Battle of the sections: Student outcomes and course feedback support combined prosection and dissection laboratory formats to maximize student success.

Gross anatomy laboratories frequently utilize dissection or prosection formats within medical curricula. Practical examination scores are consistent across the formats, yet these examinations assessed larger anatomical structures. In contrast, a single report noted improved scores when prosection was used in the hand and foot regions, areas that are more difficult to dissect. The incorporation of prosected donors within "Head and Neck" laboratories provided an opportunity to further characterize the impact of prosection in a structurally complex area. Retrospective analysis of 21 Head and Neck practical examination questions was completed to compare scores among cohorts that utilized dissection exclusively or incorporated prosection. Mean scores of practical examination questions were significantly higher in the prosection cohort (84.27% ± 12.69) as compared with the dissection cohort (75.59% ± 12.27) (p < 0.001). Of the 12 questions that performed better in the prosection cohort (88.42% ± 8.21), 10 items mapped to deeper anatomical regions. By comparison, eight of nine questions in the dissection cohort outperformed (88.44% ± 3.34) the prosection cohort (71.74% ± 18.11), and mapped to anatomically superficial regions. Despite the mean score increase with positional location of the questions, this effect was not statically significant across cohorts (p = 1.000), suggesting that structure accessibility in anatomically complex regions impacts performance. Student feedback cited structure preservation (71.5%) and time savings (55.8%) as advantages to prosection; however, dissection was the perceived superior and preferred laboratory format (88.6%). These data support combined prosection and dissection formats for improving student recognition of deeply positioned structures and maximizing student success.

Innovations Through Virtual Reality Simulation.

The need to augment standardized learner outcomes related to performance and clinical competency led to creating curricular elements that would provide instruction and assessment from multiple perspectives. The COVID-19 pandemic brought about needs for re-imagination of standardized simulated clinical experiences given the need for increased distance-learning and asynchronous formats. Our goal was to identify activities that would engage pre-clinical simulation through asynchronous virtual reality (VR) case scenarios. The intent was to provide additional resources whereby competencies could be more defined through performance metrics and standardized assessments additive to our established simulation-based curriculum throughout all curricular phases. Student reflection and metacognition identified gaps to guide future performance improvement through the VR activities. Learner outcomes encompassing history-taking, physical assessment, evidence-based clinical reasoning, and medical decision-making guided the instructional objectives. The composite data showed progressive improvements over five scenarios delivered in our second-year clinical medicine curriculum.

The orphan nuclear receptor TR4 is a vitamin A-activated nuclear receptor.

Testicular receptors 2 and 4 (TR2/4) constitute a subgroup of orphan nuclear receptors that play important roles in spermatogenesis, lipid and lipoprotein regulation, and the development of the central nervous system. Currently, little is known about the structural features and the ligand regulation of these receptors. Here we report the crystal structure of the ligand-free TR4 ligand binding domain, which reveals an autorepressed conformation. The ligand binding pocket of TR4 is filled by the C-terminal half of helix 10, and the cofactor binding site is occupied by the AF-2 helix, thus preventing ligand-independent activation of the receptor. However, TR4 exhibits constitutive transcriptional activity on multiple promoters, which can be further potentiated by nuclear receptor coactivators. Mutations designed to disrupt cofactor binding, dimerization, or ligand binding substantially reduce the transcriptional activity of this receptor. Importantly, both retinol and retinoic acid are able to promote TR4 to recruit coactivators and to activate a TR4-regulated reporter. These findings demonstrate that TR4 is a ligand-regulated nuclear receptor and suggest that retinoids might have a much wider regulatory role via activation of orphan receptors such as TR4.

Identification of COUP-TFII orphan nuclear receptor as a retinoic acid-activated receptor.

