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Abstract Moral injury has emerged as a construct of interest in healthcare workers’ (HCW) occupational stress and health. We conducted one of the first multidisciplinary, longitudinal studies evaluating the relationship between exposure to potentially morally injurious events (PMIEs), burnout, and turnover intentions. HCWs (N = 473) completed surveys in May of 2020 (T1) and again in May of 2021 (T2). Generalized Linear Models (robust Poisson regression) were used to test relative risk of turnover intentions, and burnout at T2 associated with PMIE exposure, controlling for T1 covariates. At T1, 17.67% reported they had participated in a PMIE, 41.44% reported they witnessed a PMIE and 76.61% reported feeling betrayed by healthcare or a public health organization. In models including all T1 PMIE exposures and covariates, T2 turnover intentions were increased for those who witnessed a PMIE at T1 (Relative Risk [RR] = 1.66, 95% Confidence Interval [CI] 1.17–2.34) but not those that participated or felt betrayed. T2 burnout was increased for those who participated in PMIE at T1 (RR = 1.38, 95%CI 1.03–1.85) but not those that witnessed or felt betrayed. PMIE exposure is highly prevalent among HCWs, with specific PMIEs associated with turnover intentions and burnout. Organizational interventions to reduce and facilitate recovery from moral injury should account for differences in the type of PMIE exposures that occur in healthcare work environments.

Abstract Background Measurement is one of the critical ingredients to addressing the well-being of health care professionals. However, administering an organization-wide well-being survey can be challenging due to constraints like survey fatigue, financial limitations, and other system priorities. One way to address these issues is to embed well-being items into already existing assessment tools that are administered on a regular basis, such as an employee engagement survey. The objective of this study was to assess the utility of a brief engagement survey, that included a small subset of well-being items, among health care providers working in an academic medical center. Methods In this cross-sectional study, health care providers, including physicians and advanced clinical practitioners, employed at an academic medical center completed a brief, digital engagement survey consisting of 11 quantitative items and 1 qualitative item administered by Dialogue™. The emphasis of this study was on the quantitative responses. Item responses were compared by sex and degree, domains were identified via exploratory factor analysis (EFA), and internal consistency of item responses was assessed via McDonald’s omega. Sample burnout was compared against national burnout. Results Of the 791 respondents, 158 (20.0%) were Advanced Practice Clinicians (APCs), and 633 (80.0%) were Medical Doctors (MDs). The engagement survey, with 11 items, had a high internal consistency with an omega ranging from 0.80–0.93 and was shown, via EFA, to have three domains including communication, well-being, and engagement. Significant differences for some of the 11 items, by sex and degree, in the odds of their agreement responses were found. In this study, 31.5% reported experiencing burnout, which was significantly lower than the national average of 38.2%. Conclusion Our findings indicate initial reliability, validity, and utility of a brief, digital engagement survey among health care professionals. This may be particularly useful for medical groups or health care organizations who are unable to administer their own discrete well-being survey to employees.

The mental health of healthcare workers (HCWs) is critical to their long-term well-being and future disaster preparedness. Goal 1 of this study was to identify rates of mental health problems experienced by HCWs. Goal 2 was to test a model of risk stemming from pandemic-related stressors and vulnerability factors.This cross-sectional study included HCWs (N ​= ​2,246 [1,573 clinical providers; 673 non-clinical staff]) in the Rocky Mountain West who voluntarily completed an online survey in April/May 2020. Respondents completed measures for traumatic stress symptoms, depression, anxiety, alcohol use, and sleep. Logistic regressions stratified by professional role (clinical versus non-clinical) were specified to predict clinical screening cutoff (positive/negative) as a function of five pandemic-related stressors (immunocompromised self; immunocompromised household member; care provision to infected patients; clinical management role; positive cases).Results showed that more than half of HCWs surveyed (52.5%) screened positive (above cutoff) for traumatic stress, depression, or anxiety, with ~20% reporting problematic alcohol use, and variable insufficient sleep from ~10% off shift to ~50% on shift. Clinical employees with an immunocompromised household member had increased odds of screening positive for a mental health problem. Non-clinical HCWs who were immunocompromised were at elevated risk for screening positive a mental health problem. Being female, minority status, and younger increased odds for mental health problems.Implications include alleviating a portion of the mental health burden of HCWs involved in response to the SARS-CoV-2 pandemic by considering policies to protect immunocompromised HCWs and their families (e.g., vaccine priorities, telework options).

