STRAP PTM: Software Tool for Rapid Annotation and Differential Comparison of Protein Post-Translational Modifications.

The identification of protein post-translational modifications (PTMs) is an increasingly important component of proteomics and biomarker discovery, but very few tools exist for performing fast and easy characterization of global PTM changes and differential comparison of PTMs across groups of data obtained from liquid chromatography-tandem mass spectrometry experiments. STRAP PTM (Software Tool for Rapid Annotation of Proteins: Post-Translational Modification edition) is a program that was developed to facilitate the characterization of PTMs using spectral counting and a novel scoring algorithm to accelerate the identification of differential PTMs from complex data sets. The software facilitates multi-sample comparison by collating, scoring, and ranking PTMs and by summarizing data visually. The freely available software (beta release) installs on a PC and processes data in protXML format obtained from files parsed through the Trans-Proteomic Pipeline. The easy-to-use interface allows examination of results at protein, peptide, and PTM levels, and the overall design offers tremendous flexibility that provides proteomics insight beyond simple assignment and counting.

Dynamics of enzymatic digestion of elastic fibers and networks under tension.

We study the enzymatic degradation of an elastic fiber under tension using an anisotropic random-walk model coupled with binding-unbinding reactions that weaken the fiber. The fiber is represented by a chain of elastic springs in series along which enzyme molecules can diffuse. Numerical simulations show that the fiber stiffness decreases exponentially with two distinct regimes. The time constant of the first regime decreases with increasing tension. Using a mean field calculation, we partition the time constant into geometrical, chemical and externally controllable factors, which is corroborated by the simulations. We incorporate the fiber model into a multiscale network model of the extracellular matrix and find that network effects do not mask the exponential decay of stiffness at the fiber level. To test these predictions, we measure the force relaxation of elastin sheets stretched to 20% uniaxial strain in the presence of elastase. The decay of force is exponential and the time constant is proportional to the inverse of enzyme concentration in agreement with model predictions. Furthermore, the fragment mass released into the bath during digestion is linearly related to enzyme concentration that is also borne out in the model. We conclude that in the complex extracellular matrix, feedback between the local rate of fiber digestion and the force the fiber carries acts to attenuate any spatial heterogeneity of digestion such that molecular processes manifest directly at the macroscale. Our findings can help better understand remodeling processes during development or in disease in which enzyme concentrations and/or mechanical forces become abnormal.

A human surfactant peptide-elastase inhibitor construct as a treatment for emphysema.

Two million Americans suffer from pulmonary emphysema, costing $2.5 billion/year and contributing to 100,000 deaths/year. Emphysema is thought to result from an imbalance between elastase and endogenous inhibitors of elastase, leading to tissue destruction and a loss of alveoli. Decades of research have still not resulted in an effective treatment other than stopping cigarette smoking, a highly addictive behavior. On the basis of our previous work, we hypothesize that small molecule inhibitors of human neutrophil elastase are ineffective because of rapid clearance from the lungs. To develop a long-acting elastase inhibitor with a lung pharmacodynamic profile that has minimal immunogenicity, we covalently linked an elastase inhibitor, similar to a trifluoro inhibitor that was used in clinical trials, to a 25-amino-acid fragment of human surfactant peptide B. We used this construct to prevent human neutrophil elastase-induced emphysema in a rodent model. The elastase inhibitor alone, although in a 70-fold molar excess to elastase in a mixture with <0.6% residual elastase activity, provided no protection from elastase-induced emphysema. Covalently combining an endogenous peptide from the target organ with a synthetic small molecule inhibitor is a unique way of endowing an active compound with the pharmacodynamic profile needed to create in vivo efficacy.

A computational approach for deciphering the organization of glycosaminoglycans.

BACKGROUND: Increasing evidence has revealed important roles for complex glycans as mediators of normal and pathological processes. Glycosaminoglycans are a class of glycans that bind and regulate the function of a wide array of proteins at the cell-extracellular matrix interface. The specific sequence and chemical organization of these polymers likely define function; however, identification of the structure-function relationships of glycosaminoglycans has been met with challenges associated with the unique level of complexity and the nontemplate-driven biosynthesis of these biopolymers. METHODOLOGY/PRINCIPAL FINDINGS: To address these challenges, we have devised a computational approach to predict fine structure and patterns of domain organization of the specific glycosaminoglycan, heparan sulfate (HS). Using chemical composition data obtained after complete and partial digestion of mixtures of HS chains with specific degradative enzymes, the computational analysis produces populations of theoretical HS chains with structures that meet both biosynthesis and enzyme degradation rules. The model performs these operations through a modular format consisting of input/output sections and three routines called chainmaker, chainbreaker, and chainsorter. We applied this methodology to analyze HS preparations isolated from pulmonary fibroblasts and epithelial cells. Significant differences in the general organization of these two HS preparations were observed, with HS from epithelial cells having a greater frequency of highly sulfated domains. Epithelial HS also showed a higher density of specific HS domains that have been associated with inhibition of neutrophil elastase. Experimental analysis of elastase inhibition was consistent with the model predictions and demonstrated that HS from epithelial cells had greater inhibitory activity than HS from fibroblasts. CONCLUSIONS/SIGNIFICANCE: This model establishes the conceptual framework for a new class of computational tools to use to assess patterns of domain organization within glycosaminoglycans. These tools will provide a means to consider high-level chain organization in deciphering the structure-function relationships of polysaccharides in biology.

New insights into the inhibition of human neutrophil elastase by heparin.

