Construction and validation of a medical career readiness inventory.

BACKGROUND: Medical students' preparedness for clinical practice is well researched, yet little is known on the extent to which students are being prepared for a medical career. This paper reports the construction of a short medical inventory titled eXploring medical sTudents' caReer reAdiness (XTRA) to measure students' career readiness based on Super's theory of career maturity. APPROACH: We designed an instrument consisting of a series of 5-point Likert-scale to identify participants competencies regarding career exploration and planning during their undergraduate studies. The instrument was completed by 348 medical students from 41 universities in the United Kingdom. We examined the validity and reliability of the instrument through Exploratory Factor Analysis, Cronbach's coefficient α and Pearson correlation. EVALUATION: Exploratory Factor Analysis revealed that 16 of the 20-items survey were aligned with the exploration stage of Super's theory: Crystallisation (Career goals), Specification (Career pathways) and Implementation (Career accomplishments). The four items that formed two separate statistical factors were specific to a current medical career in the UK. Internal reliability for Super's factor subscales were acceptable (α = 0.71 to α = 0.81). A significant positive relationship was found between students' overall rating of career readiness and the three factors, indicating construct validity. IMPLICATIONS: The XTRA Inventory is a short instrument with construct and content validity specifically designed to measure career readiness of medical students. Further work on its psychometric properties will help establish this inventory to be used as a guidance and career counselling tool by medical educators and educational institutions in developing career development programmes.

Huntington's disease age at motor onset is modified by the tandem hexamer repeat in TCERG1.

Huntington's disease is caused by an expanded CAG tract in HTT. The length of the CAG tract accounts for over half the variance in age at onset of disease, and is influenced by other genetic factors, mostly implicating the DNA maintenance machinery. We examined a single nucleotide variant, rs79727797, on chromosome 5 in the TCERG1 gene, previously reported to be associated with Huntington's disease and a quasi-tandem repeat (QTR) hexamer in exon 4 of TCERG1 with a central pure repeat. We developed a method for calling perfect and imperfect repeats from exome-sequencing data, and tested association between the QTR in TCERG1 and residual age at motor onset (after correcting for the effects of CAG length in the HTT gene) in 610 individuals with Huntington's disease via regression analysis. We found a significant association between age at onset and the sum of the repeat lengths from both alleles of the QTR (p = 2.1 × 10-9), with each added repeat hexamer reducing age at onset by one year (95% confidence interval [0.7, 1.4]). This association explained that previously observed with rs79727797. The association with age at onset in the genome-wide association study is due to a QTR hexamer in TCERG1, translated to a glutamine/alanine tract in the protein. We could not distinguish whether this was due to cis-effects of the hexamer repeat on gene expression or of the encoded glutamine/alanine tract in the protein. These results motivate further study of the mechanisms by which TCERG1 modifies onset of HD.

Prescribing costs of hypoglycaemic agents and associations with metabolic control in Wales; a national analysis of primary care data.

AIMS: There has been a dramatic increase in hypoglycaemic agent expenditure. We assessed the variability in prescribing costs at the practice level and the relationship between expenditure and the proportion of patients achieving target glycaemic control. METHODS: We utilized national prescribing data from 406 general practices in Wales. This was compared against glycaemic control (percentage of patients achieving a HbA1c level < 59 mmol/mol in the preceding 12 months). Analyses were adjusted for the number of patients with diabetes in each general practice and the Welsh Index of Multiple Deprivation. RESULTS: There was considerable heterogeneity in hypoglycaemic agent spend per patient with diabetes, Median = £289 (IQR 247-343) range £31.1-£1713. Higher total expenditure was not associated with improved glycaemic control B(std)  = -0.01 (95%CI -0.01, 0.002) p = 0.13. High-spend practices spent more on SGLT2 inhibitors (16 vs. 9% p < 0.001) and GLP-1 agonists (13 vs. 11% p < 0.001) and less on insulin (34 vs. 42% p < 0.001), biguanides (9 vs. 11% p = 0.001) and sulphonylureas (2 vs. 3% p < 0.001) than low spend practices. There were no differences in the pattern of drug prescribing between high spend practices with better glycaemic control (mean 68% of patients HbA1c <59 mmol/mol) and those with less good metabolic control (mean 58% of patients HbA1c <59 mmol/mol). CONCLUSIONS: Spend on hypoglycaemic agents is highly variable between practices and increased expenditure per patient is not associated with better glycaemic control. Whilst newer, more expensive agents have additional benefits, in individuals where these advantages are more marginal widespread use of these agents has important cost implications.

The Human Sodium Iodide Symporter as a Reporter Gene for Studying Middle East Respiratory Syndrome Coronavirus Pathogenesis.

