Publications by authors named "Sarah Gale"

25 Publications

  • Page 1 of 1

Loss of SNORA73 reprograms cellular metabolism and protects against steatohepatitis.

Nat Commun 2021 09 1;12(1):5214. Epub 2021 Sep 1.

Joslin Diabetes Center, Harvard Medical School, Boston, MA, USA.

Dyslipidemia and resulting lipotoxicity are pathologic signatures of metabolic syndrome and type 2 diabetes. Excess lipid causes cell dysfunction and induces cell death through pleiotropic mechanisms that link to oxidative stress. However, pathways that regulate the response to metabolic stress are not well understood. Herein, we show that disruption of the box H/ACA SNORA73 small nucleolar RNAs encoded within the small nucleolar RNA hosting gene 3 (Snhg3) causes resistance to lipid-induced cell death and general oxidative stress in cultured cells. This protection from metabolic stress is associated with broad reprogramming of oxidative metabolism that is dependent on the mammalian target of rapamycin signaling axis. Furthermore, we show that knockdown of SNORA73 in vivo protects against hepatic steatosis and lipid-induced oxidative stress and inflammation. Our findings demonstrate a role for SNORA73 in the regulation of metabolism and lipotoxicity.
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http://dx.doi.org/10.1038/s41467-021-25457-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8410784PMC
September 2021

Validation of Trifluoromethylphenyl Diazirine Cholesterol Analogues As Cholesterol Mimetics and Photolabeling Reagents.

ACS Chem Biol 2021 08 6;16(8):1493-1507. Epub 2021 Aug 6.

Aliphatic diazirine analogues of cholesterol have been used previously to elaborate the cholesterol proteome and identify cholesterol binding sites on proteins. Cholesterol analogues containing the trifluoromethylphenyl diazirine (TPD) group have not been reported. Both classes of diazirines have been prepared for neurosteroid photolabeling studies and their combined use provided information that was not obtainable with either diazirine class alone. Hence, we prepared cholesterol TPD analogues and used them along with previously reported aliphatic diazirine analogues as photoaffinity labeling reagents to obtain additional information on the cholesterol binding sites of the pentameric ligand-gated ion channel (GLIC). We first validated the TPD analogues as cholesterol substitutes and compared their actions with those of previously reported aliphatic diazirines in cell culture assays. All the probes bound to the same cholesterol binding site on GLIC but with differences in photolabeling efficiencies and residues identified. Photolabeling of mammalian (HEK) cell membranes demonstrated differences in the pattern of proteins labeled by the two classes of probes. Collectively, these date indicate that cholesterol photoaffinity labeling reagents containing an aliphatic diazirine or TPD group provide complementary information and will both be useful tools in future studies of cholesterol biology.
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http://dx.doi.org/10.1021/acschembio.1c00364DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8740335PMC
August 2021

Interleukin-22 signaling attenuates necrotizing enterocolitis by promoting epithelial cell regeneration.

Cell Rep Med 2021 Jun 15;2(6):100320. Epub 2021 Jun 15.

Department of Pediatrics, Division of Newborn Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA.

Necrotizing enterocolitis (NEC) is a deadly intestinal inflammatory disorder that primarily affects premature infants and lacks adequate therapeutics. Interleukin (IL)-22 plays a critical role in gut barrier maintenance, promoting epithelial regeneration, and controlling intestinal inflammation in adult animal models. However, the importance of IL-22 signaling in neonates during NEC remains unknown. We investigated the role of IL-22 in the neonatal intestine under homeostatic and inflammatory conditions by using a mouse model of NEC. Our data reveal that expression in neonatal murine intestine is negligible until weaning, and both human and murine neonates lack IL-22 production during NEC. Mice deficient in IL-22 or lacking the IL-22 receptor in the intestine display a similar susceptibility to NEC, consistent with the lack of endogenous IL-22 during development. Strikingly, treatment with recombinant IL-22 during NEC substantially reduces inflammation and enhances epithelial regeneration. These findings may provide a new therapeutic strategy to attenuate NEC.
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http://dx.doi.org/10.1016/j.xcrm.2021.100320DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8233697PMC
June 2021

Whole exome sequencing and functional characterization increase diagnostic yield in siblings with a 46, XY difference of sexual development (DSD).

J Steroid Biochem Mol Biol 2021 09 10;212:105908. Epub 2021 May 10.

