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Using a learning theory is key when designing simulation-based learning linked to allied health professional placements to ensure the purposeful selection of educational methods and to understand how it may assist learners in achieving desired learning outcomes. A narrative review was undertaken to identify the learning theories reported in simulation-based learning linked to allied health professional placements and how the learning outcomes aligned with the reported theories. Only eight of the 25 reviewed studies explicitly reported a learning theory, with minimal attempt to link them to the learning experience. Educators are encouraged to develop an understanding of the breadth and depth of learning theories and how they can best align these with the desired learning outcomes to allow the best fit for purpose. As a result, the authors have developed a practical guide to assist educators in designing simulation-based learning to better understand how learning theories could be incorporated and the anticipated outcomes that could be measured.

BackgroundCoccidioidomycosis, an emerging fungal disease in the western USA, exhibits seasonal patterns that are poorly understood, including periods of strong cyclicity, aseasonal intervals, and variation in seasonal timing that have been minimally characterized, and unexplained as to their causal factors. Coccidioidomycosis incidence has increased markedly in recent years, and our limited understanding of intra- and inter-annual seasonality has hindered the identification of important drivers of disease transmission, including climate conditions. In this study, we aim to characterize coccidioidomycosis seasonality in endemic regions of California and to estimate the relationship between drought conditions and coccidioidomycosis seasonal periodicity and timing.MethodsWe analysed data on all reported incident cases of coccidioidomycosis in California from 2000 to 2021 to characterize seasonal patterns in incidence, and conducted wavelet analyses to assess the dominant periodicity, power, and timing of incidence for 17 counties with consistently high incidence rates. We assessed associations between seasonality parameters and measures of drought in California using a distributed lag nonlinear modelling framework.FindingsAll counties exhibited annual cyclicity in incidence (i.e., a dominant wavelet periodicity of 12 months), but there was considerable heterogeneity in seasonal strength and timing across regions and years. On average, 12-month periodicity was most pronounced in the Southern San Joaquin Valley and Central Coast. Further, the annual seasonal cycles in the Southern San Joaquin Valley and the Southern Inland regions occurred earlier than those in coastal and northern counties, yet the timing of annual cycles became more aligned among counties by the end of the study period. Drought conditions were associated with a strong attenuation of the annual seasonal cycle, and seasonal peaks became more pronounced in the 1-2 years after a drought ended.InterpretationWe conclude that drought conditions do not increase the risk of coccidioidomycosis onset uniformly across the year, but instead promote increased risk concentrated within a specific calendar period (September to December). The findings have important implications for public health preparedness, and for how future shifts in seasonal climate patterns and extreme events may impact spatial and temporal coccidioidomycosis risk.FundingNational Institutes of Health.

A regulated stress response is essential for healthy child growth and development trajectories. We conducted a cluster-randomized trial in rural Bangladesh (funded by the Bill & Melinda Gates Foundation, ClinicalTrials.gov NCT01590095) to assess the effects of an integrated nutritional, water, sanitation, and handwashing intervention on child health. We previously reported on the primary outcomes of the trial, linear growth and caregiver-reported diarrhea. Here, we assessed additional prespecified outcomes: physiological stress response, oxidative stress, and DNA methylation (N = 759, ages 1-2 years). Eight neighboring pregnant women were grouped into a study cluster. Eight geographically adjacent clusters were block-randomized into the control or the combined nutrition, water, sanitation, and handwashing (N + WSH) intervention group (receiving nutritional counseling and lipid-based nutrient supplements, chlorinated drinking water, upgraded sanitation, and handwashing with soap). Participants and data collectors were not masked, but analyses were masked. There were 358 children (68 clusters) in the control group and 401 children (63 clusters) in the intervention group. We measured four F2-isoprostanes isomers (iPF(2α)-III; 2,3-dinor-iPF(2α)-III; iPF(2α)-VI; 8,12-iso-iPF(2α)-VI), salivary alpha-amylase and cortisol, and methylation of the glucocorticoid receptor (NR3C1) exon 1F promoter including the NGFI-A binding site. Compared with control, the N + WSH group had lower concentrations of F2-isoprostanes isomers (differences ranging from -0.16 to -0.19 log ng/mg of creatinine, P

