viupersn's blog

Nigel Farage fined after Herman Van Rompuy slur

Mr Farage used Herman Van Rompuy's maiden speech to the European Parliament last week to describe him as having the "charisma of a damp rag and the appearance of a low grade bank clerk".

He was summoned in by EU officials to explain his comments but said his only apology would be to bank clerks.

Yves Leterme, Belgium's Prime Minister, van cleef copy necklace wholesale has demanded a public apology for the "slander" and Mr Farage's added "insult" that Mr Van Rompuy was a "quiet assassin" hostile to the sovereignty of nation states because he came from the "non country" of Belgium.

Mr Farage was called to the office of Jerzy Buzek, the President of the European Parliament, and told to publicly apologise to Mr Van Rompuy and Belgium "or else".

"I said he had the charisma of a van cleef alhambra necklace replica damp rag and I challenge you to find anyone who would say any different to that," he said, "The only people I am going to apologise to are bank clerks the world over. If I have offended them I am very sorry indeed."

Following the meeting, Mr Farage was informed by Mr Buzek that he would be fined EUR3,000, to be deducted from his MEP allowances, for the comments.

Mr Buzek, a former member of the Polish Solidarity movement which fought against Communism in the 1980s, said Mr Farage had the dignity of the new EU president.

cannot accept this sort of behaviour, he said. have therefore as an expression of the seriousness of the matter van cleef necklace replica rescinded his right to ten days daily allowance as a member.

The latest incident follows an official reprimand last November for "violating the dignity of the individual" after he described the newly appointed Mr Van Rompuy and Baroness Ashton, the EU foreign minister, as "political pygmies".

"The atmosphere in the room was pretty clear, unless I apologise, what will happen to me would be severe," said Mr Farage. "I am not going to apologise if I have sparked off a debate on vital issues such as who governs the EU and are they democratically accountable. That's a good thing to have done."
Aug 30 · 0 comments
Multiplex replica Van Cleef and Arpels ring Strand Invasion Based Amplification mSIBA assay for detection of Chlamydia trachomatis and Neisseria gonorrhoeae

Views: 1,398Citations: 3More detailArticle Open

AbstractNucleic acid amplification tests have become a common method for diagnosis of STIs due to their improved sensitivity over immunoassays and traditional culture based methods. Isothermal nucleic acid amplification methods offer significant advantages over polymerase chain reaction (PCR) because they do not require sophisticated instruments needed for thermal cycling of PCR. We recently reported a novel isothermal nucleic acid amplification method, Strand Invasion Based Amplification (SIBA), which exhibited high analytical sensitivity and specificity for amplification of DNA. However, because the reactions were detected using an intercalating dye, this method was Replica Van Cleef Alhambra Pink Gold Ring only suitable for amplifying a single genomic target. Here, we report the development of multiplexed SIBA (mSIBA) that allows simultaneous detection of Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and an internal control in the same reaction tube. SIBA is compatible with probes, allowing the detection of multiple DNA targets in the same reaction tube. The IC was developed to assess the quality of the isolated DNA and the integrity of the enzyme system, as well as van cleef and arpels ring imitation to test oligonucleotides. The mSIBA assay retained high analytical sensitivity and specificity for the detection of CT and NG. The development of mSIBA enables rapid screening for CT and NG within point of care or central laboratory settings.

IntroductionChlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) infections are among the most common causative agents of sexually transmitted infections (STIs)1,2. Nucleic acid amplification tests (NAATs) are now the method of choice in clinical laboratories worldwide for routine diagnosis of CT and NG. NAATs offer superior sensitivity and specificity in comparison with immunoassays and traditional culture based methods3,4. The latter are often very time consuming, and also rely on the presence of viable organisms in the specimen. Because of the high analytical sensitivity of NAATs, direct detection of CT or NG can be performed using non invasive specimens such as urine5.

In an attempt to reduce the sample processing time and overall cost of NAATs, it is often desirable to perform multiplexed tests to simultaneously detect two or more genomic targets or organisms in a single reaction tube. Several commercially available multiplexed NAATs can simultaneously detect of CT and NG, which are frequently comorbid. These tests also include an internal control (IC) for assessing potential sample related inhibition. The existing tests are often based on multiplexed polymerase chain reaction (PCR), using specific primers and dual labeled probes for CT, NG, and IC in a single reaction tube. Despite the emergence of isothermal nucleic acid amplification platforms that obviate the use of sophisticated thermal cyclers, PCR still remains the most common platform used for NAAT methods.

We previously described a novel isothermal nucleic acid amplification method, Strand Invasion Based Amplification (SIBA), with high analytical sensitivity and specificity6. SIBA produces only target specific reaction products, which can be detected using intercalating dyes alone. However, such dyes only detect total double stranded DNA, limiting their use to detection of single targets. Here, we describe the development of an IC and a probe based method that allows SIBA reactions to be multiplexed. The IC assay was developed to allow more precise assessment of sample derived reaction inhibition. We also demonstrate the use of this multiplexed method in the simultaneous detection of CT, NG, and IC in a single reaction tube. We compared the performance of SIBA to those of two existing DNA amplification methods, real time PCR and loop mediated isothermal amplification (LAMP).

