Proteomic and Transcriptomic Analyses in the Slipper Snail Crepidula fornicata Uncover Shell Matrix Genes Expressed During Adult and Larval Biomineralization.

The gastropod shell is a composite composed of minerals and shell matrix proteins (SMPs). SMPs have been identified by proteomics in many molluscs, but few have been studied in detail. Open questions include (1) what gene regulatory networks regulate SMP expression, (2) what roles individual SMPs play in biomineralization, and (3) how the complement of SMPs changes over development. These questions are best addressed in a species in which gene perturbation studies are available; one such species is the slipper snail, Crepidula fornicata. Here, SEM and pXRD analysis demonstrated that the adult shell of C. fornicata exhibits crossed lamellar microstructure and is composed of aragonite. Using high-throughput proteomics we identified 185 SMPs occluded within the adult shell. Over half of the proteins in the shell proteome have known biomineralization domains, while at least 10% have no homologs in public databases. Differential gene expression analysis identified 20 SMP genes that are up-regulated in the shell-producing mantle tissue. Over half of these 20 SMPs are expressed during development with two, CfSMP1 and CfSMP2, expressed exclusively in the shell gland. Together, the description of the shell microstructure and a list of SMPs now sets the stage for studying the consequences of SMP gene knockdowns in molluscs.

Differences in clinical manifestations between childhood-onset lupus and adult-onset lupus: a meta-analysis.

OBJECTIVE: It is known that age at disease onset has an impact on the clinical course and outcome of systemic lupus erythematosus (SLE); however, the precise differences in the prevalence of SLE manifestations are debated. Our objective was to conduct a systematic literature review and meta-analysis of all studies that directly compare childhood-onset lupus with adult-onset lupus to determine which clinical manifestations vary with age at disease onset. METHODS: A comprehensive literature search of the MEDLINE/PubMed,EMBASE, CINAHL, and SCOPUS databases was conducted to identify relevant articles. Study quality was assessed using the STROBE checklist. Study sample characteristics and clinical manifestation event rates were extracted from each study. Pooled odds ratios (ORs) were calculated using the random effects method, and between-study heterogeneity was quantified using the I (2) statistic. RESULTS: Of the 484 studies identified by the search strategy, 16 were included in this review. The total number of patients was 5993 adults and 905 children with SLE. Study quality was on average 16/32, ranging from 8 to 29. Several statistically significant differences were found: malar rash, ulcers/mucocutaneous involvement, renal involvement, proteinuria, urinary cellular casts, seizures, thrombocytopenia, hemolytic anemia, fever, and lymphadenopathy were more common in childhood-onset SLE with ORs ranging from 1.3 to 3.7; however, Raynaud's, pleuritis, and sicca were more common in adult-onset SLE (twice as common). CONCLUSIONS: The results of this meta-analysis suggest that some clinical manifestations of lupus are different in childhood-onset SLE and adult-onset SLE.

A genome-wide analysis of biomineralization-related proteins in the sea urchin Strongylocentrotus purpuratus.

Biomineralization, the biologically controlled formation of mineral deposits, is of widespread importance in biology, medicine, and engineering. Mineralized structures are found in most metazoan phyla and often have supportive, protective, or feeding functions. Among deuterostomes, only echinoderms and vertebrates produce extensive biomineralized structures. Although skeletons appeared independently in these two groups, ancestors of the vertebrates and echinoderms may have utilized similar components of a shared genetic "toolkit" to carry out biomineralization. The present study had two goals. First, we sought to expand our understanding of the proteins involved in biomineralization in the sea urchin, a powerful model system for analyzing the basic cellular and molecular mechanisms that underlie this process. Second, we sought to shed light on the possible evolutionary relationships between biomineralization in echinoderms and vertebrates. We used several computational methods to survey the genome of the purple sea urchin Strongylocentrotus purpuratus for gene products involved in biomineralization. Our analysis has greatly expanded the collection of biomineralization-related proteins. We have found that these proteins are often members of small families encoded by genes that are clustered in the genome. Most of the proteins are sea urchin-specific; that is, they have no apparent homologues in other invertebrate deuterostomes or vertebrates. Similarly, many of the vertebrate proteins that mediate mineral deposition do not have counterparts in the S. purpuratus genome. Our findings therefore reveal substantial differences in the primary sequences of proteins that mediate biomineral formation in echinoderms and vertebrates, possibly reflecting loose constraints on the primary structures of the proteins involved. On the other hand, certain cellular and molecular processes associated with earlier events in skeletogenesis appear similar in echinoderms and vertebrates, leaving open the possibility of deeper evolutionary relationships.