Objective  While postoperative cerebrospinal fluid (CSF) leak rates of pituitary tumors have been frequently studied, there are fewer studies examining postoperative CSF leak rates for extrasellar tumors. The purpose of this study was to identify risk factors for the development of postoperative CSF leak in patients undergoing endoscopic surgery for extrasellar tumors. Methods  A retrospective chart review was done for patients who underwent endoscopic resection for extrasellar tumors between 2008 and 2020. Age, gender, tumor type, tumor location, tumor size, reconstruction technique, medical comorbidities, and other potential risk factors were identified. Data was analyzed to identify significant risk factors for development of postoperative CSF leak. Results  There were 100 patients with extrasellar tumors who developed intraoperative CSF leaks. Seventeen patients (17%) developed postoperative CSF leaks. Leaks occurred at a median of 2 days following surgery (range 0-34 days). Clival tumors had a significantly higher incidence of postoperative leak than those in other sites ( p

Natural history museums are vital repositories of specimens, samples and data that inform about the natural world; this Formal Comment revisits a Perspective that advocated for the adoption of compassionate collection practices, querying whether it will ever be possible to completely do away with whole animal specimen collection.

Abstract The International Mouse Phenotyping Consortium (IMPC) systematically produces and phenotypes mouse lines with presumptive null mutations to provide insight into gene function. The IMPC now uses the programmable RNA-guided nuclease Cas9 for its increased capacity and flexibility to efficiently generate null alleles in the C57BL/6N strain. In addition to being a valuable novel and accessible research resource, the production of 3313 knockout mouse lines using comparable protocols provides a rich dataset to analyze experimental and biological variables affecting in vivo gene engineering with Cas9. Mouse line production has two critical steps – generation of founders with the desired allele and germline transmission (GLT) of that allele from founders to offspring. A systematic evaluation of the variables impacting success rates identified gene essentiality as the primary factor influencing successful production of null alleles. Collectively, our findings provide best practice recommendations for using Cas9 to generate alleles in mouse essential genes, many of which are orthologs of genes linked to human disease.

Abstract Background To explore perspectives of work readiness, including readiness to work rurally, among health students trained in Australia during the COVID-19 pandemic. Methods Participants were allied health, medicine, and nursing students in the later years of their degree (third, fourth or final year of an undergraduate entry to practice degree, or second year of postgraduate entry to practice degree), where training is clinically immersive. These students had completed a University Department of Rural Health facilitated rural and remote placement between January 2021 and October 2022. They participated in a cross-sectional online survey (n = 426), comprising Likert-scale questions. Interested survey respondents participated in a semi-structured interview (n = 34). Multiple logistic regression was conducted to examine the predictors of work readiness within the survey, and interview data was analysed via reflexive thematic analysis. Results Among survey respondents, 69.7% felt they would be ready to be a health practitioner when the time came to graduate and 71.8% felt clinically prepared to work in a rural location. Concerns about having developed enough clinical skills on placements to competently practice on graduation and being able to continue studying their course during the pandemic were both predictive of work readiness and feeling clinically prepared to work rurally. Four themes reflecting factors impacting work readiness were developed from interview data: (1) ‘I’d estimate probably a 20–30% reduction in face-to-face handling practice over the course of all of my placements’ encompassed student concerns regarding the collective impact of cancelled placements and lower patient attendance at healthcare facilities on clinical skill development; (2) ‘Two and a half years of sitting behind a computer’ related to student experiences of superficial learning and cohort disconnectedness due to online course delivery and loss of on campus simulations; (3) ‘I’ll still need like a lot of support in my grad year’ related to students recognising the need for support and supervision post-qualifying to bridge the gaps in their learning; and (4) ‘We are the COVID nurses’ encompassed student recognition of skills gained including communication skills, competence with technology and telehealth, knowledge of infection control, and work readiness skills (e.g. adaptability and resilience) as a result of training during the global pandemic. Conclusions Universities can support work readiness during pandemic circumstances by fostering clinical skills development through continuation of quality placement experiences and face-to-face curriculum delivery. Although health graduates trained during the pandemic are likely to have a range of additional work ready skills, health services will need to proactively support their transition into the workforce in the coming years.

