Active Learning: Action Potential and Clinical Applications in Neuroscience Medical Education.

Active learning in medical education engages adult learners and increases collaborative opportunities for consolidation of concepts. An innovative learning activity was used to engage medical students in an activity about action potentials and its clinical applications, resulting in increased understanding, application, and retention of the clinical relevance of the topic.

A recurrent single-amino acid deletion (p.Glu500del) in the head domain of ß-cardiac myosin in two unrelated boys presenting with polyhydramnios, congenital axial stiffness and skeletal myopathy.

BACKGROUND: Alterations in the MYH7 gene can cause cardiac and skeletal myopathies. MYH7-related skeletal myopathies are extremely rare, and the vast majority of causal variants in the MYH7 gene are predicted to alter the rod domain of the of ß-cardiac myosin molecule, resulting in distal muscle weakness as the predominant manifestation. Here we describe two unrelated patients harboring an in-frame deletion in the MYH7 gene that is predicted to result in deletion of a single amino acid (p.Glu500del) in the head domain of ß-cardiac myosin. Both patients display an unusual skeletal myopathy phenotype with congenital axial stiffness and muscular hypertonus, but no cardiac involvement. RESULTS: Clinical data, MRI results and histopathological data were collected retrospectively in two unrelated boys (9 and 3.5 years old). Exome sequencing uncovered the same 3-bp in-frame deletion in exon 15 (c.1498_1500delGAG) of the MYH7 gene of both patients, a mutation which deletes a highly conserved glutamate residue (p.Glu500del) in the relay loop of the head domain of the ß-cardiac myosin heavy chain. The mutation occurred de novo in one patient, whereas mosaicism was detected in blood of the father of the second patient. Both boys presented with an unusual phenotype of prenatal polyhydramnios, congenital axial stiffness and muscular hypertonus. In one patient the phenotype evolved into an axial/proximal skeletal myopathy without distal involvement or cardiomyopathy, whereas the other patient exhibited predominantly stiffness and respiratory involvement. We review and compare all patients described in the literature who possess a variant predicted to alter the p.Glu500 residue in the ß-cardiac myosin head domain, and we provide in-silico analyses of potential effects on polypeptide function. CONCLUSION: The data presented here expand the phenotypic spectrum of mutations in the MYH7 gene and have implications for future diagnostics and therapeutic approaches.

Pompe disease in Austria: clinical, genetic and epidemiological aspects.

In this study, we performed a survey of infantile and late-onset Pompe disease (IOPD and LOPD) in Austria. Paediatric and neuromuscular centres were contacted to provide a set of anonymized clinical and genetic data of patients with IOPD and LOPD. The number of patients receiving enzyme replacement therapy (ERT) was obtained from the pharmaceutical company providing alglucosidase alfa. We found 25 patients in 24 families, 4 IOPD and 21 LOPD with a resulting prevalence of 1:350,914. The most frequent clinical manifestation in LOPD was a lower limb-girdle phenotype combined with axial weakness. Three patients were clinically pauci- or asymptomatic and were diagnosed because of persistent hyperCKemia. Diagnostic delay in LOPD was 7.4 ± 9.7 years. The most common mutation was c.-32-13T > G. All IOPD and 17 symptomatic LOPD patients are receiving ERT. Standardized follow-up was only available in six LOPD patients for the 6-min walk test (6minWT) and in ten for the forced vital capacity (FVC). Mean FVC did not decline (before ERT; 63.6 ± 39.7%; last evaluation during ERT: 61.9 ± 26.9%; P = 0.5) while there was a trend to decline in the mean distance covered by the 6minWT (before ERT: 373.5 ± 117.9 m; last evaluation during ERT: 308.5 ± 120.8 m; P = 0.077). The study shows a lower prevalence of Pompe disease in Austria than in other European countries and corroborates a limb-girdle phenotype with axial weakness as the most common clinical presentation, although asymptomatic hyperCKemia may be the first indication of LOPD.

Fulvestrant induces resistance by modulating GPER and CDK6 expression: implication of methyltransferases, deacetylases and the hSWI/SNF chromatin remodelling complex.

