Understanding the yeast host cell response to recombinant membrane protein production

Membrane proteins are drug targets for a wide range of diseases. Having access to appropriate samples for further research underpins the pharmaceutical industry's strategy for developing new drugs. This is typically achieved by synthesizing a protein of interest in host cells that can be cultured on a large scale, allowing the isolation of the pure protein in quantities much higher than those found in the protein's native source. Yeast is a popular host as it is a eukaryote with similar synthetic machinery to that of the native human source cells of many proteins of interest, while also being quick, easy and cheap to grow and process. Even in these cells, the production of human membrane proteins can be plagued by low functional yields; we wish to understand why. We have identified molecular mechanisms and culture parameters underpinning high yields and have consolidated our findings to engineer improved yeast host strains. By relieving the bottlenecks to recombinant membrane protein production in yeast, we aim to contribute to the drug discovery pipeline, while providing insight into translational processes.

Which yeast species shall I choose? Saccharomyces cerevisiae versus Pichia pastoris (review)

Having decided on yeast as a production host, the choice of species is often the first question any researcher new to the field will ask. With over 500 known species of yeast to date, this could pose a significant challenge. However, in reality, only very few species of yeast have been employed as host organisms for the production of recombinant proteins. The two most widely used, Saccharomyces cerevisiae and Pichia pastoris, are compared and contrasted here.

Novel strategies to improve recombinant membrane protein production in yeast

Approximately 60% of pharmaceuticals target membrane proteins; 30% of the human genome codes for membrane proteins yet they represent less than 1% of known unique crystal structures deposited in the Protein Data Bank (PDB), with 50% of structures derived from recombinant membrane proteins having been synthesized in yeasts. G protein-coupled receptors (GPCRs) are an important class of membrane proteins that are not naturally abundant in their native membranes. Unfortunately their recombinant synthesis often suffers from low yields; moreover, function may be lost during extraction and purification from cell membranes, impeding research aimed at structural and functional determination. We therefore devised two novel strategies to improve functional yields of recombinant membrane proteins in the yeast Saccharomyces cerevisiae. We used human adenosine A2A receptor (hA2AR) as a model GPRC since it is functionally and structurally well characterised.In the first strategy, we investigated whether it is possible to provide yeast cells with a selective advantage (SA) in producing the fusion protein hA2AR-Ura3p when grown in medium lacking uracil; Ura3p is a decarboxylase that catalyzes the sixth enzymatic step in the de novo biosynthesis of pyrimidines, generating uridine monophosphate. The first transformant (H1) selected using the SA strategy gave high total yields of hA2AR-Ura3p, but low functional yields as determined by radio-ligand binding, leading to the discovery that the majority of the hA2AR-Ura3p had been internalized to the vacuole. The yeast deletion strain spt3Δ is thought to have slower translation rates and improved folding capabilities compared to wild-type cells and was therefore utilised for the SA strategy to generate a second transformant, SU1, which gave higher functional yields than H1. Subsequently hA2AR-Ura3p from H1 was solubilised with n-dodecyl-β-D-maltoside and cholesteryl hemisuccinate, which yielded functional hA2AR-Ura3p at the highest yield of all approaches used. The second strategy involved using knowledge of translational processes to improve recombinant protein synthesis to increase functional yield. Modification of existing expression vectors with an internal ribosome entry site (IRES) inserted into the 5ˊ untranslated region (UTR) of the gene encoding hA2AR was employed to circumvent regulatory controls on recombinant synthesis in the yeast host cell. The mechanisms involved were investigated through the use of yeast deletion strains and drugs that cause translation inhibition, which is known to improve protein folding and yield. The data highlight the potential to use deletion strains to increase IRES-mediated expression of recombinant hA2AR. Overall, the data presented in this thesis provide mechanistic insights into two novel strategies that can increase functional membrane protein yields in the eukaryotic microbe, S. cerevisiae.

Invisible but not forgotten: The Afro-Argentine and Afro-Uruguayan experience from the sixteenth to the twentieth centuries

The problem to be examined in this thesis involves the supposedly overlooked history and contributions of Africans and their descendants in the River Plate countries of Argentina and Uruguay. Therefore, the primary purpose of the study is to narrate the social history of Afro-Argentines and Afro-Uruguayans from the sixteenth to the twentieth century. A secondary purpose, moreover, is to synthesize the academic literature on Blacks in the Rio de la Plata and their many cultural and other contributions to the current nation-states of Argentina and Uruguay. This thesis thereby challenges the regnant historiographical argument that African Argentines and African Uruguayans have been “forgotten” as historical actors by scholars both inside and outside the Rio de la Plata. By synthesizing the large body of historical and social science scholarship on Africans in the River Plate, as well as providing a thorough bibliography on the subject, this study attempts to proffer (to borrow the subtitle of Marvin Lewis' 1996 study of Afro-Argentine literature) “another dimension of the Black Diaspora” to the Americas. ^

Trends for Meetings and Expositions Industry

During the last decade, the meetings and expositions industry has flourished, even as it has struggled to cope with difficult challenges. This is a taste of things to me. In the years ahead, the global population will continue to grow and change, science and technology will tighten their hold on business and society and the world will knit itself ever more tightly into a single market. As a result, both opportunities and trials will abound.

