The histone H2A deubiquitinase Usp16 regulates hematopoiesis and hematopoietic stem cell function.

PubMed

Gu, Yue; Jones, Amanda E; Yang, Wei; Liu, Shanrun; Dai, Qian; Liu, Yudong; Swindle, C Scott; Zhou, Dewang; Zhang, Zhuo; Ryan, Thomas M; Townes, Tim M; Klug, Christopher A; Chen, Dongquan; Wang, Hengbin

2016-01-05

Epigenetic mechanisms play important regulatory roles in hematopoiesis and hematopoietic stem cell (HSC) function. Subunits of polycomb repressive complex 1 (PRC1), the major histone H2A ubiquitin ligase, are critical for both normal and pathological hematopoiesis; however, it is unclear which of the several counteracting H2A deubiquitinases functions along with PRC1 to control H2A ubiquitination (ubH2A) level and regulates hematopoiesis in vivo. Here we investigated the function of Usp16 in mouse hematopoiesis. Conditional deletion of Usp16 in bone marrow resulted in a significant increase of global ubH2A level and lethality. Usp16 deletion did not change HSC number but was associated with a dramatic reduction of mature and progenitor cell populations, revealing a role in governing HSC lineage commitment. ChIP- and RNA-sequencing studies in HSC and progenitor cells revealed that Usp16 bound to many important hematopoietic regulators and that Usp16 deletion altered the expression of genes in transcription/chromosome organization, immune response, hematopoietic/lymphoid organ development, and myeloid/leukocyte differentiation. The altered gene expression was partly rescued by knockdown of PRC1 subunits, suggesting that Usp16 and PRC1 counterbalance each other to regulate cellular ubH2A level and gene expression in the hematopoietic system. We further discovered that knocking down Cdkn1a (p21cip1), a Usp16 target and regulated gene, rescued the altered cell cycle profile and differentiation defect of Usp16-deleted HSCs. Collectively, these studies identified Usp16 as one of the histone H2A deubiquitinases, which coordinates with the H2A ubiquitin ligase PRC1 to regulate hematopoiesis, and revealed cell cycle regulation by Usp16 as key for HSC differentiation.

Somatic USP8 Gene Mutations Are a Common Cause of Pediatric Cushing Disease.

PubMed

Faucz, Fabio R; Tirosh, Amit; Tatsi, Christina; Berthon, Annabel; Hernández-Ramírez, Laura C; Settas, Nikolaos; Angelousi, Anna; Correa, Ricardo; Papadakis, Georgios Z; Chittiboina, Prashant; Quezado, Martha; Pankratz, Nathan; Lane, John; Dimopoulos, Aggeliki; Mills, James L; Lodish, Maya; Stratakis, Constantine A

2017-08-01

Somatic mutations in the ubiquitin-specific protease 8 (USP8) gene have been recently identified as the most common genetic alteration in patients with Cushing disease (CD). However, the frequency of these mutations in the pediatric population has not been extensively assessed. We investigated the status of the USP8 gene at the somatic level in a cohort of pediatric patients with corticotroph adenomas. The USP8 gene was fully sequenced in both germline and tumor DNA samples from 42 pediatric patients with CD. Clinical, biochemical, and imaging data were compared between patients with and without somatic USP8 mutations. Five different USP8 mutations (three missense, one frameshift, and one in-frame deletion) were identified in 13 patients (31%), all of them located in exon 14 at the previously described mutational hotspot, affecting the 14-3-3 binding motif of the protein. Patients with somatic mutations were older at disease presentation [mean 5.1 ± 2.1 standard deviation (SD) vs 13.1 ± 3.6 years, P = 0.03]. Levels of urinary free cortisol, midnight serum cortisol, and adrenocorticotropic hormone, as well as tumor size and frequency of invasion of the cavernous sinus, were not significantly different between the two groups. However, patients harboring somatic USP8 mutations had a higher likelihood of recurrence compared with patients without mutations (46.2% vs 10.3%, P = 0.009). Somatic USP8 gene mutations are a common cause of pediatric CD. Patients harboring a somatic mutation had a higher likelihood of tumor recurrence, highlighting the potential importance of this molecular defect for the disease prognosis and the development of targeted therapeutic options. Copyright © 2017 Endocrine Society

Synthesis and biological activities of turkesterone 11α-acyl derivatives

PubMed Central

Dinan, Laurence; Bourne, Pauline; Whiting, Pensri; Tsitsekli, Ada; Saatov, Ziyadilla; Dhadialla, Tarlochan S.; Hormann, Robert E.; Lafont, René; Coll, Josep

2003-01-01

Turkesterone is a phytoecdysteroid possessing an 11α-hydroxyl group. It is an analogue of the insect steroid hormone 20-hydroxyecdysone. Previous ecdysteroid QSAR and molecular modelling studies predicted that the cavity of the ligand binding domain of the ecdysteroid receptor would possess space in the vicinity of C-11/C-12 of the ecdysteroid. We report the regioselective synthesis of a series of turkesterone 11α-acyl derivatives in order to explore this possibility. The structures of the analogues have been unambiguously determined by spectroscopic means (NMR and low-resolution mass spectrometry). Purity was verified by HPLC. Biological activities have been determined in Drosophila melanogaster BII cell-based bioassay for ecdysteroid agonists and in an in vitro radioligand-displacement assay using bacterially-expressed D. melanogaster EcR/USP receptor proteins. The 11α-acyl derivatives do retain a significant amount of biological activity relative to the parent ecdysteroid. Further, although activity initially drops with the extension of the acyl chain length (C2 to C4), it then increases (C6 to C10), before decreasing again (C14 and C20). The implications of these findings for the interaction of ecdysteroids with the ecdysteroid receptor and potential applications in the generation of affinity-labelled and fluorescently-tagged ecdysteroids are discussed. Abbreviation: CoMFA comparative molecular field analysis DCM dichloromethane DMF dimethylformamide DMP 2,2-dimethoxypropane 4D-QSAR 4-dimensional quantitative structure-activity relationship EcR ecdysteroid receptor EcRE ecdysteroid response element HPLC high-performance liquid chromatography LBD ligand-binding domain NMR nuclear magnetic resonance ponA ponasterone A QSAR quantitative structure-activity relationship RXR retinoid X receptor SAR structure-activity relationship SPE solid-phase extraction THF tetrahydrofuran TLC thin-layer chromatography p-TsOH para-toluenesulphonic acid USP ultraspiracle UV-VIS ultraviolet-visible PMID:15841223

Usp16 contributes to somatic stem cell defects in Down syndrome

PubMed Central

Adorno, Maddalena; Sikandar, Shaheen; Mitra, Siddhartha S.; Kuo, Angera; Di Robilant, Benedetta Nicolis; Haro-Acosta, Veronica; Ouadah, Youcef; Quarta, Marco; Rodriguez, Jacqueline; Qian, Dalong; Reddy, Vadiyala M.; Cheshier, Samuel; Garner, Craig C.; Clarke, Michael F.

2013-01-01

SUMMARY Down syndrome (DS) results from full or partial trisomy of chromosome 21. However, the consequences of the underlying gene-dosage imbalance on adult tissues remain poorly understood. Here we show that in Ts65Dn mice, trisomic for 132 genes homologous to HSA21, triplication of Usp16 reduces self-renewal of hematopoietic stem cells and expansion of mammary epithelial cells, neural progenitors, and fibroblasts. Moreover, Usp16 is associated with decreased ubiquitination of Cdkn2a and accelerated senescence in Ts65Dn fibroblasts. Usp16 can remove ubiquitin from H2AK119, a critical mark for the maintenance of multiple somatic tissues. Downregulation of Usp16, either by mutation of a single normal USP16 allele or by shRNAs, largely rescues all these defects. Furthermore, in human tissues overexpression of USP16 reduces the expansion of normal fibroblasts and post-natal neural progenitors while downregulation of USP16 partially rescues the proliferation defects of DS fibroblasts. Taken together, these results suggest that USP16 plays an important role in antagonizing the self-renewal and/or senescence pathways in Down syndrome and could serve as an attractive target to ameliorate some of the associated pathologies. PMID:24025767

USP7 Attenuates Hepatic Gluconeogenesis Through Modulation of FoxO1 Gene Promoter Occupancy

PubMed Central

Hall, Jessica A.; Tabata, Mitsuhisa; Rodgers, Joseph T.

2014-01-01

Hepatic forkhead protein FoxO1 is a key component of systemic glucose homeostasis via its ability to regulate the transcription of rate-limiting enzymes in gluconeogenesis. Important in the regulation of FoxO1 transcriptional activity are the modifying/demodifying enzymes that lead to posttranslational modification. Here, we demonstrate the functional interaction and regulation of FoxO1 by herpesvirus-associated ubiquitin-specific protease 7 (USP7; also known as herpesvirus-associated ubiquitin-specific protease, HAUSP), a deubiquitinating enzyme. We show that USP7-mediated mono-deubiquitination of FoxO1 results in suppression of FoxO1 transcriptional activity through decreased FoxO1 occupancy on the promoters of gluconeogenic genes. Knockdown of USP7 in primary hepatocytes leads to increased expression of FoxO1-target gluconeogenic genes and elevated glucose production. Consistent with this, USP7 gain-of-function suppresses the fasting/cAMP-induced activation of gluconeogenic genes in hepatocyte cells and in mouse liver, resulting in decreased hepatic glucose production. Notably, we show that the effects of USP7 on hepatic glucose metabolism depend on FoxO1. Together, these results place FoxO1 under the intimate regulation of deubiquitination and glucose metabolic control with important implication in diseases such as diabetes. PMID:24694308

Overexpression of a Cytosolic Abiotic Stress Responsive Universal Stress Protein (SbUSP) Mitigates Salt and Osmotic Stress in Transgenic Tobacco Plants