BACKGROUND: Breast cancer is the leading cause of cancer death in women living in the western hemisphere. Despite major advances in first-line endocrine therapy of advanced oestrogen receptor (ER)-positive breast cancer, the frequent recurrence of resistant cancer cells represents a serious obstacle to successful treatment. Understanding the mechanisms leading to acquired resistance, therefore, could pave the way to the development of second-line therapeutics. To this end, we generated an ER-positive breast cancer cell line (MCF-7) with resistance to the therapeutic anti-oestrogen fulvestrant (FUL) and studied the molecular changes involved in resistance. METHODS: Naive MCF-7 cells were treated with increasing FUL concentrations and the gene expression profile of the resulting FUL-resistant strain (FR.MCF-7) was compared with that of naive cells using GeneChip arrays. After validation by real-time PCR and/or western blotting, selected resistance-associated genes were functionally studied by siRNA-mediated silencing or pharmacological inhibition. Furthermore, general mechanisms causing aberrant gene expression were investigated. RESULTS: Fulvestrant resistance was associated with repression of GPER and the overexpression of CDK6, whereas ERBB2, ABCG2, ER and ER-related genes (GREB1, RERG) or genes expressed in resistant breast cancer (BCAR1, BCAR3) did not contribute to resistance. Aberrant GPER and CDK6 expression was most likely caused by modification of DNA methylation and histone acetylation, respectively. Therefore, part of the resistance mechanism was loss of RB1 control. The hSWI/SNF (human SWItch/Sucrose NonFermentable) chromatin remodelling complex, which is tightly linked to nucleosome acetylation and repositioning, was also affected, because as a stress response to FUL treatment-naive cells altered the expression of five subunits within a few hours (BRG1, BAF250A, BAF170, BAF155, BAF47). The aberrant constitutive expression of BAF250A, BAF170 and BAF155 and a deviant stress response of BRG1, BAF170 and BAF47 in FR.MCF-7 cells to FUL treatment accompanied acquired FUL resistance. The regular and aberrant expression profiles of BAF155 correlated directly with that of CDK6 in naive and in FR.MCF-7 cells corroborating the finding that CDK6 overexpression was due to nucleosome alterations. CONCLUSION: The study revealed that FUL resistance is associated with the dysregulation of GPER and CDK6. A mechanism leading to aberrant gene expression was most likely unscheduled chromatin remodelling by hSWI/SNF. Hence, three targets should be conceptually addressed in a second-line adjuvant therapy: the catalytic centre of SWI/SNF (BRG1) to delay the development of FUL resistance, GPER to increase sensitivity to FUL and the reconstitution of the RB1 pathway to overcome resistance.

Long-term persistence of acquired resistance to 5-fluorouracil in the colon cancer cell line SW620.

Treatment resistance to antineoplastic drugs represents a major clinical problem. Here, we investigated the long-term stability of acquired resistance to 5-fluorouracil (FU) in an in vitro colon cancer model, using four sub-clones characterised by increasing FU-resistance derived from the cell line SW620. The resistance phenotype was preserved after FU withdrawal for 15weeks (~100 cell divisions) independent of the established level of drug resistance and of epigenetic silencing. Remarkably, resistant clones tolerated serum deprivation, adopted a CD133(+) CD44(-) phenotype, and further exhibited loss of membrane-bound E-cadherin together with predominant nuclear ß-catenin localisation. Thus, we provide evidence for a long-term memory of acquired drug resistance, driven by multiple cellular strategies (epithelial-mesenchymal transition and selective propagation of CD133(+) cells). These resistance phenomena, in turn, accentuate the malignant phenotype.

Detection of single base alterations in genomic DNA by solid phase polymerase chain reaction on oligonucleotide microarrays.

