Introducing e-learning/teaching in a physiology course for medical students: acceptance by students and subjective effect on learning.

Retrieval of information has substantially changed within the last two decades. Naturally, this has also affected learning/teaching techniques, and methods that are commonly referred to as "e-learning" have become an important part in modern education. Institutions have to decide if (and how) to implement this new form of teaching but face the problem that little subject-specific research has been published for different teaching modes and methods. The present study compares a course module of the physiology laboratory course for medical students in the preclinical phase before and after the introduction of computer-aided course instructions (CACI). Students were provided with an online questionnaire containing Likert items evaluating workspace redesign, acceptance of course instructions, incentive to actively participate in the course, and subjective gain of knowledge. CACI was clearly preferred over the previously used paper workbook. However, the questionnaire also revealed that the gain in knowledge, as subjectively perceived by the students, had not improved, which is in agreement with several studies that neglected a beneficial effect of e-learning on learning success. We conclude that the CACI meet today's student's expectations and that introducing this system seems justified from this perspective.

Combining atomic force-fluorescence microscopy with a stretching device for analyzing mechanotransduction processes in living cells.

Mechanical forces affect biological systems in their natural environment in a widespread manner. Mechanical stress may either stimulate cells or even induce pathological processes. Cells sensing mechanical stress usually respond to such stressors with proliferation or differentiation. Hence, for in vitro studies, the ability to impose a controlled mechanical stress on cells combined with appropriate analytical tools providing an immediate answer is essential to understand such fundamental processes. Here, we present a novel uniaxial motorized cell stretching device that has been integrated into a combined fluorescence microscope (FM)-atomic force microscope (AFM) system, thereby enabling high-resolution topographic and fluorescent live cell imaging. This unique tool allows the investigation of mechanotransduction processes, as the cells may be exposed to deliberately controlled mechanical stress while simultaneously facilitating fluorescence imaging and AFM studies. The developed stretching device allows applying reproducible uniaxial strain from physiologically relevant to hyperphysiological levels to cultured cells grown on elastic polydimethylsiloxane (PDMS) membranes. Exemplarily, stretching experiments are shown for transfected squamous cell carcinoma cells (SCC-25) expressing fluorescent labeled cytokeratin, whereby fluorescence imaging and simultaneously performed AFM measurements reveal the cytokeratin (CSK) network. Topographical changes and mechanical characteristics such as elasticity changes were determined via AFM while the cells were exposed to mechanical stress. By applying a cell deformation of approx. 20%, changes in the Young's modulus of the cytoskeletal network due to stretching of the cells were observed. Consequently, integrating a stretching device into the combined atomic force-fluorescence microscope provides a unique tool for dynamically analyzing structural remodeling and mechanical properties in mechanically stressed cells.

A prospective observational study of the efficacy of a novel hydroactive impregnated dressing.

OBJECTIVE: To assess the clinical efficacy, tolerance and acceptance of a novel, hydroactive-impregnated dressing (Hydrotul) in the local treatment of acute and chronic wounds. METHOD: In a prospective observational study 24 centres in France, Belgium, Germany and Austria recruited 74 patients. At each dressing change the investigators evaluated the condition of the wound, perilesional skin and patient-reported pain. Overall, five dressing changes were documented, or until complete healing occurred. The hydroactive properties of the dressing were assessed in laboratory tests by measuring fluid absorption capacity and kinetics. RESULTS: Patients were treated for an average of 17 days. The wound condition improved markedly during the observation period. The wound area covered with fibrinous slough decreased from 29% to 14%, epithelialisation increased from 19% to 54% and 22 wounds were completely healed by the end of the study. The number of patients reporting severe and moderate wound pain decreased from 35% to 19% and the proportion of patients without wound pain doubled from 27% to 60%. In laboratory tests, Hydrotul absorbed two to three times the amount of fluid compared with other impregnated wound dressings and the kinetics of absorption was much faster. CONCLUSION: The novel hydroactive impregnated dressing supports the healing process in patients with acute and chronic wounds and reduces wound pain. The dressing absorbs excess wound exudate while keeping the wound surface moist and protecting perilesional skin.

The beta 1a subunit is essential for the assembly of dihydropyridine-receptor arrays in skeletal muscle.

