Traumatic brain injury (TBI) modelled by lateral fluid percussion-induction (LFPI) in rats is a widely used experimental rodent model to explore and understand the underlying cellular and molecular alterations in the brain caused by TBI in humans. Current improvements in imaging with positron emission tomography (PET) have made it possible to map certain features of TBI-induced cellular and molecular changes equally in humans and animals. The PET imaging technique is an apt supplement to nanotheranostic-based treatment alternatives that are emerging to tackle TBI. The present study aims to investigate whether the two radioligands, [11C]PBR28 and [18F]flumazenil, are able to accurately quantify in vivo molecular-cellular changes in a rodent TBI-model for two different biochemical targets of the processes. In addition, it serves to observe any palpable variations associated with primary and secondary injury sites, and in the affected versus the contralateral hemispheres. As [11C]PBR28 is a radioligand of the 18 kD translocator protein, the up-regulation of which is coupled to the level of neuroinflammation in the brain, and [18F]flumazenil is a radioligand for GABAA-benzodiazepine receptors, whose level mirrors interneuronal activity and eventually cell death, the use of the two radioligands may reveal two critical features of TBI. An up-regulation in the [11C]PBR28 uptake triggered by the LFP in the injured (right) hemisphere was noted on day 14, while the uptake of [18F]flumazenil was down-regulated on day 14. When comparing the left (contralateral) and right (LFPI) hemispheres, the differences between the two in neuroinflammation were obvious. Our results demonstrate a potential way to measure the molecular alterations in a rodent-based TBI model using PET imaging with [11C]PBR28 and [18F]flumazenil. These radioligands are promising options that can be eventually used in exploring the complex in vivo pharmacokinetics and delivery mechanisms of nanoparticles in TBI treatment.

In this paper we present our experience in the design, modelling, implementation and evaluation of a conversational medical school tutor (MST), employing AI on the cloud. MST combines case-based tutoring with competency based curriculum review, using a natural language interface to enable an adaptive and rich learning experience. It is designed both to engage and tutor medical students through Digital Virtual Patient (DVP) interactions built around clinical reasoning activities and their application of foundational knowledge. DVPs in MST are realistic clinical cases authored by subject matter experts in natural language text. The context of each clinical case is modelled as a set of complex concepts with their associated attributes and synonyms using the UMLS ontology. The MST conversational engine understands the intent of the user’s natural language inputs by training Watson Assistant service and drives a meaningful dialogue relevant to the clinical case under investigation. The curriculum content is analysed using NLP techniques and represented as a related and cohesive graph with concepts as its nodes. The runtime application is modelled as a dynamic and adaptive flow between the case and student characteristics. We describe in detail the various challenges encountered in the design and implementation of this intelligent tutor and also present evaluation of the tutor through two field trials with third and fourth year students comprising of 90 medical students.

A typical medical curriculum is organized as a hierarchy of learning outcomes (LOs), each LO is a short text that describes a medical concept. Machine learning models have been applied to predict relatedness between LOs. These models are trained on examples of LO-relationships annotated by experts. However, medical curricula are periodically reviewed and revised, resulting in changes to the structure and content of LOs. This work addresses the problem of model adaptation under curriculum drift. First, we propose heuristics to generate reliable annotations for the revised curriculum, thus eliminating dependence on expert annotations. Second, starting with a model pre-trained on the old curriculum, we inject a task-specific transformation layer to capture nuances of the revised curriculum. Our approach makes significant progress towards reaching human-level performance.