Optimizing vaccine design for cellular processing, MHC binding and TCR recognition.

In this review we describe the methods and processes that our group have developed while aiming to test and design multiepitope vaccines for infectious diseases and cancer. Testing the performance of vaccines composed of epitopes restricted by human leukocyte antigen (HLA) molecules is accomplished by in vitro antigenicity assays, as well as in vivo immunogenicity assays in HLA transgenics. The efficiency by which multiepitope vaccines are processed is optimized by spacer residues, which are designed to facilitate generation by natural processing of the various class I- and class II-restricted epitopes. Methods and strategies to test and optimize HLA binding affinity, patient coverage from the vaccine construct, and TCR recognition of HLA/epitope complexes are also discussed.

Closed-chain rehabilitation for upper and lower extremities.

Closed-chain exercise protocols are used extensively in rehabilitation of knee injuries and are increasingly used in rehabilitation of shoulder injuries. They are felt to be preferable to other exercise programs in that they simulate normal physiologic and biomechanical functions, create little shear stress across injured or healing joints, and reproduce proprioceptive stimuli. Because of these advantages, they may be used early in rehabilitation and have been integral parts of "accelerated" rehabilitation programs. The authors review the important components of a closed-chain rehabilitation program and provide examples of specific exercises that are used for rehabilitation of knee and shoulder injuries.

Functional activation of the extensor carpi radialis muscles in humans.

OBJECTIVES: To assess activity of radial wrist extensors caused by isometric radial deviation and extension by using magnetic resonance imaging (MRI) and to assess measures that might be used to normalize T2-weighted data. DESIGN: Two-way analysis of variance (ANOVA) design. SETTING: Laboratory and children's hospital. PARTICIPANTS: Three healthy volunteers. INTERVENTIONS: Ten repetitions of 10-second randomly ordered 30% or 60% of maximum voluntary isometric contractions toward wrist extension or radial deviation. MAIN OUTCOME MEASURES: Average T2 values from T2-weighted MR images of the extensor carpi radialis brevis (ECRB) and the extensor carpi radialis longus (ECRL), flexor digitorum profundus (FDP), and radius marrow were determined across 7 sections and 4 exercise bouts and a preexercise condition. RESULTS: Significant differences across task and across sections were determined. Post hoc analysis revealed differences in activity between proximal and distal ECRB and ECRL during an exercise and differential activation of the same muscle across the 2 exercise tasks. Bone marrow and FDP did not show task-related changes. The range of average T2 values of bone marrow across sections was greater than a muscle (FDP) that was not the target of the exercise protocol. However, FDP did show small but significant differences across sections. CONCLUSIONS: T2-weighted MR images can be used to study muscle activation at 30% and 60% of maximum voluntary contractions. The use of inactive muscle and bone marrow for normalizing data requires further investigation.

Optimization of epitope processing enhances immunogenicity of multiepitope DNA vaccines.

Experimental DNA vaccines comprised of multiple minimal cytotoxic T lymphocytes (CTL) epitopes can effectively induce broad CTL responses; however, such constructs frequently exhibit significant variation in epitope immunogenicity. Antigenicity assays utilizing human cells transfected with one such multiepitope construct revealed that the epitopes with poor immunogenicity were inefficiently processed in transfected cells. Compilation of a database of 94 epitope/flanking region combinations, for which immunogenicity was measured experimentally, revealed that the type of residue immediately following the carboxyl-terminus of the epitope exerted a prominent effect on immunogenicity. Experiments utilizing a variety of HBV-specific vaccine constructs demonstrated epitope immunogenicity could be modulated by the insertion of a single amino acid and the effect on immunogenicity could be ascribed to modulation of processing efficiency. These findings demonstrate that multiepitope DNA vaccines can be engineered to enhance CTL immunogenicity by increasing processing efficiency.

