Use of differential scanning fluorimetry as a high-throughput assay to Article

Use of differential scanning fluorimetry as a high-throughput assay to identify nuclear receptor ligands - Article Example As a partial solution to this problem differential scanning fluorimetry (DSF) is normally used. This technique allows for the identification of conditions that enhance the stability of proteins. DSF is used for this process as it allows possible ligands that have not been modified to be screened as 10 µL reactions in 96-well format against partially purified protein, revealing particular interactors. The researchers aim to find out whether differential scanning fluorimetry permits screening without modification of either the protein and/or the ligand in a high-throughput fashion using a commercially-available nuclear receptor ligand candidate chemical library. The researchers attempt to identify interactors of the human estrogen receptor ? ligand binding domain (ER? LBD). They will also investigate whether compounds that come into contact with the ligand stabilize the protein and lead to an increase of the thermal denaturation point. This process will be monitored using the SYPRO d ye which is able to detect even slight environmental changes. BACKGROUND INFORMATION Nuclear receptors (NRs) are transcription factors that exist in all metazoa that control cellular activities. Incidentally, the activities of NRs are regulated by interactions that occur between small-molecule ligands and a structurally-conserved ligand-binding domain (LBD) of the NR. This interaction induce structural changes resulting in new interaction surfaces for the recruitment of transcriptional co-activators and/or co-repressors. A lot of attention has been focused on the identification of ligands that bind to NRs and this is pushed by two main factors: i. Identification of natural ligands of a given NR is important in comprehending the role of the NR within the organism, particularly when the ligand of the NR is not clearly known or understood; ii. Identification of the ligands is important in the development of drugs and has led to a rise of different techniques each with known strengths a nd weaknesses (DeSantis et al. 2). Differential scanning technologies permit identification of conditions that improve the stability of proteins. The conversion from native to denatured protein is measured in relation to temperature changes. Two differential scanning techniques that can be used are scattering (Differential Scanning Light Scattering) or the fluorescence of an environmentally-sensitive dye (Differential Scanning Fluorescence – DSF). The techniques allow for the variation of a number of variables such as pH, salt, addition of small molecules (Schulman and Heyman 642). Cell conditions that result into a shift of proteins to higher denaturation temperature are identified as â€Å"stabilizing† conditions. This knowledge has been used to create conditions that are optimal for crystal growth (Vedadi et al., 2006). Other studies have used this technique to establish that peptides can shift the denaturation point of proteins to a higher temperature. This paper a ttempts to show that DSF can be used to successfully identify known interactors of the estrogen receptor ? (ER?) from a commercially available compound library. MATERIALS AND METHODS i. Reagents and instruments His6-hER? LDB (302-552) expression vector Terrific Broth IPTG EDTA-Free Protease Inhibitor Tablets Branson Sonifier 450 Ni-NTA Superflow ?-estradiol pET15b Sypro Orange, 5000X CFX96 Real-Time PCR System Nuclear Receptor Ligand Library BML-2802 ii. Methods The first step in this study was the

Pope Rebuking Priest Using Several Modern Day Christian Thinkers Essay

Pope Rebuking Priest Using Several Modern Day Christian Thinkers - Essay Example According to the church history, a lot has changed where there has been emergence of new technology and practices altering the way religious teaching are being done. Pope Paul III has also raised concerns about the modern changes of Christian teachings; he has pointed various modern day Christian thinkers, who have used their new perceptions, ideas and understandings to alter the traditional Christian religious teachings. These characters include Immanuel Kant, Friedrich Schleiermacher, Soren Kierkegaard, Pierre Teilhard De Chardin, Rudolph Bultmann, Karl Barth, Karl Rahner, and Dietrich Bohoeffer (Brown). Rogers states that Pope Benedict XVI, who in the past few years has guided the church’s doctrinal agency, delivered his homily in St. Peter’s Basilica during the time that priests recall the swearing they made when ordained (233). He sharply condemned a team of rebellious Austrian priest and laymen for going against major teachings of Catholic Church; this group is of ficially known as Pfarrer initiative and it has posed a lot of challenges to the church on issues such as banning of female priests and priestly celibacy. During a Maundy Thursday gathering in the Vatican, Pope Benedict XVI made a speech that he was not to tolerate disobedience. He was actually referring to the current efforts made by priests in both Austria and Europe of disobeying the church. Speaking from the altar of St. Peter’s Basilica, Benedict said that despite these priests claiming their concerns for the church, they are influenced by their own ideas and preferences to create desperate actions for changing the church, and that they should instead be turned into radicalism of obedience. In the year 2006, a team of Austrian priest opened the Pfarrer initiative led by Rev.Helmut Schuller; the former leader of a Catholic aid organization, with the intentions for calling for disobedience. Their aim was to abolish priestly celibacy and opening clergy to women in order to relieve priest shortages. However, according to Italian news reports, together with priest across Europe and United states, the Pfarrer initiative claims to acquire more than 300 Austrian priest and deacons together with supporters in other nations. Its influence has therefore increased to such an extent that Vatican officials met with superior Austrian bishops with an aim of discussing on how to handle them. Benedict also mentioned that in June the same year, about 2000 Austria’s priests in the initiative, had essentially threatened a schism, claiming that Vatican’s refusal to consider their protests left them with no option but to follow their conscience and take action independently. These priests made a revised call to disobedience, stating that parishes were capable of celebrating Eucharistic services in absence of priest and that they would make public speech about female and married priesthood. Benedict rejects the dissident’s idea, made during the second Vatican committee, of fossilizing the church and reviews that on the contrary; the development of new religious society in recent decade illustrated the vitality and true rejuvenation of the church by the 1962 to 1965 Vatican II. Benedict’s message is one of the greatest – and most straight- speeches that have mostly been dominated by a scandal of sexual abuse, Vatican hierarchy in disarray and recurring tangles with other faiths. It also has made Benedict discover who has