Disparate Rates of Germline Variants in Cancer Predisposition Genes in African American/Black Compared With Non-Hispanic White Individuals Between 2015 and 2022.

PURPOSE: African American/Black (AA/B) individuals are under-represented in genomic databases and thus less likely to receive definitive information from germline genetic testing (GGT) than non-Hispanic White (NHW) individuals. With nearly 500,000 AA/B and NHW individuals having undergone multigene panel testing (MGPT) for hereditary cancer risk at a single commercial laboratory, to our knowledge, we present the largest study to date investigating cancer GGT results in AA/B and NHW individuals. METHODS: MGPT results from a retrospective cohort of AA/B (n = 48,684) and NHW (n = 444,831) patients were evaluated. Frequencies of pathogenic germline variants (PGVs) and variants of uncertain significance (VUS) were compared between AA/B and NHW individuals. Changes in frequency of VUS over time were determined. Pearson's chi-squared test was used to compare categorical variables among groups. All significance tests were two-tailed, and P < .05 was considered statistically significant. RESULTS: Between 2015 and 2022, rates of VUS decreased 2.3-fold in AA/B and 1.8-fold in NHW individuals; however, frequencies of VUS and PGV remained significantly higher (46% v 32%; P < .0001) and lower (9% v 13%; P < .0001) in AA/B compared with NHW individuals. Rates of VUS in ATM, BRCA1, BRCA2, PALB2, and PMS2 were significantly higher in AA/B compared with NHW individuals, whereas rates of PGV in BRCA1, BRCA2, and PALB2 were higher in AA/B compared with NHW individuals (P < .001). CONCLUSION: Despite reductions in VUS frequencies over time, disparities in definitive GGT results persist. Increasing inclusion of AA/B populations in both testing and research will further increase knowledge of genetic variants across these racial groups.

Isolation of human antibodies against influenza B neuraminidase and mechanisms of protection at the airway interface.

Influenza B viruses (IBVs) comprise a substantial portion of the circulating seasonal human influenza viruses. Here, we describe the isolation of human monoclonal antibodies (mAbs) that recognized the IBV neuraminidase (NA) glycoprotein from an individual following seasonal vaccination. Competition-binding experiments suggested the antibodies recognized two major antigenic sites. One group, which included mAb FluB-393, broadly inhibited IBV NA sialidase activity, protected prophylactically in vivo, and bound to the lateral corner of NA. The second group contained an active site mAb, FluB-400, that broadly inhibited IBV NA sialidase activity and virus replication in vitro in primary human respiratory epithelial cell cultures and protected against IBV in vivo when administered systemically or intranasally. Overall, the findings described here shape our mechanistic understanding of the human immune response to the IBV NA glycoprotein through the demonstration of two mAb delivery routes for protection against IBV and the identification of potential IBV therapeutic candidates.

Protocol for quantifying stem-cell-derived cardiomyocyte maturity using transcriptomic entropy score.

The inability to quantify cardiomyocyte (CM) maturation remains a significant barrier to evaluating the effects of ongoing efforts to produce adult-like CMs from pluripotent stem cells (PSCs). Here, we present a protocol to quantify stem-cell-derived CM maturity using a single-cell RNA sequencing-based metric "entropy score." We describe steps for generating an entropy score using customized R code. This tool can be used to quantify maturation levels of PSC-CMs and potentially other cell types. For complete details on the use and execution of this protocol, please refer to Kannan et al.1.

Computationally restoring the potency of a clinical antibody against Omicron.

