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Abstract Drug type and dosing recommendation have been designed and optimized based on average response in the general population. Yet, there is significant inter‐individual variability in drug response, which results in treatment inefficacy or adverse drug reactions in a subset of patients. This is partly due to genetic factors that typically affect drug metabolism or clearance. To verify the relevance and applicability of international pharmacogenetic guidelines in the Swiss population, we genotyped 1533 patients from a hospital‐based biobank who received at least 30 different drugs, as documented in their electronic health record. We then assessed the prevalence of clinically actionable variants in 13 high‐risk pharmacogenes. We compared the allele frequencies obtained in the hospital‐based cohort with those of a Swiss population‐based cohort of 4791 individuals. The prevalence of clinically actionable variants was comparable between the two cohorts, with most study participants (97.3%) carrying at least one actionable pharmacogenetic variant. We then assessed the frequency of high‐risk prescriptions due to actionable gene–drug interactions and observed that 31% of patients in the hospital‐based cohort were prescribed at least one drug for which they carried a high‐risk variant, and for which international guidelines recommend a change of drug or dosage. Our analysis confirms the high prevalence of actionable pharmacogenetic variants in the Swiss population. It also shows that a substantial minority of patients are exposed to drugs for which they carry potentially problematic variants. Implementing a genetically informed approach to drug prescribing could have a positive impact on the quality of healthcare delivery.

IntroductionThis study investigates the relationship between approaches to learning, self-perceived study burnout, and the level of knowledge among veterinary students. Veterinary educational programs are under regular development and would benefit greatly from detailed feedback on students' knowledge, proficiency, influencing factors, and coping mechanisms.MethodsThe VetRepos consortium developed and calibrated an item repository testing knowledge across the entire veterinary curriculum. Two hundred forty-eight students from seven European veterinary institutions took the VetRepos test, comprising a subset of the repository. They also responded to a questionnaire assessing deep and unreflective learning approaches and self-perceived study burnout, represented by exhaustion and cynicism. Structural equation modeling analyzed the relationship between these latent traits and the VetRepos test score.ResultsThe model failed the exact-fit test but was retained based on global fit indices, inter-item residual correlations, and standardized residual covariances. Root Mean Square Error of Approximation with robust standard errors and scaled test statistic was 0.049 (95% confidence interval 0.033–0.071), scaled and robust Comparative Fit Index 0.95 (0.90–0.98), and scaled Standardized Root Mean Square Residual 0.056 (0.049–0.071). Measurement invariance across study years was not violated (ΔCFI = 0.00, χ2 = 3.78, Δdf = 4, p = 0.44), but it could not be confirmed between genders or universities. The VetRepos test score regressed on the study year [standardized regression coefficient = 0.68 (0.62–0.73)], showed a negative regression on the unreflective learning approach [−0.25 (−0.47 to −0.03)], and a positive regression on the deep approach [0.16 (0.03–0.28)]. No direct association with perceived burnout was observed; however, a significant, medium-sized association was found between the unreflective approach and self-perceived study burnout. No significant differences in learning approaches or perceived burnout were found between study years.DiscussionThe most important source of variance in VetRepos test scores, unrelated to the study year, was the learning approach. The association between the VetRepos test score and self-perceived burnout was indirect. Future research should complement this cross-sectional approach with longitudinal and person-oriented studies, further investigating the relationship between study burnout and learning approaches.

Abstract Viral and host glycans represent an understudied aspect of host-pathogen interactions, despite potential implications for treatment of viral infections. This is due to lack of easily accessible tools for analyzing glycan function in a meaningful context. Here we generate a glycoengineered keratinocyte library delineating human glycosylation pathways to uncover roles of specific glycans at different stages of herpes simplex virus type 1 (HSV-1) infectious cycle. We show the importance of cellular glycosaminoglycans and glycosphingolipids for HSV-1 attachment, N-glycans for entry and spread, and O-glycans for propagation. While altered virion surface structures have minimal effects on the early interactions with wild type cells, mutation of specific O-glycosylation sites affects glycoprotein surface expression and function. In conclusion, the data demonstrates the importance of specific glycans in a clinically relevant human model of HSV-1 infection and highlights the utility of genetic engineering to elucidate the roles of specific viral and cellular carbohydrate structures.

