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Fast oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting (HRPF) method used to study protein structure, protein-ligand interactions, and protein-protein interactions. FPOP utilizes a KrF excimer laser at 248 nm for photolysis of hydrogen peroxide to generate hydroxyl radicals which in turn oxidatively modify solvent-accessible amino acid side chains. Recently, we expanded the use of FPOP of in vivo oxidative labeling in Caenorhabditis elegans (C. elegans), entitled IV-FPOP. The transparent nematodes have been used as model systems for many human diseases. Structural studies in C. elegans by IV-FPOP is feasible because of the animal's ability to uptake hydrogen peroxide, their transparency to laser irradiation at 248 nm, and the irreversible nature of the modification. The assembly of a microfluidic flow system for IV-FPOP labeling, IV-FPOP parameters, protein extraction, and LC-MS/MS optimized parameters are described herein.

Fast photochemical oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting method used to characterize protein structure and interactions. FPOP uses a 248 nm excimer laser to photolyze hydrogen peroxide producing hydroxyl radicals. These radicals oxidatively modify solvent exposed side chains of 19 of the 20 amino acids. Recently, this method has been used in live cells (IC-FPOP) to study protein interactions in their native environment. The study of proteins in cells accounts for intermolecular crowding and various protein interactions that are disrupted for in vitro studies. A custom single cell flow system was designed to reduce cell aggregation and clogging during IC-FPOP. This flow system focuses the cells past the excimer laser individually, thus ensuring consistent irradiation. By comparing the extent of oxidation produced from FPOP to the protein's solvent accessibility calculated from a crystal structure, IC-FPOP can accurately probe the solvent accessible side chains of proteins.

Using a national sample of youth from the U.S., this paper examines incidents of bias-based harassment by peers that include language about victims' perceived sexual orientation, race/ethnicity, religion, weight or height, or intelligence. Telephone interviews were conducted with youth who were 10-20 years old (n = 791). One in six youth (17%) reported at least one experience with bias-based harassment in the past year. Bias language was a part of over half (52%) of all harassment incidents experienced by youth. Perpetrators of bias-based harassment were similar demographically to perpetrators of non-biased harassment. However, bias-based incidents were more likely to involve multiple perpetrators, longer timeframes and multiple harassment episodes. Even controlling for these related characteristics, the use of bias language in incidents of peer harassment resulted in significantly greater odds that youth felt sad as a result of the victimization, skipped school, avoided school activities, and lost friends, compared to non-biased harassment incidents., (Copyright © 2018 The Foundation for Professionals in Services for Adolescents. Published by Elsevier Ltd. All rights reserved.)

Purpose: To determine the prevalence of youth exposure to medication or pill overdose by someone close to them, as well as how common this is within the spectrum of major stressful events and child victimization experienced by youth., Design and Methods: Data were collected as part of the Third National Survey of Children's Exposure to Violence, a nationally representative telephone survey of youth, ages 2-17years (N=3738) conducted in 2013. The analytical subset for the current paper is youth ages 10-17years (n=1959)., Results: Estimates indicate that approximately 1 in 12 youth (8%), ages 10-17 have been exposed to medication overdose by someone close to them in their lifetimes. Overdose exposure is related to recent trauma symptoms, alcohol and other substance use. However, these relationships appear to be largely driven by the co-existence of major stressful events these youth are experiencing. Alcohol use is the exception; exposure to medication overdose continues to be related to past year personal alcohol use even after adjusting for other lifetime stressful events., Conclusions: Having a close family member or friend overdose on a medication is a common experience among U.S. youth and related to high rates of co-occurring stressful events., Practice Implications: Health care providers should be aware that youth exposure to medication overdoses likely indicates exposure to other recognized adversities. Youth with a caregiver who has had an overdose may require an urgent response including referral to crisis intervention through child and family services., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Deep venous thrombosis (DVT) is common in intensive care unit (ICU) patients. It is often silent and may be complicated by pulmonary embolism and death. Thromboprophylaxis with heparin does not always prevent venous thromboembolism (VTE). Aspirin (ASA) reduces the risk of VTE in surgical and high-risk medical patients but it is unknown if ASA may prevent DVT in mechanically ventilated ICU patients. We performed a retrospective chart review of critically ill patients who received mechanical ventilation for >72 h and underwent venous ultrasonography for suspected DVT between Jan 2012 and Dec 2013. We excluded patients who were on therapeutic doses of anticoagulation or had coagulopathy. We used multivariable logistic regression to evaluate association between aspirin use and DVT during hospitalization. There were 193 patients. The mean ± SD age was 58 ± 15.7 years. Half were male. DVT was found in 49 (25.4%). DVT was found in the first 15 days of hospitalization in 67.3% of the patients. The majority (82.8%) received thromboprophylaxis with unfractionated or low molecular weight heparin. Fifty-six (29%) were on ASA. On multivariable regression analysis, ASA use was associated with a significant reduction in the odds of finding DVT (OR 0.39, 95% CI 0.16-0.94; p = 0.036). DVT is common in mechanically ventilated ICU patients despite the use of thromboprophylaxis. Aspirin may prevent DVT in such patients.

