Changing assessment practice in engineering: how can understanding lecturer perspectives help?

Assessment in engineering disciplines is typically oriented to demonstrating competence in specific tasks. Even where assessments are intended to have a formative component, little priority may be given to feedback. Engineering departments are often criticized, by their students and by external quality reviewers, for paying insufficient attention to formative assessment. The e3an project set out to build a question bank of peer-reviewed questions for use within electrical and electronic engineering. As a part of this process, a number of engineers from disparate institutions were required to work together in teams, designing a range of assessments for their subject specialisms. The project team observed that lecturers were especially keen to develop formative assessment but that their understanding of what might be required varied considerably. This paper describes the various ways in which the processes of the project have engaged lecturers in actively identifying and developing their conceptions of teaching, learning and assessment in their subject. It reports on an interview study that was conducted with a selection of participants. It is concluded that lecturers' reflections on and understanding of assessment are closely related to the nature of the subject domain and that it is essential when attempting to improve assessment practice to start from the perspective of lecturers in the discipline

Inhibition or Stimulation of Autophagy Affects Early Formation of Lipofuscin-Like Autofluorescence in the Retinal Pigment Epithelium Cell

The accumulation of lipofuscin in the retinal pigment epithelium (RPE) is dependent on the effectiveness of photoreceptor outer segment material degradation. This study explored the role of autophagy in the fate of RPE lipofuscin degradation. After seven days of feeding with either native or modified rod outer segments, ARPE-19 cells were treated with enhancers or inhibitors of autophagy and the autofluorescence was detected by fluorescence-activated cell sorting. Supplementation with different types of rod outer segments increased lipofuscin-like autofluorescence (LLAF) after the inhibition of autophagy, while the induction of autophagy (e.g., application of rapamycin) decreased LLAF. The effects of autophagy induction were further confirmed by Western blotting, which showed the conversion of LC3-I to LC3-II, and by immunofluorescence microscopy, which detected the lysosomal activity of the autophagy inducers. We also monitored LLAF after the application of several autophagy inhibitors by RNA-interference and confocal microscopy. The results showed that, in general, the inhibition of the autophagy-related proteins resulted in an increase in LLAF when cells were fed with rod outer segments, which further confirms the effect of autophagy in the fate of RPE lipofuscin degradation. These results emphasize the complex role of autophagy in modulating RPE autofluorescence and confirm the possibility of the pharmacological clearance of RPE lipofuscin by small molecules

A Splice Variant of Bardet-Biedl Syndrome 5 (BBS5) Protein that Is Selectively Expressed in Retina.

PURPOSE:Bardet-Biedl syndrome is a complex ciliopathy that usually manifests with some form of retinal degeneration, amongst other ciliary-related deficiencies. One of the genetic causes of this syndrome results from a defect in Bardet-Biedl Syndrome 5 (BBS5) protein. BBS5 is one component of the BBSome, a complex of proteins that regulates the protein composition in cilia. In this study, we identify a smaller molecular mass form of BBS5 as a variant formed by alternative splicing and show that expression of this splice variant is restricted to the retina. METHODS:Reverse transcription PCR from RNA was used to isolate and identify potential alternative transcripts of Bbs5. A peptide unique to the C-terminus of the BBS5 splice variant was synthesized and used to prepare antibodies that selectively recognized the BBS5 splice variant. These antibodies were used on immunoblots of tissue extracts to determine the extent of expression of the alternative transcript and on tissue slices to determine the localization of expressed protein. Pull-down of fluorescently labeled arrestin1 by immunoprecipitation of the BBS5 splice variant was performed to assess functional interaction between the two proteins. RESULTS:PCR from mouse retinal cDNA using Bbs5-specific primers amplified a unique cDNA that was shown to be a splice variant of BBS5 resulting from the use of cryptic splicing sites in Intron 7. The resulting transcript codes for a truncated form of the BBS5 protein with a unique 24 amino acid C-terminus, and predicted 26.5 kD molecular mass. PCR screening of RNA isolated from various ciliated tissues and immunoblots of protein extracts from these same tissues showed that this splice variant was expressed in retina, but not brain, heart, kidney, or testes. Quantitative PCR showed that the splice variant transcript is 8.9-fold (+/- 1.1-fold) less abundant than the full-length transcript. In the retina, the splice variant of BBS5 appears to be most abundant in the connecting cilium of photoreceptors, where BBS5 is also localized. Like BBS5, the binding of BBS5L to arrestin1 can be modulated by phosphorylation through protein kinase C. CONCLUSIONS:In this study we have identified a novel splice variant of BBS5 that appears to be expressed only in the retina. The BBS5 splice variant is expressed at approximately 10% of full-length BBS5 level. No unique functional or localization properties could be identified for the splice variant compared to BBS5