DNA microarray technology holds significant promise for human DNA diagnostics. A number of technical approaches directed at the parallel identification of mutations or single nucleotide polymorphisms make use of polymerase-based specificity, like minisequencing or allele-specific primer elongation. These techniques, however, require separate laborious sample amplification, preparation, and purification steps, making large-scale analyses time and cost consuming. Here, we address this challenge by applying an experimental setup using simultaneous solid and liquid phase PCR on polyethyleneimine-coated glass slides, a novel microarray support allowing on-chip amplification reactions with exquisite specificity. A gene-specific oligonucleotide tiling array contains covalently attached allele-specific primers which interrogate single nucleotide positions within a genomic region of interest. During a thermal cycling reaction amplification products remain covalently bound to the solid support and can be visualized and analyzed by the incorporation of fluorescent dyes. Using the described procedure we unequivocally defined the presence of point mutations in the human tumor suppressor gene p53 directly from a natural DNA source. This semi-multiplex solid phase amplification format allowed the rapid and correct identification of 20 nucleotide positions from minute amounts of human genomic DNA. Our results suggest that this approach might constitute a vital component of future integrated DNA chip devices used in gene analysis.

Reverse transcriptase template switching during reverse transcriptase-polymerase chain reaction: artificial generation of deletions in ribonucleotide reductase mRNA.

Using reverse transcriptase-polymerase chain reaction (RT-PCR), we have recently described a bona fide deletion within the coding sequence of the large subunit of ribonucleotide reductase (R1) mRNA in colon cancer. Consecutive studies have raised questions about the nature of this phenomenon, because the corresponding genomic alteration at the DNA level or an aberrant protein could not be detected. Thus we considered an in vitro artifact during RT-PCR as a possible explanation for this observation. In contrast to reverse transcriptase, Taq DNA polymerase or C. therm DNA polymerase did not generate the aberrant product, suggesting the demand for the template switching activity intrinsic to retroviral reverse transcriptases. In fact, virtually the same deletion was observed in RT-PCR experiments when in vitro transcribed R1 mRNA was used. Considering structural prerequisites for template switching within R1 mRNA, we show that two direct repeats adjacent to a strong stem-loop secondary structure flank the deleted region of 1851 base pairs. Because several mRNAs encoding proteins of clinical and diagnostic importance fulfill these criteria, template switching enhances the potential risk of observing artifacts when interpreting results from RT-PCR studies. As shown in the present example, this may involve the artificial generation and the misinterpretation of PCR fragments amplified from targets relevant to tumor biology or cancer pharmacology. As a possible solution, one-step PCR with C. therm polymerase should be considered. This polymerase eliminates the artificial generation of aberrant mRNA signals observed during cDNA synthesis.

CapSelect: a highly sensitive method for 5' CAP-dependent enrichment of full-length cDNA in PCR-mediated analysis of mRNAs.

Here we present CapSelect as a novel experimental approach for the selective enrichment of full-length cDNAs in PCR-mediated analysis of mRNA sequences. The method combines the 5'-CAP-dependent addition of specifically three to four non-templated dCMP residues to the 3'-end of full-length cDNAs by reverse transcriptases in the presence of manganese and the controlled ribonucleotide tailing of cDNA ends by terminal deoxynucleotidyl transferase using rATP. By virtue of the generated terminal sequence motif (5'-dC(3-4)rA(3-4)), full-length cDNAs are selectively anchored to a double-stranded DNA adapter (with a dT(3-4)dG(3)3'-overhang) by T4 DNA ligase. The technique described is highly efficient, discriminates premature termination products and enriches full-length cDNAs.

Controlled ribonucleotide tailing of cDNA ends (CRTC) by terminal deoxynucleotidyl transferase: a new approach in PCR-mediated analysis of mRNA sequences.

Controlled ribonucleotide tailing of cDNA ends (CRTC) by terminal deoxynucleotidyl transferase is a polymerase chain reaction (PCR)-mediated technique that was developed to facilitate cloning and direct sequence analysis of complete 5'-terminal unknown coding regions of rare RNA molecules. In contrast with standard tailing protocols using dNTPs as the substrate, ribo-tailing of cDNA ends is easily controllable, self-limited (from two to four rNMP incorporations) and highly efficient (>98%). By virtue of the homopolymeric ribo-tail, the modified cDNA is anchored to the 3' overhang of a double-stranded DNA-adaptor in a T4 DNA ligase-dependent ligation. PCR amplification, mediated by two sequence-specific primers, yields the desired unique product suitable for cloning and dideoxy-sequencing.