PubMed Central

Udawat, Pushpika; Jha, Rajesh K.; Sinha, Dinkar; Mishra, Avinash; Jha, Bhavanath

2016-01-01

The universal stress protein (USP) is a ubiquitous protein and plays an indispensable role in plant abiotic stress tolerance. The genome of Salicornia brachiata contains two homologs of intron less SbUSP gene which encodes for salt and osmotic responsive USP. In vivo localization reveals that SbUSP is a membrane bound cytosolic protein. The role of the gene was functionally validated by developing transgenic tobacco and compared with control [wild-type (WT) and vector control (VC)] plants under different abiotic stress condition. Transgenic lines (T1) exhibited higher chlorophyll, relative water, proline, total sugar, reducing sugar, free amino acids, polyphenol contents, osmotic potential, membrane stability, and lower electrolyte leakage and lipid peroxidation (malondialdehyde content) under stress treatments than control (WT and VC) plants. Lower accumulation of H2O2 and O2− radicals was also detected in transgenic lines compared to control plants under stress conditions. Present study confers that overexpression of the SbUSP gene enhances plant growth, alleviates ROS buildup, maintains ion homeostasis and improves the physiological status of the plant under salt and osmotic stresses. Principal component analysis exhibited a statistical distinction of plant response to salinity stress, and a significant response was observed for transgenic lines under stress, which provides stress endurance to the plant. A possible signaling role is proposed that some downstream genes may get activated by abiotic stress responsive cytosolic SbUSP, which leads to the protection of cell from oxidative damages. The study unveils that ectopic expression of the gene mitigates salt or osmotic stress by scavenging ROS and modulating the physiological process of the plant. PMID:27148338

Recurrent gain-of-function USP8 mutations in Cushing's disease

PubMed Central

Ma, Zeng-Yi; Song, Zhi-Jian; Chen, Jian-Hua; Wang, Yong-Fei; Li, Shi-Qi; Zhou, Liang-Fu; Mao, Ying; Li, Yi-Ming; Hu, Rong-Gui; Zhang, Zhao-Yun; Ye, Hong-Ying; Shen, Ming; Shou, Xue-Fei; Li, Zhi-Qiang; Peng, Hong; Wang, Qing-Zhong; Zhou, Dai-Zhan; Qin, Xiao-Lan; Ji, Jue; Zheng, Jie; Chen, Hong; Wang, Yin; Geng, Dao-Ying; Tang, Wei-Jun; Fu, Chao-Wei; Shi, Zhi-Feng; Zhang, Yi-Chao; Ye, Zhao; He, Wen-Qiang; Zhang, Qi-Lin; Tang, Qi-Sheng; Xie, Rong; Shen, Jia-Wei; Wen, Zu-Jia; Zhou, Juan; Wang, Tao; Huang, Shan; Qiu, Hui-Jia; Qiao, Ni-Dan; Zhang, Yi; Pan, Li; Bao, Wei-Min; Liu, Ying-Chao; Huang, Chuan-Xin; Shi, Yong-Yong; Zhao, Yao

2015-01-01

Cushing's disease, also known as adrenocorticotropic hormone (ACTH)-secreting pituitary adenomas (PAs) that cause excess cortisol production, accounts for up to 85% of corticotrophin-dependent Cushing's syndrome cases. However, the genetic alterations in this disease are unclear. Here, we performed whole-exome sequencing of DNA derived from 12 ACTH-secreting PAs and matched blood samples, which revealed three types of somatic mutations in a candidate gene, USP8 (encoding ubiquitin-specific protease 8), exclusively in exon 14 in 8 of 12 ACTH-secreting PAs. We further evaluated somatic USP8 mutations in additional 258 PAs by Sanger sequencing. Targeted sequencing further identified a total of 17 types of USP8 variants in 67 of 108 ACTH-secreting PAs (62.04%). However, none of these mutations was detected in other types of PAs (n = 150). These mutations aggregate within the 14-3-3 binding motif of USP8 and disrupt the interaction between USP8 and 14-3-3 protein, resulting in an elevated capacity to protect EGFR from lysosomal degradation. Accordingly, PAs with mutated USP8 display a higher incidence of EGFR expression, elevated EGFR protein abundance and mRNA expression levels of POMC, which encodes the precursor of ACTH. PAs with mutated USP8 are significantly smaller in size and have higher ACTH production than wild-type PAs. In surgically resected primary USP8-mutated tumor cells, USP8 knockdown or blocking EGFR effectively attenuates ACTH secretion. Taken together, somatic gain-of-function USP8 mutations are common and contribute to ACTH overproduction in Cushing's disease. Inhibition of USP8 or EGFR is promising for treating USP8-mutated corticotrophin adenoma. Our study highlights the potentially functional mutated gene in Cushing's disease and provides insights into the therapeutics of this disease. PMID:25675982

USP15 regulates type I interferon response and is required for pathogenesis of neuroinflammation.

PubMed

Torre, Sabrina; Polyak, Maria J; Langlais, David; Fodil, Nassima; Kennedy, James M; Radovanovic, Irena; Berghout, Joanne; Leiva-Torres, Gabriel A; Krawczyk, Connie M; Ilangumaran, Subburaj; Mossman, Karen; Liang, Chen; Knobeloch, Klaus-Peter; Healy, Luke M; Antel, Jack; Arbour, Nathalie; Prat, Alexandre; Majewski, Jacek; Lathrop, Mark; Vidal, Silvia M; Gros, Philippe

2017-01-01

Genes and pathways in which inactivation dampens tissue inflammation present new opportunities for understanding the pathogenesis of common human inflammatory diseases, including inflammatory bowel disease, rheumatoid arthritis and multiple sclerosis. We identified a mutation in the gene encoding the deubiquitination enzyme USP15 (Usp15 L749R ) that protected mice against both experimental cerebral malaria (ECM) induced by Plasmodium berghei and experimental autoimmune encephalomyelitis (EAE). Combining immunophenotyping and RNA sequencing in brain (ECM) and spinal cord (EAE) revealed that Usp15 L749R -associated resistance to neuroinflammation was linked to dampened type I interferon responses in situ. In hematopoietic cells and in resident brain cells, USP15 was coexpressed with, and functionally acted together with the E3 ubiquitin ligase TRIM25 to positively regulate type I interferon responses and to promote pathogenesis during neuroinflammation. The USP15-TRIM25 dyad might be a potential target for intervention in acute or chronic states of neuroinflammation.

CRISPR/Cas9 knockout of USP18 enhances type I IFN responsiveness and restricts HIV-1 infection in macrophages.

PubMed

Taylor, Jared P; Cash, Melanie N; Santostefano, Katherine E; Nakanishi, Mahito; Terada, Naohiro; Wallet, Mark A

2018-02-13

The IFN-stimulated gene ubiquitin-specific proteinase 18 (USP18) encodes a protein that negatively regulates T1 IFN signaling via stearic inhibition of JAK1 recruitment to the IFN-α receptor 2 subunit (IFNAR2). Here, we demonstrate that USP18 expression is induced by HIV-1 in a T1 IFN-dependent manner. Experimental depletion of USP18 by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing results in a significant restriction of HIV-1 replication in an induced pluripotent stem cell (iPSC)-derived macrophage model. In the absence of USP18, macrophages have increased responsiveness to stimulation with T1 IFNs with prolonged phosphorylation of STAT1 and STAT2 and increased expression of IFN-stimulated genes that are key for antiviral responses. Interestingly, HIV-1 requires some signaling through the T1 IFN receptor to replicate efficiently because a neutralizing antibody that inhibits T1 IFN activity reduces HIV-1 replication rate in monocyte-derived macrophages. USP18 induction by HIV-1 tunes the IFN response to optimal levels allowing for efficient transcription from the HIV-1 LTR promoter while minimizing the T1 IFN-induced antiviral response that would otherwise restrict viral replication and spread. Finally, iPSC and CRISPR/Cas9 gene targeting offer a powerful tool to study host factors that regulate innate immune responses. ©2018 Society for Leukocyte Biology.

The Gene of the Ubiquitin-Specific Protease 8 Is Frequently Mutated in Adenomas Causing Cushing's Disease.

PubMed

Perez-Rivas, Luis G; Theodoropoulou, Marily; Ferraù, Francesco; Nusser, Clara; Kawaguchi, Kohei; Stratakis, Constantine A; Faucz, Fabio Rueda; Wildemberg, Luiz E; Assié, Guillaume; Beschorner, Rudi; Dimopoulou, Christina; Buchfelder, Michael; Popovic, Vera; Berr, Christina M; Tóth, Miklós; Ardisasmita, Arif Ibrahim; Honegger, Jürgen; Bertherat, Jerôme; Gadelha, Monica R; Beuschlein, Felix; Stalla, Günter; Komada, Masayuki; Korbonits, Márta; Reincke, Martin

2015-07-01

We have recently reported somatic mutations in the ubiquitin-specific protease USP8 gene in a small series of adenomas of patients with Cushing's disease. To determine the prevalence of USP8 mutations and the genotype-phenotype correlation in a large series of patients diagnosed with Cushing's disease. We performed a retrospective, multicentric, genetic analysis of 134 functioning and 11 silent corticotroph adenomas using Sanger sequencing. Biochemical and clinical features were collected and examined within the context of the mutational status of USP8, and new mutations were characterized by functional studies. A total of 145 patients who underwent surgery for an ACTH-producing pituitary adenoma. Mutational status of USP8. Biochemical and clinical features included sex, age at diagnosis, tumor size, preoperative and postoperative hormonal levels, and comorbidities. We found somatic mutations in USP8 in 48 (36%) pituitary adenomas from patients with Cushing's disease but in none of 11 silent corticotropinomas. The prevalence was higher in adults than in pediatric cases (41 vs 17%) and in females than in males (43 vs 17%). Adults having USP8-mutated adenomas were diagnosed at an earlier age than those with wild-type lesions (36 vs 44 y). Mutations were primarily found in adenomas of 10 ± 7 mm and were inversely associated with the development of postoperative adrenal insufficiency. All the mutations affected the residues Ser718 or Pro720, including five new identified alterations. Mutations reduced the interaction between USP8 and 14-3-3 and enhanced USP8 activity. USP8 mutants diminished epidermal growth factor receptor ubiquitination and induced Pomc promoter activity in immortalized AtT-20 corticotropinoma cells. USP8 is frequently mutated in adenomas causing Cushing's disease, especially in those from female adult patients diagnosed at a younger age.