Edward Mallinckrodt Department of Pediatrics, Washington University School of Medicine and St. Louis Children's Hospital, St. Louis, MO, USA. Electronic address:

Pathogenic biallelic variants in HSD17B3 result in 17β-hydroxysteroid dehydrogenase 3 (17β-HSD3) deficiency, variable disruption of testosterone production, and phenotypic diversity among 46, XY individuals with differences of sexual development (DSDs). We performed quad whole exome sequencing (WES) on two male siblings with microphallus, perineal hypospadias, and bifid scrotum and their unaffected parents. Both male siblings were compound heterozygous for a rare pathogenic HSD17B3 variant (c.239 G > A, p.R80Q) previously identified among individuals with 17β-HSD3 deficiency and a HSD17B3 variant (c.641A > G, p.E214 G) of uncertain significance. Following WES, the siblings underwent hCG stimulation testing with measurement of testosterone, androstenedione, and dihydrotestosterone which was non-diagnostic. To confirm pathogenicity of the HSD17B3 variants, we performed transient transfection of HEK-293 cells and measured conversion of radiolabeled androstenedione to testosterone. Both HSD17B3 variants decreased conversion of radiolabeled androstenedione to testosterone. As pathogenic HSD17B3 variants are rare causes of 46, XY DSD and hCG stimulation testing may not be diagnostic for 17β-HSD3 deficiency, WES in 46, XY individuals with DSDs can increase diagnostic yield and identify genomic variants for functional characterization of disruption of testosterone production.
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http://dx.doi.org/10.1016/j.jsbmb.2021.105908DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8725205PMC
September 2021

Indole-3-Carbinol-Dependent Aryl Hydrocarbon Receptor Signaling Attenuates the Inflammatory Response in Experimental Necrotizing Enterocolitis.

Immunohorizons 2021 04 27;5(4):193-209. Epub 2021 Apr 27.

Division of Newborn Medicine, Washington University School of Medicine, St. Louis, MO;

Necrotizing enterocolitis (NEC) causes significant morbidity and mortality in premature infants; therefore, the identification of therapeutic and preventative strategies against NEC remains a high priority. The ligand-dependent transcription factor aryl hydrocarbon receptor (AhR) is well known to contribute to the regulation of intestinal microbial communities and amelioration of intestinal inflammation. However, the role of AhR signaling in NEC is unclear. Experimental NEC was induced in 4-d-old wild-type mice or mice lacking AhR expression in the intestinal epithelial cells or AhR expression in CD11c cells (AhR) by subjecting animals to twice daily hypoxic stress and gavage feeding with formula supplemented with LPS and enteric bacteria. During NEC, compared with wild-type mice treated with vehicle, littermates treated with an AhR proligand, indole-3-carbinol, had reduced expression of and a scavenger receptor that mediates dendritic cell activation and the recognition and clearance of bacterial pathogens by macrophages. Furthermore, indole-3-carbinol treatment led to the downregulation of genes involved in cytokine and chemokine, as revealed by pathway enrichment analysis. AhR expression in the intestinal epithelial cells and their cre-negative mouse littermates were similarly susceptible to experimental NEC, whereas AhR mice with NEC exhibited heightened inflammatory responses compared with their cre-negative mouse littermates. In seeking to determine the mechanisms involved in this increased inflammatory response, we identified the Tim-4 monocyte-dependent subset of macrophages as increased in AhR mice compared with their cre-negative littermates. Taken together, these findings demonstrate the potential for AhR ligands as a novel immunotherapeutic approach to the management of this devastating disease.
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http://dx.doi.org/10.4049/immunohorizons.2100018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8173979PMC
April 2021

Monitoring the itinerary of lysosomal cholesterol in Niemann-Pick Type C1-deficient cells after cyclodextrin treatment.

J Lipid Res 2020 03 27;61(3):403-412. Epub 2020 Jan 27.

Department of Medicine,Washington University School of Medicine, St. Louis, MO; Joslin Diabetes Center,Harvard Medical School, Boston, MA.

Niemann-Pick disease type C (NPC) disease is a lipid-storage disorder that is caused by mutations in the genes encoding NPC proteins and results in lysosomal cholesterol accumulation. 2-Hydroxypropyl-β-cyclodextrin (CD) has been shown to reduce lysosomal cholesterol levels and enhance sterol homeostatic responses, but CD's mechanism of action remains unknown. Recent work provides evidence that CD stimulates lysosomal exocytosis, raising the possibility that lysosomal cholesterol is released in exosomes. However, therapeutic concentrations of CD do not alter total cellular cholesterol, and cholesterol homeostatic responses at the ER are most consistent with increased ER membrane cholesterol. To address these disparate findings, here we used stable isotope labeling to track the movement of lipoprotein cholesterol cargo in response to CD in NPC1-deficient U2OS cells. Although released cholesterol was detectable, it was not associated with extracellular vesicles. Rather, we demonstrate that lysosomal cholesterol trafficks to the plasma membrane (PM), where it exchanges with lipoprotein-bound cholesterol in a CD-dependent manner. We found that in the absence of suitable extracellular cholesterol acceptors, cholesterol exchange is abrogated, cholesterol accumulates in the PM, and reesterification at the ER is increased. These results support a model in which CD promotes intracellular redistribution of lysosomal cholesterol, but not cholesterol exocytosis or efflux, during the restoration of cholesterol homeostatic responses.
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http://dx.doi.org/10.1194/jlr.RA119000571DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7053843PMC
March 2020

2-Hydroxypropyl-β-cyclodextrin is the active component in a triple combination formulation for treatment of Niemann-Pick C1 disease.