Signaling pathways are involved in key cellular functions from embryonic development to pathological conditions, with a pivotal role in tissue homeostasis and transformation. Although most signaling pathways have been intensively examined, most studies have been carried out in murine models or simple cell culture. We describe the dissection of the TGF-β signaling pathway in human tissue using CRISPR-Cas9 genetically engineered human keratinocytes (N/TERT-1) in a 3D organotypic skin model combined with quantitative proteomics and phosphoproteomics mass spectrometry. The use of human 3D organotypic cultures and genetic engineering combined with quantitative proteomics and phosphoproteomics is a powerful tool providing insight into signaling pathways in a human setting. The methods are applicable to other gene targets and 3D cell and tissue models.Key features• 3D organotypic models with genetically engineered human cells.• In-depth quantitative proteomics and phosphoproteomics in 2D cell culture.• Careful handling of cell cultures is critical for the successful formation of theorganotypic cultures.• For complete details on the use of this protocol, please refer to Ye et al. 2022.

IntroductionMultiple sclerosis (MS) is a neurodegenerative disease accumulating disabilities over time. However, the mean age of individuals with MS is increasing, consequently elevating their risk of developing comorbidities. Comorbidities' impact on MS is widely debated. Yet very few countries possess population-based registries, which provide unique opportunities for individual-level data linkage. This study aims to assess acute and chronic comorbidities among elderly patients with MS, comparing them to matched controls. Additionally, this study seeks to investigate the influence of chronic comorbidities on all-cause mortality.MethodsA nationwide register-based study using the Danish Multiple Sclerosis Registry to identify all living patients with MS older than 50 years at the reference date (January 1st, 2022). Patients were matched 1:10 with individuals from the general population. Comprehensive healthcare data within the Danish hospital system were obtained. Chronic comorbidities were classified according to the Charlson Comorbidity Index, while acute comorbidities were based on ICD-10 codes and an “acute” admission type. To investigate all-cause mortality, a Cox regression analysis was conducted.ResultsThe study encompassed a total of 8,688 individuals with MS, matched with 86,880 controls. The mean age was 63.5 years, with females constituting 68.3%. Individuals with MS exhibited a higher frequency of acute hospitalizations (OR: 2.1, 95% CI: 1.9–2.2), primarily due to various infectious diseases, along with longer median hospital stays (4 vs. 3 days, p < 0.001). When assessed using the Charlson Comorbidity Index, individuals with MS carried a significantly greater burden of chronic comorbidities (p < 0.001). The most prevalent chronic comorbidity among individuals with MS was “Uncomplicated Diabetes” (20.1%). Notably, while individuals with MS displayed an overall lower 5-year survival rate, this difference ceased to be statistically significant among those with a high Charlson Comorbidity Index score of ≥4 (p = 0.32).ConclusionThis study highlights a heightened prevalence of both acute and chronic comorbidities among individuals with MS, with chronic comorbidities significantly increasing the risk of mortality. These findings underscore the critical importance of factoring in comorbidities when devising treatment strategies for individuals living with MS.

