Synthesis of aromatic monothiols and aromatic dithiols to increase the folding rate and yield of disulfide containing proteins

Most pharmaceutically relevant proteins and many extracellular proteins contain disulfide bonds. Formation of the correct disulfide bonds is essential for stability in almost all cases. Disulfide containing proteins can be rapidly and inexpensively overexpressed in bacteria. However, the overexpressed proteins usually form aggregates inside the bacteria, called inclusion bodies, which contains inactive and non-native protein. To obtain native protein, inclusion bodies need to be isolated and resolubilized, and then the resulting protein refolded in vitro. In vitro protein folding is aided by the addition of a redox buffer, which is composed of a small molecule disulfide and/or a small molecule thiol. The most commonly used redox buffer contains reduced and oxidized glutathione. Recently, aliphatic dithiols and aromatic monothiols have been employed as redox buffers. Aliphatic dithiols improved the yield of native protein as compared to the aliphatic thiol, glutathione. Dithiols mimic the in vivo protein folding catalyst, protein disulfide isomerase, which has two thiols per active site. Furthermore, aromatic monothiols increased the folding rate and yield of lysozyme and RNase A relative to glutathione. By combining the beneficial properties of aliphatic dithiols and aromatic monothiols, aromatic dithiols were designed and were expected to increase in vitro protein folding rates and yields. Aromatic monothiols (1-4) and their corresponding disulfides (5-8), two series of ortho- and para-substituted ethylene glycol dithiols (9-15), and a series of aromatic quaternary ammonium salt dithiols (16-17) were synthesized on a multigram scale. Monothiols and disulfides (1-8) were utilized to fold lysozyme and bovine pancreatic trypsin inhibitor. Dithiols (11-17) were tested for their ability to fold lysozyme. At pH 7.0 and pH 8.0, and high protein concentration (1 mg/mL), aromatic dithiols (16, 17) and a monothiol (3) significantly enhanced the in vitro folding rate and yield of lysozyme relative to the aliphatic thiol, glutathione. Additionally, aromatic dithiols (16, 17) significantly enhance the folding yield as compared to the corresponding aromatic monothiol (3). Thus, the folding rate and yield enhancements achieved in in vitro protein folding at high protein concentration will decrease the volume of renaturation solution required for large scale processes and consequently reduce processing time and cost.

Hybrid approaches to estimating freeway travel times using point traffic detector data

The accurate and reliable estimation of travel time based on point detector data is needed to support Intelligent Transportation System (ITS) applications. It has been found that the quality of travel time estimation is a function of the method used in the estimation and varies for different traffic conditions. In this study, two hybrid on-line travel time estimation models, and their corresponding off-line methods, were developed to achieve better estimation performance under various traffic conditions, including recurrent congestion and incidents. The first model combines the Mid-Point method, which is a speed-based method, with a traffic flow-based method. The second model integrates two speed-based methods: the Mid-Point method and the Minimum Speed method. In both models, the switch between travel time estimation methods is based on the congestion level and queue status automatically identified by clustering analysis. During incident conditions with rapidly changing queue lengths, shock wave analysis-based refinements are applied for on-line estimation to capture the fast queue propagation and recovery. Travel time estimates obtained from existing speed-based methods, traffic flow-based methods, and the models developed were tested using both simulation and real-world data. The results indicate that all tested methods performed at an acceptable level during periods of low congestion. However, their performances vary with an increase in congestion. Comparisons with other estimation methods also show that the developed hybrid models perform well in all cases. Further comparisons between the on-line and off-line travel time estimation methods reveal that off-line methods perform significantly better only during fast-changing congested conditions, such as during incidents. The impacts of major influential factors on the performance of travel time estimation, including data preprocessing procedures, detector errors, detector spacing, frequency of travel time updates to traveler information devices, travel time link length, and posted travel time range, were investigated in this study. The results show that these factors have more significant impacts on the estimation accuracy and reliability under congested conditions than during uncongested conditions. For the incident conditions, the estimation quality improves with the use of a short rolling period for data smoothing, more accurate detector data, and frequent travel time updates.

