Evaluating and Enhancing Target Specificity of Gene-Editing Nucleases and Deaminases.

Programmable nucleases and deaminases, which include zinc-finger nucleases, transcription activator-like effector nucleases, CRISPR RNA-guided nucleases, and RNA-guided base editors, are now widely employed for the targeted modification of genomes in cells and organisms. These gene-editing tools hold tremendous promise for therapeutic applications. Importantly, these nucleases and deaminases may display off-target activity through the recognition of near-cognate DNA sequences to their target sites, resulting in collateral damage to the genome in the form of local mutagenesis or genomic rearrangements. For therapeutic genome-editing applications with these classes of programmable enzymes, it is essential to measure and limit genome-wide off-target activity. Herein, we discuss the key determinants of off-target activity for these systems. We describe various cell-based and cell-free methods for identifying genome-wide off-target sites and diverse strategies that have been developed for reducing the off-target activity of programmable gene-editing enzymes.

Transcriptional and chromatin profiling of human blood innate lymphoid cell subsets sheds light on HIV-1 pathogenesis.

Innate lymphoid cells (ILCs) are a diverse population of cells that include NK cells and contribute to tissue homeostasis and repair, inflammation, and provide protection from infection. The interplay between human blood ILCs, as well as their responses to HIV-1 infection, remains poorly understood. This study used transcriptional and chromatin profiling to explore these questions. Transcriptional profiling and flow cytometry analysis support that there are four main ILC subsets found in human blood. Unlike in mice, human NK cells expressed the tissue repair protein amphiregulin (AREG). AREG production was induced by TCF7/WNT, IL-2, and IL-15, and inhibited by TGFB1, a cytokine increased in people living with HIV-1. In HIV-1 infection, the percentage of AREG+ NK cells correlated positively with the numbers of ILCs and CD4+ T cells but negatively with the concentration of inflammatory cytokine IL-6. NK-cell knockout of the TGFB1-stimulated WNT antagonist RUNX3 increased AREG production. Antiviral gene expression was increased in all ILC subsets from HIV-1 viremic people, and anti-inflammatory gene MYDGF was increased in an NK-cell subset from HIV-1-infected people whose viral load was undetectable in the absence of antiretroviral therapy. The percentage of defective NK cells in people living with HIV-1 correlated inversely with ILC percentage and CD4+ T-cell counts. CD4+ T cells and their production of IL-2 prevented the loss of NK-cell function by activating mTOR. These studies clarify how ILC subsets are interrelated and provide insight into how HIV-1 infection disrupts NK cells, including an uncharacterized homeostatic function in NK cells.

Precise therapeutic gene correction by a simple nuclease-induced double-stranded break.

Current programmable nuclease-based methods (for example, CRISPR-Cas9) for the precise correction of a disease-causing genetic mutation harness the homology-directed repair pathway. However, this repair process requires the co-delivery of an exogenous DNA donor to recode the sequence and can be inefficient in many cell types. Here we show that disease-causing frameshift mutations that result from microduplications can be efficiently reverted to the wild-type sequence simply by generating a DNA double-stranded break near the centre of the duplication. We demonstrate this in patient-derived cell lines for two diseases: limb-girdle muscular dystrophy type 2G (LGMD2G)1 and Hermansky-Pudlak syndrome type 1 (HPS1)2. Clonal analysis of inducible pluripotent stem (iPS) cells from the LGMD2G cell line, which contains a mutation in TCAP, treated with the Streptococcus pyogenes Cas9 (SpCas9) nuclease revealed that about 80% contained at least one wild-type TCAP allele; this correction also restored TCAP expression in LGMD2G iPS cell-derived myotubes. SpCas9 also efficiently corrected the genotype of an HPS1 patient-derived B-lymphoblastoid cell line. Inhibition of polyADP-ribose polymerase 1 (PARP-1) suppressed the nuclease-mediated collapse of the microduplication to the wild-type sequence, confirming that precise correction is mediated by the microhomology-mediated end joining (MMEJ) pathway. Analysis of editing by SpCas9 and Lachnospiraceae bacterium ND2006 Cas12a (LbCas12a) at non-pathogenic 4-36-base-pair microduplications within the genome indicates that the correction strategy is broadly applicable to a wide range of microduplication lengths and can be initiated by a variety of nucleases. The simplicity, reliability and efficacy of this MMEJ-based therapeutic strategy should permit the development of nuclease-based gene correction therapies for a variety of diseases that are associated with microduplications.