ResultsSensitivity and specificity of SIBA, LAMP and PCR singleplex assaysWe developed singleplexed SIBA assays that detected a specific sequence from the CT cryptic plasmid or NG porA, and compared these assays with previously published LAMP and PCR assays to detect the same targets. We also developed an in house LAMP assay to detect specific sequences from porA, because the published LAMP assay was designed to detect NG ORF1 of the glutamine synthetase (glnA) gene7. Both SIBA and LAMP assays were detected using intercalating dyes, because such methods do not rely on target specific probes for detection of the target amplicon. The CT and NG PCR assays were detected using the Taqman probe chemistry8. The sensitivities of the SIBA, LAMP, and PCR assays for CT and NG were evaluated in at least three independent experiments by serially diluting the positive control DNA (constructed CT plasmid CTPlas pUC57 for CT assay and ZeptoMetrix NG control for NG assay) from 2105 copies to 2 copies in quadruplicate (Tables 1 and 2). All three DNA amplification methods (SIBA, LAMP, and PCR) were sensitive at the level of 20 copies per reaction. All three methods occasionally detected as few as 2 copies of target DNA, probably due to inconsistencies in the actual amount of DNA present at such low dilutions. CT and NG assays were further challenged with 105 copies of DNA from non gonococcal Neisseria strains (N. meningitidis, N. flavescens, N. subflava, and N. sicca) with genomes closely related to that of NG. Neither SIBA, LAMP, nor PCR assay detected DNA pooled from 10 different unrelated microorganisms in either the CT or NG assay, and neither SIBA nor PCR NG assay detected DNA from non gonococcal Neisseria strains. However, both the in house and published LAMP assays detected some of the non gonococcal Neisseria strains. This finding suggests that both SIBA and PCR offer some advantage in assays in which exclusive detection of closely related targets is desired. (Supporting information S1; alignment map of SIBA, LAMP and PCR NG primers in relation to porA gene from Neisseria gonorrhoeae and Neisseria meningitidis).

Incorporation of target specific probes in SIBABecause SIBA typically produces specific reaction products, intercalating dyes such as SYBR Green 1 are sufficient to detect amplification in SIBA reactions6. However, such dyes only detect total dsDNA, limiting their use to detection of single targets. To address this shortcoming, we developed a probe based method that could be used for mSIBA reactions. Short single stranded dual labeled probes (12 14 nucleotides) containing a mixture of DNA and LNA bases were designed to be complementary to the downstream region of the target DNA and homologous to the 5 end of the reverse primer. The probes were labeled with a fluorophore at the 5 end and a quencher at the 3 end, such that the probe's fluorescence signal increases upon hybridisation to its complementary sequence. The inclusion of a short sequence of LNA bases in the middle of the probe increased the melting temperature so that it could stably bind the complementary sequence under SIBA reaction conditions9. The inclusion of LNA bases also abolished the effect of including recombinase and single stranded DNA binding protein (T4 gp32) in the SIBA reaction, which would otherwise simulate a double stranded configuration of the probes and elicit a false signal (Supporting information S2)10.

We designed and tested probes for the SIBA CT and NG assays, which were previously detected using the intercalating dye SYBR Green 1. The CT probe was labeled with ROX and Iowa black FQ quencher, whereas the NG probe was labeled with Cy5 and Iowa black RQ quencher. The amplification signal profile of these assays when detected with either SYBR Green 1 or the labeled probes can be seen in Fig. 1B. The increase in fluorophore signal, attributed to binding of the probes to the amplicon, can be seen only in reactions containing the target DNA. The probes produced robust signals without compromising the efficiency of amplification (Fig. 1). Although a high concentration of probes might compete with the downstream primer, the probe concentration used in this study was not inhibitory, and the SIBA reactions were still efficient in the presence of the dual labeled probes. This is partially due to the ability of the primer 3 end to bind the target DNA even in the presence of a probe (Fig. 2A).

Figure 1: Detection of SIBA Neisseria gonorrhoeae (NG) and Chlamydia trachomatis (CT) using LNA probes.(A) Neisseria gonorrhoeae. (B) Chlamydia trachomatis. Probes for NG and CT assays were labeled with Cy5 and ROX, respectively. (C) Corresponding melting curve analysis for NG assay (D) corresponding melting curve analysis for CT assay. 2104 and 2102 NG and CT reactions were omitted from Fig. 1C,D for the sake of clarity.

IC developmentNext, we developed an IC assay to assess possible sample derived inhibition. This was achieved by constructing an artificial target template with a sequence assay identical to the CT target, but differing in its downstream region. Therefore, the IC assay shared the same forward primer and IO as the CT assay, and differed from the CT assay only in the reverse primer and probe used (Fig. 2A). The sensitivity of the IC assay was established by serially diluting the IC DNA template from 2106 to 200 copies per reaction. The IC assay was performed under SIBA reaction conditions using the CT forward primer, CT IO, IC reverse primer, and IC probe (FAM), and real time amplification was measured by monitoring the fluorescence produced by the probe. The increase in fluorescent signal was only detected in reactions containing IC template (Fig. 2B), and the IC assay did not cross react with either the CT or the NG template. Likewise, the CT or the NG assays are unable to amplify the IC template.

Performance of multiplexed SIBA CT and NG assayWe then developed a multiplexed SIBA assay for the detection of CT and NG in a single reaction tube. The performance of the multiplexed SIBA assay was compared with previously validated multiplexed PCR assays for the detection of CT and NG8,11. The SIBA multiplexed reaction, which consisted of a 3 plex reaction of the CT, NG, and IC assays described in Figs. 1 and 2, was performed using lysates from CT elementary bodies (EB) and NG cells. The sensitivity of the multiplexed assay was evaluated by serially diluting the lysate containing from 2104 to 2 EB or CFU of NG cells per reaction. Each reaction also contained 1000 copies of the IC template. The multiplexed CT/NG/IC assay reproducibly amplified and detected as few as 20 EB or 20 NG cells per reaction (Fig. 3). Although the assay was also capable of amplifying and detecting 2 EB or 2 NG cells per reaction, the variability at these dilutions was high, again probably due to inconsistencies in the actual amount of DNA present.

Figure 3: Multiplexed Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and internal control (IC) assay.(A) Detection of NG using Cy5 labeled LNA probe. (A) Detection of CT using ROX labeled LNA probe. (C) IC control using FAM labeled LNA probe. (D) Melt curve of CT, NG, and IC reaction product detection with SYBR Green.

