion-infected body fluids analysed by PMCA with the obtained sensitivity and specificity is shown in Table 1.
In conclusion, PMCA has certainly been a breakthrough for detection of minute amount of PrPTSE that are likely present in body fluids and therefore is a candidate method for developing sensitive tests for the diagnosis of prion diseases in animals and humans. Moreover, the amplified product of PMCA retains the PrPTSE signature of the original seed allowing the molecular diagnosis of CJD in humans and scrapie in sheep with important public health implications. In the last 10 years, PMCA has frequently been modified by addition of poly-A [20] or sulfated dextrans [37], by the use of recombinant PrP instead of brain PrPC [32], or by coupling with sensitive immunoassays [34] that have on one side improved the sensitivity of PrPTSE detection but, on the other hand, made the comparison of data produced by different laboratories difficult. PMCA coamplifies infectivity together with PrPTSE [59, 60] mimicking the disease-specific pathogenic event but requiring safety precautions in diagnostic laboratories. Finally, PrPTSE bands may appear in control preparations after several PMCA cycles [61]. This finding, whether related to de novo formation of PrPTSE [20, 60, 61] or cross-contamination of samples [22], raised concern for the reliability of PMCA in diagnostic applications. This inconvenience, however, is easily settled by using low PMCA cycles and appropriate technical tips to avoid possible prion contamination [22].
3. Quacking Induced Conversion (QuIC)
A spin-off of the PMCA method was obtained by substituting sonication with automated tube shaking for the conversion of recPrP substrates [62]. The novel "quacking induced conversion" (QuIC) protocol enables the amplification of 1 femtogram of PrPTSE of scrapie hamster brain homogenate within one day, reducing the complexity and timing of misfolding amplification. Hamster recPrP promotes the conversion of brain misfolded proteins of other species such as sheep with scrapie and humans with sporadic and variant CJDs, regardless of the primary sequence of the PrPTSE seed [41]. Some spontaneous PK-resistant fragments of less than 12 kD are occasionally observed in unseeded control samples [32], but they wane out by reducing the incubation time of the reaction [41].
One of the most significant improvements of misfolding amplification methods was achieved when western blots were replaced by a real-time fluorescent colour reaction (real-time QuIC) [40, 42]. This novel read-out system, based on a fluorescent amyloid-sensitive thioflavin dye (ThT) [63], allowed the implementation of the whole QuIC procedure to a high-throughput 96-well format. The real-time QuIC (RT-QuIC) is an efficient quantitative method for the detection of minute amount of PrPTSE with estimates of the 50% seeding dose (SD50) of hamster scrapie brain in the same order of magnitude of infectious doses (LD50) [40].
RT-QuIC protocol has been adapted to the detection of brain PrPTSE of other species such as CWD-infected deer, scrapie-infected sheep, and sporadic CJD patients by using species-specific recPrP [40, 42, 64]. Full-length human recPrP and both truncated and full-length hamster recPrPs are efficient substrates for the amplification of PrPTSE in sporadic CJD brain irrespective of the 129 codon phenotypes [43, 64]. A note of disappointment is that the efficacy of variant CJD brain in seeding RT-QuIC reaction is consistently lower than sporadic CJD samples [64].
The presence of PrPTSE in the CSF by the QuIC assay was initially revealed by Atarashi and colleagues [62] in 263K-scrapie infected hamsters and Orrú and colleagues [41] in scrapie-infected sheep. In 2010, Wilham and colleagues [40] revealed the presence of PrPTSE in the CSF of 263K scrapie-infected hamsters by RT-QuIC and estimated a titre of about 10−2 SD50 per µL. CSF samples from control animals did not revealed any presence of PrPTSE indicating a high specificity of the assay. These encouraging results on the CSF of scrapie-infected host promoted further studies in patients with various forms of prion diseases. A blinded experiment was initially performed on 30 CSF samples of definite sporadic CJD patients provided by the Australian National CJD Registry and 155 controls (25 suspected CJD cases and 130 neurological controls) achieving 87.5% specificity and 100% sensitivity. CJD cases were positive irrespectively of 129 codon genotypes [42]. Similarly, McGuire and colleagues [43] screened CSF samples from sporadic CJD patients provided by the National Creutzfeldt-Jakob Disease Research & Surveillance Unit, Edinburgh, UK, including all three 129-codon genotype and obtaining 99% specificity and 94% sensitivity. In the same study, the specificity and sensitivity of the 14-3-3 protein, a surrogate marker currently used for the diagnosis of sporadic CJD, were 65% and 94%, respectively. An example of the RT-QuIC output in the CSF of a sporadic CJD patient is given in Figure 1.
Finally, Sano and colleagues [44] reported that the RT-QuIC assay on the CSF of patients with genetic prion diseases has 78% sensitivity in GSS, 100% in FFI, 87% in E200K genetic CJD, and 100% in V203I genetic CJD, suggesting that the RT-QuIC assay for the detection of PrPTSE in the CSF might become a valid method for improving the diagnosis of patients with a clinical suspicion of human prion disease.
Besides CSF, the RT-QuIC assay revealed PrPTSE in nasal lavages from hamsters infected with the transmissible mink encephalopathy (TME) hyper strain [40] and, by using immunoaffinity beads coupled with the conformational 15B3 anti- PrPTSE antibody (enhanced QuIC), in plasma of scrapie-infected hamsters [39]. The assay showed 100% sensitivity and specificity and was able to detect a positive signal long before the appearance of clinical signs of scrapie.
Finally, the application of 15B3-conjugated beads to the QuIC protocol and the use of a hamster-sheep chimeric recPrP as substrate in the reaction increased the sensitivity (up to attogram levels) and the speed of detection (28 hrs) of variant CJD brain misfolded proteins spiked into human blood [39]. Despite this good achievement there is still no report on the use of the enhanced QuIC assay in human blood.
Overall, RT-QuIC methodology is a powerful platform for the detection and large-scale screening of misfolded PrP in both human and animals. Up to attogram levels of misfolded PrP can be detected and properly quantified within few hours by using high-throughput 96-well formats. The high levels of specificity obtained in a variety of tissues and species by using flexible recombinant substrates demonstrates the versatility of the novel method. It is of note that RT-QuIC PK-resistant products are reported to be noninfectious (quoted by [43]) and therefore likely more secure in large-scale screening diagnostic procedures. The two disadvantages of this assay are the relatively poor performance in amplifying PrPTSE from variant CJD tissues [64] and the failure to reproduce the original PrPTSE signature impeding the molecular diagnosis of sporadic CJD and the distinction between sporadic and variant CJDs.
4. Other Potential Assays for the Detection of Misfolded PrP in Blood
4.1. Immunocapillary Electrophoresis (ICE)
The assay, originally developed by Schmerr and colleagues [65] and based on a competitive immunoassay with PrP fluorescent peptides, was soon proven efficient for the detection of PrPTSE in blood of scrapie-infected sheep and elks with CWD [66]. However, these results were not confirmed in other laboratories using blood samples from CJD-infected chimpanzees or sporadic, iatrogenic, genetic, and variant CJD patients [45, 67]. It is therefore unlikely that this assay will be of any use for the diagnosis of human prion diseases.
4.2. Surface Fluorescence Intensity Distribution Analysis (Surface-FIDA)
This assay consists in the immobilization of single PrP aggregates on a capture antibody coated surface that are then visualized by the concomitant binding with two anti-PrP fluorescent antibodies and a double-laser beam scanning system (surface-FIDA). The method discriminates aggregated PrP forms from monomeric PrP without the use of the proteinase K (PK) digestion step and therefore recognizes both PK-resistant and PK-sensitive PrPTSE. Surface-FIDA enabled the counting of bovine and hamster PrP aggregates in brain homogenates and in bovine cerebrospinal fluid [47]. PrP aggregates were also blind-detected in blood of scrapie-infected sheep () with high specificity and sensitivity [46], although it remains unsettle whether the detection of PrP aggregates correlates with infectivity. It is of note that spiking of blood plasma with PrPTSE from brain was unsuccessful suggesting that the properties of PrPTSE from brain are different from endogenous blood misfolded PrP [46].
4.3. Ligand-Based Immunoassay
Terry and colleagues [48] reported the detection of PrPTSE in 55% of blood mononuclear cells (PBMC) obtained from scrapie-affected sheep () and 71% of experimentally BSE-affected sheep by a modified polyanionic ligand assay of the IDEXX HerdCheck methodology [68]. The assay resulted positive also in a subset of scrapie-infected sheep several months before the onset of clinical signs suggesting that PrPTSE can be detected in asymptomatic prion-infected hosts. However, the relatively low sensitivity observed in prion-infected sheep, the long timings of sample preparations, and the amount of blood volumes required for the purification of PBMC foretell that this assay would not be easily applicable to large-scale diagnostic scopes.
4.4. Solid-State Binding Matrix
The assay, based on the affinity that PrPTSE has for stainless steel particles [69, 70], was adapted for the detection of misfolded PrP in blood of patients with various forms of CJDs [49]. The selective absorption of PrPTSE on the metal matrix concentrates misfolded protein up to the point that the signal can be detected by an ELISA assay. Because of the selectivity of the metal matrix in binding only misfolded PrP, there is no need to pretreat samples with PK that likely removes a conspicuous fraction of PrPTSE in blood. This method was initially tested on human blood spiked with vCJD brain homogenate where misfolded particles in up to the 10−10 brain dilution were detected. Subsequently, blood of variant and sporadic CJD patients was analysed on a blinded experiment including samples from patients with other neurological diseases and controls. Only samples that were reactive in two separate assays were scored as positive. About two-third of blood samples from variant CJD but none from sporadic CJD patients and neurological or nonneurological controls yield positive signals in both assays resulting in 100% specificity for variant CJD [49].
4.5. EP-vCJD Blood Screening Assay
In 2003, Paramithiotis and colleagues [71] reported the manufacture of an antibody directed against PrP epitopes that are exposed only upon protein misfolding and therefore specific to PrPTSE. This conformational anti-PrPTSE antibody was then used for the epitope-protection (EP) vCJD-screening assay, which was later implemented by Amorfix. The high-throughput assay achieved 100% sensitivity and specificity on 1,000 blinded human plasma samples, which included samples that were spiked with variant CJD-infected and normal brains [50]. In 2009, the specificity of the method was ascertained on a large-scale screening initiated in France in over 20,000 human blood samples [51]. Results showed that on the first run 486 samples were positive (97.6% specificity), 20 of which were then confirmed positive on a second screening [51]. The repeat-reactive samples were finally considered negative on a third screening [51]. Subsequently, Amorfix tested three variant CJD blood samples provided by the National Institute for Biological Standards and Control (NIBSC, UK) that resulted negative [52]. The sensitivity of the test was therefore further improved for the detection of 1 : 5,000,000 dilution of variant CJD-infected brain spiked into blood [52]. However, despite this enhanced sensitivity, the test was still unable to detect prions in blood of variant CJD patients, and it was finally concluded that more research is required before the reevaluation of the assay [53].
4.6. Conformation-Dependent Immunoassay (CDI)
In 1998, Safar and colleagues [10] developed an ELISA-formatted, dissociation-enhanced time-resolved fluorescence detection system based on specific antibody binding to epitopes that are accessible in PrPC but that are unmasked only in denatured PrPTSE. This method does not require PK treatment and is able to recognize both sensitive and resistant PK misfolded proteins and different PrPTSE conformations. The assay, improved by incorporating a capture antibody, was able to discriminate PrPTSE signature in different molecular forms of sporadic CJDs, iatrogenic CJDs and genetic TSEs [72] and detect up to a 10−5 dilution of PrPTSE from variant CJD brain used for spiking human normal plasma [54, 73]. However, endogenous PrPTSE was undetectable in white blood cells of sporadic patients by CDI [55], but we are not aware of its use in variant CJD blood.