Club Medic

For most of us, getting sick is a good way to ruin a vacation. However, for growing numbers of people, needing to see the doctors the whole point of going abroad. When they require surgery or dental work, thy may combine treatment with a trip to the Taj Mahal, A photo safari on the African veldt, or a stay at a luxury hotel-or at a hospital that feels like one – all at bargain-basement prices. This is medical tourism, and it is one of the hottest niche markets in the hospitality industry.

Conceptualizing American power and security in a post -9/11 security context: Conflict, resistance, and global security, 2001–present

In a post-Cold War, post-9/11 world, the advent of US global supremacy resulted in the installation, perpetuation, and dissemination of an Absolutist Security Agenda (hereinafter, ASA). The US ASA explicitly and aggressively articulates and equates US national security interests with the security of all states in the international system, and replaced the bipolar, Cold War framework that defined international affairs from 1945-1992. Since the collapse of the USSR and the 11 September 2001 terrorist attacks, the US has unilaterally defined, implemented, and managed systemic security policy. The US ASA is indicative of a systemic category of knowledge (security) anchored in variegated conceptual and material components, such as morality, philosophy, and political rubrics. The US ASA is based on a logic that involves the following security components: (1) hyper militarization, (2) intimidation,(3) coercion, (4) criminalization, (5) panoptic surveillance, (6) plenary security measures, and (7) unabashed US interference in the domestic affairs of select states. Such interference has produced destabilizing tensions and conflicts that have, in turn, produced resistance, revolutions, proliferation, cults of personality, and militarization. This is the case because the US ASA rests on the notion that the international system of states is an extension, instrument of US power, rather than a system and/or society of states comprised of functionally sovereign entities. To analyze the US ASA, this study utilizes: (1) official government statements, legal doctrines, treaties, and policies pertaining to US foreign policy; (2) militarization rationales, budgets, and expenditures; and (3) case studies of rogue states. The data used in this study are drawn from information that is publicly available (academic journals, think-tank publications, government publications, and information provided by international organizations). The data supports the contention that global security is effectuated via a discrete set of hegemonic/imperialistic US values and interests, finding empirical expression in legal acts (USA Patriot ACT 2001) and the concept of rogue states. Rogue states, therefore, provide test cases to clarify the breadth, depth, and consequentialness of the US ASA in world affairs vis-à-vis the relationship between US security and global security.

Conceptualizing American power and security in a post-9/11 security context : conflict, resistance, and global security, 2001-present

In a post-Cold War, post-9/11 world, the advent of US global supremacy resulted in the installation, perpetuation, and dissemination of an Absolutist Security Agenda (hereinafter, ASA). The US ASA explicitly and aggressively articulates and equates US national security interests with the security of all states in the international system, and replaced the bipolar, Cold War framework that defined international affairs from 1945-1992. Since the collapse of the USSR and the 11 September 2001 terrorist attacks, the US has unilaterally defined, implemented, and managed systemic security policy. The US ASA is indicative of a systemic category of knowledge (security) anchored in variegated conceptual and material components, such as morality, philosophy, and political rubrics. The US ASA is based on a logic that involves the following security components: 1., hyper militarization, 2., intimidation, 3., coercion, 4., criminalization, 5., panoptic surveillance, 6., plenary security measures, and 7., unabashed US interference in the domestic affairs of select states. Such interference has produced destabilizing tensions and conflicts that have, in turn, produced resistance, revolutions, proliferation, cults of personality, and militarization. This is the case because the US ASA rests on the notion that the international system of states is an extension, instrument of US power, rather than a system and/or society of states comprised of functionally sovereign entities. To analyze the US ASA, this study utilizes: 1., official government statements, legal doctrines, treaties, and policies pertaining to US foreign policy; 2., militarization rationales, budgets, and expenditures; and 3., case studies of rogue states. The data used in this study are drawn from information that is publicly available (academic journals, think-tank publications, government publications, and information provided by international organizations). The data supports the contention that global security is effectuated via a discrete set of hegemonic/imperialistic US values and interests, finding empirical expression in legal acts (USA Patriot ACT 2001) and the concept of rogue states. Rogue states, therefore, provide test cases to clarify the breadth, depth, and consequentialness of the US ASA in world affairs vis-a-vis the relationship between US security and global security.