Homozygous zebrafish of the mutant relaxed (red(ts25)) are paralyzed and die within days after hatching. A significant reduction of intramembrane charge movements and the lack of depolarization-induced but not caffeine-induced Ca(2+) transients suggested a defect in the skeletal muscle dihydropyridine receptor (DHPR). Sequencing of DHPR cDNAs indicated that the alpha(1S) subunit is normal, whereas the beta(1a) subunit harbors a single point mutation resulting in a premature stop. Quantitative RT-PCR revealed that the mutated gene is transcribed, but Western blot analysis and immunocytochemistry demonstrated the complete loss of the beta(1a) protein in mutant muscle. Thus, the immotile zebrafish relaxed is a beta(1a)-null mutant. Interestingly, immunocytochemistry showed correct triad targeting of the alpha(1S) subunit in the absence of beta(1a). Freeze-fracture analysis of the DHPR clusters in relaxed myotubes revealed an approximately 2-fold reduction in cluster size with a normal density of DHPR particles within the clusters. Most importantly, DHPR particles in the junctional membranes of the immotile zebrafish mutant relaxed entirely lacked the normal arrangement in arrays of tetrads. Thus, our data indicate that the lack of the beta(1a) subunit does not prevent triad targeting of the DHPR alpha(1S) subunit but precludes the skeletal muscle-specific arrangement of DHPR particles opposite the ryanodine receptor (RyR1). This defect properly explains the complete deficiency of skeletal muscle excitation-contraction coupling in beta(1)-null model organisms.

Percolation and the colossal magnetoresistance of Eu-based hexaboride.

Upon substituting Ca for Eu in the local-moment ferromagnet EuB6, the Curie temperature T(C) decreases substantially with increasing dilution of the magnetic sublattice and is completely suppressed for x

Merged screening for human immunodeficiency virus Tat and Rev inhibitors.

In addition to "conventional" drug discovery targets used in modern strategies, mainly focusing on proteins, recent insights into gene regulation as a novel drug concept have begun to invite the targeting of biomolecular interactions between proteins and RNA. Because two protein-RNA interactions (Tat and trans-activation-responsive element, Rev and Rev-responsive element) are essential for any productive replication of human immunodeficiency virus, this important human pathogen was used as a model system for our studies. The design of a fluorescence-based high throughput assay, in which both targets were presented in the same vessel, enabled us to simultaneously interrogate two characteristics of a potential inhibitor: potency of interference and selectivity toward each of the interactions. Although related systems have been reported for several DNA binders, an extension into interference with transcription events would open a new dimension of cellular regulation. Here we describe the setup of the screening assay for over 110,000 compounds as well as a primary characterization of identified hits. The assay's characteristics demonstrate that a microwell-based dual screening system for RNA binders may add a powerful tool to modern drug discovery.

Selective aspects of human pathology in high-tone hearing loss of the aging inner ear.

Accompanied with aging, the thresholds for high frequency sounds may elevate and result in a progressive hearing loss described as presbycusis. Based on correlations between audiometric measures of aged patients and histologic findings garnered from postmortem examinations, four types of presbycusis have been characterized: sensory-neural, neural, strial, and conductive [Schuknecht, H.F., Gacek, M.R., 1993. Ann. Otol. Rhinol. Laryngol. 102, 1--16]. Otopathologic changes to the inner ear as a direct function of age, however, remain controversial. The focus of this investigation involves the pathological impact on remaining sensory structures in patients having sensory--neural degeneration. The current study presents seven human temporal bones extracted from patients aged 53--67 years with high-tone hearing loss and with no known history of extraordinary environmental events involving head or noise trauma, acoustic overstimulation, or ototoxicity. In previously published findings of these specimens, all but one temporal bone failed to demonstrate a meaningful correlation between audiometric measurements and loss of functional hair cell populations with secondary retrograde degeneration of nerve fibers. Using the block surface method, electron microscopic micrographs demonstrate ultrastructural changes in the cuticular plate, stereocilia, pillar cells, stria vascularis, and the spiral ligament. In all pathological specimens, the greatest incidence of degeneration was seen at the cuticular plate. Conclusively, our findings present three implications in the aging human cochlea: firstly, audiometric measures that represent a high-tone hearing loss may take various forms with respect to ultrastructural patterns of degeneration and surviving structures; secondly, the incidence of lipofuscin and lysosome granules does not correlate with the degree of hearing loss and; thirdly, as shown only in guinea pigs [Anniko, M., 1988. Scanning Microsc. 2, 1035--1041], high-tone hearing loss can be associated with deformation of the cuticular plate.