Biochim Biophys Acta Mol Cell Biol Lipids 2019 10 30;1864(10):1545-1561. Epub 2019 Apr 30.

Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA. Electronic address:

Niemann-Pick type C1 (NPC1) disease is a fatal neurovisceral disease for which there are no FDA approved treatments, though cyclodextrin (HPβCD) slows disease progression in preclinical models and in an early phase clinical trial. Our goal was to evaluate the mechanism of action of a previously described combination-therapy, Triple Combination Formulation (TCF) - comprised of the histone deacetylase inhibitor (HDACi) vorinostat/HPβCD/PEG - shown to prolong survival in Npc1 mice. In these studies, TCF's benefit was attributed to enhanced vorinostat pharmacokinetics (PK). Here, we show that TCF reduced lipid storage, extended lifespan, and preserved neurological function in Npc1 mice. Unexpectedly, substitution of an inactive analog for vorinostat in TCF revealed similar efficacy. We demonstrate that the efficacy of TCF was attributable to enhanced HPβCD PK and independent of NPC1 protein expression. We conclude that although HDACi effectively reduce cholesterol storage in NPC1-deficient cells, HDACi are ineffective in vivo in Npc1 mice.
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http://dx.doi.org/10.1016/j.bbalip.2019.04.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6679735PMC
October 2019

Synthesis and characterization of diazirine alkyne probes for the study of intracellular cholesterol trafficking.

J Lipid Res 2019 03 7;60(3):707-716. Epub 2019 Jan 7.

Departments of Medicine Washington University School of Medicine, St. Louis, MO 63110.

Cholesterol is an essential structural component of cellular membranes and precursor molecule for oxysterol, bile acid, and hormone synthesis. The study of intracellular cholesterol trafficking pathways has been limited in part due to a lack of suitable cholesterol analogues. Herein, we developed three novel diazirine alkyne cholesterol probes: LKM38, KK174, and KK175. We evaluated these probes as well as a previously described diazirine alkyne cholesterol analogue, -sterol, for their fidelity as cholesterol mimics and for study of cholesterol trafficking. LKM38 emerged as a promising cholesterol mimic because it both sustained the growth of cholesterol-auxotrophic cells and appropriately regulated key cholesterol homeostatic pathways. When presented as an ester in lipoprotein particles, LKM38 initially localized to the lysosome and subsequently trafficked to the plasma membrane and endoplasmic reticulum. LKM38 bound to diverse, established cholesterol binding proteins. Through a detailed characterization of the cellular behavior of a panel of diazirine alkyne probes using cell biological, biochemical trafficking assays and immunofluorescence approaches, we conclude that LKM38 can serve as a powerful tool for the study of cholesterol protein interactions and trafficking.
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http://dx.doi.org/10.1194/jlr.D091470DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6399506PMC
March 2019

19q13.12 microdeletion syndrome fibroblasts display abnormal storage of cholesterol and sphingolipids in the endo-lysosomal system.

Biochim Biophys Acta Mol Basis Dis 2018 06 24;1864(6 Pt A):2108-2118. Epub 2018 Mar 24.

Atlantic Research Center, Depts. of Pediatrics and Biochemistry & Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada. Electronic address:

Microdeletions in 19q12q13.12 cause a rare and complex haploinsufficiency syndrome characterized by intellectual deficiency, developmental delays, and neurological movement disorders. Variability in the size and interval of the deletions makes it difficult to attribute the complex clinical phenotype of this syndrome to an underlying gene(s). As an alternate approach, we examined the biochemical and metabolic features of fibroblasts from an affected individual to derive clues as to the molecular basis for the syndrome. Immunofluorescence and electron microscopy of affected fibroblasts revealed an abnormal endo-lysosomal compartment that was characterized by rapid accumulation of lysosomotropic dyes, elevated LAMP1 and LAMP2 expression and vacuoles containing membrane whorls, common features of lysosomal lipid storage disorders. The late endosomes-lysosomes (LE/LY) of affected fibroblasts accumulated low-density lipoprotein cholesterol, and displayed reduced cholesterol esterification and increased de novo cholesterol synthesis, indicative of defective cholesterol transport to the endoplasmic reticulum. Affected fibroblasts also had increased ceramide and sphingolipid mass, altered glycosphingolipid species and accumulation of a fluorescent lactosylceramide probe in LE/LY. Autophagosomes also accumulated in affected fibroblasts because of decreased fusion with autolysosomes, a defect associated with other lysosomal storage diseases. Attempts to correct the cholesterol/sphingolipid storage defect in fibroblasts with cyclodextrin, sphingolipid synthesis inhibitors or by altering ion transport were unsuccessful. Our data show that 19q13.12 deletion fibroblasts have abnormal accumulation of cholesterol and sphingolipids in the endo-lysosomal system that compromises organelle function and could be an underlying cause of the clinical features of the syndrome.
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http://dx.doi.org/10.1016/j.bbadis.2018.03.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6394866PMC
June 2018

Development of a bile acid-based newborn screen for Niemann-Pick disease type C.

