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The prospects for ‘Prospect Utilitarianism’
Hun Chung argues for a theory of distributive justice—‘prospect utilitarianism’—that overcomes two central problems purportedly faced by sufficientarianism: giving implausible answers in ‘lifeboat cases’, where we can save the lives of some but not all of a group, and failing to respect the axiom of continuity. Chung’s claims that prospect utilitarianism overcomes these problems, and receives empirical support from work in economics on prospect theory. This paper responds to Chung’s criticisms of sufficientarianism, showing that they are misplaced. It then shows that prospect utilitarianism faces independent problems, since it too requires a threshold, which Chung bases on the idea of ‘adequate functioning’. The paper shows that there are problems with this as a threshold, and that it is not empirically supported by prospect theory.
Epigenomic analysis reveals a dynamic and context-specific macrophage enhancer landscape associated with innate immune activation and tolerance.
BACKGROUND: Chromatin states and enhancers associate gene expression, cell identity and disease. Here, we systematically delineate the acute innate immune response to endotoxin in terms of human macrophage enhancer activity and contrast with endotoxin tolerance, profiling the coding and non-coding transcriptome, chromatin accessibility and epigenetic modifications. RESULTS: We describe the spectrum of enhancers under acute and tolerance conditions and the regulatory networks between these enhancers and biological processes including gene expression, splicing regulation, transcription factor binding and enhancer RNA signatures. We demonstrate that the vast majority of differentially regulated enhancers on acute stimulation are subject to tolerance and that expression quantitative trait loci, disease-risk variants and eRNAs are enriched in these regulatory regions and related to context-specific gene expression. We find enrichment for context-specific eQTL involving endotoxin response and specific infections and delineate specific differential regions informative for GWAS variants in inflammatory bowel disease and multiple sclerosis, together with a context-specific enhancer involving a bacterial infection eQTL for KLF4. We show enrichment in differential enhancers for tolerance involving transcription factors NFκB-p65, STATs and IRFs and prioritize putative causal genes directly linking genetic variants and disease risk enhancers. We further delineate similarities and differences in epigenetic landscape between stem cell-derived macrophages and primary cells and characterize the context-specific enhancer activities for key innate immune response genes KLF4, SLAMF1 and IL2RA. CONCLUSIONS: Our study demonstrates the importance of context-specific macrophage enhancers in gene regulation and utility for interpreting disease associations, providing a roadmap to link genetic variants with molecular and cellular functions.
Isolation and In vitro Culture of Bone Marrow-Derived Macrophages for the Study of NO-Redox Biology.
Macrophages are derived from hematopoietic progenitor cells throughout the body, are central to inflammatory processes, and participate in innate and adaptive immune responses. In vitro study of macrophages can be undertaken by ex vivo culture from the peritoneum or through differentiation of myeloid bone marrow progenitor cells to form bone marrow-derived macrophages (BMDMs). A common approach to macrophage differentiation from precursors involves the use of conditioned media from L929 cells (LCM). This media is easy to self-produce but suffers from batch variability, and its constituents are undefined. Similarly, Foetal Bovine Serum (FBS) is used to support growth but contains a vast mixture of undefined molecules that may vary between batches. These methods are not adequate for the study of nitric oxide biology and redox mechanisms as they both contain substantial amounts of small molecules that either interfere with redox mechanisms or supplement levels of cofactors, such as tetrahydrobiopterin (BH4), required for the production of NO from inducible nitric oxide synthase (iNOS). In this report, we present an optimized protocol allowing for control of the NO-redox environment by reducing the levels of exogenous biopterin while maintaining conditions suitable for cell growth and differentiation. Tight control of culture media composition helps ensure experimental reproducibility and facilitates accurate interpretation of results. In this protocol, BMDMs were obtained from a GTP cyclohydrolase (GCH)- deficient mouse model. Culture of BMDMs was performed with media containing either (i) conditioned LCM, or (ii) recombinant M-CSF and GM-CSF to produce minimal artifacts while obtaining BH4 and NO-deficient culture conditions - thus allowing for the reproducible study of NO-redox biology and immunometabolism in vitro.
A Founder Mutation in EHD1 Presents with Tubular Proteinuria and Deafness.