The chicken ovalbumin upstream promoter-transcription factors (COUP-TFI and II) make up the most conserved subfamily of nuclear receptors that play key roles in angiogenesis, neuronal development, organogenesis, cell fate determination, and metabolic homeostasis. Although the biological functions of COUP-TFs have been studied extensively, little is known of their structural features or aspects of ligand regulation. Here we report the ligand-free 1.48 A crystal structure of the human COUP-TFII ligand-binding domain. The structure reveals an autorepressed conformation of the receptor, where helix alpha10 is bent into the ligand-binding pocket and the activation function-2 helix is folded into the cofactor binding site, thus preventing the recruitment of coactivators. In contrast, in multiple cell lines, COUP-TFII exhibits constitutive transcriptional activity, which can be further potentiated by nuclear receptor coactivators. Mutations designed to disrupt cofactor binding, dimerization, and ligand binding, substantially reduce the COUP-TFII transcriptional activity. Importantly, retinoid acids are able to promote COUP-TFII to recruit coactivators and activate a COUP-TF reporter construct. Although the concentration needed is higher than the physiological levels of retinoic acids, these findings demonstrate that COUP-TFII is a ligand-regulated nuclear receptor, in which ligands activate the receptor by releasing it from the autorepressed conformation.

The role of multiple hydrogen-bonding groups in specific alcohol binding sites in proteins: insights from structural studies of LUSH.

It is now generally accepted that many of the physiological effects of alcohol consumption are a direct result of binding to specific sites in neuronal proteins such as ion channels or other components of neuronal signaling cascades. Binding to these targets generally occurs in water-filled pockets and leads to alterations in protein structure and dynamics. However, the precise interactions required to confer alcohol sensitivity to a particular protein remain undefined. Using information from the previously solved crystal structures of the Drosophila melanogaster protein LUSH in complexes with short-chain alcohols, we have designed and tested the effects of specific amino acid substitutions on alcohol binding. The effects of these substitutions, specifically S52A, T57S, and T57A, were examined using a combination of molecular dynamics, X-ray crystallography, fluorescence spectroscopy, and thermal unfolding. These studies reveal that the binding of ethanol is highly sensitive to small changes in the composition of the alcohol binding site. We find that T57 is the most critical residue for binding alcohols; the T57A substitution completely abolishes binding, while the T57S substitution differentially affects ethanol binding compared to longer-chain alcohols. The additional requirement for a potential hydrogen-bond acceptor at position 52 suggests that both the presence of multiple hydrogen-bonding groups and the identity of the hydrogen-bonding residues are critical for defining an ethanol binding site. These results provide new insights into the detailed chemistry of alcohol's interactions with proteins.

Effect of n-alcohols on the structure and stability of the Drosophila odorant binding protein LUSH.

LUSH is an odorant binding protein expressed in the olfactory organs of Drosophila melanogaster that is required for the detection of alcohol in adult flies. Here we demonstrate that, in the absence of ligand, in vitro LUSH exists in a partial molten globule state. The presence of short-chain n-alcohols at pharmacologically relevant concentrations less than 50 mM shifts the conformational equilibrium to a more compact state that exhibits reduced binding of the fluorescent dye 1-anilino-8-naphthalenesulfonic acid. Equilibrium unfolding studies of LUSH-alcohol complexes reveal that, for a series of short-chain n-alcohols, each methylene group can contribute approximately 1 K cal mol(-1) to the overall stability of the protein-alcohol complex. Using NMR spectroscopy, we have identified the regions of LUSH that show increased conformational stability on binding alcohols. These residues primarily line the alcohol-binding pocket. The results presented here provide a direct measure of the degree of stability that alcohol imparts on LUSH. These observations may represent a model for how ethanol can stabilize alternative protein conformations in alcohol-sensitive human proteins and ultimately lead to the observed changes in higher order function throughout the central nervous system.

Structure of a specific alcohol-binding site defined by the odorant binding protein LUSH from Drosophila melanogaster.

We have solved the high-resolution crystal structures of the Drosophila melanogaster alcohol-binding protein LUSH in complex with a series of short-chain n-alcohols. LUSH is the first known nonenzyme protein with a defined in vivo alcohol-binding function. The structure of LUSH reveals a set of molecular interactions that define a specific alcohol-binding site. A group of amino acids, Thr57, Ser52 and Thr48, form a network of concerted hydrogen bonds between the protein and the alcohol that provides a structural motif to increase alcohol-binding affinity at this site. This motif seems to be conserved in a number of mammalian ligand-gated ion channels that are directly implicated in the pharmacological effects of alcohol. Further, these sequences are found in regions of ion channels that are known to confer alcohol sensitivity. We suggest that the alcohol-binding site in LUSH represents a general model for alcohol-binding sites in proteins.