CRISPR-Cas9 Knock-In of T513M and G41S Mutations in the Murine β-Galactosyl-Ceramidase Gene Re-capitulates Early-Onset and Adult-Onset Forms of Krabbe Disease.

Krabbe Disease (KD) is a lysosomal storage disorder characterized by the genetic deficiency of the lysosomal enzyme β-galactosyl-ceramidase (GALC). Deficit or a reduction in the activity of the GALC enzyme has been correlated with the progressive accumulation of the sphingolipid metabolite psychosine, which leads to local disruption in lipid raft architecture, diffuse demyelination, astrogliosis, and globoid cell formation. The mouse, the most used animal model, has a nonsense mutation, which limits the study of how different mutations impact the processing and activity of GALC enzyme.

The Pathogenic Sphingolipid Psychosine is Secreted in Extracellular Vesicles in the Brain of a Mouse Model of Krabbe Disease.

Psychosine exerts most of its toxic effects by altering membrane dynamics with increased shedding of extracellular vesicles (EVs). In this study, we discovered that a fraction of psychosine produced in the brain of the Twitcher mouse, a model for Krabbe disease, is associated with secreted EVs. We evaluated the effects of attenuating EV secretion in the Twitcher brain by depleting ceramide production with an inhibitor of neutral sphingomyelinase 2, GW4869.

Waning efficacy in a long-term AAV-mediated gene therapy study in the murine model of Krabbe disease.

Neonatal AAV9-gene therapy of the lysosomal enzyme galactosylceramidase (GALC) significantly ameliorates central and peripheral neuropathology, prolongs survival, and largely normalizes motor deficits in Twitcher mice. Despite these therapeutic milestones, new observations identified the presence of multiple small focal demyelinating areas in the brain after 6-8 months. These lesions are in stark contrast to the diffuse, global demyelination that affects the brain of naive Twitcher mice.

Ion mobility-mass spectrometry for the separation and analysis of procyanidins.

Procyanidins are polymeric flavan-3-ones occurring in many plants with antioxidant and other beneficial bioactivities. They are composed of catechin and epicatechin monomeric units connected by single carbon-carbon B-type linkages or A-type linkages containing both carbon-carbon and carbon-oxygen-carbon bonds. Their polymeric structure makes analysis of procyanidin mixtures always difficult.

Interventions for undergraduate and postgraduate medical learners with academic difficulties: A BEME systematic review: BEME Guide No. 56.

Clinical teachers often struggle to report unsatisfactory trainee performance, partly because of a lack of evidence-based remediation options. To identify interventions for undergraduate (UG) and postgraduate (PG) medical learners experiencing academic difficulties, link them to a theory-based framework and provide literature-based recommendations around their use. This systematic review searched MEDLINE, CINAHL, EMBASE, ERIC, Education Source and PsycINFO (1990-2016) combining these concepts: medical education, professional competence/difficulty and educational support.

Rapid Determination of Procyanidins Using MALDI-ToF/ToF Mass Spectrometry.

Although procyanidins constitute a unique class of polymeric plant secondary metabolites with a variety of biological properties including potent antioxidant activity, structure determination has been challenging, and structures of many complex procyanidins remain uncertain. To expedite the characterization of procyanidins, negative ion matrix-assisted laser desorption ionization high-energy collision-induced dissociation tandem time-of-flight (MALDI-ToF/ToF) mass spectra of 20 isolated procyanidins containing catechin and epicatechin subunits with degrees of polymerization up to five were obtained and evaluated. Structurally significant fragmentation pathways of singly charged, deprotonated molecules were identified representing quinone methide, heterocyclic ring fission, and retro-Diels-Alder fragmentation.

α-Synuclein interacts directly but reversibly with psychosine: implications for α-synucleinopathies.

Aggregation of α-synuclein, the hallmark of α-synucleinopathies such as Parkinson's disease, occurs in various glycosphingolipidoses. Although α-synuclein aggregation correlates with deficiencies in the lysosomal degradation of glycosphingolipids (GSL), the mechanism(s) involved in this aggregation remains unclear. We previously described the aggregation of α-synuclein in Krabbe's disease (KD), a neurodegenerative glycosphingolipidosis caused by lysosomal deficiency of galactosyl-ceramidase (GALC) and the accumulation of the GSL psychosine.

Procyanidins: a comprehensive review encompassing structure elucidation via mass spectrometry.

Procyanidins are polyphenols abundant in dietary fruits, vegetables, nuts, legumes, and grains with a variety of chemopreventive biological effects. Rapid structure determination of these compounds is needed, notably for the more complex polymeric procyanidins. We review the recent developments in the structure elucidation of procyanidins with a focus on mass spectrometric approaches, especially liquid chromatography-tandem mass spectrometry (LC-MS/MS) and matrix-assisted laser desorption ionization (MALDI) MS/MS.

Long-Term Improvement of Neurological Signs and Metabolic Dysfunction in a Mouse Model of Krabbe's Disease after Global Gene Therapy.

We report a global adeno-associated virus (AAV)9-based gene therapy protocol to deliver therapeutic galactosylceramidase (GALC), a lysosomal enzyme that is deficient in Krabbe's disease. When globally administered via intrathecal, intracranial, and intravenous injections to newborn mice affected with GALC deficiency (twitcher mice), this approach largely surpassed prior published benchmarks of survival and metabolic correction, showing long-term protection of demyelination, neuroinflammation, and motor function. Bone marrow transplantation, performed in this protocol without immunosuppressive preconditioning, added minimal benefits to the AAV9 gene therapy.

Responses of Synechocystis sp. PCC 6803 to heterologous biosynthetic pathways.

