Single particle reconstructions of the transferrin-transferrin receptor complex obtained with different specimen preparation techniques

The outcome of three-dimensional (3D) reconstructions in single particle electron microscopy (EM) depends on a number of parameters. We have used the well-characterized structure of the transferrin (Tf)-transferrin receptor (TfR) complex to study how specimen preparation techniques influence the outcome of single particle EM reconstructions. The Tf-TfR complex is small (290kDa) and of low symmetry (2-fold). Angular reconstitution from images of vitrified specimens does not reliably converge on the correct structure. Random conical tilt reconstructions from negatively stained specimens are reliable, but show variable degrees of artifacts depending on the negative staining protocol. Alignment of class averages from vitrified specimens to a 3D negative stain reference model using FREALIGN largely eliminated artifacts in the resulting 3D maps, but not completely. Our results stress the need for critical evaluation of structures determined by single particle EM.     

Cryo-Electron Microscopy Reconstruction Shows Poliovirus 135S Particles Poised for Membrane Interaction and RNA Release

During infection, binding of mature poliovirus to cell surface receptors induces an irreversible expansion of the capsid, to form an infectious cell-entry intermediate particle that sediments at 135S. In these expanded virions, the major capsid proteins (VP1 to VP3) adopt an altered icosahedral arrangement to open holes in the capsid at 2-fold and quasi-3-fold axes, and internal polypeptides VP4 and the N terminus of VP1, which can bind membranes, become externalized. Cryo-electron microscopy images for 117,330 particles were collected using Leginon and reconstructed using FREALIGN. Improved rigid-body positioning of major capsid proteins established reliably which polypeptide segments become disordered or rearranged. The virus-to-135S transition includes expansion of 4%, rearrangements of the GH loops of VP3 and VP1, and disordering of C-terminal extensions of VP1 and VP2. The N terminus of VP1 rearranges to become externalized near its quasi-3-fold exit, binds to rearranged GH loops of VP3 and VP1, and attaches to the top surface of VP2. These details improve our understanding of subsequent stages of infection, including endocytosis and RNA transfer into the cytoplasm.     

Experience of an interactive program in undergraduate investigative practicals

A microcomputer program is described which has been used by first-year undergraduates throughout an investigation in microbiology, totalling five hours of practical work. The computer is used by students to check their suggestions of possible lines of investigation, the results of practical procedures, and the decisions made on the basis of this experimental evidence. The program offers advice when entries which are inadvisable or incorrect are entered thus providing guidance without usurping student control of the investigation. Evidence for the effectiveness of this program is presented and the potential for this type of consultative program in other areas is indicated. Based on this experience, advice is offered on the general design of this type of program.     

The STRESS program: A computer program for the analysis of stresses in long bones

A computer program was developed to compute torsional and bending stresses in long bones. The input format for the program is designed to be as convenient to the user as possible, allowing him to transmit to the program only as many points as he feels are required to describe the cross-sectional geometry. The program permits the user to specify by which method torsional stresses are to be computed, what type of stresses are to be computed, and to what accuracy the results are desired. Computation times are of the order of 5 sec or less (on an IBM 360), making use of the program more economical than manual computations.     

Computer treatment of gas-liquid chromatographic data, with special reference to fatty acid methyl esters

A computer program is described which analyzes output punched directly onto paper tape from a gas-liquid chromatograph. Although this program was written specifically for samples of fatty acid methyl esters derived from adipose tissue triglycerides which are eluted within 1 hr, modification of the dimension statements in the program would enable it to deal with samples which require a longer time to come off the column. The salient features of the rationale of the program are discussed in detail, particularly the procedures for base line correction and for estimating the contributions from components which are not perfectly separated in the column. Examples are given of the program in practice, of comparing the results it gives with those obtained by manual triangulation of the areas on a recorder chart, and of indicating the range of column load over which we have found that it operates satisfactorily. A sample computer print-out from the program is presented and interpreted.     