The importance of making online and distance education successful has been dramatically prioritized due to the COVID-19 pandemic. Student retention is a key performance indicator in online higher education. Evidence suggests that within online distance education the key to retaining students is to encourage an engaging and supportive online community. Online asynchronous forums are one method that has been employed to promote such engagement. This study investigated the relationship between online forum activity and student retention amongst 21 tutor groups on an Open University Level 1 module. It found that the volume of tutor group forum activity had a significant association with student retention (p<0.05 two-tailed, Pearson r=0.53), with student contributions having a greater impact on retention (p<0.01 two-tailed, Pearson r=0.628) than tutor contributions. Although tutor contributions did not appear to impact student retention directly the number of tutor and student posts were highly correlated (p<0.001, two tailed, Pearson r=0.86). Results indicate that asynchronous forums can be an effective tool in promoting student retention in an online distance learning environment. Important factors that impact on student retention within the educational medium of asynchronous forums are evidenced. The implications of these findings for educators are discussed. [Note: The page range (103-118) shown on the PDF is incorrect. The correct page range for this article is p103-117.]

Abstract Despite great advancements in the treatment of chronic airway diseases, improvements in morbidity and mortality have stalled in recent years. Asthma and chronic obstructive pulmonary disease are complex and heterogeneous diseases that require tailored management based on individual patient characteristics and needs. The Treatable Traits (TTs) approach aims to personalise and improve patient care through the identification and targeting of clinically relevant and modifiable pulmonary, extra-pulmonary and behavioural traits. In this article, we outline the rationale for TTs-based management and provide practical guidance for its application in primary care. To aid implementation, seven potential ‘prime’ traits are proposed: airflow obstruction, eosinophilic inflammation, adherence, inhaler technique, smoking, low body mass index/obesity and anxiety and depression—selected for their prevalence, recognisability and feasibility of use. Some of the key questions among healthcare professionals, that may be roadblocks to widespread application of a TTs model of care, are also addressed.

Osteogenesis imperfecta (OI) type V is the second most common form of OI, distinguished by hyperplastic callus formation and calcification of the interosseous membranes, in addition to the bone fragility. It is caused by a recurrent, dominant pathogenic variant (c.-14C>T) in interferon-induced transmembrane protein 5 (IFITM5). Here, we generated a conditional Roso26-knockin mouse model to study the mechanistic consequences of the recurrent mutation. Expression of the mutant Ifitm5 in osteo-chondroprogenitor or chondrogenic cells resulted in low bone mass and growth retardation. Mutant limbs showed impaired endochondral ossification, cartilage overgrowth, and abnormal growth plate architecture. The cartilage phenotype correlates with the pathology reported in patients with OI type V. Surprisingly, expression of mutant Ifitm5 in mature osteoblasts caused no obvious skeletal abnormalities. In contrast, earlier expression in osteo-chondroprogenitors was associated with an increase in the skeletal progenitor cell population within the periosteum. Lineage tracing showed that chondrogenic cells expressing the mutant Ifitm5 had decreased differentiation into osteoblastic cells in diaphyseal bone. Moreover, mutant IFITM5 disrupted early skeletal homeostasis in part by activating ERK signaling and downstream SOX9 protein, and inhibition of these pathways partially rescued the phenotype in mutant animals. These data identify the contribution of a signaling defect altering osteo-chondroprogenitor differentiation as a driver in the pathogenesis of OI type V., Introduction Osteogenesis imperfecta (OI) is an inherited multi-systemic disorder characterized by low bone mass, recurrent fractures, growth deficiency, and skeletal deformities (1-3). Extraskeletal manifestations may include muscle weakness, dentinogenesis imperfecta, [...]