The COVID-19 pandemic underscored the promise of monoclonal antibody-based prophylactic and therapeutic drugs1-3 and revealed how quickly viral escape can curtail effective options4,5. When the SARS-CoV-2 Omicron variant emerged in 2021, many antibody drug products lost potency, including Evusheld and its constituent, cilgavimab4-6. Cilgavimab, like its progenitor COV2-2130, is a class 3 antibody that is compatible with other antibodies in combination4 and is challenging to replace with existing approaches. Rapidly modifying such high-value antibodies to restore efficacy against emerging variants is a compelling mitigation strategy. We sought to redesign and renew the efficacy of COV2-2130 against Omicron BA.1 and BA.1.1 strains while maintaining efficacy against the dominant Delta variant. Here we show that our computationally redesigned antibody, 2130-1-0114-112, achieves this objective, simultaneously increases neutralization potency against Delta and subsequent variants of concern, and provides protection in vivo against the strains tested: WA1/2020, BA.1.1 and BA.5. Deep mutational scanning of tens of thousands of pseudovirus variants reveals that 2130-1-0114-112 improves broad potency without increasing escape liabilities. Our results suggest that computational approaches can optimize an antibody to target multiple escape variants, while simultaneously enriching potency. Our computational approach does not require experimental iterations or pre-existing binding data, thus enabling rapid response strategies to address escape variants or lessen escape vulnerabilities.

Paternal DDT exposure induces sex-specific programming of fetal growth, placenta development and offspring's health phenotypes in a mouse model.

Mounting evidence suggests that environmentally induced epigenetic inheritance occurs in mammals and that traits in the progeny can be shaped by parental environmental experiences. Epidemiological studies link parental exposure to environmental toxicants, such as the pesticide DDT, to health phenotypes in the progeny, including low birth and increased risk of chronic diseases later in life. Here, we show that the progeny of male mice exposed to DDT in the pre-conception period are born smaller and exhibit sexual dimorphism in metabolic function, with male, but not female, offspring developing severe glucose intolerance compared to controls. These phenotypes in DDT offspring were linked to reduced fetal growth and placenta size as well as placenta-specific reduction of glycogen levels and the nutrient sensor and epigenetic regulator OGT, with more pronounced phenotypes observed in male placentas. However, placenta-specific genetic reduction of OGT only partially replicates the metabolic phenotype observed in offspring of DDT-exposed males. Our findings reveal a role for paternal pre-conception environmental experiences in shaping placenta development and in fetal growth restriction. While many questions remain, our data raise the tantalizing possibility that placenta programming could be a mediator of environmentally induced intergenerational epigenetic inheritance of phenotypes and needs to be further evaluated.

Factors associated with practice readiness among newly qualified nurses in their first two years of practice.

BACKGROUND: Newly graduated nurses undergo stress and role adjustment as they transition into practice during the first year and continue to struggle beyond the first year. Determining their practice readiness can aid in the development of interventions to facilitate workplace readiness for nurses in their first two years entering the nursing profession. OBJECTIVES: To examine (i) extent of practice readiness of new nurses in their role; and (ii) associations between nurses' practice readiness and demographic and occupational variables, and reasons for choosing nursing profession. DESIGN: A cross-sectional study. SETTINGS AND PARTICIPANTS: A total of 445 registered nurses who graduated within the last two years and working in an academic medical centre in Singapore. METHODS: Participants completed an online questionnaire with questions from Casey-Fink Readiness for Practice Survey and questions related to key competencies for future practice. RESULTS: More than half (57.5 %) identified at least three skills and procedures which they were uncomfortable performing independently as they transition into the clinical practice, including: (i) responding to emergency (ii) tracheostomy care; and (iii) chest tube care. The top three reasons for choosing nursing as a career were: (i) nursing is a stable industry (54.2 %); (ii) I want to help people (52.1 %); and (iii) able to work anywhere in the world (44.3 %). Nurses were most concerned with areas of trials and tribulations (42.5 %) and clinical competency (36.6 %). When compared to nurses in their first-year post-graduation, those working in their second year reported more confidence in the ability to problem solve (p = 0.003), care for a person who is dying (p = 0.004), and less difficulties in prioritizing care needs (p = 0.04). They also perceived themselves as a good problem solver (p = 0.03). CONCLUSIONS: It is critical to continue supporting nurses' practice readiness beyond their first year of practice in their confidence and development of skills of higher complexity.