Desert Survivors: the design and implementation of a television program to enhance local scientific literacy

Outreach efforts by faculty members are oftentimes limited in scope due to hectic schedules. We developed a program to enhance science literacy in elementary school children that allows experts to reach a tremendous audience while minimizing their time commitment. The foundation of the program is a television series entitled "Desert Survivors." The episodes air on local cable access television and are available to teachers on DVD. Each episode features a guest expert who spotlights a particular organism and how that organism overcomes the myriad of hardships inherent to desert survival. Local classrooms are visited to solicit questions from students regarding the organism of interest. These videotaped questions are integrated into Desert Survivors television production and provide the guest expert with the basis to discuss the ecology, physiology, and evolutionary biology of the organism. The program is bolstered through the use of an interactive website. Assessment strategies are in place to ensure program efficacy. Herein, we describe the development of the program as a model for innovative outreach opportunities.     

Toward implementation of a regional quality assurance program in cytopathology: the Hong Kong experience

OBJECTIVE: To develop a local quality assurance program in cytopathology based on circulation of patient specimens on glass slides, with limited resources. STUDY DESIGN: A working group was set up for design and running of the program. Participation is on a laboratory basis. The scope and frequency of testing are defined. Well-documented cases (including gynecologic, nongynecologic and fine needle aspiration cytology) with commonly encountered diagnoses are collected. Consensus concerning the diagnosis, interpretive menu and scoring system is sought before the actual slide circulations using express mail. After returning their answers to the program organizer, the participating laboratories receive immediate feedback on their scores, with reference answers, explanatory notes, "whole-mount" images of glass slides and cumulative responses of peer laboratories for on-site checking. At the end of each year, an electronic file containing representative photomicrographs of all cases examined is provided to individual laboratories for their permanent records and training purposes. RESULTS: The program was launched in mid-2003. There were 24 and 27 participating laboratories from Hong Kong (and Macau) in 2003 and 2004, respectively. To date, >150 well-documented cytology cases are available in the slide pool and ready for circulation. As the revenue is mainly to cover the expenses of express mail, the program can be carried out at a relatively low cost. CONCLUSION: In order to have any cytology quality assurance program accepted by local laboratories, it has to be fair and practical. Strict confidentiality needs to be observed throughout the process. This program emphasizes both performance assessment and educational value. Adequate representation from experienced local cytology workers, detailed documentation support from authorities and assistance from dedicated staff are essential to the success of any external proficiency testing scheme. Regular review and evaluation are also necessary for continuous improvement. The Hong Kong experience can serve as an example of running a glass slide-based cytology quality assurance program in a small region with limited resources.     

A computer program for the implicit regression of the Hill equation]

A computer program for implicit regression of the Hill equation. The fundamentals for the regression of implicit functions following the Gauss method are dealt with. Basing on these fundamentals a program for the regression of the Hill equation is described. The binding constant, the Hill coefficient and end extinction can be estimated. The mean errors of parameters were calculated in the linear model. The program was written in ALGOL 60 for the computer Robotron 300. The program is available on request.     

Microcomputer experience in analysis of flow cytometric DNA distributions

The program described analyses DNA histograms obtained in flow cytometry using the Gaussians method. The program is written in BASIC to run on a low-cost microcomputer. It utilizes a simple strategy to obtain good estimates of the parameters required for reducing the problem to a task solvable with linear least-squares methods. Features of the program are flexibility, since it is possible to choose different options for parametrization and spacing of Gaussians, and the fact that the operator is not required to provide interactive inspection or inputting parameter values. The capability and velocity of the program, in all its options, are tested and compared on a series of different (not computer-simulated) histograms obtained in our flow cytometry laboratory. Our results suggest that a fresh approach to parametrization may be useful.     

Present state and possibilities for improvement of cancer prevention and early detection in the Osijek-Baranya county

Cancer morbidity and mortality are on a steady increase in Croatia. Technologic possibilities for appropriate management are available for four cancer sites, i.e. cancer of the breast, cervix uteri, colorectum and prostate, and include cancer prevention and early detection in individuals yet free from manifest signs of the disease. The magnitude of the problem, the experience acquired to date, health care personnel available, and additional resources required to launch a systematic program of early detection of the disease are presented. The program should be initially launched in a county with greatest experience in early detection of cancer, where health care service is ready to immediately start its implementation. The role of family physician, gynecologic service at primary health care level, and polyclinic-consultation hospital service in program implementation is described. The following three possible options for early detection of cancer are analyzed and proposed: minimal program (early detection every 3 years), medium program (the same individuals examined every 2 years), and optimal program proposed by the American Cancer Society and other national and international organizations.     

