Pfam is a collection of multiple alignments and profile hidden Markov models of protein domain families. Release 3.1 is a major update of the Pfam database and contains 1313 families which are ...available on the World Wide Web in Europe at http://www.sanger.ac.uk/Software/Pfam/ and http://www.cgr.ki.se/Pfam/, and in the US at http://pfam.wustl.edu/. Over 54% of proteins in SWISS-PROT-35 and SP-TrEMBL-5 match a Pfam family. The primary changes of Pfam since release 2.1 are that we now use the more advanced version 2 of the HMMER software, which is more sensitive and provides expectation values for matches, and that it now includes proteins from both SP-TrEMBL and SWISS-PROT.
Formalin-inactivated respiratory syncytial virus (RSV) vaccine preparations have been shown to cause enhanced disease in naive hosts following natural infection. In this study we demonstrate a ...similar pattern of enhanced disease severity following primary RSV infection of IFN-nonresponsive STAT1(-/-) mice. STAT1(-/-) mice showed markedly increased illness compared with wild-type BALB/c animals following RSV inoculation despite similar lung virus titers and rates of virus clearance. Histologically, STAT1(-/-) animals had eosinophilic and neutrophilic pulmonary infiltrates not present in wild-type or IFN-gamma(-/-)-infected mice. In cytokine analyses of infected lung tissue, IFN-gamma was induced in both STAT1(-/-) and wild-type mice, with preferential IL-4, IL-5, and IL-13 induction only in the STAT1(-/-) animals. Eotaxin was detected in the lungs of both wild-type and STAT1(-/-) mice following infection, with a 1.7-fold increase over wild-type in the STAT1(-/-) mice. Using a peptide epitope newly identified in the RSV fusion protein, we were able to demonstrate that wild-type memory CD4(+) T cells stimulated by this peptide produce primarily IFN-gamma, while STAT1(-/-)CD4(+) cells produce primarily IL-13. These findings suggest that STAT1 activation by both type I (alphabeta) and type II (gamma) IFNs plays an important role in establishing a protective, Th1 Ag-specific immune response to RSV infection.
To determine the risk of significant injury associated with premature graduation of young (2- to 5-year-old) children to seat belts from child restraint systems (CRS).
Advocates recommend use of ...child safety seats for children younger than age 4 and booster seats for children age 4 and older. Despite these recommendations, many children are prematurely taken out of these child restraints and placed in seat belts. Although data exist to support the use of child restraints over nonrestraint, no real-world data exist to evaluate the risk of significant injury associated with premature use of seat belts.
Partners for Child Passenger Safety includes a child-focused crash surveillance system based on a representative sample of children ages 0 to 15 years in crashes involving 1990 and newer vehicles reported to State Farm Insurance Companies in 15 states and the District of Columbia. Driver reports of crash circumstances and parent reports of child occupant injury were collected via telephone interview using validated surveys. Results were weighted based on sampling frequencies to represent the entire population.
Between December 1, 1998, and November 30, 1999, 2077 children aged 2 to 5 years were included and were weighted to represent 13 853 children. Among these young children, 98% were restrained, but nearly 40% of these children were restrained in seat belts. Compared with children in CRS, children in seat belts were more likely to suffer a significant injury (relative risk: 3.5; 95% confidence interval: 2. 4, 5.2). Children in seat belts were at particular risk of significant head injuries (relative risk: 4.2; 95% confidence interval: 2.6, 6.7) when compared with children in CRS.
Premature graduation of young children from CRS to seat belts puts them at greatly increased risk of injury in crashes. A major benefit of CRS is a reduction in head injuries, potentially attributable to a reduction in the amount of head excursion in a crash.
Top-down proteomics is capable of identifying and quantitating unique proteoforms through the analysis of intact proteins. We extended the coverage of the label-free technique, achieving differential ...analysis of whole proteins <30 kDa from the proteomes of growing and senescent human fibroblasts. By integrating improved control software with more instrument time allocated for quantitation of intact ions, we were able to collect protein data between the two cell states, confidently comparing 1577 proteoform levels. To then identify and characterize proteoforms, our advanced acquisition software, named Autopilot, employed enhanced identification efficiency in identifying 1180 unique Swiss-Prot accession numbers at 1% false-discovery rate. This coverage of the low mass proteome is equivalent to the largest previously reported but was accomplished in 23% of the total acquisition time. By maximizing both the number of quantified proteoforms and their identification rate in an integrated software environment, this work significantly advances proteoform-resolved analyses of complex systems.
Purpose: To systematically analyze evidence about the outcome and percent of newly completed ADs, focusing on the effectiveness of (a) types of educational interventions versus controls and (b) one ...educational intervention over another.
Design: Systematic review of literature based on Cochrane review criteria.
Methods: Twelve randomized and four nonrandomized studies were selected from the nursing, medical, and social work literature that met the following criteria: described educational interventions, provided information to calculate the percent of newly completed ADs as an outcome, and published between 1991 and 2009. The review focused primarily on randomized studies. Reviewers calculated the percent of newly completed ADs by determining the number of subjects per group without an AD at baseline and the percentage of those who then completed one by the end of the studies.
Findings: Findings were inconsistent regarding all types of educational interventions studied versus controls. Sufficient evidence exists to conclude that combined written and verbal educational interventions were more effective than single written interventions in increasing the percent of newly completed ADs in adult clinic outpatients and hospitalized elderly.
Conclusions: Calculating the percent of newly completed ADs was successful in allowing for study result comparisons. Overall, the evidence base regarding the effectiveness of single or combined educational interventions in increasing AD completion is weak. Randomized studies with diverse samples should be conducted against controls before more studies comparing interventions are undertaken.
