A critical event of pharyngeal swallowing is the elevation of the hyolaryngeal complex to open the upper esophageal sphincter. Current swallowing theory assigns this function to the submental and thyrohyoid muscles. However, the attachments of the long pharyngeal muscles indicate that they could contribute to this function, yet their role is uninvestigated in humans. In addition, there is evidence the posterior digastric and stylohyoid contribute to hyoid elevation. A cadaver model was used to document the structural properties of muscles. These properties were used to model muscle groups as force vectors and analyze their potential for hyolaryngeal elevation. Vector magnitude was determined using physiological cross-sectional areas (PCSAs) of muscles calculated from structural properties of muscle taken from 12 hemisected cadaver specimens. Vector direction (lines of action) was calculated from the three-dimensional coordinates of muscle attachment sites. Unit force vectors in the superior direction of submental, suprahyoid (which includes the submental muscles), long pharyngeal, and thyrohyoid muscles were derived and compared by an analysis of variance (ANOVA) to document each muscle’s potential contribution to hyolaryngeal elevation. An ANOVA with Tukey HSD post hoc analysis of unit force vectors showed no statistically significant difference between the submental (0.92 ± 0.24 cm2) and long pharyngeal (0.73 ± 0.20 cm2) muscles. Both demonstrated greater potential to elevate the hyolaryngeal complex than the thyrohyoid (0.49 ± 0.18 cm2), with P < 0.01 and P < 0.05, respectively. The suprahyoid muscles (1.52 ± 0.35 cm2) demonstrated the greatest potential to elevate the hyolaryngeal complex: greater than both the long pharyngeal muscles (P < 0.01) and the thyrohyoid (P < 0.01). The submental and thyrohyoid muscles by convention are thought to elevate the hyolaryngeal complex. This study demonstrates that structurally the long pharyngeal muscles have similar potential to contribute to this critical function, with the suprahyoid muscles having the greatest potential. If verified by functional data, these findings would amend current swallowing theory.
Deglutition; Laryngeal elevation; Physiological cross-sectional area; Structural properties; Hyolaryngeal complex; Deglutition disorders
Motivation: Sequence similarity searches performed with BLAST, SSEARCH and FASTA achieve high sensitivity by using scoring matrices (e.g. BLOSUM62) that target low identity (<33%) alignments. Although such scoring matrices can effectively identify distant homologs, they can also produce local alignments that extend beyond the homologous regions.
Results: We measured local alignment start/stop boundary accuracy using a set of queries where the correct alignment boundaries were known, and found that 7% of BLASTP and 8% of SSEARCH alignment boundaries were overextended. Overextended alignments include non-homologous sequences; they occur most frequently between sequences that are more closely related (>33% identity). Adjusting the scoring matrix to reflect the identity of the homologous sequence can correct higher identity overextended alignment boundaries. In addition, the scoring matrix that produced a correct alignment could be reliably predicted based on the sequence identity seen in the original BLOSUM62 alignment. Realigning with the predicted scoring matrix corrected 37% of all overextended alignments, resulting in more correct alignments than using BLOSUM62 alone.
Availability: RefProtDom2 (RPD2) sequences and the FASTA software are available from http://faculty.virginia.edu/wrpearson/fasta.
We examined the characteristics of adults providing regular care or assistance to friends or family members who have health problems, long-term illnesses, or disabilities (ie, caregivers). We used data from the 2009 Behavioral Risk Factor Surveillance System (BRFSS) to examine caregiver characteristics, by age and caregiving status, and compare these characteristics with those of noncaregivers. Approximately 24.7% (95% confidence interval, 24.4%–25.0%) of respondents were caregivers. Compared with younger caregivers, older caregivers reported more fair or poor health and physical distress but more satisfaction with life and lower mental distress. Understanding the characteristics of caregivers can help enhance strategies that support their role in providing long-term care.
Traumatic brain injury (TBI) can be complicated among older adults due to age-related
frailty, a greater prevalence of chronic conditions and the use of anticoagulants. We
conducted this study using the latest available, nationally-representative emergency
department (ED) data to characterize visits for TBI among older adults.
We used the 2006–2008 National Hospital Ambulatory Medical Care –
Emergency Department (NHAMCS-ED) data to examine ED visits for TBI among older adults.