Virtual patients are interactive digital simulations of clinical scenarios for the purpose of health professions education. There is no current collated evidence on the effectiveness of this form of education. The goal of this study was to evaluate the effectiveness of virtual patients compared with traditional education, blended with traditional education, compared with other types of digital education, and design variants of virtual patients in health professions education. The outcomes of interest were knowledge, skills, attitudes, and satisfaction. We performed a systematic review on the effectiveness of virtual patient simulations in pre- and postregistration health professions education following Cochrane methodology. We searched 7 databases from the year 1990 up to September 2018. No language restrictions were applied. We included randomized controlled trials and cluster randomized trials. We independently selected studies, extracted data, and assessed risk of bias and then compared the information in pairs. We contacted study authors for additional information if necessary. All pooled analyses were based on random-effects models. A total of 51 trials involving 4696 participants met our inclusion criteria. Furthermore, 25 studies compared virtual patients with traditional education, 11 studies investigated virtual patients as blended learning, 5 studies compared virtual patients with different forms of digital education, and 10 studies compared different design variants. The pooled analysis of studies comparing the effect of virtual patients to traditional education showed similar results for knowledge (standardized mean difference [SMD]=0.11, 95% CI −0.17 to 0.39, I2=74%, n=927) and favored virtual patients for skills (SMD=0.90, 95% CI 0.49 to 1.32, I2=88%, n=897). Studies measuring attitudes and satisfaction predominantly used surveys with item-by-item comparison. Trials comparing virtual patients with different forms of digital education and design variants were not numerous enough to give clear recommendations. Several methodological limitations in the included studies and heterogeneity contributed to a generally low quality of evidence. Low to modest and mixed evidence suggests that when compared with traditional education, virtual patients can more effectively improve skills, and at least as effectively improve knowledge. The skills that improved were clinical reasoning, procedural skills, and a mix of procedural and team skills. We found evidence of effectiveness in both high-income and low- and middle-income countries, demonstrating the global applicability of virtual patients. Further research should explore the utility of different design variants of virtual patients.

Senior Education Leaders in higher education settings face many challenges, not the least of which requires managing the provision of increasingly sophisticated digitised learning experiences in a world where digital innovation and transformation narratives continue to gain traction. While Learning Management Systems (LMS) and associated content authoring tools have taken root in universities and colleges throughout the world, many administrations continue to trust that they have thus met the challenge of this 'digital learning meme'. By providing faculty with an LMS, and creating ongoing training and development workshops on the use of this technology, they hope these provisions, in and of themselves, will encourage faculty to move of their accord into the digital narrative space. Characteristically, faculty movement into these highly promoted digital learning spaces follows a similar pattern: First, they are assigned a course site and then nudged or directed to transfer a percentage, e.g., 30%, of their content online. Concomitantly, they are also encouraged to adopt what has become known as the 'Blended Learning' or 'The Flipped Classroom', pedagogy, i.e., students access their learning resources online to properly prepare - before class on their own time and target - for subsequent active learning classroom activities. The more adventurous institutions aspire to be on the front line of global innovation and anticipate they are meeting the needs of their digitally savvy students by adopting MOOCs as alternative programme choices. Within this cornucopia of settings and challenges, the term 'digital learning' gets tossed around like it is a panacea, while the dream of a fully digitised undergraduate programme continues to seem like a very remote possibility. Accordingly, based on a successful implementation and using a deductive approach, this paper presents the key aspects necessary for the successful design and deployment of a fully digitised learning ecosystem. Three essential phases have been identified, and four key underpinning factors associated with the successful implementation of each phase are described, along with specific examples that must be in place in order to create and sustain the realization of a fully digitised undergraduate programme.

Synthesizing evidence from randomized controlled trials of digital health education poses some challenges. These include a lack of clear categorization of digital health education in the literature; constantly evolving concepts, pedagogies, or theories; and a multitude of methods, features, technologies, or delivery settings. The Digital Health Education Collaboration was established to evaluate the evidence on digital education in health professions; inform policymakers, educators, and students; and ultimately, change the way in which these professionals learn and are taught. The aim of this paper is to present the overarching methodology that we use to synthesize evidence across our digital health education reviews and to discuss challenges related to the process. For our research, we followed Cochrane recommendations for the conduct of systematic reviews; all reviews are reported according to the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidance. This included assembling experts in various digital health education fields; identifying gaps in the evidence base; formulating focused research questions, aims, and outcome measures; choosing appropriate search terms and databases; defining inclusion and exclusion criteria; running the searches jointly with librarians and information specialists; managing abstracts; retrieving full-text versions of papers; extracting and storing large datasets, critically appraising the quality of studies; analyzing data; discussing findings; drawing meaningful conclusions; and drafting research papers. The approach used for synthesizing evidence from digital health education trials is commonly regarded as the most rigorous benchmark for conducting systematic reviews. Although we acknowledge the presence of certain biases ingrained in the process, we have clearly highlighted and minimized those biases by strictly adhering to scientific rigor, methodological integrity, and standard operating procedures. This paper will be a valuable asset for researchers and methodologists undertaking systematic reviews in digital health education.