The majority of corticotroph adenomas are benign but some are locally invasive, demonstrate high rates of recurrence, and exhibit a relatively poor response to often repeated surgical, medical, and radiation treatment. Herein, we summarize the currently known somatic and genetic mutations and other molecular factors that influence the pathogenesis of these tumors and discuss currently available therapies. Although recent molecular studies have advanced our understanding of the pathogenesis and behavior of these refractory corticotroph adenomas, these insights do not reliably guide treatment choices at present. Development of additional diagnostic tools and novel tumor-directed therapies that offer efficacious treatment choices for patients with refractory corticotroph adenomas are needed.

Summary: Background: Coccidioidomycosis, an emerging fungal disease in the western USA, exhibits seasonal patterns that are poorly understood, including periods of strong cyclicity, aseasonal intervals, and variation in seasonal timing that have been minimally characterized, and unexplained as to their causal factors. Coccidioidomycosis incidence has increased markedly in recent years, and our limited understanding of intra- and inter-annual seasonality has hindered the identification of important drivers of disease transmission, including climate conditions. In this study, we aim to characterize coccidioidomycosis seasonality in endemic regions of California and to estimate the relationship between drought conditions and coccidioidomycosis seasonal periodicity and timing. Methods: We analysed data on all reported incident cases of coccidioidomycosis in California from 2000 to 2021 to characterize seasonal patterns in incidence, and conducted wavelet analyses to assess the dominant periodicity, power, and timing of incidence for 17 counties with consistently high incidence rates. We assessed associations between seasonality parameters and measures of drought in California using a distributed lag nonlinear modelling framework. Findings: All counties exhibited annual cyclicity in incidence (i.e., a dominant wavelet periodicity of 12 months), but there was considerable heterogeneity in seasonal strength and timing across regions and years. On average, 12-month periodicity was most pronounced in the Southern San Joaquin Valley and Central Coast. Further, the annual seasonal cycles in the Southern San Joaquin Valley and the Southern Inland regions occurred earlier than those in coastal and northern counties, yet the timing of annual cycles became more aligned among counties by the end of the study period. Drought conditions were associated with a strong attenuation of the annual seasonal cycle, and seasonal peaks became more pronounced in the 1–2 years after a drought ended. Interpretation: We conclude that drought conditions do not increase the risk of coccidioidomycosis onset uniformly across the year, but instead promote increased risk concentrated within a specific calendar period (September to December). The findings have important implications for public health preparedness, and for how future shifts in seasonal climate patterns and extreme events may impact spatial and temporal coccidioidomycosis risk. Funding: National Institutes of Health.

Background: Severe asthma pathology encompasses a wide range of pulmonary and extrapulmonary treatable traits with a high prevalence of comorbidities. Although asthma-specific health-related quality-of-life measures are most sensitive to changes in asthma control, generic measures, such as EQ-5D-5L (EuroQol 5-Dimension 5-Level questionnaire), are potentially better for capturing the impact of comorbidities. Objective: We sought to examine the impact of pulmonary and extrapulmonary treatable traits on quality of life at initial severe asthma assessment, and to compare the characteristics of those patients whose quality of life does and does not improve during follow-up at severe asthma centers. Methods: Patients’ characteristics at baseline assessment within the UK Severe Asthma Registry were compared by EQ-5D-5L utility index quartile. Patients with follow-up review data were stratified by change in EQ-5D-5L utility index from baseline to follow-up, and characteristics similarly examined. Results: Patients in the quartiles with worst dysutility at baseline were observed to exhibit more treatable traits and in particular extrapulmonary traits associated with cumulative systemic corticosteroids, including obesity, anxiety/depression, and osteoporosis. In those patients whose quality of life improved over follow-up, a reduction in exacerbations, uncontrolled symptoms, and requirement for maintenance oral corticosteroids were observed. Conclusions: Both pulmonary and extrapulmonary treatable traits are important determinants of quality of life in severe asthma. Comorbidities associated with cumulative systemic corticosteroid exposure are particularly associated with worse quality of life, emphasizing the importance of early identification and management of severe asthma before comorbidities develop.