Single photon emission computed tomography (SPECT) is frequently used in oncology and cardiology to evaluate disease progression and/or treatment efficacy. Such technology allows for real-time evaluation of disease progression and when applied to studying infectious diseases may provide insight into pathogenesis. Insertion of a SPECT-compatible reporter gene into a virus may provide insight into mechanisms of pathogenesis and viral tropism. The human sodium iodide symporter (hNIS), a SPECT and positron emission tomography reporter gene, was inserted into Middle East respiratory syndrome coronavirus (MERS-CoV), a recently emerged virus that can cause severe respiratory disease and death in afflicted humans to obtain a quantifiable and sensitive marker for viral replication to further MERS-CoV animal model development. The recombinant virus was evaluated for fitness, stability, and reporter gene functionality. The recombinant and parental viruses demonstrated equal fitness in terms of peak titer and replication kinetics, were stable for up to six in vitro passages, and were functional. Further in vivo evaluation indicated variable stability, but resolution limits hampered in vivo functional evaluation. These data support the further development of hNIS for monitoring infection in animal models of viral disease.IMPORTANCE Advanced medical imaging such as single photon emission computed tomography with computed tomography (SPECT/CT) enhances fields such as oncology and cardiology. Application of SPECT/CT, magnetic resonance imaging, and positron emission tomography to infectious disease may enhance pathogenesis studies and provide alternate biomarkers of disease progression. The experiments described in this article focus on insertion of a SPECT/CT-compatible reporter gene into MERS-CoV to demonstrate that a functional SPECT/CT reporter gene can be inserted into a virus.

Transcriptome integration analysis in hepatocellular carcinoma reveals discordant intronic miRNA-host gene pairs in expression.

Intronic miRNAs, residing in intronic regions of host genes, are thought to be co-transcribed from their host genes and present consistent expression patterns with host genes. Recent studies reported a few intronic miRNAs with discordant expression with their host genes. We therefore aimed to understand the expression pattern of intronic miRNAs and their host genes in hepatocellular carcinoma (HCC) and reveal possible associated molecular mechanisms. Our genome wide integration analysis of miRNA and mRNA transcriptomes, in three dataset from 550 patients with HCC, found that a large amount of miRNA-host gene pairs were discordantly expressed. Consistent results were also revealed in 775 breast cancer patients. Further, most of HCC-related intronic miRNAs were predicted to have distinct upstream regulators and independent proximal promoter signals from host genes. The discordant expression of representative pairs, miR-26s/CTDSPs, was validated experimentally. We have also identified the independent transcriptional start site, promoter signal, and transcriptional factor of miR-26b from its host gene. Collectively, discordant expression of intronic miRNAs and their host genes was relatively ubiquitous and the intronic miRNA "independent transcription" may partially contribute to such a phenotype.

A small stem-loop structure of the Ebola virus trailer is essential for replication and interacts with heat-shock protein A8.

Ebola virus (EBOV) is a single-stranded negative-sense RNA virus belonging to the Filoviridae family. The leader and trailer non-coding regions of the EBOV genome likely regulate its transcription, replication, and progeny genome packaging. We investigated the cis-acting RNA signals involved in RNA-RNA and RNA-protein interactions that regulate replication of eGFP-encoding EBOV minigenomic RNA and identified heat shock cognate protein family A (HSC70) member 8 (HSPA8) as an EBOV trailer-interacting host protein. Mutational analysis of the trailer HSPA8 binding motif revealed that this interaction is essential for EBOV minigenome replication. Selective 2'-hydroxyl acylation analyzed by primer extension analysis of the secondary structure of the EBOV minigenomic RNA indicates formation of a small stem-loop composed of the HSPA8 motif, a 3' stem-loop (nucleotides 1868-1890) that is similar to a previously identified structure in the replicative intermediate (RI) RNA and a panhandle domain involving a trailer-to-leader interaction. Results of minigenome assays and an EBOV reverse genetic system rescue support a role for both the panhandle domain and HSPA8 motif 1 in virus replication.

Intratracheal exposure of common marmosets to MERS-CoV Jordan-n3/2012 or MERS-CoV EMC/2012 isolates does not result in lethal disease.

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) continues to be a threat to human health in the Middle East. Development of countermeasures is ongoing; however, an animal model that faithfully recapitulates human disease has yet to be defined. A recent study indicated that inoculation of common marmosets resulted in inconsistent lethality. Based on these data we sought to compare two isolates of MERS-CoV. We followed disease progression in common marmosets after intratracheal exposure with: MERS-CoV-EMC/2012, MERS-CoV-Jordan-n3/2012, media, or inactivated virus. Our data suggest that common marmosets developed a mild to moderate non-lethal respiratory disease, which was quantifiable by computed tomography (CT), with limited other clinical signs. Based on CT data, clinical data, and virological data, MERS-CoV inoculation of common marmosets results in mild to moderate clinical signs of disease that are likely due to manipulations of the marmoset rather than as a result of robust viral replication.