BACKGROUND: The endocytic reabsorption of proteins in the proximal tubule requires a complex machinery and defects can lead to tubular proteinuria. The precise mechanisms of endocytosis and processing of receptors and cargo are incompletely understood. EHD1 belongs to a family of proteins presumably involved in the scission of intracellular vesicles and in ciliogenesis. However, the relevance of EHD1 in human tissues, in particular in the kidney, was unknown. METHODS: Genetic techniques were used in patients with tubular proteinuria and deafness to identify the disease-causing gene. Diagnostic and functional studies were performed in patients and disease models to investigate the pathophysiology. RESULTS: We identified six individuals (5-33 years) with proteinuria and a high-frequency hearing deficit associated with the homozygous missense variant c.1192C>T (p.R398W) in EHD1. Proteinuria (0.7-2.1 g/d) consisted predominantly of low molecular weight proteins, reflecting impaired renal proximal tubular endocytosis of filtered proteins. Ehd1 knockout and Ehd1R398W/R398W knockin mice also showed a high-frequency hearing deficit and impaired receptor-mediated endocytosis in proximal tubules, and a zebrafish model showed impaired ability to reabsorb low molecular weight dextran. Interestingly, ciliogenesis appeared unaffected in patients and mouse models. In silico structural analysis predicted a destabilizing effect of the R398W variant and possible inference with nucleotide binding leading to impaired EHD1 oligomerization and membrane remodeling ability. CONCLUSIONS: A homozygous missense variant of EHD1 causes a previously unrecognized autosomal recessive disorder characterized by sensorineural deafness and tubular proteinuria. Recessive EHD1 variants should be considered in individuals with hearing impairment, especially if tubular proteinuria is noted.
Tissue-Specific Roles for the Slit-Robo Pathway During Heart, Caval Vein, and Diaphragm Development.
Background Binding of Slit ligands to their Robo receptors regulates signaling pathways that are important for heart development. Genetic variants in ROBO1and ROBO4 have been linked to congenital heart defects in humans. These defects are recapitulated in mouse models with ubiquitous deletions of the Slit ligands or Robo receptors and include additional heart defects not currently linked to SLIT or ROBO mutations in humans. Given the broad expression patterns of these genes, the question remains open which tissue-specific ligand-receptor interactions are important for the correct development of different cardiac structures. Methods and Results We used tissue-specific knockout mouse models of Robo1/Robo2, Robo4, Slit2 andSlit3 and scored cardiac developmental defects in perinatal mice. Knockout of Robo2 in either the whole heart, endocardium and its derivatives, or the neural crest in ubiquitous Robo1 knockout background resulted in ventricular septal defects. Neural crest-specific removal of Robo2 in Robo1 knockouts showed fully penetrant bicuspid aortic valves (BAV). Endocardial knock-out of either Slit2or Robo4 caused low penetrant BAV. In contrast, endocardial knockout of Slit3 using a newly generated line resulted in fully penetrant BAV, while removal from smooth muscle cells also resulted in BAV. Caval vein and diaphragm defects observed in ubiquitous Slit3 mutants were recapitulated in the tissue-specific knockouts. Conclusions Our data will help understand defects observed in patients with variants in ROBO1 and ROBO4. The results strongly indicate interaction between endocardial Slit3and neural crest Robo2 in the development of BAV, highlighting the need for further studies of this connection.
Base editing and prime editing in laboratory animals.
Genome editing by programmable RNA-dependent Cas endonucleases has revolutionised the field of genome engineering, achieving targeted genomic change at unprecedented efficiencies with considerable application in laboratory animal research. Despite its ease of use and wide application, there remain concerns about the precision of this technology and a number of unpredictable consequences have been reported, mostly resulting from the DNA double-strand break (DSB) that conventional CRISPR editing induces. In order to improve editing precision, several iterations of the technology been developed over the years. Base editing is one of most successful developments, allowing for single base conversions but without the need for a DSB. Cytosine and adenine base editing are now established as reliable methods to achieve precise genome editing in animal research studies. Both cytosine and adenine base editors have been applied successfully to the editing of zygotes, resulting in the generation of animal models. Similarly, both base editors have achieved precise editing of point mutations in somatic cells, facilitating the development of gene therapy approaches. Despite rapid progress in optimising these tools, base editing can address only a subset of possible base conversions within a relatively narrow window and larger genomic manipulations are not possible. The recent development of prime editing, originally defined as a simple 'search and replace' editing tool, may help address these limitations and could widen the range of genome manipulations possible. Preliminary reports of prime editing in animals are being published, and this new technology may allow significant advancements for laboratory animal research.
