Strategies for the examination of Alzheimer's disease amyloid precursor protein isoforms.

The principal aim of this research project has been the utilisation of various proteomic techniques in the investigation of the Alzheimer's disease amyloid precursor protein (APP) isoforms, namely APP[695], APP[751] and APP[770]. One of the most noticeable pathological characteristics of Alzheimer's disease is the presence of neuritic plaques in brain tissue. The chief protein constituent of neuritic plaques is the beta amyloid peptide. This peptide is proteolytically cleaved from APP, as such the interest in APP isoforms is great and a rapid detection method for the presence of each isoform would be a huge advantage to the research effort with regards to the determination and concentration in both diseased and non-diseased states. Two-dimensional gel electrophoresis and peptide mass fingerprinting are two of the most important techniques in the proteomics arena and both are investigated fully in this work. Retinoic acid induced Ntera 2 cells, derived from a human teratocarcinoma cell line, were the in vitro source of APP. Initial isolation of APP was performed by immunoprecipitation, using a monoclonal antibody raised to amino acids 1-17 of the beta-amyloid peptide sequence, which is present in all three alpha secretase cleaved isoforms of interest. The next step was to separate whole APP into its isoform components by two-dimensional gel electrophoresis. The resulting protein spots were then subjected to peptide mass fingerprinting employing the different digest reagents, trypsin, endoproteinase Asp-N and formic acid. Initial distinction between the APP isoforms could be seen upon examination of theoretical in silica digests using the various digest reagents mentioned. The in silica digests revealed peptides unique to each isoform that in theory could be used as indicators of isoform presence

Editorial: Phytoplankton dynamics under climate change

Phytoplankton plays an important role in ocean processes, and is well-known to have an enormous positive impact on climate change or more specifically on global warming, by reducing atmospheric CO2 levels through the sinking of produced organic and inorganic matter to the deep ocean (Falkowski, 2012; Beardall and Raven, 2013). However, climate change, with consequences of elevated seawater temperatures and decreased pH levels (Beardall and Raven, 2013), influences phytoplankton dynamics, changing phytoplankton composition, geography and biomass in the oceans (Falkowski and Oliver, 2007; Boyd et al., 2015; Jonkers et al., 2019). Temperature increases could also drive temporal shifts in the onset of the regular annual blooms, their composition, duration and amplitude as well as mismatches in timing between trophic levels (Hinder et al., 2012; Mikaelyan et al., 2015). The overall impact of increased temperature on phytoplankton is not easy to assess due to variable and complex repercussions. For example, increasing temperatures can lead to more stratified waters, especially in summer months, and prevent nutrient replenishment at the ocean surface.info:eu-repo/semantics/publishedVersio

Localization of sterols and oxysterols in mouse brain reveals distinct spatial cholesterol metabolism

Dysregulated cholesterol metabolism is implicated in a number of neurological disorders. Many sterols, including cholesterol and its precursors and metabolites, are biologically active and important for proper brain function. However, spatial cholesterol metabolism in brain and the resulting sterol distributions are poorly defined. To better understand cholesterol metabolism in situ across the complex functional regions of brain, we have developed on-tissue enzyme-assisted derivatization in combination with microliquid extraction for surface analysis and liquid chromatography-mass spectrometry to locate sterols in tissue slices (10 µm) of mouse brain. The method provides sterolomic analysis at 400-µm spot diameter with a limit of quantification of 0.01 ng/mm2. It overcomes the limitations of previous mass spectrometry imaging techniques in analysis of low-abundance and difficult-to-ionize sterol molecules, allowing isomer differentiation and structure identification. Here we demonstrate the spatial distribution and quantification of multiple sterols involved in cholesterol metabolic pathways in wild-type and cholesterol 24S-hydroxylase knockout mouse brain. The technology described provides a powerful tool for future studies of spatial cholesterol metabolism in healthy and diseased tissues

Do Anti-Angiogenic VEGF (VEGFxxxb) Isoforms Exist? A Cautionary Tale

Splicing of the human vascular endothelial growth factor-A (VEGF-A) gene has been reported to generate angiogenic (VEGFxxx) and anti-angiogenic (VEGFxxxb) isoforms. Corresponding VEGFxxxb isoforms have also been reported in rat and mouse. We examined VEGFxxxb expression in mouse fibrosarcoma cell lines expressing all or individual VEGF isoforms (VEGF120, 164 or 188), grown in vitro and in vivo, and compared results with those from normal mouse and human tissues. Importantly, genetic construction of VEGF164 and VEGF188 expressing fibrosarcomas, in which exon 7 is fused to the conventional exon 8, precludes VEGFxxxb splicing from occurring. Thus, these two fibrosarcoma cell lines provided endogenous negative controls. Using RT-PCR we show that primers designed to simultaneously amplify VEGFxxx and VEGFxxxb isoforms amplified only VEGFxxx variants in both species. Moreover, only VEGFxxx species were generated when mouse podocytes were treated with TGFβ-1, a reported activator of VEGFxxxb splice selection in human podocytes. A VEGF164/120 heteroduplex species was identified as a PCR artefact, specifically in mouse. VEGFxxxb isoform-specific PCR did amplify putative VEGFxxxb species in mouse and human tissues, but unexpectedly also in VEGF188 and VEGF164 fibrosarcoma cells and tumours, where splicing to produce true VEGFxxxb isoforms cannot occur. Moreover, these products were only consistently generated using reverse primers spanning more than 5 bases across the 8b/7 or 8b/5 splice junctions. Primer annealing to VEGFxxx transcripts and amplification of exon 8b primer ‘tails’ explained the artefactual generation of VEGFxxxb products, since the same products were generated when the PCR reactions were performed with cDNA from VEGF164/VEGF188 ‘knock-in’ vectors used in the generation of single VEGF isoform-expressing transgenic mice from which the fibrosarcoma lines were developed. Collectively, our results highlight important pitfalls in data interpretation associated with detecting VEGFxxxb isoforms using current methods, and demonstrate that anti-angiogenic isoforms are not commonly expressed in mouse or human tissues