Vision Correction Habits After COVID-19 Onset in Spectacle and Contact Lens Wearers.

PURPOSE: To determine how vision correction habits changed after the 2019 coronavirus disease (COVID-19) pandemic onset. METHODS: Participants reported vision correction habits, refractive error, screen time, mask wearing time, and dry eye symptoms since the COVID-19 pandemic onset through email survey. RESULTS: A total of 133 participants completed the survey. Worsening dry eye symptoms were associated with increased screen time ( P =0.04). Hours per day of spectacle wear increased by approximately 1 hr ( P =0.001) and was associated with increased screen time ( P =0.002). Worsening dry eye symptoms were associated with increased days per week of spectacle wear ( P =0.02). Participants wore contact lenses about one day per week less than before the pandemic ( P =0.0001). Increased mask wearing time was associated with increased days per week of contact lens wear ( P =0.03). CONCLUSIONS: After pandemic onset, hours per day of spectacle wear increased, and days per week of contact lens wear decreased. Increases in hourly spectacle wear were associated with increased screen time, whereas increased daily contact lens wear was associated with increased mask wear time, suggesting that spectacles may be preferred for screen time activities and contact lenses for mask wear.

SARS-CoV-2 antibodies from children exhibit broad neutralization and belong to adult public clonotypes.

From the beginning of the COVID-19 pandemic, children have exhibited different susceptibility to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, reinfection, and disease compared with adults. Motivated by the established significance of SARS-CoV-2-neutralizing antibodies in adults, here we characterize SARS-CoV-2-specific antibody repertoires in a young cohort of individuals aged from 5 months to 18 years old. Our results show that neutralizing antibodies in children possess similar genetic features compared to antibodies identified in adults, with multiple antibodies from children belonging to previously established public antibody clonotypes in adults. Notably, antibodies from children show potent neutralization of circulating SARS-CoV-2 variants that have cumulatively resulted in resistance to virtually all approved monoclonal antibody therapeutics. Our results show that children can rely on similar SARS-CoV-2 antibody neutralization mechanisms compared to adults and are an underutilized source for the discovery of effective antibody therapeutics to counteract the ever-evolving pandemic.

Rate of Pathogenic Germline Variants in Patients With Lung Cancer.

PURPOSE: Germline genetic testing (GGT) is now recommended for all patients diagnosed with ovarian or pancreatic cancer and for a large proportion of patients based solely on a diagnosis of colorectal or breast cancer. However, GGT is not yet recommended for all patients diagnosed with lung cancer (LC), primarily because of a lack of evidence that supports a significant frequency of identifying pathogenic germline variants (PGVs) in these patients. This study characterizes GGT results in a cohort of patients with LC. METHODS: We reviewed deidentified data for 7,788 patients with GGT (2015-2022). PGV frequencies were compared to a control cohort of unaffected individuals. GGT results were stratified by genomic ancestry, history of cancer, and PGV clinical actionability per current guidelines. RESULTS: Of all patients with LC, 14.9% (1,161/7,788) had PGVs. The rate was similar when restricted to patients with no cancer family history (FH) or personal history (PH) of other cancers (14.3%). PGVs were significantly enriched in BRCA2, ATM, CHEK2, BRCA1, and mismatch repair genes compared with controls. Patients of European (EUR) genomic ancestry had the highest PGV rate (18%) and variants of uncertain significance were significantly higher in patients of non-EUR genomic ancestry. Of the PGVs identified, 61.3% were in DNA damage repair (DDR) genes and 95% were clinically actionable. CONCLUSION: This retrospective study shows a LC diagnosis identifies patients with a significant likelihood of having a cancer-predisposing PGV across genomic ancestries. Enrichment of PGVs in DDR genes suggests that these PGVs may contribute to LC cancer predisposition. The frequency of PGVs among patients with LC did not differ significantly according to FH or PH of other cancers.

Rates and Classification of Variants of Uncertain Significance in Hereditary Disease Genetic Testing.