Rational optimization of a HIV-1 Tat inhibitor: rapid progress on combinatorial lead structures.

Lead molecules identified by combinatorial chemistry approaches are preferred starting points for straightforward improvements of compound profiles. Structure-guided rationales can be supported and complemented by systematic variations based on the modular nature of the molecules. A peptoidic compound (CGP 64222), previously identified from a sequential unrandomization process, was shown to specifically inhibit the interaction between the HIV-1 trans-activator Tat and its RNA response element TAR. To improve the compound's pharmaceutical attractiveness an approach to reduce both, size and number of charges was pursued. Because this resulted in activity decrease, parallel synthesis with variations on one rationally defined position aimed at the identification of structural determinants was undertaken to regain in vitro activity in biochemical and cellular Tat-TAR interaction assays. As a result CGP74026 was identified, a drastically simplified but highly active Tat antagonist, which is able to block HIV-1 replication even in primary human cells.

An inhibitor of the Tat/TAR RNA interaction that effectively suppresses HIV-1 replication.

One of the first steps in HIV gene expression is the recruitment of Tat protein to the transcription machinery after its binding to the RNA response element TAR. Starting from a pool of 3.2 x 10(6) individual chemical entities, we were able to select a hybrid peptoid/peptide oligomer of 9 residues (CGP64222) that was able to block the formation of the Tat/TAR RNA complex in vitro at nanomolar concentrations. NMR studies demonstrated that the compound binds similarly to polypeptides derived from the Tat protein and induces a conformational change in TAR RNA at the Tat-binding site. In addition, 10-30 microM CGP64222 specifically inhibited Tat activity in a cellular Tat-dependent transactivation assay [fusion-induced gene stimulation (FIGS) assay] and blocked HIV-1 replication in primary human lymphocytes. By contrast, peptides of a comparable size and side-chain composition inhibited cell fusion in the FIGS assay and only partially inhibited HIV-1 replication in primary human lymphocytes. Thus, we have discovered a compound, CGP64222, that specifically inhibits the Tat/TAR RNA interaction, both in vitro and in vivo.

Quantitative evaluation of cochlear neurons and computer-aided three-dimensional reconstruction of spiral ganglion cells in humans with a peripheral loss of nerve fibres.

Quantitative data on human cochlear neuronal elements were collected from various regions in five patients with high-tone hearing loss due to presbycusis and in two patients with normal hearing. The number of nerve fibers was assessed in the spiral lamina and in the inner acoustic meatus together with counts of spiral ganglion cells. The results show that the number of neurons decreased peripherally, i.e., with increasing distance from the central nervous system in patients with high-tone hearing loss due to presbycusis. In two patients with normal hearing no significant difference in the number of neurons was found in the lamina spiralis as compared to the inner acoustic canal. Computer-aided 3-dimensional reconstruction of the human spiral ganglion displayed large bipolar neurons (type I cells), but also large ganglion cells with one missing axon. The results may indicate that a slow retrograde degeneration occurs from the periphery towards the spiral ganglion in presbycusis. Transmission electron microscopy analysis of freshly fixed human spiral ganglions displayed interneural connections. It is speculated whether a trophic supply from other neurons at the level of the spiral ganglion can prevent or delay further degeneration of the central axon.

A new combination of protecting groups and links for encoded synthetic libraries suited for consecutive tests on the solid phase and in solution.

A strategy for high-throughput evaluation of combinatorial compound libraries is reported, which circumvents the necessity to test complex mixtures. The method is based on a new combination of protecting groups, solid-phase linker and tags. The bulk of the library first undergoes a binding assay with the components grafted on beads. A selection of beads carrying strong ligands is stripped from the labelled target and distributed into microvessels. The ligands are cleaved and rinsed into microeluates. Subsequently, a more detailed characterization with a functional assay in solution determines the best performers, which are identified through the peptidic tag left behind on the corresponding mother bead.