Hydroxyl radical protein footprinting coupled with mass spectrometry has become an invaluable technique for protein structural characterization. In this method, hydroxyl radicals react with solvent exposed amino acid side chains producing stable, covalently attached labels. Although this technique yields beneficial information, the extensive list of known oxidation products produced make the identification and quantitation process considerably complex. Currently, the methods available for analysis either involve manual analysis steps, or limit the amount of searchable modifications or the size of sequence database. This creates a bottleneck which can result in a long and arduous analysis process, which is further compounded in a complex sample. Here, we report the use of a new footprinting analysis method for both peptide and residue-level analysis, demonstrated on the GCaMP2 synthetic construct in calcium free and calcium bound states. This method utilizes a customized multi-search node workflow developed for an on-market search platform in conjunction with a quantitation platform developed using a free Excel add-in. Moreover, the method expedites the analysis process, requiring only two post-search hours to complete quantitation, regardless of the size of the experiment or the sample complexity.

Bullying prevention is increasingly targeting education to bystanders, but more information is needed on the complexities of bystander actions across a wide variety of incidents, including both online and in-person peer harassment. The current study analyzes victim report data from a nationally representative survey of youth ages 10-20 (n = 791; 51% female). Bystander presence was common across all harassment incident types (80% of incidents). In contrast to previous research, our study found that supportive bystander behaviors occurred at relatively high rates. Unfortunately, antagonistic bystander behaviors, although less common, were predictive of higher negative impact for the victim. A large percentage of victims (76%) also disclosed the harassment to confidants, who play an important role as secondary bystanders. While friends were the most common confidant, incidents were also disclosed to adults at high rates (60%) and with mostly positive results. The findings suggest that prevention programs could increase their impact by targeting education to both direct witnesses and confidants, and considering a wider variety of peer victimization incident types.

Background: Combination therapy is commonly used for pulmonary arterial hypertension (PAH) treatment. We aimed to identify factors that may predict the need for future combination therapy., Methods: We conducted a retrospective chart review of consecutive patients with PAH in an aim to describe baseline clinical, echocardiogram, and hemodynamic characteristics of patients who eventually required combination therapy during the course of their disease and compared them to the ones who were maintained on monotherapy., Results: The monotherapy group was followed for an average of 31.8 ± 18.8 months and the combination therapy group was followed for an average of 28.7 ± 13.6 months. Among the 71 patients analyzed, a significantly higher number of patients who eventually required combination therapy belonged to World Health Organization functional class 3 (45% vs 37%) and 4 (23% vs 0) at baseline, compared with those on monotherapy (P < .05). Combination group also had a higher Registry to Evaluate Early And Long-term PAH Disease Management (REVEAL) PAH risk score at presentation. End of 6-minute walk test (6MWT), oxygen saturation (Spo 2) was also lower in the combination therapy group, 86% ± 8% versus 91% ± 7% (P < .05). Patients who eventually required combination therapy were more frequently noticed to have right ventricular enlargement, right atrial enlargement, and had a higher resting estimated right ventricular systolic pressure (RVSP). Right heart catheterization-derived hemodynamics data at baseline showed that the combination therapy group had a higher mean pulmonary artery (PA) pressure, lower pulmonary capillary wedge pressure, lower cardiac output, and higher pulmonary vascular resistance (PVR). On univariate analysis, only PVR ≥300 dyne·s/cm(5), mean PA pressure of ≥40 mm Hg, estimated RVSP ≥ 60 mm Hg, PAH risk score ≥ 10, and end of 6MWT saturation of ≤ 90% were of significance., Conclusion: Patients with PAH who require combination therapy in the course of their disease have worse hemodynamics, PAH risk score, functional class, and end of 6MWT oxygen saturation at the time of presentation compared to patients maintained on monotherapy., (© The Author(s) 2015.)

Peptides containing the oxidation products of hydroxyl radical-mediated protein footprinting experiments are typically much less abundant than their unoxidized counterparts. This is inherent to the design of the experiment as excessive oxidation may lead to undesired conformational changes or unfolding of the protein, skewing the results. Thus, as the complexity of the systems studied using this method expands, the detection and identification of these oxidized species can be increasingly difficult with the limitations of data-dependent acquisition (DDA) and one-dimensional chromatography. Here we report the application of multidimensional protein identification technology (MudPIT) in combination with hydroxyl radical footprinting as a method to increase the identification of quantifiable peptides in these experiments. Using this method led to a 37% increase in unique peptide identifications as well as a 70% increase in protein group identifications over one-dimensional data-dependent acquisition on the same samples. Furthermore, we demonstrate the combination of these methods as a means to investigate megadalton complexes.