Background/Aim: High-dose oestrogen treatment has been used to reduce growth in tall adolescent girls. The long-term safety with regard to cancer has not been clarified. Our aim was to study if this growth reduction therapy affects cancer risk later in life. Methods: A cohort study of 369 (172 treated, 197 untreated) Swedish women who in 1973-1993 were assessed for tall adolescent stature was designed. Data were collected from university hospital records, patient questionnaires, and the Swedish Cancer Register. Results: Risks are presented as odds ratios (ORs) with 95% confidence intervals comparing treated to untreated subjects. In treated subjects, the overall OR for having a tumour (malignant or non-malignant) was 1.7 (0.8-3.8). The ORs were 2.3 (0.4-12.8) for breast tumours, 0.8 (0.2-2.6) for gynaecological tumours, and 6.1 (1.04-∞) for melanoma. When limiting to malignant tumours, the crude ORs were of similar magnitude. Conclusion: The OR for any melanoma was higher in treated than in untreated women, suggesting an increased risk of melanoma associated with high-dose oestrogen treatment during adolescence. Although the risk estimates were increased for overall tumours, breast tumours, malignant gynaecological tumours, and malignant melanoma, these associations were not statistically significant. Our results need to be verified in a larger cohort.

Since the 1970s, estrogen have sometimes been used in adolescent girls to reduce very tall adult expected height. Worries about long-term effects have led to a proposal to link treatment data with cancer registers. How should one deal with informed consent for such a study? We designed a qualitative study with semi-structured telephone interviews. From 1200 women who were to be followed-up in cancer registers, we randomly selected 22 women. Major themes were a wish to be involved and a positive attitude to the proposed register research. The women did not express worry after reading the study protocol, but did convey considerable frustration that this research had not been initiated earlier. Active consent was not seen as crucial. We found strong interest in a high participation rate and a concern over missing data. The selection of information and consent or the decision to go ahead without consent in register follow-up is a delicate balancing act. Study participants wish to be contacted, but acknowledge the primary goal of answering important questions. Our study provides support for safeguarding privacy in epidemiological linkage studies and in follow-up of medical treatment without losing the scientific value by requesting for informed consent.

Biobanks will have a crucial role in the identification of genes associated with disease - a prerequisite to designing adequate diagnostic and therapeutic tools. To maximize their impact and chances of success, collaboration at a global scale is highly desirable.

Here we describe the three- dimensional crystal structures of human glucocorticoid receptor ligand- binding domain ( GR- LBD) in complex with the antagonist RU486 at 2.3 Angstrom resolution and with the agonist dexamethasone ligand together with a coactivator peptide at 2.8 Angstrom. The RU- 486 structure was solved in several different crystal forms, two with helix 12 intact ( GR1 and GR3) and one with a protease- digested C terminus ( GR2). In GR1, part of helix 12 is in a position that covers the co- activator pocket, whereas in the GR3, domain swapping is seen between the crystallographically identical subunits in the GR dimer. An arm consisting of the end of helix 11 and beyond stretches out from one molecule, and helix 12 binds to the other LBD, partly blocking the coactivator pocket of that molecule. This type of GR-LBD dimer has not been described before but might be an artifact from crystallization. Furthermore, the subunits of the GR3 dimers are covalently connected via a disulfide bond between the Cys- 736 residues in the two molecules. All three RU- 486 GR- LBD structures show that GR has a very flexible region between the end of helix 11 and the end of helix 12.

We developed a molecular genetic model to investigate glucocorticoid receptor (GR) signaling in human bronchial epithelial cells in response to the therapeutic steroid budesonide. Based on a genetic selection scheme using the human Chago K1 cell line and integrated copies of a glucocorticoid-responsive herpes simplex virus thymidine kinase gene and a green fluorescent protein gene, we isolated five Chago K1 variants that grew in media containing budesonide and ganciclovir. Three spontaneous budesonide-resistant subclones were found to express low levels of GR, whereas two mutants isolated from ethylmethane sulfonate-treated cultures contained normal levels of GR protein. Analysis of the GR coding sequence in the budesonide-resistant subclone Ch-BdE5 identified a novel Val to Met mutation at amino acid position 575 (GRV575M) which caused an 80% decrease in transcriptional regulatory functions with only a minimal effect on ligand binding activity. Homology modeling of the GR structure in this region of the hormone binding domain and molecular dynamic simulations suggested that the GRV575M mutation would have a decreased affinity for the LXXLL motif of p160 coactivators. To test this prediction, we performed transactivation and glutathione-S-transferase pull-down assays using the p160 coactivator glucocorticoid interacting protein 1 (GRIP1)/transcriptional intermediary factor 2 and found that GRV575M transcriptional activity was not enhanced by GRIP1 in transfected cells nor was it able to bind GRIP1 in vitro. Identification of the novel GRV575M variant in human bronchial epithelial cells using a molecular genetic selection scheme suggests that functional assays performed in relevant cell types could identify subtle defects in GR signaling that contribute to reduced steroid sensitivities in vivo.