Altering the Binding Properties of PRDM9 Partially Restores Fertility across the Species Boundary.
Sterility or subfertility of male hybrid offspring is commonly observed. This phenomenon contributes to reproductive barriers between the parental populations, an early step in the process of speciation. One frequent cause of such infertility is a failure of proper chromosome pairing during male meiosis. In subspecies of the house mouse, the likelihood of successful chromosome synapsis is improved by the binding of the histone methyltransferase PRDM9 to both chromosome homologs at matching positions. Using genetic manipulation, we altered PRDM9 binding to occur more often at matched sites, and find that chromosome pairing defects can be rescued, not only in an intersubspecific cross, but also between distinct species. Using different engineered variants, we demonstrate a quantitative link between the degree of matched homolog binding, chromosome synapsis, and rescue of fertility in hybrids between Mus musculus and Mus spretus. The resulting partial restoration of fertility reveals additional mechanisms at play that act to lock-in the reproductive isolation between these two species.
'Personal Health Surveillance': The Use of mHealth in Healthcare Responsibilisation.
There is an ongoing increase in the use of mobile health (mHealth) technologies that patients can use to monitor health-related outcomes and behaviours. While the dominant narrative around mHealth focuses on patient empowerment, there is potential for mHealth to fit into a growing push for patients to take personal responsibility for their health. I call the first of these uses 'medical monitoring', and the second 'personal health surveillance'. After outlining two problems which the use of mHealth might seem to enable us to overcome-fairness of burdens and reliance on self-reporting-I note that these problems would only really be solved by unacceptably comprehensive forms of personal health surveillance which applies to all of us at all times. A more plausible model is to use personal health surveillance as a last resort for patients who would otherwise independently qualify for responsibility-based penalties. However, I note that there are still a number of ethical and practical problems that such a policy would need to overcome. The prospects of mHealth enabling a fair, genuinely cost-saving policy of patient responsibility are slim.
The Ncoa7 locus regulates V-ATPase formation and function, neurodevelopment and behaviour.
Members of the Tre2/Bub2/Cdc16 (TBC), lysin motif (LysM), domain catalytic (TLDc) protein family are associated with multiple neurodevelopmental disorders, although their exact roles in disease remain unclear. For example, nuclear receptor coactivator 7 (NCOA7) has been associated with autism, although almost nothing is known regarding the mode-of-action of this TLDc protein in the nervous system. Here we investigated the molecular function of NCOA7 in neurons and generated a novel mouse model to determine the consequences of deleting this locus in vivo. We show that NCOA7 interacts with the cytoplasmic domain of the vacuolar (V)-ATPase in the brain and demonstrate that this protein is required for normal assembly and activity of this critical proton pump. Neurons lacking Ncoa7 exhibit altered development alongside defective lysosomal formation and function; accordingly, Ncoa7 deletion animals exhibited abnormal neuronal patterning defects and a reduced expression of lysosomal markers. Furthermore, behavioural assessment revealed anxiety and social defects in mice lacking Ncoa7. In summary, we demonstrate that NCOA7 is an important V-ATPase regulatory protein in the brain, modulating lysosomal function, neuronal connectivity and behaviour; thus our study reveals a molecular mechanism controlling endolysosomal homeostasis that is essential for neurodevelopment.
When the genome bluffs: a tandem duplication event during generation of a novel Agmo knockout mouse model fools routine genotyping.
