Determining the genetic bases of age-related disease remains a major challenge requiring a spectrum of approaches from human and clinical genetics to the utilization of model organism studies. Here ...we report a large-scale genetic screen in mice employing a phenotype-driven discovery platform to identify mutations resulting in age-related disease, both late-onset and progressive. We have utilized N-ethyl-N-nitrosourea mutagenesis to generate pedigrees of mutagenized mice that were subject to recurrent screens for mutant phenotypes as the mice aged. In total, we identify 105 distinct mutant lines from 157 pedigrees analysed, out of which 27 are late-onset phenotypes across a range of physiological systems. Using whole-genome sequencing we uncover the underlying genes for 44 of these mutant phenotypes, including 12 late-onset phenotypes. These genes reveal a number of novel pathways involved with age-related disease. We illustrate our findings by the recovery and characterization of a novel mouse model of age-related hearing loss.
Peroxisomal fatty acid α-oxidation is an essential pathway for the degradation of β-carbon methylated fatty acids such as phytanic acid. One enzyme in this pathway is 2-hydroxyacyl CoA lyase (HACL1), ...which is responsible for the cleavage of 2-hydroxyphytanoyl-CoA into pristanal and formyl-CoA. Hacl1 deficient mice do not present with a severe phenotype, unlike mice deficient in other α-oxidation enzymes such as phytanoyl-CoA hydroxylase deficiency (Refsum disease) in which neuropathy and ataxia are present. Tissues from wild-type and
mice fed a high phytol diet were obtained for proteomic and lipidomic analysis. There was no phenotype observed in these mice. Liver, brain, and kidney tissues underwent trypsin digestion for untargeted proteomic liquid chromatography-mass spectrometry analysis, while liver tissues also underwent fatty acid hydrolysis, extraction, and derivatisation for fatty acid gas chromatography-mass spectrometry analysis. The liver fatty acid profile demonstrated an accumulation of phytanic and 2-hydroxyphytanic acid in the
liver and significant decrease in heptadecanoic acid. The liver proteome showed a significant decrease in the abundance of Hacl1 and a significant increase in the abundance of proteins involved in PPAR signalling, peroxisome proliferation, and omega oxidation, particularly Cyp4a10 and Cyp4a14. In addition, the pathway associated with arachidonic acid metabolism was affected; Cyp2c55 was upregulated and Cyp4f14 and Cyp2b9 were downregulated. The kidney proteome revealed fewer significantly upregulated peroxisomal proteins and the brain proteome was not significantly different in
mice. This study demonstrates the powerful insight brought by proteomic and metabolomic profiling of
mice in better understanding disease mechanism in fatty acid α-oxidation disorders.
The link between mutations in collagen genes and the development of Alport Syndrome has been clearly established and a number of animal models, including knock-out mouse lines, have been developed ...that mirror disease observed in patients. However, it is clear from both patients and animal models that the progression of disease can vary greatly and can be modified genetically. We have identified a point mutation in Col4a4 in mice where disease is modified by strain background, providing further evidence of the genetic modification of disease symptoms. Our results indicate that C57BL/6J is a protective background and postpones end stage renal failure from 7 weeks, as seen on a C3H background, to several months. We have identified early differences in disease progression, including expression of podocyte-specific genes and podocyte morphology. In C57BL/6J mice podocyte effacement is delayed, prolonging normal renal function. The slower disease progression has allowed us to begin dissecting the pathogenesis of murine Alport Syndrome in detail. We find that there is evidence of differential gene expression during disease on the two genetic backgrounds, and that disease diverges by 4 weeks of age. We also show that an inflammatory response with increasing MCP-1 and KIM-1 levels precedes loss of renal function.
This paper proposes a blockchain-based mapping protocol for distributed robotic systems running on embedded hardware. This protocol was developed for a robotic system designed to locomote on lattice ...structures for space applications. A consensus mechanism, Proof of Validity, is introduced to allow the effort of mining blocks to correlate with the desired tasks the robotic system was designed for. These robots communicate using peer-to-peer LoRa radio. Options, trade-offs and considerations for implementing blockchain technology on an embedded system with wireless radio communication are explored and discussed.
