Schwartz rounds in undergraduate medical education facilitates active reflection and individual identification of learning need.

This article was migrated. The article was marked as recommended. Strategies applying Schwartz Rounds to improve wellbeing of medical students has focused on the clinical years of study. This pilot study investigates whether Schwartz Rounds could be effective in developing students' reflective practice in Year 2 undergraduates. Engagement with the Schwartz Round was high with over 50% of the students identifying learning needs through reflection on the Round. Schwartz Rounds promoted recognition of the value of reflective practice and increased self-awareness of student needs.

Genetic homogeneity of adult Langerhans cell histiocytosis lesions: Insights from BRAFV600E mutations in adult populations.

Langerhans cell histiocytosis (LCH) is a heterologous disease with a recognized disparity in incidence, affected sites and prognosis between adults and children. The recent identification of BRAFV600E mutations in LCH prompted the investigation of the frequency of these mutations in adult and childhood disease with the involvement of single or multiple sites in the present study. The study analysed the BRAFV600E status in a cohort of adult LCH patients by DNA sequencing, and performed a broader meta-analysis of BRAFV600E mutations in LCH in order to investigate any association with disease site and severity. A review of the literature revealed that ~47% of lesions from cases of adult disease (patient age, >18 years) were V600E-positive compared with 53% in those under 18 years. When single and multiple site disease was compared, there was a slight increase in the former (61 vs. 51%, respectively). A greater difference was observed when high- and low-risk organs were compared; for example, 75% of liver biopsies (a high-risk organ) were reported to bear the mutation compared with 47% of lung biopsies. In the adult LCH population, DNA sequencing identified mutations in 38% of 29 individuals, which is slightly lower than the figure identified from the meta-analysis (in which a total of 132 individuals were sampled), although we this value could not be broken down by clinical status. Thus, V600E status at presentation in itself is not predictive of tumour course, but a considerable proportion of LCH patients may respond to targeted V600E therapies.

Collagen remodeling and peripheral immune cell recruitment characterizes the cutaneous Langerhans cell histiocytosis microenvironment.

BACKGROUND: Langerhans cell histiocytosis (LCH) is a rare and potentially fatal disorder of unknown etiology arising from the accumulation of epidermal Langerhans-like cells in bone, skin, or other tissues. Tissue damage and morbidity results from lesional cytokine release, and we sought to investigate the LCH microenvironment using a combination of histological stains and immunohistochemistry. METHODS: CD1a immunoreactivity was used to identify lesional cells in archival paraffin-embedded samples of cutaneous LCH. A combined Orcein and Giemsa stain identified immune cells in general (particularly granulocytes and mast cells) and extracellular matrix (particularly elastic fibers), while CD3 and CD68 staining identified T cells and macrophages, respectively. Collagen synthesis was investigated with Herovici staining, which discriminates newly synthesized from mature collagen, while cross-polar microscopy of picrosirius-stained sections identified changes in matrix organization. RESULTS: Immune cells were consistently identified at the periphery of cutaneous LCH lesions. We quantified an increased number of thickened and disorganized elastic fibers surrounding lesions and an absence of elastic fibers within lesions. Furthermore, lesions exhibited a decrease in mature collagen fibers and a loss of supporting collagen matrix within lesions and compromised collagen integrity in adjacent tissue. CONCLUSIONS: Cutaneous LCH lesions are associated with the peripheral recruitment of a variety of immune cells and are consistently characterized by localized elastosis, collagen damage, and remodeling.

A MATLAB tool for pathway enrichment using a topology-based pathway regulation score.

BACKGROUND: Handling the vast amount of gene expression data generated by genome-wide transcriptional profiling techniques is a challenging task, demanding an informed combination of pre-processing, filtering and analysis methods if meaningful biological conclusions are to be drawn. For example, a range of traditional statistical and computational pathway analysis approaches have been used to identify over-represented processes in microarray data derived from various disease states. However, most of these approaches tend not to exploit the full spectrum of gene expression data, or the various relationships and dependencies. Previously, we described a pathway enrichment analysis tool created in MATLAB that yields a Pathway Regulation Score (PRS) by considering signalling pathway topology, and the overrepresentation and magnitude of differentially-expressed genes (J Comput Biol 19:563-573, 2012). Herein, we extended this approach to include metabolic pathways, and described the use of a graphical user interface (GUI). RESULTS: Using input from a variety of microarray platforms and species, users are able to calculate PRS scores, along with a corresponding z-score for comparison. Further pathway significance assessment may be performed to increase confidence in the pathways obtained, and users can view Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway diagrams marked-up to highlight impacted genes. CONCLUSIONS: The PRS tool provides a filter in the isolation of biologically-relevant insights from complex transcriptomic data.

Stimulation of glucose uptake in murine soleus muscle and adipocytes by 5-(4-phenoxybutoxy)psoralen (PAP-1) may be mediated by Kv1.5 rather than Kv1.3.

