Heat shock induces rapid resorption of primary cilia

Primary cilia are involved in important developmental and disease pathways, such as the regulation of neurogenesis and tumorigenesis. They function as sensory antennae and are essential in the regulation of key extracellular signalling systems. We have investigated the effects of cell stress on primary cilia. Exposure of mammalian cells in vitro, and zebrafish cells in vivo, to elevated temperature resulted in the rapid loss of cilia by resorption. In mammalian cells loss of cilia correlated with a reduction in hedgehog signalling. Heat-shock-dependent loss of cilia was decreased in cells where histone deacetylases (HDACs) were inhibited, suggesting resorption is mediated by the axoneme-localised tubulin deacetylase HDAC6. In thermotolerant cells the rate of ciliary resorption was reduced. This implies a role for molecular chaperones in the maintenance of primary cilia. The cytosolic chaperone Hsp90 localises to the ciliary axoneme and its inhibition resulted in cilia loss. In the cytoplasm of unstressed cells, Hsp90 is known to exist in a complex with HDAC6. Moreover, immediately after heat shock Hsp90 levels were reduced in the remaining cilia. We hypothesise that ciliary resorption serves to attenuate cilia-mediated signalling pathways in response to extracellular stress, and that this mechanism is regulated in part by HDAC6 and Hsp90

Mutations in NNT encoding nicotinamide nucleotide transhydrogenase cause familial glucocorticoid deficiency

This work has been supported by the Medical Research Council UK (New Investigator Research Grant G0801265 to L.A.M., Clinical Research Training Fellowship Grant G0901980 to C.R.H. and Project Grant G0700767 to P.J.K.)

Whole-Exome Sequencing in the Differential Diagnosis of Primary Adrenal Insufficiency in Children.

Adrenal insufficiency is a rare, but potentially fatal medical condition. In children, the cause is most commonly congenital and in recent years a growing number of causative gene mutations have been identified resulting in a myriad of syndromes that share adrenal insufficiency as one of the main characteristics. The evolution of adrenal insufficiency is dependent on the variant and the particular gene affected, meaning that rapid and accurate diagnosis is imperative for effective treatment of the patient. Common practice is for candidate genes to be sequenced individually, which is a time-consuming process and complicated by overlapping clinical phenotypes. However, with the availability, and increasing cost effectiveness of whole-exome sequencing, there is the potential for this to become a powerful diagnostic tool. Here, we report the results of whole-exome sequencing of 43 patients referred to us with a diagnosis of familial glucocorticoid deficiency (FGD) who were mutation negative for MC2R, MRAP, and STAR the most commonly mutated genes in FGD. WES provided a rapid genetic diagnosis in 17/43 sequenced patients, for the remaining 60% the gene defect may be within intronic/regulatory regions not covered by WES or may be in gene(s) representing novel etiologies. The diagnosis of isolated or familial glucocorticoid deficiency was only confirmed in 3 of the 17 patients, other genetic diagnoses were adrenal hypo- and hyperplasia, Triple A, and autoimmune polyendocrinopathy syndrome type I, emphasizing both the difficulty of phenotypically distinguishing between disorders of PAI and the utility of WES as a tool to achieve this

A False Start in the Race Against Doping in Sport: Concerns With Cycling’s Biological Passport

Professional cycling has suffered from a number of doping scandals. The sport’s governing bodies have responded by implementing an aggressive new antidoping program known as the biological passport. Cycling’s biological passport marks a departure from traditional antidoping efforts, which have focused on directly detecting prohibited substances in a cyclist’s system. Instead, the biological passport tracks biological variables in a cyclist’s blood and urine over time, monitoring for fluctuations that are thought to indirectly reveal the effects of doping. Although this method of indirect detection is promising, it also raises serious legal and scientific concerns. Since its introduction, the cycling community has debated the reliability of indirect biological-passport evidence and the clarity, consistency, and transparency of its use in proving doping violations. Such uncertainty undermines the legitimacy of finding cyclists guilty of doping based on this indirect evidence alone. Antidoping authorities should address these important concerns before continuing to pursue doping sanctions against cyclists solely on the basis of their biological passports

Calcium oxalate modulation of tubular epithelial cell mitochondria: oxidative vulnerability due to restricted glutathione homeostasis.

Calcium oxalate (COM) crystals are the commonest component of kidney stones. These arise mainly in the distal tubules and collecting ducts. To gain further insight for the cellular damage in terms of oxidative stress caused by COM deposition, in vitro and in vivo model studies were performed. In vitro In renal distal tubule cells, COM and free oxalate treatment caused a 3- and 2-fold increase respectively in superoxide (O2*") formation, originating from mitochondria. This was measured by lucigenin chemiluminescence in digitonin permeabilised cells. However, hydroxyapatite produced a much lower but significant enhancement of 02*", whilst other micro-particles, uric acid crystals, brushite, zymosan, and latex beads had no effect. When EDTA was omitted during O2*" monitoring, COM induced mitochondrial 02*" was ablated indicating a requirement for the release of free oxalate. Mitochondrial oxalate uptake was studied by employing different oxalate transport inhibitors. Omitting phosphate from the media or using mersalyl both of which block dicarboxylate transport, caused a significant decrease in the 02*" formation evoked by COM treatments. Using the membrane potential sensitive-probe tetramethylrhodamine methyl ester (TMRM) together with confocal microscopy, evidence is presented that in cells where COM binding had occurred a marked change in the mitochondrial membrane potential (Aij/m) occurred. COM also modulated intracellular Ca2+ signalling as demonstrated using the Ca2- sensitive dye Fura-2 AM, and this was via a non-mitochondrial mechanism. In Vivo Using a rat model of crystalluria and renal stones initiated by treatment with ethylene glycol (EG) and 1, 25-dihydroxycholecalciferol (DHC), nephrolithiasis arose in kidneys and this was linked to oxidative stress. In the EG + DHC treated animals where crystalluria was evident, this oxidative insult was manifest by a decrease in total and mitochondrial glutathione concentration, as well as an increased activity of glucose-6-phosphate dehydrogenase. Severe kidney damage at the mitochondria level was a further observation, indicated by the diminished O2 consumption resulting in a lowered O2 production. In addition, histopathological analysis revealed increased renal tubular pathology characterised by obstruction, distension and interstitial inflammation. The above findings were not observed in hyperoxaluria (EG) or calciuria (DHC) and are therefore a direct effect of crystal formation in kidney distal tubules that have implications in kidney stone disease which are discussed