We present a literature review and a toolbox to help the reader find the best method to design for and assess Sense of Embodiment (SoE) in several application scenarios. The main examples are based ...on teleoperation applications, due the challenges that these applications present. The three embodiment components that we consider to describe SoE are sense of ownership, sense of agency, and sense of self-location. We relate each embodiment component to the most often used assessment measures, test tasks, and application scenarios. The toolbox is built to efficiently design, test, and assess an embodiment experience, following seven concrete steps. We provide four main contributions: 1) a literature review of the assessment measures and strategies used to measure SoE; 2) a systematic categorization of SoE measures; 3) A categorization of the main test tasks used in SoE assessment; and 4) a toolbox consisting of seven steps as guidance to design SoE. We included several examples and tables to guide the user step by step through the design of an embodiment experience.
Accumulation of extracellular matrix in organs and tissues is a feature of both aging and disease. In the kidney, glomerulosclerosis and tubulointerstitial fibrosis accompany the decline in function, ...which current therapies cannot address, leading to organ failure. Although histologic and ultrastructural patterns of excess matrix form the basis of human disease classifications, a comprehensive molecular resolution of abnormal matrix is lacking.
Using mass spectrometry-based proteomics, we resolved matrix composition over age in mouse models of kidney disease. We compared the changes in mice with a global characterization of human kidneymatrix during aging and to existing kidney disease datasets to identify common molecular features.
Ultrastructural changes in basement membranes are associated with altered cell adhesion and metabolic processes and with distinct matrix proteomes during aging and kidney disease progression in mice. Within the altered matrix, basement membrane components (laminins, type IV collagen, type XVIII collagen) were reduced and interstitial matrix proteins (collagens I, III, VI, and XV; fibrinogens; and nephronectin) were increased, a pattern also seen in human kidney aging. Indeed, this signature of matrix proteins was consistently modulated across all age and disease comparisons, and the increase in interstitial matrix was also observed in human kidney disease datasets.
This study provides deep molecular resolution of matrix accumulation in kidney aging and disease, and identifies a common signature of proteins that provides insight into mechanisms of response to kidney injury and repair.
Abstract
Aims
Mitochondrial Complex I assembly (MCIA) is a multi-step process that necessitates the involvement of a variety of assembly factors and chaperones to ensure that the final active enzyme ...is correctly assembled. The role of the assembly factor evolutionarily conserved signalling intermediate in the toll (ECSIT) pathway was studied across various murine tissues to determine its role in this process and how this varied between tissues of varying energetic demands. We hypothesized that many of the known functions of ECSIT were unhindered by the introduction of an ENU-induced mutation, while its role in Complex I assembly was affected on a tissue-specific basis.
Methods and results
Here, we describe a mutation in the MCIA factor ECSIT that reveals tissue-specific requirements for ECSIT in Complex I assembly. MCIA is a multi-step process dependent on assembly factors that organize and arrange the individual subunits, allowing for their incorporation into the complete enzyme complex. We have identified an ENU-induced mutation in ECSIT (N209I) that exhibits a profound effect on Complex I component expression and assembly in heart tissue, resulting in hypertrophic cardiomyopathy in the absence of other phenotypes. The dysfunction of Complex I appears to be cardiac specific, leading to a loss of mitochondrial output as measured by Seahorse extracellular flux and various biochemical assays in heart tissue, while mitochondria from other tissues were unaffected.
Conclusions
These data suggest that the mechanisms underlying Complex I assembly and activity may have tissue-specific elements tailored to the specific demands of cells and tissues. Our data suggest that tissues with high-energy demands, such as the heart, may utilize assembly factors in different ways to low-energy tissues in order to improve mitochondrial output. These data have implications for the diagnosis and treatment of various disorders of mitochondrial function as well as cardiac hypertrophy with no identifiable underlying genetic cause.
