Exploration of Nitrotyrosine-Containing Proteins and Peptides by Antibody-Based Enrichment Strategies.

Nitrotyrosine, or 3-nitrotyrosine, is an oxidative post-translational modification induced by reactive nitrogen species. Although nitrotyrosine is considered a marker of oxidative stress and has been associated with inflammation, neurodegeneration, cardiovascular disease, and cancer, identification of nitrotyrosine-modified proteins remains challenging owing to its low stoichiometric levels in biological samples. To facilitate a comprehensive analysis of proteins and peptides containing nitrotyrosine, we optimized an immunoprecipitation-based enrichment workflow using a cell line model. The identification of proteins and peptides containing nitrotyrosine residues was carried out after peroxynitrite treatment of cell lysates, which generated modified nitrotyrosine residues on susceptible sites on proteins. We evaluated the efficacy of enriching nitrotyrosine-modified proteins and peptides by employing four different commercially available monoclonal antibodies directed against nitrotyrosine. LC-MS/MS analysis resulted in the identification of 1377 and 1624 nitrotyrosine-containing peptides from protein- and peptide-based enrichment experiments, respectively. Although the yield of nitrotyrosine-containing peptides was higher in experiments where peptides rather than proteins were enriched, we found a substantial proportion (37-65%) of identified nitrotyrosine-containing peptides contained nitrotyrosine at the N-terminus. However, in protein-based immunoprecipitation <9% of nitrotyrosine-containing peptides had nitrotyrosine modification at the N-terminus of the peptide. Overall, our study resulted in the identification of 2603 nitrotyrosine-containing peptides of which >2000 have not previously been reported. We synthesized 101 novel nitrotyrosine-containing peptides identified in our analysis and analyzed them by LC-MS/MS to validate our findings. We have confirmed the validity of 70% of these peptides, as they demonstrated a similarity score exceeding 0.7 when compared to peptides identified through experimental methods. Finally, we also validated the presence of nitrotyrosine modification on PKM and EF2 proteins in peroxynitrite-treated samples by immunoblot analysis. The large catalog presented in this study along with the workflow should facilitate the investigation of nitrotyrosine as an oxidative modification in a variety of settings in greater detail.

13C15N: glucagon-based novel isotope dilution mass spectrometry method for measurement of glucagon metabolism in humans.

BACKGROUND: Glucagon serves as an important regulatory hormone for regulating blood glucose concentration with tight feedback control exerted by insulin and glucose. There are critical gaps in our understanding of glucagon kinetics, pancreatic α cell function and intra-islet feedback network that are disrupted in type 1 diabetes. This is important for translational research applications of evolving dual-hormone (insulin + glucagon) closed-loop artificial pancreas algorithms and their usage in type 1 diabetes. Thus, it is important to accurately measure glucagon kinetics in vivo and to develop robust models of glucose-insulin-glucagon interplay that could inform next generation of artificial pancreas algorithms. METHODS: Here, we describe the administration of novel 13C15N heavy isotope-containing glucagon tracers-FF glucagon [(Phe 6 13C9,15N; Phe 22 13C9,15N)] and FFLA glucagon [(Phe 6 13C9,15N; Phe 22 13C9,15N; Leu 14 13C6,15N; Ala 19 13C3)] followed by anti-glucagon antibody-based enrichment and LC-MS/MS based-targeted assays using high-resolution mass spectrometry to determine levels of infused glucagon in plasma samples. The optimized assay results were applied for measurement of glucagon turnover in subjects with and without type 1 diabetes infused with isotopically labeled glucagon tracers. RESULTS: The limit of quantitation was found to be 1.56 pg/ml using stable isotope-labeled glucagon as an internal standard. Intra and inter-assay variability was < 6% and < 16%, respectively, for FF glucagon while it was < 5% and < 23%, respectively, for FFLA glucagon. Further, we carried out a novel isotope dilution technique using glucagon tracers for studying glucagon kinetics in type 1 diabetes. CONCLUSIONS: The methods described in this study for simultaneous detection and quantitation of glucagon tracers have clinical utility for investigating glucagon kinetics in vivo in humans.

DIA-Based Proteome Profiling of Nasopharyngeal Swabs from COVID-19 Patients.

