Notes

Digital spectra of ytterbium fluoride from relativistic electronic digital composition computations.
Parkinson's disease is a neurodegenerative disorder characterized by accumulation of misfolded α-synuclein within the central nervous system (CNS). Retinal manifestations have been widely described as a prodromal symptom; however, we have a limited understanding of the retinal pathology associated with Parkinson's disease. The strong similarities between the retina and the brain and the accessibility of the retina has potentiated studies to investigate retinal pathology in an effort to identify biomarkers for early detection, as well as for monitoring the progression of disease and efficacy of therapies as they become available. selleck Here, we discuss a study conducted using a transgenic mouse model of Parkinson's disease (TgM83, expressing human α-synuclein containing the familial PD-associated A53T mutation) to demonstrate the effect of the A53T α-synuclein mutation on the retina. Additionally, we show that "seeding" with brain homogenates from clinically ill TgM83 mice accelerates the accumulation of retinal α-synuclein. The work described in this chapter provides insight into retinal changes associated with Parkinson's disease and identifies retinal indicators of Parkinson's disease pathogenesis that could serve as potential biomarkers for early detection.The mammalian hippocampus shows a remarkable capacity for continued neurogenesis throughout life. Newborn neurons, generated by the radial neural stem cells (NSCs), are important for learning and memory as well as mood control. During aging, the number and responses of NSCs to neurogenic stimuli diminish, leading to decreased neurogenesis and age-associated cognitive decline and psychiatric disorders. Thus, adult hippocampal neurogenesis has been the subject of intense investigation, generating both excitement and controversy. Identifying the core molecular machinery responsible for NSC preservation is of fundamental importance if we are to use neurogenesis to halt or reverse hippocampal age-related pathology. Here, we briefly overview the most frequently used mouse models to study hippocampal neurogenesis and then focus on a unique mouse model that allows NSC-specific studies based on their unique expression of lunatic fringe (Lfng). The Lfng-eGFP and Lfng(BAC)-CreERT2;RCL-tdT transgenic mice provide us with an excellent tool to resolve long-standing questions regarding the properties of NSCs, such as their specific molecular composition, potency, and plasticity, in isolation from any other cell in the hippocampal neurogenic niche.Like bacterial and cytoplasmic ribosomes, mitoribosomes are large ribonucleoprotein complexes with molecular weights in the range of several million Daltons. Traditionally, studying the assembly of such high molecular weight complexes is done using ultracentrifugation through linear density gradients, which remains the method of choice due to its versatility and superior resolving power in the high molecular weight range. Here, we present a protocol for the analysis of mitoribosomal assembly in heart mitochondrial extracts using linear density sucrose gradients that we have previously employed to characterize the essential role of different mitochondrial proteins in mitoribosomal biogenesis. This protocol details in a stepwise manner a typical mitoribosomal assembly analysis starting with isolation of mitochondria, preparation and ultracentrifugation of the gradients, fractionation and ending with SDS-PAGE, and immunoblotting of the gradient fractions. Even though we provide an example with heart mitochondria, this protocol can be directly applied to virtually all mouse tissues, as well as cultured cells, with little to no modifications.Reporter mice transgenically expressing the bacterial (E. coli) lacZ gene encoding β-galactosidase (β-gal, EC 3.2.1.23) are a versatile and extensively used tool to study gene expression and cell lineage patterns. Enzymatic activity of the β-gal reporter can be effectively visualized at cellular resolution either histochemically using 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) or by immunofluorescent detection using a β-gal-specific antibody. Here, we summarize protocols for the localization of β-gal expressing cells in whole embryos or organs as well as in histological tissue sections of lacZ reporter mice and discuss their limitations and common pitfalls.Recent development of Easi-CRISPR (Efficient additions with ssDNA inserts-CRISPR) that utilizes long single-stranded DNA (lssDNA) of 0.2-2 kbases in length as donor templates to insert large segments of novel DNA sequences or to replace endogenous genes at precise locations in the genome has enabled CRISPR-assisted genome editing to make strides toward a more simple and rapid workflow. By leveraging the notion that short single-stranded DNA oligo ( less then 200 bases) serves as efficient donor in mouse zygotes for facilitating HDR-mediated genome editing, Easi-CRISPR expands to use lssDNA as the donor which accelerates the timeline to as little as 2 months for creating most types of genetically engineered mouse models (F0). Our lab (CGERC) has adopted Easi-CRISPR for multiple loci to generate mouse models over the past three plus years since its introduction. Here, we use two genes as examples to illustrate a step-by-step protocol for generating two commonly used models, including a knock-in (insertion of a ed to be more straightforward while applicable to generate mouse models in broader scope. (Certain figures and text passages presented in this chapter are reproduced from Shola et al. (The CRISPR J 3(2)109-122, 2020), published by Mary Ann Libert, Inc).
A multinational post-authorization safety study assessed cardiovascular safety in initiators of prucalopride for chronic constipation compared with a matched cohort of polyethylene glycol 3350 initiators. The primary safety outcome was major adverse cardiovascular events (MACE), a composite of hospitalization for acute myocardial infarction, stroke, or in-hospital cardiovascular death. We report the validation process for MACE endpoints in United Kingdom (UK) data sources Clinical Practice Research Datalink (CPRD GOLD), The Health Improvement Network (THIN), and the Information Services Division (ISD) Scotland.

Modified electronic algorithms from prior research identified potential MACE cases. Validation followed a common protocol, adapted for each database, with all information anonymized (1) direct confirmation via linkage to hospital records (CPRD GOLD); (2) requests for additional clinical information through questionnaires (CPRD GOLD), free-text (THIN), or abstraction of hospital records (ISD); (3) manual review of electronic records of potential events retrieved by the algorithm (CPRD GOLD/THIN); and (4) event adjudication by three clinicians, blinded to exposure, for all remaining events.
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