Special evolutionary properties of genes encoding a protein with a simple amino acid repeat.

We have examined the evolution of a gene, SM50, encoding a component of the spicule matrix, which plays an integral role in the formation of the echinoderm skeleton. This gene was originally characterized in Strongylocentrotus purpuratus and encodes an imperfect tandem repeat of six or seven amino acids. We have analyzed the sequence of this repeat in a number of sea urchin species and have determined that the repeat regions have undergone concerted evolution. There are differences in the repeat region between species, but the overall repeat structure is conserved, suggesting the repeat forms a structural domain important in biomineralization. The inherent conserved amino acid repeat structure promotes concerted evolution due to the high probability of misreplication and unequal crossing-over in the repeated segment of the gene. While there are constraints on the amino acids allowed in the repeat region, there are also variations, so that the sequences observed illustrate the balance between amino acid substitutions and concerted evolution. We have evidence that substitutions can alter the mechanisms of unequal crossing-over, altering the way concerted evolution occurs. The way in which concerted evolution occurred appears to be determined by the degree of sequence similarity between the repeats in a given gene, which influences how unequal crossing over may occur. We have mapped the differences in repeat regions on existing phylogenetic trees and indicate where concerted evolution has taken place. We also confirm an earlier report that Hemicentrotus pulcherrimus fits into the Strongylocentrotus genus and examine the evolution of the H. pulcherrimus SM50 repeat relative to other members of this genus.

Spreading of HIV-specific CD8+ T-cell repertoire in long-term nonprogressors and its role in the control of viral load and disease activity.

Long-term non-progressors (LTNP) represent a minority of human immunodeficiency virus (HIV) infected individuals characterized by stable or even increasing CD4+ T-cell count and by stronger immune responses against HIV than progressors. In this study, HIV-specific effector CD8+ T cells, as detected by both a sensitive ex vivo enzyme-linked immunospot (ELISPOT) assay and specific major histocompatibility complex (MHC) peptide tetramers, were at a low frequency in the peripheral blood of LTNP, and recognized a lower number of HIV peptides than their memory resting cell counterparts. Both factors may account for the lack of complete HIV clearance by LTNP, who could control the viral spread, and displayed a higher magnitude of cytotoxic T lymphocyte (CTL) responses than progressors. By combining cell purification and ELISPOT assays this study demonstrates that both effector and memory resting cells were confined to a CD8+ population with memory CD45RO+ phenotype, with the former being CD28- and the latter CD28+. Longitudinal studies highlighted a relatively stable HIV-specific effector repertoire, viremia, and CD4+ T-cell counts, which were all correlated with maintenance of nonprogressor status. In conclusion, the analysis of HIV-specific cellular responses in these individuals may help define clear correlates of protective immunity in HIV infection.

Identification and antigenicity of broadly cross-reactive and conserved human immunodeficiency virus type 1-derived helper T-lymphocyte epitopes.

Human immunodeficiency virus (HIV)-specific helper T lymphocytes (HTL) play a key role in the immune control of HIV type 1 (HIV-1) infection, and as such are an important target of potential HIV-1 vaccines. In order to identify HTL epitopes in HIV-1 that might serve as vaccine targets, conserved HIV-1-derived peptides bearing an HLA-DR binding supermotif were tested for binding to a panel of the most representative HLA-DR molecules. Eleven highly cross-reactive binding peptides were identified: three in Gag and eight in Pol. Lymphoproliferative responses to this panel of peptides, as well as to the HIV-1 p24 and p66 proteins, were evaluated with a cohort of 31 HIV-1-infected patients. All 11 peptides were recognized by peripheral blood mononuclear cells from multiple HIV-infected donors. Many of the responsive HIV-infected subjects showed recognition of multiple peptides, indicating that HIV-1-specific T-helper responses may be broadly directed in certain individuals. A strong association existed between recognition of the parental recombinant HIV-1 protein and the corresponding HTL peptides, suggesting that these peptides represent epitopes that are processed and presented during the course of HIV-1 infection. Lastly, responses to the supermotif peptides were mediated by CD4(+) T cells and were restricted by major histocompatibility complex class II molecules. The epitopes described herein are potentially important components of HIV-1 therapeutic and prophylactic vaccines.