Since the recent outbreak of COVID-19, there have been intense efforts to understand viral pathogenesis and host immune response to combat SARS-CoV-2. It has become evident that different host alterations can be identified in SARS-CoV-2 infection based on whether infected cells, animal models or clinical samples are studied. Although nasopharyngeal swabs are routinely collected for SARS-CoV-2 detection by RT-PCR testing, host alterations in the nasopharynx at the proteomic level have not been systematically investigated. Thus, we sought to characterize the host response through global proteome profiling of nasopharyngeal swab specimens. A mass spectrometer combining trapped ion mobility spectrometry (TIMS) and high-resolution QTOF mass spectrometer with parallel accumulation-serial fragmentation (PASEF) was deployed for unbiased proteome profiling. First, deep proteome profiling of pooled nasopharyngeal swab samples was performed in the PASEF enabled DDA mode, which identified 7723 proteins that were then used to generate a spectral library. This approach provided peptide level evidence of five missing proteins for which MS/MS spectrum and mobilograms were validated with synthetic peptides. Subsequently, quantitative proteomic profiling was carried out for 90 individual nasopharyngeal swab samples (45 positive and 45 negative) in DIA combined with PASEF, termed as diaPASEF mode, which resulted in a total of 5023 protein identifications. Of these, 577 proteins were found to be upregulated in SARS-CoV-2 positive samples. Functional analysis of these upregulated proteins revealed alterations in several biological processes including innate immune response, viral protein assembly, and exocytosis. To the best of our knowledge, this study is the first to deploy diaPASEF for quantitative proteomic profiling of clinical samples and shows the feasibility of adopting such an approach to understand mechanisms and pathways altered in diseases.

Synthesis of Vorinostat and cholesterol conjugate to enhance the cancer cell uptake selectivity.

Histone deacetylase (HDAC) inhibitors modulate various cellular functions including proliferation, differentiation, and apoptosis. Vorinostat (SuberAniloHydroxamic Acid, SAHA) is the first HDAC inhibitor approved by FDA for cancer treatment. However, SAHA distributes in cancer tissue and normal tissue in similar levels. It will be ideal to selectively deliver SAHA into cancer cells. Rapidly growing cancer cells have a great need of cholesterol. Low-density lipoprotein (LDL) is the major cholesterol carrier in plasma and its uptake is mediated by LDL-receptor (LDL-R), a glycoprotein overexpressed on the surface of cancer cells. Herein, we designed and synthesized a SAHA cholesterol conjugate, and further formed the conjugate containing particles with LDL as the carrier. The diameters of the particles were determined. The inhibitory activity of the particles carrying the conjugate was determined with cancer cell proliferation assay, and the hydrolysis of the conjugate by the enzymes in cancer cells was confirmed with LC-MS/MS.

International network of cancer genome projects.

The International Cancer Genome Consortium (ICGC) was launched to coordinate large-scale cancer genome studies in tumours from 50 different cancer types and/or subtypes that are of clinical and societal importance across the globe. Systematic studies of more than 25,000 cancer genomes at the genomic, epigenomic and transcriptomic levels will reveal the repertoire of oncogenic mutations, uncover traces of the mutagenic influences, define clinically relevant subtypes for prognosis and therapeutic management, and enable the development of new cancer therapies.

The Role and Influence of Faith Leaders on Health-Related Issues and Programs in their Congregation.

This qualitative study explored the influence of faith leaders on health-related issues within their congregation. Semi-structured interviewers with 24 faith leaders found that chronic conditions and poor health behaviors were the top health challenges facing their congregation. A majority mentioned health-related activities taking place at their church. Most believed they had influence on their congregation for issues related to health/wellness, most commonly in the form of increasing awareness. A majority talked about the importance of being a role model. It is important to understand how to most effectively capitalize on the strengths of and engage pastors in health promotion efforts.

Diversity of post-translational modifications and cell signaling revealed by single cell and single organelle mass spectrometry.

The rapid evolution of mass spectrometry-based single-cell proteomics now enables the cataloging of several thousand proteins from single cells. We investigated whether we could discover cellular heterogeneity beyond proteome, encompassing post-translational modifications (PTM), protein-protein interaction, and variants. By optimizing the mass spectrometry data interpretation strategy to enable the detection of PTMs and variants, we have generated a high-definition dataset of single-cell and nuclear proteomic-states. The data demonstrate the heterogeneity of cell-states and signaling dependencies at the single-cell level and reveal epigenetic drug-induced changes in single nuclei. This approach enables the exploration of previously uncharted single-cell and organellar proteomes revealing molecular characteristics that are inaccessible through RNA profiling.