CARd: Carbon distribution analysis program for protein sequences

Carbon distribution is responsible for stability and structure of proteins. Arrangement of carbon along the protein sequence is depends on how the amino acids are organized and is guided by mRNAs. An atomic level revision is important for understanding these codes. This will ultimately help in identification of disorders and suggest mutations. For this purpose a carbon distribution analysis program has been developed. This program captures the hydrophobic / hydrophilic / disordered regions in a protein. The program gives accurate results. The calculations are precise and sensitive to single amino acid resolution. This program is to help in mutational studies leading to protein stabilisation.     

Cost considerations for long-term ecological monitoring

For an ecological monitoring program to be successful over the long-term, the perceived benefits of the information must justify the cost. Financial limitations will always restrict the scope of a monitoring program, hence the program’s focus must be carefully prioritized. Clearly identifying the costs and benefits of a program will assist in this prioritization process, but this is easier said than done. Frequently, the true costs of monitoring are not recognized and are, therefore, underestimated. Benefits are rarely evaluated, because they are difficult to quantify. The intent of this review is to assist the designers and managers of long-term ecological monitoring programs by providing a general framework for building and operating a cost-effective program. Previous considerations of monitoring costs have focused on sampling design optimization. We present cost considerations of monitoring in a broader context. We explore monitoring costs, including both budgetary costs, what dollars are spent on, and economic costs, which include opportunity costs. Often, the largest portion of a monitoring program budget is spent on data collection, and other, critical aspects of the program, such as scientific oversight, training, data management, quality assurance, and reporting, are neglected. Recognizing and budgeting for all program costs is therefore a key factor in a program’s longevity. The close relationship between statistical issues and cost is discussed, highlighting the importance of sampling design, replication and power, and comparing the costs of alternative designs through pilot studies and simulation modeling. A monitoring program development process that includes explicit checkpoints for considering costs is presented. The first checkpoint occurs during the setting of objectives and during sampling design optimization. The last checkpoint occurs once the basic shape of the program is known, and the costs and benefits, or alternatively the cost-effectiveness, of each program element can be evaluated. Moving into the implementation phase without careful evaluation of costs and benefits is risky because if costs are later found to exceed benefits, the program will fail. The costs of development, which can be quite high, will have been largely wasted. Realistic expectations of costs and benefits will help ensure that monitoring programs survive the early, turbulent stages of development and the challenges posed by fluctuating budgets during implementation.     

LINKER: a program to generate linker sequences for fusion proteins

The construction of functional fusion proteins often requires a linker sequence that adopts an extended conformation to allow for maximal flexibility. Linker sequences are generally selected based on intuition. Without a reliable selection criterion, the design of such linkers is often difficult, particularly in situations where longer linker sequences are required. Here we describe a program called LINKER which can automatically generate a set of linker sequences that are known to adopt extended conformations as determined by X-ray crystallography and NMR. The only required input to the program is the desired linker sequence length. The program is specifically designed to assist in fusion protein construction. A number of optional input parameters have been incorporated so that users are able to enhance sequence selection based on specific applications. The program output simply contains a set of sequences with a specified length. This program should be a useful tool in both the biotechnology industry and biomedical research. It can be accessed through the Web page http://www.fccc. edu/research/labs/feng/linker.html.     

A flexible new computer program for handling DNA sequence data.

A compact new computer program for handling nucleic acid sequence data is presented. It consists of a number of different subsets, which may be used according to a given code system. The program is designed for the determination of restriction enzyme and other recognition sites in correlation with translation patterns, and allows tabulation of codon frequencies and protein molecular weights within specified gene boundaries. The program is especially designed for detection of overlapping genes. The language, is FORTRAN and thus the program may be used on small computers; it may also be used without any prior computer experience. Copies are available on request.     