Identification of novel HLA-A2-restricted human immunodeficiency virus type 1-specific cytotoxic T-lymphocyte epitopes predicted by the HLA-A2 supertype peptide-binding motif.

Virus-specific cytotoxic T-lymphocyte (CTL) responses are critical in the control of human immunodeficiency virus type 1 (HIV-1) infection and will play an important part in therapeutic and prophylactic HIV-1 vaccines. The identification of virus-specific epitopes that are efficiently recognized by CTL is the first step in the development of future vaccines. Here we describe the immunological characterization of a number of novel HIV-1-specific, HLA-A2-restricted CTL epitopes that share a high degree of conservation within HIV-1 and a strong binding to different alleles of the HLA-A2 superfamily. These novel epitopes include the first reported CTL epitope in the Vpr protein. Two of the novel epitopes were immunodominant among the HLA-A2-restricted CTL responses of individuals with acute and chronic HIV-1 infection. The novel CTL epitopes identified here should be included in future vaccines designed to induce HIV-1-specific CTL responses restricted by the HLA-A2 superfamily and will be important to assess in immunogenicity studies in infected persons and in uninfected recipients of candidate HIV-1 vaccines.

The development of multi-epitope vaccines: epitope identification, vaccine design and clinical evaluation.

We have developed efficient methods for epitope identification and vaccine design. Our process for epitope selection based on the combined use of motif analyses, binding assays and immunogenicity evaluations is described. We also describe how the projected population coverage and vaccine design can be optimized. Finally, it is discussed how vaccine potency is evaluated by immunogenicity and antigenicity assays.

Evaluation of predicted and actual length of stay in 22 Scottish intensive care units using the APACHE III system. Acute Physiology and Chronic Health Evaluation.

The most recent edition of the Acute Physiology and Chronic Health Evaluation provides a prediction of intensive care unit length of stay in addition to the probability of hospital mortality. Intensive care length of stay is an important determinant of intensive care costs and may be an important indicator of quality of care. Data were collected from 22 Scottish intensive care units over a 2-year period to allow comparison of actual intensive care unit length of stay with that predicted by the Acute Physiology and Chronic Health Evaluation III system. Correlation between actual and predicted stay for individual patients was poor. However, performance of the model for patients, grouped either by predicted length of stay or by intensive care unit, indicated that the model stratified patient groups appropriately while demonstrating a consistent bias. Length of stay in Scottish intensive care units was found to be consistently lower than that predicted by a model which is based on intensive care practice in the USA. Variations in severity of illness in intensive care unit populations cannot readily explain differences in intensive care unit length of stay. The availability of a model capable of predicting length of intensive care stay, based on data reflecting practice in the UK, would compliment current methods of assessing effectiveness of intensive care.

Trends in case-fatality in 117 718 patients admitted with acute myocardial infarction in Scotland.