Nonresponse and Progression of Diffuse Keloids to Dupilumab Therapy.

Th2 cytokines, including IL-4 and IL-13, have been shown to increase TGFβ, promoting fibrosis. A recent study showed keloid improvement in one patient after administration of Dupilumab, an IL-4 receptor alpha-antagonist, for his/her atopic dermatitis. Here, we present two 17-year-old patients with diffuse keloids without improvement after three months of Dupilumab treatment, with one patient experiencing clinical worsening. It is currently unknown if Th2 cytokine blockade in patients with keloids reduces TGFβ synthesis or activation. Future studies are needed to investigate the utility of Th2 cytokine blockade as a potential treatment option for keloids. J Drugs Dermatol. 2022;21(2):197-199. doi:10.36849/JDD.6252.

Editing aberrant splice sites efficiently restores ß-globin expression in ß-thalassemia.

The thalassemias are compelling targets for therapeutic genome editing in part because monoallelic correction of a subset of hematopoietic stem cells (HSCs) would be sufficient for enduring disease amelioration. A primary challenge is the development of efficient repair strategies that are effective in HSCs. Here, we demonstrate that allelic disruption of aberrant splice sites, one of the major classes of thalassemia mutations, is a robust approach to restore gene function. We target the IVS1-110G>A mutation using Cas9 ribonucleoprotein (RNP) and the IVS2-654C>T mutation by Cas12a/Cpf1 RNP in primary CD34+ hematopoietic stem and progenitor cells (HSPCs) from ß-thalassemia patients. Each of these nuclease complexes achieves high efficiency and penetrance of therapeutic edits. Erythroid progeny of edited patient HSPCs show reversal of aberrant splicing and restoration of ß-globin expression. This strategy could enable correction of a substantial fraction of transfusion-dependent ß-thalassemia genotypes with currently available gene-editing technology.

Telogen effluvium associated with COVID-19 infection.

Telogen effluvium (TE) is characterized by diffuse hair shedding 2-3 months after a stressor, and COVID-19 infection is potentially one such stressor. Those who were infected with the virus were under immense psychosocial and physiologic stress. We retrospectively reviewed electronic medical records of 552 patients who were evaluated by a Henry Ford Health System dermatologist between February 2020 and September 2020 and had a diagnosis of COVID-19 infection. Ten patients were identified with TE attributed to COVID-19 infection and described their presentations as a case series. For the ten patients selected, the mean age was 48.5 years old and 90% were female. Six of the patients were Black, one Middle Eastern, and three White. On average, the hair shedding began 50 days after the first symptom of COVID-19 infection. About 80% of these patients were treated with antibiotics, systemic corticosteroids, and/or hydroxychloroquine for their COVID-19 infection and 70% were hospitalized. The presentations of these patients suggest that COVID-19 infection may be a significant trigger of TE. TE caused by hydroxychloroquine, azithromycin or other medications cannot be ruled out, and the global pandemic itself is a source of psychosocial stress. Further studies will be needed to understand the long-term prevalence and prognosis of TE associated with COVID-19 infection.

Enhanced Cas12a editing in mammalian cells and zebrafish.

Type V CRISPR-Cas12a systems provide an alternate nuclease platform to Cas9, with potential advantages for specific genome editing applications. Here we describe improvements to the Cas12a system that facilitate efficient targeted mutagenesis in mammalian cells and zebrafish embryos. We show that engineered variants of Cas12a with two different nuclear localization sequences (NLS) on the C terminus provide increased editing efficiency in mammalian cells. Additionally, we find that pre-crRNAs comprising a full-length direct repeat (full-DR-crRNA) sequence with specific stem-loop G-C base substitutions exhibit increased editing efficiencies compared with the standard mature crRNA framework. Finally, we demonstrate in zebrafish embryos that the improved LbCas12a and FnoCas12a nucleases in combination with these modified crRNAs display high mutagenesis efficiencies and low toxicity when delivered as ribonucleoprotein complexes at high concentration. Together, these results define a set of enhanced Cas12a components with broad utility in vertebrate systems.

