What works best in a general practice specific OSCE for medical students: Mini-CEX or content-related checklists?

Aim: To develop and pilot a General Practice (GPr) OSCE assessing medical students dealing with patient encounters, which are typical for GPr and to compare different measurement instruments (global ratings, content-specific checklists).Methods: A blueprint based on Entrusted Professional Activities was used to develop prototypical OSCE stations. Four stations were tested with voluntary medical students. Students were videotaped and assessed with self-developed content-specific checklists, a global rating for communication skills, and mini-CEX. Results were compared according to students' phases of studies.Results: All three measurements were able to discriminate between clinical and pre-clinical students. Clearest results were achieved by using mini-CEX. Content-specific checklists were not able to differentiate between those groups for the more difficult stations. Inter-station reliability for the global ratings was sufficient for high-stakes exams. Students enjoyed the OSCE-setting simulating GPr consultation hours. They would prefer feedback from GPs after the OSCE and from simulated patients after each encounter.Discussion and conclusion: Although the OSCE was short, results indicate advantages for using a global rating instead of checklists. Further research should include validating these results with a larger group of students and to find the threshold during the phases of education for switching from checklists to global ratings.

Hapten-Specific Single-Cell Selection of Hybridoma Clones by Fluorescence-Activated Cell Sorting for the Generation of Monoclonal Antibodies.

The conventional hybridoma screening and subcloning process is generally considered to be one of the most critical steps in hapten-specific antibody production. It is time-consuming, monoclonality is not guaranteed, and the number of clones that can be screened is limited. Our approach employs a novel hapten-specific labeling technique of hybridoma cells. This allows for fluorescence-activated cell sorting (FACS) and single-cell deposition and thereby eliminates the above-mentioned problems. A two-step staining approach is used to detect antigen specificity and antibody expression: in order to detect antigen specificity, hybridoma cells are incubated with a hapten-horseradish peroxidase conjugate (hapten-HRP), which is subsequently incubated with a fluorophore-labeled polyclonal anti-peroxidase antibody (anti-HRP-Alexa Fluor 488). To characterize the expression of membrane-bound immunoglobulin G (IgG), a fluorophore-labeled anti-mouse IgG antibody (anti-IgG-Alexa Fluor 647) is used. Hundreds of labeled hybridoma cells producing monoclonal antibodies (mAbs) specific for a hapten were rapidly isolated and deposited from a fusion mixture as single-cell clones via FACS. Enzyme-linked immunosorbent assay (ELISA) measurements of the supernatants of the sorted hybridoma clones revealed that all hapten-specific hybridoma clones secrete antibodies against the target. There are significant improvements using this high-throughput technique for the generation of mAbs including increased yield of antibody-producing hybridoma clones, ensured monoclonality of sorted cells, and reduced development times.

Modes and regulation of endocytic membrane retrieval in mouse auditory hair cells.

Synaptic vesicle recycling sustains high rates of neurotransmission at the ribbon-type active zones (AZs) of mouse auditory inner hair cells (IHCs), but its modes and molecular regulation are poorly understood. Electron microscopy indicated the presence of clathrin-mediated endocytosis (CME) and bulk endocytosis. The endocytic proteins dynamin, clathrin, and amphiphysin are expressed and broadly distributed in IHCs. We used confocal vglut1-pHluorin imaging and membrane capacitance (Cm) measurements to study the spatial organization and dynamics of IHC exocytosis and endocytosis. Viral gene transfer expressed vglut1-pHluorin in IHCs and targeted it to synaptic vesicles. The intravesicular pH was ∼6.5, supporting only a modest increase of vglut1-pHluorin fluorescence during exocytosis and pH neutralization. Ca(2+) influx triggered an exocytic increase of vglut1-pHluorin fluorescence at the AZs, around which it remained for several seconds. The endocytic Cm decline proceeded with constant rate (linear component) after exocytosis of the readily releasable pool (RRP). When exocytosis exceeded three to four RRP equivalents, IHCs additionally recruited a faster Cm decline (exponential component) that increased with the amount of preceding exocytosis and likely reflects bulk endocytosis. The dynamin inhibitor Dyngo-4a and the clathrin blocker pitstop 2 selectively impaired the linear component of endocytic Cm decline. A missense mutation of dynamin 1 (fitful) inhibited endocytosis to a similar extent as Dyngo-4a. We propose that IHCs use dynamin-dependent endocytosis via CME to support vesicle cycling during mild stimulation but recruit bulk endocytosis to balance massive exocytosis.