Usp7 promotes medulloblastoma cell survival and metastasis by activating Shh pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhan, Meixiao; Zhuhai Precision Medicine Center, Zhuhai People's Hospital, Jinan University, Zhuhai; Sun, Xiaohan

The ubiquitin-specific protease Usp7 plays roles in multiple cellular processes through deubiquitinating and stabilizing numerous substrates, including P53, Pten and Gli. Aberrant Usp7 activity has been implicated in many disorders and tumorigenesis, making it as a potential target for therapeutic intervention. Although it is clear that Usp7 is involved in many types of cancer, its role in regulating medulloblastoma (MB) is still unknown. In this study, we show that knockdown of Usp7 inhibits the proliferation and migration of MB cells, while Usp7 overexpression exerts an opposite effect. Furthermore, we establish Usp7 knockout MB cell line using the CRISPR/Cas9 system andmore » further confirm that Usp7 knockout also blocks MB cell proliferation and metastasis. In addition, we reveal that knockdown of Usp7 compromises Shh pathway activity and decrease Gli protein levels, while P53 level and P53 target gene expression have no obvious changes. Finally, we find that Usp7 inhibitors apparently inhibit MB cell viability and migration. Taken together, our findings suggest that Usp7 is important for MB cell proliferation and metastasis by activating Shh pathway, and is a putative therapeutic target for MBs. - Highlights: • Loss of usp7 blocks the proliferation and metastasis of MB cells. • Usp7 regulates MB cell growth and migration through stimulating Shh pathway. • Usp7 inhibitors hamper MB cell proliferation and migration. • Usp7 inhibitors could attenuate Shh pathway activity.« less

Isolation and characterization of a novel pollen-specific promoter in maize (Zea mays L.).

PubMed

Wang, He; Fan, Mingxia; Wang, Guohong; Zhang, Chunyu; Shi, Lei; Wei, Zhengyi; Ma, Wenjuan; Chang, Jing; Huang, Senxin; Lin, Feng

2017-06-01

ZmSTK2_USP, located on the long arm of chromosome 4, belongs to the serine/threonine kinase gene in maize. The sequence analysis of 2100 bp upstream from the start codon ATG has shown that it contains cis-element motifs and two types of anther/pollen-specific promoter elements (GTGA and AGAAA), suggesting that it is the pollen-specific promoter. To investigate the function of ZmSTK2_USP promoter, the GUS gene fusion system was employed. In proZmSTK2_USP-GUS genetically modified plants, GUS activity was detected in mature pollen grains and pollen tubes but not found in other floral and vegetative tissues. These results show that proZmSTK2_USP is the pollen-specific promoter and drives pollen-specific activity during the middle stage of pollen development until pollen maturation.

USP22 regulates oncogenic signaling pathways to drive lethal cancer progression.

PubMed

Schrecengost, Randy S; Dean, Jeffry L; Goodwin, Jonathan F; Schiewer, Matthew J; Urban, Mark W; Stanek, Timothy J; Sussman, Robyn T; Hicks, Jessica L; Birbe, Ruth C; Draganova-Tacheva, Rossitza A; Visakorpi, Tapio; DeMarzo, Angelo M; McMahon, Steven B; Knudsen, Karen E

2014-01-01

Increasing evidence links deregulation of the ubiquitin-specific proteases 22 (USP22) deubitiquitylase to cancer development and progression in a select group of tumor types, but its specificity and underlying mechanisms of action are not well defined. Here we show that USP22 is a critical promoter of lethal tumor phenotypes that acts by modulating nuclear receptor and oncogenic signaling. In multiple xenograft models of human cancer, modeling of tumor-associated USP22 deregulation demonstrated that USP22 controls androgen receptor accumulation and signaling, and that it enhances expression of critical target genes coregulated by androgen receptor and MYC. USP22 not only reprogrammed androgen receptor function, but was sufficient to induce the transition to therapeutic resistance. Notably, in vivo depletion experiments revealed that USP22 is critical to maintain phenotypes associated with end-stage disease. This was a significant finding given clinical evidence that USP22 is highly deregulated in tumors, which have achieved therapeutic resistance. Taken together, our findings define USP22 as a critical effector of tumor progression, which drives lethal phenotypes, rationalizing this enzyme as an appealing therapeutic target to treat advanced disease.

Nodular fasciitis: a novel model of transient neoplasia induced by MYH9-USP6 gene fusion.

PubMed

Erickson-Johnson, Michele R; Chou, Margaret M; Evers, Barbara R; Roth, Christopher W; Seys, Amber R; Jin, Long; Ye, Ying; Lau, Alan W; Wang, Xiaoke; Oliveira, Andre M

2011-10-01

Nodular fasciitis (NF) is a relatively common mass-forming and self-limited subcutaneous pseudosarcomatous myofibroblastic proliferation of unknown pathogenesis. Due to its rapid growth and high mitotic activity, NF is often misdiagnosed as a sarcoma. While studying the USP6 biology in aneurysmal bone cyst and other mesenchymal tumors, we identified high expression levels of USP6 mRNA in two examples of NF. This finding led us to further examine the mechanisms underlying USP6 overexpression in these lesions. Upon subsequent investigation, genomic rearrangements of the USP6 locus were found in 92% (44 of 48) of NF. Rapid amplification of 5'-cDNA ends identified MYH9 as the translocation partner. RT-PCR and direct sequencing revealed the fusion of the MYH9 promoter region to the entire coding region of USP6. Control tumors and tissues were negative for this fusion. Xenografts of cells overexpressing USP6 in nude mice exhibited clinical and histological features similar to human NF. The identification of a sensitive and specific abnormality in NF holds the potential to be used diagnostically. Considering the self-limited nature of the lesion, NF may represent a model of 'transient neoplasia', as it is, to our knowledge, the first example of a self-limited human disease characterized by a recurrent somatic gene fusion event. © 2011 USCAP, Inc All rights reserved

Developmental Regulation of Genes Encoding Universal Stress Proteins in Schistosoma mansoni

PubMed Central

Isokpehi, Raphael D.; Mahmud, Ousman; Mbah, Andreas N.; Simmons, Shaneka S.; Avelar, Lívia; Rajnarayanan, Rajendram V.; Udensi, Udensi K.; Ayensu, Wellington K.; Cohly, Hari H.; Brown, Shyretha D.; Dates, Centdrika R.; Hentz, Sonya D.; Hughes, Shawntae J.; Smith-McInnis, Dominique R.; Patterson, Carvey O.; Sims, Jennifer N.; Turner, Kelisha T.; Williams, Baraka S.; Johnson, Matilda O.; Adubi, Taiwo; Mbuh, Judith V.; Anumudu, Chiaka I.; Adeoye, Grace O.; Thomas, Bolaji N.; Nashiru, Oyekanmi; Oliveira, Guilherme

2011-01-01

The draft nuclear genome sequence of the snail-transmitted, dimorphic, parasitic, platyhelminth Schistosoma mansoni revealed eight genes encoding proteins that contain the Universal Stress Protein (USP) domain. Schistosoma mansoni is a causative agent of human schistosomiasis, a severe and debilitating Neglected Tropical Disease (NTD) of poverty, which is endemic in at least 76 countries. The availability of the genome sequences of Schistosoma species presents opportunities for bioinformatics and genomics analyses of associated gene families that could be targets for understanding schistosomiasis ecology, intervention, prevention and control. Proteins with the USP domain are known to provide bacteria, archaea, fungi, protists and plants with the ability to respond to diverse environmental stresses. In this research investigation, the functional annotations of the USP genes and predicted nucleotide and protein sequences were initially verified. Subsequently, sequence clusters and distinctive features of the sequences were determined. A total of twelve ligand binding sites were predicted based on alignment to the ATP-binding universal stress protein from Methanocaldococcus jannaschii. In addition, six USP sequences showed the presence of ATP-binding motif residues indicating that they may be regulated by ATP. Public domain gene expression data and RT-PCR assays confirmed that all the S. mansoni USP genes were transcribed in at least one of the developmental life cycle stages of the helminth. Six of these genes were up-regulated in the miracidium, a free-swimming stage that is critical for transmission to the snail intermediate host. It is possible that during the intra-snail stages, S. mansoni gene transcripts for universal stress proteins are low abundant and are induced to perform specialized functions triggered by environmental stressors such as oxidative stress due to hydrogen peroxide that is present in the snail hemocytes. This report serves to catalyze the formation of a network of researchers to understand the function and regulation of the universal stress proteins encoded in genomes of schistosomes and their snail intermediate hosts. PMID:22084571

The Gene of the Ubiquitin-Specific Protease 8 Is Frequently Mutated in Adenomas Causing Cushing's Disease

PubMed Central

Perez-Rivas, Luis G.; Theodoropoulou, Marily; Ferraù, Francesco; Nusser, Clara; Kawaguchi, Kohei; Stratakis, Constantine A.; Faucz, Fabio Rueda; Wildemberg, Luiz E.; Assié, Guillaume; Beschorner, Rudi; Dimopoulou, Christina; Buchfelder, Michael; Popovic, Vera; Berr, Christina M.; Tóth, Miklós; Ardisasmita, Arif Ibrahim; Honegger, Jürgen; Bertherat, Jerôme; Gadelha, Monica R.; Beuschlein, Felix; Stalla, Günter; Komada, Masayuki; Korbonits, Márta

2015-01-01

Context: We have recently reported somatic mutations in the ubiquitin-specific protease USP8 gene in a small series of adenomas of patients with Cushing's disease. Objective: To determine the prevalence of USP8 mutations and the genotype-phenotype correlation in a large series of patients diagnosed with Cushing's disease. Design: We performed a retrospective, multicentric, genetic analysis of 134 functioning and 11 silent corticotroph adenomas using Sanger sequencing. Biochemical and clinical features were collected and examined within the context of the mutational status of USP8, and new mutations were characterized by functional studies. Patients: A total of 145 patients who underwent surgery for an ACTH-producing pituitary adenoma. Main Outcomes Measures: Mutational status of USP8. Biochemical and clinical features included sex, age at diagnosis, tumor size, preoperative and postoperative hormonal levels, and comorbidities. Results: We found somatic mutations in USP8 in 48 (36%) pituitary adenomas from patients with Cushing's disease but in none of 11 silent corticotropinomas. The prevalence was higher in adults than in pediatric cases (41 vs 17%) and in females than in males (43 vs 17%). Adults having USP8-mutated adenomas were diagnosed at an earlier age than those with wild-type lesions (36 vs 44 y). Mutations were primarily found in adenomas of 10 ± 7 mm and were inversely associated with the development of postoperative adrenal insufficiency. All the mutations affected the residues Ser718 or Pro720, including five new identified alterations. Mutations reduced the interaction between USP8 and 14-3-3 and enhanced USP8 activity. USP8 mutants diminished epidermal growth factor receptor ubiquitination and induced Pomc promoter activity in immortalized AtT-20 corticotropinoma cells. Conclusions: USP8 is frequently mutated in adenomas causing Cushing's disease, especially in those from female adult patients diagnosed at a younger age. PMID:25942478

USP39 promotes the growth of human hepatocellular carcinoma in vitro and in vivo.