In the normal feedback mechanism of injury and repair in the lung, fragmented heparan sulfate proteoglycans (HSPGs) from damaged extracellular matrix and cells are believed to interact with elastases to limit their activity. An imbalance in the HSPG-elastase response may play an important role in situations where uncontrolled lung injury leads to diseases such as emphysema. To gain insight into this complex process of heparin and heparan sulfate regulation of elastases, an experimental study was undertaken to resolve the mechanism and structural requirements of heparin inhibition of human neutrophil elastase (HNE). Kinetic analyses were completed using in vitro assays with synthetic and insoluble elastin substrates in the presence of HNE and various heparin preparations (14-15 kDa; 17-19 kDa), heparin-derived oligosaccharides (4-22 saccharides), and chemically modified heparins (2-O-, 6-O-, O-, and N-desulfated). Results showed that heparin inhibits HNE by a tight-binding, hyperbolic, competitive mechanism, contrary to previous reports in the literature. A minimum length of at least 12-14 saccharides is required for inhibition, after which inhibitory activity increases with chain length (or molecular mass). Although all N- and O-sulfate groups contribute to inhibition, 2-O-sulfate groups are less critical than either N- or 6-O-sulfate groups, indicating that inhibitory activity is dependent upon the heparin fine structure. Molecular-docking simulations support the kinetic results and provide a plausible model for the size requirement, whereby positively charged, clamp-like regions at the ends of the interdomain crevice (elastase fold) are used by heparin to bridge the active site and inhibit activity.

Contrasting perspectives on pain following hand injury.

The purposes of this article are 1) to compare contrasting ways of documenting pain, 2) to identify the usefulness of information each method provides, and 3) to examine implications for hand therapy. Participants are tracked for 12 months in an ongoing study of Adaptation to Hand Injury that incorporates quantitative measures of both intensity and functional impact of pain, as well as semistructured qualitative Adaptation Interviews that elicit and document patients' responses to pain and injury experience. Each method of documenting pain tells us something important. Numeric measures of pain intensity (Visual Analog Scale) allow us to document change over time and identify relationships between intensity and self-reported changes in daily life as documented in a structured questionnaire (Disabilities of Arm, Shoulder, and Hand). Qualitative interviews provide an individual perspective on responses to pain and disability and the personal beliefs and experiences that influenced the responses of each unique individual. Findings support the importance of studying more about the clinical behaviors of our patients for better treatment outcomes and recommend the use of qualitative research in helping us to uncover this knowledge.

Examining the Community Integration Measure (CIM): a replication study with life satisfaction.

OBJECTIVE: To examine instrument reliability, validity, factor structure, and conceptual underpinnings of the Community Integration Measure (CIM) with the Community Integration Questionnaire Revised (CIQ-R) and the Satisfaction with Life Scale (SWLS). DESIGN: A replication study. SETTING: Community living. PARTICIPANTS: Ninety-one participants: 51 individuals with a brain injury, and 40 without brain injury. MAIN OUTCOMES: Internal consistency, criterion validity, construct validity, discriminant validity factor structure. RESULTS: CIM items produced standardized alphas ranging from 0.72 to 0.83. Significant positive correlations were found among the CIM and both the CIQ-R and SWLS, with the CIM performing better with the SWLS than did the CIQ-R. The CIM discriminated between subject samples as well as by living arrangement. The factor loading solution revealed a three-factor model that explained 63.72 percent of the variance. CONCLUSION: The CIM shows good promise for capturing an individual's perception of community integration. The factor structure supports the original Model of Community Integration. Further research is needed to examine the influence of objective items on the CIM and Model of Community Integration.

Adaptation to hand injury: an evolving experience.

OBJECTIVES: Objectives of this study were to examine similarities and differences in physical recovery and psychosocial adaptation, engagement in occupations and relationships, perceived outcomes and expectations, and adaptive issues and strategies of 5 participants from an ongoing longitudinal study of adaptation to hand injury. METHODS: Participants were tracked for 12 months using quantitative measures of physical recovery and psychosocial adaptation, and qualitative adaptation interviews focused on the impact of an injury experience in clients' daily lives. A computerized graphic format documented changes over time in key quantitative and qualitative indicators in individual Adaptation Trajectories. RESULTS: Findings included the importance of motivating occupations and relationships, changes over time in expectations for the future, and differences between independent and interdependent adaptive strategies following hand injury. DISCUSSION: Connections between the International Classification of Functioning and Disability domains of body systems, activity capabilities, and social participation were examined. Findings support the value of individualized, occupation-based therapy that addresses the mind and spirit as well as physical recovery in occupational therapy practice with hand injury clients.

Functional outcomes and daily life activities of African-American elders after hospitalization.

OBJECTIVE: African-American elders were recruited from a transitional unit after hospitalization and tracked for 6 months in the community after discharge to (a) examine functional outcomes on the unit and in the community and (b) identify patterns of participation in daily life activities. The International Classification of Impairments, Disabilities, and Handicaps (ICIDH-2) framework provided the structure to examine the connections among body systems, functional outcomes, and social participation for this population that has been underrepresented in past research. METHOD: A mixed design combined qualitative and quantitative methods, including qualitative interviews to document personal adaptive experience, a standardized functional assessment to identify functional outcomes, and a structured format to record activity participation. RESULTS: Findings revealed that 11 of the 17 participants improved their functional outcomes after discharge. Three patterns of activity participation identified were self-care, self-care and household management, and mixed activities. Contextual influences were diverse family support arrangements. CONCLUSION: Complex relationships were identified among body systems, functional outcomes, and daily life activities that were influenced by individual values and support arrangements.