The IC template was detected in reactions that did not contain CT EB. The CT assay was designed to amplify more efficiently than the IC assay. The presence of the EB in the reaction and consequent positive CT test result leads to the consumption of CT forward primers, which are required for IC template amplification. Therefore, the IC is a conditional control that ensures the negative reaction is not a result of sample inhibition; consequently, it is not needed in a positive assay. The specificity of the multiplexed CT/NG/IC assay was established by challenging the reaction with a pool of cell lysate from 10 different unrelated microorganisms (see Table 3 for a list of the microorganisms used). Another pool of cell lysate from four different non gonococcal Neisseria strains was also used to establish assay specificity. Neither the unrelated microorganisms nor the non gonococcal Neisseria strains cross reacted with the multiplexed CT/NG/IC assay. Furthermore, the EB and NG could only be detected via their corresponding probe channels (ROX and Cy5, respectively). The multiplexed CT/NG/IC assay was also used to detect EB and NG in presence of nucleic acids from 14 unrelated microorganisms (103 copies from each microorganism per reaction, Table 3). No reduction in assay efficiency was observed. The assay was also able to detect CT and NG cells from a low positive clinical control specimen (The amplification curves can be seen in figure supporting information S3). These results demonstrate that the multiplexed CT/NG/IC assay was highly specific. The analytical sensitivity and specificity of the multiplexed CT/NG/IC were found to be similar to the previously validated multiplexed PCR assay for the detection of CT and NG8,11. However, the mSIBA assay displayed a shorter detection time than the multiplexed PCR assay. In particular, we assessed the ability of the assay to detect low levels of EB in the presence of high levels of NG cells, and vice versa (Fig. 4). The presence of high levels of NG cells in the reaction did not result in any significant reduction in amplification from a small number of EBs. Similarly, the presence of large numbers of EBs in the reaction did not significantly reduce amplification from small numbers of NG cells. This suggests that the SIBA multiplexed CT/NG assay would be suitable for detecting these targets in patient samples co infected with CT and NG.

Figure 4: Competition between Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) in multiplexed reactions.(A) Detection of CT elementary body (EB) in the presence of a high background of NG. (B) Detection of NG in presence of a high background of CT. Average detection from eight replicates. The detection time reported was the time at which the probe fluorescent signal exceeded the background signal.

DiscussionThe results of this study demonstrated the ability of SIBA to detect NG and CT together in the presence of an IC in a single reaction tube. SIBA was previously shown to exhibit high analytical sensitivity and specificity for amplification of genomic DNA6. However, because the reactions were detected using an intercalating dye, the method was only suitable for amplifying a single genomic target. SIBA was also found to be compatible with probes and ICs such that multiple target DNAs could be simultaneously amplified and detected in a single reaction tube without loss of assay integrity. Furthermore, the multiplexed assays performed with a sensitivity and specificity similar to those of the singleplexed assay.

SIBA relies on a recombinase (UvsX) and a single stranded binding protein for dissociation of the target duplex. This allows target specific primers to bind and extend the target via the action of a DNA polymerase. The use of dual labeled probes in SIBA also presented some challenges in assay design because of the presence of single strand binding (SSB) proteins in the reaction mixture. SSB proteins bind to ssDNA, simulating a double stranded configuration in terms of the quencher/fluorophore labeled probes, limiting their ability to discriminate between a complementary and non complementary target template. Because SSB proteins do not bind efficiently to LNA, this problem was solved by incorporating LNA bases in the dual labeled probes10.

The ICs included in most nucleic acid amplification methods are often housekeeping genes, but these do not test for the functionality of the primers used in the test reaction. By contrast, the ICs developed here can assess DNA isolation and the integrity of the enzyme system, as well as test oligonucleotides. Previous studies showed that the efficiency of the SIBA reaction is influenced by the length of the target region peripheral to IO invasion, which forms a part of the primer binding site6. A longer region, typically greater than 14bp, leads to a reduction in amplification efficiency. Based on these previous findings, we designed the IC assay to have a peripheral region longer than 14bp to ensure that the assay was less efficient than the CT or NG assays. This approach ensures that the IC assay does not compete with the CT or NG assays when the reactions are performed in the same reaction tube. Consequently, the detection time of the IC assay was longer than those of the CT and NG assays (Fig. 2B). Because a low level of IC template was used in the multiplex assay, the IC template was only detected after small numbers of copies of CT or NG were detected in the reaction. This further ensured that the IC assay did not compete with the CT assay for primers and IO. Although the IC assay was designed to use the same forward primer and IO as the CT assay, it can also be constructed to use the same forward primer and IO as the NG assay. This makes it possible to have multiple ICs (one for each test analyte) in the same reaction tube. Such an approach could further improve assay robustness and reliability.

The multiplexed SIBA CT/NG/IC assay could only be compared directly with a previously published multiplexed PCR assay. To our knowledge, no multiplexed LAMP assay for the detection of CT and NG has been reported in the literature. CT and NG detection have, however, been multiplexed by a number of other nucleic acid amplification methods5,12,13. The unavailability of a multiplex CT/NG LAMP assay may be due to the use of intercalating dye based detection14 or turbidimetry15 in LAMP assays; these detection methods are limited to monitoring of a single target. Furthermore, multiplexing in LAMP could also prove problematic because of the use of numerous large primers that lead to unwanted primer interactions, resulting in generation of unwanted false positives. In the case of SIBA, the specificity of the reaction is mainly driven by the non extendible IO rather than the primers. A template that differs by two or more nucleotides in the region homologous to the IO will amplify poorly or not at all6, allowing multiple primers to be included in the same reaction for the purpose of multiplexing. This may also be the reason why SIBA NG assay did not cross react with other commensal Neisseria strains, a problem seen in some NAAT commercial kits16. Although real time PCR is a robust NAAT platform for multiplexing several diagnostic analytes, the requirement for thermal cyclers limits the use of PCR for field or point of care applications. In this study, we used a qPCR device for convenience to perform multiple reaction conditions and replicates. Several small and portable low cost devices have been developed for the fluorescent detection of common isothermal DNA amplification reactions17. These devices can also be used for the detection of mSIBA reactions and are more convenient for field or point of care applications.