IntroductionProgress testing in education is an assessment principle for the measurement of students’ progress over time, e.g., from start to graduation. Progress testing offers valid longitudinal formative measurement of the growth in the cognitive skills of the individual students within the subjects of the test as well as a tool for educators to monitor potential educational gaps and mismatches within the curriculum in relation to the basic veterinary learning outcomes.MethodsSix veterinary educational establishments in Denmark, Finland, Germany (Hannover), the Netherlands, Norway, and Sweden established in cooperation with the European Association of Establishments for Veterinary Education (EAEVE) a common veterinary item repository that can be used for progress testing in European Veterinary Education Establishments (VEEs), linear as well as computer adaptive, covering the EAEVE veterinary subjects and theoretical “Day One Competencies.” First, a blueprint was created, suitable item formats were identified, and a quality assurance process for reviewing and approving items was established. The items were trialed to create a database of validated and calibrated items, and the responses were subsequently psychometrically analyzed according to Modern Test Theory.ResultsIn total, 1,836 items were submitted of which 1,342 were approved by the reviewers for trial testing. 1,119 students from all study years and all partners VEEs participated in one or more of six item trials, and 1,948 responses were collected. Responses were analyzed using Rasch Modeling (analysis of item-fit, differential item function, item-response characteristics). A total of 821 calibrated items of various difficulty levels matching the veterinary students’ abilities and covering the veterinary knowledge domains have been banked.DiscussionThe item bank is now ready to be used for formative progress testing in European veterinary education. This paper presents and discusses possible pitfalls, problems, and solutions when establishing an international veterinary progress test.

Information about O-glycosylation site regulation and occupancy in the human proteome is limited. Here, the authors identify GalNAc transferase-specific glycan sites in human keratinocytes and describe their occupancy.

Summary: Galectins are a group of carbohydrate-binding proteins with a presumed immunomodulatory role and an elusive function on antigen-presenting cells. Here we analyzed the expression of galectin-1 and found upregulation of galectin-1 in the extracellular matrix across multiple tumors. Performing an in-depth and dynamic proteomic and phosphoproteomic analysis of human macrophages stimulated with galectin-1, we show that galectin-1 induces a tumor-associated macrophage phenotype with increased expression of key immune checkpoint protein programmed cell death 1 ligand 1 (PD-L1/CD274) and immunomodulator indoleamine 2,3-dioxygenase-1 (IDO1). Galectin-1 induced IDO1 and its active metabolite kynurenine in a dose-dependent manner through JAK/STAT signaling. In a 3D organotypic tissue model system equipped with genetically engineered tumorigenic epithelial cells, we analyzed the cellular source of galectin-1 in the extracellular matrix and found that galectin-1 is derived from epithelial and stromal cells. Our results highlight the potential of targeting galectin-1 in immunotherapeutic treatment of human cancers.

Introduction/objectivesMultiple sclerosis (MS) leads to physical and cognitive disability, which in turn impacts the socioeconomic status of the individual. The altered socioeconomic trajectory combined with the critical role of aging in MS progression could potentially lead to pronounced differences between MS patients and the general population. Few nations have the ability to connect long-term clinical and socioeconomic data at the individual level, and Denmark's robust population-based registries offer unique insights. This study aimed to examine the socioeconomic aspects of elderly Danish MS patients in comparison to matched controls from the general population.MethodsA nationwide population-based study in Denmark was conducted, comprising all living MS patients aged 50 years or older as of 1 January 2021. Patients were matched 1:10 based on sex, age, ethnicity, and residence with a 25% sample of the total Danish population. Demographic and clinical information was sourced from the Danish Multiple Sclerosis Registry, while socioeconomic data were derived from national population-based registries containing details on education, employment, social services, and household characteristics. Univariate comparisons between MS patients and matched controls were then carried out.ResultsThe study included 8,215 MS patients and 82,150 matched individuals, with a mean age of 63.4 years (SD: 8.9) and a 2:1 female-to-male ratio. For those aged 50–64 years, MS patients demonstrated lower educational attainment (high education: 28.3 vs. 34.4%, P < 0.001) and fewer received income from employment (46.0 vs. 78.9%, P < 0.001), and working individuals had a lower annual income (48,500 vs. 53,500€, P < 0.001) in comparison to the controls. Additionally, MS patients within this age group were more likely to receive publicly funded practical assistance (14.3 vs. 1.6%, P < 0.001) and personal care (10.5 vs. 0.8%, P < 0.001). Across the entire population, MS patients were more likely to live alone (38.7 vs. 33.8%, P < 0.001) and less likely to have one or more children (84.2 vs. 87.0%, P < 0.001).ConclusionMS presents significant socioeconomic challenges among the elderly population, such as unemployment, reduced income, and increased dependence on social care. These findings underscore the pervasive impact of MS on an individual's life course, extending beyond the clinical symptoms of cognitive and physical impairment.

