Analytical methods. United States Renal Data System.
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Centers for Medicare and Medicaid Services, U.S.↗
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Complement regulation at the molecular level: the structure of decay-accelerating factor.
The human complement regulator CD55 is a key molecule protecting self-cells from complement-mediated lysis. X-ray diffraction and analytical ultracentrifugation data reveal a rod-like arrangement of four short consensus repeat (SCR) domains in both the crystal and solution. The stalk linking the four SCR domains to the glycosylphosphatidylinositol anchor is extended by the addition of 11 highly charged O-glycans and positions the domains an estimated 177 A above the membrane. Mutation mapping and hydrophobic potential analysis suggest that the interaction with the convertase, and thus complement regulation, depends on the burial of a hydrophobic patch centered on the linker between SCR domains 2 and 3.
CD55 Antigens↗
Regulation of gene expression in the mammalian eye and its relevance to eye disease.
We used expression quantitative trait locus mapping in the laboratory rat (Rattus norvegicus) to gain a broad perspective of gene regulation in the mammalian eye and to identify genetic variation relevant to human eye disease. Of >31,000 gene probes represented on an Affymetrix expression microarray, 18,976 exhibited sufficient signal for reliable analysis and at least 2-fold variation in expression among 120 F(2) rats generated from an SR/JrHsd x SHRSP intercross. Genome-wide linkage analysis with 399 genetic markers revealed significant linkage with at least one marker for 1,300 probes (alpha = 0.001; estimated empirical false discovery rate = 2%). Both contiguous and noncontiguous loci were found to be important in regulating mammalian eye gene expression. We investigated one locus of each type in greater detail and identified putative transcription-altering variations in both cases. We found an inserted cREL binding sequence in the 5' flanking sequence of the Abca4 gene associated with an increased expression level of that gene, and we found a mutation of the gene encoding thyroid hormone receptor beta2 associated with a decreased expression level of the gene encoding short-wavelength sensitive opsin (Opn1sw). In addition to these positional studies, we performed a pairwise analysis of gene expression to identify genes that are regulated in a coordinated manner and used this approach to validate two previously undescribed genes involved in the human disease Bardet-Biedl syndrome. These data and analytical approaches can be used to facilitate the discovery of additional genes and regulatory elements involved in human eye disease.
Analysis of Variance↗
Binding to chaperones allows import of a purified mitochondrial precursor into mitochondria.
Refolding of the acid-unfolded precursor to mitochondrial aspartate aminotransferase (pmAAT) is inhibited when cytosolic Hsc70 is included in the refolding reaction (Artigues, A., Iriarte, A., and Martinez-Carrion, M. (1997) J. Biol. Chem. 272, 16852-16861). At low molar excess of Hsc70 pmAAT is recovered in insoluble aggregates containing equal amounts of Hsc70. However, in the presence of a large excess of Hsc70, refolding of pmAAT is still arrested, but the enzyme remains in solution. Similar behavior was observed with two other cytosolic chaperones, bovine Hsp90 and yeast Ydj1. Coimmunoprecipitation of pmAAT using Hsc70 antibodies confirmed the formation of soluble Hsc70-pmAAT complexes at high concentrations of the chaperone. Data from analytical centrifugation, sedimentation in glycerol gradients, and partial purification of the soluble complexes indicate that multiple Hsc70 molecules bind per pmAAT polypeptide chain. The absence of catalytic activity together with the protease susceptibility of pmAAT bound to Hsc70, Hsp90, or Ydj1 suggest that these chaperones bind and maintain pmAAT in a partially unfolded state, analogous to the import-competent conformation of the protein synthesized in cell-free extracts. Remarkably, the purified pmAAT bound to Hsc70 or Ydj1, but not to Hsp90, is imported by isolated mitochondria in a reticulocyte lysate-dependent manner. Thus, both Hsc70 and Ydj1 can trap an import-competent folding intermediate of pmAAT, but productive binding and import into mitochondria require the collaboration of additional cytosolic factors from the lysate.