Bright or dark immune complexes of anti-TAMRA antibodies for adapted fluorescence-based bioanalysis.

Fluorescence labels, for example fluorescein or rhodamin derivatives, are widely used in bioanalysis applications including lateral-flow assays, PCR, and fluorescence microscopy. Depending on the layout of the particular application, fluorescence quenching or enhancement may be desired as the detection principle. Especially for multiplexed applications or high-brightness requirements, a tunable fluorescence probe can be beneficial. The alterations in the photophysics of rhodamine derivatives upon binding to two different anti-TAMRA antibodies were investigated by absorption and fluorescence-spectroscopy techniques, especially determining the fluorescence decay time and steady-state and time-resolved fluorescence anisotropy. Two monoclonal anti-TAMRA antibodies were generated by the hybridoma technique. Although surface-plasmon-resonance measurements clearly proved the high affinity of both antibodies towards 5-TAMRA, the observed effects on the fluorescence of rhodamine derivatives were very different. Depending on the anti-TAMRA antibody either a strong fluorescence quenching (G71-DC7) or a distinct fluorescence enhancement (G71-BE11) upon formation of the immune complex was observed. Additional rhodamine derivatives were used to gain further information on the binding interaction. The data reveal that such haptens as 5-TAMRA could generate different paratopes with equal binding affinities but different binding interactions, which provide the opportunity to adapt bioanalysis methods including immunoassays for optimized detection principles for the same hapten depending on the specific requirements.

A missense mutation in a highly conserved alternate exon of dynamin-1 causes epilepsy in fitful mice.

Dynamin-1 (Dnm1) encodes a large multimeric GTPase necessary for activity-dependent membrane recycling in neurons, including synaptic vesicle endocytosis. Mice heterozygous for a novel spontaneous Dnm1 mutation--fitful--experience recurrent seizures, and homozygotes have more debilitating, often lethal seizures in addition to severe ataxia and neurosensory deficits. Fitful is a missense mutation in an exon that defines the DNM1a isoform, leaving intact the alternatively spliced exon that encodes DNM1b. The expression of the corresponding alternate transcripts is developmentally regulated, with DNM1b expression highest during early neuronal development and DNM1a expression increasing postnatally with synaptic maturation. Mutant DNM1a does not efficiently self-assemble into higher order complexes known to be necessary for proper dynamin function, and it also interferes with endocytic recycling in cell culture. In mice, the mutation results in defective synaptic transmission characterized by a slower recovery from depression after trains of stimulation. The DNM1a and DNM1b isoform pair is highly conserved in vertebrate evolution, whereas invertebrates have only one isoform. We speculate that the emergence of more specialized forms of DNM1 may be important in organisms with complex neuronal function.

Antibody mediated fluorescence enhancement of nucleoside analogue 1,3-diaza-2-oxophenoxazine (tC°).

We report on the generation and analytical application of the monoclonal antibody G93-ED2 raised against the tricyclic fluorescent nucleoside analogue 1,3-diaza-2-oxophenoxazine (tC°). G93-ED2 is specifically binding this deoxycytidine analogue and was found to raise its fluorescence intensity by a factor of 5. This unique feature makes it a valuable tool in fluorescence dependent immunoassays. G93-ED2 was successfully applied in a homogeneous fluorescence quenching immunoassay (DNA-Q) for the sequence specific determination of DNA.

Specific DNA detection using antibody mediated fluorescence quenching.

First homogenous immunoassay for sequence-specific nucleic acid detection is developed. The assay bases on our finding that a fluorophore inserted into a DNA probe instead of one of the internal nucleotides may get protected from fluorescence quenching caused by an anti-fluorophore antibody, if the probe is hybridized with the target sequence. This ensures a positive signal in the antibody presence. The assay enables quantitative detection and may have potential for development of homogenous high-throughput platforms.

Secretory leukocyte protease inhibitor (SLPI) might contaminate murine monoclonal antibodies after purification on protein G.

The large scale production of a monoclonal anti-progesterone antibody in serum free medium followed by affinity chromatography on protein G lead to a contamination of the antibody sample with a protein of about 14 kDa. This protein was identified by mass spectrometry as secretory leukocyte protease inhibitor (SLPI). This SLPI contamination lead to a failure of the fiber-optic based competitive fluorescence assay to detect progesterone in milk. Purification of the monoclonal antibody using protein A columns circumvented this problem.