Industry Education: The Merger Continues

In the discussion - Industry Education: The Merger Continues - by Rob Heiman Assistant Professor Hospitality Food Service Management at Kent State University, the author originally declares, “Integrating the process of an on-going catering and banquet function with that of selected behavioral academic objectives leads to an effective, practical course of instruction in catering and banquet management. Through an illustrated model, this article highlights such a merger while addressing a variety of related problems and concerns to the discipline of hospitality food service management education.” The article stresses the importance of blending the theoretical; curriculum based learning process with that of a hands-on approach, in essence combining an in-reality working program, with academics, to develop a well rounded hospitality student. “How many programs are enjoying the luxury of excessive demand for students from industry [?],” the author asks in proxy for, and to highlight the immense need for qualified personnel in the hospitality industry. As the author describes it, “An ideal education program concerns itself with the integration of theory and simulation with hands-on experience to teach the cognitive as well as the technical skills required to achieve the pre-determined hospitality education objectives.” In food service one way to achieve this integrated learning curve is to have the students prepare foods and then consume them. Heiman suggests this will quickly illustrate to students the rights and wrongs of food preparation. Another way is to have students integrating the academic program with feeding the university population. Your author offers more illustrations on similar principles. Heiman takes special care in characterizing the banquet and catering portions of the food service industry, and he offers empirical data to support the descriptions. It is in these areas, banquet and catering, that Heiman says special attention is needed to produce qualified students to those fields. This is the real focus of the discussion, and it is in this venue that the remainder of the article is devoted. “Based on the perception that quality education is aided by implementing project assignments through the course of study in food service education, a model description can be implemented for a course in Catering and Banquet Management and Operations. This project model first considers the prioritized objectives of education and industry and then illustrates the successful merging of resources for mutual benefits,” Heiman sketches. The model referred to above is also the one aforementioned in the thesis statement at the beginning of the article. This model is divided into six major components; Heiman lists and details them. “The model has been tested through two semesters involving 29 students,” says Heiman. “Reaction by all participants has been extremely positive. Recent graduates of this type of program have received a sound theoretical framework and demonstrated their creative interpretation of this theory in practical application,” Heiman says in summation.

Making a Case for Global Education: Revisiting Pre-Service Teacher Lesson Planning Methods

Globalization has tightened the bonds of interconnectedness and interdependence among nation-states. Students are destined for careers that are profoundly international in nature. They need to develop global literacy aided by a global education. This paper mandates the need for global education as part of the pre-service teacher-training programs.

A reforma psiquiátrica e a política de saúde mental do Rio Grande do Norte: papéis e funções de profissionais e gestores

This study arose from an interest in knowing the reality of mental health care in Rio Grande do Norte (RN) on the advances and challenges in the intersectoral agreements paths and consolidation of the Psychosocial Care Network (RAPS) from the state. Considering problematic and concerns were defined as objectives: Identify the knowledge of managers of Rio Grande do Norte on the National Mental Health Policy (PNSM) in the RN State; Describe the activities developed by health professionals in the individual service offered in the CAPS from RN; Understanding the relationship of managers’ knowledge on national mental health policy in professionals’ practice working in the the CAPS from the countryside. It is a descriptive study with a quantitative and qualitative approach, carried out in 30 CAPS from RN’s countryside, where 183 professionals answered a structured questionnaire with closed questions about the activities they do in individual care; and 19 mental health coordinators of municipalities and the state coordinator of RAPS were interviewed about their knowledge on the Mental Health Policy. Data were collected after approval by the Research Ethics Committee of the Federal University of Rio Grande do Norte, with the number 508.430 CAAE: 25851913.7.0000.5537 from August through October of 2014 in 26 municipalities with CAPS from the state. Quantitative data were tabulated and analyzed using a descriptive statistics aided by the software Statistical Package for the Social Scienses (SPSS) version 20.0. The qualitative data were prepared in a corpus and analyzed through software Analyse Lexicale par Contexte d’um Ensemble de Segments de Texte (ALCESTE) that allow to perform textual statistical analysis and categorization from their comments, submitted to Bardin content analysis. Five categories were generated approaching the managers’ knowledge, namely: Back to society: leadership and users’ role and autonomy; The gap between policy and practice; Barriers that affect the service; Structuring the Psychosocial Care Network; Multidisciplinary team: attribuitios and activities. The CAPS professionals’ ages ranged from 20 to 58 years, prevailing females, with 76.5% of the total, the majority were social workers (16.8%), psychologists (15.3%), nurses (14.8%) and nursing technicians (14.8%). The results showed precariousness in care associated with physical workload regard to high workload and low wages of the CAPS professionals' and, also, it was possible to observe a large involvement of professionals in care delivery, despite the difficulties encountered in services. It was found little knowledge in managers regarding the National Mental Health Policy having as causes of this reality the poor education and training of these professionals. The responses of professionals working in care reveals strong consistency with what is expected of a psychosocial care service. Points up as a thesis of this study that the psychiatric reform and mental health policy in Rio Grande do Norte is following a structural expansion process, but with precariousness of services from a still unprepared management to act in a psychosocial context.