4.7. Misfolded Protein Diagnostic Assay (MPD)
This technique is based on a pyrene-labeled palindromic sequence of prion peptides that converts to β-sheets in the presence of PrPTSE [56, 74]. This process induces an excimeric signal from the conjugated pyrenes that propagates to other peptides with the final goal to amplify the PrPTSE signal. MPD assay detects PrPTSE in brain of 263K scrapie-infected hamsters during the preclinical and clinical stages of disease [74] and in small volumes of plasma from prion-infected mice and sheep with sensitivity up to 1 infectious dose per mL [56]. The same assay discriminated in blinded small-scale experiments control plasma from that of patients with sporadic CJD and squirrel monkeys with experimental CJD with 100% specificity and sensitivity [56].
4.8. Multimer Detection System (MDS)
This technique is a modified ELISA assay that recognizes only multimeric forms of PrPTSE without using any pretreatment with proteinases, which might remove PrPTSE forms likely present in body fluids [57]. This assay uses the same principle previously described by Pan and colleagues [75] and is based on the use of two monoclonal antibodies that share overlapping epitopes. Monomers (PrPC) are captured by an antibody attached to the surface of a plate and are not detected by the second antibody due to the absence of any exposed epitopes. On the other hand, multimers (PrPTSE) are easily recognized by the second antibody because they expose more copies of the same epitope. The assay was tested on plasma samples of nine scrapie-infected and nine control hamsters resulting in 100% specificity and sensitivity [57]. This simple assay, however, requires validation in other laboratories and more basic work for determining whether the multimeric forms detected by the MDS assay are related to infectivity.
5. Final Remarks
It is unquestionable that in the last 15 years there has been an outstanding progress in improving the detection of PrPTSE for developing sensitive and specific diagnostic assays. These sophisticated and highly sensitive methods successfully detect up to attogram levels of PrPTSE in body fluids of different species (Table 1). A major breakthrough is the development of the RT-QuIC technology for the detection of PrPTSE in the CSF of patients with sporadic [43, 64] and genetic [44] TSEs that as soon as is validated by other groups will change the diagnostic criteria of human prion diseases.
Endogenous PrPTSE has been identified in blood of scrapie-infected hamster by PMCA [27, 28] and RT-QuIC [39] assays and of patients with variant CJD by the solid-state binding matrix assay [49]. Despite these successful observations, however, there are no published reports on the application of either PMCA or enhanced RT-QuIC on blood samples of patients with any form of prion diseases suggesting that both assays still need substantial improvement before their use in the diagnostic setting. Although the development of the RT-QuIC technology for the detection of PrPTSE in blood samples is more recent than PMCA, an extra impediment of the RT-QuIC assay might come from the interference of blood molecules with the ThT reading. On the other hand, the solid-state binding matrix assay might be a valid alternative for the development of a blood test for variant CJD, but the relative low sensitivity (71%) and the finding that some control samples resulted positive in one of the two runs [49] make the use of this assay a remote ambition.
What remains elusive is the reproducible detection of endogenous PrPTSE in blood despite the successful identification of minute amounts of spiked brain PrPTSE into healthy blood. It becomes more and more evident that the properties of PrPTSE in brain are different from those in blood and that some components of blood both inhibit and interfere with PrPTSE detection causing false positive and negative results and compromising the reproducibility of the assay [46, 51, 76]. A clear example is given by the failure of the EP-vCJD assay that had excellent and reproducible performances on spiked blood but then completely failed to identify positive and negative human blood samples [50–53].
These findings pose the question on whether the criteria delineated by the National Institute for Biological Standards and Control (NIBSC, UK) [77] for prion diagnostic assay validation in terms of satisfactory sensitivity and specificity on spiked blood and for the request of variant CJD blood samples are still relevant for defining the best condition of success of potential prion test in blood.
We think that the principles for assay validation and accessibility to variant CJD blood samples should rather focus on reproducible and large scale blinded studies on blood taken from animal models of prion diseases, such as scrapie or BSE in sheep followed by a large scale screening of healthy blood donors to ascertain a sufficient level of specificity.
Finally, our impression is that the research on prion detection in blood does not really need further sensitive assays but rather requires further work aiming to the identification of interfering blood components and understanding prion metabolism in blood.
The authors thank Anna Ladogana, Anna Poleggi, and Michele Equestre for kindling provide them data on the detection of PrPTSE in the CSF of a patient with sporadic CJD by the RT-QuIC assay. Part of this work was supported by the Joint Program of Neurodegenerative Disease (JPND) research on "Optimisation, harmonisation and standardisation of CSF RT-QuIC analysis for the diagnosis of sporadic CJD".
- H. J. T. Ward, D. Everington, S. N. Cousens et al., "Risk factors for variant Creutzfeldt-Jakob disease: a case-control study," Annals of Neurology, vol. 59, no. 1, pp. 111–120, 2006. View at Publisher · View at Google Scholar · View at Scopus
- J. W. Wilesmith, G. A. Wells, M. P. Cranwell, and J. B. Ryan, "Bovine spongiform encephalopathy: epidemiological studies," Veterinary Record, vol. 123, no. 25, pp. 638–644, 1988. View at Scopus
- P. Brown, J. P. Brandel, T. Sato et al., "Iatrogenic Creutzfeldt-Jakob disease, final assessment," Emerging Infectious Diseases, vol. 18, no. 6, pp. 901–907, 2012. View at Publisher · View at Google Scholar
- R. Knight, "The risk of transmitting prion disease by blood or plasma products," Transfusion and Apheresis Science, vol. 43, no. 3, pp. 387–391, 2010. View at Publisher · View at Google Scholar · View at Scopus
- A. Ladogana, M. Puopolo, D. Tiple, S. Graziano, and M. Pocchiari, "Creutzfeldt-Jakob disease: the public health perception," European Journal of Neurodegenerative Diseases, vol. 1, no. 1, pp. 101–113, 2012.
- The National CJD Research and Surveillance Unit, http://www.cjd.ed.ac.uk/documents/figs.pdf.
- Advisory Committee on Dangerous Pathogens, Annual Report for 2012, ACDP/100/P7a, 2013, http://www.hse.gov.uk/aboutus/meetings/committees/acdp/ar2012.pdf.
- P. Brown, "Blood infectivity, processing and screening tests in transmissible spongiform encephalopathy," Vox Sanguinis, vol. 89, no. 2, pp. 63–70, 2005. View at Publisher · View at Google Scholar · View at Scopus
- E. Campisi, F. Cardone, S. Graziano, R. Galeno, and M. Pocchiari, "Role of proteomics in understanding prion infection," Expert Reviews in Proteomics, vol. 9, no. 6, pp. 649–666, 2012. View at Publisher · View at Google Scholar
- J. Safar, H. Wille, V. Itri et al., "Eight prion strains have PrP(Sc) molecules with different conformations," Nature Medicine, vol. 4, no. 10, pp. 1157–1165, 1998. View at Publisher · View at Google Scholar · View at Scopus
- P. Brown, L. Cervenáková, L. M. McShane, P. Barber, R. Rubenstein, and W. N. Drohan, "Further studies of blood infectivity in an experimental model of transmissible spongiform encephalopathy, with an explanation of why blood components do not transmit Creutzfeldt-Jakob disease in humans," Transfusion, vol. 39, no. 11-12, pp. 1169–1178, 1999. View at Publisher · View at Google Scholar · View at Scopus
- P. Piccardo, J. C. Manson, D. King, B. Ghetti, and R. M. Barron, "Accumulation of prion protein in the brain that is not associated with transmissible disease," Proceedings of the National Academy of Sciences of the United States of America, vol. 104, no. 11, pp. 4712–4717, 2007. View at Publisher · View at Google Scholar · View at Scopus
- C. I. Lasmézas, J. Deslys, O. Robain et al., "Transmission of the BSE agent to mice in the absence of detectable abnormal prion protein," Science, vol. 275, no. 5298, pp. 402–405, 1997. View at Publisher · View at Google Scholar · View at Scopus
- P. Gambetti, I. Cali, S. Notari, Q. Kong, W. Zou, and W. K. Surewicz, "Molecular biology and pathology of prion strains in sporadic human prion diseases," Acta Neuropathologica, vol. 121, no. 1, pp. 79–90, 2011. View at Publisher · View at Google Scholar · View at Scopus
- G. P. Saborio, B. Permanne, and C. Soto, "Sensitive detection of pathological prion protein by cyclic amplification of protein misfolding," Nature, vol. 411, no. 6839, pp. 810–813, 2001. View at Publisher · View at Google Scholar · View at Scopus
- C. Soto, L. Anderes, S. Suardi et al., "Pre-symptomatic detection of prions by cyclic amplification of protein misfolding," FEBS Letters, vol. 579, no. 3, pp. 638–642, 2005. View at Publisher · View at Google Scholar · View at Scopus
- Y. Murayama, M. Yoshioka, H. Horii et al., "Protein misfolding cyclic amplification as a rapid test for assessment of prion inactivation," Biochemical and Biophysical Research Communications, vol. 348, no. 2, pp. 758–762, 2006. View at Publisher · View at Google Scholar · View at Scopus
- T. D. Kurt, M. R. Perrott, C. J. Wilusz et al., "Efficient in vitro amplification of chronic wasting disease PrPRES," Journal of Virology, vol. 81, no. 17, pp. 9605–9608, 2007. View at Publisher · View at Google Scholar · View at Scopus
- C. Meyerett, B. Michel, B. Pulford et al., "In vitro strain adaptation of CWD prions by serial protein misfolding cyclic amplification," Virology, vol. 382, no. 2, pp. 267–276, 2008. View at Publisher · View at Google Scholar · View at Scopus
- L. Thorne and L. A. Terry, "In vitro amplification of PrPSc derived from the brain and blood of sheep infected with scrapie," The Journal of General Virology, vol. 89, no. 12, pp. 3177–3184, 2008. View at Publisher · View at Google Scholar · View at Scopus
- N. J. Haley, D. M. Seelig, M. D. Zabel, G. C. Telling, and E. A. Hoover, "Detection of CWD prions in urine and saliva of deer by transgenic mouse bioassay," PLoS ONE, vol. 4, no. 3, Article ID e4848, 2009. View at Publisher · View at Google Scholar · View at Scopus
- G. M. Cosseddu, R. Nonno, G. Vaccari et al., "Ultra-efficient PrPSc amplification highlights potentialities and pitfalls of PMCA technology," PLoS Pathogens, vol. 7, no. 11, Article ID e1002370, 2011. View at Publisher · View at Google Scholar · View at Scopus
- N. J. Haley, C. K. Mathiason, S. Carver, G. C. Telling, M. D. Zabel, and E. A. Hoover, "Sensitivity of protein misfolding cyclic amplification versus immunohistochemistry in ante-mortem detection of chronic wasting disease," The Journal of General Virology, vol. 93, no. 5, pp. 1141–1150, 2012. View at Publisher · View at Google Scholar · View at Scopus
- M. Jones, A. H. Peden, C. V. Prowse et al., "In vitro amplification and detection of variant Creutzfeldt-Jakob disease PrPSc," Journal of Pathology, vol. 213, no. 1, pp. 21–26, 2007. View at Publisher · View at Google Scholar · View at Scopus
- M. Jones, A. H. Peden, D. Wight et al., "Effects of human PrPSc type and PRNP genotype in an in-vitro conversion assay," NeuroReport, vol. 19, no. 18, pp. 1783–1786, 2008. View at Publisher · View at Google Scholar · View at Scopus
- M. Jones, A. H. Peden, H. Yull et al., "Human platelets as a substrate source for the in vitro amplification of the abnormal prion protein (PrPSc) associated with variant Creutzfeldt-Jakob disease," Transfusion, vol. 49, no. 2, pp. 376–384, 2009. View at Publisher · View at Google Scholar · View at Scopus
- J. Castilla, P. Saá, and C. Soto, "Detection of prions in blood," Nature Medicine, vol. 11, no. 9, pp. 982–985, 2005. View at Publisher · View at Google Scholar · View at Scopus
- P. Saá, J. Castilla, and C. Soto, "Presymptomatic detection of prions in blood," Science, vol. 313, no. 5783, pp. 92–94, 2006. View at Publisher · View at Google Scholar · View at Scopus
- Y. Murayama, M. Yoshioka, T. Yokoyama et al., "Efficient in vitro amplification of a mouse-adapted scrapie prion protein," Neuroscience Letters, vol. 413, no. 3, pp. 270–273, 2007. View at Publisher · View at Google Scholar · View at Scopus