Quantitative evaluation of myelinated nerve fibres and hair cells in cochleae of humans with age-related high-tone hearing loss.

In this study 9 human temporal bones from 8 individuals were fixed with Karnovsky solution by perilymphatic perfusion within 1-3 h after death and examined using the "block-surface method' (Spoendlin and Brun, 1974; Spoendlin and Schrott, 1987) and the "micro-dissection method' (Johnsson and Hawkins, 1967). The audiogram of 7 individuals showed high-tone hearing loss, typical for sensory-neural presbycusis. The inner (IHC) and outer hair cells (OHC) and the myelinated nerve fibers in the osseous spiral lamina were counted to correlate audiometric curves with hair-cell and nerve-fibre densities. The "block-surface' method allows accurate hair-cell and myelinated nerve-fibre enumeration with maximal preservation of cochlear structures. The most significant change in the cochlea was not the expected loss of hair cells but an evident loss of nerve fibres in the spiral lamina along the entire length of the cochlea. This loss of nerve fibres was found to be age-related. Reductions up to 30-40% in comparison to normal-hearing middle-aged persons were found in cochleae from persons older than 60 years. In 2 cases only 13% of the fibres remained in some regions of the cochlea. The hair-cell counts showed a reduction of approximately 80% of the OHCs, mainly in the apical parts of the cochlea, and only little differences in the number of IHCs as compared with a group of normal-hearing middle-aged persons. We conclude that neither loss of hair cells nor primary degeneration of nerve fibres alone can fully explain the high-tone loss. Probably injuries of hair cells or neuronal elements at the cellular level can cause threshold elevation.

Integrating cultural diversity theoretical concepts into the educational preparation of the advanced practice nurse: the Cultural Diversity Practice Model.

Educational preparation of the Advanced Practice Nurse (APN) for the challenging role of providing available and accessible health care to ethnic diverse populations requires that cultural diversity be included in the expanded education. The Cultural Diversity Practice Model is offered for that purpose.

Synthesis of an N-3-guanidinopropylglycine (Narg) derivative as a versatile building block for solid-phase peptide and peptoid synthesis.

N-(2,2,5,7,8-Pentamethylchroman-6-sulfonyl)-N'-3- (N-9-fluorenylmethoxycarbonyl-glycinyl)propylguanidine (1) was prepared and utilized as an arginine surrogate (Narg) building block compatible with solid-phase synthesis according to the Fmoc methodology. Narg is potentially useful in the assembly of combinatorial compound libraries or in the preparation of modified peptides. The applicability of this building block was demonstrated by its incorporation into an analogue of Thr-Arg-Ser-Ala-Trp, a pentapeptide for which inhibition of osteoclastic bone resorption was claimed. The modified pentapeptide showed an increased proteolytic stability when compared to the original inhibitor.

Modulation of human prorenin gene expression by antisense oligonucleotides in transfected CHO cells.

Four phosphorothioate oligonucleotides whose sequences are complementary to the 5' untranslated region, the initiation codon or the coding region of human prorenin mRNA, were studied for their capacity to inhibit gene expression in stably transfected Chinese hamster ovary (CHO) cells constitutively producing human prorenin. In contrast to oligomers complementary to the initiation codon and the coding region, antisense oligomers directed towards the 5' untranslated region have no inhibitory effects. The intracellular delivery of a biotinylated phosphorothioate oligonucleotide (biotin-CATCCATGCTTCCCTC) was monitored in immunofluorescence studies. In the absence of a cationic liposome preparation, Lipofectin, the oligomer failed to penetrate the cells. In the presence of Lipofectin, the 35S-labelled oligomer entered the cells and was distributed in proportions of 54% to the nuclei and 35% to the cytosol. The effects of regular oligonucleotides and of 3'-end and/or 5'-end-modified phosphodiester oligonucleotides on prorenin production were tested. Terminal modification by biotinylation at the 5'-end and/or 3'-dodecyl esterification stabilized oligonucleotides towards exonucleases, but did not translate into a significant inhibition of prorenin production and did not improve the intracellular delivery and or stability of the oligomers. We have shown that it is possible to inhibit prorenin production intracellularly using specific antisense oligonucleotides. Stability and delivery are crucial factors in the design of potent and specific compounds directed at prorenin mRNA.