Avaliação do controle da diagênese na qualidade de reservatório siliciclástico do cenomaniano superior da formação Açu, sudoeste da bacia potiguar

The distribution of diagenetic alterations in Late Cenomanian siliciclastic reservoirs from Potiguar Basin was influenced by the stratigraphic framework and the depositional system. Seismic sections and geophysical logs of two wells drilled in the SW portion of the mentioned basin above register regional stratigraphic surfaces representing maximum floods related to a transgressive event. The sequential analysis of 80 m of drill core (~450 m deep) recognized nine depositional facies with an upwards granodecrescent standard piling that limits cycles with an erosional conglomeratic base (lag) overlain by intercalations of medium to very fine sandstones showing cross bedding (channel, planar and low angled) and horizontal bedding (plane-parallel , wave and flaser). The top of the cycles is marked by the deposition of pelites and the development of paleosoils and lagoons. The correlation of genetically related facies reveals associations of channel fillings, crevasse, and flood plains deposited in a transgressive system. Detailed descriptions of seventy nine thin sections aided by MEV-EBSD/EDS, DRX and stable isotope analyses in sandstones revealed an arcosian composition and complex textural arrays with abundant smectite fringes continuously covering primary components, mechanically infiltrated cuticles and moldic and intragrain pores. K-feldspar epitaxial overgrowth covers microcline and orthoclase grains before any other phase. Abundant pseudomatrix due to the compactation of mud intraclasts concentrate along the stratification planes, locally replaced by macrocristalline calcite and microcrystalline and framboidal pyrite. Kaolinite (booklets and vermicular), microcrystalline smectite, microcrystalline titanium minerals and pyrite replace the primary components. The intergrain porosity prevails over the moldic, intragrain and contraction porosities. The pores are poorly connected due to the presence of intergranular smectite, k-feldspar overgrowth, infiltrated mud and pseudomatrix. The sandstones were subjected to eodiagenetic conditions next to the surface and shallow burial mesodiagenetic conditions. The diagenetic alterations reduced the porosity and the permeability mainly due to the precipitation of smectite fringes, compactation of mud intraclasts onto the pseudomatrix and cementing by poikilotopic calcite characterizing different reservoir petrofacies. These diagenetic products acted as barriers and detours to the flow of fluids thus reducing the quality of the reservoir.

Major ion chemistry of snow cores along a transect in central Dronning Maud Land, Antarctica

Among the large variety of particulates in the atmosphere, calcic mineral dust particles have highly reactive surfaces that undergo heterogeneous reactions with nitrogen oxides contiguously. The association between Ca2+, an important proxy indicator of mineral dust and NO3-, a dominant anion in the Antarctic snow pack was analysed. A total of 41 snow cores (~ 1 m each) that represent snow deposited during 2008-2009 were studied along coastal-inland transects from two different regions - the Princess Elizabeth Land (PEL) and central Dronning Maud Land (cDML) in East Antarctica. Correlation statistics showed a strong association (at 99 % significance level) between NO3- and Ca2+ at the near-coastal sections of both PEL (r = 0.72) and cDML (r = 0.76) transects. Similarly, a strong association between these ions was also observed in snow deposits at the inland sections of PEL (r = 0.8) and cDML (r = 0.85). Such systematic associations between Ca2+ and NO3- is attributed to the interaction between calcic mineral dust and nitrogen oxides in the atmosphere, leading to the possible formation of calcium nitrate (Ca(NO3)2). Forward and back trajectory analyses using HYSPLIT model v. 4 revealed that Southern South America (SSA) was an important dust emitting source to the study region, aided by the westerlies. Particle size distribution showed that over 90 % of the dust was in the range < 4 µm, indicating that these dust particles reached the Antarctic region via long range transport from the SSA region. We propose that the association between Ca2+ and NO3- occurs during the long range transport due to the formation of Ca(NO3)2. The Ca(NO3)2 thus formed in the atmosphere undergo deposition over Antarctica under the influence of anticyclonic polar easterlies. However, influence of local dust sources from the nunataks in cDML evidently mask such association in the mountainous region. The study indicates that the input of dust-bound NO3- may contribute a significant fraction of the total NO3- deposited in Antarctic snow.