Self-reported tattoo prevalence and motivations in transgender adults: a cross-sectional survey.

PURPOSE: To assess the prevalence and motivations for obtaining tattoos among transgender persons. METHODS: A survey of 696 transgender persons recruited from the Study of Transition, Outcomes, and Gender (STRONG) cohort evaluated the prevalence of tattoos and motivations for acquiring tattoos. RESULTS: Transmasculine persons were more likely than transfeminine persons to have tattoos (66.5% versus 24.0%, P<0.05). Most commonly reported motivators were personal preference, aesthetics, and/or symbolism (61.8%). Scar coverage and replacement of anatomic features accounted for 10.2% of responses. CONCLUSION: Future studies should look into the relationship between tattoos and health status in the transgender population.

Dermatologic care for lesbian, gay, bisexual, and transgender persons: Terminology, demographics, health disparities, and approaches to care.

More than 10 million lesbian, gay, bisexual, and transgender (LGBT) persons live in the United States. Improving their health is a public health priority. LGBT persons have specific health concerns and face health care disparities. Awareness of those issues and disparities can enable dermatologists to provide medically appropriate and culturally competent care to LGBT patients. This review highlights terminology important in caring for LGBT persons, LGBT demographics in the United States, health care disparities faced by LGBT persons, and approaches to caring for LGBT patients.

Dermatologic care for lesbian, gay, bisexual, and transgender persons: Epidemiology, screening, and disease prevention.

Lesbian, gay, bisexual, and transgender (LGBT) persons face important health issues relevant to dermatologists. Men who have sex with men (MSM) are at higher risk of certain infectious diseases, including HIV, syphilis and other sexually transmitted diseases (STDs), methicillin-resistant Staphylococcus aureus infections, and invasive meningococcal disease, and might be at higher risk of non-infectious conditions, including skin cancer. Recommendations for preventive health care, including screening for HIV and other STDs, sexual health-related vaccinations, and HIV pre-exposure prophylaxis, differ for MSM compared with non-MSM. Women who have sex with women experience disparities in STDs, including chlamydia and HPV. Transgender patients have unique, and often unmet, dermatologic needs during gender transition (also called gender affirmation), related to hormonal therapy and gender-affirming surgery. Familiarity with LGBT health issues and disease-prevention guidelines can enable dermatologists to provide medically appropriate and culturally competent care to LGBT persons.

Diagnostic accuracy of whole slide imaging for cutaneous, soft tissue, and melanoma sentinel lymph node biopsies with and without immunohistochemistry.

BACKGROUND: Diagnostic accuracy with whole slide imaging (WSI) for complex inpatient and outpatient dermatopathology cases with immunohistochemistry (IHC) is unknown. METHODS: WSI (Leica Aperio AT2 Digital Pathology scanner, N = 151 cases) was performed for Emory inpatient and outpatient skin (N = 105), soft tissue (N = 30), and melanoma sentinel lymph node biopsies (N = 16) collected between 2000 and 2016. Resultant images were uploaded to an online cloud storage system for review by 2 board-certified dermatopathologists (reviewers 1 and 2) with greater than 5 years of dermatopathology experience and 1 dermatopathology fellow (reviewer 3). RESULTS: Reviewers 1 (diagnostic accuracy = 97%) and 2 (diagnostic accuracy = 95%) demonstrated high diagnostic accuracy with WSI. Diagnostic accuracy was greater than 90% for inpatient biopsies, melanocytic lesions, melanoma sentinel lymph node biopsies, and cases with immunohistochemistry, but was slightly lower for soft tissue cases (reviewer 1 = 89%; reviewer 2 = 89%). The dermatopathology fellow (reviewer 3) demonstrated lower diagnostic accuracy (84%). CONCLUSIONS: Diagnostic accuracy with WSI for skin, soft tissue, and melanoma sentinel lymph node biopsies with and without immunohistochemistry was greater than 95% for 2 reviewers with greater than 5 years of dermatopathology experience. Professional experience signing out dermatopathology cases may impact diagnostic accuracy with WSI.