CT and NG are major pathogens, and over 6 million tests were performed in 2007 in the USA18. Effective and early diagnosis plays an important role in treatment and preventing spread of these infections. It is desirable to perform routine screening in physicians' offices or polyclinics, rather than in central laboratory settings. Therefore, the development of a robust multiplex isothermal amplification method could enable detection of CT and NG within these settings, because such methods would be compatible with low cost and low footprint devices.

Materials and MethodsMaterials and reagentsThe oligonucleotides used in this study were purchased from Eurofins Genomics (Germany) or Integrated DNA Technologies (Belgium) and purified by the manufacturer using reverse phase HPLC (for primers and probes) and PAGE (for the invasion oligonucleotides). Sequences are shown in Table 4. Sucrose and sucrose phosphorylase were purchased from Sigma Aldrich (USA).

Bacterial strains and DNA preparationA 993 bp template harboring positions 1020 1380, 3315 3785, and 6530 6690 from CT plasmid pCHL1 (NC_001372) was clo
Aug 29 · 0 comments
Maple Leafs playoff berth a tale of two turnarounds

TORONTO At about this point last year Leo Komarov had his bags packed and plane ticket home to Finland almost ready to go, his Toronto Maple Leafs wrapping up a 30th place season.

Even Komarov, one of the longer serving Leafs, didn't expect his team to leap back into the post season so soon the Leafs clinching their first such berth in four years and second since 2004 with a 5 3 win over the Penguins Saturday night.

"If I'm really honest, maybe not," said Komarov. "But the thing when we started playing, when you see the young kids, you see they've got a lot of skills and when we start winning you think we've got a pretty good chance. The closer we get the bigger chance we have so you kind of start of replica van Cleef and Arpels ring thinking 'Maybe, we've got a chance to get in here'."

The Leafs playoff berth is really a tale of two turnarounds from last season, and from three years before that when a vastly different Toronto entity last cracked the post season.

Mike Babcock promised "pain" when he was hired as the Leafs head coach in May 2015, but that pain was really short lived. The end result of one last place season was the best odds for Toronto to land Auston Matthews, the best rookie in the 100 year history of the franchise.

It was Matthews, Mitch Marner, William Nylander and the emergence of a historic rookie class that truly catapulted the Leafs from last season's dull misery into the post season. The mostly steady goaltending of Frederik Andersen who was injured against Pittsburgh helped, as did career years from Nazem Kadri, Jake Gardiner and Tyler Bozak. But it was the instantaneous contributions of the rookies that made the biggest difference from one year to the other along with Babcock behind the bench.

Matthews became only the fourth rookie under 20 in NHL history to score 40 goals, his 40th into an empty net sealing the Leafs wild playoff clinching win over Pittsburgh. That goal paled in importance to fellow rookie Connor Brown, whose 20th of the season Saturday gave the Leafs a late regulation lead over the Penguins.

Then there was Kasperi Kapanen, his first NHL goal and point a few minutes earlier tying the game at three.

Babcock said he told his coaching staff at the beginning of the year that if his club was to get into the post season it would take until Game 82 which comes Sunday against Columbus.

"But to be honest with you, I didn't know the kids could be this good," Babcock said.

The Leafs return to the post season isn't just about the jump from last year though, but from four years earlier when they last cracked the playoffs. Toronto of those days was built tenuously around veterans like Phil Kessel and Dion Phaneuf, ultimately unravelling in Game 7 of a first round series against Boston.

Coaches, managers, and scouts were fired in the ensuing turbulence, the organization overhauled in almost every way imaginable. Brendan Shanahan was brought in as president, Babcock as coach, and Lou Lamoriello as general manager. And of course, the roster was altered considerably, suddenly built around skilled young players like Matthews and Marner.

The club revamped its logo and jerseys too.

Just about everything has changed save for those five players that still remain: Komarov, Bozak, Kadri, Gardiner, and James van Riemsdyk.

"It's definitely been some ups and downs over the course of that," van Riemsdyk said.

"That was a good year too and a good team and now it's different," Komarov added. "You've got younger fake Van Cleef & Arpels ring guys who just came to the league and it's great for them, like they've got a chance to win right away. And if not, they still get a lot of experience."

It wasn't the smoothest effort that got them into the post season, Brown's late goal saving the club from defeat against a Penguins squad that had Sidney Crosby, Kessel and few other regulars Pittsburgh opting to rest a handful of players ahead of the playoffs.

Momentous in the win was the early exit of Andersen, bumped in the head by Tom Sestito and unable to return. It was the second such exit for the 27 year old in the last two weeks, his status now a real question mark ahead of a post season matchup against either Ottawa or Washington.

Babcock had no update on Andersen other than to suggest that "ideally" he would start the finale against the Blue Jackets.

The Leafs coach described the playoff berth as momentous for not only the city and fans, but those young players who live on their phones Van Cleef & Arpels ring knock off and "know what everyone thinks and how excited everyone was".

Babcock said he spent the first two years of his Toronto experience with rarely a peep of interaction among his neighbours and now people were talking to him "every single second for the last week so it must be a big deal."

"This is a great situation for everyone," Komarov said. "For us older guys we've got a chance to win and for the youngest guys they battle for it right away."
Aug 29 · 0 comments
n van a menudo de la mano

Williams igual que los actores britnicos Stephen Fry, Hugh Laurie y John Cleese o los estadounidenses Jim Carrey, Owen Wilson y Ben Stiller fue uno ms de los humoristas que en los ltimos aos reconoci pblicamente haber batallado contra la depresin o la adiccin a las drogas y al alcohol.

Pese a que los expertos en psiquiatra hacen hincapi en que no se puede generalizar a la hora de vincular determinadas enfermedades mentales con un cierto tipo de personalidad, lo cierto es que la imagen de los comediantes como seres depresivos que exorcizan sus demonios a travs del humor lo que algunos han bautizado informalmente como el sndrome del payaso triste se ha vuelto un lugar comn.

