isolation-vitro-culture-bone-marrow-derived-macrophages-for-study-no

Isolation and <em>In vitro</em> 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 ...

University of Oxford

&#x7C98;&#x9644; GPCRs &#x63D0;&#x4F9B;&#x57FA;&#x672C;&#x5355;&#x5143;&#x683C;&#x548C;&#x5355;&#x5143;&#x683C;&#x77E9;&#x9635;&#x4E92;&#x52A8;&#x53D1;&#x5C55;&#xFF0C;&#x548C;&#x7EE7;&#x627F;&#x4EBA;&#x7C7B;&#x75BE;&#x75C5;&#x50CF;&#x8FCE;&#x6765;&#x7EFC;&#x5408;&#x5F81;&#x548C;&#x53CC;&#x8FB9; frontoparietal polymicrogyria &#x6709;&#x7275;&#x8FDE;&#x3002;&#x4ED6;&#x4EEC;&#x662F;&#x810A;&#x690E;&#x52A8;&#x7269;&#xFF0C;&#x8DE8;&#x8D8A;&#x4E03;&#x8DE8;&#x819C;&#x86CB;&#x767D;&#x7684;&#x7B2C;&#x4E8C;&#x5927;&#x4E9A;&#x79D1;&#xFF0C;&#x4F46;&#x5927;&#x90E8;&#x5206;&#x7684;&#x8FD9;&#x4E9B;&#x53D7;&#x4F53;&#x7684;&#x4F5C;&#x7528;&#x4ECD;&#x7136;&#x4E0D;&#x7406;&#x89E3;&#x3002;&#x5B64;&#x513F;&#x7C98;&#x9644;&#x6C14;&#x76F8; GPR126 &#x6700;&#x8FD1;&#x5DF2;&#x7ECF;&#x8868;&#x73B0;&#x51FA;&#x5728;&#x6591;&#x9A6C;&#x9C7C;&#x7684;&#x5916;&#x5468;&#x795E;&#x7ECF;&#x9AD3;&#x9798;&#x4E2D;&#x53D1;&#x6325;&#x7740;&#x91CD;&#x8981;&#x7684;&#x4F5C;&#x7528;&#x3002;&#x540C;&#x65F6;&#xFF0C;&#x5168;&#x57FA;&#x56E0;&#x7EC4;&#x5173;&#x8054;&#x7814;&#x7A76;&#x6709;&#x7275;&#x8FDE;&#x5230;&#x8EAB;&#x9AD8;&#x7684;&#x4EBA;&#x53E3;&#x4F5C;&#x4E3A;&#x51B3;&#x5B9A;&#x56E0;&#x7D20; GPR126 &#x8F68;&#x8FF9;&#x53D8;&#x5316;&#x3002;GPR126 &#x5728;&#x54FA;&#x4E73;&#x52A8;&#x7269;&#x4E2D;&#x7684;&#x751F;&#x7406;&#x529F;&#x80FD;&#x4ECD;&#x7136;&#x672A;&#x77E5;&#x3002;&#x6211;&#x4EEC;&#x63CF;&#x8FF0;&#x9F20;&#x6807;&#xFF0C;&#x6709;&#x9488;&#x5BF9;&#x6027;&#x5730;&#x7684;&#x7A81;&#x53D8;&#x7684; GPR126 &#x548C;&#x663E;&#x793A; GPR126 &#x662F;&#x80DA;&#x80CE;&#x7684;&#x53EF;&#x884C;&#x6027;&#x548C;&#x5FC3;&#x8840;&#x7BA1;&#x53D1;&#x5C55;&#x6240;&#x5FC5;&#x9700;&#x7684;&#x3002;

&#x5B64;&#x513F;&#x7C98;&#x9644;&#x6C14;&#x76F8; GPR126 &#x662F;&#x5FC5;&#x9700;&#x7684;&#x80DA;&#x80CE;&#x53D1;&#x80B2;&#x7684;&#x9F20;&#x6807;&#x3002;

Adhesion-GPCRs provide essential cell-cell and cell-matrix interactions in development, and have been implicated in inherited human diseases like Usher Syndrome and bilateral frontoparietal polymicrogyria. They are the second largest subfamily of seven-transmembrane spanning proteins in vertebrates, but the function of most of these receptors is still not understood. The orphan Adhesion-GPCR GPR126 has recently been shown to play an essential role in the myelination of peripheral nerves in zebrafish. In parallel, whole-genome association studies have implicated variation at the GPR126 locus as a determinant of body height in the human population. The physiological function of GPR126 in mammals is still unknown. We describe a targeted mutation of GPR126 in the mouse, and show that GPR126 is required for embryonic viability and cardiovascular development.

The orphan adhesion-GPCR GPR126 is required for embryonic development in the mouse.

Adh&eacute;rence-RCPG fournit des cellules essentielles et les interactions cellule-matrice dans le d&eacute;veloppement et ont &eacute;t&eacute; impliqu&eacute;s dans des maladies h&eacute;r&eacute;ditaires humaines comme le Syndrome d&#39;Usher et polymicrogyria fronto-pari&eacute;tal bilat&eacute;ral. Ils sont la deuxi&egrave;me plus grande sous-famille des prot&eacute;ines s&#39;&eacute;tend sur sept domaines transmembranaires chez les vert&eacute;br&eacute;s, mais la fonction de la plupart de ces r&eacute;cepteurs n&#39;est toujours pas comprise. L&#39;orphelin GPR126 adh&eacute;rence-GPCR a r&eacute;cemment d&eacute;montr&eacute; &agrave; jouer un r&ocirc;le essentiel dans la my&eacute;linisation des nerfs p&eacute;riph&eacute;riques chez le poisson z&egrave;bre. En parall&egrave;le, des &eacute;tudes du g&eacute;nome entier association ont mis en cause au locus GPR126 comme d&eacute;terminant la variation de la hauteur du corps chez l&#39;homme. La fonction physiologique de GPR126 chez les mammif&egrave;res est encore inconnue. Nous d&eacute;crivons une mutation cibl&eacute;e de GPR126 chez la souris et montrent que GPR126 est n&eacute;cessaire pour la viabilit&eacute; embryonnaire et d&eacute;veloppement cardiovasculaire.

Le GPR126 d&#39;adh&eacute;rence-GPCR orphelins est n&eacute;cessaire pour le d&eacute;veloppement embryonnaire chez la souris.

Adh&auml;sion-GPCRs bieten wesentliche Zell-Zell und Zelle-Matrix-Interaktionen in der Entwicklung und haben im menschlichen Erbkrankheiten wie Usher-Syndrom und bilaterale frontoparietale Polymikrogyrie impliziert worden. Sie sind der zweitgr&ouml;&szlig;te Unterfamilie der sieben-spannender Transmembranproteine der Wirbeltiere, aber die Funktion der meisten dieser Rezeptoren ist immer noch nicht verstanden. Vor kurzem hat der verwaisten Adh&auml;sion-GPCR GPR126 spielen eine wesentliche Rolle in der Myelinisierung der peripheren Nerven im Zebrafisch gezeigt. Parallel haben ganze-Genom Association Studies Variation in der GPR126-Locus als bestimmender Faktor der K&ouml;rpergr&ouml;&szlig;e in der menschlichen Bev&ouml;lkerung verwickelt. Die physiologische Funktion des GPR126 bei S&auml;ugetieren ist noch unbekannt. Wir beschreiben eine gezielte Mutation des GPR126 in der Maus, und zeigen, dass GPR126 f&uuml;r embryonale Lebensf&auml;higkeit und Herz-Kreislauf-Entwicklung erforderlich ist.

Die verwaisten Adh&auml;sion-GPCR-GPR126 ist f&uuml;r die Embryonalentwicklung der Maus erforderlich.

Adesione-GPCR fornire essenziale cellula-cellula e le interazioni cellula-matrice in fase di sviluppo e sono stati implicati in malattie ereditarie umane come la sindrome di Usher e polimicrogiria bilaterale frontoparietale. Essi sono la seconda pi&ugrave; grande sottofamiglia delle sette transmembrane spanning proteine nei vertebrati, ma la funzione della maggior parte di questi recettori non &egrave; ancora capita. L&#39;orfano adesione-GPCR GPR126 recentemente ha dimostrato di giocare un ruolo essenziale nella mielinizzazione dei nervi periferici in zebrafish. In parallelo, whole genome association studies hanno implicato la variazione presso il locus GPR126 come un determinante di altezza corpo nella popolazione umana. La funzione fisiologica del GPR126 nei mammiferi &egrave; ancora sconosciuta. Ci descrivono una mutazione mirata di GPR126 nel topo e dimostrare che GPR126 &egrave; necessaria per lo sviluppo cardiovascolare e vitalit&agrave; embrionale.

