The ABRF Proteomics Research Group studies: educational exercises for qualitative and quantitative proteomic analyses

Resource (core) facilities have played an ever-increasing role in furnishing the scientific community with specialized instrumentation and expertise for proteomics experiments in a cost-effective manner. The Proteomics Research Group (PRG) of the Association of Biomolecular Resource Facilities (ABRF) has sponsored a number of research studies designed to enable participants to try new techniques and assess their capabilities relative to other laboratories analyzing the same samples. Presented here are results from three PRG studies representing different samples that are typically analyzed in a core facility, ranging from simple protein identification to targeted analyses, and include intentional challenges to reflect realistic studies. The PRG2008 study compares different strategies for the qualitative characterization of proteins, particularly the utility of complementary methods for characterizing truncated protein forms. The use of different approaches for determining quantitative differences for several target proteins in human plasma was the focus of the PRG2009 study. The PRG2010 study explored different methods for determining specific constituents while identifying unforeseen problems that could account for unanticipated results associated with the different samples, and included (15) N-labeled proteins as an additional challenge. These studies provide a valuable educational resource to research laboratories and core facilities, as well as a mechanism for establishing good laboratory practices.     

Epithermal neutron beams for clinical studies of boron neutron capture therapy: a dosimetric comparison of seven beams

A comparison of seven epithermal neutron beams used in clinical studies of boron neutron capture therapy (BNCT) in Sweden (Studsvik), Finland (Espoo), Czech Republic (ReZ), The Netherlands (Petten) and the U.S. (Brookhaven and Cambridge) was performed to facilitate sharing of preclinical and clinical results. The physical performance of each beam was measured using a common dosimetry method under conditions pertinent to brain irradiations. Neutron fluence and absorbed dose measurements were performed with activation foils and paired ionization chambers on the central axis both in air and in an ellipsoidal water phantom. The overall quality of each beam was assessed by figures of merit determined from the total weighted dose profiles that assumed the presence of boron in tissue. The in-air specific beam contamination from both fast neutrons and gamma rays ranged between 8 and 65 x 10(-11) cGy(w) cm2 for the different beams and the epithermal neutron flux intensities available at the patient position differed by more than a factor of 20 from 0.2-4.3 x 10(9) n cm(-2) s(-1). Percentage depth dose profiles measured in-phantom for the individual photon, thermal and fast-neutron dose components differed only subtly in shape between facilities. Assuming uptake characteristics consistent with the currently used boronated phenylalanine, all the epithermal beams exhibit a useful penetration of 8 cm or greater that is sufficient to irradiate a lesion seated at the brain midline. The performance of the existing facilities will benefit from the introduction of advanced compounds through improved beam penetrability. This could increase by as much as 2 cm for the purest of beams, although the beam intensities generally need to be increased to between 2-5 x 10(9) n cm(-2) s(-1) to maintain manageable irradiation times. These data provide the first consistent measurement of beam performance at the different centers and will enable a preliminary normalization of the calculated patient dosimetry.     

Development of Monte Carlo based real-time treatment planning system with fast calculation algorithm for boron neutron capture therapy

PurposeWe simulated the effect of patient displacement on organ doses in boron neutron capture therapy (BNCT). In addition, we developed a faster calculation algorithm (NCT high-speed) to simulate irradiation more efficiently.MethodsWe simulated dose evaluation for the standard irradiation position (reference position) using a head phantom. Cases were assumed where the patient body is shifted in lateral directions compared to the reference position, as well as in the direction away from the irradiation aperture.For three groups of neutron (thermal, epithermal, and fast), flux distribution using NCT high-speed with a voxelized homogeneous phantom was calculated. The three groups of neutron fluxes were calculated for the same conditions with Monte Carlo code. These calculated results were compared.ResultsIn the evaluations of body movements, there were no significant differences even with shifting up to 9 mm in the lateral directions. However, the dose decreased by about 10% with shifts of 9 mm in a direction away from the irradiation aperture.When comparing both calculations in the phantom surface up to 3 cm, the maximum differences between the fluxes calculated by NCT high-speed with those calculated by Monte Carlo code for thermal neutrons and epithermal neutrons were 10% and 18%, respectively. The time required for NCT high-speed code was about 1/10th compared to Monte Carlo calculation.ConclusionsIn the evaluation, the longitudinal displacement has a considerable effect on the organ doses.We also achieved faster calculation of depth distribution of thermal neutron flux using NCT high-speed calculation code.     