Kv1 channels are shaker-related potassium channels that influence insulin sensitivity. Kv1.3(-/-) mice are protected from diet-induced insulin resistance and some studies suggest that Kv1.3 inhibitors provide similar protection. However, it is unclear whether blockade of Kv1.3 in adipocytes or skeletal muscle increases glucose uptake. There is no evidence that the related channel Kv1.5 has any influence on insulin sensitivity and its expression in adipose tissue has not been reported. PAP-1 is a selective inhibitor of Kv1.3, with 23-fold, 32-fold and 125-fold lower potencies as an inhibitor of Kv1.5, Kv1.1 and Kv1.2 respectively. Soleus muscles from wild-type and genetically obese ob/ob mice were incubated with 2-deoxy[1-(14)C]-glucose for 45 min and formation of 2-deoxy[1-(14)C]-glucose-6-phosphate was measured. White adipocytes were incubated with D-[U-(14)C]-glucose for 1 h. TNFα and Il-6 secretion from white adipose tissue pieces were measured by enzyme-linked-immunoassay. In the absence of insulin, a high concentration (3 µM) of PAP-1 stimulated 2-deoxy[1-14C]-glucose uptake in soleus muscle of wild-type and obese mice by 30% and 40% respectively, and in adipocytes by 20% and 50% respectively. PAP-1 also stimulated glucose uptake by adipocytes at the lower concentration of 1 µM, but at 300 nM, which is still 150-fold higher than its EC50 value for inhibition of the Kv1.3 channel, it had no effect. In the presence of insulin, PAP-1 (3 µM) had a significant effect only in adipocytes from obese mice. PAP-1 (3 µM) reduced the secretion of TNFα by adipose tissue but had no effect on the secretion of IL-6. Expression of Kv1.1, Kv1.2, Kv1.3 and Kv1.5 was determined by RT-PCR. Kv1.3 and Kv1.5 mRNA were detected in liver, gastrocnemius muscle, soleus muscle and white adipose tissue from wild-type and ob/ob mice, except that Kv1.3 could not be detected in gastrocnemius muscle, nor Kv1.5 in liver, of wild-type mice. Expression of both genes was generally higher in liver and muscle of ob/ob mice compared to wild-type mice. Kv1.5 appeared to be expressed more highly than Kv1.3 in soleus muscle, adipose tissue and adipocytes of wild-type mice. Expression of Kv1.2 appeared to be similar to that of Kv1.3 in soleus muscle and adipose tissue, but Kv1.2 was undetectable in adipocytes. Kv1.1 could not be detected in soleus muscle, adipose tissue or adipocytes. We conclude that inhibition of Kv1 channels by PAP-1 stimulates glucose uptake by adipocytes and soleus muscle of wild-type and ob/ob mice, and reduces the secretion of TNFα by adipose tissue. However, these effects are more likely due to inhibition of Kv1.5 than to inhibition of Kv1.3 channels.

Toll-like receptor 2 activation and comedogenesis: implications for the pathogenesis of acne.

BACKGROUND: Acne is a common disorder of the human pilosebaceous unit, yet the mechanisms underlying hyperkeratinisation and subsequent inflammation (comedogenesis) remain to be determined, although cutaneous pathogens are implicated. Previously, it was reported that the release of the cytokine interleukin-1α (IL-1α) by keratinocytes of the sebaceous duct was pivotal in the life cycle of the comedone, mediating both its development and its spontaneous resolution. Toll-like receptors are a family of molecules that recognise pathogen associated molecular patterns (PAMPs) presented by microorganisms, initiating a signalling cascade terminating in the release of antimicrobial compounds and cytokines. METHODS: We used ex vivo sebaceous gland and primary monolayer keratinocyte culture, alongside ELISAs, immunohistochemistry, Western blotting and RT-PCR to investigate the contribution of TLR activation to acne pathogenesis. RESULTS: We found TLR2 to be expressed in basal and infundibular keratinocytes, and sebaceous glands, and its activation provoked the release of IL-1α from primary human keratinocytes in vitro. The exposure of microdissected human sebaceous glands to PAMPs specific for TLR2 in vitro resulted in a pattern of IL-1α like cornification after seven days of exposure. CONCLUSIONS: TLR activation and secretion of IL-1α from keratinocytes may be initiating steps in comedogenesis and, therefore, critical to the pathophysiology of acne.

A novel method to assess collagen architecture in skin.