Background: There are an increasing number of studies regarding genetic manipulation of cyanobacteria to produce commercially interesting compounds. The majority of these works study the expression and optimization of a selected heterologous pathway, largely ignoring the wholeness and complexity of cellular metabolism. Regulation and response mechanisms are largely unknown, and even the metabolic pathways themselves are not fully elucidated.

Come and Knock on Our Door: Offenders' Perspectives on Home Visits Through Ecological Theory.

The use of home visits has a long and storied history in the United States from different disciplines, such as nursing, prenatal mothers, young families, health promotion, and community corrections. Ecological theory explains how formal actors play a role in the promotion in the health field through home visits, but does not address community corrections home visits. Through the use of 30 semi-structured interviews, this research seeks to expand the understanding of ecological theory by capturing the perceptions of offenders sentenced to home visits conducted by a sheriff's office.

Hop (Humulus lupulus L.) Extract and 6-Prenylnaringenin Induce P450 1A1 Catalyzed Estrogen 2-Hydroxylation.

Humulus lupulus L. (hops) is a popular botanical dietary supplement used by women as a sleep aid and for postmenopausal symptom relief. In addition to its efficacy for menopausal symptoms, hops can also modulate the chemical estrogen carcinogenesis pathway and potentially protect women from breast cancer.

Modeling complexometric titrations of natural water samples.

Complexometric titrations are the primary source of metal speciation data for aquatic systems, yet their interpretation in waters containing humic and fulvic acids remains problematic. In particular, the accuracy of inferred ambient free metal ion concentrations and parameters quantifying metal complexation by natural ligands has been challenged because of the difficulties inherent in calibrating common analytical methods and in modeling the diverse array of ligands present. This work tests and applies a new method of modeling titration data that combines calibration of analytical sensitivity (S) and estimation of concentrations and stability constants for discrete natural ligand classes ([Li]T and Ki) into a single step using nonlinear regression and a new analytical solution to the one-metal/two-ligand equilibrium problem.

Photochemical cycling of iron in the surface ocean mediated by microbial iron(III)-binding ligands.

Iron is a limiting nutrient for primary production in large areas of the oceans. Dissolved iron(III) in the upper oceans occurs almost entirely in the form of complexes with strong organic ligands presumed to be of biological origin. Although the importance of organic ligands to aquatic iron cycling is becoming clear, the mechanism by which they are involved in this process remains uncertain.

High-resolution comparative physical mapping of mouse chromosome 10 in the region of homology with human chromosome 21.

Comparative mapping of human and mouse chromosomes can be used to predict locations of homologous loci between the species, provides the substrate to examine the process of chromosomal evolution, and facilitates the continuing development of mouse genetic models for human disorders. A YAC contig of the region of mouse Chromosome (Chr) 10 (MMU10) that demonstrates conserved linkage with the distal portion of human Chr 21 (HSA21) has been constructed. The contig contains all known genes mapped in both species, defines the proximal region of homology between MMU10 and HSA22, and contains the evolutionary junction between HSA21 and HSA22 on MMU10.

Physical and comparative mapping of distal mouse chromosome 16. 5 p5.

Distal mouse Chromosome 16 (Chr. 16) includes a region of conserved linkage with human Chromosome 21 (Chr. 21).

Characterization and localization of Mox2, the gene encoding the murine homolog of the rat MRC OX-2 membrane glycoprotein.

MRC OX-2 is a rat type I membrane glycoprotein and a member of the immunoglobulin gene superfamily that has recently been shown to be able to costimulate murine T cell proliferation (Borriello et al. J. Immunol, 158, 4548, 1997).

High-resolution recombinational map of mouse chromosome 16.

Five intersubspecific backcrosses and an intercross were used to establish a sex-averaged recombinational map spanning 56 cM across most of mouse Chromosome 16 (Chr 16). A total of 123 markers were ordered using an interval mapping approach to identify 425 recombination sites in a collection of 1154 meioses from 1155 progeny generated in the six crosses. The markers include the 10 "classic" Chr 16 reference markers, 26 additional genes or transcripts including two phenotypic markers (Pit1dw and Kcnj6wv), and 87 simple sequence length polymorphisms (SSLPs).

Dimerization, DNA binding, and transactivation properties of hypoxia-inducible factor 1.

Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric basic helix-loop-helix transcription factor that regulates hypoxia-inducible genes including the human erythropoietin (EPO) gene. In this study, we report structural features of the HIF-1alpha subunit that are required for heterodimerization, DNA binding, and transactivation. The HIF-1alpha and HIF-1beta (ARNT; aryl hydrocarbon receptor nuclear translocator) subunits were coimmunoprecipitated from nuclear extracts, indicating that these proteins heterodimerize in the absence of DNA.

Assignment of the hypoxia-inducible factor 1alpha gene to a region of conserved synteny on mouse chromosome 12 and human chromosome 14q.

Hypoxia-inducible factor 1 (HIF-1) is a basic helix-loop-helix transcription factor that mediates homeostatic responses to hypoxia. HIF-1 is a heterodimer consisting of HIF-1alpha, which is encoded by the HIF1A gene, complexed with HIF-1beta, which is encoded by the ARNT gene. In this paper we report the assignment of Hif1a and HIF1A to mouse chromosome 12 and human chromosome 14, respectively.

Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension.

Hypoxia-inducible factor 1 (HIF-1) is found in mammalian cells cultured under reduced O2 tension and is necessary for transcriptional activation mediated by the erythropoietin gene enhancer in hypoxic cells. We show that both HIF-1 subunits are basic-helix-loop-helix proteins containing a PAS domain, defined by its presence in the Drosophila Per and Sim proteins and in the mammalian ARNT and AHR proteins. HIF-1 alpha is most closely related to Sim.