Here we describe the three-dimensional crystal structures of human glucocorticoid receptor ligand-binding domain (GR-LBD) in complex with the antagonist RU-486 at 2.3 Å resolution and with the agonist dexamethasone ligand together with a coactivator peptide at 2.8 Å. The RU-486 structure was solved in several different crystal forms, two with helix 12 intact (GR1 and GR3) and one with a protease-digested C terminus (GR2). In GR1, part of helix 12 is in a position that covers the co-activator pocket, whereas in the GR3, domain swapping is seen between the crystallographically identical subunits in the GR dimer. An arm consisting of the end of helix 11 and beyond stretches out from one molecule, and helix 12 binds to the other LBD, partly blocking the coactivator pocket of that molecule. This type of GR-LBD dimer has not been described before but might be an artifact from crystallization. Furthermore, the subunits of the GR3 dimers are covalently connected via a disulfide bond between the Cys-736 residues in the two molecules. All three RU-486 GR-LBD structures show that GR has a very flexible region between the end of helix 11 and the end of helix 12.

Nuclear receptors bind to DNA and activate in general the transcription of sets of genes in response to the binding of cognate ligands, usually small lipophilic molecules such as steroids, vitamins, and fatty acid derivatives.About 50 nuclear receptor genes have been identified in the human genome, but most of them have no known ligand and are therefore called orphans; their biological functions are also poorly understood.Among the orphan nuclear receptors, some appear to be constitutively active.We have solved the structure of the ligand-binding domain (LBD) of two such receptors: RORβ and ERR3.The human RORβ LBD was found in the transcriptionally active conformation thanks to the combined action of stearate, a fortuitous ligand, and a coactivator peptide.Mutagenesis based on the structure showed that RORβ transcriptional activity is in fact ligand-dependent, and thus RORβ constitutive activity is only apparent.The human ERR3 LBD in complex with a coactivator peptide was also found in the transcriptionally active conformation, but this time in the absence of any ligand, suggesting that ERR3 is indeed constitutively active.

Glucocorticoid resistance is a problem in the treatment of many diseases. One possible factor involved in the modulation of a glucocorticoid response is the export of glucocorticoids out of the cell. It has been shown that multidrug resistance protein 1 (MDR1, ABCB1), a member of the ABC family, is capable of transporting some glucocorticoids. This paper uses a mouse cell line, LMCAT in which the glucocorticoid response can be modulated by inhibitors of multidrug resistance proteins. Glucocorticoids fall into three categories. Firstly, those that are transported by an Abcb1a/Abcb1b transporter and whose transport can be inhibited by inhibitors of ABCB1 activity. Functional Abcb1a/Abcb1b was detected by inhibition of rhodamine efflux by these drugs and mRNA for Abcb1a and Abcb1b were detected in these cells. Secondly, those that are not transported. Finally, those that are transported by an Abcc1a transporter. Calcein transport out of these cells was blocked by treatment with probenecid indicating a functional Abcc1a transporter. Abcc1a mRNA was also detected in these cells. Thus, this paper provides insight into the mechanisms of glucocorticoid transport in cells and demonstrates a diversity of two independent mechanisms of transport of glucocorticoids by Abcb1a/Abcb1b and Abcc1a with individual patterns of steroid specificity.