Sci Transl Med 2016 05;8(337):337ra63

Diabetic Cardiovascular Disease Center, Washington University School of Medicine, St. Louis, MO 63110, USA.

Niemann-Pick disease type C (NPC) is a fatal, neurodegenerative, cholesterol storage disorder. With new therapeutics in clinical trials, it is imperative to improve diagnostics and facilitate early intervention. We used metabolomic profiling to identify potential markers and discovered three unknown bile acids that were increased in plasma from NPC but not control subjects. The bile acids most elevated in the NPC subjects were identified as 3β,5α,6β-trihydroxycholanic acid and its glycine conjugate, which were shown to be metabolites of cholestane-3β,5α,6β-triol, an oxysterol elevated in NPC. A high-throughput mass spectrometry-based method was developed and validated to measure the glycine-conjugated bile acid in dried blood spots. Analysis of dried blood spots from 4992 controls, 134 NPC carriers, and 44 NPC subjects provided 100% sensitivity and specificity in the study samples. Quantification of the bile acid in dried blood spots, therefore, provides the basis for a newborn screen for NPC that is ready for piloting in newborn screening programs.
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http://dx.doi.org/10.1126/scitranslmed.aaf2326DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5316294PMC
May 2016

A novel intrinsically fluorescent probe for study of uptake and trafficking of 25-hydroxycholesterol.

J Lipid Res 2015 Dec 24;56(12):2408-19. Epub 2015 Oct 24.

Diabetic Cardiovascular Disease Center, Washington University School of Medicine, St. Louis, MO 63110

Cholesterol homeostasis is regulated not only by cholesterol, but also by oxygenated cholesterol species, referred to as oxysterols. Side-chain oxysterols, such as 25-hydroxycholesterol (25-HC), regulate cholesterol homeostasis through feedback inhibition and feed-forward activation of transcriptional pathways that govern cholesterol synthesis, uptake, and elimination, as well as through direct nongenomic actions that modulate cholesterol accessibility in membranes. Elucidating the cellular distribution of 25-HC is required to understand its biological activity at the molecular level. However, studying oxysterol distribution and behavior within cells has proven difficult due to the lack of fluorescent analogs of 25-HC that retain its chemical and physical properties. To address this, we synthesized a novel intrinsically fluorescent 25-HC mimetic, 25-hydroxycholestatrienol (25-HCTL). We show that 25-HCTL modulates sterol homeostatic responses in a similar manner as 25-HC. 25-HCTL associates with lipoproteins in media and is taken up by cells through LDL-mediated endocytosis. In cultured cells, 25-HCTL redistributes among cellular membranes and, at steady state, has a similar distribution as cholesterol, being enriched in both the endocytic recycling compartment as well as the plasma membrane. Our findings indicate that 25-HCTL is a faithful fluorescent 25-HC mimetic that can be used to investigate the mechanisms through which 25-HC regulates sterol homeostatic pathways.
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http://dx.doi.org/10.1194/jlr.D064287DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4655980PMC
December 2015

snoRNA U17 regulates cellular cholesterol trafficking.

Cell Metab 2015 Jun 14;21(6):855-67. Epub 2015 May 14.

Diabetic Cardiovascular Disease Center and Department of Medicine, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, USA. Electronic address:

Cholesterol is required for the growth and viability of mammalian cells and is an obligate precursor for steroid hormone synthesis. Using a loss-of-function screen for mutants with defects in intracellular cholesterol trafficking, a Chinese hamster ovary cell mutant with haploinsufficiency of the U17 snoRNA was isolated. U17 is an H/ACA orphan snoRNA, for which a function other than ribosomal processing has not previously been identified. Through expression profiling, we identified hypoxia-upregulated mitochondrial movement regulator (HUMMR) mRNA as a target that is negatively regulated by U17 snoRNA. Upregulation of HUMMR in U17 snoRNA-deficient cells promoted the formation of ER-mitochondrial contacts, decreasing esterification of cholesterol and facilitating cholesterol trafficking to mitochondria. U17 snoRNA and HUMMR regulate mitochondrial synthesis of steroids in vivo and are developmentally regulated in steroidogenic tissues, suggesting that the U17 snoRNA-HUMMR pathway may serve a previously unrecognized, physiological role in gonadal tissue maturation.
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http://dx.doi.org/10.1016/j.cmet.2015.04.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4456254PMC
June 2015

Side-chain oxysterols modulate cholesterol accessibility through membrane remodeling.