PubMed

Yuan, Xianwen; Sun, Xitai; Shi, Xiaolei; Jiang, Chunping; Yu, Decai; Zhang, Weiwei; Guan, Wenxian; Zhou, Jianxin; Wu, Yafu; Qiu, Yudong; Ding, Yitao

2015-08-01

Ubiquitin specific protease 39 (USP39) plays an important role in mRNA splicing. In the present study, we investigated the role of USP39 in regulating the growth of hepatocellular carcinoma (HCC). We detected USP39 expression in more than 100 HCC clinical samples. The USP39 expression was significantly higher in the tumor tissues compared to the adjacent normal tissues, and was strongly associated with the pathological grade of HCC. USP39 knockdown inhibited cell proliferation and colony formation in vitro in the HepG2 cells, while upregulation of USP39 promoted tumor cell growth. FCM assay showed that USP39 knockdown led to G2/M arrest and induced apoptosis in the HepG2 cells. USP39 knockdown by shRNA inhibited xenograft tumor growth in nude mice. Moreover, USP39 knockdown led to the upregulation of p-Cdc2 and downregulation of p-Cdc25c and p-myt1, while the expression of total Cdc2, Cdc25c and myt1 was not changed in the USP39-knockdown cells. We also found that p-Cdc2 was decreased in the USP39-overexpressing cells and was upregulated in the xenografted tumors derived from the HepG2/KD cells from nude mice. Meanwhile, the expression levels of FoxM1 and its target genes PLK1 and cyclin B1 were decreased in the USP39-knockdown cells. These results suggest that USP39 may contribute to FoxM1 splicing in HCC tumor cells. Our data indicate that USP39 knockdown inhibited the growth of HCC both in vitro and in vivo through G2/M arrest, which was partly achieved via the inhibition of FoxM1 splicing.

USP22 acts as an oncoprotein to maintain glioma malignancy through deubiquitinating BMI1 for stabilization.

PubMed

Qiu, Guan-Zhong; Mao, Xiao-Yuan; Ma, Yue; Gao, Xing-Chun; Wang, Zhen; Jin, Ming-Zhu; Sun, Wei; Zou, Yong-Xiang; Lin, Jing; Fu, Hua-Lin; Jin, Wei-Lin

2018-05-22

USP22 is a member of "death-from-cancer" signature, which plays a key role in cancer progression. Although previous evidence has shown that USP22 is overexpressed and correlated with poor prognosis in glioma. The effect and mechanism of USP22 in glioma malignancy especially cancer stemness remain elusive. Here, we find USP22 is more enriched in stem-like tumorspheres than differentiated glioma cells. USP22 knockdown inhibits cancer stemness in glioma cell lines. With a cell-penetrating TAT-tag protein, BMI1, a robust glioma stem-cell marker, is found to mediate the effect of USP22 on glioma stemness. By immunofluorescence, USP22 and BMI1 are found to share similar intranuclear expression in glioma cells. By analysis with immunohistochemistry and bioinformatics, USP22 is found to positively correlated with BMI1 only in the post-translational level rather than transcriptional level. By immunoprecipitation and in vivo deubiquitination assay, USP22 is found to interact with and deubiquitinate BMI1 for protein stabilization. Microarray analysis reveals that USP22 and BMI1 mutually regulate a series of genes involved in glioma stemness such as POSTN, HEY2, PDGFRA and ATF3. In vivo study with nude mice confirms the role of USP22 in promoting glioma tumorigenesis by regulating BMI1. All these findings indicate USP22 as a novel deubiquitinase of BMI1 in glioma. We propose a working model of USP22-BMI1 axis, which promotes glioma stemness and tumorigenesis through oncogenic activation. Thus, targeting USP22 might be an effective strategy to treat glioma especially those with elevated BMI1 expression. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Progressive Hearing Loss in Mice Carrying a Mutation in Usp53

PubMed Central

Kazmierczak, Marcin; Harris, Suzan L.; Kazmierczak, Piotr; Shah, Prahar; Starovoytov, Valentin; Ohlemiller, Kevin K.

2015-01-01

Disordered protein ubiquitination has been linked to neurodegenerative disease, yet its role in inner ear homeostasis and hearing loss is essentially unknown. Here we show that progressive hearing loss in the ethylnitrosourea-generated mambo mouse line is caused by a mutation in Usp53, a member of the deubiquitinating enzyme family. USP53 contains a catalytically inactive ubiquitin-specific protease domain and is expressed in cochlear hair cells and a subset of supporting cells. Although hair cell differentiation is unaffected in mambo mice, outer hair cells degenerate rapidly after the first postnatal week. USP53 colocalizes and interacts with the tight junction scaffolding proteins TJP1 and TJP2 in polarized epithelial cells, suggesting that USP53 is part of the tight junction complex. The barrier properties of tight junctions of the stria vascularis appeared intact in a biotin tracer assay, but the endocochlear potential is reduced in adult mambo mice. Hair cell degeneration in mambo mice precedes endocochlear potential decline and is rescued in cochlear organotypic cultures in low potassium milieu, indicating that hair cell loss is triggered by extracellular factors. Remarkably, heterozygous mambo mice show increased susceptibility to noise injury at high frequencies. We conclude that USP53 is a novel tight junction-associated protein that is essential for the survival of auditory hair cells and normal hearing in mice, possibly by modulating the barrier properties and mechanical stability of tight junctions. SIGNIFICANCE STATEMENT Hereditary hearing loss is extremely prevalent in the human population, but many genes linked to hearing loss remain to be discovered. Forward genetics screens in mice have facilitated the identification of genes involved in sensory perception and provided valuable animal models for hearing loss in humans. This involves introducing random mutations in mice, screening the mice for hearing defects, and mapping the causative mutation. Here, we have identified a mutation in the Usp53 gene that causes progressive hearing loss in the mambo mouse line. We demonstrate that USP53 is a catalytically inactive deubiquitinating enzyme and a novel component of tight junctions that is necessary for sensory hair cell survival and inner ear homeostasis. PMID:26609154

N- and C-terminal degradation of ecdysteroid receptor isoforms, when transiently expressed in mammalian CHO cells, is regulated by the proteasome and cysteine and threonine proteases.

PubMed

Schauer, S; Burster, T; Spindler-Barth, M

2012-06-01

Transcriptional activity of nuclear receptors is the result of transactivation capability and the concentration of the receptor protein. The concentration of ecdysteroid receptor (EcR) isoforms, constitutively expressed in mammalian CHO cells, is dependent on a number of factors. As shown previously, ligand binding stabilizes receptor protein concentration. In this paper, we investigate the degradation of EcR isoforms and provide evidence that N-terminal degradation is modulated by isoform-specific ubiquitination sites present in the A/B domains of EcR-A and -B1. This was demonstrated by the increase in EcR concentration by treatment with carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132), an inhibitor of ubiquitin-mediated proteasomal degradation and by deletion of ubiquitination sites. In addition, EcR is degraded by the peptidyl-dipeptidase cathepsin B (CatB) and the endopeptidase cathepsin S (CatS) at the C-terminus in an isoform-specific manner, despite identical C-termini. Ubiquitin-proteasome-mediated degradation and the proteolytic action are modulated by heterodimerization with Ultraspiracle (USP). The complex regulation of receptor protein concentration offers an additional opportunity to regulate transcriptional activity in an isoform- and target cell-specific way and allows the temporal limitation of hormone action. © 2012 The Authors. Insect Molecular Biology © 2012 The Royal Entomological Society.

The Promoter of AtUSP Is Co-regulated by Phytohormones and Abiotic Stresses in Arabidopsis thaliana.

PubMed

Bhuria, Monika; Goel, Parul; Kumar, Sanjay; Singh, Anil K

2016-01-01

Universal stress proteins (USPs) are known to be expressed in response to various abiotic stresses in a wide variety of organisms, such as bacteria, archaebacteria, protists, algae, fungi, plants, and animals. However, in plants, biological function of most of the USPs still remains obscure. In the present study, Arabidopsis USP gene ( AtUSP ) showed induction in response to abscisic acid (ABA) and various abiotic stresses viz . heat, dehydration, salt, osmotic, and cold stresses. Additionally, in silico analysis of AtUSP promoter identified several cis -elements responsive to phytohormones and abiotic stresses such as ABRE, ERE, DRE, and HSE, etc. To functionally validate the AtUSP promoter, the 1115 bp region of promoter was characterized under phytohormone and abiotic stress treatments. Deletion analysis of promoter was carried out by cloning the full length promoter (D0) and its three 5' deletion derivatives, D1 (964 bp), D2 (660 bp), and D3 (503 bp) upstream of the β-glucuronidase (GUS) reporter gene, which were then stably transformed in Arabidopsis plants. The AtUSP promoter (D0) showed minimal activity under non-stress conditions which was enhanced in response to phytohormone treatments (ABA and ACC) and abiotic stresses such as dehydration, heat, cold, salt, and osmotic stresses. The seedlings harboring D1 and D2 deletion fragments showed constitutive GUS expression even under control condition with increased activity almost under all the treatments. However, D3 seedlings exhibited complete loss of activity under control condition with induction under ACC treatment, dehydration, heat, oxidative, salt, and osmotic stresses. Thus, present study clearly showed that AtUSP promoter is highly inducible by phytohormones and multiple abiotic stresses and it can be exploited as stress inducible promoter to generate multi-stress tolerant crops with minimal effects on their other important traits.

USP22 knockdown enhanced chemosensitivity of hepatocellular carcinoma cells to 5-Fu by up-regulation of Smad4 and suppression of Akt

PubMed Central

Li, Jiazhi; Yang, Xiaozhou; Yin, Huimin; Xiao, Congshu; Sheng, Jie; Li, Yang; Tang, Bo; Li, Rongkuan

2017-01-01

USP22, a member of the deubiquitinases (DUBs) family, is known to be a key subunit of the human Spt-Ada-Gcn5 acetyltransferase (hSAGA) transcriptional cofactor complex. Within hSAGA, USP22 removes ubiquitin from histone proteins, thus regulating the transcription and expression of downstream genes. USP22 plays important roles in many cancers; however, its effect and the mechanism underlying HCC chemoresistance remain unclear. In the present study, we found that USP22 was highly expressed in chemoresistant HCC tissues and cells and was correlated with the prognosis of HCC patients who received chemotherapy. Silencing USP22 in chemoresistant HCC Bel/Fu cells dramatically inhibited proliferation, migration, invasion and epithelial-mesenchymal transition in vitro; suppressed tumorigenic and metastatic capacities in vivo; and inhibited drug resistance-related proteins (MDR1, LRP, MRP1). Mechanistically, we found that USP22 knockdown exerts its function through down-regulating PI3K and activating Smad4, which inhibited phosphorylation of Akt. Silencing Smad4 blocked USP22 knockdown-induced Akt inhibition in Bel/Fu cells. Our results, for the first time, provide evidence that USP22 plays a critical role in the development of chemoresistant HCC cells and that high USP22 expression serves as a molecular marker for the prognosis of HCC patients who undergo chemotherapy. PMID:28445968

USP22 knockdown enhanced chemosensitivity of hepatocellular carcinoma cells to 5-Fu by up-regulation of Smad4 and suppression of Akt.