Graphical Abstract
Graphical Abstract
In this paper we present a Multimodal Echoborg interface to explore the effect of different embodiments of an Embodied Conversational Agent (ECA) in an interaction. We compared an interaction where ...the ECA was embodied as a virtual human (VH) with one where it was embodied as an Echoborg, i.e, a person whose actions are covertly controlled by a dialogue system. The Echoborg in our study not only shadowed the speech output of the dialogue system but also its non-verbal actions. The interactions were structured as a debate between three participants on an ethical dilemma. First, we collected a corpus of debate sessions with three humans debaters. This we used as baseline to design and implement our ECAs. For the experiment, we designed two debate conditions. In one the participant interacted with two ECAs both embodied by virtual humans). In the other the participant interacted with one ECA embodied by a VH and the other by an Echoborg. Our results show that a human embodiment of the ECA overall scores better on perceived social attributes of the ECA. In many other respects the Echoborg scores as poorly as the VH except
copresence
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Nephrotic syndrome is characterized by severe proteinuria, hypoalbuminaemia, edema and hyperlipidaemia. Genetic studies of nephrotic syndrome have led to the identification of proteins playing a ...crucial role in slit diaphragm signaling, regulation of actin cytoskeleton dynamics and cell-matrix interactions. The laminin α5 chain is essential for embryonic development and, in association with laminin β2 and laminin γ1, is a major component of the glomerular basement membrane, a critical component of the glomerular filtration barrier. Mutations in LAMA5 were recently identified in children with nephrotic syndrome. Here, we have identified a novel missense mutation (E884G) in the uncharacterized L4a domain of LAMA5 where homozygous mice develop nephrotic syndrome with severe proteinuria with histological and ultrastructural changes in the glomerulus mimicking the progression seen in most patients. The levels of LAMA5 are reduced in vivo and the assembly of the laminin 521 heterotrimer significantly reduced in vitro. Proteomic analysis of the glomerular extracellular fraction revealed changes in the matrix composition. Importantly, the genetic background of the mice had a significant effect on aspects of disease progression from proteinuria to changes in podocyte morphology. Thus, our novel model will provide insights into pathologic mechanisms of nephrotic syndrome and pathways that influence the response to a dysfunctional glomerular basement membrane that may be important in a range of kidney diseases.
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Abstract
Aims
Cardiac arrhythmias comprise a major health and economic burden and are associated with significant morbidity and mortality, including cardiac failure, stroke, and sudden cardiac death ...(SCD). Development of efficient preventive and therapeutic strategies is hampered by incomplete knowledge of disease mechanisms and pathways. Our aim is to identify novel mechanisms underlying cardiac arrhythmia and SCD using an unbiased approach.
Methods and results
We employed a phenotype-driven N-ethyl-N-nitrosourea mutagenesis screen and identified a mouse line with a high incidence of sudden death at young age (6–9 weeks) in the absence of prior symptoms. Affected mice were found to be homozygous for the nonsense mutation Bcat2p.Q300*/p.Q300* in the Bcat2 gene encoding branched chain amino acid transaminase 2. At the age of 4–5 weeks, Bcat2p.Q300*/p.Q300* mice displayed drastic increase of plasma levels of branch chain amino acids (BCAAs—leucine, isoleucine, valine) due to the incomplete catabolism of BCAAs, in addition to inducible arrhythmias ex vivo as well as cardiac conduction and repolarization disturbances. In line with these findings, plasma BCAA levels were positively correlated to electrocardiogram indices of conduction and repolarization in the German community-based KORA F4 Study. Isolated cardiomyocytes from Bcat2p.Q300*/p.Q300* mice revealed action potential (AP) prolongation, pro-arrhythmic events (early and late afterdepolarizations, triggered APs), and dysregulated calcium homeostasis. Incubation of human pluripotent stem cell-derived cardiomyocytes with elevated concentration of BCAAs induced similar calcium dysregulation and pro-arrhythmic events which were prevented by rapamycin, demonstrating the crucial involvement of mTOR pathway activation.
Conclusions
Our findings identify for the first time a causative link between elevated BCAAs and arrhythmia, which has implications for arrhythmogenesis in conditions associated with BCAA metabolism dysregulation such as diabetes, metabolic syndrome, and heart failure.
Graphical Abstract
Graphical Abstract
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