Processos de construção do sentido por portadores da Síndrome de Asperger

Para la lingüística Cognitiva, la construcción del sentido darse a través del accionamiento de procesos mentales específicos que están directamente ligados a las experiencias socioculturales, sensorio motores y perceptuales. En portadores de la Síndrome de Asperger, el comprometimiento de estos procesos, debido a características específicas dela propia síndrome, puede estar directamente relacionado a problemas de construcción de sentido por estos individuos. En este sentido, el objetivo de esta investigación es investigar los procesos cognitivos que direccionan la construcción del sentido en estos portadores de trastornos neurales. La investigación es realizada con alumnos de la APPARN (Associação de país e amigos dos autistas do Rio Grande do Norte), teniendo como grupo de control alumnos neurotípicos de otras instituciones, siendo los portadores de la síndrome practicantes de la lengua materna o grupo experimental de la búsqueda. Para elucidar el proceso de construcción del sentido por estos alumno, fueron desarrollados testes contiendo rasgos linguisticos norteadores responsables por el accionamiento de algunos procesos cognitivos como los esquemas (JOHNSON, 1987), frames (MINSKY, 1974), affordances (GIBSON, 1979) y simulación mental (BARSALOU, 1999). Los testes fueron basados en los presupuestos teóricos de la Lingüística Cognitiva, más precisamente, de la Teoria Neural del Linguaje. Los resultados apuntan para una posible justificativa para el comprometimiento de la construcción del sentido en portadores de la Síndrome de Asperger, que comprende directamente la relevancia intersubjetiva de la infersencias recibidas y construidas socialmente por los participantes específicamente de este análisis.

Narratividade e estruturas cognitivas: o encontro entre a linguagem, a cognição e a experiência sociocultural

This work is within the framework of Cognitive Linguistics, which, as opposed to generative modular approach posits that language is not autonomous but part of human cognition manifest mental processing, socio-cultural and bodily experiences. Our goal is to describe and analyze cognitive mechanisms of understanding who work in the formal and meaningful organization of the narrative. In order to study and verification of this phenomenon, this project was based in the theoretical framework of Rapaport et al (1994) with the treatment of deictic center, Zwaan (1999) and Zwaan and Radvansky (1998) with situation models, Minsky (1974) with the concept frame, Johnson (1987) and Duke and Costa (2012) with pictorial schemes. To this end, we focused on the deictic perspective (WHERE, WHEN, WHO), social cognitive structures (frames) and body (pictorial diagrams) and the situation of models built by compreendedor from these cognitive structures. Methodologically, it is a qualitative research (BAUER and GASKELL, 2002), of interpretive base (MOITA LOPES, 1994), based on introspection (Talmy, 2005). The corpus selected is a sample of twelve texts written by 8th grade students, whose production consists of fictional narrative, the production of diary pages. The analyses were conducted by cognitive structures known as constructional blocks (BCs)(SANTOS, 2011), which guid the discussion about how we build understanding and creation of meanings in narratives. The result shows that the narrative events are mentally represented by the understander that conceives a deictic center and that, guided by it, has access to understanding and construction of meaning in narrative by means of cognitive domains established by bodily and socio-cultural experiences.

Os youtubers e a representação do certo, errado, adequado e inadequado no trabalho com a variação linguística em sala de aula

Some of the current discussions in the teaching of Portuguese Language (LP) pertain to how the school should deal with the phenomenon of language variation in the classroom. In 2010, for example, an explosion of talk took over the academic corridors: a book, entitled "Por uma vida melhor", the collection "Viver, Aprender", published by the MEC (Ministry of Education and Culture) to students EJA (Youth and Adults) brought notions regarding linguistic variation, even in their first chapter. In it is clear the notion that it is possible to make use of structures as "pretty boy", instead of "pretty boys", depending on the context in which such use is insert. Therefore, the discussions focused around the notions of variety cultivated, standard and popular measuring them to the possibilities of linguistic appropriateness. The community was surprised by the defense of the "power" to use, since it would be the school space to teach a standard "default", and not the possibility of legitimate use of grammatical patterns that clashed with those recommended in traditional grammars. The television media has been responsible for a major blaze that MEC had endorsed the use in schools of a book that legitimized such linguistic patterns. The quarrel was released on Youtube and in that space, netizens expressed themselves for or against the proposal of LD often directing the discussion to questions of a purely political. We observed that, on one side, loomed arguments related to Sociolinguistics (BAGNO , 2002, 2003, 2007, 2009; BAGNO, M.; STUBBS, M., Gagne, G., 2006; Bortoni - RICARDO, S.M., 2008; Tarallo, F., 1982; U. Weinreich, MARVIN I. HERZOG, Labov, W., 1968, Labov 1972, etc.); another, arguments concentrated on defending the school is the area of language teaching standard, and not fit to bring certain discussions within an LD. It was from these words, that this research was born. Interested in the particular way that the community media, which seemed to have no training in linguistics, understand the concepts of right, wrong, appropriate and inappropriate, so intimate in academic circles. Our thoughts take as reference the theoretical studies on the question of sociolinguistic variation and education, official documents that guide the "work" with the Portuguese language in the classroom, like the NCP (National Curriculum) and Curriculum Proposal for Education Youth and Adult (PCEJA). In our analysis, we found that LD" For a better life "makes no apology for teaching the "error", but it raises discussions about the possibility of "change", linked to factors and different order. We realize how significant it is to observe how speakers of a language are positioned in relation to language teaching which they are not speakers and scholars. Our study showed that certain issues regarding the teaching of the Portuguese language, as is the case of linguistic variation, points are far from being resolved, either for linguists and/or grammarians, whether for language speakers.