Population-level estimates of triage immediacy, receipt of a head computed tomography
(CT) and/or head magnetic resonance imaging (MRI), and hospital admission by type were
used to characterize 1,561 sample visits, stratified by age <65 and ≥65
years of age.
Of ED visits made by persons ≥65 years of age, 29.1% required attention
from a physician within 15 minutes of arrival; 82.1% required a head CT, and
20.9% required hospitalization. Persons ≥65 years of age were 3 times
more likely to receive a head CT or MRI compared to younger patients presenting with TBI
(adjusted odds ratio [aOR] 3.2; 95% confidence interval
[CI], 1.8–5.8), and were 4 times more likely to be admitted to
an intensive care unit, step-down unit, or surgery (aOR 4.1; 95% CI
2.1–8.0) compared to younger patients presenting with TBI, while controlling for
sex and race.
Results demonstrate increased emergent service delivery for older persons presenting
with TBI. As the United States population ages and continues to grow, TBI will become an
even more important public health issue that will place a greater demand on the
Summary: Iterative similarity searches with PSI-BLAST position-specific score matrices (PSSMs) find many more homologs than single searches, but PSSMs can be contaminated when homologous alignments are extended into unrelated protein domains—homologous over-extension (HOE). PSI-Search combines an optimal Smith–Waterman local alignment sequence search, using SSEARCH, with the PSI-BLAST profile construction strategy. An optional sequence boundary-masking procedure, which prevents alignments from being extended after they are initially included, can reduce HOE errors in the PSSM profile. Preventing HOE improves selectivity for both PSI-BLAST and PSI-Search, but PSI-Search has ~4-fold better selectivity than PSI-BLAST and similar sensitivity at 50% and 60% family coverage. PSI-Search is also produces 2- for 4-fold fewer false-positives than JackHMMER, but is ~5% less sensitive.
Availability and implementation: PSI-Search is available from the authors as a standalone implementation written in Perl for Linux-compatible platforms. It is also available through a web interface (www.ebi.ac.uk/Tools/sss/psisearch) and SOAP and REST Web Services (www.ebi.ac.uk/Tools/webservices).
To examine whether childhood traumatic stress increased the risk of developing autoimmune diseases as an adult.
Retrospective cohort study of 15,357 adult health maintenance organization members enrolled in the Adverse Childhood Experiences (ACEs) Study from 1995 to 1997 in San Diego, California, and eligible for follow-up through 2005. ACEs included childhood physical, emotional, or sexual abuse; witnessing domestic violence; growing up with household substance abuse, mental illness, parental divorce, and/or an incarcerated household member. The total number of ACEs (ACE Score range = 0–8) was used as a measure of cumulative childhood stress. The outcome was hospitalizations for any of 21 selected autoimmune diseases and 4 immunopathology groupings: T- helper 1 (Th1) (e.g., idiopathic myocarditis); T-helper 2 (Th2) (e.g., myasthenia gravis); Th2 rheumatic (e.g., rheumatoid arthritis); and mixed Th1/Th2 (e.g., autoimmune hemolytic anemia).
Sixty-four percent reported at least one ACE. The event rate (per 10,000 person-years) for a first hospitalization with any autoimmune disease was 31.4 in women and 34.4 in men. First hospitalizations for any autoimmune disease increased with increasing number of ACEs (p < .05). Compared with persons with no ACEs, persons with ≥2 ACEs were at a 70% increased risk for hospitalizations with Th1, 80% increased risk for Th2, and 100% increased risk for rheumatic diseases (p < .05).
Childhood traumatic stress increased the likelihood of hospitalization with a diagnosed autoimmune disease decades into adulthood. These findings are consistent with recent biological studies on the impact of early life stress on subsequent inflammatory responses.