Early Systolic Dysfunction Following Traumatic Brain Injury: A Cohort Study.

OBJECTIVE: Prior studies have suggested that traumatic brain injury may affect cardiac function. Our study aims were to determine the frequency, longitudinal course, and admission risk factors for systolic dysfunction in patients with moderate-severe traumatic brain injury. DESIGN: Prospective cohort study. SETTING: Level 1 trauma center. MEASUREMENTS: Transthoracic echocardiogram within 1 day and over the first week after moderate-severe traumatic brain injury; transthoracic echocardiogram within 1 day after mild traumatic brain injury (comparison group). MEASUREMENTS AND MAIN RESULTS: Systolic function was assessed by transthoracic echocardiogram, and systolic dysfunction was defined as fractional shortening less than 25%. Multivariable Poisson regression models examined admission risk factors for systolic dysfunction. Systolic function in 32 patients with isolated moderate-severe traumatic brain injury and 32 patients with isolated mild traumatic brain injury (comparison group) was assessed with transthoracic echocardiogram. Seven (22%) moderate-severe traumatic brain injury and 0 (0%) mild traumatic brain injury patients had systolic dysfunction within the first day after injury (p < 0.01). All patients with early systolic dysfunction recovered in 1 week. Younger age (relative risk, 0.87; 95% CI, 0.79-0.94; for 1 yr increase in age) and lower admission Glasgow Coma Scale score (relative risk, 0.34; 95% CI, 0.20-0.58; for one unit increase in Glasgow Coma Scale) were independently associated with the development of systolic dysfunction among moderate-severe traumatic brain injury patients. CONCLUSIONS: Early systolic dysfunction can occur in previously healthy patients with moderate-severe traumatic brain injury, and it is reversible over the first week of hospitalization. Younger age and lower admission Glasgow Coma Scale score are independently associated with the development of systolic dysfunction after moderate-severe traumatic brain injury.

Racial disparities in the impact of chronic pruritus: A cross-sectional study on quality of life and resource utilization in United States veterans.

BACKGROUND: Chronic pruritus has a lifetime prevalence of up to 26% in the worldwide population. Research has shown that the incidence and quality of life (QoL) impact of chronic pruritus varies by race. OBJECTIVE: We sought to explore the effects of race on specific pruritus-related QoL factors and resource utilization. METHODS: We performed a cross-sectional, national telephone survey of 6000 US veterans randomly sampled from the Veterans Hospital Patient Database. We administered surveys to assess QoL impact and resource utilization of chronic pruritus. RESULTS: Nonwhites overall reported higher levels of burning and scarring with their pruritus. African Americans had a significantly greater emotional impact and use of special soaps, lotions, and clothes. African Americans were also more likely to visit their primary care provider for pruritus (P = .03), yet had similar numbers of specialty care visits. LIMITATIONS: Because our sample was drawn from a veteran population, generalizability may be limited. CONCLUSION: The data indicate a racial disparity in specific QoL impact and resource utilization from pruritus. These findings merit further exploration into explanations, such as access, communication, trust of the medical system, and biologic differences.

Association of low-density lipoprotein cholesterol-related genetic variants with aortic valve calcium and incident aortic stenosis.