Professional well-being in health care is critical to the success of academic medical centers inpatient care, educating trainees, serving communities, and pursuing research missions. The COVID-19 pandemic, caused by SARS-COV-2, has stretched health care teams and individuals in unique ways, leading to high levels of persistent stress with concern for longer term mental health implications. The pandemic is a catalyst to grow and strengthen support for those who work in health professions. Using one academic health center as a model, this paper reviews how professional well-being can be approached comprehensively at a system level while considering the needs of diverse employees during a time of increased need. This ramping up of services has the opportunity to build community and support a trajectory of post traumatic growth.

K-12 online students are increasingly communicating and collaborating with their peers; however, research on the topic is limited—especially research examining students’ perceptions and experiences. Guided by the Adolescent Community of Engagement framework’s concept of peer engagement, this case study examined learner-learner interactions at a cyber charter high school. Ten students were selected by teachers based on their ability to independently engage in learning activities—five students were more independent and five required more support from others. Each student took part in two, hour-long interviews for a total of 20 interviews. The interviewers covered students’ perceptions an experiences regarding teachers, parents, and peers. This report only focuses on peers. Students found that their interactions with peers allowed them to develop friendships, improve their motivation, receive peer instruction, and collaborate effectively with others. Challenges are also covered. The article concludes with recommendations for research and practice.

Abstract Streptococcus pyogenes Cas9 (SpCas9) and derived enzymes are widely used as genome editors, but their promiscuous nuclease activity often induces undesired mutations and chromosomal rearrangements. Several strategies for mapping off-target effects have emerged, but they suffer from limited sensitivity. To increase the detection sensitivity, we develop an off-target assessment workflow that uses Duplex Sequencing. The strategy increases sensitivity by one order of magnitude, identifying previously unknown SpCas9’s off-target mutations in the humanized PCSK9 mouse model. To reduce off-target risks, we perform a bioinformatic search and identify a high-fidelity Cas9 variant of the II-B subfamily from Parasutterella secunda (PsCas9). PsCas9 shows improved specificity as compared to SpCas9 across multiple tested sites, both in vitro and in vivo, including the PCSK9 site. In the future, while PsCas9 will offer an alternative to SpCas9 for research and clinical use, the Duplex Sequencing workflow will enable a more sensitive assessment of Cas9 editing outcomes.

Summary: Primary human lung organoid-derived air-liquid interface (ALI) cultures serve as a physiologically relevant model to study human airway epithelium in vitro. Here, we present a protocol for establishing these cultures from cryopreserved human lung tissue. We describe steps for lung tissue cryostorage, tissue dissociation, lung epithelial organoid generation, and ALI culture differentiation. We also include quality control steps and technical readouts for monitoring virus response. This protocol demonstrates severe acute respiratory syndrome coronavirus 2 infection in these cultures as an example of their utility.For complete details on the use and execution of this protocol, please refer to Diana Cadena Castaneda et al. (2023).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: The COVID-19 pandemic continues to be a health crisis with major unmet medical needs. The early responses from airway epithelial cells, the first target of the virus regulating the progression toward severe disease, are not fully understood. Primary human air-liquid interface cultures representing the broncho-alveolar epithelia were used to study the kinetics and dynamics of SARS-CoV-2 variants infection. The infection measured by nucleoprotein expression, was a late event appearing between day 4–6 post infection for Wuhan-like virus. Other variants demonstrated increasingly accelerated timelines of infection. All variants triggered similar transcriptional signatures, an “early” inflammatory/immune signature preceding a “late” type I/III IFN, but differences in the quality and kinetics were found, consistent with the timing of nucleoprotein expression. Response to virus was spatially organized: CSF3 expression in basal cells and CCL20 in apical cells. Thus, SARS-CoV-2 virus triggers specific responses modulated over time to engage different arms of immune response.