Background The long-term outcomes of COVID-19 hospitalisation in individuals with pre-existing airway diseases are unknown. Methods Adult participants hospitalised for confirmed or clinically suspected COVID-19 and discharged between 5 March 2020 and 31 March 2021 were recruited to the Post-hospitalisation COVID-19 (PHOSP-COVID) study. Participants attended research visits at 5 months and 1 year post discharge. Clinical characteristics, perceived recovery, burden of symptoms and health-related quality of life (HRQoL) of individuals with pre-existing airway disease (i.e., asthma, COPD or bronchiectasis) were compared to the non-airways group. Results A total of 615 out of 2697 (22.8%) participants had a history of pre-existing airway diseases (72.0% diagnosed with asthma, 22.9% COPD and 5.1% bronchiectasis). At 1 year, the airways group participants were less likely to feel fully recovered (20.4% versus 33.2%, p

Abstract A regulated stress response is essential for healthy child growth and development trajectories. We conducted a cluster-randomized trial in rural Bangladesh (funded by the Bill & Melinda Gates Foundation, ClinicalTrials.gov NCT01590095) to assess the effects of an integrated nutritional, water, sanitation, and handwashing intervention on child health. We previously reported on the primary outcomes of the trial, linear growth and caregiver-reported diarrhea. Here, we assessed additional prespecified outcomes: physiological stress response, oxidative stress, and DNA methylation (N = 759, ages 1–2 years). Eight neighboring pregnant women were grouped into a study cluster. Eight geographically adjacent clusters were block-randomized into the control or the combined nutrition, water, sanitation, and handwashing (N + WSH) intervention group (receiving nutritional counseling and lipid-based nutrient supplements, chlorinated drinking water, upgraded sanitation, and handwashing with soap). Participants and data collectors were not masked, but analyses were masked. There were 358 children (68 clusters) in the control group and 401 children (63 clusters) in the intervention group. We measured four F2-isoprostanes isomers (iPF(2α)-III; 2,3-dinor-iPF(2α)-III; iPF(2α)-VI; 8,12-iso-iPF(2α)-VI), salivary alpha-amylase and cortisol, and methylation of the glucocorticoid receptor (NR3C1) exon 1F promoter including the NGFI-A binding site. Compared with control, the N + WSH group had lower concentrations of F2-isoprostanes isomers (differences ranging from −0.16 to −0.19 log ng/mg of creatinine, P

Abstract Severe asthma is associated with an increased risk for exacerbations, reduced lung function, fixed airflow obstruction, and substantial morbidity and mortality. The concept of remission in severe asthma as a new treatment goal has recently gained attention due to the growing use of monoclonal antibody therapies, which target specific pathologic pathways of inflammation. This review evaluates the current definitions of asthma remission and unveils some of the barriers for achieving this state in the severe asthma population. Although there is no unified definition, the concept of clinical remission in asthma should be based on a sustained period of symptom control, elimination of oral corticosteroid exposure and exacerbations, and stabilization of pulmonary function. The conjugation of these criteria seems a realistic treatment target in a minority of asthmatic patients. Some unmet needs in severe asthma may affect the achievement of clinical remission. Late intervention with targeted therapies in the severe asthma population may increase the risk of corticosteroid exposure and the development of irreversible structural airway changes. Moreover, airway infection is an important component in persistent exacerbations in patients on biologic therapies. Phenotyping exacerbations may be useful to guide therapy decisions and to avoid the liberal use of oral corticosteroids. Another challenge associated with the aim of clinical remission in severe asthma is the multifaceted interaction between the disease and its associated comorbidities. Behavioural factors should be evaluated in case of persistent symptoms despite optimised treatment, and assessing biomarkers and targeting treatable traits may allow for a more objective way of reaching remission. The concept of clinical remission will benefit from an international consensus to establish unifying criteria for its assessment, and it should be addressed in the future management guidelines.