OBJECTIVES: To analyse short- and long-term case-fatality trends following admission to hospital with a first acute myocardial infarction, in men and women between 1986 and 1995, after adjusting for risk factors known to influence survival. DESIGN: A Scottish-wide retrospective cohort study. SETTING: The Linked Scottish Morbidity Record Database was analysed. This contains accurate data on all hospital admissions since 1981, for the Scottish population of 5.1 million. It is linked to the Registrar General's death certificate data. SUBJECTS: All 117 718 patients admitted to Scottish hospitals with a principal diagnosis of first acute myocardial infarction (ICD-9 code 410) between 1986 and 1995. MAIN OUTCOME MEASURES: The outcome was death, both in and out of hospital, from any cause, at 30 days, 1 year, 5 and 10 years. RESULTS: Overall case-fatality following hospital admission with acute myocardial infarction was 22. 2%, 31.4%, 51.1% and 64.0% at 1 month, 1 year, 5 and 10 years, respectively. Multivariate analyses identified statistically significant independent prognostic factors. Thirty day mortality increased twofold for each decade of increasing age, and increased with any prior admission to hospital. When comparing the most deprived category to that of the most affluent, men had a 10% increased mortality (P<0.01), whilst women had an increased mortality of 4% (not significant). After adjustment for age, sex, deprivation and prior admission to hospital, case-fatality rates fell significantly between 1986 and 1995. Short-term case-fatality fell by 46% in men (27% in women) and long-term by 34% in men (30% in women) (both P<0.001). CONCLUSIONS: Population-based case-fatality rates in Scotland have fallen dramatically since 1986, particularly in men. The increasing survival in patients admitted to hospital suggests that the trial-based efficacy of modern therapies is now translating into population-based effectiveness. However, an individual's life expectancy still halves after a diagnosis of acute myocardial infarction. Of the variables that we could examine, age was the most powerful predictor of prognosis.

A sea urchin genome project: sequence scan, virtual map, and additional resources.

Results of a first-stage Sea Urchin Genome Project are summarized here. The species chosen was Strongylocentrotus purpuratus, a research model of major importance in developmental and molecular biology. A virtual map of the genome was constructed by sequencing the ends of 76,020 bacterial artificial chromosome (BAC) recombinants (average length, 125 kb). The BAC-end sequence tag connectors (STCs) occur an average of 10 kb apart, and, together with restriction digest patterns recorded for the same BAC clones, they provide immediate access to contigs of several hundred kilobases surrounding any gene of interest. The STCs survey >5% of the genome and provide the estimate that this genome contains approximately 27,350 protein-coding genes. The frequency distribution and canonical sequences of all middle and highly repetitive sequence families in the genome were obtained from the STCs as well. The 500-kb Hox gene complex of this species is being sequenced in its entirety. In addition, arrayed cDNA libraries of >10(5) clones each were constructed from every major stage of embryogenesis, several individual cell types, and adult tissues and are available to the community. The accumulated STC data and an expanding expressed sequence tag database (at present including >12, 000 sequences) have been reported to GenBank and are accessible on public web sites.

Assessment of the performance of five intensive care scoring models within a large Scottish database.

OBJECTIVE: To assess and compare the performance of five severity of illness scoring systems used commonly for intensive care unit (ICU) patients in the United Kingdom. The five models analyzed were versions II and III of the Acute Physiology and Chronic Health Evaluation (APACHE) system, a version of APACHE II using United Kingdom (UK)-derived coefficients (UK APACHE II), version II of the Simplified Acute Physiology Score (SAPS), and version II of the Mortality Probability Model, computed at admission (MPM0) and after 24 hrs in the ICU (MPM24). DESIGN: A 2-yr prospective cohort study of consecutive admissions to intensive care units. SETTING: A total of 22 general ICUs in Scotland PATIENTS: A total of 13,291 admissions to the study, which after prospectively agreed exclusions left a total of 10,393 patients for the analysis. OUTCOME MEASURES: Death or survival at hospital discharge. MEASUREMENTS AND MAIN RESULTS: All the models showed reasonable discrimination using the area under the receiver operating characteristic curve (APACHE III, 0.845; APACHE II, 0.805; UKAPACHE II, 0.809; SAPS II, 0.843; MPM0, 0.785; MPM24, 0.799). The levels of observed mortality were significantly different than that predicted by all models, using the Hosmer-Lemeshow goodness-of-fit C test (p < .001), with the results of the test being confirmed by calibration curves. When excluding patients discharged in the first 24 hrs to allow for comparisons using the same patient group, APACHE III, MPM24, and SAPS II (APACHE III, 0.795; MPM24, 0.791; SAPS II, 0.784) showed significantly better discrimination than APACHE II, UK APACHE II, and MPM0 (APACHE II, 0.763; UK APACHE II, 0.756; MPM0, 0.741). However, calibration changed little for all models with observed mortality still significantly different from that predicted by the scoring systems (p < .001). For equivalent data sets, APACHE II demonstrated superior calibration to all the models using the chi-squared value from the Hosmer-Lemeshow test for both populations (APACHE III, 366; APACHE II, 67; UKAPACHE II, 237; SAPS II, 142; MPM0, 452; MPM24, 101). CONCLUSIONS: SAPS II demonstrated the best overall performance, but the superior calibration of APACHE II makes it the most appropriate model for comparisons of mortality rates in different ICUs. The significance of the Hosmer-Lemeshow C test in all the models suggest that new logistic regression coefficients should be generated and the systems retested before they could be used with confidence in Scottish ICUs.