Importance: Variants of uncertain significance (VUSs) are rampant in clinical genetic testing, frustrating clinicians, patients, and laboratories because the uncertainty hinders diagnoses and clinical management. A comprehensive assessment of VUSs across many disease genes is needed to guide efforts to reduce uncertainty. Objective: To describe the sources, gene distribution, and population-level attributes of VUSs and to evaluate the impact of the different types of evidence used to reclassify them. Design, Setting, and Participants: This cohort study used germline DNA variant data from individuals referred by clinicians for diagnostic genetic testing for hereditary disorders. Participants included individuals for whom gene panel testing was conducted between September 9, 2014, and September 7, 2022. Data were analyzed from September 1, 2022, to April 1, 2023. Main Outcomes and Measures: The outcomes of interest were VUS rates (stratified by age; clinician-reported race, ethnicity, and ancestry groups; types of gene panels; and variant attributes), percentage of VUSs reclassified as benign or likely benign vs pathogenic or likely pathogenic, and enrichment of evidence types used for reclassifying VUSs. Results: The study cohort included 1 689 845 individuals ranging in age from 0 to 89 years at time of testing (median age, 50 years), with 1 203 210 (71.2%) female individuals. There were 39 150 Ashkenazi Jewish individuals (2.3%), 64 730 Asian individuals (3.8%), 126 739 Black individuals (7.5%), 5539 French Canadian individuals (0.3%), 169 714 Hispanic individuals (10.0%), 5058 Native American individuals (0.3%), 2696 Pacific Islander individuals (0.2%), 4842 Sephardic Jewish individuals (0.3%), and 974 383 White individuals (57.7%). Among all individuals tested, 692 227 (41.0%) had at least 1 VUS and 535 385 (31.7%) had only VUS results. The number of VUSs per individual increased as more genes were tested, and most VUSs were missense changes (86.6%). More VUSs were observed per sequenced gene in individuals who were not from a European White population, in middle-aged and older adults, and in individuals who underwent testing for disorders with incomplete penetrance. Of 37 699 unique VUSs that were reclassified, 30 239 (80.2%) were ultimately categorized as benign or likely benign. A mean (SD) of 30.7 (20.0) months elapsed for VUSs to be reclassified to benign or likely benign, and a mean (SD) of 22.4 (18.9) months elapsed for VUSs to be reclassified to pathogenic or likely pathogenic. Clinical evidence contributed most to reclassification. Conclusions and Relevance: This cohort study of approximately 1.6 million individuals highlighted the need for better methods for interpreting missense variants, increased availability of clinical and experimental evidence for variant classification, and more diverse representation of race, ethnicity, and ancestry groups in genomic databases. Data from this study could provide a sound basis for understanding the sources and resolution of VUSs and navigating appropriate next steps in patient care.

User-friendly microfluidic system reveals native-like morphological and transcriptomic phenotypes induced by shear stress in proximal tubule epithelium.

Drug-induced nephrotoxicity is a leading cause of drug attrition, partly due to the limited relevance of pre-clinical models of the proximal tubule. Culturing proximal tubule epithelial cells (PTECs) under fluid flow to mimic physiological shear stress has been shown to improve select phenotypes, but existing flow systems are expensive and difficult to implement by non-experts in microfluidics. Here, we designed and fabricated an accessible and modular flow system for culturing PTECs under physiological shear stress, which induced native-like cuboidal morphology, downregulated pathways associated with hypoxia, stress, and injury, and upregulated xenobiotic metabolism pathways. We also compared the expression profiles of shear-dependent genes in our in vitro PTEC tissues to that of ex vivo proximal tubules and observed stronger clustering between ex vivo proximal tubules and PTECs under physiological shear stress relative to PTECs under negligible shear stress. Together, these data illustrate the utility of our user-friendly flow system and highlight the role of shear stress in promoting native-like morphological and transcriptomic phenotypes in PTECs in vitro, which is critical for developing more relevant pre-clinical models of the proximal tubule for drug screening or disease modeling.

