Double Cysteine Mutations in Staphylococcal Nuclease: The effect of Artificially Introduced Disulfide Bonds on Protein Structure and Stability

Since a protein\u27s function depends on its structure, basic research in protein structure facilitates the solution of many practical problems, such as the synthesis of more effective medicines. With this larger goal in sight, the purpose of this research project is to understand better the chemical principles that underlie protein structure and stability. Disulfide bonds are a potentially stabilizing feature of many proteins. They may form between cysteine residues in close proximity to one another if the orientation is favorable. Often found in proteins produced by organisms that grow at high temperatures, disulfide bonds may anchor side chains together, making a protein resistant to thermal or chemical denaturation. In order to provide a better understanding of the stabilizing effects of disulfide bonds, disulfides are artificially introduced into the protein staphylococcal nuclease to create mutant versions of the protein. Wild-type S. nuclease has no cysteine residues, so disulfide bonds must be engineered by substituting cysteines for pairs of amino acid residues in the wild-type protein. To synthesize these double mutants, successive rounds of site-directed mutagenesis are performed on bacteriophage DNA using the Kunkel method. After transformation with the modified DNA, E. coli bacteria are used to synthesize the mutant proteins for analysis. Biophysical techniques such as solvent and thermal denaturation provide essential thermodynamic data for characterizing the stabilities of the mutants. On the basis of the data obtained from the S. nuclease mutants, generalized predictions about protein structure and stability can be established

Medicine and Health Care in Later Medieval Europe: Hospitals, Public Health,, and Minority Medical Practitioners in English and German Cities, 1250-1450

Hospitals and individual caregivers helped meet the physical and psychological needs of medieval people, just as they do today. My overall objective is to explain social and individual responses to disease within the context of Christian theology and the urban community, focusing on England and Germany in the period between 1250 and 1450. First I investigate social responses to disease, including hospitals and public health ordinances. Christianity mandated the care of the afflicted, yet physical and mental illness was associated with sin and divine punishment. Urban authorities often attempted to deal with plague outbreaks by imposing quarantines and strict regulations on minorities and outsiders. In addition to these more immediate concerns, the experience of plague permeated every aspect of medieval European culture, from the philosophy of health care to artistic representation. Next I discuss individual encounters with disease, focusing on the ambivalent positions that female and Jewish physicians occupied within the medical profession. Women were perceived as nurturers with natural healing abilities. In spite of restrictions on formal university education, many women trained privately under male physicians. Jewish physicians exerted a considerable influence on the medical profession, even though religious and racial discourse pervaded popular perceptions of their work in the medieval urban community. Yet municipal authorities occasionally engaged the services of Jewish physicians, and Jewish doctors often treated Christian patients. The roles of minority medical practitioners illustrate tolerance as well as prejudice, one aspect of the ambiguity that characterizes medieval views of health care and disease

Are methodological quality and completeness of reporting associated with citation-based measures of publication impact? A secondary analysis of a systematic review of dementia biomarker studies

Objective: To determine whether methodological and reporting quality are associated with surrogate measures of publication impact in the field of dementia biomarker studies. Methods: We assessed dementia biomarker studies included in a previous systematic review in terms of methodological and reporting quality using the Quality Assessment of Diagnostic Accuracy Studies (QUADAS) and Standards for Reporting of Diagnostic Accuracy (STARD), respectively. We extracted additional study and journal-related data from each publication to account for factors shown to be associated with impact in previous research. We explored associations between potential determinants and measures of publication impact in univariable and stepwise multivariable linear regression analyses. Outcome measures: We aimed to collect data on four measures of publication impact: two traditional measures—average number of citations per year and 5-year impact factor of the publishing journal and two alternative measures—the Altmetric Attention Score and counts of electronic downloads. Results: The systematic review included 142 studies. Due to limited data, Altmetric Attention Scores and electronic downloads were excluded from the analysis, leaving traditional metrics as the only analysed outcome measures. We found no relationship between QUADAS and traditional metrics. Citation rates were independently associated with 5-year journal impact factor (β=0.42; p<0.001), journal subject area (β=0.39; p<0.001), number of years since publication (β=-0.29; p<0.001) and STARD (β=0.13; p<0.05). Independent determinants of 5-year journal impact factor were citation rates (β=0.45; p<0.001), statement on conflict of interest (β=0.22; p<0.01) and baseline sample size (β=0.15; p<0.05). Conclusions: Citation rates and 5-year journal impact factor appear to measure different dimensions of impact. Citation rates were weakly associated with completeness of reporting, while neither traditional metric was related to methodological rigour. Our results suggest that high publication usage and journal outlet is not a guarantee of quality and readers should critically appraise all papers regardless of presumed impact

