Hydroxyl Radical Protein Footprinting: A Mass Spectrometry-Based Structural Method for Studying the Higher Order Structure of Proteins.

Hydroxyl radical protein footprinting (HRPF) coupled to mass spectrometry has been successfully used to investigate a plethora of protein-related questions. The method, which utilizes hydroxyl radicals to oxidatively modify solvent-accessible amino acids, can inform on protein interaction sites and regions of conformational change. Hydroxyl radical-based footprinting was originally developed to study nucleic acids, but coupling the method with mass spectrometry has enabled the study of proteins. The method has undergone several advancements since its inception that have increased its utility for more varied applications such as protein folding and the study of biotherapeutics. In addition, recent innovations have led to the study of increasingly complex systems including cell lysates and intact cells. Technological advances have also increased throughput and allowed for better control of experimental conditions. In this review, we provide a brief history of the field of HRPF and detail recent innovations and applications in the field.

Efficient Analysis of Proteome-Wide FPOP Data by FragPipe.

Monitoring protein structure before and after environmental alterations (e.g., different cell states) can give insights into the role and function of proteins. Fast photochemical oxidation of proteins (FPOP) coupled with mass spectrometry (MS) allows for monitoring of structural rearrangements by exposing proteins to OH radicals that oxidize solvent-accessible residues, indicating protein regions undergoing movement. Some of the benefits of FPOP include high throughput and a lack of scrambling due to label irreversibility. However, the challenges of processing FPOP data have thus far limited its proteome-scale uses. Here, we present a computational workflow for fast and sensitive analysis of FPOP data sets. Our workflow, implemented as part of the FragPipe computational platform, combines the speed of the MSFragger search with a unique hybrid search method to restrict the large search space of FPOP modifications. Together, these features enable more than 10-fold faster FPOP searches that identify 150% more modified peptide spectra than previous methods. We hope this new workflow will increase the accessibility of FPOP to enable more protein structure and function relationships to be explored.

Men and women experience different barriers to care seeking for fecal incontinence.

BACKGROUND: Despite effective treatments for fecal incontinence (FI), FI remains underdiagnosed and undertreated. This study sought to characterize and compare rates of, delays in, experiences with, and barriers to care seeking for FI among men and women. METHODS: This study was a secondary analysis of electronic survey data collected from adults with FI. The survey included the use of a validated instrument to assess FI severity and questions that now comprise a validated instrument for assessing barriers to care seeking for FI in women. Descriptive analyses characterized differences between men and women. χ2 testing for categorical variables and t-testing for normally distributed continuous variables compared statistical differences. RESULTS: The sample (N = 548) was predominately female (84%), non-Hispanic White (90%), and insured (96%), with a mean Vaizey score (13.4 ± 5.3) consistent with moderate or severe FI. Care seeking rates (p = 0.81) and symptom duration before care seeking (p = 0.23) did not differ between women and men, but women were more likely than men to be told that effective treatments exist; most male and female respondents who sought care were offered treatment. Very few respondents had been asked about FI by a healthcare provider. Whereas normative thinking, limited life impact, and believing that a healthcare provider could not help were more common barriers to care seeking among men, avoidance, fear, and discouragement were more common in women. CONCLUSIONS: Men and women with FI seek care at similar rates and after experiencing symptoms for a similar duration of time. Very few patients with FI have been screened for it by a healthcare provider. Barriers to FI care seeking are different for women and men, and men are less likely than women to be informed about effective treatments by a healthcare provider.

The mental health of officials who regularly examine child sexual abuse material: strategies for harm mitigation.

BACKGROUND: The current study aims to better understand the mental health and subjective well-being of investigators and forensic examiners exposed to child sexual abuse material (CSAM) by examining which components of this work are associated with elevated mental health conditions and decreased well-being, as well as the intra-personal and organizational variables that may mitigate harm and improve well-being. METHODS: Police investigators, forensic examiners, and others connected with the criminal justice system from across the United States who were exposed to CSAM as part of their professions (N = 500) completed an anonymous online survey. Participants were recruited through connections with the National Criminal Justice Training Center. RESULTS: Duration, frequency, amount, and content of CSAM exposure was not related to poorer mental health with the exception of exposure to violent CSAM which was related to elevated post-traumatic stress symptoms. Several agency-level practices and policies, such as the availability of an Officer Wellness Program and more frequently knowing the final case resolution, were related to better mental health and well-being. Harm mitigation strategies, such as talking to other officers investigating the case and taking breaks from the material being viewed, were also related to better mental health. CONCLUSIONS: Findings indicate that police agencies have options for implementing agency-level procedures and practices that have the potential to reduce the negative impact of CSAM investigations. Additionally, many investigators use strategies that are correlated with greater well-being, suggesting opportunities for improving training programs.