Animals↗
All four members of the Ten-m/Odz family of transmembrane proteins form dimers.
Ten-m/Odz/teneurins are a new family of four distinct type II transmembrane molecules. Their extracellular domains are composed of an array of eight consecutive EGF modules followed by a large globular domain. Two of the eight modules contain only 5 instead of the typical 6 cysteine residues and have the capability to dimerize in a covalent, disulfide-linked fashion. The structural properties of the extracellular domains of all four mouse Ten-m proteins have been analyzed using secreted, recombinant molecules produced by mammalian HEK-293 cells. Electron microscopic analysis supported by analytical ultracentrifugation data revealed that the recombinant extracellular domains of all Ten-m proteins formed homodimers. SDS-PAGE analysis under nonreducing conditions as well as negative staining after partial denaturation of the molecules indicated that the globular COOH-terminal domains of Ten-m1 and -m4 contained subdomains with a pronounced stability against denaturing agents, especially when compared with the homologous domains of Ten-m2 and -m3. Cotransfection experiments of mammalian cells with two different extracellular domains revealed that Ten-m molecules have also the ability to form heterodimers, a property that, combined with alternative splicing events, allows the formation of a multitude of molecules with different characteristics from a limited set of genes.
Amino Acid Sequence↗
Free human mitochondrial GrpE is a symmetric dimer in solution.
The co-chaperone GrpE is essential for the activities of the Hsp70 system, which assists protein folding. GrpE is present in several organisms, and characterization of homologous GrpEs is important for developing structure-function relationships. Cloning, producing, and conformational studies of the recombinant human mitochondrial GrpE are reported here. Circular dichroism measurements demonstrate that the purified protein is folded. Thermal unfolding of human GrpE measured both by circular dichroism and differential scanning calorimetry differs from that of prokaryotic GrpE. Analytical ultracentrifugation data indicate that human GrpE is a dimer, and the sedimentation coefficient agrees with an elongated shape model. Small angle x-ray scattering analysis shows that the protein possesses an elongated shape in solution and demonstrates that its envelope, determined by an ab initio method, is similar to the high resolution envelope of Escherichia coli GrpE bound to DnaK obtained from single crystal x-ray diffraction. However, in these conditions, the E. coli GrpE dimer is asymmetric because the monomer that binds DnaK adopts an open conformation. It is of considerable importance for structural GrpE research to answer the question of whether the GrpE dimer is only asymmetric while bound to DnaK or also as a free dimer in solution. The low resolution structure of human GrpE presented here suggests that GrpE is a symmetric dimer when not bound to DnaK. This information is important for understanding the conformational changes GrpE undergoes on binding to DnaK.
Amino Acid Sequence↗
Staphylococcus aureus aminopeptidase S is a founding member of a new peptidase clan.
Staphylococcus aureus aminopeptidase S (AmpS) has been named for its predicted, but experimentally untested, aminopeptidase activity. The enzyme is homologous to biochemically characterized aminopeptidases that contain two cobalt or zinc ions in their active centers, but it is unrelated to all structurally characterized metallopeptidases. Here, we demonstrate AmpS aminopeptidase activity experimentally, and we present the 1.8-A crystal structure of the enzyme. Two metal ions with full occupancy and a third metal ion with low occupancy are present in the active site. A water molecule and Glu-319 serve as bridging ligands to the two metals with full occupancy. One of these metal ions is additionally coordinated by Glu-253 and His-348 and the other by His-381 and Asp-383. In addition, the metals are involved in weak metal-donor interactions to a water molecule and to Tyr-355. In the crystal, AmpS forms a dimer with a large internal cavity. The active sites are located at opposite ends of this internal cavity and are essentially inaccessible from the outside, suggesting that an inactive conformation was crystallized. Because gel filtration and analytical ultracentrifugation data also suggest dimer formation, the problem of substrate access to the active site cavity remains unresolved.