The landscape of reported VUS in multi-gene panel and genomic testing: Time for a change.

PURPOSE: Variants of uncertain significance (VUS) are a common result of diagnostic genetic testing and can be difficult to manage with potential misinterpretation and downstream costs, including time investment by clinicians. We investigated the rate of VUS reported on diagnostic testing via multi-gene panels (MGPs) and exome and genome sequencing (ES/GS) to measure the magnitude of uncertain results and explore ways to reduce their potentially detrimental impact. METHODS: Rates of inconclusive results due to VUS were collected from over 1.5 million sequencing test results from 19 clinical laboratories in North America from 2020 to 2021. RESULTS: We found a lower rate of inconclusive test results due to VUSs from ES/GS (22.5%) compared with MGPs (32.6%; P < .0001). For MGPs, the rate of inconclusive results correlated with panel size. The use of trios reduced inconclusive rates (18.9% vs 27.6%; P < .0001), whereas the use of GS compared with ES had no impact (22.2% vs 22.6%; P = ns). CONCLUSION: The high rate of VUS observed in diagnostic MGP testing warrants examining current variant reporting practices. We propose several approaches to reduce reported VUS rates, while directing clinician resources toward important VUS follow-up.

Impact of Mask-Associated Dry Eye on Symptom Score.

OBJECTIVES: Owing to widespread mask use during the COVID-19 pandemic and clinical reports tying mask use with dryness, this study endeavors to determine if mask use is linked to symptoms of dry eye. METHODS: A prospective, cross-sectional survey study was performed. The survey used a modified Standard Patient Evaluation of Eye Dryness Questionnaire (SPEED, TearScience, Morrisville, NC) within 15 min of the beginning and discontinuation of mask wear. The survey also asked about mask wear time, mask style, visual correction, age, and gender. RESULTS: The change in SPEED scores was statistically significant ( P =0.03) between participants with mild SPEED score at baseline (0-9) versus severe SPEED score at baseline (10-28) (n=77: 59 female, 16 male, 1 nonbinary, and 1 declined to answer; range 22-55 years old). Participants in the severe group used masks with nose wire more than the mild group ( P =0.03). CONCLUSIONS: In this sample, dry eye symptoms were most exacerbated with mask wear in those that had mild initial symptom scores compared with those with severe symptom scores at baseline. The use of nose wire masks may be protective, as the severe group used this type more and had significantly less exacerbation of symptoms postmask wear.

Trajectory reconstruction identifies dysregulation of perinatal maturation programs in pluripotent stem cell-derived cardiomyocytes.

A limitation in the application of pluripotent stem cell-derived cardiomyocytes (PSC-CMs) is the failure of these cells to achieve full functional maturity. The mechanisms by which directed differentiation differs from endogenous development, leading to consequent PSC-CM maturation arrest, remain unclear. Here, we generate a single-cell RNA sequencing (scRNA-seq) reference of mouse in vivo CM maturation with extensive sampling of previously difficult-to-isolate perinatal time periods. We subsequently generate isogenic embryonic stem cells to create an in vitro scRNA-seq reference of PSC-CM-directed differentiation. Through trajectory reconstruction, we identify an endogenous perinatal maturation program that is poorly recapitulated in vitro. By comparison with published human datasets, we identify a network of nine transcription factors (TFs) whose targets are consistently dysregulated in PSC-CMs across species. Notably, these TFs are only partially activated in common ex vivo approaches to engineer PSC-CM maturation. Our study can be leveraged toward improving the clinical viability of PSC-CMs.

Systematic analysis of human antibody response to ebolavirus glycoprotein shows high prevalence of neutralizing public clonotypes.