Biochemistry 2014 May 1;53(18):3042-51. Epub 2014 May 1.

Diabetic Cardiovascular Disease Center, Washington University School of Medicine , St. Louis, Missouri 63110, United States.

Side-chain oxysterols, such as 25-hydroxycholesterol (25-HC), are key regulators of cholesterol homeostasis. New evidence suggests that the alteration of membrane structure by 25-HC contributes to its regulatory effects. We have examined the role of oxysterol membrane effects on cholesterol accessibility within the membrane using perfringolysin O (PFO), a cholesterol-dependent cytolysin that selectively binds accessible cholesterol, as a sensor of membrane cholesterol accessibility. We show that 25-HC increases cholesterol accessibility in a manner dependent on the membrane lipid composition. Structural analysis of molecular dynamics simulations reveals that increased cholesterol accessibility is associated with membrane thinning, and that the effects of 25-HC on cholesterol accessibility are driven by these changes in membrane thickness. Further, we find that the 25-HC antagonist LY295427 (agisterol) abrogates the membrane effects of 25-HC in a nonenantioselective manner, suggesting that agisterol antagonizes the cholesterol-homeostatic effects of 25-HC indirectly through its membrane interactions. These studies demonstrate that oxysterols regulate cholesterol accessibility, and thus the availability of cholesterol to be sensed and transported throughout the cell, by modulating the membrane environment. This work provides new insights into how alterations in membrane structure can be used to relay cholesterol regulatory signals.
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http://dx.doi.org/10.1021/bi5000096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4020583PMC
May 2014

Box C/D small nucleolar RNA (snoRNA) U60 regulates intracellular cholesterol trafficking.

J Biol Chem 2013 Dec 30;288(50):35703-13. Epub 2013 Oct 30.

From the Diabetic Cardiovascular Disease Center, Washington University School of Medicine, St. Louis, Missouri 63110.

Mobilization of plasma membrane (PM) cholesterol to the endoplasmic reticulum is essential for cellular cholesterol homeostasis. The mechanisms regulating this retrograde, intermembrane cholesterol transfer are not well understood. Because mutant cells with defects in PM to endoplasmic reticulum cholesterol trafficking can be isolated on the basis of resistance to amphotericin B, we conducted an amphotericin B loss-of-function screen in Chinese hamster ovary (CHO) cells using insertional mutagenesis to identify genes that regulate this trafficking mechanism. Mutant line A1 displayed reduced cholesteryl ester formation from PM-derived cholesterol and increased de novo cholesterol synthesis, indicating a deficiency in retrograde cholesterol transport. Genotypic analysis revealed that the A1 cell line contained one disrupted allele of the U60 small nucleolar RNA (snoRNA) host gene, resulting in haploinsufficiency of the box C/D snoRNA U60. Complementation and mutational studies revealed the U60 snoRNA to be the essential feature from this locus that affects cholesterol trafficking. Lack of alteration in predicted U60-mediated site-directed methylation of 28 S rRNA in the A1 mutant suggests that the U60 snoRNA modulates cholesterol trafficking by a mechanism that is independent of this canonical function. Our study adds to a growing body of evidence for participation of small noncoding RNAs in cholesterol homeostasis and is the first to implicate a snoRNA in this cellular function.
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http://dx.doi.org/10.1074/jbc.M113.488577DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3861622PMC
December 2013

Tumoral calcinosis: a dental literature review and case report.

Dent Update 2012 Jul-Aug;39(6):416-8, 421

Birmingham Dental Hospital, Birmingham B4 6NN, UK.

Unlabelled: Tumoral calcinosis (TC) is a rare familial disease characterized by abnormal peri-articular calcification in affected joints, without any associated renal, metabolic or collagen vascular disease. It is characterized by usual hyperphosphataemia with normal serum calcium and alkaline phosphatase values. There are only a few reported cases ofTC patients with dental findings. This article reviews the dental literature and describes progressive gingival, alveolar and mandibular tori enlargement in a 41-year-old female from Zimbabwe with tumoral calcinosis.

Clinical Relevance: Tumoral calcinosis is a rare disorder of mineral metabolism with oral manifestations.
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http://dx.doi.org/10.12968/denu.2012.39.6.416DOI Listing
September 2012

Cholesterol oxidation products are sensitive and specific blood-based biomarkers for Niemann-Pick C1 disease.

Sci Transl Med 2010 Nov;2(56):56ra81

Program in Developmental Endocrinology and Genetics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Department of Health and Human Services, Bethesda, MD 20892, USA.