L&#39;adesione-GPCR orfano GPR126 &egrave; necessaria per lo sviluppo embrionale nel topo.

&#x63A5;&#x7740; Gpcr &#x4E0D;&#x53EF;&#x6B20;&#x306A;&#x7D30;&#x80DE;&#x3068;&#x7D30;&#x80DE;-&#x30DE;&#x30C8;&#x30EA;&#x30C3;&#x30AF;&#x30B9;&#x306E;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x306E;&#x958B;&#x767A;&#x3001;&#x63D0;&#x4F9B;&#x3001;&#x30A2;&#x30C3;&#x30B7;&#x30E3;&#x30FC;&#x75C7;&#x5019;&#x7FA4;&#x3068;&#x4E21;&#x5074;&#x524D;&#x982D;&#x982D;&#x9802;&#x30CD;&#x30C3;&#x30C8;&#x30EF;&#x30FC;&#x30AF; polymicrogyria &#x306E;&#x3088;&#x3046;&#x306A;&#x7D99;&#x627F;&#x3055;&#x308C;&#x305F;&#x3072;&#x3068;&#x75BE;&#x60A3;&#x306B;&#x95A2;&#x4E0E;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x5F7C;&#x3089;&#x306F;&#x3001;&#x810A;&#x690E;&#x52D5;&#x7269;&#x3067; 7 &#x56DE;&#x819C;&#x8CAB;&#x901A;&#x578B;&#x30B9;&#x30D1;&#x30CB;&#x30F3;&#x30B0; &#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306E;&#x4E8C;&#x756A;&#x76EE;&#x306B;&#x5927;&#x304D;&#x3044;&#x4E9C;&#x79D1;&#x304C;&#x3053;&#x308C;&#x3089;&#x306E;&#x53D7;&#x5BB9;&#x4F53;&#x306E;&#x307B;&#x3068;&#x3093;&#x3069;&#x306E;&#x6A5F;&#x80FD;&#x306F;&#x307E;&#x3060;&#x89E3;&#x660E;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x305B;&#x3093;&#x3002;&#x30AA;&#x30FC;&#x30D5;&#x30A1;&#x30F3; GPCR &#x63A5;&#x7740; GPR126 &#x306F;&#x30BC;&#x30D6;&#x30E9;&#x30D5;&#x30A3;&#x30C3;&#x30B7;&#x30E5;&#x672B;&#x68A2;&#x795E;&#x7D4C;&#x306E;&#x8133;&#x9AC4;&#x9798;&#x5F62;&#x6210;&#x306B;&#x91CD;&#x8981;&#x306A;&#x5F79;&#x5272;&#x3092;&#x518D;&#x751F;&#x3059;&#x308B;&#x6700;&#x8FD1;&#x793A;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x4E26;&#x5217;&#x3067;&#x5168;&#x30B2;&#x30CE;&#x30E0;&#x95A2;&#x9023;&#x7814;&#x7A76;&#x4EBA;&#x9593;&#x306E;&#x4EBA;&#x53E3;&#x306E;&#x8EAB;&#x9577;&#x306E;&#x6C7A;&#x5B9A;&#x8981;&#x56E0;&#x3068;&#x3057;&#x3066; GPR126 &#x5EA7;&#x5909;&#x52D5;&#x95A2;&#x4E0E;&#x3057;&#x3066;&#x304F;&#x3060;&#x3055;&#x3044;&#x3002;GPR126 &#x306E;&#x751F;&#x7406;&#x6A5F;&#x80FD;&#x306E;&#x54FA;&#x4E73;&#x985E;&#x306F;&#x307E;&#x3060;&#x4E0D;&#x660E;&#x3067;&#x3059;&#x3002;&#x6211;&#x3005; &#x306F; GPR126 &#x306F;&#x30DE;&#x30A6;&#x30B9;&#x3067;&#x30BF;&#x30FC;&#x30B2;&#x30C3;&#x30C8;&#x3092;&#x7D5E;&#x3063;&#x305F;&#x7A81;&#x7136;&#x5909;&#x7570;&#x3092;&#x8AAC;&#x660E;&#x3057;&#x3066; GPR126 &#x304C;&#x80DA;&#x306E;&#x751F;&#x5B58;&#x304A;&#x3088;&#x3073;&#x5FC3;&#x8840;&#x7BA1;&#x306E;&#x958B;&#x767A;&#x306B;&#x5FC5;&#x8981;&#x3067;&#x3042;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002;