Equivalency and Reciprocity of Qualifications for LIS Professionals in a Web 2.0 Environment

In the age of Web 2.0 and globalization of information, the challenge of information professionals included the determination of equivalent educational experiences as professionals move more freely in the international information environment. Reciprocity of degrees among recognized LIS education program is one possible solution, but the establishment of an international program of reciprocity has been difficult up to this time. The authors discuss the background of efforts over a 30 year period to develop acceptable guidelines for international equivalency and reciprocity of qualifications for LIS professionals by IFLA and other library interests. The challenges of the latest IFLA effort our detailed and options provided in a 2.0 web environment are explored. The possibility that applying the principles of interactivity of the web in the 21st Century to provide a solution to the equivalency and reciprocity problem are analyzed and specific proposal are presented for discussion. The results of surveys of library education professionals are presented and specific proposal for the future are outlined

Barriers to infection of human cells by feline leukemia virus: insights into resistance to zoonosis

The human genome displays a rich fossil record of past gamma-retrovirus infections, yet no current epidemic is evident, despite environmental exposure to viruses that infect human cells in vitro. Feline leukemia viruses (FeLVs) rank high on this list, but domestic or workplace exposure has not been associated with detectable serological responses. Non-specific inactivation of gamma-retroviruses by serum factors appears insufficient to explain these observations. To investigate further we explored the susceptibility of primary and established human cell lines to FeLV-B, the most likely zoonotic variant. Fully permissive infection was common in cancer-derived cell lines, but was also a feature of non-transformed keratinocytes and lung fibroblasts. Cells of haematopoietic origin were less generally permissive and formed discrete groups on the basis of high or low intracellular protein expression and virion release. Potent repression was observed in primary human blood mononuclear cells and a subset of leukemia cell lines. However, the early steps of reverse transcription and integration appear to be unimpaired in non-permissive cells. FeLV-B was subject to G->A hypermutation with a predominant APOBEC3G signature in partially permissive cells but was not mutated in permissive cells or in non-permissive cells that block secondary viral spread. Distinct cellular barriers that protect primary human blood cells are likely to be important in protection against zoonotic infection with FeLV

Quantifying Golgi structure using EM : combining volume-SEM and stereology for higher throughput

John Lucocq was supported by a Programme grant from the Wellcome Trust (Number 045404). Sophie Ferguson was a recipient of a 600th anniversary studentship from the University of St Andrews.Investigating organelles such as the Golgi complex depends increasingly on high-throughput quantitative morphological analyses from multiple experimental or genetic conditions. Light microscopy (LM) has been an effective tool for screening but fails to reveal fine details of Golgi structures such as vesicles, tubules and cisternae. Electron microscopy (EM) has sufficient resolution but traditional transmission EM (TEM) methods are slow and inefficient. Newer volume scanning EM (volume-SEM) methods now have the potential to speed up 3D analysis by automated sectioning and imaging. However, they produce large arrays of sections and/or images, which require labour-intensive 3D reconstruction for quantitation on limited cell numbers. Here, we show that the information storage, digital waste and workload involved in using volume-SEM can be reduced substantially using sampling-based stereology. Using the Golgi as an example, we describe how Golgi populations can be sensed quantitatively using single random slices and how accurate quantitative structural data on Golgi organelles of individual cells can be obtained using only 5–10 sections/images taken from a volume-SEM series (thereby sensing population parameters and cell–cell variability). The approach will be useful in techniques such as correlative LM and EM (CLEM) where small samples of cells are treated and where there may be variable responses. For Golgi study, we outline a series of stereological estimators that are suited to these analyses and suggest workflows, which have the potential to enhance the speed and relevance of data acquisition in volume-SEM.Publisher PDFPeer reviewe

Double Cysteine Mutations in Staphylococcal Nuclease: The effect of Artificially Introduced Disulfide Bonds on Protein Structure and Stability