Formation of lipofuscin-like material in the RPE Cell by different components of rod outer segments

The mechanisms that control the natural rate of lipofuscin accumulation in the retinal pigment epithelial (RPE) cell and its stability over time are not well understood. Similarly, the contributions of retinoids, phospholipids and oxidation to the rate of accumulation of lipofuscin are uncertain. The experiments in this study were conducted to explore the individual contribution of rod outer segments (ROS) components to lipofuscin formation and its accumulation and stability over time. During the period of 14 days incubation of ROS, lipofuscin-like autofluorescence (LLAF) determined at two wavelengths (530 and 585 nm) by fluorescence-activated cell sorting (FACS) was measured from RPE cells. The autofluorescence increased in an exponential manner with a strong linear component between days 1 and 7. The magnitude of the increase was larger in cells incubated with 4-hydroxynonenal (HNE-ROS) compared with cells incubated with either bleached or unbleached ROS, but with a different spectral profile. A small (10-15%) decrease in LLAF was observed after stopping the ROS feeding for 14 days. The phagocytosis rate of HNE-ROS was higher than that of either bleached or unbleached ROS during the first 24 h of supplementation. Among the different ROS components, the increase of LLAF was highest in cells incubated with all-trans-retinal. Surprisingly, incubation with 11-cis-retinal and 9-cis-retinal also resulted in strong LLAF increase, comparable to the increase induced by all-trans-retinal. Supplementation with liposomes containing phosphatidylethanolamine (22: 6-PE) and phosphatidylcholine (18:1-PC) also increased LLAF, while incubation with opsin had little effect. Cells incubated with retinoids demonstrated strong dose-dependence in LLAF increase, and the magnitude of the increase was 2-3 times higher at 585 nm compared to 530 nm, while cells incubated with liposomes showed little dose-dependence and similar increase at both wavelengths. Very little difference in LLAF was noted between cells incubated with either unbleached or bleached ROS under any conditions. In summary, results from this study suggest that supplementation with various ROS components can lead to an increase in LLAF, although the autofluorescence generated by the different classes of components has distinct spectral profiles, where the autofluorescence induced by retinoids results in a spectral profile closest to the one observed from human lipofuscin. Future fluorescence characterization of LLAF in vitro would benefit from an analysis of multiple wavelengths to better match the spectral characteristics of lipofuscin in vivo

The mouse Bbs5 gene and alternative transcripts.

<p>(A) Structure of the murine <i>Bbs5</i> gene; locations for sense (S) and anti-sense (A) primers used to test for alternative transcripts are indicated above the gene structure, along with oligo(dT) and 5’RACE primers (5’R). (B) PCR amplification of reverse-transcribed murine retinal poly(A)⁺ RNA with S2 and A5 primers separated by agarose electrophoresis; arrowhead indicates a DNA product larger than the expected 216 bp product. (C) DNA sequence of intron 7 of <i>Bbs5</i> with the alternatively spliced region indicated in red (potential splice donor/acceptor sites are bold faced). (D) Sequence chromatogram of the alternative Bbs5 transcript (BBS5L) with the beginning of the cryptic exon 7a (arrow) and stop codon (asterisk) indicated. (E) Gene structure of exons 7, 7a, and 8 showing the two splicing patterns detected for the Bbs5 transcript (the location of the stop codon in Exon 7a is indicated with an asterisk).</p

Characterization of a polyclonal antibody specific for BBS5L.

<p>(A) Conceptual translation of the alternative Bbs5 transcript (Bbs5L) in comparison with Bbs5 (the unique C-terminus is colored in red). (B) Immunoblots of murine retinal extracts and heterologously expressed BBS5L (rBBS5L) and BBS5 (rBBS5) were probed with either anti-BBS5#7–15 monoclonal antibody (left half) or with anti-BBS5L polyclonal serum (right half); arrowhead indicates BBS5 or rBBS5, arrow indicates BBS5L or rBBS5L. (C) Immunoblot of aqueous-soluble extracts prepared from the indicated mouse tissue probed with anti-BBS5L polyclonal serum identifies anti-BBS5L immunoreactivity only in the retinal extract (upper panel; arrow); an antibody that recognizes both the BBS5 and BBS5L reacts with BBS5 in all extracts, and BBS5L only in retinal extract (middle panel); inset below the blot shows a replicate blot of the tissue extracts probed with anti-beta-tubulin as a loading control.</p