As therapeutic monoclonal antibodies (mAbs) become a major focus in biotechnology and a source of the next-generation drugs, new analytical methods or combination methods are needed for monitoring changes in higher order structure and effects of post-translational modifications. The complexity of these molecules and their vulnerability to structural change provide a serious challenge. We describe here the use of complementary mass spectrometry methods that not only characterize mutant mAbs but also may provide a general framework for characterizing higher order structure of other protein therapeutics and biosimilars. To frame the challenge, we selected members of the IgG2 subclass that have distinct disulfide isomeric structures as a model to evaluate an overall approach that uses ion mobility, top-down MS sequencing, and protein footprinting in the form of fast photochemical oxidation of proteins (FPOP). These three methods are rapid, sensitive, respond to subtle changes in conformation of Cys → Ser mutants of an IgG2, each representing a single disulfide isoform, and may be used in series to probe higher order structure. The outcome suggests that this approach of using various methods in combination can assist the development and quality control of protein therapeutics.

Two recent chapters in professional books have criticized children's advocacy centers for creating role conflict for mental health professionals because of their work with criminal justice and child protection professionals in children's advocacy centers as part of a coordinated response to child abuse. This article argues that these critiques misunderstand children's advocacy center practice and overestimate the risk of role conflict. Children's advocacy center standards set a boundary between forensic interviewing and therapy, which in most children's advocacy centers are done by separate professionals and never by the same professional for a given child. Many mental health professionals serve children's advocacy centers as consultants with no treatment role. Children's advocacy center therapists are rarely involved in investigation, and their participation in multidisciplinary teams focuses on children's interests and well-being.

This article explores the variety of ways in which the Internet is used to facilitate the commercial sexual exploitation of children (CSEC) and provides national incidence estimates for the number of arrests involving such technology-facilitated crimes in 2006. The National Juvenile Online Victimization Study is a nationally representative longitudinal study of more than 2,500 local, county, state, and federal law enforcement agencies across the United States. The current article utilizes Wave 2 data, which surveyed arrests in 2006 for Internet-related sex crimes against minors. Detailed data were collected via telephone interviews with investigators about 1,051 individual arrest cases. Findings show that an estimated 569 arrests for Internet-facilitated commercial sexual exploitation of children (IF-CSEC) occurred in the United States in 2006. Offenders in IF-CSEC cases fell into two main categories: (1) those who used the Internet to purchase or sell access to identified children for sexual purposes including child pornography (CP) production (36% of cases), and (2) those who used the Internet to purchase or sell CP images they possessed but did not produce (64% of cases). Offenders attempting to profit from child sexual exploitation were more likely than those who were purchasing to have (a) prior arrests for sexual and nonsexual offenses, (b) a history of violence, (c) produced CP, (d) joined forces with other offenders, and (e) involved female offenders. Although the number of arrests for IF-CSEC crimes is relatively small, the victims of these crimes are a high-risk subgroup of youth, and the offenders who try to profit from these crimes are particularly concerning from a child welfare perspective.

Fast photochemical oxidation of proteins (FPOP) is a mass spectrometry-based protein footprinting method that modifies proteins on the microsecond time scale. Highly reactive (•)OH, produced by laser photolysis of hydrogen peroxide, oxidatively modifies the side chains of approximately one-half the common amino acids on this time scale. Because of the short labeling exposure, only solvent-accessible residues are sampled. Quantification of the modification extent for the apo and holo states of a protein-ligand complex provides structurally sensitive information at the amino-acid level to compare the structures of unknown protein complexes with known ones. We report here the use of FPOP to monitor the structural changes of calmodulin in its established binding to M13 of the skeletal muscle myosin light chain kinase. We use the outcome to establish the unknown structures resulting from binding with melittin and mastoparan. The structural comparison follows a comprehensive examination of the extent of FPOP modifications as measured by proteolysis and LC-MS/MS for each protein-ligand equilibrium. The results not only show that the three calmodulin-peptide complexes have similar structures but also reveal those regions of the protein that became more or less solvent-accessible upon binding. This approach has the potential for relatively high throughput, information-dense characterization of a series of protein-ligand complexes in biochemistry and drug discovery when the structure of one reference complex is known, as is the case for calmodulin and M13 of the skeletal muscle myosin light chain kinase, and the structures of related complexes are not.