Aminopeptidases↗
Isolation of human alpha-fetoprotein in two fractionation steps and demonstration of homogeneity.
Human alpha-fetoprotein (hAFP) has been isolated from cord serum in 40% yield using an isolation procedure consisting of only two major steps: affinity chromatography followed by preparative polyacrylamide gel electrophoresis (PAGE). The final product appeared homogeneous on the basis of five independent criteria for purity. Sodium dodecyl sulfate gel electrophoresis (SDS-PAGE) demonstrated a single polypeptide chain with molecular weight of 71,000. The protein exhibited an apparent isoelectric point (pI') of 4.85, molecular radius of 3.0 nm and a valence (net H+/molecule) of 21.9 derived from computation of analytical PAGE data. The two-step isolation procedure made it possible for a single operator to isolate milligram amounts of hAFP in a matter of weeks.
Buffers↗
Key elements of food contamination monitoring programmes.
The key elements of programmes for monitoring food contamination are reviewed in relation to their purposes, scope and priorities. Attention is drawn to the requirements and quality assurance of procedures for sampling and analysis, and methods of handling and processing analytical surveillance data. The benefits to be derived from national monitoring programmes are improved food safety, warning of problems of contamination, provision of intake data for evaluation of health hazards, better management and use of natural resources, supply of data on environmental pollution and reliable information on food safety for the public, and provision of a means of checking the effectiveness of regulatory mechanisms and planning technological developments.
Food Contamination↗
The mortality of bacteriophage containing assimilated radioactive phosphorus.
The bacteriophage T4 containing assimilated radioactive phosphorus is inactivated at a rate proportional to the specific radioactivity of the constituent phosphorus. The beta radiation from the phosphorus makes a negligible contribution to this effect. The inactivation is therefore a direct consequence of the nuclear reaction, which kills the phage with an efficiency of about 1/12. Several phages related to T4 behave similarly. When radioactive phage is grown from a seed of non-radioactive phage, all of the phage progeny are subject to killing by radioactive decay. The phage is killed by beta radiation from P(32) with an efficiency of about 1/100 per ionization within the particle volume. Bacteriophage T4 and its relatives contain about 500,000 atoms of phosphorus per infective particle. Virtually all this phosphorus is adsorbed to bacteria with the specificity characteristic of the infective particles, and none of it can be removed from the particles by the enzyme desoxyribonuclease. The phosphorus content per particle, together with the published data on analytical composition, indicates a particle diameter close to 110 mmicro for the varieties of phage studied.
Bacteriophages↗
nf-core/pacsomatic: a scalable somatic analytic pipeline using PacBio HiFi data.
MOTIVATION: Pacific Biosciences (PacBio) HiFi long-read sequencing enables robust characterization of complex genomic regions, repetitive elements, and structural variants (SVs) that are often inaccessible to short-read technologies. To fully leverage HiFi reads to advance cancer genomics and epigenetics, researchers require an end-to-end, scalable and optimized bioinformatics workflow. The nf-core framework meets this need by providing rigorously tested, community-curated pipelines that ensure reproducibility, transparency, and broad compatibility across computational environments. RESULTS: We present nf-core/pacsomatic, an automated Nextflow DSL2 pipeline designed for comprehensive paired tumor-normal somatic analysis using PacBio HiFi data. The workflow includes steps for read alignments against reference genome, somatic SNV/indel, SV, and CNV calling, CpG methylation profiling and differential methylation region (DMR) detection. Additional downstream modules support functional annotation, mutational signature analysis, tumor purity and ploidy estimation, and homologous recombination deficiency (HRD) assessment. Utilizing nf-core's modular design and containerized execution, nf-core/pacsomatic provides a stable framework for the reproducible discovery of biological insights. AVAILABILITY: nf-core/pacsomatic is available under the MIT License at nf-core (https://nf-co.re/pacsomatic) and github (https://github.com/nf-core/pacsomatic).