Leading their flocks to health? Clergy health and the role of clergy in faith-based health promotion interventions.

Faith-based organizations are a frequent partner in health promotion due to their large and expansive reach across multiple demographics of the United States. These faith-based organizations are led by clergy members who have a strong influence over their institutions and who shape the physical and social environments of their institutions for health-related matters. The purpose of this review was to examine current issues associated with the health, behaviors, and well-being of clergy, highlight the literature on the role clergy play in delivering effective health promotion interventions, and present recommendations for improving clergy health and the involvement of clergy in faith-based initiatives.

Perceptions of Midwest rural women related to their physical activity and eating behaviors.

The study aim was to describe the perceptions of 65 Midwestern rural women related to healthy eating, physical activity, and weight management. A semistructured interview guide was used to elicit data. Theory of planned behavior constructs were used to categorize the data into 4 predominant themes related to healthy lifestyle behaviors, (a) knowledge and attitudes, (b) rural cultural influences, (c) facilitators, and (d) barriers. Analyses revealed that facilitators and barriers consisted of social and environmental factors, and personal life situations. Results suggest key elements for developing and implementing effective physical activity and weight management interventions for Midwestern rural women.

The NIH Human Microbiome Project.

The Human Microbiome Project (HMP), funded as an initiative of the NIH Roadmap for Biomedical Research (http://nihroadmap.nih.gov), is a multi-component community resource. The goals of the HMP are: (1) to take advantage of new, high-throughput technologies to characterize the human microbiome more fully by studying samples from multiple body sites from each of at least 250 "normal" volunteers; (2) to determine whether there are associations between changes in the microbiome and health/disease by studying several different medical conditions; and (3) to provide both a standardized data resource and new technological approaches to enable such studies to be undertaken broadly in the scientific community. The ethical, legal, and social implications of such research are being systematically studied as well. The ultimate objective of the HMP is to demonstrate that there are opportunities to improve human health through monitoring or manipulation of the human microbiome. The history and implementation of this new program are described here.

The mammalian gene function resource: the International Knockout Mouse Consortium.

In 2007, the International Knockout Mouse Consortium (IKMC) made the ambitious promise to generate mutations in virtually every protein-coding gene of the mouse genome in a concerted worldwide action. Now, 5 years later, the IKMC members have developed high-throughput gene trapping and, in particular, gene-targeting pipelines and generated more than 17,400 mutant murine embryonic stem (ES) cell clones and more than 1,700 mutant mouse strains, most of them conditional. A common IKMC web portal (www.knockoutmouse.org) has been established, allowing easy access to this unparalleled biological resource. The IKMC materials considerably enhance functional gene annotation of the mammalian genome and will have a major impact on future biomedical research.

One theoretical framework for cardiovascular disease prevention in women.

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality for women in the United States. Vulnerable and underserved women are even less likely to participate in healthy lifestyle behaviors to prevent CVD than the general US population. Many women are not aware that they are at risk for CVD and do not modify unhealthy lifestyle behaviors to reduce their risks for CVD. Healthcare providers may not promote awareness of CVD or consistently counsel female patients concerning lifestyle behavior modifications to reduce their risks for CVD. "The Evidence-Based Guidelines for the Prevention of Cardiovascular Disease in Women 2011 Update: A Guideline From the American Heart Association" provides a guide for healthcare providers to help women lower their risk for heart disease. By using a theoretical approach, nurses can facilitate implementation of these evidence-based guidelines to help women reduce their risks for CVD. Commonly used intrapersonal, interpersonal, and organizational- and community-level health behavior change theories that can be used to effectively promote CVD risk reduction in women will be discussed. The theoretical basis for changing CVD risk behaviors in women can be applied by using the PRECEDE-PROCEED Model to guide planning, implementing, and evaluating health behavior programs. The purpose of this article was to help nurses improve healthcare delivery by using the PRECEDE-PROCEED Model as a theoretical framework to guide CVD risk reduction efforts for women.