- D. Gonzalez-Romero, M. A. Barria, P. Leon, R. Morales, and C. Soto, "Detection of infectious prions in urine," FEBS Letters, vol. 582, no. 21-22, pp. 3161–3166, 2008. View at Publisher · View at Google Scholar · View at Scopus
- B. Chen, R. Morales, M. A. Barria, and C. Soto, "Estimating prion concentration in fluids and tissues by quantitative PMCA," Nature Methods, vol. 7, no. 7, pp. 519–520, 2010. View at Publisher · View at Google Scholar · View at Scopus
- R. Atarashi, R. A. Moore, V. L. Sim et al., "Ultrasensitive detection of scrapie prion protein using seeded conversion of recombinant prion protein," Nature Methods, vol. 4, no. 8, pp. 645–650, 2007. View at Publisher · View at Google Scholar · View at Scopus
- C. Lacroux, D. Vilette, N. Fernández-Borges et al., "Prionemia and leukocyte-platelet-associated infectivity in sheep transmissible spongiform encephalopathy models," Journal of Virology, vol. 86, no. 4, pp. 2056–2066, 2012. View at Publisher · View at Google Scholar · View at Scopus
- R. Rubenstein, B. Chang, P. Gray et al., "A novel method for preclinical detection of PrPSc in blood," The Journal of General Virology, vol. 91, no. 7, pp. 1883–1892, 2010. View at Publisher · View at Google Scholar · View at Scopus
- R. Rubenstein, B. Chang, P. Gray et al., "Prion disease detection, PMCA kinetics, and IgG in urine from sheep naturally/experimentally infected with scrapie and deer with preclinical/clinical chronic wasting disease," Journal of Virology, vol. 85, no. 17, pp. 9031–9038, 2011. View at Publisher · View at Google Scholar · View at Scopus
- B. C. Maddison, H. C. Raes, C. A. Baker et al., "Prions are secreted into the oral cavity in sheep with preclinical scrapie," Journal of Infectious Diseases, vol. 201, no. 11, pp. 1672–1676, 2010. View at Publisher · View at Google Scholar · View at Scopus
- Y. Murayama, M. Yoshioka, K. Masujin et al., "Sulfated dextrans enhance in vitro amplification of bovine spongiform encephalopathy PrPSc and enable ultrasensitive detection of bovine PrPSc," PLoS ONE, vol. 5, no. 10, Article ID e13152, 2010. View at Publisher · View at Google Scholar · View at Scopus
- T. A. Nichols, T. R. Spraker, T. Gidlewski et al., "Detection of prion protein in the cerebrospinal fluid of elk (Cervus canadensis nelsoni) with chronic wasting disease using protein misfolding cyclic amplification," Journal of Veterinary Diagnostic Investigation, vol. 24, no. 4, pp. 746–749, 2012. View at Publisher · View at Google Scholar
- C. D. Orrú, J. M. Wilham, L. D. Raymond et al., "Prion disease blood test using immunoprecipitation and improved quaking-induced conversion," mBio, vol. 2, no. 3, pp. e00078–e00011, 2011. View at Publisher · View at Google Scholar · View at Scopus
- J. M. Wilham, C. D. Orrú, R. A. Bessen et al., "Rapid end-point quantitation of prion seeding activity with sensitivity comparable to bioassays," PLoS Pathogens, vol. 6, no. 12, Article ID e1001217, 2010. View at Publisher · View at Google Scholar · View at Scopus
- C. D. Orrú, J. M. Wilham, A. G. Hughson et al., "Human variant Creutzfeldt-Jakob disease and sheep scrapie PrPres detection using seeded conversion of recombinant prion protein," Protein Engineering, Design and Selection, vol. 22, no. 8, pp. 515–521, 2009. View at Publisher · View at Google Scholar · View at Scopus
- R. Atarashi, K. Satoh, K. Sano et al., "Ultrasensitive human prion detection in cerebrospinal fluid by real-time quaking-induced conversion," Nature Medicine, vol. 17, no. 2, pp. 175–178, 2011. View at Publisher · View at Google Scholar · View at Scopus
- L. I. McGuire, A. H. Peden, C. D. Orrú et al., "Real time quaking-induced conversion analysis of cerebrospinal fluid in sporadic Creutzfeldt-Jakob disease," Annals of Neurology, vol. 72, no. 2, pp. 278–285, 2012. View at Publisher · View at Google Scholar
- K. Sano, K. Satoh, R. Atarashi et al., "Early detection of abnormal prion protein in genetic human prion diseases now possible using real-time QUIC assay," PLoS ONE, vol. 8, no. 1, Article ID e54915, 2013.
- P. C. Lourenco, M. J. Schmerr, I. MacGregor, R. G. Will, J. W. Ironside, and M. W. Head, "Application of an immunocapillary electrophoresis assay to the detection of abnormal prion protein in brain, spleen and blood speciemens from patients with variant Creuzfeldt-Jakob disease," The Journal of General Virology, vol. 87, no. 10, pp. 3119–3124, 2006. View at Publisher · View at Google Scholar · View at Scopus
- O. Bannach, E. Birkmann, E. Reinartz et al., "Detection of prion protein particles in blood plasma of scrapie infected sheep," PLoS ONE, vol. 7, no. 5, Article ID e36620, 2012. View at Publisher · View at Google Scholar · View at Scopus
- E. Birkmann, F. Henke, N. Weinmann et al., "Counting of single prion particles bound to a capture-antibody surface (surface-FIDA)," Veterinary Microbiology, vol. 123, no. 4, pp. 294–304, 2007. View at Publisher · View at Google Scholar · View at Scopus
- L. A. Terry, L. Howells, J. Hawthorn et al., "Detection of PrPsc in blood from sheep infected with the scrapie and bovine spongiform encephalopathy agents," Journal of Virology, vol. 83, no. 23, pp. 12552–12558, 2009. View at Publisher · View at Google Scholar · View at Scopus
- J. A. Edgeworth, M. Farmer, A. Sicilia et al., "Detection of prion infection in variant Creutzfeldt-Jakob disease: a blood-based assay," The Lancet, vol. 377, no. 9764, pp. 487–493, 2011. View at Publisher · View at Google Scholar · View at Scopus
- Amorfix Life Sciences, Press Release, "Amorfix vCJD assay achieves 100% sensitivity and 100% specificity on 1,000 fresh samples from uk blood donors," 2008, http://www.amorfix.com/pdf_press/pr_2008/2008_10_17_AMF_vCJD_NIBSC_Fresh_Results.pdf.
- P. Guntz, C. Walter, P. Schosseler, P. Morel, J. Coste, and J. Cazenave, "Feasibility study of a screening assay that identifies the abnormal prion protein PrPTSE in plasma: Initial results with 20,000 samples," Transfusion, vol. 50, no. 5, pp. 989–995, 2010. View at Publisher · View at Google Scholar · View at Scopus
- Amorfix Life Sciences, Press Release, "Amorfix announces third quarter, 2010 results," 2010, http://www.amorfix.com/pdf_press/pr_2010/2009_02_08_amf_q3_results_2010.pdf.
- Amorfix Life Sciences, Press Release, "Corporate update on vCJD test development," 2010, http://www.amorfix.com/pdf_press/pr_2010/2010_05_31_corporate_update_on_vCJD.pdf.
- J. K. Cooper, K. Ladhani, and P. Minor, "Comparison of candidate vCJD in vitro diagnostic assays using identical sample sets," Vox Sanguinis, vol. 102, no. 2, pp. 100–109, 2012. View at Publisher · View at Google Scholar · View at Scopus
- E. M. Choi, M. D. Geschwind, C. Deering et al., "Prion proteins in subpopulations of white blood cells from patients with sporadic Creutzfeldt-Jakob disease," Laboratory Investigation, vol. 89, no. 6, pp. 624–635, 2009. View at Publisher · View at Google Scholar · View at Scopus
- T. Pan, J. Sethi, C. Nelsen et al., "Detection of misfolded prion protein in blood with conformationally sensitive peptides," Transfusion, vol. 47, no. 8, pp. 1418–1425, 2007. View at Publisher · View at Google Scholar · View at Scopus
- S. S. A. An, K. T. Lim, H. J. Oh et al., "Differentiating blood samples from scrapie infected and non-infected hamsters by detecting disease-associated prion proteins using multimer detection system," Biochemical and Biophysical Research Communications, vol. 392, no. 4, pp. 505–509, 2010. View at Publisher · View at Google Scholar · View at Scopus
- N. J. Haley, C. K. Mathiason, S. Carver, M. Zabel, G. C. Telling, and E. A. Hoover, "Detection of chronic wasting disease prions in salivary, urinary, and intestinal tissues of deer: potential mechanisms of prion shedding and transmission," Journal of Virology, vol. 85, no. 13, pp. 6309–6318, 2011. View at Scopus
- J. Castilla, P. Saá, C. Hetz, and C. Soto, "In vitro generation of infectious scrapie prions," Cell, vol. 121, no. 2, pp. 195–206, 2005. View at Publisher · View at Google Scholar · View at Scopus
- N. R. Deleault, B. T. Harris, J. R. Rees, and S. Supattapone, "Formation of native prions from minimal components in vitro," Proceedings of the National Academy of Sciences of the United States of America, vol. 104, no. 23, pp. 9741–9746, 2007. View at Publisher · View at Google Scholar · View at Scopus
- M. A. Barria, A. Mukherjee, D. Gonzalez-Romero, R. Morales, and C. Soto, "De novo generation of infectious prions in vitro produces a new disease phenotype," PLoS Pathogens, vol. 5, no. 5, Article ID e1000421, 2009. View at Publisher · View at Google Scholar · View at Scopus
- R. Atarashi, J. M. Wilham, L. Christensen et al., "Simplified ultrasensitive prion detection by recombinant PrP conversion with shaking," Nature Methods, vol. 5, no. 3, pp. 211–212, 2008. View at Publisher · View at Google Scholar · View at Scopus
- D. W. Colby, Q. Zhang, S. Wang et al., "Prion detection by an amyloid seeding assay," Proceedings of the National Academy of Sciences of the United States of America, vol. 104, no. 52, pp. 20914–20919, 2007. View at Publisher · View at Google Scholar · View at Scopus
- A. H. Peden, L. I. McGuire, N. E. J. Appleford et al., "Sensitive and specific detection of sporadic Creutzfeldt-Jakob disease brain prion protein using real-time quaking-induced conversion," The Journal of General Virology, vol. 93, no. 2, pp. 438–449, 2012. View at Publisher · View at Google Scholar · View at Scopus
- M. J. Schmerr, K. R. Goodwin, and R. C. Cutlip, "Capillary electrophoresis of the scrapie prion protein from sheep brain," Journal of Chromatography A, vol. 680, no. 2, pp. 447–453, 1994. View at Publisher · View at Google Scholar · View at Scopus
- M. J. Schmerr, A. L. Jenny, M. S. Bulgin et al., "Use of capillary electrophoresis and fluorescent labeled peptides to detect the abnormal prion protein in the blood of animals that are infected with a transmissible spongiform encephalopathy," Journal of Chromatography A, vol. 853, no. 1-2, pp. 207–214, 1999. View at Publisher · View at Google Scholar · View at Scopus
- L. Cervenakova, P. Brown, S. Soukharev et al., "Failure of immunocompetitive capillary electrophoresis assay to detect disease-specific prion protein in buffy coat from humans and chimpanzees with Creutzfeldt-Jakob disease," Electrophoresis, vol. 24, no. 5, pp. 853–859, 2003. View at Publisher · View at Google Scholar · View at Scopus
- European Food Safety Authority (EFSA), "Scientific report on the evaluation of rapid post mortem TSE tests intended for small ruminants," EFSA Journal, vol. 49, pp. 1–16, 2005.
- E. Zobeley, E. Flechsig, A. Cozzio, M. Enari, and C. Weissmann, "Infectivity of scrapie prions bound to a stainless steel surface," Molecular Medicine, vol. 5, no. 4, pp. 240–243, 1999. View at Scopus
- J. A. Edgeworth, G. S. Jackson, A. R. Clarke, C. Weissmann, and J. Collinge, "Highly sensitive, quantitative cell-based assay for prions adsorbed to solid surfaces," Proceedings of the National Academy of Sciences of the United States of America, vol. 106, no. 9, pp. 3479–3483, 2009. View at Publisher · View at Google Scholar · View at Scopus
- E. Paramithiotis, M. Pinard, T. Lawton et al., "A prion protein epitope selective for the pathologically misfolded conformation," Nature Medicine, vol. 9, no. 7, pp. 893–899, 2003. View at Publisher · View at Google Scholar · View at Scopus
- J. G. Safar, M. D. Geschwind, C. Deering et al., "Diagnosis of human prion disease," Proceedings of the National Academy of Sciences of the United States of America, vol. 102, no. 9, pp. 3501–3506, 2005. View at Publisher · View at Google Scholar · View at Scopus
- A. Bellon, W. Seyfert-Brandt, W. Lang, H. Baron, A. Gröner, and M. Vey, "Improved conformation-dependent immunoassay: suitability for human prion detection with enhanced sensitivity," The Journal of General Virology, vol. 84, no. 7, pp. 1921–1925, 2003. View at Publisher · View at Google Scholar · View at Scopus
- A. Grosset, K. Moskowitz, C. Nelsen, T. Pan, E. Davidson, and C. S. Orser, "Rapid presymptomatic detection of PrPSc via conformationally responsive palindromic PrP peptides," Peptides, vol. 26, no. 11, pp. 2193–2200, 2005. View at Publisher · View at Google Scholar · View at Scopus
- T. Pan, B. Chang, P. Wong et al., "An aggregation-specific enzyme-linked immunosorbent assay: detection of conformational differences between recombinant PrP protein dimers and PrPSc aggregates," Journal of Virology, vol. 79, no. 19, pp. 12355–12364, 2005. View at Publisher · View at Google Scholar · View at Scopus
- M. H. Tattum, S. Jones, S. Pal, A. Khalili-Shirazi, J. Collinge, and G. S. Jackson, "A highly sensitive immunoassay for the detection of prion-infected material in whole human blood without the use of proteinase K," Transfusion, vol. 50, no. 12, pp. 2619–2627, 2010. View at Publisher · View at Google Scholar · View at Scopus
- National Institute for Biological Standards and Control (NIBSC), "Flow chart for the evaluation of potential tests for CJD for access to rare samples (such as plasma) from vCJD patients," http://www.nibsc.org/pdf/CJDtest-draft1.pdf.