Diatom oxygen and silicon isotopes data from IODP Hole 323-U1341B in the Bering Sea through the late Pliocene and over the onset of major Northern Hemisphere Glaciation

The Bering Sea is one of the most biologically productive regions in the marine system and plays a key role in regulating the flow of waters to the Arctic Ocean and into the subarctic North Pacific Ocean. Cores from Integrated Ocean Drilling Program (IODP) Expedition 323 to the Bering Sea provide the first opportunity to obtain reconstructions from the region that extend back to the Pliocene. Previous research at Bowers Ridge, south Bering Sea, has revealed stable levels of siliceous productivity over the onset of major Northern Hemisphere Glaciation (NHG) (circa 2.85-2.73 Ma). However, diatom silica isotope records of oxygen (d18Odiatom) and silicon (d30Sidiatom) presented here demonstrate that this interval was associated with a progressive increase in the supply of silicic acid to the region, superimposed on shift to a more dynamic environment characterized by colder temperatures and increased sea ice. This concluded at 2.58 Ma with a sharp increase in diatom productivity, further increases in photic zone nutrient availability and a permanent shift to colder sea surface conditions. These transitions are suggested to reflect a gradually more intense nutrient leakage from the subarctic northwest Pacific Ocean, with increases in productivity further aided by increased sea ice- and wind-driven mixing in the Bering Sea. In suggesting a linkage in biogeochemical cycling between the south Bering Sea and subarctic Northwest Pacific Ocean, mainly via the Kamchatka Strait, this work highlights the need to consider the interconnectivity of these two systems when future reconstructions are carried out in the region.

Strength of the Iberian intraplate lithosphere: Cenozoic deformations and seismicity

Previous studies about the strength of the lithosphere in the Iberia centre fail to resolve the depth of earthquakes because of the rheological uncertainties. Therefore, new contributions are considered (the crustal structure from a density model) and several parameters (tectonic regime, mantle rheology, strain rate) are checked in this paper to properly examine the role of lithospheric strength in the intraplate seismicity and the Cenozoic evolution. The strength distribution with depth, the integrated strength, the effective elastic thickness and the seismogenic thickness have been calculated by a finite element modelling of the lithosphere across the Central System mountain range and the bordering Duero and Madrid sedimentary basins. Only a dry mantle under strike-slip/extension and a strain rate of 10-15 s-1, or under extension and 10-16 s-1, causes a strong lithosphere. The integrated strength and the elastic thickness are lower in the mountain chain than in the basins. These anisotropies have been maintained since the Cenozoic and determine the mountain uplift and the biharmonic folding of the Iberian lithosphere during the Alpine deformations. The seismogenic thickness bounds the seismic activity in the upper–middle crust, and the decreasing crustal strength from the Duero Basin towards the Madrid Basin is related to a parallel increase in Plio–Quaternary deformations and seismicity. However, elasto–plastic modelling shows that current African–Eurasian convergence is resolved elastically or ductilely, which accounts for the low seismicity recorded in this region.

The current and potential applications of Ambient Mass Spectrometry in detecting food fraud

The adulteration of food has received substantial amounts of media attention in the last few years, with events such as the European horsemeat scandal in 2013 sending shockwaves through society. Almost all cases are motivated by the pursuit of profits and are often aided by long and complex supply chains. In the past few years, the rapid growth of ambient mass spectrometry (AMS) has been remarkable, with over thirty different ambient ionisation techniques available. Due to the increasing concerns of the food industry and regulators worldwide, AMS is now being utilised to investigate whether or not it can generate results which are faster yet comparable to those of conventional techniques. This article reviews some aspects of the adulteration of food and its impact on the economy and the public's health, the background to ambient mass spectrometry and the studies that have been undertaken to detect food adulteration using this technology.