PubMed

Zhang, Jing; Luo, Nan; Tian, Yu; Li, Jiazhi; Yang, Xiaozhou; Yin, Huimin; Xiao, Congshu; Sheng, Jie; Li, Yang; Tang, Bo; Li, Rongkuan

2017-04-11

USP22, a member of the deubiquitinases (DUBs) family, is known to be a key subunit of the human Spt-Ada-Gcn5 acetyltransferase (hSAGA) transcriptional cofactor complex. Within hSAGA, USP22 removes ubiquitin from histone proteins, thus regulating the transcription and expression of downstream genes. USP22 plays important roles in many cancers; however, its effect and the mechanism underlying HCC chemoresistance remain unclear. In the present study, we found that USP22 was highly expressed in chemoresistant HCC tissues and cells and was correlated with the prognosis of HCC patients who received chemotherapy. Silencing USP22 in chemoresistant HCC Bel/Fu cells dramatically inhibited proliferation, migration, invasion and epithelial-mesenchymal transition in vitro; suppressed tumorigenic and metastatic capacities in vivo; and inhibited drug resistance-related proteins (MDR1, LRP, MRP1). Mechanistically, we found that USP22 knockdown exerts its function through down-regulating PI3K and activating Smad4, which inhibited phosphorylation of Akt. Silencing Smad4 blocked USP22 knockdown-induced Akt inhibition in Bel/Fu cells. Our results, for the first time, provide evidence that USP22 plays a critical role in the development of chemoresistant HCC cells and that high USP22 expression serves as a molecular marker for the prognosis of HCC patients who undergo chemotherapy.

Decoding the genetic basis of Cushing's disease: USP8 in the spotlight.

PubMed

Theodoropoulou, Marily; Reincke, Martin; Fassnacht, Martin; Komada, Masayuki

2015-10-01

Cushing's disease (CD) arises from pituitary-dependent glucocorticoid excess due to an ACTH-secreting corticotroph tumor. Genetic hits in oncogenes and tumor suppressor genes that afflict other pituitary tumor subtypes are not found in corticotrophinomas. Recently, a somatic mutational hotspot was found in up to half of corticotrophinomas in the USP8 gene that encodes a protein that impairs the downregulation of the epidermal growth factor receptor (EGFR) and enables its constitutive signaling. EGF is an important regulator of corticotroph function and its receptor is highly expressed in Cushing's pituitary tumors, where it leads to increased ACTH synthesis in vitro and in vivo. The mutational hotspot found in corticotrophinomas hyper-activates USP8, enabling it to rescue EGFR from lysosomal degradation and ensure its stimulatory signaling. This review presents new developments in the study of the genetics of CD and focuses on the USP8-EGFR system as trigger and target of corticotroph tumorigenesis. © 2015 European Society of Endocrinology.

Ubiquitin specific protease 2 acts as a key modulator for the regulation of cell cycle by adiponectin and leptin in cancer cells.

PubMed

Nepal, Saroj; Shrestha, Anup; Park, Pil-Hoon

2015-09-05

Adiponectin and leptin, both produced from adipose tissue, cause cell cycle arrest and progression, respectively in cancer cells. Ubiquitin specific protease-2 (USP-2), a deubiquitinating enzyme, is known to impair proteasome-induced degradation of cyclin D1, a critical cell cycle regulator. Herein, we investigated the effects of these adipokines on USP-2 expression and its potential role in the modulation of cell cycle. Treatment with globular adiponectin (gAcrp) decreased, whereas leptin increased USP-2 expression both in human hepatoma and breast cancer cells. In addition, overexpression or gene silencing of USP-2 affected cyclin D1 expression and cell cycle progression/arrest by adipokines. Adiponectin and leptin also modulated in vitro proteasomal activity, which was partially dependent on USP-2 expression. Taken together, our results reveal that modulation of USP-2 expression plays a crucial role in cell cycle regulation by adipokines. Thus, USP-2 would be a promising therapeutic target for the modulation of cancer cell growth by adipokines. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

USP48 restrains resection by site-specific cleavage of the BRCA1 ubiquitin mark from H2A.

PubMed

Uckelmann, Michael; Densham, Ruth M; Baas, Roy; Winterwerp, Herrie H K; Fish, Alexander; Sixma, Titia K; Morris, Joanna R

2018-01-15

BRCA1-BARD1-catalyzed ubiquitination of histone H2A is an important regulator of the DNA damage response, priming chromatin for repair by homologous recombination. However, no specific deubiquitinating enzymes (DUBs) are known to antagonize this function. Here we identify ubiquitin specific protease-48 (USP48) as a H2A DUB, specific for the C-terminal BRCA1 ubiquitination site. Detailed biochemical analysis shows that an auxiliary ubiquitin, an additional ubiquitin that itself does not get cleaved, modulates USP48 activity, which has possible implications for its regulation in vivo. In cells we reveal that USP48 antagonizes BRCA1 E3 ligase function and in BRCA1-proficient cells loss of USP48 results in positioning 53BP1 further from the break site and in extended resection lengths. USP48 repression confers a survival benefit to cells treated with camptothecin and its activity acts to restrain gene conversion and mutagenic single-strand annealing. We propose that USP48 promotes genome stability by antagonizing BRCA1 E3 ligase function.

USP15 regulates dynamic protein–protein interactions of the spliceosome through deubiquitination of PRP31

PubMed Central

Das, Tanuza; Park, Joon Kyu; Park, Jinyoung; Kim, Eunji; Rape, Michael

2017-01-01

Abstract Post-translational modifications contribute to the spliceosome dynamics by facilitating the physical rearrangements of the spliceosome. Here, we report USP15, a deubiquitinating enzyme, as a regulator of protein–protein interactions for the spliceosome dynamics. We show that PRP31, a component of U4 snRNP, is modified with K63-linked ubiquitin chains by the PRP19 complex and deubiquitinated by USP15 and its substrate targeting factor SART3. USP15SART3 makes a complex with USP4 and this ternary complex serves as a platform to deubiquitinate PRP31 and PRP3. The ubiquitination and deubiquitination status of PRP31 regulates its interaction with the U5 snRNP component PRP8, which is required for the efficient splicing of chromosome segregation related genes, probably by stabilizing the U4/U6.U5 tri-snRNP complex. Collectively, our data suggest that USP15 plays a key role in the regulation of dynamic protein–protein interactions of the spliceosome. PMID:28088760

The nucleolar ubiquitin-specific protease USP36 deubiquitinates and stabilizes c-Myc

PubMed Central

Sun, Xiao-Xin; He, Xia; Yin, Li; Komada, Masayuki; Sears, Rosalie C.; Dai, Mu-Shui

2015-01-01

c-Myc protein stability and activity are tightly regulated by the ubiquitin-proteasome system. Aberrant stabilization of c-Myc contributes to many human cancers. c-Myc is ubiquitinated by SCFFbw7 (a SKP1-cullin-1-F-box complex that contains the F-box and WD repeat domain-containing 7, Fbw7, as the F-box protein) and several other ubiquitin ligases, whereas it is deubiquitinated and stabilized by ubiquitin-specific protease (USP) 28. The bulk of c-Myc degradation appears to occur in the nucleolus. However, whether c-Myc is regulated by deubiquitination in the nucleolus is not known. Here, we report that the nucleolar deubiquitinating enzyme USP36 is a novel c-Myc deubiquitinase. USP36 interacts with and deubiquitinates c-Myc in cells and in vitro, leading to the stabilization of c-Myc. This USP36 regulation of c-Myc occurs in the nucleolus. Interestingly, USP36 interacts with the nucleolar Fbw7γ but not the nucleoplasmic Fbw7α. However, it abolished c-Myc degradation mediated both by Fbw7γ and by Fbw7α. Consistently, knockdown of USP36 reduces the levels of c-Myc and suppresses cell proliferation. We further show that USP36 itself is a c-Myc target gene, suggesting that USP36 and c-Myc form a positive feedback regulatory loop. High expression levels of USP36 are found in a subset of human breast and lung cancers. Altogether, these results identified USP36 as a crucial and bono fide deubiquitinating enzyme controlling c-Myc’s nucleolar degradation pathway. PMID:25775507

Inhibition of Ubiquitin-specific Peptidase 8 Suppresses Adrenocorticotropic Hormone Production and Tumorous Corticotroph Cell Growth in AtT20 Cells

PubMed Central

Jian, Fang-Fang; Li, Yun-Feng; Chen, Yu-Fan; Jiang, Hong; Chen, Xiao; Zheng, Li-Li; Zhao, Yao; Wang, Wei-Qing; Ning, Guang; Bian, Liu-Guan; Sun, Qing-Fang

2016-01-01

Background: Two recent whole-exome sequencing researches identifying somatic mutations in the ubiquitin-specific protease 8 (USP8) gene in pituitary corticotroph adenomas provide exciting advances in this field. These mutations drive increased epidermal growth factor receptor (EGFR) signaling and promote adrenocorticotropic hormone (ACTH) production. This study was to investigate whether the inhibition of USP8 activity could be a strategy for the treatment of Cushing's disease (CD). Methods: The anticancer effect of USP8 inhibitor was determined by testing cell viability, colony formation, apoptosis, and ACTH secretion. The immunoblotting and quantitative reverse transcription polymerase chain reaction were conducted to explore the signaling pathway by USP8 inhibition. Results: Inhibition of USP8-induced degradation of receptor tyrosine kinases including EGFR, EGFR-2 (ERBB2), and Met leading to a suppression of AtT20 cell growth and ACTH secretion. Moreover, treatment with USP8 inhibitor markedly induced AtT20 cells apoptosis. Conclusions: Inhibition of USP8 activity could be an effective strategy for CD. It might provide a novel pharmacological approach for the treatment of CD. PMID:27569239