childhood abuse; traumatic stress; autoimmune diseases; stress; inflammatory response
Superior and anterior hyoid movements are important events in pharyngeal deglutition. This cross-sectional study uses a cadaver model to document the structural properties of the muscles underlying these movements in an effort to understand how their morphology influences function. Measurements to determine physiological cross-sectional areas (PCSAs) of swallowing muscles were taken from hemisected head and neck formalin-fixed cadaver specimens (n = 13). Coordinates of muscle attachment sites and PCSAs were used to calculate î and ĵ unit force vectors, where î and ĵ represent anterior-posterior and superior-inferior directions, respectively. The suprahyoid muscle subsamples were grouped for analysis as follows: digastric (DG), geniohyoid (GH), mylohyoid (MH), and stylohyoid (SH). The ANOVA with Tukey HSD post hoc analysis of unit force vectors showed the following results: GH (−0.44 ± 0.15 cm2) >MH (−0.02 ± 0.21 cm2), DG (−0.05 ± 0.11 cm2), SH (0.14 ± 0.04 cm2), with negative values representing the anterior direction (p < 0.01); and MH (0.91 ± 0.28 cm2) >DG (0.29 ± 0.14 cm2), SH (0.22 ± 0.08 cm2), GH (12 ± 0.08 cm2), with positive values representing the superior direction (p < 0.01). The morphology of the suprahyoid muscles suggests that based on structural properties, the geniohyoid has the most potential to displace the hyoid in the anterior direction and the mylohyoid has the most potential to displace the hyoid in the superior direction. These data in complement with physiological findings may provide greater insight into these movements for those developing novel treatments for dysphagia.
Deglutition; Suprahyoid muscles; Hyoid movement; Physiological cross-sectional area; Structural properties; Deglutition disorders
MACiE (which stands for Mechanism, Annotation and Classification in Enzymes) is a database of enzyme reaction mechanisms, and can be accessed from http://www.ebi.ac.uk/thornton-srv/databases/MACiE/. This article presents the release of Version 3 of MACiE, which not only extends the dataset to 335 entries, covering 182 of the EC sub-subclasses with a crystal structure available (∼90%), but also incorporates greater chemical and structural detail. This version of MACiE represents a shift in emphasis for new entries, from non-homologous representatives covering EC reaction space to enzymes with mechanisms of interest to our users and collaborators with a view to exploring the chemical diversity of life. We present new tools for exploring the data in MACiE and comparing entries as well as new analyses of the data and new searches, many of which can now be accessed via dedicated Perl scripts.
Profile hidden Markov models (profile HMMs) and probabilistic inference methods have made important contributions to the theory of sequence database homology search. However, practical use of profile HMM methods has been hindered by the computational expense of existing software implementations. Here I describe an acceleration heuristic for profile HMMs, the “multiple segment Viterbi” (MSV) algorithm. The MSV algorithm computes an optimal sum of multiple ungapped local alignment segments using a striped vector-parallel approach previously described for fast Smith/Waterman alignment. MSV scores follow the same statistical distribution as gapped optimal local alignment scores, allowing rapid evaluation of significance of an MSV score and thus facilitating its use as a heuristic filter. I also describe a 20-fold acceleration of the standard profile HMM Forward/Backward algorithms using a method I call “sparse rescaling”. These methods are assembled in a pipeline in which high-scoring MSV hits are passed on for reanalysis with the full HMM Forward/Backward algorithm. This accelerated pipeline is implemented in the freely available HMMER3 software package. Performance benchmarks show that the use of the heuristic MSV filter sacrifices negligible sensitivity compared to unaccelerated profile HMM searches. HMMER3 is substantially more sensitive and 100- to 1000-fold faster than HMMER2. HMMER3 is now about as fast as BLAST for protein searches.
Searching sequence databases is one of the most important applications in computational molecular biology. The main workhorse in the field is the BLAST suite of programs. Since the introduction of BLAST in the 1990's, important theoretical advances in homology search methodology have been made using probabilistic inference methods and hidden Markov models (HMMs). However, previous software implementations of these newer probabilistic methods were slower than BLAST by about 100-fold. This hindered their utility, because computation speed is so critical with the rapidly increasing size of modern sequence databases. Here I describe the acceleration methods I implemented in a new, freely available profile HMM software package, HMMER3. HMMER3 makes profile HMM searches about as fast as BLAST, while retaining the power of using probabilistic inference technology.
Motivation: To test whether protein folding constraints and secondary structure sequence preferences significantly reduce the space of amino acid words in proteins, we compared the frequencies of four- and five-amino acid word clumps (independent words) in proteins to the frequencies predicted by four random sequence models.