Neutron microscopy. The low-damage imaging of specialized organic materials

It is shown that, insofar as radiation damage is concerned, transmission neutron microscopy using neutrons in the energy range approximately 0.0001-1.0 eV is extremely attractive for the imaging of specialized organic materials. By "specialized organic materials" is meant organic specimens composed entirely of specific isotopes that have been selected on the basis of their favorable properties with regard to radiation damage. In connection with such specimens, it is demonstrated that at a resolution of, for example, 100 A, neutrons will have an advantage over soft X-rays in terms of radiation damage, provided that the inherent (neutron) bright field image contrast turns out to be greater than 10(-5). Suggestions relating to (a) the comprehensive calculation of the radiation damage sustained by specialized organic specimens under slow neutron irradiation, (b) the construction of a theory of image formation in the neutron microscope, (c) the development of neutron lenses/focusing devices, and (d) the development of a brighter neutron source (essential for neutron microscopy) are outlined in some detail. The paper concludes with two appendices, which provide important background material.     

Noninvasive techniques for measuring body elemental composition

In the past 20 yr, in vivo analysis of body elements by neutron activation has become an important tool in medical research. In particular, it provides a much needed means to make quantitative assessments of body composition of human beings in vivo. The data are useful both for basic physiological understanding and for diagnosis and management of a variety of diseases and disorders. This paper traces the development of the in vivo neutron activation technique from basic systems to the present state of the art facilities. A scan of some of the numerous clinical applications that have been made with this technique reveals the broad potentialities of in vivo neutron activation. The paper also considers alternative routes of future development and raises some of the questions now faced in making the technique more widely available to both medical practitioners and medical investigtors. In vivo neutron activation has opened a new era of both clinical diagnosis and therapy evaluation, and investigation into the modeling of body composition. The techniques are new, but it is already clear that considerable strides can be made in increasing accuracy and precision, increasing the number of elements susceptible to measurement, and reducing the dose required for the measurement.     

Neutron microscopy

It is shown that, insofar as radiation damage is concerned, transmission neutron microscopy using neutrons in the energy range ≈0.0001–1.0 eV is extremely attractive for the imaging of specialized organic materials. By “specialized organic materials” is meant organic specimens composed entirely of specific isotopes that have been selected on the basis of their favorable properties with regard to radiation damage. In connection with such specimens, it is demonstrated that at a resolution of, for example, 100 Å, neutrons will have an advantage over soft X-rays in terms of radiation damage, provided that the inherent (neutron) bright field image contrast turns out to be greater than 10^?5. Suggestions relating to (a) the comprehensive calculation of the radiation damage sustained by specialized organic specimens under slow neutron irradiation, (b) the construction of a theory of image formation in the neutron microscope, (c) the development of neutron lenses/focusing devices, and (d) the development of a brighter neutron source (essential for neutron microscopy) are outlined in some detail. The paper concludes with two appendices, which provide important background material.     

Research at the European Synchrotron Radiation Facility medical beamline.

The application of synchrotron radiation in medical research has become a mature field of research at synchrotron facilities worldwide. In the relatively short time that synchrotrons have been available to the scientific community, their characteristic beams of UV and X-ray radiation have been applied to virtually all areas of medical science which use ionizing radiation. The ability to tune intense monochromatic beams over wide energy ranges differentiates these sources from standard clinical and research tools. At the European Synchrotron Radiation Facility (Grenoble, France), a major research facility is operational on an advanced wiggler radiation beamport, ID17. The beamport is designed to carry out a broad range of research ranging from cell radiation biology to in vivo human studies. Medical imaging programs at ID17 include transvenous coronary angiography, computed tomography, mammography and bronchography. In addition, a major research program on microbeam radiation therapy is progressing. This paper will present a very brief overview of the beamline and the imaging and therapy programs.     