Downhole logging of the AND-2A borehole, Victoria Land Basin, McMurdo Sound, Antarctica

Under the framework of the ANDRILL Southern McMurdo Sound (SMS) Project successful downhole experiments were conducted in the 1138.54 metre (m)-deep AND-2A borehole. Wireline logs successfully recorded were: magnetic susceptibility, spectral gamma ray, sonic velocity, borehole televiewer, neutron porosity, density, calliper, geochemistry, temperature and dipmeter. A resistivity tool and its backup both failed to operate, thus resistivity data were not collected. Due to hole conditions, logs were collected in several passes from the total depth at ~1138 metres below sea floor (mbsf) to ~230 mbsf, except for some intervals that were either inaccessible due to bridging or were shielded by the drill string. Furthermore, a Vertical Seismic Profile (VSP) was created from ~1000 mbsf up to the sea floor. The first hydraulic fracturing stress measurements in Antarctica were conducted in the interval 1000-1138 mbsf. This extensive data set will allow the SMS Science Team to reach some of the ambitious objectives of the SMS Project. Valuable contributions can be expected for the following topics: cyclicity and climate change, heat flux and fluid flow, seismic stratigraphy in the Victoria Land Basin, and structure and state of the modern crustal stress field.

Conventional and 40Ar/39Ar K-Ar ages of volcanic rocks at DSDP Leg 55 Holes

Conventional K-Ar, 40Ar/39Ar total fusion, and 40Ar/39Ar incremental heating data on hawaiite and tholeiitic basalt samples from Ojin (Site 430), alkalic basalt samples from Nintoku (Site 432), and alkalic and tholeiitic basalt samples from Suiko (Site 433) seamounts in the Emperor Seamount chain give the following best ages for these volcanoes: Ojin = 55.2 ± 0.7 m.y., Nintoku = 56.2 ± 0.6 m.y., and Suiko = 64.7 ± 1.1 m.y. These new data bring to 27 the number of dated volcanoes in the Hawaiian-Emperor volcanic chain. The new dates prove that the age progression from Kilauea Volcano on Hawaii (0 m.y.) through the Hawaiian-Emperor bend (- 43 m.y.) to Koko Seamount (48.1 m.y.) in the southernmost Emperor Seamounts continues more than halfway up the Emperor chain to Suiko Seamount. The age versus distance data for the Hawaiian-Emperor chain are consistent with the kinematic hot-spot hypothesis, which predicts that the volcanoes are progressively older west and north away from the active volcanoes of Kilauea and Mauna Loa. The data are consistent with an average volcanic propagation velocity of either 8 cm/year from Suiko to Kilauea or of 6 cm/year from Suiko to Midway followed by a velocity of 9 cm/year from Midway to Kilauea, but it appears that the change in direction that formed the Hawaiian- Emperor bend probably was not accompanied by a major change in velocity.

(Table 1, page 1169) Chemical composition of land (fossil) manganese deposits rich in todorokite

Chemical, x-ray and other data are given for todorokite, (Mn, Mg, Ca, Ba, Na, K)2.Mn5O12.3H2O, from Charco Redondo, Cuba, Farragudo, Portugal, and Hüttenberg, Austria. Additional localities at Romanèche, France, Saipan Island, Bahia, Brazil and Sterling Hill, New Jersey, are noted. Delatorreite of Simon and Straczek (1958) is identical with todorokite.

Nanotechnology in the food industry I: applications

Nanotechnology is a multidisciplinary science that is having a boom today, providing new products with attractive physicochemical properties for many applications. In agri/feed/food sector, nanotechnology offers great opportunities for obtaining products and innovative applications for agriculture and livestock, water treatment and the production, processing, storage and packaging of food. To this end, a wide variety of nanomaterials, ranging from metals and inorganic metal oxides to organic nanomaterials carrying bioactive ingredients are applied. This review shows an overview of current and future applications of nanotechnology in the food industry. Food additives and materials in contact with food are now the main applications, while it is expected that in the future are in the field of nano-encapsulated and nanocomposites in applications as novel foods, additives, biocides, pesticides and materials food contact.