Results: While the human proteome has many overrepresented word clumps, these words come from large protein families with biased compositions (e.g. Zn-fingers). In contrast, in a non-redundant sample of Pfam-AB, only 1% of four-amino acid word clumps (4.7% of 5mer words) are 2-fold overrepresented compared with our simplest random model [MC(0)], and 0.1% (4mers) to 0.5% (5mers) are 2-fold overrepresented compared with a window-shuffled random model. Using a false discovery rate q-value analysis, the number of exceptional four- or five-letter words in real proteins is similar to the number found when comparing words from one random model to another. Consensus overrepresented words are not enriched in conserved regions of proteins, but four-letter words are enriched 1.18- to 1.56-fold in α-helical secondary structures (but not β-strands). Five-residue consensus exceptional words are enriched for α-helix 1.43- to 1.61-fold. Protein word preferences in regular secondary structure do not appear to significantly restrict the use of sequence words in unrelated proteins, although the consensus exceptional words have a secondary structure bias for α-helix. Globally, words in protein sequences appear to be under very few constraints; for the most part, they appear to be random.
Supplementary information: Supplementary data are available at Bioinformatics online.
Background. The Hispanic population in the United States is growing, and disparities in the receipt of healthcare services as a result of limited English proficiency have been demonstrated. We set out to determine if Spanish language preference was a barrier to receiving influenza vaccinations among Hispanic persons 65 years and older in the USA. Methods. Differences in the receipt of vaccinations by language preference were tested with both Chi-square analyses and adjusted logistic regression analyses. Results. Findings suggest that elderly Hispanic persons, 65 years of age and older, who prefer to communicate in Spanish instead of English, are significantly less likely to have received influenza vaccinations when compared to their Hispanic counterparts who prefer to communicate in English. Conclusions. Influenza infections can more often be fatal in older persons and may disparately affect minority populations such as Hispanic persons. Therefore, understanding barriers to the receipt of effective preventive health measures is necessary.
Summary: RefProtDom provides a set of divergent query domains, originally selected from Pfam, and full-length proteins containing their homologous domains, with diverse architectures, for evaluating pair-wise and iterative sequence similarity searches. Pfam homology and domain boundary annotations in the target library were supplemented using local and semi-global searches, PSI-BLAST searches, and SCOP and CATH classifications.
Availability: RefProtDom is available from http://faculty.virginia.edu/wrpearson/fasta/PUBS/gonzalez09a
Contact: email@example.com; firstname.lastname@example.org
Significant differences in health outcomes have been documented among Hispanic persons, the fastest-growing demographic segment of the United States. The objective of this study was to examine trends in population growth and the collection of health data among Hispanic persons, including issues of language preference and survey completion using a national health survey to highlight issues of measurement of an increasingly important demographic segment of the United States.
Data from the 2003-2007 United States Census and the Behavioral Risk Factor Surveillance System were used to compare trends in population growth and survey sample size as well as differences in survey response based on language preference among a Hispanic population. Percentages of item non-response on selected survey questions were compared for Hispanic respondents choosing to complete the survey in Spanish and those choosing to complete the survey in English. The mean number of attempts to complete the survey was also compared based on language preference among Hispanic respondents.
The sample size of Hispanic persons in the Behavioral Risk Factor Surveillance System saw little growth compared to the actual growth of the Hispanic population in the United States. Significant differences in survey item non-response for nine of 15 survey questions were seen based on language preference. Hispanic respondents choosing to complete the survey in Spanish had a significantly fewer number of call attempts for survey completion compared to their Hispanic counterparts choosing to communicate in English.
Including additional measures of acculturation and increasing the sample size of Hispanic persons in a national health survey such as the Behavioral Risk Factor Surveillance System may result in more precise findings that could be used to better target prevention and health care needs for an ethnic minority population.
Modern sequence alignment algorithms are used routinely to identify homologous proteins, proteins that share a common ancestor. Homologous proteins always share similar structures and often have similar functions. Over the past 20 years, sequence comparison has become both more sensitive, largely because of profile-based methods, and more reliable, because of more accurate statistical estimates. As sequence and structure databases become larger, and comparison methods become more powerful, reliable statistical estimates will become even more important for distinguishing similarities that are due to homology from those that are due to analogy (convergence). The newest sequence alignment methods are more sensitive than older methods, but more accurate statistical estimates are needed for their full power to be realized.
While the pairwise alignments produced by sequence similarity searches are a powerful tool for identifying homologous proteins - proteins that share a common ancestor and a similar structure; pairwise sequence alignments often fail to represent accurately the structural alignments inferred from three-dimensional coordinates. Since sequence alignment algorithms produce optimal alignments, the best structural alignments must reflect suboptimal sequence alignment scores. Thus, we have examined a range of suboptimal sequence alignments and a range of scoring parameters to understand better which sequence alignments are likely to be more structurally accurate.