Choose to Move for Positive Living: physical activity program for obese women.

The Choose to Move for + (Positive) Living program was implemented to increase physical activity among obese women. A holistic approach was used to promote stage of health behavior change, social support, and quality of life and reduce depression. Within 6 months, physical fitness improved and depressive symptoms decreased.

Self-Care for Advanced Practice Nursing Students in Rural Primary Care.

BACKGROUND: Advanced practice RNs (APRNs) working in rural primary care provide a spectrum of health care needs that can lead to professional burnout. As a preventative for burnout, the Institute of Medicine developed a strategy focused on self-care. Understanding the importance of self-care as a preventive for building professional resilience to manage workplace stressors during students' academic years may improve retention. METHOD: Twenty-two APRN students participated in a rural primary care immersion course with a specific component on self-care. Preand postsurveys, student journaling, class discussions, and a posttraining focus group were used to assess students' progress. RESULTS: Students reported strengthening patient and team relationships, enhanced interpersonal communication, and increased self-awareness of stressors for managing their emotions. These self-imposed processes improved student confidence, job satisfaction, and workplace resilience. CONCLUSION: Heath-promoting behaviors via self-care during APRN students' formal education may assist them in their clinical practice as rural primary care providers. [J Nurs Educ. 2022;61(4):187-191.].

Decreasing pressure ulcers across a healthcare system: moving beneath the tip of the iceberg.

The authors describe a pressure ulcer prevention program implemented across a large healthcare system in Minnesota. An interprofessional team of representatives from 10 hospitals developed a bundle of interventions directed at measurement standardization, provider education, patient/family education and point-of-care resources for providers, timely nutritional assessment, and a novel Skin Day event intended to increase awareness. The number of pressure ulcers reported to the State of Minnesota decreased 33% after implementation of the program with a potential cost savings of up to $430,000.

The uptake of Human Papillomavirus (HPV) vaccine among adolescent females in the United States: a review of the literature.

A comprehensive review of literature was conducted to identify the barriers and facilitators, from the parents'/guardians' and primary care providers' (PCPs) perspective, that are associated with the uptake of Human Papillomavirus (HPV) vaccine among adolescent females. Findings from 14 peer-reviewed articles indicate that 37% of adolescent females, 9 to 17 years of age, initiated the HPV vaccine, compared to the national average of 13- to 15-years-olds, which is 50%. The overall uptake and completion rates of the vaccine were low across all racial and ethnic groups. Barriers included parents'/guardians' knowledge, perceptions, and attitudes toward the HPV disease and the vaccine, along with the convenience in receiving the vaccine. Six studies reported that HPV vaccine initiation was highly likely if the parents/guardians had received a doctor's recommendation. Learning what interventions school nurses currently utilize could positively affect the HPV vaccine uptake and completion rates by other nursing disciplines realizing this ongoing effort.

Evaluation of the Heart and Soul Physical Activity Program by African American women.

The purpose of this study was to determine the perceptions of African American (AA) women regarding an active lifestyle, and to evaluate the Heart and Soul Physical Activity Program (HSPAP) as a potential strategy to promote physical activity. The HSPAP is a church-based physical activity intervention conceptualized in appraisal, belonging, tangible and self-esteem domains of social support. Seven midlife, sedentary AA women from a Midwestern urban church participated in the group discussion after completing the HSPAP. Guiding questions were utilized to solicit their attitudes about physical activity and the HSPAP. The study participants believed that physical activity improves health and prevents chronic diseases however, their primary responsibility is to family and jobs, leaving little time or energy for their personal health needs. They further believed that physical activity would increase if recommended by health professionals and encouraged by family, friends, and church members; and, that spiritual messages and prayer would strengthen their commitment to attain an active lifestyle.

Digging deeper into the immunopeptidome: characterization of post-translationally modified peptides presented by MHC I.