BACKGROUND: Genome editing in mice using either classical approaches like homologous recombination or CRISPR/Cas9 has been reported to harbor off target effects (insertion/deletion, frame shifts or gene segment duplications) that lead to mutations not only in close proximity to the target site but also outside. Only the genomes of few engineered mouse strains have been sequenced. Since the role of the ether-lipid cleaving enzyme alkylglycerol monooxygenase (AGMO) in physiology and pathophysiology remains enigmatic, we created a knockout mouse model for AGMO using EUCOMM stem cells but unforeseen genotyping issues that did not agree with Mendelian distribution and enzyme activity data prompted an in-depth genomic validation of the mouse model. RESULTS: We report a gene segment tandem duplication event that occurred during the generation of an Agmo knockout-first allele by homologous recombination. Only low homology was seen between the breakpoints. While a single copy of the recombinant 18 kb cassette was integrated correctly around exon 2 of the Agmo gene, whole genome nanopore sequencing revealed a 94 kb duplication in the Agmo locus that contains Agmo wild-type exons 1-3. The duplication fooled genotyping by routine PCR, but could be resolved using qPCR-based genotyping, targeted locus amplification sequencing and nanopore sequencing. Despite this event, this Agmo knockout mouse model lacks AGMO enzyme activity and can therefore be used to study its physiological role. CONCLUSIONS: A duplication event occurred at the exact locus of the homologous recombination and was not detected by conventional quality control filters such as FISH or long-range PCR over the recombination sites. Nanopore sequencing provides a cost convenient method to detect such underrated off-target effects, suggesting its use for additional quality assessment of gene editing in mice and also other model organisms.
Replacement of surgical vasectomy through the use of wild-type sterile hybrids.
For the production and rederivation of mouse strains, pseudopregnant female mice are used for embryo transfer and serve as surrogate mothers to support embryo development to term. Vasectomized males are commonly used to render pseudopregnancy in females, generated by surgical procedures associated with considerable pain and discomfort. Genetically modified mouse strains with a sterility phenotype provide a non-surgical replacement and represent an important application of the 3Rs (Replacement, Reduction, Refinement). However, the maintenance of such genetically modified mouse strains requires extensive breeding and genotyping procedures, which are regulated procedures under national legislation. As an alternative, we have explored the use of sterile male hybrids that result when two wild-type mouse subspecies, Mus musculus musculus and Mus musculus domesticus, interbreed. We find the male STUSB6F1 hybrid, resulting from the mating of female STUS/Fore with male C57BL/6J, ideally suited and demonstrate that its performance for the production of oviduct and uterine transfer recipients is indistinguishable when compared to surgically vasectomized mice. The use of these sterile hybrids avoids the necessity for surgical procedures or the breeding of sterile genetically modified lines and can be generated by the simple mating of two wild-type laboratory strains-a non-regulated procedure. Furthermore, in contrast with the breeding of genetically sterile mice, all male offspring are sterile and suitable for the generation of pseudopregnancy, allowing their efficient production with minimal breeding pairs.
Genome-wide CRISPR/Cas9-knockout in human induced Pluripotent Stem Cell (iPSC)-derived macrophages.
Genome engineering using CRISPR/Cas9 technology enables simple, efficient and precise genomic modifications in human cells. Conventional immortalized cell lines can be easily edited or screened using genome-wide libraries with lentiviral transduction. However, cell types derived from the differentiation of induced Pluripotent Stem Cells (iPSC), which often represent more relevant, patient-derived models for human pathology, are much more difficult to engineer as CRISPR/Cas9 delivery to these differentiated cells can be inefficient and toxic. Here, we present an efficient, lentiviral transduction protocol for delivery of CRISPR/Cas9 to macrophages derived from human iPSC with efficiencies close to 100%. We demonstrate CRISPR/Cas9 knockouts for three nonessential proof-of-concept genes-HPRT1, PPIB and CDK4. We then scale the protocol and validate for a genome-wide pooled CRISPR/Cas9 loss-of-function screen. This methodology enables, for the first time, systematic exploration of macrophage involvement in immune responses, chronic inflammation, neurodegenerative diseases and cancer progression, using efficient genome editing techniques.
Phenotype of a transient neonatal diabetes point mutation (SUR1-R1183W) in mice.