We compared near-optimal protein sequence alignments produced by the Zuker algorithm and a set of probabilistic alignments produced by the probA program with structural alignments produced by four different structure alignment algorithms. There is significant overlap between the solution spaces of structural alignments and both the near-optimal sequence alignments produced by commonly used scoring parameters for sequences that share significant sequence similarity (E-values < 10-5) and the ensemble of probA alignments. We constructed a logistic regression model incorporating three input variables derived from sets of near-optimal alignments: robustness, edge frequency, and maximum bits-per-position. A ROC analysis shows that this model more accurately classifies amino acid pairs (edges in the alignment path graph) according to the likelihood of appearance in structural alignments than the robustness score alone. We investigated various trimming protocols for removing incorrect edges from the optimal sequence alignment; the most effective protocol is to remove matches from the semi-global optimal alignment that are outside the boundaries of the local alignment, although trimming according to the model-generated probabilities achieves a similar level of improvement. The model can also be used to generate novel alignments by using the probabilities in lieu of a scoring matrix. These alignments are typically better than the optimal sequence alignment, and include novel correct structural edges. We find that the probA alignments sample a larger variety of alignments than the Zuker set, which more frequently results in alignments that are closer to the structural alignments, but that using the probA alignments as input to the regression model does not increase performance.
The pool of suboptimal pairwise protein sequence alignments substantially overlaps structure-based alignments for pairs with statistically significant similarity, and a regression model based on information contained in this alignment pool improves the accuracy of pairwise alignments with respect to structure-based alignments.
Mathematically optimal alignments do not always properly align active site residues or well-recognized structural elements. Most near-optimal sequence alignment algorithms display alternative alignment paths, rather than the conventional residue-by-residue pairwise alignment. Typically, these methods do not provide mechanisms for finding effectively the most biologically meaningful alignment in the potentially large set of options.
We have developed Web-based software that displays near optimal or alternative alignments of two protein or DNA sequences as a continuous moving picture. A WWW interface to a C++ program generates near optimal alignments, which are sent to a Java Applet, which displays them in a series of alignment frames. The Applet aligns residues so that consistently aligned regions remain at a fixed position on the display, while variable regions move. The display can be stopped to examine alignment details.
Available at http://fasta.bioch.virginia.edu/ noptalign. For source code contact the authors at email@example.com
We have characterized a novel type of PSI-BLAST error, homologous over-extension (HOE), using embedded PFAM domain queries on searches against a reference library containing Pfam-annotated UniProt sequences and random synthetic sequences. PSI-BLAST makes two types of errors: alignments to non-homologous regions and HOE alignments that begin in a homologous region, but extend beyond the homology into neighboring sequence regions. When the neighboring sequence region contains a non-homologous domain, PSI-BLAST can incorporate the unrelated sequence into its position specific scoring matrix, which then finds non-homologous proteins with significant expectation values. HOE accounts for the largest fraction of the initial false positive (FP) errors, and the largest fraction of FPs at iteration 5. In searches against complete protein sequences, 5–9% of alignments at iteration 5 are non-homologous. HOE frequently begins in a partial protein domain; when partial domains are removed from the library, HOE errors decrease from 16 to 3% of weighted coverage (hard queries; 35–5% for sampled queries) and no-error searches increase from 2 to 58% weighed coverage (hard; 16–78% sampled). When HOE is reduced by not extending previously found sequences, PSI-BLAST specificity improves 4–8-fold, with little loss in sensitivity.
Lack of health insurance coverage for working-age adults is one of the most pressing issues facing the U.S. population, and it continues to be a concern for a large number of people. In the absence of a national solution, the states and municipalities are left to address this need. We examined the disparities in uninsurance prevalence by state and metropolitan areas in the U.S. and among racial/ethnic groups.
Data from the 2006 Behavioral Risk Factor Surveillance System (BRFSS) were analyzed for working-age adults 18 to 64 years of age.
In 2006, according to the BRFSS data, overall 18.6% (standard error = 0.20) of working-age adults were without health insurance coverage; by state, this proportion ranged from 9.7% to 29.0%. Health insurance coverage varied by state and metropolitan area and racial/ethnic group, and a higher age-adjusted prevalence of uninsurance was observed for non-Hispanic black and Hispanic respondents.