Peptides presented by MHC molecules on the cell surface, or the immunopeptidome, play an important role in the adaptive arm of the immune response. Antigen processing for MHC class I molecules is a ubiquitous pathway present in all nucleated cells which generates and presents peptides of both self and non-self-origin. Peptides with post-translational modifications represent one category of peptides presented by MHC class I molecules. However, owing to the complexity of self-peptides presented by cells, the diversity of peptides with post-translational modifications is not well-studied. In this study, we carried out MHC Class I immunopeptidomics analysis of Loucy T-cell leukemia and A375 malignant melanoma cell line to characterize the diversity of post-translational modifications of MHC class I-bound peptides. Using high resolution mass spectrometry, we identified 25,761 MHC-bound peptides across both cell lines using Bolt and Sequest search engines. The enrichment method was highly specific as ~ 90% of the peptides were of typical length (8-12 amino acids long) and the motifs were expected based on previously reported motifs for MHC I alleles. Among the MHC-bound peptides, we identified phosphorylation as a major post-translational modification followed by deamidation. We observed site-specific localization of these post-translational modifications, at position P4 for phosphorylated peptides and position P3 for deamidated peptides. We identified a smaller number of peptides with acetylated and methylated lysine, possibly due to very low stoichiometric levels of these PTMs compared to phosphorylation and deamidation. Using PEAKS de novo sequencing algorithm, we identified spliced peptides that accounted for ~ 5-7% of MHC-bound peptides that were otherwise similar in their features as normal MHC-bound peptides. We validated the identity of several post-translationally modified peptides and spliced peptides through mass spectrometric analysis of synthetic peptides. Our study confirms post-translationally modified peptides to be present at low stoichiometric levels along with unusual spliced peptides through unbiased identification using high resolution mass spectrometry. Supplementary Information: The online version contains supplementary material available at 10.1007/s42485-021-00066-x.

A mass spectrometry-based targeted assay for detection of SARS-CoV-2 antigen from clinical specimens.

BACKGROUND: The COVID-19 pandemic caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has overwhelmed health systems worldwide and highlighted limitations of diagnostic testing. Several types of diagnostic tests including RT-PCR-based assays and antigen detection by lateral flow assays, each with their own strengths and weaknesses, have been developed and deployed in a short time. METHODS: Here, we describe an immunoaffinity purification approach followed a by high resolution mass spectrometry-based targeted qualitative assay capable of detecting SARS-CoV-2 viral antigen from nasopharyngeal swab samples. Based on our discovery experiments using purified virus, recombinant viral protein and nasopharyngeal swab samples from COVID-19 positive patients, nucleocapsid protein was selected as a target antigen. We then developed an automated antibody capture-based workflow coupled to targeted high-field asymmetric waveform ion mobility spectrometry (FAIMS) - parallel reaction monitoring (PRM) assay on an Orbitrap Exploris 480 mass spectrometer. An ensemble machine learning-based model for determining COVID-19 positive samples was developed using fragment ion intensities from the PRM data. FINDINGS: The optimized targeted assay, which was used to analyze 88 positive and 88 negative nasopharyngeal swab samples for validation, resulted in 98% (95% CI = 0.922-0.997) (86/88) sensitivity and 100% (95% CI = 0.958-1.000) (88/88) specificity using RT-PCR-based molecular testing as the reference method. INTERPRETATION: Our results demonstrate that direct detection of infectious agents from clinical samples by tandem mass spectrometry-based assays have potential to be deployed as diagnostic assays in clinical laboratories, which has hitherto been limited to analysis of pure microbial cultures. FUNDING: This study was supported by DBT/Wellcome Trust India Alliance Margdarshi Fellowship grant IA/M/15/1/502023 awarded to AP and the generosity of Eric and Wendy Schmidt.

A qualitative comparison of parent and adolescent views regarding substance use.

Substance use is a major cause of adolescent morbidity and mortality. By age 14, 70% of adolescents have consumed alcohol and half of 12th graders report having used marijuana. The purpose of this study was to increase the understanding of parent and adolescent perceptions regarding adolescent use of alcohol, tobacco, and other drugs (ATOD) to enhance the development of effective prevention programs. Six adolescent focus groups and one parent focus group participated. Results were derived following elicitation of adolescents' and parents' beliefs regarding adolescent ATOD use and current prevention programs. Findings indicate that current prevention programs are ineffective; ATOD use is normalized by schools, community, and family; positive adult role models deter use; and programs should involve youth, parents, schools, and community. Focus group discussions can strengthen the development of tailored ATOD prevention programs. School nurses can foster collaboration between families, schools, and communities to reduce adolescent substance use.