With the advent of CRISPR-Cas and the ability to easily modify the genome of diverse organisms, rat models are being increasingly developed to interrogate the genetic events underlying mammary development and tumorigenesis. Protocols for the isolation and characterization of mammary epithelial cell subpopulations have been thoroughly developed for mouse and human tissues, yet there is an increasing need for rat-specific protocols. To date, there are no standard protocols for isolating rat mammary epithelial subpopulations. Analyzing changes in the rat mammary hierarchy will help us elucidate the molecular events in breast cancer, the cells of origin for breast cancer subtypes, and the impact of the tumor microenvironment. Here we describe several methods developed for 1) rat mammary epithelial cell isolation; 2) rat mammary fibroblast isolation; 3) culturing rat mammary epithelial cells; and characterization of rat mammary cells by 4) flow cytometric analysis; and 5) immunofluorescence. Cells derived from this protocol can be used for many purposes, including RNAseq, drug studies, functional assays, gene/protein expression analyses, and image analysis.

Skimmed milk powder (SMP) and whey protein concentrate (WPC) were manufactured from fresh milk collected from cows producing high or low Immunoglobulin (Ig) A levels in their milk. In addition commercial products were purchased for use as diluent or control treatments. A murine enteric disease model (Citrobacter rodentium) was used to assess whether delivery of selected bioactive molecules (IgA, IgG, Lactoferrin (Lf)) or formulation delivery matrix (SMP, WPC) affected faecal shedding of bacteria in C. rodentium infected mice. In trial one, faecal pellets collected from mice fed SMP containing IgA (0.007–0.35 mg/mL), IgG (0.28–0.58 mg/mL) and Lf (0.03–0.1 mg/mL) contained fewer C. rodentium (cfu) compared to control mice fed water (day 8, p < 0.04, analysis of variance (ANOVA) followed by Fisher’s unprotected least significant difference (ULSD)). In trial two, WPC containing IgA (0.35–1.66 mg/mL), IgG (0.58–2.36 mg/mL) and Lf (0.02–0.45 mg/mL) did not affect C. rodentium shedding, but SMP again reduced faecal C. rodentium levels (day 12, p < 0.04, ANOVA followed by Fisher’s ULSD). No C. rodentium was detected in sham phosphate-buffered saline inoculated mice. Mice fed a commercial WPC shed significantly greater numbers of C. rodentium over 4 consecutive days (Fishers ULSD test), compared to control mice fed water. These data indicate that SMP, but not WPC, modulates faecal shedding in C. rodentium-infected mice and may impact progression of C. rodentium infection independently of selected bioactive concentration. This suggests that food matrix can impact biological effects of foods.

Results from large multicentre epidemiological studies suggest an association between the consumption of raw milk and a reduced incidence of allergy and asthma in children. Although the underlying mechanisms for this association are yet to be confirmed, researchers have investigated whether bacteria or bacterial components that naturally occur in cow’s milk are responsible for modulating the immune system to reduce the risk of allergic diseases. Previous research in human and mice suggests that bacterial components derived from the maternal intestine are transported to breast milk through the bloodstream. The aim of our study was to assess whether a similar mechanism of bacterial trafficking could occur in the cow. Through the application of culture-independent methodology, we investigated the microbial composition and diversity of milk, blood and feces of healthy lactating cows. We found that a small number of bacterial OTUs belonging to the genera Ruminococcus and Bifidobacterium, and the Peptostreptococcaceae family were present in all three samples from the same individual animals. Although these results do not confirm the hypothesis that trafficking of intestinal bacteria into mammary secretions does occur in the cow, they support the existence of an endogenous entero-mammary pathway for some bacterial components during lactation in the cow. Further research is required to define the specific mechanisms by which gut bacteria are transported into the mammary gland of the cow, and the health implications of such bacteria being present in milk.