"Doloroso"El productor britnico John Lloyd, responsable de la exitosa serie cmica de la BBC de los aos 80 "Blackadder" y a quien se le diagnostic una bipolaridad, asegura que los humoristas suelen tener personalidades que van de un extremo al otro porque ellos mismos "son gente extrema" que no est satisfecha con el van cleef necklace yellow gold replica mundo que les rodea.

"Robin Williams era un genio y tuvo una carrera muy prolfica. No puedes hacer eso si ests deprimido. Es ms habitual que eso suceda en las personas bipolares, que tienen enormes estallidos de creatividad", seala Lloyd en conversacin con la BBC.

"Pero todo tiene un precio. A menudo, van cleef & arpels necklace replica y lo s por haber sido productor de televisin, cuando acabas una serie, despus de un tiempo bajo los efectos del subidn que produce la adrenalina, te hundes y llegas muy abajo. Es doloroso", apunta Lloyd.

La sensacin que describe Lloyd es, segn la psicoterapeuta estadounidense Amy Alpine, algo muy comn entre los humoristas.

"Para muchos comediantes, el humor es una va de escape. Toman algo que les preocupa o molesta y hacen broma con ello para intentar superarlo. La recompensa es la risa del pblico", apunta Alpine en conversacin con BBC Mundo.

"Muchos cmicos tienen una personalidad introvertida y para compensarlo utilizan el humor. Pero el tener que ser divertido todo el rato puede ser muy cansado. La gente te ve y espera que les hagas rer y eso no es posible todo el tiempo", seala la experta.

"Cuando estn solos no estn haciendo rer a otros y tampoco se ren consigo mismos. As que no tienen el mecanismo para volver los pensamientos negativos en algo positivo".

"Adems, la depresin es fruto de un mecanismo qumico en el cerebro. Cuando estn solos no tienen el subidn de adrenalina que les da el estar sobre el escenario frente al pblico. Y cuando te falta esa adrenalina el cerebro est bajo el efecto de sustancias que causan depresin".

Drogas como escapeSegn Alpine, es por eso que "hay muchos casos de humoristas que utilizan drogas como la cocana cuando no estn actuando, ya que esa sustancia estimula los centros cerebrales del placer y les hace sentir bien".

"Intentan obtener a travs de las drogas el placer que les da estar sobre el escenario. As que estn constantemente en un ciclo que va de la euforia que produce la adrenalina a la depresin que produce el cortisol que sus cerebros segregan cuando estn solos. Las drogas les ayudan a producir dopamina, que les relaja y les ayuda momentneamente a olvidar su depresin. Es un ciclo muy destructivo".

Muchos cmicos tienen una personalidad introvertida y para compensarlo utilizan el humor. Pero el tener que ser divertido todo el rato puede ser muy cansado. La gente te ve y espera que les hagas rer y eso no es posible todo el tiempoAmy Alpine, psicoterapeuta

El psiquiatra Roger Montenegro, miembro del consejo de direccin de la Fundacin Mundial para la Salud Mental (WFMH, por sus siglas en ingls), coincide con Alpine en que existe un vnculo fake van alhambra long necklace entre el abuso de sustancias y las enfermedades mentales.

"El alcohol y las drogas hacen ms llevable la enfermedad pero no sacan al enfermo de la depresin, sino que terminan acentuando la sensacin de culpa o de no tener futuro", explica Montenegro en conversacin con BBC Mundo.

"Eso sucede tambin con pacientes con esquizofrenia que pretenden aliviar sus sntomas con el consumo de sustancias", seala el experto.

El pasado enero cientficos de la Universidad de Oxford, en Reino Unido, publicaron un estudio en la Revista Britnica de Psiquiatra en el que participaron ms de 500 comediantes de Australia, Gran Bretaa y Estados Unidos.

Los investigadores concluyeron que los actores que se ganan la vida haciendo rer a los dems presentan ms caractersticas psicticas en su personalidad que los actores que no practican el humor o las personas que no tienen trabajos creativos como los suyos.

Esos rasgos incluyen una tendencia hacia el comportamiento impulsivo y antisocial y a evitar la intimidad.

Depresin y humorSegn Gordon Claridge, jefe del Departamento de Psicologa Experimental de la Universidad de Oxford, "los elementos creativos necesarios para producir humor son sorprendentemente similares a los que caracterizan el estilo cognitivo de las personas con esquizofrenia o trastorno bipolar".

"Por un lado, algunos humoristas son ms bien introvertidos, depresivos e incluso esquizoides. Y por el otro, son ms bien extrovertidos y manacos", seal el experto.

"Probablemente la comedia, el lado extrovertido, es una manera de lidiar con el lado depresivo. Por supuesto ese no es el caso de todos los humoristas", aclar Claridge.

El doctor Nick Maguire, especialista en psicologa clnica de la Universidad de Southampton, en Inglaterra, asegura que puede que haya una conexin entre depression y comedia, aunque en su opinion "no es tan fuerte".