Inactivation and safety testing of Middle East Respiratory Syndrome Coronavirus.

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is a recently emerged virus that has caused a number of human infections and deaths, primarily in the Middle East. The transmission of MERS-CoV to humans has been proposed to be as a result of contact with camels, but evidence of human-to-human transmission also exists. In order to work with MERS-CoV in a laboratory setting, the US Centers for Disease Control and Prevention (CDC) has determined that MERS-CoV should be handled at a biosafety level (BSL) 3 (BSL-3) biocontainment level. Many processes and procedures used to characterize MERS-CoV and to evaluate samples from MERS-CoV infected animals are more easily and efficiently completed at BSL-2 or lower containment. In order to complete experimental work at BSL-2, demonstration or proof of inactivation is required before removal of specimens from biocontainment laboratories. In the studies presented here, we evaluated typical means of inactivating viruses prior to handling specimens at a lower biocontainment level. We found that Trizol, AVL buffer and gamma irradiation were effective at inactivating MERS-CoV, that formaldehyde-based solutions required at least 30 min of contact time in a cell culture system while a mixture of methanol and acetone required 60 min to inactivate MERS-CoV. Together, these data provide a foundation for safely inactivating MERS-CoV, and potentially other coronaviruses, prior to removal from biocontainment facilities.

Real-time sequence-validated loop-mediated isothermal amplification assays for detection of Middle East respiratory syndrome coronavirus (MERS-CoV).

The Middle East respiratory syndrome coronavirus (MERS-CoV), an emerging human coronavirus, causes severe acute respiratory illness with a 35% mortality rate. In light of the recent surge in reported infections we have developed asymmetric five-primer reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays for detection of MERS-CoV. Isothermal amplification assays will facilitate the development of portable point-of-care diagnostics that are crucial for management of emerging infections. The RT-LAMP assays are designed to amplify MERS-CoV genomic loci located within the open reading frame (ORF)1a and ORF1b genes and upstream of the E gene. Additionally we applied one-step strand displacement probes (OSD) for real-time sequence-specific verification of LAMP amplicons. Asymmetric amplification effected by incorporating a single loop primer in each assay accelerated the time-to-result of the OSD-RT-LAMP assays. The resulting assays could detect 0.02 to 0.2 plaque forming units (PFU) (5 to 50 PFU/ml) of MERS-CoV in infected cell culture supernatants within 30 to 50 min and did not cross-react with common human respiratory pathogens.

Transcriptomic profiling reveals hepatic stem-like gene signatures and interplay of miR-200c and epithelial-mesenchymal transition in intrahepatic cholangiocarcinoma.

UNLABELLED: Intrahepatic cholangiocellular carcinoma (ICC) is the second most common type of primary liver cancer. However, its tumor heterogeneity and molecular characteristics are largely unknown. In this study, we conducted transcriptomic profiling of 23 ICC and combined hepatocellular cholangiocarcinoma tumor specimens from Asian patients using Affymetrix messenger RNA (mRNA) and NanoString microRNA microarrays to search for unique gene signatures linked to tumor subtypes and patient prognosis. We validated the signatures in an additional 68 ICC cases derived from Caucasian patients. We found that both mRNA and microRNA expression profiles could independently classify Asian ICC cases into two main subgroups, one of which shared gene expression signatures with previously identified hepatocellular carcinoma (HCC) with stem cell gene expression traits. ICC-specific gene signatures could predict survival in Asian HCC cases and independently in Caucasian ICC cases. Integrative analyses of the ICC-specific mRNA and microRNA expression profiles revealed that a common signaling pathway linking miR-200c signaling to epithelial-mesenchymal transition (EMT) was preferentially activated in ICC with stem cell gene expression traits. Inactivation of miR-200c resulted in an induction of EMT, whereas activation of miR-200c led to a reduction of EMT including a reduced cell migration and invasion in ICC cells. We also found that miR-200c and neural cell adhesion molecule 1 (NCAM1) expression were negatively correlated and their expression levels were predictive of survival in ICC samples. NCAM1, a known hepatic stem/progenitor cell marker, was experimentally demonstrated to be a direct target of miR-200c. CONCLUSION: Our results indicate that ICC and HCC share common stem-like molecular characteristics and poor prognosis. We suggest that the specific components of EMT may be exploited as critical biomarkers and clinically relevant therapeutic targets for an aggressive form of stem cell-like ICC.

Proteomic profiling of H-Ras-G12V induced hypertrophic cardiomyopathy in transgenic mice using comparative LC-MS analysis of thin fresh-frozen tissue sections.

