Yeast NPI46 encodes a novel prolyl cis-trans isomerase that is located in the nucleolus

We have identified a gene (NPI46) encoding a new prolyl cis-trans isomerase within the nucleolus of the yeast Saccharomyces cerevisiae. The protein encoded by NPI46 was originally found by us in a search for proteins that recognize nuclear localization sequences (NLSs) in vitro. Thus, NPI46 binds to affinity columns that contain a wild-type histone H2B NLS but not a mutant H2B NLS that is incompetent for nuclear localization in vivo. NPI46 has two domains, a highly charged NH2 terminus similar to two other mammalian nucleolar proteins, nucleolin and Nopp140, and a COOH terminus with 45% homology to a family of mammalian and yeast proline isomerases. NPI46 is capable of catalyzing the prolyl cis-trans isomerization of two small synthetic peptides, succinyl-Ala-Leu-Pro-Phe-p-nitroanilide and succinyl-Ala-Ala-Pro-Phe-p- nitroanilide, as measured by a chymotrypsin-coupled spectrophotometric assay. By indirect immunofluorescence we have shown that NPI46 is a nucleolar protein. NPI46 is not essential for cell viability.     

Making British cortisone: Glaxo and the development of corticosteroids in Britain in the 1950s-1960s

Following the announcement in 1949 in the USA that cortisone offered rheumatoid arthritis sufferers effective treatment for their crippling disease, the Ministry of Health came under considerable pressure from the medical profession and the public to make cortisone available in Britain. The Ministry, therefore, urged British companies to start manufacturing cortisone. Among the several pharmaceutical firms responding to the Ministry's request, Glaxo's expertise in the field of vitamins gave them a head start. This paper describes the varied and flexible strategy that enabled Glaxo to maintain this head start, and the scientific and technical capabilities which the company subsequently built up, enabling them to dominate the market for corticosteroids in Britain. Among the drugs to emerge out of the Glaxo project to manufacture cortisone, which began in 1950 and later became a wider R&D programme on steroids, was the topical steroid Betnovate, launched in 1963, which remains a best-seller today. However, although it led to successful new products, Glaxo's programme had limitations. The paper identifies a missed opportunity, in the shape of the biosynthetic route to steroid drugs, often considered as a milestone in the development of the new biotechnology. Whether or not this missed opportunity proved costly to the company is uncertain. However, it illustrates the role of technological path-dependence, and the importance of the integration between different scientific disciplines, in this case chemistry and biology, in pharmaceutical innovation.     

Making British Cortisone: Glaxo and the development of Corticosteroids in Britain in the 1950s–1960s

Following the announcement in 1949 in the USA that cortisone offered rheumatoid arthritis sufferers effective treatment for their crippling disease, the Ministry of Health came under considerable pressure from the medical profession and the public to make cortisone available in Britain. The Ministry, therefore, urged British companies to start manufacturing cortisone. Among the several pharmaceutical firms responding to the Ministry’s request, Glaxo’s expertise in the field of vitamins gave them a head start. This paper describes the varied and flexible strategy that enabled Glaxo to maintain this head start, and the scientific and technical capabilities which the company subsequently built up, enabling them to dominate the market for corticosteroids in Britain.Among the drugs to emerge out of the Glaxo project to manufacture cortisone, which began in 1950 and later became a wider R&D programme on steroids, was the topical steroid Betnovate, launched in 1963, which remains a best-seller today. However, although it led to successful new products, Glaxo’s programme had limitations. The paper identifies a missed opportunity, in the shape of the biosynthetic route to steroid drugs, often considered as a milestone in the development of the new biotechnology. Whether or not this missed opportunity proved costly to the company is uncertain. However, it illustrates the role of technological path-dependence, and the importance of the integration between different scientific disciplines, in this case chemistry and biology, in pharmaceutical innovation.     