Diffusion effects of a sexual violence prevention program leveraging youth-adult partnerships.

The purpose of the current study was to examine the diffusion effects of a youth-led sexual violence prevention program (i.e., Youth Voices in Prevention [Youth VIP]). Specifically, social network analysis was used to measure the extent to which Youth VIP changed behaviors for 1172 middle and high school youth who did not attend program events but were friends with Youth VIP participants and completed the first and final survey (approximately 2 years apart). Findings suggest that there was considerable interpersonal communication about Youth VIP among the students generated by program participation. Specifically, youth with friends who participated in Youth VIP were more likely to report hearing their friends talk about Youth VIP and reported talking to their friends about Youth VIP compared with those not connected to Youth VIP participants. However, there were no diffusion effects found for behavioral outcomes (i.e., bystander intervention behavior, violence victimization, and perpetration). Given the mixed findings, further research is needed to determine the extent to which youth-led sexual violence prevention initiatives lead to changes in broader community-wide changes in youths' behaviors.

THE MAKING OF A FOOTPRINT IN PROTEIN FOOTPRINTING: A REVIEW IN HONOR OF MICHAEL L. GROSS.

Within the past decade protein footprinting in conjunction with mass spectrometry has become a powerful and versatile means to unravel the higher order structure of proteins. Footprinting-based approaches has demonstrated the capacity to inform on interaction sites and dynamic regions that participate in conformational changes. These findings when set in a biological perspective inform on protein folding/unfolding, protein-protein interactions, and protein-ligand interactions. In this review, we will look at the contribution of Dr. Michael L. Gross to protein footprinting approaches such as hydrogen deuterium exchange mass spectrometry and hydroxyl radical protein footprinting. This review details the development of novel footprinting methods as well as their applications to study higher order protein structure. © 2020 The Authors. Mass Spectrometry Reviews published by John Wiley & Sons Ltd. Mass Spec Rev.

Voices of Youth and Emerging Adults on Suicide Prevention: Experiences and Expectations.

Self-directed violence (SDV) is a significant public health issue for adolescents and emerging adults, and yet youth exposure to prevention messaging and youth perspectives on SDV prevention needs are understudied. The current study sought to better understand the ways in which a national sample of youth and emerging adults were exposed to suicide prevention programs or conversations. A sample of 1031 young people ages 13-23 were recruited nationally through social media. Survey questions asked about SDV prevention exposure. Open-ended questions asked youth to suggest additional information they desired about SDV. A majority of participants (87%) reported that they had received prevention exposure from at least one source (i.e., family, online, attending a talk, or formal program) with few differences by demographic characteristics. However, sexual and gender minority (SGM) youth reported accessing more SDV prevention information online compared to other youth. Overall, youth had many ideas about what additional information they needed, including how to help someone at risk for SDV and how to access information about mental health. While the majority of youth are receiving some SDV prevention messages, there is variation in how they get this information, and survey participants still felt they were missing important information. Findings highlight the need to resource more comprehensive SDV prevention for youth and young adults.

Evaluating the Impact of a Youth-Led Sexual Violence Prevention Program: Youth Leadership Retreat Outcomes.

Involving youth in developing and implementing prevention programs to reduce sexual violence (SV) has the potential to improve prevention outcomes. However, there has been little focus on youth-led SV prevention programs, and limited evaluation research to help guide efforts. The current study examined the effectiveness of Youth Voices in Prevention (Youth VIP) leadership retreats on SV victimization and perpetration, forms of violence related to SV (e.g., bullying), SV bystander behaviors and readiness, and perceptions of norms related to SV prevention. Results identified mixed findings for program impact, with variations in outcomes that can help guide future youth-led prevention program initiatives. Youth attending a large "kick-off" leadership retreat (that was less youth-led that subsequent smaller retreats) later reported more bystander behaviors, but also reported increased perpetration and victimization, compared to non-attending youth. However, youth attending smaller, more focused leadership retreats held during the school year, reported reductions in sexual harassment perpetration and improved bystander behaviors and attitudes compared to non-attending youth. Evaluation of moderator variables suggests that program impact was generally stronger for younger participants, sexual minority youth, and non-White youth (which were largely Native American youth in this sample). Findings suggest promise for youth-led prevention work but also highlight the need for testing the impact of different training structures and modalities. Clinical trials number: NCT03207386.

