There has been rapid growth in the range of models available for teaching veterinary clinical skills. To promote further uptake, particularly in lower-income settings and for students to practice at home, factors to consider include cost, availability of materials and ease of construction of the model. Two models were developed to teach suturing: a silicon skin pad, and a tea towel (with a check pattern) folded and stapled to represent an incision. The models were reviewed by seven veterinarians, all of whom considered both suitable for teaching, with silicon rated as more realistic. The learning outcome of each model was compared after students trained to perform a simple interrupted suture. Thirty-two second-year veterinary students with no prior suturing experience were randomly assigned to three training groups: silicon skin pad or tea towel (both self-directed with an instruction booklet), or watching a video. Following training, all students undertook an Objective Structured Clinical Examination (OSCE), placing a simple interrupted suture in piglet cadaver skin. The OSCE pass rates of the three groups were silicon skin pad, 10/11; tea towel, 9/10; and video, 1/11. There was no significant difference between the model groups, but the model groups were significantly different from the video group ( p < .017). In conclusion, the tea towel was as effective as the silicon skin pad, but it was cheaper, simpler to make, and the materials were more readily available. In addition, both models were used effectively with an instruction booklet illustrating the value of self-directed learning to complement taught classes.
Alzheimers-disease (AD) is an age-related neurodegenerative condition and the most common type of dementia, characterised by pathological accumulation of extracellular plaques and intracellular neurofibrillary tangles that mainly consist of amyloid-beta (A beta) and hyperphosphorylated tau aggregates, respectively. Previous studies in mouse models with a targeted knock-out of the microtubule-associated protein tau (Mapt) gene demonstrated that A beta-driven toxicity is tau-dependent. However, human cellular models with chronic tau lowering remain unstudied. In this study, we generated stable tau-depleted human induced pluripotent stem cell (iPSC) isogenic panels from two healthy individuals using CRISPR-Cas9 technology. We then differentiated these iPSCs into cortical neurons in vitro in co-culture with primary rat cortical astrocytes before conducting electrophysiological and imaging experiments for a wide range of disease-relevant phenotypes. Both AD brain derived and recombinant A beta were used in this study to elicit toxic responses from the iPSC-derived cortical neurons. We showed that tau depletion in human iPSC-derived cortical neurons caused considerable reductions in neuronal activity without affecting synaptic density. We also observed neurite outgrowth impairments in two of the tau-depleted lines used. We found axonal transport of mitochondria, mitochondrial function, and cortical neuron differentiation propensity remained unaffected regardless of tau expression levels. Finally, tau depletion protected neurons from adverse effects mitigating the impact of exogenous Aβ-induced hyperactivity, deficits in retrograde axonal transport of mitochondria, and neurodegeneration. Our study established stable human iPSC isogenic panels with chronic tau depletion from two healthy individuals. Cortical neurons derived from these iPSC lines showed that tau is essential in A beta-driven hyperactivity, axonal transport deficits, and neurodegeneration, consistent with studies conducted in Mapt-/- mouse models. These findings highlight the protective effects of chronic tau lowering strategies in AD pathogenesis and reinforce the potential in clinical settings. The tau-depleted human iPSC models can now be applied at scale to investigate the involvement of tau in disease-relevant pathways and cell types.
The molecular mechanisms of reduced frataxin (FXN) expression in Friedreich’s ataxia (FRDA) are linked to epigenetic modification of the FXN locus caused by the disease-associated GAA expansion. Here, we identify that SUV4-20 histone methyltransferases, specifically SUV4-20 H1, play an important role in the regulation of FXN expression and represent a novel therapeutic target. Using a human FXN-GAA-Luciferase repeat expansion genomic DNA reporter model of FRDA, we screened the Structural Genomics Consortium epigenetic probe collection. We found that pharmacological inhibition of the SUV4-20 methyltransferases by the tool compound A-196 increased the expression of FXN by approximately 1.5-fold in the reporter cell line. In several FRDA cell lines and patient-derived primary peripheral blood mononuclear cells A-196 increased FXN expression by up to 2-fold, an effect not seen in wild-type cells. SUV4-20 inhibition was accompanied by a reduction in H4K20me2 and H4K20me3 and an increase in H4K20me1, but only modest (1.4–7.8%) perturbation in genome-wide expression was observed. Finally, based on the structural activity relationship and crystal structure of A-196, novel small molecule A-196 analogues were synthesized and shown to give a 20-fold increase in potency for increasing FXN expression. Overall, our results suggest that histone methylation is important in the regulation of FXN expression, and highlight SUV4-20 H1 as a potential novel therapeutic target for FRDA.
This protocol describes the capture of eGFP-L10a-tagged ribosomes and mRNA from DAT-expressing cells in mouse ventral midbrain.
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