BACKGROUND: Texture within biological specimens may reveal critical insights, while being very difficult to quantify. This is a particular problem in histological analysis. For example, cross-polar images of picrosirius stained skin reveal exquisite structure, allowing changes in the basketweave conformation of healthy collagen to be assessed. Existing techniques measure gross pathological changes, such as fibrosis, but are not sufficiently sensitive to detect more subtle and progressive pathological changes in the dermis, such as those seen in ageing. Moreover, screening methods for cutaneous therapeutics require accurate, unsupervised and high-throughput image analysis techniques. RESULTS: By analyzing spectra of images post Gabor filtering and Fast Fourier Transform, we were able to measure subtle changes in collagen fibre orientation intractable to existing techniques. We detected the progressive loss of collagen basketweave structure in a series of chronologically aged skin samples, as well as in skin derived from a model of type 2 diabetes mellitus. CONCLUSIONS: We describe a novel bioimaging approach with implications for the evaluation of pathology in a broader range of biological situations.

A novel automated image analysis method for accurate adipocyte quantification.

Increased adipocyte size and number are associated with many of the adverse effects observed in metabolic disease states. While methods to quantify such changes in the adipocyte are of scientific and clinical interest, manual methods to determine adipocyte size are both laborious and intractable to large scale investigations. Moreover, existing computational methods are not fully automated. We, therefore, developed a novel automatic method to provide accurate measurements of the cross-sectional area of adipocytes in histological sections, allowing rapid high-throughput quantification of fat cell size and number. Photomicrographs of H&E-stained paraffin sections of murine gonadal adipose were transformed using standard image processing/analysis algorithms to reduce background and enhance edge-detection. This allowed the isolation of individual adipocytes from which their area could be calculated. Performance was compared with manual measurements made from the same images, in which adipocyte area was calculated from estimates of the major and minor axes of individual adipocytes. Both methods identified an increase in mean adipocyte size in a murine model of obesity, with good concordance, although the calculation used to identify cell area from manual measurements was found to consistently over-estimate cell size. Here we report an accurate method to determine adipocyte area in histological sections that provides a considerable time saving over manual methods.

A topology-based score for pathway enrichment.

Investigators require intuitive tools to rationalize complex datasets generated by transcriptional profiling experiments. Pathway analysis methods, in which differentially expressed genes are mapped to databases of reference pathways to facilitate assessment of relative enrichment, lead investigators more effectively to biologically testable hypotheses. However, once a set of differentially expressed genes is isolated, pathway analysis approaches tend to ignore rich gene expression information and, moreover, do not exploit relationships between transcripts. In this article, we report the development of a new method in which both pathway topology and the magnitude of gene expression changes inform the scoring system, thereby providing a powerful filter in the enrichment of biologically relevant information. When four sample datasets were evaluated with this method, literature mining confirmed that those pathways germane to the physiological process under investigation were highlighted by our method relative to z-score overrepresentation calculations. Moreover, non-relevant processes were downgraded using the method described herein. The inclusion of expression and topological data in the calculation of a pathway regulation score (PRS) facilitated discrimination of key processes in real biological datasets. Specifically, by combining fold-change data for those transcripts exceeding a significance threshold, and by taking into account the potential for altered gene expression to impact upon downstream transcription, one may readily identify those pathways most relevant to pathophysiological processes.

Exhaustive identification of human class II basic helix-loop-helix proteins by virtual library screening.

Cellular proliferation, specification and differentiation in developing tissues are tightly coordinated by groups of transcription factors in response to extrinsic and intrinsic signals. Furthermore, renewable pools of stem cells in adult tissues are subject to similar regulation. Basic helix-loop-helix (bHLH) proteins are a group of transcription factors that exert such a determinative influence on a variety of developmental pathways from C. elegans to humans, and we wished to exclusively identify novel members from within the whole human bHLH family. We have, therefore, developed an 'empirical custom fingerprint', to define the class II bHLH domain and exclusively identify these proteins in silico. We have identified nine previously uncharacterised human class II proteins, four of which were novel, by interrogating conceptual translations of the GenBank HTGS database. RT-PCR and mammalian 2-hybrid analysis of a subset of the factors demonstrated that they were indeed expressed, and were able to interact with an appropriate binding partner in vitro. Thus, we are now approaching an almost complete listing of human class II bHLH factors.

Exhaustive identification of human class II basic helix-loop-helix proteins by virtual library screening.

Cellular proliferation, specification and differentiation in developing tissues are tightly coordinated by groups of transcription factors in response to extrinsic and intrinsic signals. Furthermore, renewable pools of stem cells in adult tissues are subject to similar regulation. Basic helix-loop-helix (bHLH) proteins are a group of transcription factors that exert such a determinative influence on a variety of developmental pathways from C. elegans to humans, and we wished to exclusively identify novel members from within the whole human bHLH family. We have, therefore, developed an 'empirical custom fingerprint', to define the class II bHLH domain and exclusively identify these proteins in silico. We have identified nine previously uncharacterised human class II proteins, four of which were novel, by interrogating conceptual translations of the GenBank HTGS database. RT-PCR and mammalian 2-hybrid analysis of a subset of the factors demonstrated that they were indeed expressed, and were able to interact with an appropriate binding partner in vitro. Thus, we are now approaching an almost complete listing of human class II bHLH factors.