Commentary: comparing efficiency in aquatic and terrestrial animal production systems

First paragraph: Aquaculture is receiving increased attention from a variety of stakeholders. This is largely due to its current role in the global food system of supplying more than half of the seafood consumed, and also because the industry continues to steadily expand (UN Food and Agriculture Organization 2018). A recent article in Environmental Research Letters, 'Feed conversion efficiency in aquaculture: do we measure it correctly?', by Fry et al (2018a) found that measuring feed conversion efficiency of selected aquatic and terrestrial farmed animals using protein and calorie retention resulted in species comparisons (least to most efficient) and overlap among species dissimilar from comparisons based on widely used weight-based feed conversion ratio (FCR) values. The study prompted spirited discussions among researchers, industry representatives, and others. A group assembled to write a standard rebuttal, but during this process, decided it was best to engage the study's original authors to join the discourse. Through this collaboration, we provide the resultant additional context relevant to the study in order to advance conversations and research on the use of efficiency measures in aquatic and terrestrial animal production systems

Novel application of behavioral assays allows dissociation of joint pathology from systemic extra-articular alterations induced by inflammatory arthritis

Introduction: Although rheumatoid arthritis (RA) is a disease of articular joints, patients often suffer from co-morbid neuropsychiatric changes, such as anxiety, that may reflect links between heightened systemic inflammation and abnormal regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Here, we apply behavioral neuroscience methods to assess the impact of antigen-induced arthritis (AIA) on behavioral performance in wild type (WT) and interleukin-10 deficient (Il10-/-) mice. Our aim was to identify limb-specific motor impairments, as well as neuropsychological responses to inflammatory arthritis. Methods: Behavioral testing was performed longitudinally in WT and Il10-/- mice before and after the induction of arthritic joint pathology. Footprint analysis, beam walking and open field assessment determined a range of motor, exploratory and anxiety-related parameters. Specific gene changes in HPA axis tissues were analyzed using qPCR. Results: Behavioral assessment revealed transient motor and exploratory impairments in mice receiving AIA, coinciding with joint swelling. Hind limb coordination deficits were independent of joint pathology. Behavioral impairments returned to baseline by 10 days post-AIA in WT mice. Il10-/- mice demonstrated comparable levels of swelling and joint pathology as WT mice up to 15 days post-AIA, but systemic differences were evident in mRNA expression in HPA axis tissues from Il10-/- mice post-AIA. Interestingly, the behavioral profile of Il10-/- mice revealed a significantly longer time post-AIA for activity and anxiety-related behaviors to recover. Conclusions: The novel application of sensitive behavioral tasks has enabled dissociation between behaviors that occur due to transient joint-specific pathology and those generated by more subtle systemic alterations that manifest post-AIA

Label-free Quantitative Proteomics and Substrate Based Mass Spectrometry Imaging of Xenobiotic Metabolizing Enzymes in ex Vivo Human Skin and a Human Living Skin Equivalent Model.

We report for the first time label-free quantification of xenobiotic metabolizing enzymes (XME), transporters, redox enzymes, proteases and nucleases in six human skin explants and a 3D living skin equivalent model from LabSkin. We aimed to evaluate the suitability of LabSkin as an alternative to animal testing for the development of topical formulations. More than 2000 proteins were identified and quantified from total cellular protein. Alcohol dehydrogenase 1C (ADH1C), the most abundant phase I XME in human skin, and glutathione S-transferase pi 1 (GSTP1), the most abundant phase II XME in human skin, were present in similar abundance in LabSkin. Several esterases were quantified and esterase activity was confirmed in LabSkin using substrate-based mass spectrometry imaging. No cytochrome P450 (CYP) activity was observed for the substrates tested, in agreement with the proteomics data, where the cognate CYPs were absent in both human skin and LabSkin. Label-free protein quantification allowed insights into other related processes such as redox homeostasis and proteolysis. For example, the most abundant antioxidant enzymes were thioredoxin (TXN) and peroxiredoxin-1 (PRDX1). This systematic determination of functional equivalence between human skin and LabSkin is a key step towards the construction of a representative human in vitro skin model, which can be used as an alternative to current animal-based tests for chemical safety and for predicting dosage of topically administered drugs. Significance Statement The use of label-free quantitative mass spectrometry to elucidate the abundance of xenobiotic metabolizing enzymes, transporters, redox enzymes, proteases and nucleases in human skin enhance our understanding of the skin physiology and biotransformation of topical drugs and cosmetics. This will help develop mathematical models to predict drug metabolism in human skin and to develop more robust in vitro engineered human skin tissue as alternatives to animal testing

Visualizing Cholesterol in the Brain by On-Tissue Derivatization and Quantitative Mass Spectrometry Imaging.

Despite being a critical molecule in the brain, mass spectrometry imaging (MSI) of cholesterol has been under-reported compared to other lipids due to the difficulty in ionizing the sterol molecule. In the present work, we have employed an on-tissue enzyme-assisted derivatization strategy to improve detection of cholesterol in brain tissue sections. We report distribution and levels of cholesterol across specific structures of the mouse brain, in a model of Niemann-Pick type C1 disease, and during brain development. MSI revealed that in the adult mouse, cholesterol is the highest in the pons and medulla and how its distribution changes during development. Cholesterol was significantly reduced in the corpus callosum and other brain regions in the Npc1 null mouse, confirming hypomyelination at the molecular level. Our study demonstrates the potential of MSI to the study of sterols in neuroscience

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts