Person: Stillman, Isaac
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Stillman
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Isaac
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Stillman, Isaac
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Publication Nanoscale imaging of clinical specimens using pathology-optimized expansion microscopy(2017) Zhao, Yongxin; Bucur, Octavian; Irshad, Humayun; Chen, Fei; Weins, Astrid; Stancu, Andreea; Oh, Eun-Young; DiStasio, Marcello; Torous, Vanda; Glass, Benjamin; Stillman, Isaac; Schnitt, Stuart; Beck, Andrew; Boyden, Edward S.Expansion microscopy (ExM), a method for improving the resolution of light microscopy by physically expanding the specimen, has not been applied to clinical tissue samples. Here we report a clinically optimized form of ExM that supports nanoscale imaging of human tissue specimens that have been fixed with formalin, embedded in paraffin, stained with hematoxylin and eosin (H&E), and/or fresh frozen. The method, which we call expansion pathology (ExPath), converts clinical samples into an ExM-compatible state, then applies an ExM protocol with protein anchoring and mechanical homogenization steps optimized for clinical samples. ExPath enables ~70 nm resolution imaging of diverse biomolecules in intact tissues using conventional diffraction-limited microscopes, and standard antibody and fluorescent DNA in situ hybridization reagents. We use ExPath for optical diagnosis of kidney minimal-change disease, which previously required electron microscopy (EM), and demonstrate high-fidelity computational discrimination between early breast neoplastic lesions that to date have challenged human judgment. ExPath may enable the routine use of nanoscale imaging in pathology and clinical research.Publication Minimal-change renal disease and Graves’ disease: a case report and literature review(Oxford University Press, 2011) Hasnain, Wirasat; Stillman, Isaac; Bayliss, George P.Objective: To describe a possible association between Graves' disease and nephrotic syndrome secondary to minimal change renal disease and to review the literature related to renal diseases in patients with Graves' disease. Methods: The clinical, laboratory, and renal biopsy findings in a patient with Graves' disease and minimal change renal disease are discussed. In addition, the pertinent English-language literature published from 1966 to 2009, determined by means of a MEDLINE search, is reviewed. Results: A 63-year-old man who was admitted to the hospital with nephrotic syndrome. Patient had a history of Graves' disease for last 2 years with associated ophthalmopathy. He had been treated with methimazole and low low-dose steroids (prednisone 5 mg daily). Examination revealed generalized oedemaedema and exophthalmoses. Laboratory tests showed 6.62 g/day of proteinuria. Antinuclear antibodies, anti-glomerular basement membrane antibodies, antineutrophil cytoplasmic antibody (ANCA), serum complement levels, cryoglobulin, hepatitis screen and serum and urine protein electrophoreses were normal. A kidney biopsy revealed features consistent with minimal change disease on light, immunofluorescence, and electron microscopy. The patient had an excellent clinical and laboratory response after treatment with steroids and near total thyroidectomy. Conclusions: To the best of our knowledge, this is the fourth report of the occurrence of minimal change disease in a patient with Graves' disease in the absence of any other immunologic disorder known to be associated with minimal change renal disease.Publication Congenital chloride-losing diarrhea in a Mexican child with the novel homozygous SLC26A3 mutation G393W(Frontiers Media S.A., 2015) Reimold, Fabian Raoul; Balasubramanian, Savithri; Doroquez, David B.; Shmukler, Boris; Zsengeller, Zsuzsanna; Saslowsky, David E.; Thiagarajah, Jay; Stillman, Isaac; Lencer, Wayne; Wu, Bai-Lin; Villalpando-Carrion, Salvador; Alper, SethCongenital chloride diarrhea is an autosomal recessive disease caused by mutations in the intestinal lumenal membrane Cl−/HCO−3 exchanger, SLC26A3. We report here the novel SLC26A3 mutation G393W in a Mexican child, the first such report in a patient from Central America. SLC26A3 G393W expression in Xenopus oocytes exhibits a mild hypomorphic phenotype, with normal surface expression and moderately reduced anion transport function. However, expression of HA-SLC26A3 in HEK-293 cells reveals intracellular retention and greatly decreased steady-state levels of the mutant polypeptide, in contrast to peripheral membrane expression of the wildtype protein. Whereas wildtype HA-SLC26A3 is apically localized in polarized monolayers of filter-grown MDCK cells and Caco2 cells, mutant HA-SLC26A3 G393W exhibits decreased total polypeptide abundance, with reduced or absent surface expression and sparse punctate (or absent) intracellular distribution. The WT protein is similarly localized in LLC-PK1 cells, but the mutant fails to accumulate to detectable levels. We conclude that the chloride-losing diarrhea phenotype associated with homozygous expression of SLC26A3 G393W likely reflects lack of apical surface expression in enterocytes, secondary to combined abnormalities in polypeptide trafficking and stability. Future progress in development of general or target-specific folding chaperonins and correctors may hold promise for pharmacological rescue of this and similar genetic defects in membrane protein targeting.Publication PGC1α-dependent NAD biosynthesis links oxidative metabolism to renal protection(2016) Tran, Mei T.; Zsengeller, Zsuzsanna; Berg, Anders; Khankin, Eliyahu; Bhasin, Manoj; Kim, Wondong; Clish, Clary B.; Stillman, Isaac; Karumanchi, Subbian; Rhee, Eugene; Parikh, SamirThe energetic burden of continuously concentrating solutes against gradients along the tubule may render the kidney especially vulnerable to ischemia. Indeed, acute kidney injury (AKI) affects 3% of all hospitalized patients.1,2 Here we show that the mitochondrial biogenesis regulator, PGC1α,3,4 is a pivotal determinant of renal recovery from injury by regulating NAD biosynthesis. Following renal ischemia, PGC1α−/− mice developed local deficiency of the NAD precursor niacinamide (Nam), marked fat accumulation, and failure to re-establish normal function. Remarkably, exogenous Nam improved local NAD levels, fat accumulation, and renal function in post-ischemic PGC1α−/− mice. Inducible tubular transgenic mice (iNephPGC1α) recapitulated the effects of Nam supplementation, including more local NAD and less fat accumulation with better renal function after ischemia. PGC1α coordinately upregulated the enzymes that synthesize NAD de novo from amino acids whereas PGC1α deficiency or AKI attenuated the de novo pathway. Nam enhanced NAD via the enzyme NAMPT and augmented production of the fat breakdown product beta-hydroxybutyrate (β-OHB), leading to increased prostaglandin PGE2, a secreted autocoid that maintains renal function.5 Nam treatment reversed established ischemic AKI and also prevented AKI in an unrelated toxic model. Inhibition of β-OHB signaling or prostaglandins similarly abolished PGC1α-dependent renoprotection. Given the importance of mitochondrial health in aging and the function of metabolically active organs, the results implicate Nam and NAD as key effectors for achieving PGC1α-dependent stress resistance.Publication Transcriptional Patterns in Peritoneal Tissue of Encapsulating Peritoneal Sclerosis, a Complication of Chronic Peritoneal Dialysis(Public Library of Science, 2013) Reimold, Fabian Raoul; Braun, Niko; Zsengellér, Zsuzsanna K.; Stillman, Isaac; Karumanchi, Subbian; Toka, Hakan; Latus, Joerg; Fritz, Peter; Biegger, Dagmar; Segerer, Stephan; Alscher, M. Dominik; Bhasin, Manoj; Alper, SethEncapsulating peritoneal sclerosis (EPS) is a devastating complication of peritoneal dialysis (PD), characterized by marked inflammation and severe fibrosis of the peritoneum, and associated with high morbidity and mortality. EPS can occur years after termination of PD and, in severe cases, leads to intestinal obstruction and ileus requiring surgical intervention. Despite ongoing research, the pathogenesis of EPS remains unclear. We performed a global transcriptome analysis of peritoneal tissue specimens from EPS patients, PD patients without EPS, and uremic patients without history of PD or EPS (Uremic). Unsupervised and supervised bioinformatics analysis revealed distinct transcriptional patterns that discriminated these three clinical groups. The analysis identified a signature of 219 genes expressed differentially in EPS as compared to PD and Uremic groups. Canonical pathway analysis of differentially expressed genes showed enrichment in several pathways, including antigen presentation, dendritic cell maturation, B cell development, chemokine signaling and humoral and cellular immunity (P value<0.05). Further interactive network analysis depicted effects of EPS-associated genes on networks linked to inflammation, immunological response, and cell proliferation. Gene expression changes were confirmed by qRT-PCR for a subset of the differentially expressed genes. EPS patient tissues exhibited elevated expression of genes encoding sulfatase1, thrombospondin 1, fibronectin 1 and alpha smooth muscle actin, among many others, while in EPS and PD tissues mRNAs encoding leptin and retinol-binding protein 4 were markedly down-regulated, compared to Uremic group patients. Immunolocalization of Collagen 1 alpha 1 revealed that Col1a1 protein was predominantly expressed in the submesothelial compact zone of EPS patient peritoneal samples, whereas PD patient peritoneal samples exhibited homogenous Col1a1 staining throughout the tissue samples. The results are compatible with the hypothesis that encapsulating peritoneal sclerosis is a distinct pathological process from the simple peritoneal fibrosis that accompanies all PD treatment.