Abstract Cellular responses to the steroid hormones, estrogen (E2), and progesterone (P4) are governed by their cognate receptor’s transcriptional output. However, the feed-forward mechanisms that shape cell-type-specific transcriptional fulcrums for steroid receptors are unidentified. Herein, we found that a common feed-forward mechanism between GREB1 and steroid receptors regulates the differential effect of GREB1 on steroid hormones in a physiological or pathological context. In physiological (receptive) endometrium, GREB1 controls P4-responses in uterine stroma, affecting endometrial receptivity and decidualization, while not affecting E2-mediated epithelial proliferation. Of mechanism, progesterone-induced GREB1 physically interacts with the progesterone receptor, acting as a cofactor in a positive feedback mechanism to regulate P4-responsive genes. Conversely, in endometrial pathology (endometriosis), E2-induced GREB1 modulates E2-dependent gene expression to promote the growth of endometriotic lesions in mice. This differential action of GREB1 exerted by a common feed-forward mechanism with steroid receptors advances our understanding of mechanisms that underlie cell- and tissue-specific steroid hormone actions.

Abstract Background High levels of physical activity are associated with reduced risk of the blood cancer multiple myeloma (MM). MM is preceded by the asymptomatic stages of monoclonal gammopathy of undetermined significance (MGUS) and smouldering multiple myeloma (SMM) which are clinically managed by watchful waiting. A case study (N = 1) of a former elite athlete aged 44 years previously indicated that a multi-modal exercise programme reversed SMM disease activity. To build from this prior case study, the present pilot study firstly examined if short-term exercise training was feasible and safe for a group of MGUS and SMM patients, and secondly investigated the effects on MGUS/SMM disease activity. Methods In this single-arm pilot study, N = 20 participants diagnosed with MGUS or SMM were allocated to receive a 16-week progressive exercise programme. Primary outcome measures were feasibility and safety. Secondary outcomes were pre- to post-exercise training changes to blood biomarkers of MGUS and SMM disease activity– monoclonal (M)-protein and free light chains (FLC)– plus cardiorespiratory and functional fitness, body composition, quality of life, blood immunophenotype, and blood biomarkers of inflammation. Results Fifteen (3 MGUS and 12 SMM) participants completed the exercise programme. Adherence was 91 ± 11%. Compliance was 75 ± 25% overall, with a notable decline in compliance at intensities > 70% V̇O2PEAK. There were no serious adverse events. There were no changes to M-protein (0.0 ± 1.0 g/L, P =.903), involved FLC (+ 1.8 ± 16.8 mg/L, P =.839), or FLC difference (+ 0.2 ± 15.6 mg/L, P =.946) from pre- to post-exercise training. There were pre- to post-exercise training improvements to diastolic blood pressure (− 3 ± 5 mmHg, P =.033), sit-to-stand test performance (+ 5 ± 5 repetitions, P =.002), and energy/fatigue scores (+ 10 ± 15%, P =.026). Other secondary outcomes were unchanged. Conclusions A 16-week progressive exercise programme was feasible and safe, but did not reverse MGUS/SMM disease activity, contrasting a prior case study showing that five years of exercise training reversed SMM in a 44-year-old former athlete. Longer exercise interventions should be explored in a group of MGUS/SMM patients, with measurements of disease biomarkers, along with rates of disease progression (i.e., MGUS/SMM to MM). Registration https://www.isrctn.com/ISRCTN65527208 (14/05/2018).