Microsphere-based binding assays for organic pollutants

In this thesis the “proof of principle” of a preliminary “prototype kit” for the screening of dioxin-like PCBs, PBDEs and PAHs in fish combined with two different simplified generic cleanup procedures, according to the fish fat content, is presented. Toxicants such as the PCBs, PBDEs and PAHs and POPs mixtures (such as the technical mixtures of PCBs (Aroclors)) can be detected at relative low levels (with IC50 values 55±15, 2±0.4 and 4±0.5 ppb for the PCB77, PBDE47 and BaP, respectively) in fish samples in a multiplex, simple and inexpensive manner compared to existing techniques. The 3-plex immunoassay can be performed in two different detection platforms: the traditional flow cytometer- and the new imaging-based bead analyzers. The latter system offers a cheaper analysis and it is an easier transportable platform than the flow cytometer.</p

Oxidative stress and adrenocortical insufficiency

This work has been supported by the Wellcome Trust (Clinical Research Training Fellowship grant number WT095984AIA to R P) and the Medical Research Council UK (project grant number MR/K020455/1 to L A M

Adapted qPCR methodology to detect telomere oxidative damage (TOD) associated with telomere attrition (TA) in murine tissue

Numerous Studies suggest that oxidative stress (OS) is associated with telomere attrition (TA) but they rarely directly assay telomeric oxidative damage (TOD). TOD can alter shelterin binding, induce replication fork stalling and inhibit telomerase activity. We used a previously published method 1 to measure TOD and adapted it for use in murine tissue. Our model is a C57BL/6J mouse strain and we used qPCR and a formamidopyrimidine DNA glycosylase (FPG) enzyme-based qPCR method to directly determine TOD. FPG analysis compares reaction efficiencies before and after treatment. FPG is normally involved in base excision repairs pathways, removing oxidized Purines (notably 8-Oxo Guanine). A reduction in the efficiency of the qPCR indicates the presence of 8-oxo guanine in the telomeric tract ie TOD

Loss of Nnt Increases Expression of Oxidative Phosphorylation Complexes in C57BL/6J Hearts

Nicotinamide nucleotide transhydrogenase (NNT) is a proton pump in the inner mitochondrial membrane that generates reducing equivalents in the form of NAPDH, which can be used for anabolic pathways or to remove reactive oxygen species (ROS). A number of studies have linked NNT dysfunction to cardiomyopathies and increased risk of atherosclerosis; however, biallelic mutations in humans commonly cause a phenotype of adrenal insufficiency, with rare occurrences of cardiac dysfunction and testicular tumours. Here, we compare the transcriptomes of the hearts, adrenals and testes from three mouse models: the C57BL/6N, which expresses NNT; the C57BL/6J, which lacks NNT; and a third mouse, expressing the wild-type NNT sequence on the C57BL/6J background. We saw enrichment of oxidative phosphorylation genes in the C57BL/B6J in the heart and adrenal, possibly indicative of an evolved response in this substrain to loss of Nnt. However, differential gene expression was mainly driven by mouse background with some changes seen in all three tissues, perhaps reflecting underlying genetic differences between the C57BL/B6J and -6N substrains

School architecture from the perspective of autism: a reflection on visual perception

Este estudo busca compreender como as particularidades do processamento sensorial, de pessoas com Transtorno do Espectro do Autismo (TEA), impactam a maneira como estes usuários extraem as informações visuais do ambiente construído e como tais características influenciam o projeto de arquitetura escolar. A relevância desta investigação baseia-se no grande número de crianças e adolescentes que são diagnosticados com TEA no Brasil e no mundo a cada ano. A partir do método correlacional, os temas chaves da pesquisa, que são oriundos de diferentes campos de conhecimentos como: arquitetura, educação e psicologia, foram relacionados a partir de revisões bibliográficas. A metodologia qualitativa, através da lógica argumentativa, possibilitou a apropriação de informações do âmbito do neurodesenvolvimento, a fim de construir respostas arquitetônicas para as questões observadas. Assim, a partir da compreensão do processamento sensorial atípico, dos autistas, foram estruturadas observações projetuais, que visam tornar os projetos arquitetônicos escolares, com foco no conforto visual dos educandos com TEA, sensível às necessidades destes usuários.This study seeks to understand how the particularities of sensory processing, of people with Autism Spectrum Disorder (ASD), impact the way these users extract visual information from the built environment and how these characteristics influence the school architecture project. The relevance of this investigation is based on the large number of children and adolescents who are diagnosed with ASD in Brazil and worldwide each year. From the correlational method, the key themes of the research, which come from different fields of knowledge such as: architecture, education and psychology, were related from bibliographic reviews. The qualitative methodology, through argumentative logic, enabled the appropriation of information from the scope of neurodevelopment, in order to build architectural answers to the observed questions. Thus, from the understanding of the atypical sensory processing of autistic people, design observations were structured, which aim to make school architectural projects, focusing on the visual comfort of students with ASD, sensitive to the needs of these users