Software↗
SGMD: the Soybean Genomics and Microarray Database.
The Soybean Genomics and Microarray Database (SGMD) attempts to provide an integrated view of the interaction of soybean with the soybean cyst nematode and contains genomic, EST and microarray data with embedded analytical tools allowing correlation of soybean ESTs with their gene expression profiles. SGMD provides analytical tools to mine the microarray data quickly by integrating many analysis methods within the database itself. The expression profiles of genes at time intervals during the first 8 days of nematode invasion is searchable by gene name or GenBank accession number. Recent developments include the addition of a searchable database for soybean cyst nematode ESTs and photographs of the invasion process at time points examined using microarrays. SGMD is completely accessible from the web at: http://psi081.ba.ars.usda.gov/SGMD/default.htm.
Computational Biology↗
The ribosomal database project (RDP-II): introducing myRDP space and quality controlled public data.
Substantial new features have been implemented at the Ribosomal Database Project in response to the increased importance of high-throughput rRNA sequence analysis in microbial ecology and related disciplines. The most important changes include quality analysis, including chimera detection, for all available rRNA sequences and the introduction of myRDP Space, a new web component designed to help researchers place their own data in context with the RDP's data. In addition, new video tutorials describe how to use RDP features. Details about RDP data and analytical functions can be found at the RDP-II website (http://rdp.cme.msu.edu/).
Databases, Nucleic Acid↗
Puumala virus infections in Finland: increased occupational risk for farmers.
Puumala hantavirus, transmitted by bank voles (Clethrionomys glareolus), causes a mild-type hemorrhagic fever with renal syndrome. The disease is common in Finland and is considered an occupational hazard for farmers, but the actual risk has not been assessed by analytical studies. Data on 5,132 serologically confirmed Puumala virus infections during 1989-1994 were analyzed, and cases among farmers and the population living in similar conditions were compared. The farmers contracted the disease earlier and more often than did the comparison group. In the province of Mikkeli with the highest incidence (70/100,000), the risk ratio was 5.1 (95% confidence interval (CI) 3.0-8.4) for 20- to 29-year-old farmers; in the older age groups, the risk was still increased but the risk ratios were lower. The peak incidence in the comparison group was 10 years later (age group 30-39 years). For the whole country, the result was similar although less marked. The average risk ratio adjusted by age, sex, and geographic variation was 1.7 (95% CI 1.5-1.8) for the whole country and 1.9 (95% CI 1.5-2.3) for the Mikkeli province, where 80% of Puumala virus infections among young farmers could be estimated to be attributable to occupation.
Adult↗
The validity of self-reported health-care utilization by AIDS patients.
OBJECTIVES: To examine the validity of self-reported health-care utilization among persons with AIDS. DESIGN: A comparison of survey data with information collected from medical and financial records. METHODS: Personal interviews provided information on utilization within a 4-month period for inpatient admissions (n = 296), ambulatory visits (n = 284), and hours of homecare (n = 106). Risk group, socioeconomic characteristics, disease stage, functional status, memory, and respondent's recall ability were also measured. Reporting error was defined as the difference between self reports and medical/financial records. Variations among subgroups of patients were examined using t tests and multiple regression. To determine whether reporting errors affected analysis of utilization data, we compared coefficients from parallel utilization models using each data source to predict use/non-use and total utilization. RESULTS: Mean overall reporting errors were small and not significantly different from zero. Reporting errors were lowest for hospital admissions and highest for homecare. High utilizers underreported all types of services. The interviewer evaluation of recall was an independent and significant predictor of reporting errors for admissions and ambulatory visits. Reporting errors varied by selected subgroup characteristics, but the direction and significance of the error depended on the type of utilization measured. In the parallel utilization models, few differences appeared between models using self-reports and medical/financial records to identify correlates of use/non-use, but some differences between the models of total utilization were apparent. CONCLUSIONS: Self-reports of utilization by AIDS patients with a recall period of 4 months or less provide, on average, valid data for analytic purposes. However, caution should be applied to reports by high or low users or by respondents judged by interviewers to have major recall problems.