IMPORTANCE: Plasma low-density lipoprotein cholesterol (LDL-C) has been associated with aortic stenosis in observational studies; however, randomized trials with cholesterol-lowering therapies in individuals with established valve disease have failed to demonstrate reduced disease progression. OBJECTIVE: To evaluate whether genetic data are consistent with an association between LDL-C, high-density lipoprotein cholesterol (HDL-C), or triglycerides (TG) and aortic valve disease. DESIGN, SETTING, AND PARTICIPANTS: Using a Mendelian randomization study design, we evaluated whether weighted genetic risk scores (GRSs), a measure of the genetic predisposition to elevations in plasma lipids, constructed using single-nucleotide polymorphisms identified in genome-wide association studies for plasma lipids, were associated with aortic valve disease. We included community-based cohorts participating in the CHARGE consortium (n = 6942), including the Framingham Heart Study (cohort inception to last follow-up: 1971-2013; n = 1295), Multi-Ethnic Study of Atherosclerosis (2000-2012; n = 2527), Age Gene/Environment Study-Reykjavik (2000-2012; n = 3120), and the Malmö Diet and Cancer Study (MDCS, 1991-2010; n = 28,461). MAIN OUTCOMES AND MEASURES: Aortic valve calcium quantified by computed tomography in CHARGE and incident aortic stenosis in the MDCS. RESULTS: The prevalence of aortic valve calcium across the 3 CHARGE cohorts was 32% (n = 2245). In the MDCS, over a median follow-up time of 16.1 years, aortic stenosis developed in 17 per 1000 participants (n = 473) and aortic valve replacement for aortic stenosis occurred in 7 per 1000 (n = 205). Plasma LDL-C, but not HDL-C or TG, was significantly associated with incident aortic stenosis (hazard ratio [HR] per mmol/L, 1.28; 95% CI, 1.04-1.57; P = .02; aortic stenosis incidence: 1.3% and 2.4% in lowest and highest LDL-C quartiles, respectively). The LDL-C GRS, but not HDL-C or TG GRS, was significantly associated with presence of aortic valve calcium in CHARGE (odds ratio [OR] per GRS increment, 1.38; 95% CI, 1.09-1.74; P = .007) and with incident aortic stenosis in MDCS (HR per GRS increment, 2.78; 95% CI, 1.22-6.37; P = .02; aortic stenosis incidence: 1.9% and 2.6% in lowest and highest GRS quartiles, respectively). In sensitivity analyses excluding variants weakly associated with HDL-C or TG, the LDL-C GRS remained associated with aortic valve calcium (P = .03) and aortic stenosis (P = .009). In instrumental variable analysis, LDL-C was associated with an increase in the risk of incident aortic stenosis (HR per mmol/L, 1.51; 95% CI, 1.07-2.14; P = .02). CONCLUSIONS AND RELEVANCE: Genetic predisposition to elevated LDL-C was associated with presence of aortic valve calcium and incidence of aortic stenosis, providing evidence supportive of a causal association between LDL-C and aortic valve disease. Whether earlier intervention to reduce LDL-C could prevent aortic valve disease merits further investigation.

Can High-Fidelity Patient Simulation Be Used for Skill Development in Junior Undergraduate Students: A Quasi-Experimental Study.

High-fidelity patient simulation (HFPS) is widely used in professional training to enhance students' competence in clinical management. A guideline for HFPS provides a systematic approach to direct students to learning during the simulation process. Problem-solving (PS) and clinical reasoning (CR) skills are essential to developing students' professional competence in safe and effective care. These two skills should be initiated in the early training. A structured guideline was developed for HFPS. This study aimed to investigate the effects of the structured HFPS guideline on the development of PS and CR skills in junior nursing students. The students were required to go through four sessions, pre-briefing, simulation design, facilitation, and debriefing, for the HFPS; the study utilized the Problem-Solving Inventory (PSI) and the Nurses' Clinical Reasoning Scale (NCRS) to measure PS and CR abilities before and after HFPS. Bivariate analysis, a one-sample t-test, and an independent t-test were performed to evaluate the performance of the PS and CR skills during the two study periods. A total of 189 students were recruited, with 92 in the intervention group and 97 in the control group. The research assistant was responsible for student recruitment through email invitations and allocating the students into the control group or the intervention group. A Wilcoxon analysis was performed and revealed significant differences in PS and CR between the two groups (p < 0.001). The analytic results showed that the PSI, particularly in domains of Problem-Solving Confidence (PSC) (p < 0.001) and overall PS (p < 0.001), and the CR (p < 0.001) had significant improvement after HFPS, particularly in the intervention group. The study concluded that the structured HFPS guideline significantly improved the students' problem-solving and clinical reasoning abilities. Nurse educators play an important role in providing explicit learning instructions in a simulation guideline that directs and guides students to learn at each stage of HFPS. The students can be directed to be engaged in their learning through HFPS to enhance their competence in knowledge and skill development (PS and CR) for their personal and professional development.

Generation and application of endogenously floxed alleles for cell-specific knockout in zebrafish.