"La gente a menudo se asla. Una manera de mitigar temporalmente las emociones negativas es hacer que los otros se ran, para que les gustes", seala Maguire.
Aug 29 · 0 comments
Meet the Press replica van cleef necklace with David Gregory

newsmakers. President John F. Kennedy once called Meet the Press van cleef ans arpels Pendant replica the "fifty first state." Since then, every man who has occupied the Oval Office has appeared on the program. More FAQs Airtimes

Intern at MTPWant to work at Meet the Press?Wehave internship positions available throughout the year. You must be a college junior or senior and be able to receive school credit for your work. Discuss it here. 2: Denis McDonough, Tim Scott, Julian Assange, Rich Lowry, Robert Gibbs, Gwen Ifill, Doris Kearns Goodwin, Chuck Todd, Alan Schwarz, Tony Dungy

January 26: Rand Paul, Dick Durbin, Michael Chertoff, Jesselyn Radack, Carolyn Ryan, Michael Powell, Mike Murphy, Loretta Sanchez, Chuck Todd

January 19: Dianne Feinstein, Mike Rogers, Alexis Ohanian, John Wisniewski, Rudy Giuliani, Robert Gates, Newt Gingrich, Andrea Mitchell, Harold Ford Jr., Nia Malika Henderson

January 12: Reince Preibus, Mark Halperin, Chuck Todd, Stephanie Rawlings Blake, Kim Strassel, Maria Shriver, Rick Santorum, Jeffrey Goldberg, Jane Harman, Chris Matthews, Harry Smith

January 5: Janet Napolitano, Gene Sperling, Jim Cramer, Delos Cosgrove, John Noseworthy, Steve Schmidt, Donna Edwards, Judy Woodruff, Chuck Todd

Dec. 29: David Kirkpatrick, Darrell Issa, Joaquin Castro, Ben Wizner, Eugene Robinson, Andrea Mitchell, Robin Wright, Elliot Abrams, Peter Stearns

Dec. 15: Paul Ryan, Patty Murray, Michael Hayden, Bill Richardson, Nancy Gibbs, Kathleen Parker, Steve Inskeep, Scott Walker

Dec. 8: Charlayne Hunter Gault, Tom Brokaw, Jesse Jackson, Richard Stengel, Charles Ogletree, Al Sharpton, van cleef & arpels onyx necklace replica Katty Kay, Paul Gigot, Harry Smith

Dec. 1: Mike Rogers, Chris Van Hollen, Timothy Dolan, Ezekiel Emanuel, David Brooks, Stephanie Rawlings Blake, Andrea Mitchell, Chuck Todd, Harry Smith
Aug 29 · 0 comments
Money Gets Lewis Out of Way for Tyson Fight

The handlers of Mike Tyson couldn't win in court, so they paid Lennox Lewis, who agreed Thursday to step aside and let Tyson fight World Boxing Assn. champion Bruce Seldon on July 13 in Las Vegas.

The New Jersey Supreme Court had blocked Tyson's bid to fight Seldon after Lewis had gone to court, claiming that he should be fighting for Tyson's World Boxing Council title, rather than Seldon. Attempts by Tyson copy Van Cleef & Arpels jewelry and promoter Don King to overturn that decision failed, and that crown will not be at stake when Tyson fights Seldon.

Though terms of the agreement were not announced, Lewis reportedly will be paid an amount in seven figures and is guaranteed a fight with Tyson by Sept.

An ESPN report said Czyz complained of an injured back between the fifth and sixth rounds of the bout at Madison Square Garden, but his copy Van cleef arpels clover bracelet trainer, Tommy Parks, insisted to the fighter that it was his eye that was hurt, presumably because Holyfield had liniment on his gloves.

"It's not your back," Parks is heard to say on the videotape. "I said it's your eye. Let me do it. Just let me do it. Let me do it. It's your eye. If I say it's your eye, that's what it is. You ain't fighting no more."

The referee then comes over to the fighter's corner and Czyz is heard to say, "There's something in my eye. It's burning like crazy, burning like crazy."Bryan and Joel Glazer, sons of Tampa Bay Buccaneer owner Malcolm Glazer, and Buccaneer General Manager Rich McKay are in Southern California, investigating the prospects for moving the team here. They were scheduled to meet with Hollywood Park officials and with agent Leigh Steinberg, who has actively promoted Orange County as potential site for an NFL team.

The Buccaneers have a "walk a way" lease that would allow them to buy their freedom for $6 million and move elsewhere should a Sept. 3 Hillsboro County referendum to build a new stadium through a sales tax increase fail.

The team has also talked to Orlando area officials about a move there. The Buccaneers must pay a $35 million penalty to the Culverhouse trust if they leave central Florida.

Northwestern Coach Gary Barnett, who agreed to a new contract shortly after the Wildcats played in the Rose Bowl, finally signed the deal. Terms weren't disclosed, but the contract reportedly is for 12 years, at $500,000 per season. 54 in the world rankings, upset second seeded Goran Ivanisevic, 6 4, 6 2, and top seeded Thomas Muster beat the last American in the field, Todd Martin, 1 6, 6 4, 6 2, to reach the quarterfinals of the Italian Open in Rome.

Second seeded Arantxa Sanchez Vicario was tested for nearly three hours before defeating Ludmila Richterova, 6 7 (9 7), 6 3, 7 6 (7 1), and reaching the quarterfinals of the German Open in Berlin. Fifth seeded Mary Pierce lost to Karina Habsudova, 6 7 (7 4), 6 2, 7 6 (7 1).

Last year, Moss was sentenced to 30 days in jail to be spread over 18 months so he could play football, but he failed a drug test last week as he began serving the final 27 days of the sentence.

The commissioners of the NFL, NBA and NHL urged Congress Van Cleef and Arpels bracelet fake to defeat a bill they said would unconstitutionally force their leagues to grant expansion franchises to cities that lose teams to relocation. "No other business is subject to legislation that would determine how many operating units it must maintain or whom it must accept as an owner or partner," NFL Commissioner Paul Tagliabue told a House subcommittee.

Todd Bodine was picked to take over the Winston Cup driving duties of injured Bill Elliott, beginning with the Coca Cola 600 on May 26 at Charlotte Motor Speedway. Harry Gant is coming out of retirement to drive Elliott's Ford Thunderbird in Saturday night's Winston Select all star race at Charlotte. Elliott broke his left thighbone in an April 28 crash at Talladega, Ala.
Aug 29 · 0 comments
MPs back calls for English national anthem

Jerusalem was chosen as the anthem to be played for English athletes competing at the 2010 Commonwealth Games in Delhi, and was used again in 2014 in Glasgow

MPs have given initial support to the idea of England adopting an official national anthem.