Niemann-Pick type C1 (NPC1) disease is a rare progressive neurodegenerative disorder characterized by accumulation of cholesterol in the endolysosomes. Previous studies implicating oxidative stress in NPC1 disease pathogenesis raised the possibility that nonenzymatic formation of cholesterol oxidation products could serve as disease biomarkers. We measured these metabolites in the plasma and tissues of the Npc1(-/-) mouse model and found several cholesterol oxidation products that were elevated in Npc1(-/-) mice, were detectable before the onset of symptoms, and were associated with disease progression. Nonenzymatically formed cholesterol oxidation products were similarly increased in the plasma of all human NPC1 subjects studied and delineated an oxysterol profile specific for NPC1 disease. This oxysterol profile also correlated with the age of disease onset and disease severity. We further show that the plasma oxysterol markers decreased in response to an established therapeutic intervention in the NPC1 feline model. These cholesterol oxidation products are robust blood-based biochemical markers for NPC1 disease that may prove transformative for diagnosis and treatment of this disorder, and as outcome measures to monitor response to therapy.
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http://dx.doi.org/10.1126/scitranslmed.3001417DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3170139PMC
November 2010

Common things are common: a case series of oral foreign bodies in paediatric patients.

Dent Update 2009 Jan-Feb;36(1):53-4, 57-8

Birmingham Dental Hospital, Birmingham, UK.

Reports of foreign bodies in the oral cavity are few in number. Three cases of children of varying ages, presenting with oral foreign bodies, and their subsequent diagnosis and management, are described. The importance of considering foreign bodies, as part of a differential diagnosis in paediatric patients, where aetiology is uncertain and clinical appearance is unusual, is highlighted.
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http://dx.doi.org/10.12968/denu.2009.36.1.53DOI Listing
June 2009

Side chain oxygenated cholesterol regulates cellular cholesterol homeostasis through direct sterol-membrane interactions.

J Biol Chem 2009 Jan 6;284(3):1755-64. Epub 2008 Nov 6.

Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

Side chain oxysterols exert cholesterol homeostatic effects by suppression of sterol regulatory element-binding protein maturation and promoting degradation of hydroxymethylglutaryl-CoA reductase. To examine whether oxysterol-membrane interactions contribute to the regulation of cellular cholesterol homeostasis, we synthesized the enantiomer of 25-hydroxycholesterol. Using this unique oxysterol probe, we provide evidence that oxysterol regulation of cholesterol homeostatic responses is not mediated by enantiospecific oxysterol-protein interactions. We show that side chain oxysterols, but not steroid ring-modified oxysterols, exhibit membrane expansion behavior in phospholipid monolayers and bilayers in vitro. This behavior is non-enantiospecific and is abrogated by increasing the saturation of phospholipid acyl chain constituents. Moreover, we extend these findings into cultured cells by showing that exposure to saturated fatty acids at concentrations that lead to endoplasmic reticulum membrane phospholipid remodeling inhibits oxysterol activity. These studies implicate oxysterol-membrane interactions in acute regulation of sterol homeostatic responses and provide new insights into the mechanism through which oxysterols regulate cellular cholesterol balance.
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http://dx.doi.org/10.1074/jbc.M807210200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2615513PMC
January 2009

Niemann-Pick type C1 I1061T mutant encodes a functional protein that is selected for endoplasmic reticulum-associated degradation due to protein misfolding.

J Biol Chem 2008 Mar 23;283(13):8229-36. Epub 2008 Jan 23.

Center for Cardiovascular Research, Department of Internal Medicine, Washington University School of Medicine, St Louis, MO 63110, USA.

Over 200 disease-causing mutations have been identified in the NPC1 gene. The most prevalent mutation, NPC1(I1061T), is predicted to lie within the cysteine-rich luminal domain and is associated with the classic juvenile-onset phenotype of Niemann-Pick type C disease. To gain insight into the molecular mechanism by which the NPC1(I1061T) mutation causes disease, we examined expression of the mutant protein in human fibroblasts homozygous for the NPC1(I1061T) mutation. Despite similar NPC1 mRNA levels between wild type and NPC1(I1061T) fibroblasts, NPC1 protein levels are decreased by 85% in NPC1(I1061T) cells. Metabolic labeling studies demonstrate that unlike wild type protein, which undergoes a glycosylation pattern shift from Endo H-sensitive to Endo H-resistant species, NPC1(I1061T) protein remains almost exclusively Endo H-sensitive and exhibits a reduced half-life (t((1/2)) 6.5 h) versus wild type Endo H-resistant species (t((1/2)) 42 h). Treatment with chemical chaperones, growth at permissive temperature, or inhibition of proteasomal degradation increases NPC1(I1061T) protein levels, indicating that the mutant protein is likely targeted for endoplasmic reticulum-associated degradation (ERAD) due to protein misfolding. Overexpression of NPC1(I1061T) in NPC1-deficient cells results in late endosomal localization of the mutant protein and complementation of the NPC mutant phenotype, likely due to a small proportion of the nascent NPC1(I1061T) protein that is able to fold correctly and escape the endoplasmic reticulum quality control checkpoints. Our findings provide the first description of an endoplasmic reticulum trafficking defect as a mechanism for human NPC disease, shedding light on the mechanism by which the NPC1(I1061T) mutation causes disease and suggesting novel approaches to treat NPC disease caused by the NPC1(I1061T) mutation.
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http://dx.doi.org/10.1074/jbc.M708735200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2276376PMC
March 2008