Objective Primary glucocorticoid resistance Is characterized by decreased sensitivity to cortisol signalling. We have performed genetic analysis of the glucocorticoid receptor (GR) gene in 12 unrelated patients with primary cortisol resistance as defined by a pathological dexamethasone suppression test.Methods Exon specific polymerase chain reaction amplification of the GR gene and sequencing of each exon was carried out. The two mutations were characterized in vitro in terms of glucocorticoid driven reporter gene activity in a transient transfection assay and in a ligand binding assay. Molecular modelling of the R477H mutant was performed based on the X-ray structure of the GR-DNA binding domain.Results Two novel mutations in the GR gene were found: R477H in the DNA-binding domain which is the first reported mutation in that region of the human GR gene and G679S in the ligand binding domain. The R477H mutation showed no transactivating capacity, whereas the G679S mutation had reduced transactivation capacity compared to the wild-type (wt) GR. When tested for ligand binding capacity, the G679S mutation had 50% binding affinity compared to the wt GR. The effect of the point mutation R477H was deduced by a comparison between the wt structure and the model of the mutant. The wt GR has direct and water mediated contact with the phosphate groups of the glucocorticoid responsive element (GRE) whereas, in the model, the mutation R477H has no contact with the GRE. The G679S mutation is located on the surface of the ligand binding domain, at a distance from the steroid-binding site. A previously reported polymorphism, AAT to AAC at amino acid position 766, was found in four of the patients.Conclusions In two of 12 patients with clinical glucocorticoid resistance, mutant forms of GR could be found. The glucocorticoid resistance in vivo in these two patients corresponds to impaired function of the two mutated GR forms in two in vitro assays. The relevance of the conservative polymorphism for the glucocorticoid insensitivity noted in these patients remains to be clarified.

Estrogens exert their principal biological effects through the actions of two different intracellular estrogen receptor (ER) proteins, ERα and ERβ. Following the binding of steroid, the protein undergoes a conformational change that results in a transcriptionally active form. The receptor protein is locked into an active state by estradiol, which results in the transition of the receptor through a signal transduction cascade of events, ultimately resulting in the activation of specific genes, thereby inducing the biological events specific for that type of target cell. There is a large variation in the relative expression levels of the two ER isoforms in different target tissues and in different stages of development. In addition, variant forms of the two ER isoforms, the result of splice variation, have been described. ERα and ERβ have been shown to differ in specific aspects within the various stages of the signal transduction pathway. Thus, there is a broad spectrum of estrogen response mechanisms as a result of an infinite number of possible combinations of all these factors. In addition, there are gene regulatory mechanisms that are the result of ER–protein interactions instead of ER–DNA interactions. Steroid binding is the key initiating action of the whole pathway, which, in terms of cell biology, is a relatively slow process. The response induced through the action of ER induction can be shown to be dependent on the total dose exposure rather than estradiol concentrations at subsaturating levels.

To elucidate which amino acids in the glucocorticoid receptor ligand-binding domain might be involved in determining steroid binding specificity by interaction with the D-ring of glucocorticoids, we have performed site-directed mutagenesis of the four amino acids Met-560, Met-639, Gln-642, and Thr-739 based on their proximity to the steroid in a model structure. Mutations of these residues affected steroid binding affinity, specificity, and/or steroid-dependent transactivation. The results indicate that these residues are located in close proximity to the ligand and appear to play a role in steroid recognition and/or transactivating sensitivity, possibly by changes in the steroid-dependent conformational change of this region, resulting in the formation of the AF-2 site. Mutation of Gln-642 resulted in a marked decrease in affinity for steroids containing a 17 alpha-OH group. This effect was alleviated by the presence of a 16 alpha-CH3 group to a varying degree. Thr-739 appears to form a hydrogen bond with the 21-OH group of the steroid, as well as possibly forming hydrophobic interactions with the steroid, Met-EGO and Met-639 appear to form hydrophobic interactions with the D-ring of the steroid, although the nature of these interactions cannot be characterized in more detail at this point.

The involvement of phospholipase A 2 (PLA 2 ) enzymes, the 85 kDa cytosolic PLA 2 (cPLA 2 ) and the 14 kDa secretory PLA 2 (sPLA 2 ), on PGE 2 production in human gingival fibroblasts was investigated. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the inflammatory mediators interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα) induce the mRNA expression of cPLA 2 in gingival fibroblasts. In addition, treatment of the cells with calcium ionophore, A23187, or the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), also induced cPLA 2 mRNA expression, accompanied by enhanced PGE 2 production. The anti-inflammatory steroid dexamethasone (DEX) reduced basal cPLA 2 mRNA expression as well as blocked the induction of cPLA 2 mRNA by IL-1β and TNFα in gingival fibroblasts. In contrast to cPLA 2 , the expression of sPLA 2 mRNA was not detected either in untreated or in treated gingival fibroblasts. The study demonstrates that cPLA 2 mRNA expression is upregulated by IL-1β and TNFα in human gingival fibroblasts suggesting an important role for the enzyme cPLA 2 in the cytokine-induced PGE 2 production. Furthermore, the enzyme cPLA 2 , rather than sPLA 2 , may be involved in the pathogenesis of periodontal disease by mediating PGE 2 production in gingival fibroblasts.