Should the pre-sedation Glasgow Coma Scale value be used when calculating Acute Physiology and Chronic Health Evaluation scores for sedated patients? Scottish Intensive Care Society Audit Group.

OBJECTIVE: To assess the effect on the performance of Acute Physiology and Chronic Health Evaluation (APACHE) II and APACHE III of two different approaches to scoring the Glasgow Coma Scale (GCS) in sedated patients. The first approach was to assume that the GCS score was normal, and the second was to use the GCS value recorded before the patient was sedated. DESIGN: Prospective cohort study over 2 yrs. SETTING: Twenty-two general adult intensive care units in Scotland. PATIENTS: 13,291 consecutive admissions to the participating intensive care units. MEASUREMENTS AND MAIN RESULTS: After exclusion of patients according to standard, predefined criteria, the Acute Physiology and Chronic Health Evaluation II and III systems were used to calculate the probability of hospital mortality for patients included in the study. In patients whose GCS scores could not be assessed accurately during the first 24 hrs, the APACHE II and III predictions were calculated twice: first, assuming that the GCS score was normal; and second, substituting the GCS score recorded before sedation. This generated two different databases for each system, and the predictions for both were compared with the observed hospital mortality rate. The effect of the two different approaches to the GCS on the performance of both APACHE II and APACHE III was assessed using measures of discrimination (area under the receiver operating characteristic curve) and goodness of fit (calibration curves and the Hosmer-Lemeshow statistic). Analysis was undertaken for both the entire cohort and for the group of patients whose APACHE scores were altered. There was a wide variation in the number of patients who had their scores altered between participating units. There were also differences between diagnostic groups. Overall, however, 50% of the patients were sedated and 22% had their scores altered. Using the presedation GCS score increased the discrimination of both APACHE II and APACHE III. The calibration of APACHE III was also improved but that of APACHE II deteriorated. The calibration improved, however, in those patients with altered scores, suggesting that the overall deterioration is attributable to other limitations in the fit of the model to these data. Although changes had the greatest effect in patients with a neurologic or trauma diagnosis, the changes were important in most diagnostic groups. CONCLUSIONS: The GCS is an important component of both APACHE II and APACHE III. It should be assessed directly whenever possible. When patients are sedated, using the GCS score recorded before sedation is preferable to the assumption of normality. The variations between different units and different diagnostic groups highlight the possible effects of case mix on the performance of prognostic scoring systems.

HLA-binding peptides as a therapeutic approach for chronic HIV infection.

Herein, we describe the Epimmune approach to prophylaxis and development of a multi-epitope vaccine for immunotherapy of HIV-1 infection. The central strategy of our program is to induce cellular immune responses, cytotoxic T-lymphocytes (CTL) and helper Tlymphocytes (HTL), specific for conserved epitopes from both structural and regulatory proteins of HIV-1. The HIV-1 derived and HLA-restricted CTL and HTL epitopes needed to design and construct the experimental vaccines are now known and allow for broad and non-ethnically biased coverage of the human population. The design optimization of an epitope-based DNA vaccine and evaluating methods for various DNA vaccine delivery technologies for possible use in clinical trials are addressed.