Predictors of Multidisciplinary Team Sustainability in Work With Child Sex Trafficking Cases.

A coordinated response by a trained multidisciplinary team (MDT) can help support child sex trafficking (CST) victims, but little is known about factors that influence the development and sustainability of MDTs in this work. An online survey was conducted with 171 professionals who attended a Multidisciplinary Team Child Sex Trafficking (MDT-CST) training to identify factors related to team growth. Increased MDT success was related to: (1) the presence of a CST-specific advocacy organization in the community; (2) other community agencies active in supporting CST victims (e.g., SANE nurses, faith-based organizations, and runaway shelters); (3) a greater breadth of professional representation on the MDT; and (4) agency leadership support for the CST action plan. Most of the MDTs sustained and increased their coordination with other community agencies over time, but the study identified that growth is improved when administrators support team efforts and there are resources and supports for CST victims elsewhere in the community.

The utilization of the search engine, Bolt, to decrease search time and increase peptide identifications in hydroxyl radical protein footprinting-based workflows.

Hydroxyl radical protein footprinting (HRPF) utilizes hydroxyl radicals to covalently modify solvent exposed regions of proteins. When coupled with mass spectrometry, HRPF can provide insightful information on protein structural changes including inside cells. However, the complex mixture of proteins and modifications makes identification a complicated task. To search all of the HRPF-induced modification combinations across the full proteome, requires substantial computational power and still can take days to search. To drastically decrease processing time and improve identifications, a novel cloud-based search engine, Bolt, was used to search for HRPF modifications in comparison to a commonly used search engine, Sequest. A 35% increase in the identification of modified peptides was observed in Bolt compared to Sequest with a decrease in computation time.

Factors Related to Providers Screening Children for Behavioral Health Risks in Primary Care Settings.

PURPOSE: To provide information from a large sample of pediatric and family medicine primary care providers on practices in screening children for behavioral health risks. DESIGN AND METHODS: Participants were a sample of physicians (n=319) and nurse practitioners (n=292) from across the U.S. who completed a confidential online survey about screening practices through a computer-assisted self-interview. RESULTS: Almost all respondents (89%) reported screening children for depression/anxiety and behavior problems. Child substance use (82%), family social support (74%), significant household changes (73%), bullying (72%), child abuse (62%) and domestic violence (52%) were also asked about regularly, although with high rates of informal screening methods. Caregiver mental health (49%), caregiver substance use (35%), family financial strain (33%) and transportation difficulties (27%) were screened less frequently. Screening was associated with higher rates of referral for risk-related problems, and was more likely when providers reported greater confidence providing support to clients, perceived community resource availability as higher, and worked in systems with integrated primary care and behavioral health. CONCLUSIONS: Findings suggest a great amount of diversity in how providers screen for behavioral health risks. There is reluctance to screen when options for addressing the problems are seen as limited. Research is needed to better guide healthcare providers in determining the right context and methods for screening social risks. PRACTICE IMPLICATIONS: Protocols for screening adverse childhood events (ACES) and other social risk factors should be accompanied by adequate training and efforts to improve community resource and support networks.

Insights on the Conformational Ensemble of Cyt C Reveal a Compact State during Peroxidase Activity.

Cytochrome c (cyt c) is known for its role in the electron transport chain but transitions to a peroxidase-active state upon exposure to oxidative species. The peroxidase activity ultimately results in the release of cyt c into the cytosol for the engagement of apoptosis. The accumulation of oxidative modifications that accompany the onset of the peroxidase function are well-characterized. However, the concurrent structural and conformational transitions of cyt c remain undercharacterized. Fast photochemical oxidation of proteins (FPOP) coupled with mass spectrometry is a protein footprinting technique used to structurally characterize proteins. FPOP coupled with native ion mobility separation shows that exposure to H2O2 results in the accumulation of a compact state of cyt c. Subsequent top-down fragmentation to localize FPOP modifications reveals changes in heme coordination between conformers. A time-resolved functional assay suggests that this compact conformer is peroxidase active. Altogether, combining FPOP, ion mobility separation, and top-down and bottom-up mass spectrometry allows us to discern individual conformations in solution and obtain a better understanding of the conformational ensemble and structural transitions of cyt c as it transitions from a respiratory role to a proapoptotic role.

Chemical Penetration Enhancers Increase Hydrogen Peroxide Uptake in C. elegans for In Vivo Fast Photochemical Oxidation of Proteins.