Alzheimer’s disease (AD) is the most common cause of dementia, with no current cure. Consequently, alternative approaches focusing on early pathological events in specific neuronal populations, besides targeting the well-studied amyloid beta (Aβ) accumulations and Tau tangles, are needed. In this study, we have investigated disease phenotypes specific to glutamatergic forebrain neurons and mapped the timeline of their occurrence, by implementing familial and sporadic human induced pluripotent stem cell models as well as the 5xFAD mouse model. We recapitulated characteristic late AD phenotypes, such as increased Aβ secretion and Tau hyperphosphorylation, as well as previously well documented mitochondrial and synaptic deficits. Intriguingly, we identified Golgi fragmentation as one of the earliest AD phenotypes, indicating potential impairments in protein processing and post-translational modifications. Computational analysis of RNA sequencing data revealed differentially expressed genes involved in glycosylation and glycan patterns, whilst total glycan profiling revealed minor glycosylation differences. This indicates general robustness of glycosylation besides the observed fragmented morphology. Importantly, we identified that genetic variants in Sortilin-related receptor 1 (SORL1) associated with AD could aggravate the Golgi fragmentation and subsequent glycosylation changes. In summary, we identified Golgi fragmentation as one of the earliest disease phenotypes in AD neurons in various in vivo and in vitro complementary disease models, which can be exacerbated via additional risk variants in SORL1.

Coronary heart disease (CHD) is one of the most pressing health problems of our time and a major cause of preventable death. CHD results from complex interactions between genetic and environmental factors. Using multiplex serological testing for persistent or frequently recurring infections and genome-wide analysis in a prospective population study, we delineate the respective and combined influences of genetic variation, infections, and low-grade inflammation on the risk of incident CHD. Study participants are enrolled in the CoLaus|PsyCoLaus study, a longitudinal, population-based cohort with baseline assessments from 2003 through 2008 and follow-up visits every 5 years. We analyzed a subgroup of 3459 individuals with available genome-wide genotyping data and immunoglobulin G levels for 22 persistent or frequently recurring pathogens. All reported CHD events were evaluated by a panel of specialists. We identified independent associations with incident CHD using univariable and multivariable stepwise Cox proportional hazards regression analyses. Of the 3459 study participants, 210 (6.07%) had at least one CHD event during the 12 years of follow-up. Multivariable stepwise Cox regression analysis, adjusted for known cardiovascular risk factors, socioeconomic status, and statin intake, revealed that high polygenic risk (hazard ratio [HR] 1.31, 95% CI 1.10–1.56, p=2.64 × 10−3) and infection with Fusobacterium nucleatum (HR 1.63, 95% CI 1.08–2.45, p=1.99 × 10−2) were independently associated with incident CHD. In a prospective, population-based cohort, high polygenic risk and infection with F. nucleatum have a small, yet independent impact on CHD risk.

Drug type and dosing recommendation have been designed and optimized based on average response in the general population. Yet, there is significant inter‐individual variability in drug response, which results in treatment inefficacy or adverse drug reactions in a subset of patients. This is partly due to genetic factors that typically affect drug metabolism or clearance. To verify the relevance and applicability of international pharmacogenetic guidelines in the Swiss population, we genotyped 1533 patients from a hospital‐based biobank who received at least 30 different drugs, as documented in their electronic health record. We then assessed the prevalence of clinically actionable variants in 13 high‐risk pharmacogenes. We compared the allele frequencies obtained in the hospital‐based cohort with those of a Swiss population‐based cohort of 4791 individuals. The prevalence of clinically actionable variants was comparable between the two cohorts, with most study participants (97.3%) carrying at least one actionable pharmacogenetic variant. We then assessed the frequency of high‐risk prescriptions due to actionable gene–drug interactions and observed that 31% of patients in the hospital‐based cohort were prescribed at least one drug for which they carried a high‐risk variant, and for which international guidelines recommend a change of drug or dosage. Our analysis confirms the high prevalence of actionable pharmacogenetic variants in the Swiss population. It also shows that a substantial minority of patients are exposed to drugs for which they carry potentially problematic variants. Implementing a genetically informed approach to drug prescribing could have a positive impact on the quality of healthcare delivery. [ABSTRACT FROM AUTHOR]