Background: The K ATP channel plays a key role in glucose homeostasis by coupling metabolically generated changes in ATP to insulin secretion from pancreatic beta-cells. Gain-of-function mutations in either the pore-forming (Kir6.2) or regulatory (SUR1) subunit of this channel are a common cause of transient neonatal diabetes mellitus (TNDM), in which diabetes presents shortly after birth but remits within the first few years of life, only to return in later life. The reasons behind this time dependence are unclear. Methods: In an attempt to understand the mechanism behind diabetes remission and relapse, we generated mice expressing the common TNDM mutation SUR1-R1183W. We employed Cre/LoxP technology for both inducible and constitutive expression of SUR1-R1183W specifically in mouse beta-cells, followed by investigation of their phenotype using glucose tolerance tests and insulin secretion from isolated islets. Results: We found that the R1183W mutation impaired inhibition of K ATP channels by ATP when heterologously expressed in human embryonic kidney cells. However, neither induced nor constitutive expression of SUR1-R1183W in mice resulted in changes in blood glucose homeostasis, compared to littermate controls. When challenged with a high fat diet, female mice expressing SUR1-R1183W showed increased weight gain, elevated blood glucose and impaired glycaemic control, but glucose-stimulated insulin secretion from pancreatic islets appeared unchanged. Conclusions: The mouse model of TNDM did not recapitulate the human phenotype. We discuss multiple potential reasons why this might be the case. Based on our findings, we recommend future TNDM mouse models employing a gain-of-function SUR1 mutation should be created using the minimally invasive CRISPR/Cas technology, which avoids many potential pitfalls associated with the Cre/LoxP system.
Fluorescent PSC-Derived Cardiomyocyte Reporter Lines: Generation Approaches and Their Applications in Cardiovascular Medicine.
Recent advances have made pluripotent stem cell (PSC)-derived cardiomyocytes an attractive option to model both normal and diseased cardiac function at the single-cell level. However, in vitro differentiation yields heterogeneous populations of cardiomyocytes and other cell types, potentially confounding phenotypic analyses. Fluorescent PSC-derived cardiomyocyte reporter systems allow specific cell lineages to be labelled, facilitating cell isolation for downstream applications including drug testing, disease modelling and cardiac regeneration. In this review, the different genetic strategies used to generate such reporter lines are presented with an emphasis on their relative technical advantages and disadvantages. Next, we explore how the fluorescent reporter lines have provided insights into cardiac development and cardiomyocyte physiology. Finally, we discuss how exciting new approaches using PSC-derived cardiomyocyte reporter lines are contributing to progress in cardiac cell therapy with respect to both graft adaptation and clinical safety.
Electroporation and genetic supply of Cas9 increase the generation efficiency of CRISPR/Cas9 knock-in alleles in C57BL/6J mouse zygotes.
CRISPR/Cas9 machinery delivered as ribonucleoprotein (RNP) to the zygote has become a standard tool for the development of genetically modified mouse models. In recent years, a number of reports have demonstrated the effective delivery of CRISPR/Cas9 machinery via zygote electroporation as an alternative to the conventional delivery method of microinjection. In this study, we have performed side-by-side comparisons of the two RNP delivery methods across multiple gene loci and conclude that electroporation compares very favourably with conventional pronuclear microinjection, and report an improvement in mutagenesis efficiency when delivering CRISPR via electroporation for the generation of simple knock-in alleles using single-stranded oligodeoxynucleotide (ssODN) repair templates. In addition, we show that the efficiency of knock-in mutagenesis can be further increased by electroporation of embryos derived from Cas9-expressing donor females. The maternal supply of Cas9 to the zygote avoids the necessity to deliver the relatively large Cas9 protein, and high efficiency generation of both indel and knock-in allele can be achieved by electroporation of small single-guide RNAs and ssODN repair templates alone. Furthermore, electroporation, compared to microinjection, results in a higher rate of embryo survival and development. The method thus has the potential to reduce the number of animals used in the production of genetically modified mouse models.
The Configuration of RPA, RAD51, and DMC1 Binding in Meiosis Reveals the Nature of Critical Recombination Intermediates.
Meiotic recombination proceeds via binding of RPA, RAD51, and DMC1 to single-stranded DNA (ssDNA) substrates created after formation of programmed DNA double-strand breaks. Here we report high-resolution in vivo maps of RPA and RAD51 in meiosis, mapping their binding locations and lifespans to individual homologous chromosomes using a genetically engineered hybrid mouse. Together with high-resolution microscopy and DMC1 binding maps, we show that DMC1 and RAD51 have distinct spatial localization on ssDNA: DMC1 binds near the break site, and RAD51 binds away from it. We characterize inter-homolog recombination intermediates bound by RPA in vivo, with properties expected for the critical displacement loop (D-loop) intermediates. These data support the hypothesis that DMC1, not RAD51, performs strand exchange in mammalian meiosis. RPA-bound D-loops can be resolved as crossovers or non-crossovers, but crossover-destined D-loops may have longer lifespans. D-loops resemble crossover gene conversions in size, but their extent is similar in both repair pathways.