&#x5B64;&#x7ACB;&#x3057;&#x305F;&#x63A5;&#x7740; GPCR GPR126 &#x306F;&#x30DE;&#x30A6;&#x30B9;&#x840C;&#x82BD;&#x671F;&#x306E;&#x958B;&#x767A;&#x304C;&#x5FC5;&#x8981;&#x3067;&#x3059;&#x3002;

&#xC811;&#xCC29; GPCRs &#xD544;&#xC218;&#xC801;&#xC778; &#xC140; &#xBC0F; &#xC140; &#xB9E4;&#xD2B8;&#xB9AD;&#xC2A4; &#xAC1C;&#xBC1C;, &#xC0C1;&#xD638; &#xC791;&#xC6A9;&#xC744; &#xC81C;&#xACF5; &#xD558; &#xACE0; &#xC5B4; &#xC154; &#xC99D;&#xD6C4;&#xAD70;&#xACFC; &#xC591;&#xC790; frontoparietal polymicrogyria &#xAC19;&#xC740; &#xC0C1;&#xC18D; &#xB41C; &#xC778;&#xAC04;&#xC758; &#xC9C8;&#xBCD1;&#xC5D0; &#xC5F0;&#xB8E8; &#xB418;&#xC5B4;. &#xADF8;&#xB4E4;&#xC740; &#xCC99;&#xCD94; &#xB3D9;&#xBB3C;, &#xC2A4;&#xD328;&#xB2DD; 7 &#xB9C9; &#xD6A1;&#xB2E8; &#xB2E8;&#xBC31;&#xC9C8;&#xC758; &#xB450; &#xBC88;&#xC9F8;&#xB85C; &#xD070; &#xC544; &#xD558;&#xC9C0;&#xB9CC; &#xC774;&#xB7EC;&#xD55C; &#xC218;&#xC6A9; &#xCCB4;&#xC758; &#xB300;&#xBD80;&#xBD84;&#xC758; &#xD568;&#xC218;&#xB294; &#xC774;&#xD574; &#xB418;&#xC9C0; &#xC54A;&#xC2B5;&#xB2C8;&#xB2E4;. &#xACE0;&#xC544; GPCR &#xC811;&#xCC29; GPR126 &#xCD5C;&#xADFC; zebrafish &#xC8FC;&#xBCC0; &#xC2E0;&#xACBD; myelination&#xC5D0; &#xD544;&#xC218;&#xC801;&#xC778; &#xC5ED;&#xD560;&#xC744; &#xBCF4;&#xC5EC;&#xC918; &#xC654;&#xB2E4;. &#xB3D9;&#xC2DC;&#xC5D0; &#xC804;&#xCCB4; &#xAC8C;&#xB188; &#xD611;&#xD68C; &#xC5F0;&#xAD6C; &#xC778;&#xAC04;&#xC758; &#xC778;&#xAD6C;&#xC5D0; &#xC788;&#xB294; &#xBAB8; &#xB192;&#xC774;&#xC758; &#xACB0;&#xC815;&#xC73C;&#xB85C; GPR126 &#xB85C;&#xCEE4; &#xC2A4;&#xC5D0;&#xC11C; &#xBCC0;&#xD615;&#xC744; &#xC5F0;&#xB8E8; &#xC788;&#xB2E4;. &#xD3EC;&#xC720;&#xB958;&#xC5D0;&#xC11C; gpr126&#xC758; &#xC0DD;&#xB9AC; &#xAE30;&#xB2A5; &#xC544;&#xC9C1;&#xB3C4; &#xC54C; &#xC218; &#xC788;&#xB2E4;. &#xC6B0;&#xB9AC;&#xB294; &#xB9C8;&#xC6B0;&#xC2A4;&#xC5D0;&#xC11C; gpr126&#xC758; &#xD0C0;&#xAC9F;&#xB41C; &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &#xC124;&#xBA85; &#xD558; &#xACE0; gpr126&#xB294; &#xBC30;&#xC544; &#xC0DD;&#xC874; &#xB2A5;&#xB825; &#xBC0F; &#xC2EC;&#xC7A5; &#xD608;&#xAD00; &#xAC1C;&#xBC1C;&#xC5D0; &#xD544;&#xC694;&#xD55C; &#xBCF4;&#xC5EC;&#xC90D;&#xB2C8;&#xB2E4;.