The zebrafish is amenable to a variety of genetic approaches. However, lack of conditional deletion alleles limits stage- or cell-specific gene knockout. Here, we applied an existing protocol to establish a floxed allele for gata2a but failed to do so due to off-target integration and incomplete knockin. To address these problems, we applied simultaneous co-targeting with Cas12a to insert loxP sites in cis, together with transgenic counterscreening and comprehensive molecular analysis, to identify off-target insertions and confirm targeted knockins. We subsequently used our approach to establish endogenously floxed alleles of foxc1a, rasa1a, and ruvbl1, each in a single generation. We demonstrate the utility of these alleles by verifying Cre-dependent deletion, which yielded expected phenotypes in each case. Finally, we used the floxed gata2a allele to demonstrate an endothelial autonomous requirement in lymphatic valve development. Together, our results provide a framework for routine generation and application of endogenously floxed alleles in zebrafish.

Comparison of three Roche hepatitis B virus viral load assay formats.

Two FDA-approved (in vitro diagnostic [IVD]) hepatitis B virus (HBV) viral load assays, the manual Cobas TaqMan HBV Test for use with the High Pure System (HP) and the automated Cobas AmpliPrep/Cobas TaqMan HBV Test v2.0 (CAP/CTM), were compared to a modified (not FDA-approved) version of the HP assay by automating the DNA extraction using the Total Nucleic Acid Isolation (TNAI) kit on the Cobas AmpliPrep. On average, CAP/CTM measurements were 0.08 log IU/ml higher than HP results (n = 206), and TNAI results were 0.17 log IU/ml higher than HP results (n = 166). The limit of detection (LOD), as determined by probit analysis using dilutions of the 2nd HBV international standard, was 10.2 IU/ml for CAP/CTM. The data sets for HP and TNAI were insufficient for probit analysis; however, there was 100% detection at ≥ 5 or ≥ 10 IU/ml for TNAI and HP, respectively. Linearity was demonstrated between 60 and 2,000,000 IU/ml, with slopes between 0.95 and 0.99 and R(2) values of >0.99 for all assays. Total precision (log percent coefficient of variance [CV]) was between 0.8% and 2.1% at 4.3 log IU/ml and between 1.4% and 4.9% at 2.3 log IU/ml. Correlation of samples, reproducibility, linearity, and LOD were acceptable and similar in all assays. The CAP/CTM assay and, to a lesser extent, the TNAI assay reduced hands-on time due to automation. There were no instances of contamination detected in negative samples during the course of the study, despite testing several samples up to 9.6 log IU/ml. The incidence of false-positive negative controls in HP and CAP/CTM clinical testing was <0.5% over 6 to 7 months of testing.

Moderating Role of Coping Style on the Relationship between Stress and Psychological Well-Being in Hong Kong Nursing Students.

AIMS AND OBJECTIVES: To examine whether coping styles moderate the influence of stressors and psychological well-being in Hong Kong nursing students. BACKGROUND: Stress could contribute to psychological distress in nursing students. Coping strategies are essential to mitigate psychological distress. So far, the moderating effects of coping between stressors and psychological well-being has not been thoroughly investigated. DESIGN: This is a cross-sectional study conducted at four higher education institutions in Hong Kong. METHODS: We recruited a convenience sample of 293 nursing students in February 2018. The Stressors in Nursing Students Scale-Chinese version (SINS-CN), Brief Cope Inventory-Chinese version (Brief COPE-C), and the Chinese version of the General Health Questionnaire-12 (C-GHQ-12) were used to measure the stressors, coping styles, and psychological well-being, respectively. Three multiple hierarchical linear regression models were used to identify the associations between the variables. RESULTS: The stressors related to clinical learning, confidence, and personal problems were significant in explaining the psychological well-being. The coping strategies also predicted the psychological well-being and explained 44.5% of the variance. The coping strategy-accommodation-moderated the relationship between personal problems and psychological well-being. CONCLUSION: Problem-solving and accommodation types of coping were adaptive to stress and effective in promoting psychological well-being. However, using accommodation to cope with stressors related to personal problems will exacerbate the negative effects of the personal problems on the psychological well-being. RELEVANCE TO CLINICAL PRACTICE: This study reveals the relationships between stressors, coping, and psychological well-being. Nurse educators must be aware of nursing student coping styles so they may devise strategies to promote effective coping to reduce the psychological distress among nursing students.