Proteomics data repositories: Providing a safe haven for your data and acting as a springboard for further research

Despite the fact that data deposition is not a generalised fact yet in the field of proteomics, several mass spectrometry (MS) based proteomics repositories are publicly available for the scientific community. The main existing resources are: the Global Proteome Machine Database (GPMDB), PeptideAtlas, the PRoteomics IDEntifications database (PRIDE), Tranche, and NCBI Peptidome. In this review the capabilities of each of these will be described, paying special attention to four key properties: data types stored, applicable data submission strategies, supported formats, and available data mining and visualization tools. Additionally, the data contents from model organisms will be enumerated for each resource. There are other valuable smaller and/or more specialized repositories but they will not be covered in this review. Finally, the concept behind the ProteomeXchange consortium, a collaborative effort among the main resources in the field, will be introduced.     

A radiobiological model for the relative biological effectiveness of high-dose-rate 252Cf brachytherapy

While there is significant clinical experience using both low- and high-dose-rate 252Cf brachytherapy, there are minimal data regarding values for the neutron relative biological effectiveness (RBE) with both modalities. The aim of this research was to derive a radiobiological model for 252Cf neutron RBE and to compare these results with neutron RBE values used clinically in Russia. The linear-quadratic (LQ) model was used as the basis to characterize cell survival after irradiation, with identical cell killing rates (S(N) = S(gamma)) between 252Cf neutrons and photons used for derivation of RBE. Using this equality, a relationship among neutron dose and LQ radiobiological parameter (i.e., alpha(N), beta(N), alpha(gamma), beta(gamma)) was obtained without the need to specify the photon dose. These results were used to derive the 252Cf neutron RBE, which was then compared with Russian neutron RBE values. The 252Cf neutron RBE was determined after incorporating the LQ radiobiological parameters obtained from cell survival studies with fast neutrons and teletherapy photons. For single-fraction high-dose-rate neutron doses of 0.5, 1.0, 1.5 and 2.0 Gy, the total biologically equivalent doses were 1.8, 3.4, 4.7 and 6.0 RBE Gy with 252Cf neutron RBE values of 3.2, 2.9, 2.7 and 2.5, respectively. Using clinical data for late-responding reactions from 252Cf, Russian investigators created an empirical model that predicted high-dose-rate 252Cf neutron RBE values ranging from 3.6 to 2.9 for similar doses and fractionation schemes and observed that 252Cf neutron RBE increases with the number of treatment fractions. Using these relationships, our results were in general concordance with high-dose-rate 252Cf RBE values obtained from Russian clinical experience.     

CellDepot: A Unified Repository for scRNA-seq Data and Visual Exploration

CellDepot containing over 270 datasets from 8 species and many tissues serves as an integrated web application to empower scientists in exploring single-cell RNA-seq (scRNA-seq) datasets and comparing the datasets among various studies through a user-friendly interface with advanced visualization and analytical capabilities. To begin with, it provides an efficient data management system that users can upload single cell datasets and query the database by multiple attributes such as species and cell types. In addition, the graphical multi-logic, multi-condition query builder and convenient filtering tool backed by MySQL database system, allows users to quickly find the datasets of interest and compare the expression of gene(s) across these. Moreover, by embedding the cellxgene VIP tool, CellDepot enables fast exploration of individual dataset in the manner of interactivity and scalability to gain more refined insights such as cell composition, gene expression profiles, and differentially expressed genes among cell types by leveraging more than 20 frequently applied plotting functions and high-level analysis methods in single cell research. In summary, the web portal available at http://celldepot.bxgenomics.com, prompts large scale single cell data sharing, facilitates meta-analysis and visualization, and encourages scientists to contribute to the single-cell community in a tractable and collaborative way. Finally, CellDepot is released as open-source software under MIT license to motivate crowd contribution, broad adoption, and local deployment for private datasets.     

Porphyrin-mediated boron neutron capture therapy: a preclinical evaluation of the response of the oral mucosa