&#xACE0;&#xC544; &#xC811;&#xCC29; GPCR gpr126&#xB294; &#xB9C8;&#xC6B0;&#xC2A4; &#xBC30;&#xC544; &#xBC1C;&#xB2EC;&#xC5D0; &#xB300; &#xD55C; &#xD544;&#xC694; &#xD569;&#xB2C8;&#xB2E4;.

Adherencia-GPCRs proporcionan esencial c&eacute;lula-c&eacute;lula y c&eacute;lula-matriz interacciones en desarrollo y han sido implicados en enfermedades humanas hereditarias como el s&iacute;ndrome de Usher y Polimicrogiria frontoparietal bilateral. Son la segunda subfamilia de prote&iacute;nas que abarca siete-transmembrana en vertebrados, pero todav&iacute;a no se entiende la funci&oacute;n de la mayor&iacute;a de estos receptores. El hu&eacute;rfano adherencia-GPCR GPR126 se ha demostrado recientemente para jugar un papel esencial en la mielinizaci&oacute;n de los nervios perif&eacute;ricos en el pez cebra. Paralelamente, estudios de Asociaci&oacute;n de genoma completo han implicado la variaci&oacute;n en el locus GPR126 como determinante de la altura del cuerpo en la poblaci&oacute;n humana. La funci&oacute;n fisiol&oacute;gica de GPR126 en mam&iacute;feros se desconoce. Describimos una mutaci&oacute;n espec&iacute;fica de GPR126 en el rat&oacute;n y mostrar que la GPR126 es necesario para la viabilidad embrionaria y desarrollo cardiovascular.

El GPR126 de adherencia-GPCR hu&eacute;rfano es necesaria para el desarrollo embrionario en el rat&oacute;n.

The male reproductive tract expresses a diverse array of adhesion-GPCRs, many in a highly specific and regulated manner. Despite this specificity of expression, little is known about the function of this receptor family in male reproductive physiology. Insights into function are beginning to emerge with the increasing availability of genetically modified mice harbouring mutations in these genes. Gpr64 is the best characterised of the adhesion-GPCRs in the male reproductive system and the phenotype of Gpr64 knock-out mice implicates this receptor in the regulation of fluid absorption in the efferent ducts and proximal epididymis. This chapter summarizes recent data concerning this receptor and other family members in the male reproductive system.

Adhesion-GPCRs in the male reproductive tract.

Oxidative stress is a common etiological feature of neurological disorders, although the pathways that govern defence against reactive oxygen species (ROS) in neurodegeneration remain unclear. We have identified the role of oxidation resistance 1 (Oxr1) as a vital protein that controls the sensitivity of neuronal cells to oxidative stress; mice lacking Oxr1 display cerebellar neurodegeneration, and neurons are less susceptible to exogenous stress when the gene is over-expressed. A conserved short isoform of Oxr1 is also sufficient to confer this neuroprotective property both in vitro and in vivo. In addition, biochemical assays indicate that Oxr1 itself is susceptible to cysteine-mediated oxidation. Finally we show up-regulation of Oxr1 in both human and pre-symptomatic mouse models of amyotrophic lateral sclerosis, indicating that Oxr1 is potentially a novel neuroprotective factor in neurodegenerative disease.

Oxr1 is essential for protection against oxidative stress-induced neurodegeneration.

Development of RNAi-based therapeutics has the potential to revolutionize treatment options for a range of human diseases. However, as with gene therapy, a major barrier to progress is the lack of methods to achieve and measure efficient delivery for systemic administration. We have developed a positive-readout pharmacodynamic transgenic reporter mouse model allowing noninvasive real-time assessment of siRNA activity. The model combines a luciferase reporter gene under the control of regulatory elements from the lac operon of Escherichia coli. Introduction of siRNA targeting lac repressor results in increased luciferase expression in cells where siRNA is biologically active. Five founder luciferase-expressing and three founder Lac-expressing lines were generated and characterized. Mating of ubiquitously expressing luciferase and lac lines generated progeny in which luciferase expression was significantly reduced compared with the parental line. Administration of isopropyl β-D-1-thiogalactopyranoside either in drinking water or given intraperitoneally increased luciferase expression in eight of the mice examined, which fell rapidly when withdrawn. Intraperitoneal administration of siRNA targeting lac in combination with Lipofectamine 2000 resulted in increased luciferase expression in the liver while control nontargeting siRNA had no effect. We believe a sensitive positive readout pharmacodynamics reporter model will be of use to the research community in RNAi-based vector development.Molecular Therapy-Nucleic Acids (2013) 2, e133; doi:10.1038/mtna.2013.63; published online 19 November 2013. 