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Data Access Layer in C# using Repository and Data Mapper – Part 4. The customer repository inherits the base repository and uses the data mapper instance for object creation.
All the CRUD procedures defined in the first post of this series are called in this class. The query for accessing and persisting data in the relational model is formed in the Customer repository. protected override BaseMapper<Customer, long> Mapper get if (___Mapper == default(CustomerMapper)) ___Mapper = new CustomerMapper("CustomerIntId"); An Elegant C# Data Access Layer using the Template Pattern and Generics.
The GOF Template pattern coupled with .NET 2.0 Framework generics provides an awesome synergistic alliance.
This article demonstrates how to drastically reduce the amount of code required in building a data access layer. Less code to debug... less code to break... less code to maintain... what could be better? Implementing a Data Access Layer in C# A Data Access Layer is an important layer in the architecture of any software.
This layer is responsible for communicating with the underlying database. Making this layer provider independent can ensure multi database support with ease. This article discusses implementation of a provider independent Data Access Layer in C#. Interfaces + Factory pattern = Decoupled architecture. Interfaces + Factory pattern = Decoupled architecture Introduction In this tutorial we will try to understand how we can use interfaces and factory pattern to create a truly decoupled architecture framework.
In this sample we will take up a simple three tier architecture and apply interfaces and factory pattern to see how we can transform the three tier in to a truly decoupled architecture. For past some days I have been writing and recording videos in design patterns, UML, FPA, Enterprise blocks and lot you can watch the videos at. Practical Entity Framework for C#: Explore Entity Framework. Entity Framework Support. The latest ADO.NET evolution focuses on raising the level of abstraction from the logical (relational) level to the conceptual (entity) level.
For this purpose Microsoft introduces the Entity Framework, designed to simplify data - object conversion and embed data access queries into program code. The Devart dotConnect product line supports the ADO.NET Entity Framework for Oracle, MySQL, PostgreSQL, SQLite, DB2, and Salesforce. dotConnect data providers support all the Entity Framework versions - Entity Framework v1, v4, v5, and v6. NHibernate Forge. Devart Product. Click on a price to enter the secure purchasing site and begin your order.
As soon as we have been notified that your order has been processed, we will send you an email with your license number and further download information. dotConnect for Oracle can be registered under three types of licenses. You can get Single License for one developer, Team License for up to four developers, and Site License for an unlimited number of developers within a single company at one physical address. If you have other questions, please read our FAQ on License Agreement. NHibernate. Feature summary[edit] NHibernate's primary feature is mapping from .NET classes to database tables (and from CLR data types to SQL data types).
NHibernate also provides data query and retrieval facilities. NHibernate generates the SQL commands and relieves the developer from manual data set handling and object conversion, keeping the application portable to most SQL databases, with database portability delivered at very little performance overhead. NHibernate provides transparent persistence for Plain Old CLR Objects (POCOs). The only strict requirement for a persistent class is a no-argument constructor, which does not have to be public. Best way to improve performance of NHibernate?