Acquired Immunodeficiency Syndrome↗
Best practices in NCLEX-RN readiness preparation for baccalaureate student success.
Success for first-time takers of the NCLEX-RN has implications for the students, faculty, and nursing programs. As the passing standard for the NCLEX-RN has risen, some programs have experienced a corresponding decrease in their graduates' first-time pass rates. This article describes one baccalaureate program's journey from low first-time NCLEX-RN pass rates in 2001 to greater student and program success using a data-based, analytical approach. Although passing an exit exam was a program requirement for 20 years, the change to computerized testing and more in-depth test preparation has enhanced opportunities for success for students and reduced stress among faculty. The resulting best practices for preparing students for NCLEX-RN success are described.
Benchmarking↗
Influence of definition and location of hypotension on outcome following severe pediatric traumatic brain injury.
OBJECTIVE: To examine the influence of definition and location (field, emergency department, or pediatric intensive care unit) of hypotension on outcome following severe pediatric traumatic brain injury. DESIGN: Retrospective cohort study. SETTING: Harborview Medical Center (level I pediatric trauma center), Seattle, WA, over a 5-yr period between 1998 and 2003. PATIENTS: Ninety-three children <14 yrs of age with traumatic brain injury following injury, head Abbreviated Injury Score > or = 3, and pediatric intensive care unit admission Glasgow Coma Scale score <9 formed the analytic sample. Data sources included the Harborview Trauma Registry and hospital records. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The relationship between hypotension and outcome was examined comparing two definitions of hypotension: a) systolic blood pressure <5th percentile for age; and b) systolic blood pressure <90 mm Hg. Hospital discharge Glasgow Outcome Score <4 or disposition of either death or discharge to a skilled nursing facility was considered a poor outcome. Pediatric intensive care unit and hospital length of stay were also examined. Systolic blood pressure <5th percentile for age was more highly associated with poor hospital discharge Glasgow Outcome Score (p = .001), poor disposition (p = .02), pediatric intensive care unit length of stay (rate ratio 9.5; 95% confidence interval 6.7-12.3), and hospital length of stay (rate ratio 18.8; 95% confidence interval 14.0-23.5) than systolic blood pressure <90 mm Hg. Hypotension occurring in either the field or emergency department, but not in the pediatric intensive care unit, was associated with poor Glasgow Outcome Score (p = .008), poor disposition (p = .03), and hospital length of stay (rate ratio 18.7; 95% confidence interval 13.1-24.2). CONCLUSIONS: Early hypotension, defined as systolic blood pressure <5th percentile for age in the field and/or emergency department, was a better predictor of poor outcome than delayed hypotension or the use of systolic blood pressure <90 mm Hg.
Abbreviated Injury Scale↗
Genetics and genomics in practice: the continuum from genetic disease to genetic information in health and disease.
This article reviews how the continuum and gradual shift from genetics (study of genes) to genomics (study of the whole genome) in medicine and public health will require reassessment of the traditional approach to delivery of genetic information, namely genetic services. A more general approach is needed to assess the value-added of genetic information for promoting health and for diagnosing, treating, predicting, and preventing all diseases, not only "genetic diseases." The article also discusses how family history can serve as a bridge from genetics to genomics in practice because it reflects the presence, not only of single-gene disorders, but also of shared genes, shared environments, and complex gene-environment interactions. Because of the expected volume of new genetic information, evidence-based practice should increasingly rely on scientific data on analytic performance of such information, its validity in predicting health outcomes, and its utility in improving health and preventing disease beyond approaches that do not use genetic information.
Environment↗