Development of a Positive-readout Mouse Model of siRNA Pharmacodynamics.

Amyotrophic lateral sclerosis is a devastating neurodegenerative disorder characterized by the progressive loss of spinal motor neurons. While the aetiological mechanisms underlying the disease remain poorly understood, oxidative stress is a central component of amyotrophic lateral sclerosis and contributes to motor neuron injury. Recently, oxidation resistance 1 (OXR1) has emerged as a critical regulator of neuronal survival in response to oxidative stress, and is upregulated in the spinal cord of patients with amyotrophic lateral sclerosis. Here, we tested the hypothesis that OXR1 is a key neuroprotective factor during amyotrophic lateral sclerosis pathogenesis by crossing a new transgenic mouse line that overexpresses OXR1 in neurons with the SOD1(G93A) mouse model of amyotrophic lateral sclerosis. Interestingly, we report that overexpression of OXR1 significantly extends survival, improves motor deficits, and delays pathology in the spinal cord and in muscles of SOD1(G93A) mice. Furthermore, we find that overexpression of OXR1 in neurons significantly delays non-cell-autonomous neuroinflammatory response, classic complement system activation, and STAT3 activation through transcriptomic analysis of spinal cords of SOD1(G93A) mice. Taken together, these data identify OXR1 as the first neuron-specific antioxidant modulator of pathogenesis and disease progression in SOD1-mediated amyotrophic lateral sclerosis, and suggest that OXR1 may serve as a novel target for future therapeutic strategies. 

Neuron-specific antioxidant OXR1 extends survival of a mouse model of amyotrophic lateral sclerosis.

The cost to develop a new drug from target discovery to market is a staggering $1.8 billion, largely due to the very high attrition rate of drug candidates and the lengthy transition times during development. Open access is an emerging model of open innovation that places no restriction on the use of information and has the potential to accelerate the development of new drugs.

Open Access Could Transform Drug Discovery: A Case Study of JQ1.

 For many patients with kidney failure, the cause and underlying defect remain unknown. Here, we describe a novel mechanism of a genetic order characterized by renal Fanconi syndrome and kidney failure. We clinically and genetically characterized members of five families with autosomal dominant renal Fanconi syndrome and kidney failure. We performed genome-wide linkage analysis, sequencing, and expression studies in kidney biopsy specimens and renal cells along with knockout mouse studies and evaluations of mitochondrial morphology and function. Structural studies examined the effects of recognized mutations. The renal disease in these patients resulted from monoallelic mutations in the gene encoding glycine amidinotransferase (GATM), a renal proximal tubular enzyme in the creatine biosynthetic pathway that is otherwise associated with a recessive disorder of creatine deficiency.  analysis showed that the particular  mutations, identified in 28 members of the five families, create an additional interaction interface within the GATM protein and likely cause the linear aggregation of GATM observed in patient biopsy specimens and cultured proximal tubule cells. GATM aggregates-containing mitochondria were elongated and associated with increased ROS production, activation of the NLRP3 inflammasome, enhanced expression of the profibrotic cytokine IL-18, and increased cell death. In this novel genetic disorder, fully penetrant heterozygous missense mutations in  trigger intramitochondrial fibrillary deposition of GATM and lead to elongated and abnormal mitochondria. We speculate that this renal proximal tubular mitochondrial pathology initiates a response from the inflammasome, with subsequent development of kidney fibrosis.

Glycine Amidinotransferase (GATM), Renal Fanconi Syndrome, and Kidney Failure.