Neuropsychiatric symptoms of dementia: psychometric aspects of the Dutch Neuropsychiatric Inventory (NPI)

Behavioral and psychological symptoms are highly prevalent in dementia. The Neuropsychiatric Inventory was constructed to measure these symptoms. Data from three studies are presented concerning psychometric aspects of the NPI Dutch version. The NPI was compared to the Revised Memory and Behavioral Problems Checklist (RMBPC) and the Mini Mental State Examination (MMSE). In the three selected patient samples prevalence of behavioral or psychological symptoms was as high as 90%. Interrater agreement (n = 19) was very high (kappa > .90). Factor analysis (n = 199) supports NPI construct validity. The NPI items correlated reasonably high (R = .35 - .60) with the relevant RMBPC subscales (n = 24). Although some NPI items did, the NPI total score was not significantly related to the MMSE. The NPI Dutch version can be scored objectively and it is a valid rating scale for measuring a wide range of behavioral and psychological symptoms of dementia.     

新生猪胰岛保存运输的实验研究

探索新生猪胰岛较长时间有效保存运输的方法,使不同胰岛应用单位间分享胰岛资源成为可能.用胶原酶消化、分离得到新生猪胰岛,培养5 d后收集,将所得胰岛置于自行设计制作的保存运输系统内,分别于置入后2、4、5、7 h,分次取出胰岛进行双硫腙(DTZ)染色、吖啶橙荧光染色、体外葡萄糖刺激胰岛素分泌实验,确定胰岛细胞的功能和活性,并使用流式细胞仪检测凋亡情况.使用本研究的保存运输方法,在5 h以内,胰岛的功能和活性无明显受损,超过5 h,胰岛明显凋亡,功能、活性显著降低.在脱离培养箱环境情况下,本方法5 h内可以有效保存胰岛.     

Automated site-directed drug design: a general algorithm for knowledge acquisition about hydrogen-bonding regions at protein surfaces

This is the first of four papers that begin to explore the possibility of automated site-directed drug design. A general outline is given of the logical steps involved in approaching the problem. The starting point is the process of knowledge acquisition about the site. An algorithm is described here for the construction of a map of hydrogen-bonding regions at protein surfaces directly from the Brookhaven Protein Data Bank coordinates. Hydrogen-bonding atoms are located, intramolecular bonds are searched for, hydrogen-bonding atoms at the surface are found and hydrogen-bonding regions are computed at the accessible surface. A grid is placed within each region discovered and the probability of hydrogen bonding at each grid point is computed. The output of the program is a map of hydrogen-bonding regions displayed within a user-defined window. This information can be used as part of a knowledge base for the automatic construction of novel ligands to fit specified binding sites.     

Molecular dynamics in a grid computing environment: experiences using DL_POLY_3 within the eMinerals escience project

We use the example of a study of the compressibility anomaly in amorphous silica to illustrate how molecular-scale simulations can be performed using grid computing. The potential for running many simulations within a single study requires the use of new data management methods, which are discussed in this paper. The example of silica highlights the advantages of the use of grid computing for studying subtle effects.     

Increasing Precision in Greenhouse Gas Accounting Using Real‐Time Emission Factors

For many companies, the greenhouse gas (GHG) emissions associated with their purchased and consumed electricity form one of the largest contributions to the GHG emissions that result from their activities. Currently, hourly variations in electricity grid emissions are not considered by standard GHG accounting protocols, which apply a national grid emission factor (EF), potentially resulting in erred estimates for the GHG emissions. In this study, a method is developed that calculates GHG emissions based on real‐time data, and it is shown that the use of hourly electricity grid EFs can significantly improve the accuracy of the GHG emissions that are attributed to the purchased and consumed electricity of a company. A model analysis for the electricity delivered to the Spanish grid in 2012 reveals that, for companies operating during the day, GHG emissions calculated by the real‐time method are estimated to be up to 5% higher (and in some special cases up to 9% higher) than the emissions calculated by the conventional method in which a national grid EF is applied, whereas for companies operating during nightly hours, GHG emissions are estimated to be as low as 3% below the GHG emissions determined by the conventional method. A significant error can therefore occur in the organizational carbon footprint (CF) of a company and, consequently, also in the product CF. It is recommended that hourly EFs be developed for other countries and power grids.     