Disruption of endoplasmic reticulum structure and integrity in lipotoxic cell death.

J Lipid Res 2006 Dec 7;47(12):2726-37. Epub 2006 Sep 7.

Center for Cardiovascular Research, Division of Cardiology, Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA.

Cell dysfunction and death induced by lipid accumulation in nonadipose tissues, or lipotoxicity, may contribute to the pathogenesis of obesity and type 2 diabetes. However, the mechanisms leading to lipotoxic cell death are poorly understood. We recently reported that, in Chinese hamster ovary (CHO) cells and in H9c2 cardiomyoblasts, lipid overload induced by incubation with 500 muM palmitate leads to intracellular accumulation of reactive oxygen species, which subsequently induce endoplasmic reticulum (ER) stress and cell death. Here, we show that palmitate also impairs ER function through a more direct mechanism. Palmitate was rapidly incorporated into saturated phospholipid and triglyceride species in microsomal membranes of CHO cells. The resulting membrane remodeling was associated with dramatic dilatation of the ER and redistribution of protein-folding chaperones to the cytosol within 5 h, indicating compromised ER membrane integrity. Increasing beta-oxidation, through the activation of AMP-activated protein kinase, decreased palmitate incorporation into microsomes, decreased the escape of chaperones to the cytosol, and decreased subsequent caspase activation and cell death. Thus, palmitate rapidly increases the saturated lipid content of the ER, leading to compromised ER morphology and integrity, suggesting that impairment of the structure and function of this organelle is involved in the cellular response to fatty acid overload.
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http://dx.doi.org/10.1194/jlr.M600299-JLR200DOI Listing
December 2006

Pregnane X receptor (PXR) activation: a mechanism for neuroprotection in a mouse model of Niemann-Pick C disease.

Proc Natl Acad Sci U S A 2006 Sep 29;103(37):13807-12. Epub 2006 Aug 29.

Center for Cardiovascular Research, Department of Internal Medicine, and Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, MO 63110, USA.

Niemann-Pick type C1 (NPC1) disease is a fatal neurodegenerative disease characterized by neuronal lipid storage and progressive Purkinje cell loss in the cerebellum. We investigated whether therapeutic approaches to bypass the cholesterol trafficking defect in NPC1 disease might delay disease progression in the npc1(-/-) mouse model. We show that the neurosteroid allopregnanolone (ALLO) and T0901317, a synthetic oxysterol ligand, act in concert to delay onset of neurological symptoms and prolong the lifespan of npc1(-/-) mice. ALLO and T0901317 therapy preserved Purkinje cells, suppressed cerebellar expression of microglial-associated genes and inflammatory mediators, and reduced infiltration of activated microglia in the cerebellar tissue. To establish whether the mechanism of neuroprotection in npc1(-/-) mice involves GABA(A) receptor activation, we compared treatment of natural ALLO and ent-ALLO, a stereoisomer that has identical physical properties of natural ALLO but is not a GABA(A) receptor agonist. ent-ALLO provided identical functional and survival benefits as natural ALLO in npc1(-/-) mice, strongly supporting a GABA(A) receptor-independent mechanism for ALLO action. On the other hand, the efficacy of ALLO, ent-ALLO, and T0901317 therapy correlated with the ability of these compounds to activate pregnane X receptor-dependent pathways in vivo. These findings suggest that treatment with pregnane X receptor ligands may be useful clinically in delaying the progressive neurodegeneration in human NPC disease.
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http://dx.doi.org/10.1073/pnas.0606218103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1564205PMC
September 2006

A regulatory role for 1-acylglycerol-3-phosphate-O-acyltransferase 2 in adipocyte differentiation.

J Biol Chem 2006 Apr 22;281(16):11082-9. Epub 2006 Feb 22.

Center for Cardiovascular Research and Department of Internal Medicine, School of Medicine, Washington University, St. Louis, Missouri 63110-1010, USA.