Physiological effects of cellular hypoxia are sensed by prolyl hydroxylase (PHD) enzymes which regulate HIFs. Genetic interventions on HIF/PHD pathways reveal multiple phenotypes that extend the known biology of hypoxia. Recent studies unexpectedly implicate HIF in aspects of multiple immune and inflammatory pathways. However such studies are often limited by systemic lethal effects and/or use tissue-specific recombination systems, which are inherently irreversible, un-physiologically restricted and difficult to time. To study these processes better we developed recombinant mice which express tetracycline-regulated shRNAs broadly targeting the main components of the HIF/PHD pathway, permitting timed bi-directional intervention. We have shown that stabilization of HIF levels in adult mice through PHD2 enzyme silencing by RNA interference, or inducible recombination of floxed alleles, results in multi-lineage leukocytosis and features of autoimmunity. This phenotype was rapidly normalized on re-establishment of the hypoxia-sensing machinery when shRNA expression was discontinued. In both situations these effects were mediated principally through the Hif2a isoform. Assessment of cells bearing regulatory T cell markers from these mice revealed defective function and pro-inflammatory effects in vivo. We believe our findings have shown a new role for the PHD2/Hif2a couple in the reversible regulation of T cell and immune activity.

Systemic silencing of PHD2 causes reversible immune regulatory dysfunction.

There is significant controversy over whether patients have a 'right not to know' information relevant to their health. Some arguments for limiting such a right appeal to potential burdens on others that a patient's avoidable ignorance might generate. This paper develops this argument by extending it to cases where refusal of relevant information may generate greater demands on a publicly funded healthcare system. In such cases, patients may have an 'obligation to know'. However, we cannot infer from the fact that a patient has an obligation to know that she does not also have a right not to know. The right not to know is held against medical professionals at a formal institutional level. We have reason to protect patients' control over the information that they receive, even if in individual instances patients exercise this control in ways that violate obligations.

The right not to know and the obligation to know.

'The right not to know and the obligation to know', response to commentaries.

The idea of using responsibility in the allocation of healthcare resources has been criticized for, among other things, too readily abandoning people who are responsible for being very badly off. One response to this problem is that while responsibility can play a role in resource allocation, it cannot do so if it will leave those who are responsible below a "sufficiency" threshold. This paper considers first whether a view can be both distinctively sufficientarian and allow responsibility to play a role even for those who will be left with very poor health. It then draws several further distinctions that may affect the application of responsibility at this level. We conclude that a more plausible version of the sufficientarian view is to allow a role for responsibility where failure to do so will leave someone else who is not responsible below the sufficiency threshold. However, we suggest that individuals must exhibit "sufficient responsibility" in order for this to apply, involving both a sufficient level of control and an avoidable failure to respond adequately to reasons for action.

From Sufficient Health to Sufficient Responsibility.

Grow the pie, or the resource shuffle? Commentary on Munthe, Fumagalli and Malmqvist.

Healthcare systems need to consider not only how to prevent error, but how to respond to errors when they occur. In the United Kingdom's National Health Service, one strand of this latter response is the 'No Blame Culture', which draws attention from individuals and towards systems in the process of understanding an error. Defences of the No Blame Culture typically fail to distinguish between  someone and holding them responsible. This article argues for a 'responsibility culture', where healthcare professionals are held responsible in cases of foreseeable and avoidable errors. We demonstrate how healthcare professionals can justifiably be held responsible for their errors even though they work in challenging circumstances. We then review the idea of 'responsibility without blame', applying this to cases of error in healthcare. Sensitive to the undesirable effects of blaming healthcare professionals and to the moral significance of holding individuals accountable, we argue that a responsibility culture has significant advantages over a No Blame Culture due to its capacity to enhance patient safety and support medical professionals in learning from their mistakes, while also recognising and validating the legitimate sense of responsibility that many medical professionals feel following avoidable error, and motivating medical professionals to report errors.

No Blame No Gain? From a No Blame Culture to a Responsibility Culture in Medicine.

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.

Genome-wide CRISPR/Cas9-knockout in human induced Pluripotent Stem Cell (iPSC)-derived macrophages.

There is an ongoing debate in medicine about whether patients have a 'right not to know' pertinent medical information, such as diagnoses of life-altering diseases. While this debate has employed various ethical concepts, probably the most widely-used by both defenders and detractors of the right is autonomy. Whereas defenders of the right not to know typically employ a 'liberty' conception of autonomy, according to which to be autonomous involves doing what one wants to do, opponents of the right not to know often employ a 'duty' understanding, viewing autonomy as involving an obligation to be self-governing. The central contribution of this paper is in showing that neither view of autonomy can reasonably be said to support the extreme stances on the right not to know that they are sometimes taken to. That is, neither can a liberty view properly defend a right not to know without limits, nor can a duty view form the basis of an absolute rejection of the right not to know. While there is still theoretical distance between these two approaches, we conclude that the views are considerably closer on this issue than they first appear, opening the way for a possible compromise.