Utility of sarcoma-specific fusion gene analysis in paraffin-embedded material for routine diagnosis at a specialist centre.

AIMS: Diagnosis of soft tissue sarcomas can be difficult. It can be aided by detection of specific genetic aberrations in many cases. This study assessed the utility of a molecular genetics/cytogenetics service as part of the routine diagnostic service at the Royal Marsden Hospital. METHODS: A retrospective audit was performed over a 15-month period to evaluate the diagnostic usefulness for soft tissue sarcomas with translocations of fluorescence in situ hybridisation (FISH) and reverse-transcriptase PCR (RT-PCR) in paraffin-embedded (PE) material. Results were compared with histology, and evaluated. RESULTS: Molecular investigations were performed on PE material in 158 samples (total 194 RT-PCR and 174 FISH tests), of which 85 were referral cases. Synovial sarcoma, Ewing sarcoma and low-grade fibromyxoid sarcoma were the most commonly tested tumours. Myxoid liposarcoma showed the best histological and molecular concordance, and alveolar rhabdomyosarcoma showed the best agreement between methods. FISH had a higher sensitivity for detecting tumours (73%, compared with 59% for RT-PCR) with a better success rate than RT-PCR, although the latter was specific in identifying the partner gene for each fusion. In particular, referral blocks in which methods of tissue fixation and processing were not certain resulted in higher RT-PCR failure rates. CONCLUSIONS: FISH and RT-PCR on PE tissue are practical and effective ancillary tools in the diagnosis of soft tissue sarcomas. They are useful in confirming doubtful histological diagnoses and excluding malignant diagnoses. PCR is less sensitive than FISH, and the use of both techniques is optimal for maximising the detection rate of translocation-positive sarcomas.

Flexible strategies for association analysis with genomic phenotypes

Thesis (Ph.D.)--University of Washington, 2016-08

‘The Emergence of the Operatic’

The chapter discusses the notion of the ‘operatic’ with particular reference to movement. Through observation of singers in rehearsal and through interviews with singers over two years, the research posits that ‘operatic’ movement may be identified and explored as a discrete quality of movement in performance and recognised as an, albeit underappreciated, aspect of the artistry of the opera singer. The paper explores the effects of the demands of singing on the dramatic expressivity of the body and the strategies employed by singers to navigate, the sometimes conflicting demands of the composer and the director. ‘Operatic’ movement is regarded as that which is not generated through character or narrative (positioned as a normative approach to acting) but through the negotiation of the physical constrictions and artificiality of breath associated with operatic singing, combined with sensitivity towards opera’s non-normative performance conventions. This position is aided by an interrogation of opera through the lens of gender theory. The chapter makes two propositions, that the ‘operatic’, which does not form part of the formal pedagogy of singers, is an emergent property, especially evident at the intersection of the various creative disciplines that contribute to opera, such as when the orchestra is introduced to the rehearsal process, and that ‘operaticness’ is acquired and passed on through a process of kinaesthetic empathy.

Da cozinha ao terreiro em Mutamba da Caieira (RN) : cotidiano, espaço doméstico e sociabilidade

In this work we thought about the social use of the domestic space and, specifically, the space destined to the preparation of food. The kitchen is a creative space of social relationships. There the families exchange daily interaction with the neighbors, in a collective of the space reserved to the making of food. Our empiric research was in the Mutamba da Caieira community, located in the county of Assu (RN). The data was collected through the etnographic method aided by the photographic resources. In the discussion, we delimitated at the house three types of kitchens: one reserved to the family, another kitchen in the terrace and yet another kitchen in the yard. They are kitchens that link amongst themselves with activities and differentiated temporalities. In the daily social exchange, the kitchen imposes itself as a social space through the conditions that it offers for the production of the foods, the circulation of domestic objects, the communication of knowledge around the cookery, operating a group of symbolic and ritual actions combined with appropriate techniques for the transformation of the foods. The study reveals that the domestic chores accomplished amon relatives and friends form webs of intracommunitary relationships in the use of the three kitchens, and that the social network is updated in a singular moment that usually happens in the community, the Game of Pacará

A dicionarização de termos em língua brasileira de sinais (Libras) para o ensino de biologia: uma atitude empreendedora

Acompanha: Glossário de termos biológicos em língua brasileira de sinais (GBLI)