subset: commoncrawl


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WorleyParsons wins $US115m contract
PUBLISHED: 12 Jan 2012 11:59:06 | UPDATED: 12 Jan 2012 11:59:06PRINT EDITION: 12 Jan 2012
Engineering giant WorleyParsons has won a contract for work in Alaska with ExxonMobil estimated to be worth $US115 million ($111.80 million).
WorleyParsons will provide project management and engineering and construction work for the Point Thomson Initial Production System project on the Beaufort Sea.
The project includes gas processing facilities.
If the project progressed as currently planned, WorleyParsons' portion of re-imbursable project services would be worth about $US115 million over five years, the company said in a statement on Thursday.
Read more
Companies | Worleyparsons Ltd |
Topics | Construction & Engineering, Energy & Utilities, Energy & Utilities /Oil |


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The purpose of the Center for Learning And Student Success (CLASS) is to promote academic and personal achievement by engaging students, forming partnerships and creating programs and services to foster student success and the retention of undergraduate students.
This is done by:
- coordinating retention activities between academic and student affairs divisions;
- supporting and monitoring students' academic progress;
- coordinating with academic advisement units;
- providing collaborative learning opportunities;
- providing referrals to campus resources and support services;
- instilling a sense of the FAU community in our students and coordinating programs that foster the academic success of FAU students.
Visit us in General Classroom South (GS) Room 223
E-mail us @ firstname.lastname@example.com
Call us @ 561.297.0906


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Digital Serve: Day 10 preview
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By Kat Giantis
Will Kristen Stewart soon become the glummest glowing bride ever? In a sure sign that promotion for "Twilight: Breaking Dawn Part 2" is kicking into high gear, engagement rumors are floating around the smile-stingy actress, 22, and her on- and off-screen leading man, Robert Pattinson, 26.
According to In Touch, K.Stew was recently spotted browsing the glittery wares at Los Angeles jeweler-to-the-stars Neil Lane.
"She was trying on gold rings with various colored diamonds -- nothing traditional," a spy told the tab.
On Wednesday, Beverly Hills-based Claude Morady Estate Jewelry raised eyebrows when it posted a link to the story on Facebook with the message, "We can neither confirm nor deny firsthand knowledge of this rumor.... :)."
The question-popping speculation comes hot on the heels of an Us Weekly report that claims Robsten, who were first linked in 2009, are eager to take a cue from Edward and Bella by procreating.
"He is over-the-moon in love with Kristen," gushes a source. "For him, she's The One, so it's just a matter of when they decide to have kids."
Stewart is supposedly on board with the domestic bliss plan, starting with the betrothal.
"She loves Rob, and she's finally ready," oversells a source to In Touch. "An engagement is a logical next step. In the past year, they've really heated up: moving in together, traveling together, introducing each other to friends."
In light of their together-time and recent touchy-feeliness at the Cannes Film Festival, a bigger commitment wouldn't be outside the realm of possibility for the low-key pair.
But Gossip Cop throws some cold water on the rumblings, with sources insisting K.Stew didn't shop at Neil Lane and dismissing the notion that the couple is currently courting a stork visit.
Rob and Kristen attend a New Jersey wedding
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Time released its 11th annual list of the 100 most influential people in the world. Take a look at which artists, leaders, titans, pioneers and icons made this year's list.
The views expressed are not those of this site, this station or its affiliated companies. By posting your comments you agree to accept our terms of use.blog comments powered by Disqus


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sunblocking curtain/shade for large sliding door
winniebooNovember 14, 2012
We have just finished renovations on a condo in Sarasota, FL. and are moving there from CT. The dilemma I have is this...we face the west and have beautiful sunsets every night, that's the good news! But that comes with a price of very hot western sun streaming into our living area through a wall of sliding glass doors. I hate the vertical blinds from the 80's,but I'm reluctant to get sunshades that roll down, due to ease of coming in and out. Any suggestions?
PH Interiors, LLC
I prefer drapery in this situation. I'd recommend semi-sheer linen (or poly look alike for durability) on rods & rings for easy open and close. Extending the rod beyond the width of the sliders by about 10" on each side, you should be able to stack them back so they don't cover too much of the winodw. Semi-sheers will still allow for a glow of light but also provide a bit of protection from the heat. If you need more thermal insulation, you could go with lined linen draperies.
I have the exact same problem! Last year at 7 PM it hit 113 degrees, in the shade. I had the vinyl verticals but couldn't stand them, so I purchased 6 thermal panels and sewed them all together to make one long (really long!) grommet topped panel. Living rooms that face the west are a challenge, but better than the bedroom facing west!
If your windows are not already tinted, you can buy the smoky window film, it's easy to put on and it really does help.
If your windows are not already tinted, you can buy the smoky window film, it's easy to put on and it really does help.
I installed triple honeycomb blinds on my windows. Down, they provide great insulation from heat and cold; up, they almost disappear. They are on west windows, so if the sun is in our eyes when we are at the dining room table, I pull them just partway down. I still have my old vertical blinds over them because I like to have the space of the window broken up. I wouldn't say that they are particularly beautiful, but they solve the insulation problem nicely.