Understanding the human antibody response to emerging viral pathogens is key to epidemic preparedness. As the size of the B cell response to a pathogenic-virus-protective antigen is poorly defined, we perform deep paired heavy- and light-chain sequencing in Ebola virus glycoprotein (EBOV-GP)-specific memory B cells, allowing analysis of the ebolavirus-specific antibody repertoire both genetically and functionally. This approach facilitates investigation of the molecular and genetic basis for the evolution of cross-reactive antibodies by elucidating germline-encoded properties of antibodies to EBOV and identification of the overlap between antibodies in the memory B cell and serum repertoire. We identify 73 public clonotypes of EBOV, 20% of which encode antibodies with neutralization activity and capacity to protect mice in vivo. This comprehensive analysis of the public and private antibody repertoire provides insight into the molecular basis of the humoral immune response to EBOV GP, which informs the design of vaccines and improved therapeutics.

Environmentally Induced Sperm RNAs Transmit Cancer Susceptibility to Offspring in a Mouse Model.

DNA sequence accounts for the majority of disease heritability, including cancer. Yet, not all familial cancer cases can be explained by genetic factors. It is becoming clear that environmentally induced epigenetic inheritance occurs and that the progeny's traits can be shaped by parental environmental experiences. In humans, epidemiological studies have implicated environmental toxicants, such as the pesticide DDT, in intergenerational cancer development, including breast and childhood tumors. Here, we show that the female progeny of males exposed to DDT in the pre-conception period have higher susceptibility to developing aggressive tumors in mouse models of breast cancer. Sperm of DDT-exposed males exhibited distinct patterns of small non-coding RNAs, with an increase in miRNAs and a specific surge in miRNA-10b levels. Remarkably, embryonic injection of the entire sperm RNA load of DDT-exposed males, or synthetic miRNA-10b, recapitulated the tumor phenotypes observed in DDT offspring. Mechanistically, miR-10b injection altered the transcriptional profile in early embryos with enrichment of genes associated with cell differentiation, tissue and immune system development. In adult DDT-derived progeny, transcriptional and protein analysis of mammary tumors revealed alterations in stromal and in immune system compartments. Our findings reveal a causal role for sperm RNAs in environmentally induced inheritance of cancer predisposition and, if confirmed in humans, this could help partially explain some of the "missing heritability" of breast, and other, malignancies.

Palliative Ventilator Withdrawal Practices in an Inpatient Hospice Unit.

BACKGROUND: Palliative ventilator withdrawal (PVW) involves removal of mechanical ventilation in patients not expected to survive to allow a peaceful death. This process traditionally occurs in Intensive Care Units (ICU) and recently has evolved to occur in Inpatient Hospice and Palliative Care Units (IPU). OBJECTIVES: To describe the process and response of patients undergoing PVW in an IPU setting. METHODS: This is a longitudinal observational cohort study of adult patients who underwent PVW in an IPU from January 2021 through March 2022. RESULTS: Among 25 enrolled subjects, median age was 68 (IQR 62.5-76.5) years and 14 (56%) were females. Median time from PVW to death was 16.8 (IQR 2.6-100) hours. A registered nurse and attending physician were present in all the cases, while a respiratory therapist was present in 20 (80%) and chaplain in 9 (36%) of the cases. Before PVW, opioids and benzodiazepines were administered to 24 (96%) patients. Post PVW, respiratory distress was noted among 16 (64%) patients and medication was given to 15 (60%) patients for respiratory distress. There was a significant association between the presence of respiratory distress and administration of medication within 30 minutes after PVW (P = .009). The rituals performed during PVW were reciting prayers for 11 (44%), playing music for 8 (32%), and observing silence for 6 (24%) of the patients. CONCLUSION: This study describes the PVW practices in an IPU setting where a multidisciplinary team was present during PVW for most of the cases and two-third of the patients undergoing PVW experienced respiratory distress immediately after PVW.

Computationally restoring the potency of a clinical antibody against SARS-CoV-2 Omicron subvariants.