Fast photochemical oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting method that covalently labels solvent-accessible amino acids by photolysis of hydrogen peroxide. Recently, we expanded the use of FPOP for in vivo (IV-FPOP) covalent labeling in C. elegans. In initial IV-FPOP studies, 545 proteins were oxidatively modified in all body systems within the worm. Here, with the use of chemical penetration enhancers (CPEs), we increased the number of modified proteins as well as the number of modifications per protein to gain more structural information. CPEs aid in the delivery of hydrogen peroxide inside C. elegans by disturbing the highly ordered lipid bilayer of the worm cuticle without affecting worm viability. IV-FPOP experiments performed using the CPE azone showed an increase in oxidatively modified proteins and peptides. This increase correlated with greater hydrogen peroxide uptake by C. elegans quantified using a chemical fluorophore demonstrating the efficacy of using CPEs with IV-FPOP. Mass spectrometry proteomics data are available via ProteomeXchange with identifier PXD019290.

Fast photochemical oxidation of proteins (FPOP): A powerful mass spectrometry-based structural proteomics tool.

Fast photochemical oxidation of proteins (FPOP) is a MS-based method that has proved useful in studies of protein structures, interactions, conformations, and protein folding. The success of this method relies on the irreversible labeling of solvent-exposed amino acid side chains by hydroxyl radicals. FPOP generates these radicals through laser-induced photolysis of hydrogen peroxide. The data obtained provide residue-level resolution of protein structures and interactions on the microsecond timescale, enabling investigations of fast processes such as protein folding and weak protein-protein interactions. An extensive comparison between FPOP and other footprinting techniques gives insight on their complementarity as well as the robustness of FPOP to provide unique structural information once unattainable. The versatility of this method is evidenced by both the heterogeneity of samples that can be analyzed by FPOP and the myriad of applications for which the method has been successfully used: from proteins of varying size to intact cells. This review discusses the wide applications of this technique and highlights its high potential. Applications including, but not limited to, protein folding, membrane proteins, structure elucidation, and epitope mapping are showcased. Furthermore, the use of FPOP has been extended to probing proteins in cells and in vivo These promising developments are also presented herein.

In Vivo Fast Photochemical Oxidation of Proteins Using Enhanced Multiplexing Proteomics.

In vivo fast photochemical oxidation of proteins (IV-FPOP) is a hydroxyl radical protein footprinting method used to study protein structure and protein-protein interactions. Oxidatively modified proteins by IV-FPOP are analyzed by mass spectrometry (MS), and the extent of oxidation is quantified by label-free MS. Peptide oxidation changes yield useful information about protein structure, due to changes in solvent accessibility. However, the sample size necessary for animal studies requires increased sample preparation and instrument time. Here, we report the combined application of IV-FPOP and the enhanced multiplexing strategy combined precursor isotopic labeling and isobaric tagging (cPILOT) for higher-throughput analysis of oxidative modifications in C. elegans. Key differences in the performance of label-free MS and cPILOT were identified. The addition of oxygen (+16) was the most abundant modification identified among all known possible FPOP modifications. This study presents IV-FPOP coupled with enhanced multiplexing strategies such as cPILOT to increase throughput of studies seeking to examine oxidative protein modifications.

Implementing In-Cell Fast Photochemical Oxidation of Proteins in a Platform Incubator with a Movable XY Stage.

Fast photochemical oxidation of proteins (FPOP) is a protein footprinting technique that is being increasingly used in MS-based proteomics. FPOP is utilized to study protein-protein interactions, protein-ligand interactions, and protein conformational dynamics. This method has recently been extended to protein labeling in live cells (IC-FPOP), allowing the study of protein conformations in the complex cellular environment. Traditionally, IC-FPOP has been executed using a single cell flow system, in which hydrodynamic focusing drives cells along in a single file line, keeping the cells from clumping and thus ensuring equal exposure to the laser irradiation required for photochemical oxidation. Here, we introduce a novel platform that allows IC-FPOP to occur in a sterile incubation system complete with a mobile stage for XY movement, peristaltic pumps equipped with perfusion lines for chemical transport, and mirrors for laser beam guidance. This new system, called Platform Incubator with movable XY stage (PIXY), also utilizes software enabling automated communication between equipment and execution of the entire system. Further, comparison with a standard IC-FPOP flow system results reveal that this platform can successfully be used in lieu of the flow system while also decreasing the time to complete analysis of a single sample.

Mass spectrometry-based methods for structural biology on a proteome-wide scale.