Since a protein\u27s function depends on its structure, basic research in protein structure facilitates the solution of many practical problems, such as the synthesis of more effective medicines. With this larger goal in sight, the purpose of this research project is to understand better the chemical principles that underlie protein structure and stability. Disulfide bonds are a potentially stabilizing feature of many proteins. They may form between cysteine residues in close proximity to one another if the orientation is favorable. Often found in proteins produced by organisms that grow at high temperatures, disulfide bonds may anchor side chains together, making a protein resistant to thermal or chemical denaturation. In order to provide a better understanding of the stabilizing effects of disulfide bonds, disulfides are artificially introduced into the protein staphylococcal nuclease to create mutant versions of the protein. Wild-type S. nuclease has no cysteine residues, so disulfide bonds must be engineered by substituting cysteines for pairs of amino acid residues in the wild-type protein. To synthesize these double mutants, successive rounds of site-directed mutagenesis are performed on bacteriophage DNA using the Kunkel method. After transformation with the modified DNA, E. coli bacteria are used to synthesize the mutant proteins for analysis. Biophysical techniques such as solvent and thermal denaturation provide essential thermodynamic data for characterizing the stabilities of the mutants. On the basis of the data obtained from the S. nuclease mutants, generalized predictions about protein structure and stability can be established

Challenges in lifestyle and community interventions research

Earlier this year the BMC portfolio was enriched by a new journal BMC Obesity. Here, we present the aims and objectives of the section on Lifestyle and Community Interventions. Innovative research is needed. Preventing or managing obesity requires addressing different determinants across multiple levels where diverse levers and stakeholders can play a critical role. Interactions of these determinants within and between systems need to be studied. How to leverage, manage and measure this complexity underlies the innovation that is needed in the next generation of obesity interventions. The ambition of the Lifestyle and Community Interventions section is to provide a space for innovative research, including research that falls outside the traditional comfort zone. We welcome studies of heterogeneous designs, including those of qualitative, quantitative, mixed and systems methodologies. Studies of interest include not only outcomes research of interventions but also process evaluation, cost effectiveness or cost-benefit analysis, and implementation and dissemination research. Innovations that integrate diverse intervention levers or combine primary and secondary levels of prevention are particularly encouraged. The general aim of BMC Obesity’ s Lifestyle and Community Interventions section is to advance our ability to decide on what combinations of approaches will be required to effectively and equitably prevent obesity

Doing gender locally: The importance of ‘place’ in understanding marginalised masculinities and young men’s transitions to ‘safe’ and successful futures

Observable anxieties have been developing about the position of boys and young men in contemporary society in recent years. This is expressed as a crisis of masculinity, in which place is often implicitly implicated, but is rarely considered for its role in the shaping of young men’s practices, trajectories and aspirations. Drawing on research conducted with young people who accessed a range of social care support services, this article argues that transition means different things for young men in different locales and that local definitions of masculinity are required to better understand young men’s lives and the opportunities available to them. The authors argue that home life, street life, individual neighbourhoods, regions and nations all shaped the young men’s identities and the practices they (and the staff working with them) drew on in order to create successful futures and ‘safe’ forms of masculinity. It is suggested that this place-based approach has the potential to re-shape the ‘crisis’ discourse surrounding masculinity and the anxieties associated with young men

A proposed music education curriculum for the small college based on a survey of curriculums in selected colleges

Thesis (Ph.D.)--Boston UniversityThe purposes of the study were (1) to analyze and evaluate present practices in the education of school music teachers; {2) to discover any differences that might exist between large and small colleges; (3) to determine any advantages that might characterize the small college; (4) to propose a music education curriculum~ in the light of data derived from a questionnaire and other sources~ for a small college with enrollment up to 2,000 students. Of the five hundred and twenty-five distributed questionnaires, two hundred and eight served as the source of primary data for this study. The responding institutions were classified as conservatories, liberal arts colleges, universities, and teachers colleges, located in forty-two states under both private and public control, and limited to enrollment of 2,000 students. Supplementary data were supplied through examination of one hundred catalogues; through conferences with thirty-five music educators and supervisors of public school music; and an informal inquiry sheet as to musical readiness in five hundred high school seniors. The data from the questionnaire analyzed and evaluated in terms of the National Music Association, the American Association of Teachers Colleges, Music Educators National Conference, and Music Teachers National Association, were tabulated according to the main headings of the questionnaire . Appropriate tabulation and outline presentation were made of the supplementary data. The proposed curriculum, including as an innovation, a special course in musical experiences and incorporating pertinent suggestions from all aforementioned sources and from the literature reviewed, was presented in outline form with explanatory tabulations. [TRUNCATED