The COVID-19 pandemic underscored the promise of monoclonal antibody-based prophylactic and therapeutic drugs1-3, but also revealed how quickly viral escape can curtail effective options4,5. With the emergence of the SARS-CoV-2 Omicron variant in late 2021, many clinically used antibody drug products lost potency, including Evusheld™ and its constituent, cilgavimab4,6. Cilgavimab, like its progenitor COV2-2130, is a class 3 antibody that is compatible with other antibodies in combination4 and is challenging to replace with existing approaches. Rapidly modifying such high-value antibodies with a known clinical profile to restore efficacy against emerging variants is a compelling mitigation strategy. We sought to redesign COV2-2130 to rescue in vivo efficacy against Omicron BA.1 and BA.1.1 strains while maintaining efficacy against the contemporaneously dominant Delta variant. Here we show that our computationally redesigned antibody, 2130-1-0114-112, achieves this objective, simultaneously increases neutralization potency against Delta and many variants of concern that subsequently emerged, and provides protection in vivo against the strains tested, WA1/2020, BA.1.1, and BA.5. Deep mutational scanning of tens of thousands pseudovirus variants reveals 2130-1-0114-112 improves broad potency without incurring additional escape liabilities. Our results suggest that computational approaches can optimize an antibody to target multiple escape variants, while simultaneously enriching potency. Because our approach is computationally driven, not requiring experimental iterations or pre-existing binding data, it could enable rapid response strategies to address escape variants or pre-emptively mitigate escape vulnerabilities.

Emotional impact of compassionate extubation on respiratory therapists and nurses: A pilot study.

Background: Compassionate extubation (CE) refers to withdrawing mechanical ventilation and allowing a patient to die peacefully at the end of life. The primary objective of this pilot study was to quantify the emotional impact of CE on Respiratory Therapists (RT) and Registered Nurses (RNs). Methods: This pilot survey was conducted between March and April 2021 at an academic medical center among RTs and RNs. It included questions on participants' demographics, work characteristics, and Impact of Events (IES) scale to assess the subjective stress caused by CE. Data were analyzed using descriptive and χ2 statistics. Results: Among 20 participants, 18 (90%) were females, 12 (60%) were in the 20-40-year age group, 12 (60%) were RTs, and 8 (40%) RNs. Around 15 (75%) participants worked day shifts with a weekly average of 3-4 shifts, and 14 (70%) performed/observed CE within 1 month before taking this survey. CE performed/observed in a month was ≤2 among 15 (75%) and 3-5 among 4 (20%) participants. Mean total IES score was 16.7 (12.3) among all participants representing 7 (35%) having low, 6 (30%) moderate, and 7 (35%) high emotional impact when performing CE. Risk of developing post-traumatic stress disorder (PTSD) was present in 6 (30%) participants. A significantly higher number of participants in the low impact group were satisfied with the institutional CE process (p = 0.043) than those in the medium/high impact group. Conclusion: This pilot study findings reveal that RTs and RNs experience moderate to high levels of subjective stress when performing CE. One-third of the survey participants were at risk of developing PTSD.

Bulk gDNA Sequencing of Antibody Heavy-Chain Gene Rearrangements for Detection and Analysis of B-Cell Clone Distribution: A Method by the AIRR Community.

In this method we illustrate how to amplify, sequence, and analyze antibody/immunoglobulin (IG) heavy-chain gene rearrangements from genomic DNA that is derived from bulk populations of cells by next-generation sequencing (NGS). We focus on human source material and illustrate how bulk gDNA-based sequencing can be used to examine clonal architecture and networks in different samples that are sequenced from the same individual. Although bulk gDNA-based sequencing can be performed on both IG heavy (IGH) or kappa/lambda light (IGK/IGL) chains, we focus here on IGH gene rearrangements because IG heavy chains are more diverse, tend to harbor higher levels of somatic hypermutations (SHM), and are more reliable for clone identification and tracking. We also provide a procedure, including code, and detailed instructions for processing and annotation of the NGS data. From these data we show how to identify expanded clones, visualize the overall clonal landscape, and track clonal lineages in different samples from the same individual. This method has a broad range of applications, including the identification and monitoring of expanded clones, the analysis of blood and tissue-based clonal networks, and the study of immune responses including clonal evolution.