Metastable states, phase transitions, and persistent neural activity

A new mathematical model to describe the spiking rate of a neural population is derived, which considers both the mean and the variance of the activity. Bifurcation analysis identifies a critical interval of parameter values in which the standard bistability regime coexists with an additional third attractor corresponding to the metastable state of bounded mean activity and high variance. To understand the structure of spatio-temporal activity in the metastable state, we study a simple discrete-time model of binary elements with random noise locally coupled on the grid, which produces rich dynamics including metastability. A critical value of the noise amplitude is identified; in the vicinity of this value the system is flexible and can easily generate transitions between UP and DOWN metastable states, either autonomously or in response to a control process. These metastable states and phase transitions provide a proper basis for the modelling of persistent neural activity reported in many experimental studies.     

NPI1, an essential yeast gene involved in induced degradation of Gap1 and Fur4 permeases, encodes the Rsp5 ubiquitin—protein ligase

When yeast cells growing on a poor nitrogen source are supplied with NH₄⁺ ions, several nitrogen permeases including the general amino acid permease (Gap1p) are rapidly and completely inactivated. This report shows that inactivation by NH₄⁺ of the Gap1 permease is accompanied by its degradation. A functional NPI1 gene product is required for both inactivation and degradation of Gap1p. Molecular analysis of the NPI1 gene showed that it is identical to RSP5 . The RSP5 product is a ubiquitin—protein ligase (E3 enzyme) whose physiological function was, however, unknown. Its C-terminal region is very similar to that of other members of the E6-AP-like family of ubiquitin-protein ligases. Its N-terminal region contains a single C₂ domain that may be a Ca²⁺-dependent phospholipid interaction motif, followed by several copies of a recently identified domain called WW(P). The Npi1/Rsp5 protein has a homologue both in humans and in mice, the latter being involved in brain development. Stress-induced degradation of the uracil permease (Fur4p), a process in which ubiquitin is probably involved, was also found to require a functional NPI1/RSP5 product. Chromosomal deletion of NPI1/RSP5 showed that this gene is essential for cell viability. In the viable np1/rsp5 strain, expression of NPI1/RSP5 is reduced as a result of insertion of a Ty1 element in its 5' region. Our results show that the Npi1/Rsp5 ubiquitin-protein ligase participates in induced degradation of at least two permeases, Gap1p and Fur4p, and probably also other proteins.     

Improving the Prognostic Ability through Better Use of Standard Clinical Data - The Nottingham Prognostic Index as an Example

Background

Prognostic factors and prognostic models play a key role in medical research and patient management. The Nottingham Prognostic Index (NPI) is a well-established prognostic classification scheme for patients with breast cancer. In a very simple way, it combines the information from tumor size, lymph node stage and tumor grade. For the resulting index cutpoints are proposed to classify it into three to six groups with different prognosis. As not all prognostic information from the three and other standard factors is used, we will consider improvement of the prognostic ability using suitable analysis approaches.

Methods and Findings

Reanalyzing overall survival data of 1560 patients from a clinical database by using multivariable fractional polynomials and further modern statistical methods we illustrate suitable multivariable modelling and methods to derive and assess the prognostic ability of an index. Using a REMARK type profile we summarize relevant steps of the analysis. Adding the information from hormonal receptor status and using the full information from the three NPI components, specifically concerning the number of positive lymph nodes, an extended NPI with improved prognostic ability is derived.

Conclusions

The prognostic ability of even one of the best established prognostic index in medicine can be improved by using suitable statistical methodology to extract the full information from standard clinical data. This extended version of the NPI can serve as a benchmark to assess the added value of new information, ranging from a new single clinical marker to a derived index from omics data. An established benchmark would also help to harmonize the statistical analyses of such studies and protect against the propagation of many false promises concerning the prognostic value of new measurements. Statistical methods used are generally available and can be used for similar analyses in other diseases.     