Introduction: This study investigates the relationship between approaches to learning, self-perceived study burnout, and the level of knowledge among veterinary students. Veterinary educational programs are under regular development and would benefit greatly from detailed feedback on students' knowledge, proficiency, influencing factors, and coping mechanisms. Methods: The VetRepos consortium developed and calibrated an item repository testing knowledge across the entire veterinary curriculum. Two hundred forty-eight students from seven European veterinary institutions took the VetRepos test, comprising a subset of the repository. They also responded to a questionnaire assessing deep and unreflective learning approaches and self-perceived study burnout, represented by exhaustion and cynicism. Structural equation modeling analyzed the relationship between these latent traits and the VetRepos test score. Results: The model failed the exact-fit test but was retained based on global fit indices, inter-item residual correlations, and standardized residual covariances. Root Mean Square Error of Approximation with robust standard errors and scaled test statistic was 0.049 (95% confidence interval 0.033-0.071), scaled and robust Comparative Fit Index 0.95 (0.90-0.98), and scaled Standardized Root Mean Square Residual 0.056 (0.049-0.071). Measurement invariance across study years was not violated (ΔCFI = 0.00, χ² = 3.78, Δdf = 4, p = 0.44), but it could not be confirmed between genders or universities. The VetRepos test score regressed on the study year [standardized regression coefficient = 0.68 (0.62-0.73)], showed a negative regression on the unreflective learning approach [-0.25 (-0.47 to -0.03)], and a positive regression on the deep approach [0.16 (0.03-0.28)]. No direct association with perceived burnout was observed; however, a significant, medium-sized association was found between the unreflective approach and self-perceived study burnout. No significant differences in learning approaches or perceived burnout were found between study years. Discussion: The most important source of variance in VetRepos test scores, unrelated to the study year, was the learning approach. The association between the VetRepos test score and self-perceived burnout was indirect. Future research should complement this cross-sectional approach with longitudinal and person-oriented studies, further investigating the relationship between study burnout and learning approaches. [ABSTRACT FROM AUTHOR]

The complex carbohydrate structures decorating human proteins and lipids, also called glycans, are abundantly present at cell surfaces and in the secretome. Glycosylation is vital for biological processes including cell–cell recognition, immune responses, and signaling pathways. Therefore, the structural and functional characterization of the human glycome is gaining more and more interest in basic biochemistry research and in the context of developing new therapies, diagnostic tools, and biotechnology applications. For glycomics to reach its full potential in these fields, it is critical to appreciate the specific factors defining the function of the human glycome. Here, we review the glycosyltransferases (the writers) that form the glycome and the glycan-binding proteins (the readers) with an essential role in decoding glycan functions. While abundantly present throughout different cells and tissues, the function of specific glycosylation features is highly dependent on their context. In this review, we highlight the relevance of studying the glycome in the context of specific carrier proteins, cell types, and subcellular locations. With this, we hope to contribute to a richer understanding of the glycome and a more systematic approach to identifying the roles of glycosylation in human physiology. [ABSTRACT FROM AUTHOR]