Mass spectrometry (MS) has long been used to study proteins mainly via sequence identification and quantitation of expression abundance. In recent years, MS has emerged as a tool for structural biology. Intact protein structural analysis has been enabled by the development of methods such as native MS, top-down proteomics, and ion mobility MS. Other MS-based structural methods include affinity purification MS, chemical cross-linking, and protein footprinting. These methods have enabled the study of protein-protein and protein-ligand interactions and regions of conformational change. The coupling of MS with liquid chromatography has permitted the analysis of complex samples. This bottom-up proteomics workflow enables the study of protein structure in the native cellular environment and provides structural information across the proteome. It has been demonstrated that the crowded environment of the cell affects protein binding interactions and affinities. Performing studies in this complex environment is essential for understanding the functional roles of proteins. MS-based structural methods permit analysis of samples such as cell lysates, intact cells, and tissue to provide a more physiological view of protein structure. This mini-review discusses the various MS-based methods that can be used for proteome-wide structural studies and highlights some of their application.

Image-guided hypofractionated double-scattering proton therapy in the management of centrally-located early-stage non-small cell lung cancer.

BACKGROUND: The treatment of centrally-located early-stage non-small cell lung cancer (NSCLC) with image-guided stereotactic body radiotherapy (SBRT) is challenging due to the proximity of critical normal structures to the tumor target. The purpose of this study was to report the results of our experience in treating centrally-located early-stage NSCLC with hypofractionated proton therapy (PT). MATERIAL AND METHODS: Between 2009 and 2018, 23 patients with T1-T2N0M0 NSCLC (T1, 46%; T2, 54%) were treated with image-guided hypofractionated double-scattering PT. The median age at the time of treatment was 74 years (range, 58-88). Patients underwent 4-dimensional computed tomography (CT) simulation following fiducial marker placement, and daily image guidance was performed. All patients were treated with 60 GyRBE in 10 fractions. Patients were assessed for CTCAEv4 toxicities weekly during treatment, and at regular follow-up intervals with CT imaging for tumor assessment. Overall survival, cause-specific survival, local control, regional control, and metastases-free survival were evaluated using cumulative incidence with competing risks. RESULTS: Median follow-up for all patients was 3.2 years (range, 0.2-9.2 years). Overall survival rates at 3 and 5 years were 81% and 50% (95% CI, 27-79%), respectively. Cause-specific survival rates at 3 and 5 years were 81% and 71% (95% CI, 46-92%). The 3-year local, regional, and distant control rates were 90%, 81%, and 87%, respectively. Three patients (13%) experienced local recurrences as their first recurrence, at a median time of 28 months from completion of radiation (range, 18-61 months). Two patients (9%) experienced late grade 3 toxicities, including 1 patient who developed a bronchial stricture that required stent placement. CONCLUSION: Image-guided hypofractionated PT for centrally-located early-stage NSCLC provides excellent local control with low rates of grade ≥3 toxicities. For tumors in sensitive locations, PT may provide safer treatment than photon-based treatments due to its dosimetric advantages.

Exposure to Multiple Forms of Bias Victimization on Youth and Young Adults: Relationships with Trauma Symptomatology and Social Support.

Research has documented that a significant portion of youth are exposed to bias victimization. However, less is known about whether experiencing certain types of bias victimization (e.g., sexual orientation bias) is more or less likely to be related to a more extensive bias victimization history (i.e., experiencing multiple types of bias victimization) and whether exposure to multiple types of bias victimization explains any relationships between specific types of bias victimization and negative outcomes. To address these gaps, the current study explores relationships between exposure to multiple types of bias-motivated victimization, trauma symptomatology and perceived social support. Participants were 854 youth and young adults (60.9% female) from three higher risk communities who completed a survey on personal experiences with bias-related victimization. The average age of participants was 16.6 years; 28.5% of the sample described themselves as Black or African American; 13.4% as Hispanic or Latino (any race); 45.3% as White, and 12.8% as another race. Sixty-nine percent of the sample described their sexual orientation as heterosexual; 8.9% as gay, lesbian, or homosexual; 12.5% as bisexual; and 9.5% as another sexual orientation. Sixty-three percent of participants reported at least one type of bias victimization in their lifetime, and more than one in three youth (38.7%) experienced two or more types of bias victimization in their lifetimes (18.1% two types, 12.1% three types, and 8.5% four or more types). Experiencing multiple types of bias victimization was related to higher trauma symptomatology and less perceived social support. Experiencing multiple types of bias victimization attenuated or eliminated the association between individual types of bias victimization and well-being. The findings contribute to a growing body of research demonstrating the damaging mental health effects of occupying multiple marginalized statuses, and points to the cumulation of bias victimization experiences as an important factor contributing to significant differences in well-being and support among youth and young adults.