The importance of ecological research for ecosystem management: The case of browsing by swamp wallabies (Wallabia bicolor) in commercially harvested native forests

Summary Ecosystem management often proceeds within the context of sub‐optimal relationships between ecologists and ecosystem managers, and management outcomes could be improved with greater collaboration between members of these disciplines. This paper identifies an ecosystem management problem resulting from the interaction between timber harvesting and browsing wallabies, and this case study is used to exemplify how ecological data and expertise can contribute to the process of ecosystem management. It is argued that appropriate use of existing ecological data, establishment of strategic new research and the implementation of management actions as experimental hypothesis tests can facilitate achievement of management objectives, but greater collaboration between ecologists and managers is required before this can occur. Reasons for sub‐optimal relationships are outlined, and the potential for structural change within large State‐run ecosystem management agencies to improve interactions between managers and ecologists is discussed.     

Setting priorities for possum control at a regional level

Abstract

Because funds for possum control are always insufficient to carry out control in all the areas that may require it, it is important that the funds available are allocated to those areas that most deserve it. This can be achieved only by implementing a process that first identifies the resource types likely to be degraded by possums, and secondly ranks areas for control within each resource type. If necessary, high priority areas between resource types can also be ranked. A method for undertaking such a process is described.     

The pleated sheet: an unusual negative-staining method for transmission electron microscopy of biological macromolecules

A novel method for preparing negatively stained specimens is described which appears to improve the routine resolution of biological structure in direct images obtained by transmission electron microscopy. In the new method, which we term the pleated sheet technique, macromolecules are adsorbed to a carbon film by the Valentine procedure (R. Valentine, B. Shapiro, and E. Stadtman (1968) Biochemistry, 7, 2143-2152), and the film then carefully pleated while in contact with a 1% uranyl formate solution to trap stain within the folds of pleats. A grid is placed on the compressed film, and film plus grid retrieved with a Saran Wrap drum. Subsequent dehydration produces a filmed grid containing negatively stained macromolecules within the folds of pleated regions and positively stained macromolecules in single sheet regions. The effect of sandwiching sample and stain between carbon layers is to produce exceedingly uniform negative staining so that stain contours more accurately and more reproducibly reflect true molecular contours. Electron micrographs of IgG and IgA molecules prepared by these methods are exhibited that permit unambiguous comparison of structure imaged in the electron microscope against known structures solved by single-crystal X-ray diffraction. Correlation is excellent; the smallest resolvable element in micrographs is an immunoglobulin domain, whose molecular weight is 12 000.     

A framework for increasing the availability of life cycle inventory data based on the role of multinational companies

Purpose

The aim of the paper is to assess the role and effectiveness of a proposed novel strategy for Life Cycle Inventory (LCI) data collection in the food sector and associated supply chains. The study represents one of the first of its type and provides answers to some of the key questions regarding the data collection process developed, managed and implemented by a multinational food company across the supply chain.

Methods

An integrated LCI data collection process for confectionery products was developed and implemented by Nestlé, a multinational food company. Some of the key features includes (1) management and implementation by a multinational food company; (2) types of roles to manage, provide and facilitate data exchange; (3) procedures to identify key products, suppliers and customers; (4) LCI questionnaire and cover letter and (5) data quality management based on the pedigree matrix. Overall, the combined features in an integrated framework provide a new way of thinking about the collection of LCI data from the perspective of a multinational food company.

Results and discussion

The integrated LCI collection framework spanned across 5 months and resulted in 87 new LCI datasets for confectionery products from raw material, primary resource use, emission and waste release data collected from suppliers across 19 countries. The data collected was found to be of medium to high quality compared with secondary data. However, for retailers and waste service companies, only partially completed questionnaires were returned. Some of the key challenges encountered during the collection and creation of data included lack of experience, identifying key actors, communication and technical language, commercial compromise, confidentiality protection and complexity of multi-tiered supplier systems. A range of recommendations are proposed to reconcile these challenges which include standardisation of environmental data from suppliers, concise and targeted LCI questionnaires and visualising complexity through drawings.

Conclusions

The integrated LCI data collection process and strategy has demonstrated the potential role of a multinational company to quickly engage and act as a strong enabler to unlock latent data for various aspects of the confectionery supply chain. Overall, it is recommended that the research findings serve as the foundations to transition towards a standardised procedure which can practically guide other multinational companies to considerably increase the availability of LCI data.