Desmopressin is a synthetic analog of the endogenous peptide hormone arginine vasopressin, or antidiuretic hormone. The drug was initially developed in 1967 with the intention of creating a molecule that could treat central diabetes insipidus, a condition characterized by a deficiency of ADH and results in the production of large volumes of dilute urine (Zaoral et al., 1967). In the years since, therapeutic use of desmopressin has expanded beyond central diabetes insipidus to include the treatment of many other conditions, notably nocturnal polyuria in children with enuresis (bed wetting), adult nocturia, congenital bleeding disorders, and more. The purpose of the present narrative review is to recount the history of desmopressin, review its key therapeutic indications, and discuss important pharmacologic and pharmacokinetic considerations of this medication.

The frequency and severity of wildfires in the Western United States have increased over recent decades, motivating hypotheses that wildfires contribute to the incidence of coccidioidomycosis, an emerging fungal disease in the Western United States with sharp increases in incidence observed since 2000. While coccidioidomycosis outbreaks have occurred among wildland firefighters clearing brush, it remains unknown whether fires are associated with an increased incidence among the general population.MethodsWe identified 19 wildfires occurring within California's highly endemic San Joaquin Valley between 2003 and 2015. Using geolocated surveillance records, we applied a synthetic control approach to estimate the effect of each wildfire on the incidence of coccidioidomycosis among residents that lived within a hexagonal buffer of 20 km radii surrounding the fire.ResultsWe did not detect excess cases due to wildfires in the 12 months (pooled estimated percent change in cases: 2.8%; 95% confidence interval [CI] = -29.0, 85.2), 13-24 months (7.9%; 95% CI = -27.3, 113.9), or 25-36 months (17.4%; 95% CI = -25.1, 157.1) following a wildfire. When examined individually, we detected significant increases in incidence following three of the 19 wildfires, all of which had relatively large adjacent populations, high transmission before the fire, and a burn area exceeding 5,000 acres.DiscussionWe find limited evidence that wildfires drive increases in coccidioidomycosis incidence among the general population. Nevertheless, our results raise concerns that large fires in regions with ongoing local transmission of Coccidioides may be associated with increases in incidence, underscoring the need for field studies examining Coccidioides spp. in soils and air pre- and post-wildfires.

Genome editing with CRISPR-associated (Cas) proteins holds exceptional promise for "correcting" variants causing genetic disease. To realize this promise, off-target genomic changes cannot occur during the editing process. Here, we use whole genome sequencing to compare the genomes of 50 Cas9-edited founder mice to 28 untreated control mice to assess the occurrence of S. pyogenes Cas9-induced off-target mutagenesis. Computational analysis of whole-genome sequencing data detects 26 unique sequence variants at 23 predicted off-target sites for 18/163 guides used. While computationally detected variants are identified in 30% (15/50) of Cas9 gene-edited founder animals, only 38% (10/26) of the variants in 8/15 founders validate by Sanger sequencing. In vitro assays for Cas9 off-target activity identify only two unpredicted off-target sites present in genome sequencing data. In total, only 4.9% (8/163) of guides tested have detectable off-target activity, a rate of 0.2 Cas9 off-target mutations per founder analyzed. In comparison, we observe ~1,100 unique variants in each mouse regardless of genome exposure to Cas9 indicating off-target variants comprise a small fraction of genetic heterogeneity in Cas9-edited mice. These findings will inform future design and use of Cas9-edited animal models as well as provide context for evaluating off-target potential in genetically diverse patient populations.