TEFOOL/2: a program for theoretical drug design on microcomputers

TEFOOL/2, a program written in BASIC, is presented in this paper.The purpose of TEFOOL12 is to provide people interested in drugdesign with an easy-to-handle program where some of the mostimportant techniques in QSAR are included. The program permitsthe selection of the training series, performs regression calculationsand searches for optimum substituents. The latter is achievedby using either a Hansch's strategy or geometrical procedures.The program is interactive and can be implemented on an IBM-PCor compatible microcomputer. Although TEFOOL/2 has been developedfor its application in drug design studies, its great flexibilitymakes it suitable for application to any experimental designor optimization process. Received on November 22, 1988; accepted on March 21, 1989     

PREDITOP: A program for antigenicity prediction

A program (PREDITOP) for predicting the location of antigenic regions (or epitopes) on proteins is described. This program and the associated ones are written in Turbo Pascal and run on IBM-PC compatibles. The program contains 22 normalized scales, corresponding to hydrophilicity, accessibility, flexibility, or secondary structure propensities. New scales are easily implemented. An hydrophobic moment procedure has also been implemented in order to determine amphiphilic helices. The program generates a result file where the values represent a particular physicochemical aspect of the studied protein. PREDITOP can display one or several result files by simple graphical superimposition. Curve combinations can be done by the ADDITIO or MULTIPLI routines which create a new result file by adding or multiplying previously calculated files representing several propensities. The program is useful and efficient for identifying potential antigenic regions in a protein with the aim of raising antibodies against synthesized peptides which cross-react with the native protein.     

The Education and Training of Biology Teachers

A technique for the numerical identification of bacteria using normalized likelihoods calculated from a probabilistic database is described, and the principles of the technique are explained. A simple computer program, which can be used to perform the calculations and identifications on a microcomputer in undergraduate classes, is presented. The program is annotated so that the steps in the calculation can be linked to the equivalent steps in the program to assist in teaching the principles of programming. Specimen results from the program, and examples of how they should be interpreted and explained, are given. It is expected that the program will be of use to teachers of undergraduates to teach the principles of numerical identification and an important use of computers in biology.     

LILLY—A linear least squares curve fitting program for one independent variable

A program was written to perform a linear least squares curve fitting on data. It includes facilities to report the usual statistics and digital plotter output. Seven types of curves are available for fitting the data. Other features of LILLY include provision of facilities for the selection of subsets in different symbols and separate curve fitting for these subsets. The program also provides a confidence region about the fitted line and the prediction interval for data points. Examples of the use of the program are described.     

ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences

A computer program, ARAGORN, identifies tRNA and tmRNA genes. The program employs heuristic algorithms to predict tRNA secondary structure, based on homology with recognized tRNA consensus sequences and ability to form a base-paired cloverleaf. tmRNA genes are identified using a modified version of the BRUCE program. ARAGORN achieves a detection sensitivity of 99% from a set of 1290 eubacterial, eukaryotic and archaeal tRNA genes and detects all complete tmRNA sequences in the tmRNA database, improving on the performance of the BRUCE program. Recently discovered tmRNA genes in the chloroplasts of two species from the ‘green’ algae lineage are detected. The output of the program reports the proposed tRNA secondary structure and, for tmRNA genes, the secondary structure of the tRNA domain, the tmRNA gene sequence, the tag peptide and a list of organisms with matching tmRNA peptide tags.     

Trends and values of ‘Land for Wildlife’ programs for private land conservation

The Land for Wildlife program started in Victoria in 1981 as a voluntary program with the broad aim of supporting landholders in providing habitat for wildlife on their property. The program has since spread across Australia and is implemented in a range of guises, through a variety of governance approaches. This research collected qualitative and quantitative data on Land for Wildlife programs across Australia to conduct the first national review. Data were gathered on changes in program membership to assess different participation trends. In addition, phone interviews with Land for Wildlife coordinators throughout Australia were conducted to explore how the programs are positioned in delivering biodiversity outcomes in a range of different regions. Over 14,000 properties covering 2.3 million ha are currently registered under Land for Wildlife programs. with at least 500,000 ha of habitat managed for conservation. Limited resources present a large challenge faced by a number of programs, with generally low funding and staffing resulting in restricted biodiversity focus and conservation outcomes. We suggest options to enhance the programs and propose future research directions.