Preclinical studies are in progress to determine the potential of boron neutron capture therapy (BNCT) for the treatment of carcinomas of the head and neck. Recently, it has been demonstrated that various boronated porphyrins can target a variety of tumor types. Of the porphyrins evaluated so far, copper tetracarboranylphenyl porphyrin (CuTCPH) is potentially a strong candidate for clinical use. In the present investigation, the response of the oral mucosa to CuTCPH-mediated boron neutron capture (BNC) irradiation was assessed using the ventral surface of the tongue of adult male Fischer 344 rats, a standard rodent model. CuTCPH was administered by intravenous infusion, at a dose of 200 mg/kg body weight, over a 48-h period. Three days after the end of the administration of CuTCPH, biodistribution studies indicated very low levels of boron (<2 microg/g) in the blood. Levels of boron in tongue tissue were 39.0 +/- 3.8 microg/g at this time. This was the time selected for irradiation with single doses of thermal neutrons from the Brookhaven Medical Research Reactor. The estimated level of boron-10 in the oral mucosa was used in the calculation of the physical radiation doses from the 10B(n,alpha)7Li reaction. This differs from the approach using the present generation of clinical boron carriers, where boron levels in blood at the time of irradiation are used for this calculation. Dose-response curves for the incidence of mucosal ulceration were fitted using probit analysis, and the doses required to produce a 50% incidence of the effect (ED50 +/- SE) were calculated. Analysis of the dose-effect data for CuTCPH-mediated BNC irradiation, compared with those for X rays and thermal neutrons alone, gave a compound biological effectiveness (CBE) factor of approximately 0.04. This very low CBE factor would suggest that there was relatively low accumulation of boron in the key target epithelial stem cells of the oral mucosa. As a consequence, with low levels of boron (<2 microg/g) in the blood, the response of the oral mucosa to CuTCPH-mediated BNCT will be governed primarily by the radiation effects of the thermal neutron beam and not from the boron neutron capture reaction [10B(n,alpha)7Li].     

RBE of the MIT epithermal neutron beam for crypt cell regeneration in mice

The RBE of the new MIT fission converter epithermal neutron capture therapy (NCT) beam has been determined using intestinal crypt regeneration in mice as the reference biological system. Female BALB/c mice were positioned separately at depths of 2.5 and 9.7 cm in a Lucite phantom where the measured total absorbed dose rates were 0.45 and 0.17 Gy/ min, respectively, and irradiated to the whole body with no boron present. The gamma-ray (low-LET) contributions to the total absorbed dose (low- + high-LET dose components) were 77% (2.5 cm) and 90% (9.7 cm), respectively. Control irradiations were performed with the same batch of animals using 6 MV photons at a dose rate of 0.83 Gy/min as the reference radiation. The data were consistent with there being a single RBE for each NCT beam relative to the reference 6 MV photon beam. Fitting the data according to the LQ model, the RBEs of the NCT beams were estimated as 1.50 +/- 0.04 and 1.03 +/- 0.03 at depths of 2.5 and 9.7 cm, respectively. An alternative parameterization of the LQ model considering the proportion of the high- and low-LET dose components yielded RBE values at a survival level corresponding to 20 crypts (16.7%) of 5.2 +/- 0.6 and 4.0 +/- 0.7 for the high-LET component (neutrons) at 2.5 and 9.7 cm, respectively. The two estimates are significantly different (P = 0.016). There was also some evidence to suggest that the shapes of the curves do differ somewhat for the different radiation sources. These discrepancies could be ascribed to differences in the mechanism of action, to dose-rate effects, or, more likely, to differential sampling of a more complex dose-response relationship.     

Calculation of dose components in head phantom for boron neutron capture therapy.

Application of neutrons to cancer treatment has been a subject of considerable clinical and research interest since the discovery of the neutron by Chadwick in 1932 (3). Boron neutron capture therapy (BNCT) is a technique of radiation oncology which is used in treating brain cancer (glioblastoma multiform) or melanoma and that consists of preferentially loading a compound containing 10B into the tumor location, followed by the irradiation of the patient with a beam of neutron. Dose distribution for BNCT is mainly based on Monte Carlo simulations. In this work, the absorbed dose spatial distribution resultant from an idealized neutron beam incident upon ahead phantom is investigated using the Monte Carlo N-particles code, MCNP 4B. The phantom model used is based on the geometry of a circular cylinder on which sits an elliptical cylinder capped by half an ellipsoid representing the neck and head, both filled with tissue-equivalent material. The neutron flux and the contribution of individual absorbed dose components, as a function of depths and of radial distance from the beam axis (dose profiles) in phantom model, is presented and discussed. For the studied beam the maximum thermal neutron flux is at a depth of 2 cm and the maximum gamma dose at a depth of 4 cm.     