Determination of disease-relevant proteomic profiles from limited tissue specimens, such as pathological biopsies and tissues from small model organisms, remains an analytical challenge and a much needed clinical goal. In this study, a transgenic mouse disease model of cardiac-specific H-Ras-G12V induced hypertrophic cardiomyopathy provided a system to explore the potential of using mass spectrometry (MS)-based proteomics to obtain a disease-relevant molecular profile from amount-limited specimens that are routinely used in pathological diagnosis. Our method employs a two-stage methanol-assisted solubilization to digest lysates prepared from 8-µm-thick fresh-frozen histological tissue sections of diseased/experimental and normal/control hearts. Coupling this approach with a nanoflow reversed-phase liquid chromatography (LC) and a hybrid linear ion trap/Fourier transform-ion cyclotron resonance MS resulted in the identification of 704 and 752 proteins in hypertrophic and wild-type (control) myocardium, respectively. The disease driving H-Ras protein along with vimentin were unambiguously identified by LC-MS in hypertrophic myocardium and cross-validated by immunohistochemistry and western blotting. The pathway analysis involving proteins identified by MS showed strong association of proteomic data with cardiovascular disease. More importantly, the MS identification and subsequent cross-validation of Wnt3a and ß-catenin, in conjunction with IHC identification of phosphorylated GSK-3ß and nuclear localization of ß-catenin, provided evidence of Wnt/ß-catenin canonical pathway activation secondary to Ras activation in the course of pathogenic myocardial hypertrophic transformation. Our method yields results indicating that the described proteomic approach permits molecular discovery and assessment of differentially expressed proteins regulating H-Ras induced hypertrophic cardiomyopathy. Selected proteins and pathways can be further investigated using immunohistochemical techniques applied to serial tissue sections of similar or different origin.

Molecular carcinogenesis of hepatocellular carcinoma and intrahepatic cholangiocarcinoma: one step closer to personalized medicine?

Hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) are the two major forms of primary liver cancers (PLC), accounting for approximately 90% and 5% respectively. The incidence of each is increasing rapidly in the western world, however our knowledge of the underlying mechanisms remains limited and the outcome, dismal. The etiologies of each vary geographically; nevertheless, chronic inflammation has been identified in more than 80% of the cases and appears to be a key mediator in altering the liver microenvironment, increasing the risk of carcinogenesis. However, since not all HCC and especially ICC cases have a recognized risk factor, there are currently two proposed models for liver carcinogenesis. The clonal evolution model demonstrates a multi-step process of tumor development from precancerous lesions to metastatic carcinoma, arising from the accumulation of genetic and epigenetic changes in a cell in the setting of chronic inflammation. While the majority of cases do occur as a consequence of chronic inflammation, most individuals with chronic infection do not develop PLC, suggesting the involvement of individual genetic and environmental factors. Further, since hepatocytes and cholangiocytes both have regenerative potential and arise from the same bi-potential progenitor cell, the more recently proposed cancer stem cell model is gaining its due attention. The integration of these models and the constant improvement in molecular profiling platforms is enabling a broader understanding of the mechanisms underlying these two devastating malignancies, perhaps moving us closer to a new world of molecularly-informed personalized medicine.

Capacity for resolution of Ras-MAPK-initiated early pathogenic myocardial hypertrophy modeled in mice.

Activation of Ras signaling in cardiomyocytes has been linked to pathogenic myocardial hypertrophy progression and subsequent heart failure. Whether cardiomyopathy can regress once initiated needs to be established more fully. A 'tet-off' system was used to regulate expression of H-Ras-G12V in myocardium to examine whether Ras-induced pathogenic myocardial hypertrophy could resolve after removal of Ras signaling in vivo. Ras activation at weaning for 2 wk caused hypertrophy, whereas activation for 4 to 8 wk led to cardiomyopathy and heart failure. Discontinuing H-Ras-G12V transgene expression after cardiomyopathy onset led to improved survival and cardiomyopathy lesion scores, with reduced heart:body weight ratios, demonstrating the reversibility of early pathogenic hypertrophy. Activation of Ras and downstream ERK 1/2 was associated with elevated expression of proliferating cell nuclear antigen and cyclins B1 and D1, indicating cell-cycle activation and reentry. Coordinate elevation of broad-spectrum cyclin-dependent kinase inhibitors (p21, p27, and p57) and Tyr15 phosphorylation of cdc2 signified the activation of cell-cycle checkpoints; absence of cell-cycle completion and cardiomyocyte replication were documented by using immunohistochemistry for mitosis and cytokinesis markers. After resolution of cardiomyopathy, cell-cycle activators and inhibitors examined returned to basal levels, a change that we interpreted as exit from the cell cycle. Cardiac cell-cycle regulation plays a role in recovery from pathogenic hypertrophy. The model we present provides a means to further explore the underlying mechanisms governing cell-cycle capacity in cardiomyocytes, as well as progression and regression of pathogenic cardiomyocyte hypertrophy.