However, Chesterfield MP Toby Perkins believes van cleef bracelet imitaiton England needs its own anthem and presented his case in the House of Commons as a ten minute rule motion.

His English National Anthem Bill was adopted by the House.

The idea will be debated again at a second reading on 4 copy Van Cleef & Arpels jewelry March.

Media captionJerusalem, based on the William Blake poem "And did those feet in ancient time", has come out as favourite in previous polls

Mr Perkins said it "seemed incongruous" for the English to sing about Britain when their teams play against other home nations, while the Welsh and Scots sing an anthem that reflects their nation's identity.

"It reflects a sense that we see Britain and England as synonymous," he said.

"This not only denies us English an opportunity to celebrate the nation that is being represented but is also a cause for resentment amongst other countries within the British Isles who feel that England have requisitioned a British song."

But his proposal for an English anthem was opposed by Jacob Rees Mogg, Conservative MP for North East Somerset.

"When the honourable gentleman for Chesterfield said that now English crowds take St George's flag rather than the Union Jack, to me that's a matter of pity, of shame, that we have given up viewing ourselves as one United Kingdom, whether we are supporting England, Scotland, Wales or Northern Ireland," he said.

"These expressions of the individual nationalism are a disuniting factor in our country, in a country that we ought to want to make more united," he said.

Mr Perkins said there had been a lot of interest in choosing an anthem when he spoke on radio stations across England.

"I won't say which area it was that thought the most appropriate choice for an English national anthem should be Heaven Knows I'm Miserable Now," he said.

"It will remain a secret between myself and the listeners of BBC Humberside, but it was perhaps reflective that each local area has its own sense of what Englishness means."

Campaign group England In My Heart wants Jerusalem to be adopted and arranged for a van with loudspeakers to drive around Parliament playing the song ahead of the debate.

"It's about time England was able to celebrate being English at sporting events," said group member Eddie Bone.

"Let the Scots and the Welsh celebrate theirs and then we come together at the end and sing God Save the Queen."

Another campaign group, Anthem 4 England, said Jerusalem has come out as favourite in previous polls.

Member Gareth Young said it was his personal favourite too.

"It's a beautiful song and it actually mentions and is about England, unlike its competitors," he said.

He said people wrongly think it's a hymn and also "object to the fact that it references a Middle Eastern city" but said these objections were "largely based on ignorance" as Jerusalem was a metaphor for a better place.

Using the UK anthem as the English anthem was an "unnecessary strain on relations between England and Scotland" in the wake of the independence referendum, he added.

"God Save the Queen is the British anthem and should belong equally to the English, Scottish, Welsh and Northern Irish," he said.

Prime Minister David Cameron has van cleef and arpels clover bracelet fake said that Jerusalem would be his choice for England's national anthem, according to the ConservativeHome website.

Jerusalem was chosen as the anthem to be played for English athletes competing at the 2010 Commonwealth Games in Delhi following a month long public vote.

The England cricket team have used Jerusalem as an unofficial anthem for more than a decade but still also sing God Save The Queen before many matches.

Land of Hope and Glory which was used by England at the Commonwealth Games before 2010 often comes up as second choice, behind Jerusalem, in public polls.
Aug 29 · 0 comments
Marco Muzzo apologizes

They stood and walked out of the courtroom.

Ed Lake and his wife, Jennifer Neville Lake, always said there was nothing they wanted to hear from the young man who killed all their children, who wiped out an entire generation of their family with his selfish decision to drink and drive on that sunny afternoon of Sept. 27, 2015.

Dressed in a sober black suit and shirt, Marco Muzzo left the prisoner box and nervously walked to the witness stand to address them for the first time since he ran that stop sign and T boned the van carrying Daniel Neville Lake, 9, brother Harrison, 5, and sister Milagros, 2, as well as their grandfather Gary Neville. In his trembling hands was a page long statement of apology but his words never reached them. He shook and choked back tears, but they saw none of it.

He ripped their lives apart. It is too soon to expect them to listen to what he has to say.

stand here before you today with great remorse, sympathy and unimaginable regret, Muzzo began. I listened with horror yesterday to the details of the catastrophic consequences of my actions, I knew that my words would be of no consolation. Ever since the tragedy that occurred as a result of my inexcusable conduct, I have wanted to say that I am sorry and apologize to your family from the bottom of my heart. can weep and apologize and pledge to spend the rest of his life trying to make up for the horror he has done, and as heartfelt as it was and it seemed genuine it not enough for the family he has decimated. Nothing short of miraculously bringing their kids back to life will do.

don want to listen to the man who is responsible for killing my children, Neville Lake said fiercely outside the courthouse. nothing he can say, his actions spoke louder than words. I don see why I should have to put myself through that. Paul Tait said replica van Yellow Gold necklace Muzzo driving with almost three times the legal limit of alcohol in his system was akin to someone going down the road shooting a gun. He could have taken a limo or a taxi home that day. Instead it was selfishness to get behind the wheel and drive while impaired, he said.

The Crown told Justice Michelle Fuerst that deterrence should be paramount and she can set a national precedent by imposing a tougher than average sentence. is time to send a message, Tait said, 10 to 12 years for Mr. Muzzo sends such a message. lawyer Brian Greenspan filed 92 letters of support for Muzzo and reminded the judge that his client has no criminal record, has taken responsibility for the deadly crash, and spared the family a trial by pleading guilty at the earliest opportunity. He urged her to impose an eight year term.

Fuerst will deliver her sentence Van Cleef & Arpels necklace imitation March 29. How much will she be swayed by Muzzo plea for forgiveness?

know that there are no actions that can ever change what has happened. I know that there is no step that I can take to bring back your children, he said. pray that I could but I cannot. by the grief and pain he caused them, he pledged to the rest of my life attempting to atone for my conduct by devoting myself to educating the public of the disastrous consequences of drinking and driving. not something they can hear right now or in the near future. They asked that Muzzo not try to contact them while he serves his sentence.