Clinical Relevance: This article provides nurses with a summary of research related to educational interventions and AD completion and identifies where future study is needed.
Advances in genetics and sequencing have identified a plethora of disease-associated and disease-causing genetic alterations. To determine causality between genetics and disease, accurate models for ...molecular dissection are required; however, the rapid expansion of transcriptional populations identified through single-cell analyses presents a major challenge for accurate comparisons between mutant and wild-type cells. Here we generate mouse models of human severe congenital neutropenia (SCN) using patient-derived mutations in the GFI1 transcription factor. To determine the effects of SCN mutations, we generated single-cell references for granulopoietic genomic states with linked epitopes
, aligned mutant cells to their wild-type equivalents and identified differentially expressed genes and epigenetic loci. We find that GFI1-target genes are altered sequentially, as cells go through successive states of differentiation. These insights facilitated the genetic rescue of granulocytic specification but not post-commitment defects in innate immune effector function, and underscore the importance of evaluating the effects of mutations and therapy within each relevant cell state.
Despite significant reductions in the number of children killed in motor vehicle crashes over the past decade, crashes continue to be the leading cause of death for children 4 years and older. ...Therefore, the American Academy of Pediatrics continues to recommend inclusion of child passenger safety anticipatory guidance at every health-supervision visit. This technical report provides a summary of the evidence in support of 5 recommendations for best practices to optimize safety in passenger vehicles for children from birth through adolescence that all pediatricians should know and promote in their routine practice. These recommendations are presented in the revised policy statement on child passenger safety in the form of an algorithm that is intended to facilitate their implementation by pediatricians with their patients and families. The algorithm is designed to cover the majority of situations that pediatricians will encounter in practice. In addition, a summary of evidence on a number of additional issues that affect the safety of children in motor vehicles, including the proper use and installation of child restraints, exposure to air bags, travel in pickup trucks, children left in or around vehicles, and the importance of restraint laws, is provided. Finally, this technical report provides pediatricians with a number of resources for additional information to use when providing anticipatory guidance to families.
Knowledge of the complete genomic DNA sequence of an organism allows a systematic approach to defining its genetic components. The genomic sequence provides access to the complete structures of all ...genes, including those without known function, their control elements, and, by inference, the proteins they encode, as well as all other biologically important sequences. Furthermore, the sequence is a rich and permanent source of information for the design of further biological studies of the organism and for the study of evolution through cross-species sequence comparison. The power of this approach has been amply demonstrated by the determination of the sequences of a number of microbial and model organisms. The next step is to obtain the complete sequence of the entire human genome. Here we report the sequence of the euchromatic part of human chromosome 22. The sequence obtained consists of 12 contiguous segments spanning 33.4 megabases, contains at least 545 genes and 134 pseudogenes, and provides the first view of the complex chromosomal landscapes that will be found in the rest of the genome.
The stability of antibody–drug conjugates (ADCs) in circulation is critical for maximum efficacy and minimal toxicity. An ADC reaching the intended target intact can deliver the highest possible drug ...load to the tumor and reduce off-target toxicity from free drug in the blood. As such, assessment of ADC stability is a vital piece of data during development. However, traditional ADC stability assays can be manually intensive, low-throughput, and require large quantities of ADC material. Here, we introduce an automated, high-throughput plasma stability assay for screening drug release and aggregation over 144 h for up to 40 ADCs across five matrices simultaneously. The amount of ADC material during early drug development is often limited, so this assay was implemented in 384-well format to minimize material requirements to <100 μg of each ADC and 100 μL of plasma per species type. Drug release and aggregation output were modeled using nonlinear regression equations to calculate formation rates for each data type. A set of 15 ADCs with different antibodies and identical valine–citrulline–p-aminobenzylcarbamate–monomethylauristatin E linker-drug payloads was tested and formation rates were compared across ADCs and between species, revealing several noteworthy trends. In particular, a wide range in aggregation was found when altering only the antibody, suggesting a key role for plasma stability screening early in the development process to find and remove antibody candidates with the potential to create unstable ADCs. The assay presented here can be leveraged to provide stability data on new chemistry and antibody screening initiatives, select the best candidate for in vivo studies, and provide results that highlight stability issues inherent to particular ADC designs throughout all stages of ADC development.
Obtaining extensive sequencing of an intact protein is essential in order to simultaneously determine both the nature and exact localization of chemical and genetic modifications which distinguish ...different proteoforms arising from the same gene. To effectively achieve such characterization, it is necessary to take advantage of the analytical potential offered by the top-down mass spectrometry approach to protein sequence analysis. However, as a protein increases in size, its gas-phase dissociation produces overlapping, low signal-to-noise fragments. The application of advanced ion dissociation techniques such as electron transfer dissociation (ETD) and ultraviolet photodissociation (UVPD) can improve the sequencing results compared to slow-heating techniques such as collisional dissociation; nonetheless, even ETD- and UVPD-based approaches have thus far fallen short in their capacity to reliably enable extensive sequencing of proteoforms ≥30 kDa. To overcome this issue, we have applied proton transfer charge reduction (PTCR) to limit signal overlap in tandem mass spectra (MS2) produced by ETD (alone or with supplemental ion activation, EThcD). Compared to conventional MS2 experiments, following ETD/EThcD MS2 with PTCR MS3 prior to m/z analysis of deprotonated product ions in the Orbitrap mass analyzer proved beneficial for the identification of additional large protein fragments (≥10 kDa), thus improving the overall sequencing and in particular the coverage of the central portion of all four analyzed proteins spanning from 29 to 56 kDa. Specifically, PTCR-based data acquisition led to 39% sequence coverage for the 56 kDa glutamate dehydrogenase, which was further increased to 44% by combining fragments obtained via HCD followed by PTCR MS3.
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