RESUMO
The degree to which non-human animals can be used to model Alzheimer's disease is a contentious issue, particularly as there is still widespread disagreement regarding the pathogenesis of this neurodegenerative dementia. The currently popular transgenic models are based on artificial expression of genes mutated in early onset forms of familial Alzheimer's disease (EOfAD). Uncertainty regarding the veracity of these models led us to focus on heterozygous, single mutations of endogenous genes (knock-in models) as these most closely resemble the genetic state of humans with EOfAD, and so incorporate the fewest assumptions regarding pathological mechanism. We have generated a number of lines of zebrafish bearing EOfAD-like and non-EOfAD-like mutations in genes equivalent to human PSEN1, PSEN2, and SORL1. To analyze the young adult brain transcriptomes of these mutants, we exploited the ability of zebrafish to produce very large families of simultaneous siblings composed of a variety of genotypes and raised in a uniform environment. This "intra-family" analysis strategy greatly reduced genetic and environmental "noise" thereby allowing detection of subtle changes in gene sets after bulk RNA sequencing of entire brains. Changes to oxidative phosphorylation were predicted for all EOfAD-like mutations in the three genes studied. Here we describe some of the analytical lessons learned in our program combining zebrafish genome editing with transcriptomics to understand the molecular pathologies of neurodegenerative disease.
RESUMO
DNA size markers (also known as 'molecular weight markers' or 'DNA ladders') are an essential tool when using gel electrophoresis to identify and purify nucleic acids. However, the cost of these DNA ladders is not insignificant and, over time, impinges on the funds available for research and training in molecular biology. Here, we describe a method for the generation of 'pHAPE', a plasmid from which a variety of DNA ladders can be generated via simple restriction enzyme digestions. The pHAPE plasmid can be generated by mutagenesis of the commonly used pBluescript II SK+ phagemid followed by insertion of a 7141 bp sequence (comprised of three smaller, synthetic fragments). Our use of pHAPE allows us some small relief from the ever-rising costs of performing molecular biology experiments ('Don't worry, pHAPE').
RESUMO
OBJECTIVE: To determine whether patients with moderate to severe acne who were treated with isotretinoin experienced significant increases in depressive symptoms over a 3- to 4-month period compared with patients who received conservative acne therapy. DESIGN: Cohort study. SETTING: Hospital-affiliated and community-based clinics in St Louis, Mo. PARTICIPANTS: One hundred thirty-two subjects aged 12 to 19 years with moderate to severe acne. MAIN OUTCOME MEASURES: Depressive symptoms were assessed using the Center for Epidemiological Studies Depression Scale (CES-D), a standardized self-reported instrument. Mean CES-D scores were compared between treatment groups, as were the prevalence and incidence of scores suggestive of clinically significant depression (CES-D score >16). RESULTS: A total of 101 subjects completed the study. At follow-up, CES-D scores (adjusted for baseline CES-D score and sex of patient) suggestive of clinically significant depression were no more prevalent in the isotretinoin group than in the conservative therapy group. Similarly, the incidence (new onset) of depressive symptoms suggestive of clinical significance also was not significantly different between the treatment groups. CONCLUSIONS: The use of isotretinoin in the treatment of moderate-severe acne in adolescents did not increase symptoms of depression. On the contrary, treatment of acne either with conservative therapy or with isotretinoin was associated with a decrease in depressive symptoms.