Perhaps one day, Neville Lake said, she might be ready to read his words in a transcript. For now, she doesn believe his remorse is real. She might have changed her mind if she heard his final words for herself.

will forever be haunted by the reality of what I have done, the drunk driver said in a voice shaking with emotion. I am truly sorry. isn enough. But it a van cleef and arpels fake necklace start. That and a stiff sentence to show what real atonement should be.

HE'S NOT A SPOILED RICH KID, PALS INSIST

In the 92 letters filed on behalf of Marco Muzzo, friends, family and employees insisted the media has unfairly made him out to be a spoiled rich kid. Instead, they said he is a humble and kind man who worked his way up in his family billion dollar construction business.

Neighbour Anna Bellisimo called Muzzo down to earth and always willing to help her. media has presented a version of Marco that is so contrary to the next door neighbour that I have known all these years. famous photo of him in a Ferrari shirt and sitting in a red sports car? His uncle Marc Muzzo, head of the family business, insists the car belonged to Muzzo late father, Robert, and in his memory, his son drives it just once a year to the family fundraiser, Motoamore, which has raised $4 million for cancer research.

media portrayal of Marco couldn be further from the truth. He is a hard worker. is not the type of person to show off. He really is a humble human being and those who know him can attest to that. 2013 Motoamore celebrated its 10th anniversary at the Rogers Centre with a full sized ferris wheel, dinner, dancing and private concert from Lenny Kravitz.
Aug 29 · 0 comments
Memories linger but Oakville's Van Koeverden focused on present at world championships

It's a venue that has fond memories fake Van Cleef and Arpels necklace for Adam Van Koeverden, but instead of basking in the past the three time Olympic medallist prefers to concentrate on the present.

Van Koeverden will lead a 33 member Canadian team into the canoe and kayak world championships which begin Thursday in Poznan, Poland, and ends Sunday. It's the same venue where Van Koeverden competed in his first senior world championships back in 2001.

A lot of water has flowed over Van Koeverden's paddle since then. He has experienced triumph and disappointment. The man might have changed but the significance of the competition remains the same for the Oakville native.

"It's not like any other event," Van Koeverden, 28, said Wednesday in a telephone interview from Poznan. "It's more important than a World Cup.

"The world championships only comes three times every four years. It's special. I'm glad I fake Van Cleef & Arpels Malachite necklace have quite a few of these under my belt. It's why I train for 11 1/2 months a year. I'm pretty focused on doing well."

Van Koeverden will race in the K 1 500 and 1,00 metre events. During his career he has won six world championship medals (one gold, three silver and two bronze). That helped make amends for his van cleef fake necklace disappointing eighth place finish in the same distance at the 2008 Beijing Olympics.

With the 2012 Summer Olympics just two years away, Van Koeverden sees this year's world championships as another step along the road to London.

"The first step was my first race after the Olympics," he said.

"I see every event we do as we approach London as another opportunity to learn and grow and improve as an athlete. We have one more world championship to go before the Olympics and a couple more World Cups."

Van Koeverden has won three World Cup medals this season. He's used the 10 week break since the end of the World Cup season to train and stave off the staleness he sometimes feels at this point of the season.

"I approach my season hoping to peak at the end of the season," he said. "The break is good for some reasons and it's something to accommodate for others.

"For the most part I'm very proud of the training I've been doing for the last month."

These will be the first world championships since the International Canoe Federation decided to add the 200 metre K 1 sprint to the Olympics. That has resulted in the 500 metre being dropped from the London Games.

It's a move that still upsets Van Koeverden, who won gold in the 500 metres at the 2004 Athens Games and silver in Beijing.

"It's very frustrating to have my best event removed from the Olympic program," he said. "I'm very disappointed with the direction the international federation is headed.

"At the same time, it allows me to concentre on the 1,000 metres."

In men's canoe, Mark Oldershaw of Burlington is expected to contend in the C 1 1,000 metre after winning four medals on the World Cup circuit.

For the first time, Paracanoe for athletes with a disabilities will be official events at the world championships.

Canada had its best ever showing at last year's world championships, winning one gold and three bronze medals.

Van Koeverden thinks Canadian paddlers can do well again this year.

"We have a lot of new events and some very fast sprinters," he said. "One thing that Canada has always done very well is be strong in all disciplines."
Aug 29 · 0 comments
Museum uncovers Van Gogh painting of his brother

If true, it would be the only known painting of Theo, although Vincent made several sketches of his brother, who supported him Van Cleef & Arpels necklace imitation financially and was his lifelong confidant and friend.

"People have often thought it was funny that there were no portraits of Theo, given that they were so close," said museum spokeswoman Linda Snoek.

She said the portrait was made in 1887 while the pair lived together in Paris a lesser known period of Van Gogh's life, since the bulk of information about Vincent is derived from letters he sent to Theo.

The painting has long been in storage, but went on display at the museum in Amsterdam on Tuesday as part of an exhibition on new findings about the painter's time spent in Antwerp and Paris in 1885 1888.

Though the brothers resembled each other physically, scholars determined the painting represents Theo by a number of factors.

Head researcher Louis van Tilborgh compared two paintings from 1887 with similar looking Van Cleef & Arpels Clover imitation necklace men in suits set against a blue background.

"They are two small, detailed portraits that when you see them you think: they belong together," Van Tilborgh said in an interview with Dutch state broadcaster NOS.

The portrait of Theo shows he had rounder ears than Vincent did. The other portrait shows Vincent with long, angular ears, consistent with other artists' paintings of Vincent. That's before he famously self mutilated one of his ears in December 1888.

In addition, Theo's goatee is more yellow brown than Vincent's dark red beard, and Theo has shaven cheeks, consistent with photographs of him from van cleef necklace fake the same period, while Vincent painted himself sporting mutton chop sideburns.
Aug 29 · 0 comments
Pages: «« « ... 2 3 4 5 6 ... » »»

News

Problem with reading feeds