20-Hydroxyecdysone (20E) Primary Response Gene E93 Modulates 20E Signaling to Promote Bombyx Larval-Pupal Metamorphosis*

PubMed Central

Liu, Xi; Dai, Fangyin; Guo, Enen; Li, Kang; Ma, Li; Tian, Ling; Cao, Yang; Zhang, Guozheng; Palli, Subba R.; Li, Sheng

2015-01-01

As revealed in a previous microarray study to identify genes regulated by 20-hydroxyecdysone (20E) and juvenile hormone (JH) in the silkworm, Bombyx mori, E93 expression in the fat body was markedly low prior to the wandering stage but abundant during larval-pupal metamorphosis. Induced by 20E and suppressed by JH, E93 expression follows this developmental profile in multiple silkworm alleles. The reduction of E93 expression by RNAi disrupted 20E signaling and the 20E-induced autophagy, caspase activity, and cell dissociation in the fat body. Reducing E93 expression also decreased the expression of the 20E-induced pupal-specific cuticle protein genes and prevented growth and differentiation of the wing discs. Importantly, the two HTH domains in E93 are critical for inducing the expression of a subset of 20E response genes, including EcR, USP, E74, Br-C, and Atg1. By contrast, the LLQHLL and PLDLSAK motifs in E93 inhibit its transcriptional activity. E93 binds to the EcR-USP complex via a physical association with USP through its LLQHLL motif; and this association is enhanced by 20E-induced EcR-USP interaction, which attenuates the transcriptional activity of E93. E93 acts through the two HTH domains to bind to GAGA-containing motifs present in the Atg1 promoter region for inducing gene expression. In conclusion, E93 transcriptionally modulates 20E signaling to promote Bombyx larval-pupal metamorphosis. PMID:26378227

20-Hydroxyecdysone (20E) Primary Response Gene E93 Modulates 20E Signaling to Promote Bombyx Larval-Pupal Metamorphosis.

PubMed

Liu, Xi; Dai, Fangyin; Guo, Enen; Li, Kang; Ma, Li; Tian, Ling; Cao, Yang; Zhang, Guozheng; Palli, Subba R; Li, Sheng

2015-11-06

As revealed in a previous microarray study to identify genes regulated by 20-hydroxyecdysone (20E) and juvenile hormone (JH) in the silkworm, Bombyx mori, E93 expression in the fat body was markedly low prior to the wandering stage but abundant during larval-pupal metamorphosis. Induced by 20E and suppressed by JH, E93 expression follows this developmental profile in multiple silkworm alleles. The reduction of E93 expression by RNAi disrupted 20E signaling and the 20E-induced autophagy, caspase activity, and cell dissociation in the fat body. Reducing E93 expression also decreased the expression of the 20E-induced pupal-specific cuticle protein genes and prevented growth and differentiation of the wing discs. Importantly, the two HTH domains in E93 are critical for inducing the expression of a subset of 20E response genes, including EcR, USP, E74, Br-C, and Atg1. By contrast, the LLQHLL and PLDLSAK motifs in E93 inhibit its transcriptional activity. E93 binds to the EcR-USP complex via a physical association with USP through its LLQHLL motif; and this association is enhanced by 20E-induced EcR-USP interaction, which attenuates the transcriptional activity of E93. E93 acts through the two HTH domains to bind to GAGA-containing motifs present in the Atg1 promoter region for inducing gene expression. In conclusion, E93 transcriptionally modulates 20E signaling to promote Bombyx larval-pupal metamorphosis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

High expression of ubiquitin-specific peptidase 39 is associated with the development of vascular remodeling

PubMed Central

He, Shuai; Zhong, Wei; Yin, Li; Wang, Yifei; Qiu, Zhibing; Song, Gang

2017-01-01

Vascular remodeling is the primary cause underlying the failure of angioplasty surgeries, including vascular stenting, transplant vasculopathy and vein grafts. Multiple restenosis-associated proteins and genes have been identified to account for this. In the present study, the functions of ubiquitin-specific peptidase 39 (USP39) were investigated in the context of two vascular remodeling models (a mouse common carotid artery ligation and a pig bilateral saphenous vein-carotid artery interposition graft). USP39 has previously been observed to be upregulated in ligated arteries, and this result was confirmed in the pig vein graft model. In addition, Transwell assay results demonstrated that vascular smooth muscle cell (VSMC) migration was suppressed by lentiviral vector-mediated downregulation of USP39 and enhanced by upregulation of USP39. Furthermore, knockdown of USP39 inhibited VSMC cell proliferation and the expression of cyclin D1 and cyclin-dependent kinase 4, as analyzed via cell counting, MTT assay and western blotting. These results suggest that USP39 may represent a novel therapeutic target for treating vascular injury and preventing vein-graft failure. PMID:28447728

[Morphological spectrum of USP6 rearranged lesions].

PubMed

Mechtersheimer, G; Werner, M

2018-03-01

USP6, also known as Tre-2 and TRE17, is an ubiquitase-specific proteinase that was identified more than two decades ago as a potential oncogene when it exhibited transforming properties upon overexpression in NIH 3T3 cells. Until recently, however, little was known about the function and the oncogenetic activation of USP6. The identification of rearrangements of the USP6 gene in aneurysmal bone cyst and in nodular fasciitis has not only led to a better understanding of the pathogenesis of these entities, but is also a useful tool in their diagnosis and differential diagnostic delineation from morphological mimics. In this review, the clinical, pathomorphological, and molecular genetic aspects of aneurysmal bone cyst and of nodular fasciitis, as well as from related lesions, are presented and discussed.

Acinetobacter baumannii universal stress protein A plays a pivotal role in stress response and is essential for pneumonia and sepsis pathogenesis.

PubMed

Elhosseiny, Noha M; Amin, Magdy A; Yassin, Aymen S; Attia, Ahmed S

2015-01-01

Acinetobacter baumannii is one of the most significant threats to global public health. This threat is compounded by the fact that A. baumannii is rapidly becoming resistant to all relevant antimicrobials. Identifying key microbial factors through which A. baumannii resists hostile host environment is paramount to the development of novel antimicrobials targeting infections caused by this emerging pathogen. An attractive target could be a molecule that plays a role in the pathogenesis and stress response of A. baumannii. Accordingly, the universal stress protein A (UspA) was chosen to be fully investigated in this study. A platform of A. baumannii constructs, expressing various levels of the uspA gene ranging from zero to thirteen folds of wild-type level, and a recombinant E. coli strain, were employed to investigate the role of UspA in vitro stress and in vivo pathogenesis. The UspA protein plays a significant role in protecting A. baumannii from H(2)O(2), low pH, and the respiratory toxin 2,4-DNP. A. baumannii UspA protein plays an essential role in two of the deadliest types of infection caused by A. baumannii; pneumonia and sepsis. This distinguishes A. baumannii UspA from its closely related homolog, the Staphylococcus aureus Usp2, as well as from the less similar Burkholderia glumae Usps. Heterologous and overexpression experiments suggest that UspA mediates its role via an indirect mechanism. Our study highlights the role of UspA as an important contributor to the A. baumannii stress and virulence machineries, and polishes it as a plausible target for new therapeutics. Copyright © 2014 Elsevier GmbH. All rights reserved.

The ubiquitin-specific protease USP15 promotes RIG-I-mediated antiviral signaling by deubiquitylating TRIM25.

PubMed

Pauli, Eva-Katharina; Chan, Ying Kai; Davis, Meredith E; Gableske, Sebastian; Wang, May K; Feister, Katharina F; Gack, Michaela U

2014-01-07

Ubiquitylation is an important mechanism for regulating innate immune responses to viral infections. Attachment of lysine 63 (Lys(63))-linked ubiquitin chains to the RNA sensor retinoic acid-inducible gene-I (RIG-I) by the ubiquitin E3 ligase tripartite motif protein 25 (TRIM25) leads to the activation of RIG-I and stimulates production of the antiviral cytokines interferon-α (IFN-α) and IFN-β. Conversely, Lys(48)-linked ubiquitylation of TRIM25 by the linear ubiquitin assembly complex (LUBAC) stimulates the proteasomal degradation of TRIM25, thereby inhibiting the RIG-I signaling pathway. Here, we report that ubiquitin-specific protease 15 (USP15) deubiquitylates TRIM25, preventing the LUBAC-dependent degradation of TRIM25. Through protein purification and mass spectrometry analysis, we identified USP15 as an interaction partner of TRIM25 in human cells. Knockdown of endogenous USP15 by specific small interfering RNA markedly enhanced the ubiquitylation of TRIM25. In contrast, expression of wild-type USP15, but not its catalytically inactive mutant, reduced the Lys(48)-linked ubiquitylation of TRIM25, leading to its stabilization. Furthermore, ectopic expression of USP15 enhanced the TRIM25- and RIG-I-dependent production of type I IFN and suppressed RNA virus replication. In contrast, depletion of USP15 resulted in decreased IFN production and markedly enhanced viral replication. Together, these data identify USP15 as a critical regulator of the TRIM25- and RIG-I-mediated antiviral immune response, thereby highlighting the intricate regulation of innate immune signaling.

The Ubiquitin-Specific Protease USP15 Promotes RIG-I–Mediated Antiviral Signaling by Deubiquitylating TRIM25

PubMed Central

Pauli, Eva-Katharina; Chan, Ying Kai; Davis, Meredith E.; Gableske, Sebastian; Wang, May K.; Feister, Katharina F.; Gack, Michaela U.