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St Patrick's Day Celebrations in Benalmadena: Here's the Plan….
St Patrick's Day is a popular celebration in Benalmadena. Last year more than 1000 Irish locals joined in the parade in the centre of town; add to that the holiday makers that visit the popular resort and you've got the makings of a great party!
It's no secret that Sunset Beach Club is an Irish-owned hotel, therefore, the 17th March is an important day on our calendar. Every year we prepare our "Sunset Style" celebrations which include consuming vast amounts of Guinness at the Panorama Bar and a fantastic "Irish Themed" set menu for all our guests in the Oasis Restaurant!
If you're looking for a plan for St Patrick's day, here's what we recommend you do:
1) When you wake up, head down to our Oasis Restaurant and tuck into the hot buffet breakfast; you're going to need something solid in your stomach before venturing out and enjoying the festivities
2) At around mid-day, you might want to make your way into the centre of Benalmadena (Arroyo de la Miel) and join in the St Patrick's Day parade (starts at the Catholic Church in arroyo de la Miel and ends up at the Feria ground in Benalmadena) Weather permitting, you're guaranteed a great afternoon of singing, dancing and, of-course, Guinness!!!
3) At 6:30pm, make sure you head back to Sunset Beach Club to enjoy our Happy Hour in the Panorama Bar. Half price drinks until 7:30pm as well as free canapés and giveaways (hats, etc).
4) Dinner at the Oasis Restaurant: It's time for a delicious St Patrick's Day Themed Dinner prepared by our Head Chef for the occasion. Here's the menu:
Cream of leek and potato soup with chives
Salad Nicoise
Marinated melon balls with amaretto
Irish lamb stew
Goujons of Rosada with an alioli sauce
Pasta with rocket, pesto and prawns
Irish coffee cheese cake with a baileys and chocolate sauce
Lemon mousse meringue with red berries sauce
So what do you think? Sounds like a good plan doesn't it? Hurry up and make your booking by calling the restaurant on extension 3000. Local residents who are not staying in the hotel are more than welcome to come and join in the fun! (please call 952 579400 – ext 3000 for reservations)


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Why CTE?Career and Technical Education (CTE) programs provide students with a foundation for a wide range of careers that reflect the contemporary workplace. College and Career Readiness - CTE programs provide quality work-based learning programs, relevant academic skills, and the employability skills and workforce behaviors necessary for postsecondary success and careers. Academic & Technical Skills - promotes life-long learning in a global society. Work-Based Learning - enhances business and education partnerships, provides students with opportunities to reinforce skills and behaviors to be effective in the workforce. Leadership and 21st Century Skills through Career and Technical Student Organizations (CTSOs) - CTE programs support skills such as communication, critical thinking, problem solving, entrepreneurship and leadership through CTSOs. |
TwitterTweet us @ WisconsinCTE using #wicte throughout the year!
YouTubeWI CTE has joined Youtube!
Facebook CTE has joined Facebook! Come and join us at: Wisconsin Career & Technical Education
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Sara Baird Assistant Director Email: email@example.com Phone: 608-266-9609 |
Christine Smith |
What's New in Wisconsin Career & Technical Education Wisconsin Technical Excellence Scholarships (TES) CTE Technical Incentive Grants Course Options overview letter to district administrators The 2013-14 Industry Certifications Listing The 2014-15 Skills Standards Online Application is now available! |


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Item# PBWT2015OP
Availability :
- Suede Upper with Wing Tip Detail
- Lace- Up Closure with Tonal Laces
- Leather Sole
- Cushioned Man-Made Foot-bed
- Imported
Rated 3 out of 5 by 2
Rated 2 out of 5 by hash Not a leather sole
Not a leather sole. Almost all my dress shoes are leather soled, and these my friends have a layer of leather in the heel (light area), but it is not a full leather sole. Buy 1/2 size larger too.
May 20, 2013
Rated 4 out of 5 SHOE FROM THE GREAT GATSBY!
The shoe is an exceptional puchase, and I expect to wear them for many many years. That's all folks!
May 9, 2013
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About Kate Earl
My name is Kate Earl. I've had a few incarnations.
I started out as a church girl from a gas station in Chugiak, Alaska. My first record garnered the review of "folk chanteuse" by Rolling Stone. For the second album I journeyed as a kind of pop artist with a promising #1 on I-tunes and a single on the Billboard Hot 100. Simultaneously, I discovered that I was pregnant.
Naturally, my life quickly shifted to welcome my greatest treasure into this world. I eventually returned to the music scene as what radio called an alt-rock maven. But through each period of musical collaboration, I would write soul and blues tunes for my personal gratification, too intimate to release into the wild unknown. Until now.
Without the confidence to unveil myself, these songs were originally written for private expression and catharsis. I would show folks here and there. And many times I was told by friends and fans alike, that I should really just do me. By the time I approached each of my labels with it, it just didn't fit in with the projects that were underway. This thing I was doing seemed like one-off songs because I had treated them as such. And then one day it hit me. This style of music keeps returning to me. And I've been too busy asking people what they thought even though my spirit felt best in soul and the blues.
I'm a lover of classic rock, doo-wop, pop and jazz alike thanks to my generation spanning, culturally diverse family and gospel infused upbringing. Being signed 3 times has given me the awareness that I indeed have something to offer. Maybe I just haven't been offering all of myself. Perhaps the experience of making albums, touring from bars to stadiums, writing hundreds of songs, cultivating musicianship, relationships and performance skills, let alone my new role as a mama lion, has now made me capable of executing the ideas that pull from my hidden self, the music that secret Kate was inspired by. I then promised myself to be true. To let out this creature I'd spent my whole life trying to tame.
During my Jamaica sessions this past february, I was introduced to Gary Noble. I called him to tell him how inspired I was by his work with Amy Winehouse and he accepted my invitation to co-produce Ransom with me. His talents span the genres of rock, soul, blues and r&b as heard in his work with Santana, Winehouse, Joss Stone and the Fugees to name a few. We unearthed my Jamaica tracks at Rak studio's in London and magic ensued there, blending the styles I've been so inspired to work on.. I look forward to unveiling the sounds of the collaboration.
So here lies the culmination of one girl's heart and soul, kept in a cage for so long, because I'd once believed that I couldn't be free. Or else that I just wasn't ready. The time has come to let the world see me as I am. This EP is made up of my first soul recordings. It is my Ransom.


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William Simon Age: 72USOC president from 1981-84 and former Treasury secretary and energy expert in President Richard Nixon's administration; USOC vice president and treasurer (1976-80); gave influential speech in support of President Carter's boycott of the 1980 Summer Games; first chairman of the U.S. Olympic Foundation, the financial source for American amateur sports, and a member of the board of trustees until his death; inducted into U.S. Olympic Hall of Fame in 1991; won 1999 Douglas MacArthur Award for his lifetime contributions to the U.S. Olympic movement; headed the Federal Energy Office in 1973; replaced George Shultz as Treasury secretary in 1974. He died of complications from lung disease. Died: Santa Barbara, Calif., June 3Information Please® Database, © 2007 Pearson Education, Inc. All rights reserved.
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Hanging Folder Insert, 11136 Avery Blank Templates for Microsoft Word
Great for creating custom and personalized hanging file folder inserts
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This review is from AVERY Hanging Folder Insert, 11136:
Tried for a couple of hours using the online template at Avery site (it printed landscape with no way to change), trying the template in MS Word 2011 for Mac, Pages and trying to create a custom template in Word --- absolutely nothing worked. Also tried the pdf idea from another reviewer but couldn't get that working. The ONLY thing that worked - with a glitch - was to use the template included in Word 2011 - Avery Standard, Hanging Folder Inserts 1/5 Cut. It will let you put in 10 tabs on the left, not 20 (that is, it only prints 2 across not 4). So I printed the 10, reversed the paper and printed the second 10 on the other side. What a pain! Avery, you can do better than this and please don't forget about the Mac users. We print and make purchases too! I can't even select my OS or software from your drop down lists. I'm using OS Mavericks, MS Word 2011 for Mac
This review is from AVERY Hanging Folder Insert, 11136:
I don't usually write online reviews, but I read a lot of them, so I figured it was time to give back. After chatting online with two Avery customer service reps and on the phone with another two, I finally figured out how to print on these tabs correctly.
Here's the trick. I used the online design program found on this website. I then printed the pdf (as instructed) and then saved it as a pdf to my desktop. From there I printed it to my printer , making sure to un-check "rotate and center" on my printer dialog box. I hope this helps someone out there and they won't be as frustrated as I was for days.
This review is from AVERY Hanging Folder Insert, 11136:
For those of you who got this template to work, please share how you did. I used to use this template years ago and it work beautifully. I had to recently download it again and it does not work at all. Please advise. Thank you.
This review is from AVERY Hanging Folder Insert, 11136:
I found this template easy to use and really effective. I'm confused by all the bad reviews. Once the template opened in Microsoft word, I just clicked on table Tools > Layout > Show Gridlines. Once you have the gridlines, I just typed my labels. I copy and pasted each row to the next row so I could have front and back labels. Loaded the label paper, hit print, and they look fabulous!
This review is from AVERY Hanging Folder Insert, 11136:
I have tried several times to use you template for 11136. I have ruined 2 complete packages and have still not completed my task. The product is great but the software is now very confusing. No matter which way I put in the labels they come out wrong....and they no longer double print so that they can be read front and back.
PLEASE redo the software for this product. I used to LOVE it - now I hate it since the software upgrades.
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Rugby Union
Select a team
Six Nations
- England
- France
- Ireland
- Italy
- Scotland
- Wales
Aviva Premiership
- Bath
- Exeter
- Gloucester
- Harlequins
- Leicester
- London Irish
- London Welsh
- Newcastle
- Northampton
- Sale
- Saracens
- Wasps
The Rugby Championship
- Australia
- New Zealand
- South Africa
Guinness PRO12
- Cardiff Blues
- Connacht
- Dragons
- Edinburgh
- Glasgow
- Leinster
- Munster
- Ospreys
- Scarlets
- Treviso
- Ulster
- Zebre
Top 14
- Bayonne
- Bordeaux-Begles
- Brive
- Castres
- Clermont Auvergne
- Lyon
- Montpellier
- Racing Metro
- Stade Francais
- Toulon
- Toulouse
- Grenoble
- La Rochelle
- Oyonnax
Home team | Away Team | |
19 - 24 |
Match Links
- Super Rugby
- Overall Group
- 9th Mar 2012
- Ground: AMI Stadium, Christchurch
Chiefs hold off Crusaders
Messam and Taumalolo both score to seal victory
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The Chiefs held on to clinch a nail-biting 24-19 victory over the Crusaders at McLean Park in Napier on Friday.
The Waikato franchise silenced criticisms over the strength of their pack as they took the fight to the seven-time champions.
However a lack of discipline allowed the Crusaders to remain in touch.
Liam Messam and Arizona Taumalolo both drove over for tries for the Chiefs, with 14 points coming from the misfiring boot of Aaron Cruden.
The Crusaders, though, responded with 14 points from rising star Tyler Bleyendaal - while a second-half try from Robbie Fruean made it interesting.
The Chiefs got off to the ideal start as Cruden slotted over a regulation penalty in just the second minute, with Bleyendaal missing a long-range effort to level matters.
Bleyendaal was successful with his second effort on 10 minutes - however the Chiefs should have pulled clear but Cruden failed with two relatively straight-forward shots at goal.
Cruden finally rewarded the Chiefs for their dominance during the opening period with a penalty, however the visitors deserved much more from all their efforts.
The Chiefs, though, finally crossed the whitewash when Messam powered over - with the TMO needing to verify the grounding. Cruden converted.
The Crusaders responded well and, after Andy Ellis had a try chalked off for offside, Bleyendaal cut the deficit with his second penalty of the game.
However, Cruden restored the 10-point cushion before the break with a penalty.
The second half saw the Crusaders come out all guns blazing - and they we rewarded with a try through Fruean, with the centre profiting after Kieran Read had barged through the Chiefs defence. Bleyendaal converted.
The Crusaders couldn't build on their onslaught and the Chiefs were able to reassert their dominance - with Taumalolo burrowing over from close range after a series of drives. Again the TMO was needed to spot the ball under the pile-up.
The boot of Bleyendaal, though, kept the home side in touch - with the fly-half punishing the indiscipline with two efforts to cut the lead to 21-19.
Cruden settled the Chiefs' nerves with a late penalty - and despite Read having half a chance to break their hearts, the visitors held on.
Super 15. Click here to bet.Experts & Blogs
Phil Edwards
Read the thoughts and opinions of Phil Edwards with skysports.com