Neutron-induced gamma dose from a reactor beam filter for boron neutron capture therapy

For the boron neutron capture therapy (NCT) of deep-seated metastatic melanoma, an epithermal (up to a few keV energy) neutron beam from a reactor horizontal facility could be useful if the inherent contamination from fast neutrons and gamma rays could be minimised. Calculations for ANSTO's 10 MW research reactor HIFAR have shown that, even though a filter material such as AlF3 attenuates the fast neutron dose, the beam quality improvement is counteracted by a relative increase in the gamma dose because of the gammas arising from neutron captures in the filter material, particularly the aluminium. The aluminium gammas, most of which arise from thermal neutron capture, are hard and cannot be attenuated by lead or bismuth without comparable attenuation of the epithermal neutron flux. Addition of an absorber such as 6Li to the AlF3 filter was investigated as a means of reducing the hard gamma dose, but the improvement in beam quality was small and at considerable cost to dose intensity. Dose characteristics calculations confirmed the superiority of a tangential beam over a radial beam with better results from an unfiltered tangential beam than from an AlF3 filter in a radial beam. This study showed conclusively that assessments of filter assemblies based on the effect of individual components on either the neutron or gamma dose in isolation are inadequate. In assessing any epithermal neutron filter, thermal neutron shield, and gamma shield combination, the total effect of each on the neutron, gamma, and boron-10 dose must be considered.     

Optimization of Boron and Neutron Delivery for Neutron Capture Therapy

A number of groups in the United States have received funding that will permit evaluation of the clinical efficacy of the neutron capture therapy (NCT) procedure. Various reactors are being modified to allow the construction of an epithermal neutron beam. At the Brookhaven Medical Research Reactor (BMRR), the patient irradiation facility is being modified to produce an optimized epithermal neutron beam. An 80-cm-thick Al-D₂O mixture (184 g/cm², 25% D₂O by volume) is being installed in the shutter assembly. One-dimensional calculations indicate that this configuration should provide an epithermal neutron flux density of ～1 × 10⁹ n/cm²/sec at 3 MW and a concomitant fast neutron dose rate of ～2 × 10^?11 rad per epithermal neutron (assuming a homogeneous Al-D₂O mixture). The actual geometry will be an inhomogeneous array of D₂O and Al layers producing parameters somewhat less favorable than those listed above; experimental verification is in progress. Significant gains have recently been made in selectively targeting B to melanoma with various melanaffinic compounds, including p-boronophenylalanine, and with boronated porphyrins that may be applicable to a variety of tumors. Neutron capture radiographs have been obtained with the above compounds, and efforts have been made to quantitate boron uptake in growing and quiescent or necrotic regions of tumor via double-labeling techniques obtained with tritiated thymidine. A correlation between therapeutic efficacy and the ability to deliver boron to viable areas of tumor has been observed.     

Comparison of a modified shell vial culture procedure with conventional mouse inoculation for rabies virus isolation

Rabies is a neurotropic disease that is often lethal. The early diagnosis of rabies infection is important and requires methods that allow for the isolation of the virus from animals and humans. The present study compared a modified shell vial (MSV) procedure using 24-well tissue culture plates with the mouse inoculation test (MIT), which is considered the gold standard for rabies virus isolation. Thirty brain samples (25 positive and 5 negative by the fluorescent antibody test) obtained from different animal species at the National Institute of Hygiene Rafael Rangel in Caracas, Venezuela, were studied by the MIT and MSV assays. Nine samples (36%) were positive at 24 h, 10 (40%) were positive at 48 h and six (24%) were positive at 72 h by the MSV assay. With the MIT assay, 76% were positive at six days post inoculation and 12% were positive at 12 and 18 days post inoculation. One sample that was negative according to the MSV assay was positive with MIT on the 12th day. The MSV procedure exhibited a sensitivity of 96.2%, a specificity of 100%, a positive predictive value of 100% and a negative predictive value 80%. This procedure allowed for rapid rabies virus detection. MIT can be employed as an alternative method in laboratories without tissue culture facilities.     