Mutations in the 1-acylglycerol-3-phosphate-O-acyltransferase 2 (AGPAT2) gene have been identified in individuals affected with congenital generalized lipodystrophy (CGL). AGPAT2 catalyzes acylation of lysophosphatidic acid to phosphatidic acid, a precursor for both triacylglycerol (TAG) and phospholipid synthesis. Recent studies suggest that reduced AGPAT2 enzymatic activity may underlie the CGL clinical phenotype. To gain insight into how altered AGPAT2 activity causes lipodystrophy, we examined the effect of knockdown of AGPAT2 expression in preadipocytes on TAG synthesis and storage, and on adipocyte differentiation. We show that AGPAT2 mRNA expression is induced 30-fold during adipocyte differentiation and that AGPAT2 enzymatic activity is required for TAG mass accumulation in mature adipocytes. We demonstrate that small interference RNA-mediated knockdown of AGPAT2 expression prevents appropriate early induction of C/EBPbeta and PPARgamma, key transcriptional activators of the adipogenic program, and delays expression of multiple adipocyte-related genes. The unexpected finding, that levels of several phospholipid species, including phosphatidic acid (PA), are elevated in TAG-depleted adipocytes with AGPAT2 knockdown, suggests that impaired AGPAT2 activity affects availability of PA for TAG synthesis but not overall PA synthesis nor utilization of PA for phospholipid synthesis. These findings underscore the importance of an AGPAT2-mediated metabolic pathway in adipocyte differentiation.
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http://dx.doi.org/10.1074/jbc.M509612200DOI Listing
April 2006

The sterol-sensing domain of the Niemann-Pick C1 (NPC1) protein regulates trafficking of low density lipoprotein cholesterol.

J Biol Chem 2005 Aug 20;280(31):28581-90. Epub 2005 May 20.

Center for Cardiovascular Research, Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110-1010, USA.

The Niemann-Pick C1 (NPC1) protein is a key participant in intracellular sterol trafficking and regulation of cholesterol homeostasis. NPC1 contains a pentahelical region that is evolutionarily related to sterol-sensing domains found in other polytopic proteins involved in sterol interactions or sterol metabolism, including sterol regulatory element-binding protein cleavage-activating protein and hydroxymethylglutaryl-CoA reductase. To gain insight into the role of the sterol-sensing domain of NPC1, we examined the effect of point mutations in the NPC1 sterol-sensing domain on the trafficking of low density lipoprotein-derived cholesterol and sphingolipids. We show that an NPC1 P692S loss of function mutation results in decreased cholesterol delivery to the plasma membrane and endoplasmic reticulum. By contrast, NPC1 proteins carrying a L657F or D787N point mutation, which correspond to the activating SCAP L315F and D443N mutations, respectively, exhibit a gain of function phenotype. Specifically, cell lines expressing the NPC1 L657F or D787N mutations show a nearly 2-fold increase in the rates of low density lipoprotein cholesterol trafficking to the plasma membrane and to the endoplasmic reticulum, and more rapid suppression of sterol regulatory element-binding protein-dependent gene expression. Trafficking of sphingolipids is intact in the D787N and L657F cell lines. Our finding that D787N and L657F are activating NPC1 mutations provide evidence for a conserved mechanism for the sterol-sensing domain among cholesterol homeostatic proteins.
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http://dx.doi.org/10.1074/jbc.M414024200DOI Listing
August 2005

Day 3 and day 5 morphological predictors of embryo viability.

Reprod Biomed Online 2003 Apr-May;6(3):323-31

Department of Obstetrics and Gynecology, Brigham and Women's Hospital, Harvard Medical School, 75 Francis Street, ASB 1+3, Room 082, Boston, MA 02115, USA.

Controlling multiple pregnancies in patients undergoing artificial reproductive procedures requires consideration of single embryo transfers. Therefore, refinements for embryo evaluation are needed that select for the most developmentally competent embryo. The present study was designed to identify day 3 and day 5 morphological predictors of viability following transfers in which the morphology and fate of each embryo was precisely determined. Assessments on day 3 included cell number, and the extent of fragmentation and asymmetry, and on day 5, the developmental stage. Embryos resulting in a viable fetus at 11 weeks gestation were considered developmentally competent. The relationships among individual and collective embryo morphological characteristics were evaluated. Analysis of the interactions among morphological characteristics of embryos transferred on day 3 enabled identification of a multivariable selection order. Assessment of day 5 embryos revealed that expanding and expanded blastocysts exhibited comparable developmental potential that was superior to that of either morulae or early blastocysts. However, expanding or expanded blastocysts derived from 7-cell or 8-cell embryos were developmentally superior to those derived from other cleavage stages, regardless of fragmentation or asymmetry. Collectively, these findings further understanding of morphological predictors of viability, thereby improving the ability to select the most viable embryo for transfer.
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http://dx.doi.org/10.1016/s1472-6483(10)61852-4DOI Listing
December 2003
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