A substantial proportion of working-age Americans remain without health insurance coverage. Disparities in health insurance coverage were observed by population and geographic groups. Overall, black and Hispanic populations fared far worse in terms of lack of health-care coverage than working-age white Americans.
Obesity is associated with morbidity, mortality, and increased health care costs. Few studies have examined the impact of obesity on outpatient office visits. The purpose of this study was to determine if outpatient visits by obese persons required more time with the provider and more prescription medication management compared to visits made by non-obese persons.
Obesity status was determined for 9,280 patient visits made by persons aged 18 years or older in the 2006 National Ambulatory Medical Care Survey. Multivariate analyses compared obese and non-obese visits, stratified by sex, for duration of the visit and the number of medications mentioned at the visit.
Average duration of visit was higher among visits with patients determined to be obese. However, these differences were not considered significant after statistical testing. Visits made by obese female patients were significantly more likely to involve more than two prescription medications (OR 1.26, 95% CI 1.05 - 1.51) and visits made by obese male patients were significantly more likely to involve more than two prescription medications (OR 1.46, 95% CI 1.16 - 1.83) as compared to visits made by non-obese patients.
Time spent with the provider was found to be greater among visits with obese patients, but not significantly different from visits with non-obese patients. The number of medications for each visit was found to be significantly greater for visits where the patient was considered to be obese. Increased time for the visit and increased numbers of medication for each visit translate into increased costs. These findings document the impact of obesity on our health care system and have great implications on medical care cost and planning.
Alcohol consumption is pervasive in the United States, and extent of alcohol consumption for the growing US Hispanic population needs further study. We examined the association between language chosen for a national health survey and alcohol use among Hispanic adults.
Hispanic participants aged 18 years and older (N = 20,234) from the 2005 Behavioral Risk Factor Surveillance System were stratified by choice of language (English, n = 13,035; Spanish, n = 7,199) for completing the survey. Differences for these 2 groups in current alcohol use, heavy alcohol use, and binge drinking were determined by using χ2 analyses and logistic regression models.
In bivariate associations, current drinking (P < .001), heavy drinking (P < .001), and binge drinking (P = .002) were significantly higher among participants who chose to complete the survey in English than among those who elected to complete the survey in Spanish. After controlling for demographic characteristics, associations between language choice and drinking behaviors were found to be greatest among women. Compared with women who chose to complete the survey in Spanish, women who chose to complete the survey in English were more than twice as likely to report current drinking (odds ratio [OR] = 2.42, 95% confidence interval [CI] = 2.02-2.91), heavy drinking (OR = 3.82, 95% CI = 1.44-10.10), and binge drinking (OR = 2.51, 95% CI = 1.64-3.84).
This study suggests that language choice when completing a health survey is a predictor of high levels of alcohol use among Hispanic adults in the United States and that differences in drinking behaviors based on language choice for a survey are more profound among women.
A formal synthesis of the Aspidosperm a alkaloids aspidospermidine, aspidospermine, and quebrachamine is reported through an efficient preparation of Stork’s penultimate intermediate. The key step of the sequence involved an intramolecular [3 + 2] cycloaddition of the 2-azapentadienyllithium 21 formed in situ from the corresponding imine 1, which after N-alkylation of the resulting cycloadduct, provided 2 in excellent yield. The synthesis represents a new disconnection of the classical tricyclic ketone used for appendage of the requisite indole.
The CRP (Cleavage of Radiolabeled Phosphoproteins) program guides the design and interpretation of experiments to identify protein phosphorylation sites by Edman sequencing of unseparated peptides. Traditionally, phosphorylation sites are determined by cleaving the phosphoprotein and separating the peptides for Edman 32P-phosphate release sequencing. CRP analysis of a phosphoprotein's sequence accelerates this process by omitting the separation step: given a protein sequence of interest, the CRP program performs an in silico proteolytic cleavage of the sequence and reports the predicted Edman cycles in which radioactivity would be observed if a given serine, threonine or tyrosine were phosphorylated. Experimentally observed cycles containing 32P can be compared with CRP predictions to confirm candidate sites and/or explore the ability of additional cleavage experiments to resolve remaining ambiguities. To reduce ambiguity, the phosphorylated residue (P-Tyr, P-Ser or P-Thr) can be determined experimentally, and CRP will ignore sites with alternative residues. CRP also provides simple predictions of likely phosphorylation sites using known kinase recognition motifs. The CRP interface is available at http://fasta.bioch.virginia.edu/crp.