     

Comprehensive mutagenesis of the C-terminal domain of the M13 gene-3 minor coat protein: the requirements for assembly into the bacteriophage particle

Filamentous bacteriophage assemble at the host membrane in a non-lytic process; the gene-3 minor coat protein (P3) is required for release from the membrane and subsequently, for recognition and infection of a new host. P3 contains at least three distinct domains: two N-terminal domains that mediate host recognition and infection, and a C-terminal domain (P3-C) that is required for release from the host cell following phage assembly and contributes to the structural stability of the phage particle. A comprehensive mutational analysis of the 150 residue P3-C revealed that only 24 side-chains, located within the last 70 residues of sequence, were necessary for efficient incorporation into a wild-type coat. The results reveal that the requirements for the assembly of P3 into the phage particle are quite lax and involve only a few key side-chains. These findings shed light on the functional and structural requirements for filamentous phage assembly, and they may provide guidelines for the engineering of improved coat proteins as scaffolds for phage display technology.     

Guidelines for establishing a culture collection within a biotechnology company

Culture collections within large pharmaceutical and biological companies act as depositories for preserving and maintaining important strains owned by, on the premises of, or sought on behalf of, the company. Other functions of a culture collection include: (a) regulating the flow of cultures into and out of the company; (b) recording and documenting such transfers; and (c) characterizing and/or identifying strains used for research, publications and patents. A culture collection within a biotechnology company is often required to maintain a diverse group of both prokaryote and eukaryote recombinant and non-recombinant strains. All strains and plasmids must be carefully characterized and preserved. A microbiologist with a strong background in microbial physiology, genetics and taxonomy is usually responsible for supervising the culture collection. This article focuses on guidelines for establishing a culture collection in biotechnology companies to serve the needs of both the scientist and the company.     

Identifying the rooted species tree from the distribution of unrooted gene trees under the coalescent

Gene trees are evolutionary trees representing the ancestry of genes sampled from multiple populations. Species trees represent populations of individuals—each with many genes—splitting into new populations or species. The coalescent process, which models ancestry of gene copies within populations, is often used to model the probability distribution of gene trees given a fixed species tree. This multispecies coalescent model provides a framework for phylogeneticists to infer species trees from gene trees using maximum likelihood or Bayesian approaches. Because the coalescent models a branching process over time, all trees are typically assumed to be rooted in this setting. Often, however, gene trees inferred by traditional phylogenetic methods are unrooted. We investigate probabilities of unrooted gene trees under the multispecies coalescent model. We show that when there are four species with one gene sampled per species, the distribution of unrooted gene tree topologies identifies the unrooted species tree topology and some, but not all, information in the species tree edges (branch lengths). The location of the root on the species tree is not identifiable in this situation. However, for 5 or more species with one gene sampled per species, we show that the distribution of unrooted gene tree topologies identifies the rooted species tree topology and all its internal branch lengths. The length of any pendant branch leading to a leaf of the species tree is also identifiable for any species from which more than one gene is sampled.     

Basic faced alpha-helices are widespread in the peptide extensions of the eukaryotic aminoacyl-tRNA synthetases

Three aminoacyl-tRNA synthetases from yeast, one from plants and one from mammals possess unusual structures at their N termini, namely alpha helices with basic residues distributed asymmetrically, on a single face of the helix. It is unknown if these 'basic faced' alpha helices (BFAHs) are unique to the aminoacyl-tRNA synthetases. Analysis of the amino acid sequences of these five aminoacyl-tRNA synthetases using the hydrophobic moment algorithm failed to accurately identify the BFAHs. A new algorithm was therefore developed, called the 'basic moment'. This is a Fourier analysis procedure that predicts the distribution of basic residues within protein secondary structure. The basic moment identifies with a high degree of accuracy the five known BFAHs and also identifies further potential BFAHs at evolutionarily conserved positions in the peptide extensions of aspartyl-, lysyl- and valyl- tRNA synthetases from a range of eukaryotic species. In addition, the algorithm identifies the two-helix pair tRNA binding domain of alanyl-tRNA synthetase, implying that the domain includes a BFAH. The functional and evolutionary aspects of these structural features are discussed.