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CFCU's online banking platform has more choice, freedom and convenience than ever. Customize the view on your online banking home page to give you quick access to the features that matter most to you.
Highlights include:
The home page is completely customizable! Choose between a two or three column layout and add, move or delete content boxes to give you quick access to the activities that matter most to you.
Click through the tabs to get familiar with everything we offer. If you don't see what you're looking for, visit the Services tab. Here, you can take advantage of the many services that we offer to give you the convenience of being in control of your finances and make managing your money easier than ever before.
Click here to log on to U@Home!
Not enrolled in U@Home? Click here to sign up today!
Click here for CFCU's Fee Schedule to review fees related to online banking.
Christian Financial Credit Union is excited to announce AlertMe, a credit-monitoring service designed to help members fight identity theft which is a growing problem that affects millions of Americans. AlertMe gives early warning of activity on your credit report. Since credit report activity can be a sign of possible identity theft, early detection can help you catch problems quickly to minimize losses.
AlertMe monitors your Experian credit file daily. New accounts, credit cards over the credit limit, and address changes are just a few of the items AlertMe monitors. If an activity occurs, AlertMe will send you an email alerting you to the activity. You can log into the AlertMe Web site to read details about the activity and determine if it is suspicious. AlertMe will also send an email to remind you to order your annual free credit reports.
The AlertMe cost is $4.25 month. Quarterly and annual plans are available at a reduced rate. To enroll or learn more, simply click on AlertMe in the Services menu inside of U@Home.


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- GetAway Feat. Sean Kingston
- Beamer, Benz & Bentley
- One Night Feat. Pleasure p & Flo Rida
- Show Out Feat. Schife
- Nothin On You Remix
- 01First Song
- 02Track Number Two
- 03Third Song
- 04Composition #Four
- 055th
- 06Song
New Breed which consists of identical twin brothers Mario Johnson (Marvles) & Antonio Johnson (Cezz) were born on March 6th 1988. Since childhood New Breed Knew they wanted to be entertainers in the music business. New Breed began to indulge into the music world around the age of 13. It started as a friendly competition between the two brothers, trying to see who can rhyme better than the other, faster, and more creative than the next. They would write down certain topics and force each other to only use words relating to the topics. At first things were not as serious but as they got older and kept writing they started to notice that they could actually have something in the works. At that time they formed their first group "G-BLOCK", 4which consisted of the brothers and 4 other childhood friends. After leaving the group " G Block" Antonio & Mario decided they wanted to give it another shot and formed the group New Breed. New Breed linked up with producer Russ Swain and moved to Atlanta, Where they dropped a mixtape that pushed well over 14,000 copies. New breed decided they wanted to further their career and decided they need a real manager. They met the co-owner of P.S Management group who saw a lot of raw talent in the brothers. After P.S Management signed the twins and everything went through New Breed recorded their first buzz single "One Night". Once the recording was finished their manager's thought it would be perfect to add Pleasure P & Flo Rida to the song. "One Night " received over 300 plays on radio and gained a lot of buzz for the twins on the Internet and in the streets. Mario & Antonio currently just released their first official single "GetAway" which features Sean Kingston. While the song is starting to get airplay on radio the brothers are in the studio working on their new mixtape "15 Minutes Till Fame " and their debut album. Working with producers from Kane Beatz, Raw Smoov, Finatik n Zac, Cool & Dre, & many more. New Breed brings a different style of music to the industry. With a cross over of rap & pop music the two brothers have what it takes to go far in the music industry. Keep an eye out on them they are definitely one to watch over the next 12 months.


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Kansas to ban local wage rules starting in July
Published on -4/17/2013, 6:46 AM
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TOPEKA, Kan. (AP) -- Cities and counties in Kansas will be barred from requiring private contractors to pay union-scale wages on public projects, starting in July.
Gov. Sam Brownback signed a bill into law Tuesday to prohibit local governments from requiring private firms to pay employees more than the federal minimum wage for work on public contracts.
The legislation was aimed at local policies requiring contractors to pay so-called prevailing wages. The federal Department of Labor sets those rates, and critics say they're often based on union scales.
Wyandotte County has a prevailing wage policy that will be repealed by the new law.
Brownback's fellow Republicans said such a law would lower the cost of public projects. Democrats said prevailing wage policies don't boost costs and ensure workers earn a decent living.


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| Home | Advertise with Us | Circulation | Contact Us | About Us | Send a Letter to the Editor |
Laura Ingalls Wilder (1867-1957) speaks in a kindred voice, from another time. From "Missouri Ruralist," she muses, "...I walked to the church through the beauties of the sunny spring Sundays. I have forgotten what I was taught on those days also."
"But I can still plainly see the grass and the trees and the path winding ahead, flocked with sunshine and shadow and the beautiful golden-hearted daisies scattered all along the way."
Dusk was settling-in, through welcomed cooler breezes, one July evening as Pat and I were headed from our daughter's on County 25. Nearing town, lights of Lewiston, from tall elevators and streetlights, broke through my blank thoughts.
For a brief moment I was back in my teens; the excitement and anticipation of joining chums for girl talk, in lengthening moonlit shadows, pulsed through me. Beams from giant poles once brought the ball field and high school athletes to life, during charged football games.
Briefly the scent of rich grass, that din of a crowd in rhythm with spirited cheerleaders, and youthful faces from the old school, swept me back in a nostalgic aura. As intensely as we clasp them, those sensations soon fade.
Several stimuli can trigger hints of youthful Christmases at home. Sometimes it's the way red and green lights meld in a soft, fluid halo. The spicy aroma of mincemeat and the scent of black walnuts (which Mother always put in her divinity fudge).
The way steam rises from a platter of sliced ham and a mound of riced potatoes. Evergreen nodding in a forest of poignant, fecund patterns. Flannel pajamas peeking from festive holiday wrapping paper.
I brought home a bag of bing cherries this week, as fetching as they are tasty. Immediately, my Great Uncle Dick and Aunt Helen Wollin came to mind.
Every summer, a wooden box would arrive at our post office, from Kalispell, Montana. The charming couple tended fruit groves at their wooded property on Flathead Lake. I can picture her wispy, white hair enhancing a peaches and cream face, and his warm, chuckling demeanor.
The Wollin's guest cabins were nestled near a lakeshore spread of smooth, earth-toned stones. That crystal clear water swept us four kids "out to sea," as we frolicked with cousins.
Uncle Dick Wollin hailed from the Bethany area, ten miles north of Lewiston, before raising a family, as a high school superintendent, in Montana. Their yearly gift of bing cherries was a savory way to keep in touch.
Hiking in the forest up on Eagle Bluff near Lanesboro, I bent to examine strange, orange-colored lichen beside a dead tree trunk. As I spotted the foliage nearby, my heart skipped a beat.
My fingers danced across the cool, semi-circular, serrated fingers of "maidenhair fern." These are prevalent in Whitewater Valley, north of Utica, in wilderness spaces where precious snippets of my time were once spent exploring, before the steep, treacherous hill became too difficult to tackle.
Music can carry you away. Reminiscing can warm the heart, as well as invoke images of loss and regrets. Every time I hear "Rock of Ages" I wander back to sensuous, Bethany Moravian harmony, so smooth and flowing it brings tears to the eyes. Only the cemetery remains on its country plot.
Daydreaming and woolgathering bring to surface the most unexpected recollections. Then there are those rare instants of understanding, which graze your awareness so briefly that they slip from the clutches of our gray matter before they can flood us with TRUTH.
We are, as the song goes, "Aging with time...like yesterday's wine." So much forgotten...a full lifetime to recall. Dream on!