2014-01-01

Ubiquitylation is an important mechanism for regulating innate immune responses to viral infections. Attachment of lysine 63 (Lys63)–linked ubiquitin chains to the RNA sensor retinoic acid–inducible gene-I (RIG-I) by the ubiquitin E3 ligase tripartite motif protein 25 (TRIM25) leads to the activation of RIG-I and stimulates production of the antiviral cytokines interferon-α (IFN-α) and IFN-β. Conversely, Lys48-linked ubiquitylation of TRIM25 by the linear ubiquitin assembly complex (LUBAC) stimulates the proteasomal degradation of TRIM25, thereby inhibiting the RIG-I signaling pathway. Here, we report that ubiquitin-specific protease 15 (USP15) deubiquitylates TRIM25, preventing the LUBAC-dependent degradation of TRIM25. Through protein purification and mass spectrometry analysis, we identified USP15 as an interaction partner of TRIM25 in human cells. Knockdown of endogenous USP15 by specific small interfering RNA markedly enhanced the ubiquitylation of TRIM25. In contrast, expression of wild-type USP15, but not its catalytically inactive mutant, reduced the Lys48-linked ubiquitylation of TRIM25, leading to its stabilization. Furthermore, ectopic expression of USP15 enhanced the TRIM25- and RIG-I–dependent production of type I IFN and suppressed RNA virus replication. In contrast, depletion of USP15 resulted in decreased IFN production and markedly enhanced viral replication. Together, these data identify USP15 as a critical regulator of the TRIM25- and RIG-I–mediated antiviral immune response, thereby highlighting the intricate regulation of innate immune signaling. PMID:24399297

Behavioral Characteristics of Ubiquitin-Specific Peptidase 46-Deficient Mice

PubMed Central

Imai, Saki; Kano, Makoto; Nonoyama, Keiko; Ebihara, Shizufumi

2013-01-01

We have previously identified Usp46, which encodes for ubiquitin-specific peptidase 46, as a quantitative trait gene affecting the immobility time of mice in the tail suspension test (TST) and forced swimming test. The mutation that we identified was a 3-bp deletion coding for lysine (Lys 92), and mice with this mutation (MT mice), as well as Usp46 KO mice exhibited shorter TST immobility times. Behavioral pharmacology suggests that the gamma aminobutyric acid A (GABAA) receptor is involved in regulating TST immobility time. In order to understand how far Usp46 controls behavioral phenotypes, which could be related to mental disorders in humans, we subjected Usp46 MT and KO mice to multiple behavioral tests, including the open field test, ethanol preference test, ethanol-induced loss of righting reflex test, sucrose preference test, novelty-suppressed feeding test, marble burying test, and novel object recognition test. Although behavioral phenotypes of the Usp46 MT and KO mice were not always identical, deficiency of Usp46 significantly affected performance in all these tests. In the open field test, activity levels were lower in Usp46 KO mice than wild type (WT) or MT mice. Both MT and KO mice showed lower ethanol preference and shorter recovery times after ethanol administration. Compared to WT mice, Usp46 MT and KO mice exhibited decreased sucrose preference, took longer latency periods to bite pellets, and buried more marbles in the sucrose preference test, novelty-suppressed feeding test, and marble burying test, respectively. In the novel object recognition test, neither MT nor KO mice showed an increase in exploration of a new object 24 hours after training. These findings indicate that Usp46 regulates a wide range of behavioral phenotypes that might be related to human mental disorders and provides insight into the function of USP46 deubiquitinating enzyme in the neural system. PMID:23472206

Use of the usp45 lactococcal secretion signal sequence to drive the secretion and functional expression of enterococcal bacteriocins in Lactococcus lactis.

PubMed

Borrero, Juan; Jiménez, Juan J; Gútiez, Loreto; Herranz, Carmen; Cintas, Luis M; Hernández, Pablo E

2011-01-01

Replacement of the signal peptide (SP) of the bacteriocins enterocin P (EntP) and hiracin JM79 (HirJM79), produced by Enterococcus faecium P13 and Enterococcus hirae DCH5, respectively, by the signal peptide of Usp45 (SP(usp45)), the major Sec-dependent protein secreted by Lactococcus lactis, permits the production, secretion, and functional expression of EntP and HirJM79 by L. lactis. Chimeric genes encoding the SP(usp45) fused to either mature EntP (entP), with or without the immunity gene (entiP) or to mature HirJM79 (hirJM79), with or without the immunity gene (hiriJM79), were cloned into the expression vector pMG36c, carrying the P(32) constitutive promoter, and into pNZ8048 under control of the inducible PnisA promoter. The production of EntP and HirJM79 by most of the L. lactis recombinant strains was 1.5- to 3.7-fold higher and up to 3.6-fold higher than by the E. faecium P13 and E. hirae DCH5 control strains, respectively. However, the specific antimicrobial activity of the recombinant EntP was 1.1- to 6.2-fold higher than that produced by E. faecium P13, while that of the HirJM79 was a 40% to an 89% of that produced by E. hirae DCH5. Chimeras of SP(usp45) fused to mature EntP or HirJM79 drive the production and secretion of these bacteriocins in L. lactis in the absence of specific immunity and secretion proteins. The supernatants of the recombinant L. lactis NZ9000 strains, producers of EntP, showed a much higher antimicrobial activity against Listeria spp. than that of the recombinant L. lactis NZ9000 derivatives, producers of HirJM79.

Two females with mutations in USP9X highlight the variable expressivity of the intellectual disability syndrome.

PubMed

Au, P Y B; Huang, L; Broley, S; Gallagher, L; Creede, E; Lahey, D; Ordorica, S; Mina, K; Boycott, K M; Baynam, G; Dyment, D A

2017-07-01

The genetic causes of intellectual disability (ID) are heterogeneous and include both chromosomal and monogenic etiologies. The X-chromosome is known to contain many ID-related genes and males show a marked predominance for intellectual disability. Here we report two females with syndromic intellectual disability. The first individual was relatively mild in her presentation with mild-moderate intellectual disability, hydronephrosis and altered pigmentation along the lines of Blaschko without additional congenital anomalies. A second female presented shortly after birth with dysmorphic facial features, post-axial polydactyly and, on follow-up assessment, demonstrated moderate intellectual disability. Chromosomal studies for Individual 1 identified an X-chromosome deletion due to a de novo pericentric inversion; the inversion breakpoint was associated with deletion of the 5'UTR of the USP9X, a gene which has been implicated in a syndromic intellectual disability affecting females. The second individual had a de novo frameshift mutation detected by whole-exome sequencing that was predicted to be deleterious, NM_001039590.2 (USP9X): c.4104_4105del (p.(Arg1368Serfs*2)). Haploinsufficiency of USP9X in females has been associated with ID and congenital malformations that include heart defects, scoliosis, dental abnormalities, anal atresia, polydactyly, Dandy Walker malformation and hypoplastic corpus callosum. The extent of the congenital malformations observed in Individual 1 was less striking than Individual 2 and other individuals previously reported in the literature, and suggests that USP9X mutations in females can have a wider spectrum of presentation than previously appreciated. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

The USP8 mutational status may predict drug susceptibility in corticotroph adenomas of Cushing's disease.

PubMed

Hayashi, Kyohei; Inoshita, Naoko; Kawaguchi, Kohei; Ibrahim Ardisasmita, Arif; Suzuki, Hisanori; Fukuhara, Noriaki; Okada, Mitsuo; Nishioka, Hiroshi; Takeuchi, Yasuhiro; Komada, Masayuki; Takeshita, Akira; Yamada, Shozo

2016-02-01

Somatic mutations in the ubiquitin-specific peptidase USP8 gene were recently detected in one- to two-third(s) of corticotroph adenomas of Cushing's disease (CD). These mutations may lead to the deubiquitination of EGFR, thereby increasing EGFR signaling, which has been implicated in ACTH hypersecretion. Our objective was to determine the impact of USP8 mutations on the clinicopathological features of CD. USP8 mutations as well as clinicopathological characteristics were examined in 60 corticotroph adenomas including 15 Crooke's cell adenomas (CCAs), a rare histological variant presenting with generally aggressive behavior, using qRT-PCR and/or immunohistochemistry. USP8 mutations were exclusively detected in women, except for one case, with a prevalence of 42.2% in non-CCA and 13.3% in CCA (overall 35%). Clinically well-behaved presentations including microadenoma and curative resection were more common in mutated cases. The expression of EGFR was not associated with the mutation status. In contrast, mutated tumors expressed significantly higher levels of POMC, SSTR5, and MGMT. Microadenomas that strongly express POMC were common among mutated tumors, which may lead to the mechanisms by which very small adenomas secrete excess ACTH to present overt CD. While USP8 mutations were less likely to enhance tumorous ACTH hypersecretion via EGFR-mediated activation, the presence of USP8 mutations may predict favorable responses to the somatostatin analog pasireotide, which exhibits high affinity for SSTR5. In contrast, non-mutated aggressive tumors such as CCA may respond better to the alkylating agent temozolomide because of their significantly weak expression of MGMT. © 2016 European Society of Endocrinology.

USP8 regulates mitophagy by removing K6-linked ubiquitin conjugates from parkin

PubMed Central

Durcan, Thomas M; Tang, Matthew Y; Pérusse, Joëlle R; Dashti, Eman A; Aguileta, Miguel A; McLelland, Gian-Luca; Gros, Priti; Shaler, Thomas A; Faubert, Denis; Coulombe, Benoit; Fon, Edward A

2014-01-01

Mutations in the Park2 gene, encoding the E3 ubiquitin-ligase parkin, are responsible for a familial form of Parkinson's disease (PD). Parkin-mediated ubiquitination is critical for the efficient elimination of depolarized dysfunctional mitochondria by autophagy (mitophagy). As damaged mitochondria are a major source of toxic reactive oxygen species within the cell, this pathway is believed to be highly relevant to the pathogenesis of PD. Little is known about how parkin-mediated ubiquitination is regulated during mitophagy or about the nature of the ubiquitin conjugates involved. We report here that USP8/UBPY, a deubiquitinating enzyme not previously implicated in mitochondrial quality control, is critical for parkin-mediated mitophagy. USP8 preferentially removes non-canonical K6-linked ubiquitin chains from parkin, a process required for the efficient recruitment of parkin to depolarized mitochondria and for their subsequent elimination by mitophagy. This work uncovers a novel role for USP8-mediated deubiquitination of K6-linked ubiquitin conjugates from parkin in mitochondrial quality control. PMID:25216678

USP7 Is a Tumor-Specific WNT Activator for APC-Mutated Colorectal Cancer by Mediating β-Catenin Deubiquitination.

PubMed

Novellasdemunt, Laura; Foglizzo, Valentina; Cuadrado, Laura; Antas, Pedro; Kucharska, Anna; Encheva, Vesela; Snijders, Ambrosius P; Li, Vivian S W

2017-10-17

The tumor suppressor gene adenomatous polyposis coli (APC) is mutated in most colorectal cancers (CRCs), resulting in constitutive Wnt activation. To understand the Wnt-activating mechanism of the APC mutation, we applied CRISPR/Cas9 technology to engineer various APC-truncated isogenic lines. We find that the β-catenin inhibitory domain (CID) in APC represents the threshold for pathological levels of Wnt activation and tumor transformation. Mechanistically, CID-deleted APC truncation promotes β-catenin deubiquitination through reverse binding of β-TrCP and USP7 to the destruction complex. USP7 depletion in APC-mutated CRC inhibits Wnt activation by restoring β-catenin ubiquitination, drives differentiation, and suppresses xenograft tumor growth. Finally, the Wnt-activating role of USP7 is specific to APC mutations; thus, it can be used as a tumor-specific therapeutic target for most CRCs. Copyright © 2017 The Francis Crick Institute. Published by Elsevier Inc. All rights reserved.