Microbiological etiology has been associated with surgery for infective endocarditis (IE) during admission, especially Staphylococcus aureus. We aimed to compare patient characteristics, microbiological characteristics, and outcomes by treatment choice (surgery or not). We identified patients with first-time IE between 2010 and 2020 and examined the microbiological etiology of IE according to treatment choice. To identify factors associated with surgery during initial admission, we used the Aalen–Johansen estimator and an adjusted cause-specific Cox model. One-year mortality stratified by microbiological etiology and treatment choice was assessed using unadjusted Kaplan–Meier estimates and an adjusted Cox proportional hazard model. A total of 6255 patients were included, of which 1276 (20.4%) underwent surgery during admission. Patients who underwent surgery were younger (65 vs. 74 years) and less frequently had cerebrovascular disease, cardiovascular disease, diabetes, and chronic kidney disease. Patients with Staphylococcus aureus IE were less likely to undergo surgery during admission (13.6%) compared to all other microbiological etiologies. One-year mortality according to microbiological etiology in patients who underwent surgery was 7.0%, 5.3%, 5.5%, 9.6%, 13.2, and 11.2% compared with 24.2%, 19.1%, 27,6%, 25.2%, 21%, and 16.9% in patients who received medical therapy for Staphylococcus aureus, Streptococcus spp., Enterococcus spp., coagulase-negative Staphylococci, “other microbiological etiologies”, and blood culture-negative infective endocarditis, respectively. Patients with IE who underwent surgery differed in terms of microbiology, more often having Streptococci than those who received medical therapy. Contrary to expectations, Staphylococcus aureus was more common among patients who received medical therapy only.

There is great potential in making assessment and learning complementary. In this study, we investigated the feasibility of developing a desktop virtual reality (VR) laboratory simulation on the topic of genetics, with integrated assessment using multiple choice questions based on item response theory (IRT) and feedback based on the cognitive theory of multimedia learning. A pre-test post-test design was used to investigate three research questions related to: (1) students' perceptions of assessment in the form of MC questions within the VR genetics simulation; (2) the fit of the MC questions to the assumptions of the partial credit model (PCM) within the framework of IRT; and (3) if there was a significant increase in intrinsic motivation, self-efficacy, and transfer from pre- to post-test after using the VR genetics simulation as a classroom learning activity. The sample consisted of 208 undergraduate students taking a medical genetics course. The results showed that assessment items in the form of gamified multiple-choice questions were perceived by 97% of the students to lead to higher levels of understanding, and only 8% thought that they made the simulation more boring. Items within a simulation were found to fit the PCM and the results showed that the sample had a small significant increase in intrinsic motivation and self-efficacy, and a large significant increase in transfer following the genetics simulation. It was possible to develop assessments for online educational material and retain the relevance and connectedness of informal assessment while simultaneously serving the communicative and credibility-based functions of formal assessment, which is a great challenge facing education today.

Abundant research has pointed to the importance of non-cognitive skills for success in life. This paper describes the development and validation of the "UiL", designed to measure 19 non-cognitive skills that have been identified as being important for school children in Denmark. First, we describe the development of the scales, and then report a preliminary validation with 1560 students from Grades 4-9. Second, we present a validation of the final UiL, which was revised and re-administered to the same sample of students. The final validation sample consisted of 1373 students (48.6% boys, ages 9-16 years). Results from the Confirmatory Factor Analysis indicated that the UiL had acceptable discriminant and convergent validity. The results from a RASCH partial credit analysis indicated that four of the 19 had excellent fit, with other scales needing nuanced interpretation because of some item misfit, local dependence, multidimensionality, and DIF by grade.

Background: Coronary tortuosity (CorT) is frequently observed in invasive angiography, though its aetiology and clinical significance remain ambiguous. Prior research has indicated possible links between CorT and factors such as hypertension, age, and calcium scores in the left anterior descending (LAD) artery. The aim of this study was to examine and optimize the usage of coronary computed tomography angiography (CCTA) with vessel tracking to explore these associations. Methods: Observational sub‐study of the single centre randomised controlled CATCH‐trial. From the original study 600 participants, who underwent CCTA, 250 were randomly selected. Clinical data and patient risk factors were sourced from medical records and structured interviews. Tortuosity of the LAD was quantified by calculating the ratio of the actual vessel‐length to the straight‐line distance. Results: The final study population comprised 194 patients (56 patients were excluded due to poor image quality or inability to perform adequate vessel tracking). After adjusting for confounding variables, tortuosity was significantly associated with hypertension (p < 0.001), female gender (p = 0.01), and increasing age (p = 0.045). No significant correlation was observed between CorT and calcium scores. Univariate analysis indicated that higher CorT levels were linked to lower metabolic equivalents of task (METs) in bicycle tests (p = 0.003); however, this relationship became nonsignificant (p = 0.97) upon adjustment for age, gender, and hypertension. Conclusions: Our findings suggest that increased CorT is most prevalent in patients with hypertension, advancing age, and female gender. Although higher tortuosity levels did not significantly impact METs during physical activity, further research is warranted to explore the underlying mechanisms of this relationship. [ABSTRACT FROM AUTHOR]