Transposable group II introns in fission and budding yeast. Site-specific genomic instabilities and formation of group II IVS plDNAs.

The recent report on RNA-mediated group II intron (IVS, intervening sequence) transposition in mitochondria (mt) of Saccharomyces cerevisiae and Podospora anserina and the demonstration of reverse transcriptase (RT) activity encoded by the mobile S. cerevisiae intron cox1-aI1 suggests that group II introns constitute a new class of site-specific retro-like (retroid) elements. This is supported by the finding that the mitochondrial cob1-bI1 intron from the fission yeast Schizosaccharomyces pombe, encoding an RT-like open reading frame, is transposed in mtDNA populations. In agreement with the involvement of an RNA-intermediate in IVS transposition: First, the insertion sites were preceded by at least an IBS1-like (intron binding site) motif, which corresponds to the upstream exon and suffices to form the IBS1/EBS1 (EBS: exon binding site) base-pairing interactions. Second, intron transposition was conservative with respect to sequences flanking the insertion sites. We formulated the hypothesis that transient IVS insertion at non-allelic sites followed by recombination can be viewed as a general molecular mechanism, applicable equally well to site-specific genomic instabilities involving splice-site borders of group II introns and to the formation of extra-genomic IVS plasmid DNAs (plDNAs). We used polymerase chain reaction (PCR) techniques to detect infrequent rearrangements in mtDNA and report here on duplicative IVS transposition, twintron formation (e.g. bI1 insertion into another bI1 intron), and IVS insertions at canonical 5' exon-intron borders in S. pombe (cob1-bI1) and in S. cerevisiae (cox1-aI1). These data substantiate the concept that group II intron homing, IVS transposition and circular IVS plDNA formation involve a common RNA-mediated mechanism. Finally, the findings suggest that extra-genomic group II IVS copies are not restricted to senescence mycelia of P. anserina, but constitute natural components of group II IVS-containing genomes.

On the question of the carcinogenic action of hydrazine--evaluation on the basis of new experimental results.

Hydrazine has not previously been shown to have any carcinogenic action in man. After the administration of probably clearly toxic doses (the data in earlier publications are very fragmentary) or doses severely irritating to the sensitive nasal epithelium of the rodent over a large part of its life, hydrazine was shown in the majority of the studies described in the literature to be carcinogenic in rodents. Even under these severe experimental conditions, however, the carcinogenic action was not very pronounced or was even very weak. In the study in mice described below, which was carried out according to modern guidelines, no carcinogenic action was detected for hydrazine even after the administration of toxic doses over the entire lifespan of the animals. Administration of a still higher dose would have conflicted with all current recommendations. On the basis of the results available in the literature, indirect alkylation of DNA was assumed to be the mechanism of the carcinogenic action of hydrazine. According to the current level of knowledge, this effect, like the mutagenic and the carcinogenic action of the substance, is closely linked with the toxic activity of hydrazine. Overall, hydrazine should be regarded as a substance having a probably indirect weakly carcinogenic action after toxic doses administered over the entire lifespan.

A differentiated approach to testing skin sensitization. Proposal for a new test guideline skin sensitization.

The present EEC and OECD Guidelines for testing skin sensitization have been reviewed in light of scientific evidence demonstrating that those methods which use Freund's Complete Adjuvant (FCA) are likely to be more accurate in predicting a probable skin-sensitizing effect of a new substance in humans than those methods not employing Freund's Complete Adjuvant. In this new test guideline, therefore, the primary testing of a substance should be carried out using one of the recommended Adjuvant methods. In special cases a non-adjuvant method may be performed in addition. Not all of the seven methods in the EEC Guideline or eight methods in the OECD Guideline have been included, but in a proposal for an updated test protocol two Adjuvant tests (Maximization test by Magnusson and Kligman and Optimization test by Maurer), and two non-Adjuvant tests (Open Epicutaneous test by Klecak and Buehler test) are suggested. The criteria for selecting these methods are based on the fact that they are well validated and widely used on a broad basis by the scientific community. Furthermore, it is considered appropriate to permit the use of a lower number of animals than presently recommended for the testing of skin sensitization. This is also in agreement with aspects of animal welfare.