BackgroundMicrophthalmia, anophthalmia, and coloboma (MAC) spectrum disease encompasses a group of eye malformations which play a role in childhood visual impairment. Although the predominant cause of eye malformations is known to be heritable in nature, with 80% of cases displaying loss-of-function mutations in the ocular developmental genes OTX2 or SOX2, the genetic abnormalities underlying the remaining cases of MAC are incompletely understood. This study intended to identify the novel genes and pathways required for early eye development. Additionally, pathways involved in eye formation during embryogenesis are also incompletely understood. This study aims to identify the novel genes and pathways required for early eye development through systematic forward screening of the mammalian genome.ResultsQuery of the International Mouse Phenotyping Consortium (IMPC) database (data release 17.0, August 01, 2022) identified 74 unique knockout lines (genes) with genetically associated eye defects in mouse embryos. The vast majority of eye abnormalities were small or absent eyes, findings most relevant to MAC spectrum disease in humans. A literature search showed that 27 of the 74 lines had previously published knockout mouse models, of which only 15 had ocular defects identified in the original publications. These 12 previously published gene knockouts with no reported ocular abnormalities and the 47 unpublished knockouts with ocular abnormalities identified by the IMPC represent 59 genes not previously associated with early eye development in mice. Of these 59, we identified 19 genes with a reported human eye phenotype. Overall, mining of the IMPC data yielded 40 previously unimplicated genes linked to mammalian eye development. Bioinformatic analysis showed that several of the IMPC genes colocalized to several protein anabolic and pluripotency pathways in early eye development. Of note, our analysis suggests that the serine-glycine pathway producing glycine, a mitochondrial one-carbon donator to folate one-carbon metabolism (FOCM), is essential for eye formation.ConclusionsUsing genome-wide phenotype screening of single-gene knockout mouse lines, STRING analysis, and bioinformatic methods, this study identified genes heretofore unassociated with MAC phenotypes providing models to research novel molecular and cellular mechanisms involved in eye development. These findings have the potential to hasten the diagnosis and treatment of this congenital blinding disease.

BackgroundFK506 binding protein 51 (FKBP5) is a co-chaperone regulator of the glucocorticoid receptor (GR). Recent studies have reported increased FKBP5 mRNA in the circulation from patients with Cushing disease (CD) which returned to comparable levels seen in healthy controls following successful trans-nasal trans-sphenoidal (TNTS) surgical corticotroph tumor removal. However, the expression of circulating FKBP5 mRNA levels in other pituitary tumor subtypes and its specificity to corticotroph tumors is unknown.MethodsPre-operative blood was collected from consecutive patients undergoing TNTS for pituitary tumors (n = 57) at our center between 2015 and 2019. Total RNA was isolated from whole blood using RiboPure blood RNA isolation kit and real-time qPCR was used to quantitate circulating FKBP5 mRNA expression.ResultsConsistent with the prior report, higher circulating FKBP5 mRNA levels were observed in 20 patients with CD prior to surgical tumor removal, compared to 21 healthy controls (p

OBJECTIVE: This extended evaluation (EE) of the SONICS study assessed the effects of levoketoconazole for an additional 6 months following open-label, 6-month maintenance treatment in endogenous Cushings syndrome. DESIGN/METHODS: SONICS included dose-titration (150-600 mg BID), 6-month maintenance, and 6-month EE phases. Exploratory efficacy assessments were performed at months 9 and 12 (relative to the start of maintenance). For pituitary MRI in patients with Cushings disease, a threshold of ≥2 mm denoted change from baseline in the largest tumor diameter. RESULTS: Sixty patients entered EE at month 6; 61% (33/54 with data) exhibited normal mean urinary free cortisol (mUFC). At months 9 and 12, respectively, 55% (27/49) and 41% (18/44) of patients with data had normal mUFC. Mean fasting glucose, total and LDL-cholesterol, body weight, BMI, abdominal girth, hirsutism, CushingQoL, and Beck Depression Inventory-II scores improved from the study baseline at months 9 and 12. Forty-six patients completed month 12; four (6.7%) discontinued during EE due to adverse events. The most common adverse events in EE were arthralgia, headache, hypokalemia, and QT prolongation (6.7% each). No patient experienced alanine aminotransferase or aspartate aminotransferase >3× upper limit of normal, Fridericia-corrected QT interval >460 ms, or adrenal insufficiency during EE. Of 31 patients with tumor measurements at baseline and month 12 or follow-up, the largest tumor diameter was stable in 27 (87%) patients, decreased in one, and increased in three (largest increase 4 mm). CONCLUSION: In the first long-term levoketoconazole study, continued treatment through a 12-month maintenance period sustained the early clinical and biochemical benefits in most patients completing EE, without new adverse effects.