BackgroundStudies have demonstrated an increasing mean age of the population with multiple sclerosis (MS). The association between increased age and socioeconomic outcomes has been investigated sparsely.ObjectiveThe purpose of this study is to describe the demographic and socioeconomic status of the current Danish population of patients with MS according to age and to assess the age-related risks of no income or losing all income from earnings or receiving disability pension.MethodsThe nationwide population-based Danish Multiple Sclerosis Registry provided data linked with the Danish Income Statistics Register and the Danish Rational Economic Agents Model (DREAM) database. The prevalence of socioeconomic milestones of the current MS population was compared with healthy controls and the risks of reaching socioeconomic milestones were assessed using cause-specific Cox models and cumulative incidence functions compared to healthy controls.ResultsThe current Danish population of patients with MS of working age (18–65 years of age) consists of 11,287 patients, of which 29.3% was older than 55 years. In 2018, 38.0% of all patients and 18.9% of controls had no income from earnings, whereas 30.5% of all patients and 7.7% of controls received disability pension. The risk of losing all income from earnings was higher for patients with MS with a hazard ratio (HR) peaking at of 4.0 (95% CI, 3.8–4.2) for the ages of 45–54 years. The risk of receiving disability pension was much higher for patients with MS peaking at a HR of 22.6 (95% CI, 20.9–24.4) for the ages of 25–34 years. Likewise, the absolute risks of both outcomes were higher for the patients with MS at all ages.ConclusionDanish patients with MS are at a higher risk of losing all income from earnings and at a much higher risk of receiving disability pension compared with healthy controls.

Summary: Glycosylation is one of the most common protein modifications in living organisms and has important regulatory roles in animal tissue development and homeostasis. Here, we present a protocol for generation of 3D organotypic skin models using CRISPR-Cas9 genetically engineered human keratinocytes (N/TERT-1) to study the role of glycans in epithelial tissue formation. This strategy is also applicable to other gene targets and organotypic tissue models. Careful handling of the cell cultures is critical for the successful formation of the organoids.For complete details on the use and execution of this protocol, please refer to Dabelsteen et al. (2020).

Abstract Background CRISPR/Cas9 is widely used for precise genetic editing in various organisms. CRISPR/Cas9 editing may in many plants be hampered by the presence of complex and high ploidy genomes and inefficient or poorly controlled delivery of the CRISPR/Cas9 components to gamete cells or cells with regenerative potential. Optimized strategies and methods to overcome these challenges are therefore in demand. Results In this study we investigated the feasibility of improving CRISPR/Cas9 editing efficiency by Fluorescence Activated Cell Sorting (FACS) of protoplasts. We used Agrobacterium infiltration in leaves of Nicotiana benthamiana for delivery of viral replicons for high level expression of gRNAs designed to target two loci in the genome, NbPDS and NbRRA, together with the Cas9 nuclease in fusion with the 2A self-splicing sequence and GFP (Cas9-2A-GFP). Protoplasts isolated from the infiltrated leaves were then subjected to FACS for selection of GFP enriched protoplast populations. This procedure resulted in a 3–5 fold (from 20 to 30% in unsorted to more than 80% in sorted) increase in mutation frequencies as evidenced by restriction enzyme analysis and the Indel Detection by Amplicon Analysis, which allows for high throughput profiling and quantification of the generated mutations. Conclusions FACS of protoplasts expressing GFP tagged CRISPR/Cas9, delivered through A. tumefaciens leaf infiltration, facilitated clear CRISPR/Cas9 mediated mutation enrichment in selected protoplast populations.