Redox-Specialized Bacterioplankton Metacommunity in a Temperate Estuary

This study explored the spatiotemporal dynamics of the bacterioplankton community composition in the Gulf of Finland (easternmost sub-basin of the Baltic Sea) based on phylogenetic analysis of 16S rDNA sequences acquired from community samples via pyrosequencing. Investigations of bacterioplankton in hydrographically complex systems provide good insight into the strategies by which microbes deal with spatiotemporal hydrographic gradients, as demonstrated by our research. Many ribotypes were closely affiliated with sequences isolated from environments with similar steep physiochemical gradients and/or seasonal changes, including seasonally anoxic estuaries. Hence, one of the main conclusions of this study is that marine ecosystems where oxygen and salinity gradients co-occur can be considered a habitat for a cosmopolitan metacommunity consisting of specialized groups occupying niches universal to such environments throughout the world. These niches revolve around functional capabilities to utilize different electron receptors and donors (including trace metal and single carbon compounds). On the other hand, temporal shifts in the bacterioplankton community composition at the surface layer were mainly connected to the seasonal succession of phytoplankton and the inflow of freshwater species. We also conclude that many relatively abundant populations are indigenous and well-established in the area.     

Comparative kinetics of the early repair of intestines and lung in mice after fast neutron and gamma-ray irradiation

Early repair (Elkind) after d(50) + Be neutron and gamma irradiation is assessed by determining the additional dose Dr necessary to reach a given biological effect when a single fraction Ds is split into 2 equal fractions 2Di separated by a time interval "i". LD50 at 180 days after thoracic irradiation is used as an evaluation of late pulmonary tolerance; LD50 at 5 days after abdominal irradiation is used as an evaluation of early intestinal tolerance. Dr is reduced but still important after neutron irradiation as compared to gamma irradiation. For LD50/180, after fast neutron irradiation Dr reaches 66, 90, 64, 162, 195, 150 cGy for "i" = 1, 2, 3, 5, 4, 12, and 24 hours respectively; after gamma irradiation, Field and Hornsey reported Dr = 390, 530, and 376 cGy for "i" = 2, 6, and 24 hours respectively; after neutron irradiation, they reported Dr = 190 cGy for "i" = 24 hours. For LD50/5, after fast neutron irradiation, Dr = 14, 45, 43, and 133 cGy for "i" = 1,5, 3,5, 5,5 and 24 hours respectively. Early repair is faster after gamma irradiation: Dr reaches a maximum for "i" = 3-4 hours. For neutrons, Dr reaches its maximum at 24 hours for both criteria.     

Biodistribution of sodium borocaptate (BSH) for boron neutron capture therapy (BNCT) in an oral cancer model

Boron neutron capture therapy (BNCT) is based on selective accumulation of ¹⁰B carriers in tumor followed by neutron irradiation. We previously proved the therapeutic success of BNCT mediated by the boron compounds boronophenylalanine and sodium decahydrodecaborate (GB-10) in the hamster cheek pouch oral cancer model. Based on the clinical relevance of the boron carrier sodium borocaptate (BSH) and the knowledge that the most effective way to optimize BNCT is to improve tumor boron targeting, the specific aim of this study was to perform biodistribution studies of BSH in the hamster cheek pouch oral cancer model and evaluate the feasibility of BNCT mediated by BSH at nuclear reactor RA-3. The general aim of these studies is to contribute to the knowledge of BNCT radiobiology and optimize BNCT for head and neck cancer. Sodium borocaptate (50 mg ¹⁰B/kg) was administered to tumor-bearing hamsters. Groups of 3–5 animals were killed humanely at nine time-points, 3–12 h post-administration. Samples of blood, tumor, precancerous pouch tissue, normal pouch tissue and other clinically relevant normal tissues were processed for boron measurement by optic emission spectroscopy. Tumor boron concentration peaked to therapeutically useful boron concentration values of 24–35 ppm. The boron concentration ratio tumor/normal pouch tissue ranged from 1.1 to 1.8. Pharmacokinetic curves showed that the optimum interval between BSH administration and neutron irradiation was 7–11 h. It is concluded that BNCT mediated by BSH at nuclear reactor RA-3 would be feasible.     

The Molecular Biology Database Collection: 2003 update

The Molecular Biology Database Collection is an online resource listing key databases of value to the biological community. This Collection is intended to bring fellow scientists' attention to high-quality databases that are available throughout the world, rather than just be a lengthy listing of all available databases. As such, this up-to-date listing is intended to serve as the jumping-off point from which to find specialized databases that may be of use in advancing biological research. The databases included in this Collection provide new value to the underlying data by virtue of curation, new data connections or other innovative approaches. Short, searchable summaries and updates for each of the databases included in this Collection are available through the Nucleic Acids Research Web site at http://nar.oupjournals.org.