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Not to toot my own horn, but rather to help everyone understand the situation....
This person was flagged, automatically, for 4 different suspicious things (I can't say which). They were manually flagged by the photo reviewer, and then additionally flagged by Mike. Had this scammer attempted to pay, they would have been automatically denied access to full member access until reviewed by our staff. The account was removed as soon as someone started reviewing the flagged accounts.
We remove literally dozens of these accounts per day, but most of them don't last more than a few hours.
That being said, my system isn't perfect, so if you DO see something odd, please report it from their profile. And if anyone, for any reason, says that they need money to get home from the ocean oil rig that they're on so they can visit their dying daughter, please don't. Just report it.
Just want to say thank you for all that you do to ensure our safety!!! I have reported a couple of people I wondered about and the response has always been quick and helpful! Kudos to the staff of Catholic Match!


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Velvet is a name that couldn't possibly be softer or more luxuriant. Many people have fond memories of it via the character of Velvet Brown, played by the young Elizabeth Taylor in National Velvet, an intrepid young woman willing to masquerade as a boy to race her horse in a dangerous steeplechase.
Velvet actually had a brief sojourn on the Social Security list, from 1961 to 1964. Now, double trending as both a 'V' name and a word name might give Velvet a boost.


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Today, U.S. Senator Richard Burr (R-NC) issued the following statement on the Senate Health, Education, Labor and Pensions (HELP) Committee's mark-up of the Food and Drug Administration Safety and Innovation Act:
"I am pleased that many of the key provisions of the Burr-Coburn PATIENTS' FDA Act are included in the manager's package we are considering today, and I am even more pleased that we found a bipartisan path forward to increase transparency and accountability at FDA. These provisions support meaningful congressional oversight that will help ensure timely decisions on behalf of America's patients.
"America's patients deserve access to the latest, most effective life-saving treatments, but red-tape at the FDA is stifling scientific investment and innovation, which is a disservice to medical innovators and can delay patients' access to medical therapies.
"I am also pleased that the Advancing Breakthrough Therapies for Patients Act, a bipartisan bill I introduced with Senators Bennett and Hatch, was also included in today's package. We owe it to patients to provide a clear pathway for expediting the development and review of breakthrough therapies so promising innovation can reach patients as soon as possible.
"I look forward to working with my colleagues to find a workable path forward on bipartisan legislation to secure the pharmaceutical distribution supply chain integrity."
04/10/12 | Senator Burr Announces Events for April 11-12 |
04/17/12 | Tax Day Statement |
04/18/12 | Senators Burr and Coburn Introduce PATIENTS' FDA Act |
04/24/12 | Congressional VA Committees Request Administration's Assistance in Caring for Lejeune Veterans |
04/25/12 | Senator Richard Burr's Statement on Mark-Up of FDA Safety and Innovation Act |
04/26/12 | Burr Bill Would Allow Off-Road Vehicle Use on Cape Hatteras National Seashore |
04/30/12 | MEDIA ADVISORY: Senator Burr Announces Events for May 1 - 3, 2012 |


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An expected 500 restaurateurs from across the country will convene in Washington, D.C., April 17-18 for the National Restaurant Association's 27th annual Public Affairs Conference.
The 2013 public affairs conference theme, "Carry the Message — America Works Here," is part of the NRA's ongoing campaign to tell the industry's story as a leading job creator to opinion leaders. The restaurant industry is the nation's second-largest private sector employer of more than 13 million individuals.
During Capitol Hill meetings, conference attendees will provide members of Congress and staff with firsthand experiences, articulating the need for pro-growth policies, including tax and immigration reforms, as well as reforms to the 2010 health care law.
The conference agenda also includes the Gala Restaurant Industry Awards Program honoring the winners of the 2013 Faces of Diversity and Restaurant Neighbor awards.
Read more about operations management.
Topics: National Restaurant Association , Operations Management


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Have a romantic adventure-date; kayak the Chicago River
BY WENDY ALTSCHULER Special Columnist
Article Extras
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the parenting column ·
Meet Charlie Portis, owner of Wateriders: Chicago Kayak Tours and Rentals. This man, and his team of affable urban cowboys, will guide you over powerboat-created wakes, under iron bridges and down the Chicago River alongside some of Chicago's most impressive architecture. While on the tour, you'll stop at a few key spots to learn about Chicago history, structural design and the dark mysterious stories that make our city so interesting. Slicing through the water makes you feel like you've really accomplished something. Kayaking is a bit of a workout, the duck-view of the city is incredible and the insider's information is brilliant: no run of the mill tour spiel here. All in all, this water-lovers jaunt is a perfect date.
"Even after almost 20 years of paddling downtown, I still find it thrilling to paddle the Chicago River," Portis said. "The visual experience is ever changing: day to night, spring to summer and with new buildings arising in waves of development."
Portis has about 19 years of paddling experience under his belt; he started the journey with a folding kayak that he bought for home use and for trips around the world to Hawaii, Alaska, Thailand, Jamaica, Florida and San Francisco, among other places.
"I discovered that whenever I paddled the Chicago River, people expressed curiosity about paddling downtown," Portis said. "So, I created the tour by reading books, talking to architects and drawing on knowledge I had gained after ten years in Chicago commercial real estate, leasing buildings like the Sears Tower."
Portis has found a way to combine his two great passions: kayaking and Chicago architecture; his love of the two really translates into inimitable enjoyment for those on the tour.
"It is a genuine experience that puts paddlers in the seat of the Native Americans paddling here hundreds of years ago. And, to experience Chicago from the peaceful platform of a sea kayak is to view our magnificent architecture from a truly unique and special vista."
From tyro to expert, this tour is for everyone.
"It's a great thrill to teach a novice kayaker paddler the necessary skills and watch them successfully navigate a paddle downtown," Portis said.
If you're looking for a fun date idea, this is it. Book your trip for the two-and-a-half-hour Wateriders Classic Chicago Kayak Tour or the nighttime Shady Chicago: Ghosts and Gangsters of Hustlertown Tour. The season ends as soon as the nice weather does. For more information, visit www.wateriders.com.
"Paddling on one of our tours is a great choice for couples nights or date nights and it's a great way to see the city. In short, it's a rewarding adventure," Portis said.


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I am looking forward to representing Network Solutions at the Mashable Connect 2011 event at Walt Disney World® in Florida May 12 th to 14th. I must confess I have never been to a event at Disney World before. By the time you read this post I may already be on my way there.
According to Adam Hirsch COO of Mashable:
Google+The event will bring together digital leaders across a variety of industries — including marketing, consumer electronics, entertainment and more — to make connections and exchange ideas around how brands, organizations and communities are connecting through social and digital tools, and innovation. The agenda is very interesting http://mashable.com/connect/agenda/ and I hope to be able to report back some of the learning.
I am looking forward to meeting a lot of my peers- a few names that come to mind are Rohit Bhargava whose keynote topic sounds very intriguing "Likeonomics: How to Survive the Modern Believability Crisis", Betsy Weber of TechSmith makers of Snagit, Dave Peck, Social Media Strategist at Meshin, Christina Warren @film_girl, and Brian Dresher Mashable's Director of Business Development and good buddy. I am sure I am going to be meeting and reconnecting with several other friends and fellow attendees. Having attended several Mashable events I am certain that there will be surprises in store (pleasant of course)! Mashable connect is also using Pathable for the attendees to talk to each other and connect even before the conference which is a great way to converse with fellow attendees early on. GroupMe is another tool that is being used for attendees unofficially to connect.
If you are reading this and attending Mashable Connect hope you will say "Hi".
After Mashable Connect the Network Solutions' team is looking forward to Blog World Expo in New York on May 24th to 26th.
Brought to you by Network Solutions®, a Web.com® service.


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Texas falls out of AP poll
SEP 08, 2013 1:16p ET
Alabama is still No. 1 after an off week, heading into a much-anticipated rematch with No. 7 Texas A&M on Saturday.
Oregon is No. 2. Clemson moved up a spot to No. 3 and Ohio State slipped one to No. 4. Stanford remains No. 5.
The Longhorns came into the weekend ranked 15th, but a 40-21 loss at BYU in which Texas allowed a school-record 550 yards rushing has Mack Brown's team unranked.
Southern California was No. 25 before a 10-7 loss at home to Washington State.
Miami is ranked for the first time since November 2010.