The Right Not to Know: some Steps towards a Compromise.

 The K  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.  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.   We found that the R1183W mutation impaired inhibition of K  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.  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.

Phenotype of a transient neonatal diabetes point mutation (SUR1-R1183W) in mice.

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.

'Personal Health Surveillance': The Use of mHealth in Healthcare Responsibilisation.

How should we respond to patients who do not wish to take on the responsibility and burdens of making decisions about their own care? In this paper, we argue that existing models of decision-making in modern healthcare are ill-equipped to cope with such patients and should be supplemented by an "appointed fiduciary" model where decision-making authority is formally transferred to a medical professional. Healthcare decisions are often complex and for patients can come at time of vulnerability. While this does not undermine their capacity, it can be excessively burdensome. Most existing models of decision-making mandate that patients with capacity must retain ultimate responsibility for decisions. An appointed fiduciary model provides a formalized mechanism through which those few patients who wish to defer responsibility can hand over decision-making authority. By providing a formal structure for deferring to an appointed fiduciary, the confusions and risks of the informal transfers that can occur in practice are avoided. Finally, we note how appropriate governance and law can provide safeguards against risks to the welfare of patients and medical professionals.

Article	Year
The orphan adhesion-GPCR GPR126 is required for embryonic development in the mouse. PloS one\| PubMed ID: 21124978	2010
Adhesion-GPCRs in the male reproductive tract. Advances in experimental medicine and biology\| PubMed ID: 21618837	2010
Oxr1 is essential for protection against oxidative stress-induced neurodegeneration. PLoS genetics\| PubMed ID: 22028674	2011
Development of a Positive-readout Mouse Model of siRNA Pharmacodynamics. Molecular therapy. Nucleic acids\| PubMed ID: 24253258	2013
Neuron-specific antioxidant OXR1 extends survival of a mouse model of amyotrophic lateral sclerosis. Brain : a journal of neurology\| PubMed ID: 25753484	2015
Open Access Could Transform Drug Discovery: A Case Study of JQ1. Expert opinion on drug discovery\| PubMed ID: 26791045	2016
Glycine Amidinotransferase (GATM), Renal Fanconi Syndrome, and Kidney Failure. Journal of the American Society of Nephrology : JASN\| PubMed ID: 29654216	2018
Systemic silencing of PHD2 causes reversible immune regulatory dysfunction. The Journal of clinical investigation\| PubMed ID: 31162141	2019
The right not to know and the obligation to know. Journal of medical ethics\| PubMed ID: 32350031	2020
'The right not to know and the obligation to know', response to commentaries. Journal of medical ethics\| PubMed ID: 32350033	2020
From Sufficient Health to Sufficient Responsibility. Journal of bioethical inquiry\| PubMed ID: 32696113	2020
Grow the pie, or the resource shuffle? Commentary on Munthe, Fumagalli and Malmqvist. Journal of medical ethics\| PubMed ID: 33335069	2021
No Blame No Gain? From a No Blame Culture to a Responsibility Culture in Medicine. Journal of applied philosophy\| PubMed ID: 33362325	2020
Genome-wide CRISPR/Cas9-knockout in human induced Pluripotent Stem Cell (iPSC)-derived macrophages. Scientific reports\| PubMed ID: 33608581	2021
The Right Not to Know: some Steps towards a Compromise. Ethical theory and moral practice : an international forum\| PubMed ID: 34335078	2021
Phenotype of a transient neonatal diabetes point mutation (SUR1-R1183W) in mice. Wellcome open research\| PubMed ID: 34368464	2020
'Personal Health Surveillance': The Use of mHealth in Healthcare Responsibilisation. Public health ethics\| PubMed ID: 34899983	2021
Doctors as Appointed Fiduciaries: A Supplemental Model for Medical Decision-Making. Cambridge quarterly of healthcare ethics : CQ : the international journal of healthcare ethics committees\| PubMed ID: 35049458	2022

Ben Davies

Biography

JoVE Journal Publications

Other Publications