Quantitative analysis by next generation sequencing of hematopoietic stem and progenitor cells (LSK) and of splenic B cells transcriptomes from wild-type and Usp3-knockout mice.

PubMed

Lancini, Cesare; Gargiulo, Gaetano; van den Berk, Paul C M; Citterio, Elisabetta

2016-03-01

The data described here provide genome-wide expression profiles of murine primitive hematopoietic stem and progenitor cells (LSK) and of B cell populations, obtained by high throughput sequencing. Cells are derived from wild-type mice and from mice deficient for the ubiquitin-specific protease 3 (USP3; Usp3Δ/Δ). Modification of histone proteins by ubiquitin plays a crucial role in the cellular response to DNA damage (DDR) (Jackson and Durocher, 2013) [1]. USP3 is a histone H2A deubiquitinating enzyme (DUB) that regulates ubiquitin-dependent DDR in response to DNA double-strand breaks (Nicassio et al., 2007; Doil et al., 2008) [2], [3]. Deletion of USP3 in mice increases the incidence of spontaneous tumors and affects hematopoiesis [4]. In particular, Usp3-knockout mice show progressive loss of B and T cells and decreased functional potential of hematopoietic stem cells (HSCs) during aging. USP3-deficient cells, including HSCs, display enhanced histone ubiquitination, accumulate spontaneous DNA damage and are hypersensitive to ionizing radiation (Lancini et al., 2014) [4]. To address whether USP3 loss leads to deregulation of specific molecular pathways relevant to HSC homeostasis and/or B cell development, we have employed the RNA-sequencing technology and investigated transcriptional differences between wild-type and Usp3Δ/Δ LSK, naïve B cells or in vitro activated B cells. The data relate to the research article "Tight regulation of ubiquitin-mediated DNA damage response by USP3 preserves the functional integrity of hematopoietic stem cells" (Lancini et al., 2014) [4]. The RNA-sequencing and analysis data sets have been deposited in NCBI׳s Gene Expression Omnibus (Edgar et al., 2002) [5] and are accessible through GEO Series accession number GSE58495 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE58495). With this article, we present validation of the RNA-seq data set through quantitative real-time PCR and comparative analysis.

RXR function requires binding to an endogenous terpenoid ligand

USDA-ARS?s Scientific Manuscript database

The issue of whether the nuclear receptor RXR must bind to an endogenous, nanomolar affinity ligand in order to perform its natural function is still unsettled (1). On the basis of our previous studies establishing that the Drosophilamelanogaster ortholog of the retinoid X receptor ("ultraspiracle,"...

CSN-associated USP48 confers stability to nuclear NF-κB/RelA by trimming K48-linked Ub-chains.

PubMed

Schweitzer, Katrin; Naumann, Michael

2015-02-01

Diligent balance of nuclear factor kappa B (NF-κB) activity is essential owing to NF-κB's decisive role in cellular processes including inflammation, immunity and cell survival. Ubiquitin/proteasome-system (UPS)-dependent degradation of activated NF-κB/RelA involves the cullin-RING-ubiquitin-ligase (CRL) ECS(SOCS1). The COP9 signalosome (CSN) controls ubiquitin (Ub) ligation by CRLs through the removal of the CRL-activating Ub-like modifier NEDD8 from their cullin subunits and through deubiquitinase (DUB) activity of associated DUBs. However, knowledge about DUBs involved in the regulation of NF-κB activity within the nucleus is scarce. In this study we observed that USP48, a DUB of hitherto ill-defined function identified through a siRNA screen, associates with the CSN and RelA in the nucleus. We show that USP48 trims rather than completely disassembles long K48-linked free and substrate-anchored Ub-chains, a catalytic property only shared with ataxin-3 (Atx3) and otubain-1 (OTU1), and that USP48 Ub-chain-trimming activity is regulated by casein-kinase-2 (CK2)-mediated phosphorylation in response to cytokine-stimulation. Functionally, we demonstrate for the first time the CSN and USP48 to cooperatively stabilize the nuclear pool of RelA, thereby facilitating timely induction and shutoff of NF-κB target genes. In summary, this study demonstrates that USP48, a nuclear DUB regulated by CK2, controls the UPS-dependent turnover of activated NF-κB/RelA in the nucleus together with the CSN. Thereby USP48 contributes to a timely control of immune responses. Copyright © 2014 Elsevier B.V. All rights reserved.

Crystal structure of the protein At3g01520, a eukaryotic universal stress protein-like protein from Arabidopsis thaliana in complex with AMP.

PubMed

Kim, Do Jin; Bitto, Eduard; Bingman, Craig A; Kim, Hyun-Jung; Han, Byung Woo; Phillips, George N

2015-07-01

Members of the universal stress protein (USP) family are conserved in a phylogenetically diverse range of prokaryotes, fungi, protists, and plants and confer abilities to respond to a wide range of environmental stresses. Arabidopsis thaliana contains 44 USP domain-containing proteins, and USP domain is found either in a small protein with unknown physiological function or in an N-terminal portion of a multi-domain protein, usually a protein kinase. Here, we report the first crystal structure of a eukaryotic USP-like protein encoded from the gene At3g01520. The crystal structure of the protein At3g01520 was determined by the single-wavelength anomalous dispersion method and refined to an R factor of 21.8% (Rfree = 26.1%) at 2.5 Å resolution. The crystal structure includes three At3g01520 protein dimers with one AMP molecule bound to each protomer, comprising a Rossmann-like α/β overall fold. The bound AMP and conservation of residues in the ATP-binding loop suggest that the protein At3g01520 also belongs to the ATP-binding USP subfamily members. © 2015 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.

Ubiquitin-Specific Protease 2 Regulates Hepatic Gluconeogenesis and Diurnal Glucose Metabolism Through 11β-Hydroxysteroid Dehydrogenase 1

PubMed Central

Molusky, Matthew M.; Li, Siming; Ma, Di; Yu, Lei; Lin, Jiandie D.

2012-01-01

Hepatic gluconeogenesis is important for maintaining steady blood glucose levels during starvation and through light/dark cycles. The regulatory network that transduces hormonal and circadian signals serves to integrate these physiological cues and adjust glucose synthesis and secretion by the liver. In this study, we identified ubiquitin-specific protease 2 (USP2) as an inducible regulator of hepatic gluconeogenesis that responds to nutritional status and clock. Adenoviral-mediated expression of USP2 in the liver promotes hepatic glucose production and exacerbates glucose intolerance in diet-induced obese mice. In contrast, in vivo RNA interference (RNAi) knockdown of this factor improves systemic glycemic control. USP2 is a target gene of peroxisome proliferator–activated receptor γ coactivator-1α (PGC-1α), a coactivator that integrates clock and energy metabolism, and is required for maintaining diurnal glucose homeostasis during restricted feeding. At the mechanistic level, USP2 regulates hepatic glucose metabolism through its induction of 11β-hydroxysteroid dehydrogenase 1 (HSD1) and glucocorticoid signaling in the liver. Pharmacological inhibition and liver-specific RNAi knockdown of HSD1 significantly impair the stimulation of hepatic gluconeogenesis by USP2. Together, these studies delineate a novel pathway that links hormonal and circadian signals to gluconeogenesis and glucose homeostasis. PMID:22447855

The downregulation of Mcl-1 via USP9X inhibition sensitizes solid tumors to Bcl-xl inhibition

PubMed Central

2012-01-01

Background It has been shown in many solid tumors that the overexpression of the pro-survival Bcl-2 family members Bcl-xL and Mcl-1 confers resistance to a variety of chemotherapeutic agents. Mcl-1 is a critical survival protein in a variety of cell lineages and is critically regulated via ubiquitination. Methods The Mcl-1, Bcl-xL and USP9X expression patterns in human lung and colon adenocarcinomas were evaluated via immunohistochemistry. Interaction between USP9X and Mcl-1 was demonstrated by immunoprecipitation-western blotting. The protein expression profiles of Mcl-1, Bcl-xL and USP9X in multiple cancer cell lines were determined by western blotting. Annexin-V staining and cleaved PARP western blotting were used to assay for apoptosis. The cellular toxicities after various treatments were measured via the XTT assay. Results In our current analysis of colon and lung cancer samples, we demonstrate that Mcl-1 and Bcl-xL are overexpressed and also co-exist in many tumors and that the expression levels of both genes correlate with the clinical staging. The downregulation of Mcl-1 or Bcl-xL via RNAi was found to increase the sensitivity of the tumor cells to chemotherapy. Furthermore, our analyses revealed that USP9X expression correlates with that of Mcl-1 in human cancer tissue samples. We additionally found that the USP9X inhibitor WP1130 promotes Mcl-1 degradation and increases tumor cell sensitivity to chemotherapies. Moreover, the combination of WP1130 and ABT-737, a well-documented Bcl-xL inhibitor, demonstrated a chemotherapeutic synergy and promoted apoptosis in different tumor cells. Conclusion Mcl-1, Bcl-xL and USP9X overexpression are tumor survival mechanisms protective against chemotherapy. USP9X inhibition increases tumor cell sensitivity to various chemotherapeutic agents including Bcl-2/Bcl-xL inhibitors. PMID:23171055

Genetic and microbial factors modulating the ubiquitin proteasome system in inflammatory bowel disease.

PubMed

Cleynen, Isabelle; Vazeille, Emilie; Artieda, Marta; Verspaget, Hein W; Szczypiorska, Magdalena; Bringer, Marie-Agnès; Lakatos, Peter L; Seibold, Frank; Parnell, Kirstie; Weersma, Rinse K; Mahachie John, Jestinah M; Morgan-Walsh, Rebecca; Staelens, Dominiek; Arijs, Ingrid; De Hertogh, Gert; Müller, Stefan; Tordai, Atilla; Hommes, Daniel W; Ahmad, Tariq; Wijmenga, Cisca; Pender, Sylvia; Rutgeerts, Paul; Van Steen, Kristel; Lottaz, Daniel; Vermeire, Severine; Darfeuille-Michaud, Arlette

2014-08-01

Altered microbiota composition, changes in immune responses and impaired intestinal barrier functions are observed in IBD. Most of these features are controlled by proteases and their inhibitors to maintain gut homeostasis. Unrestrained or excessive proteolysis can lead to pathological gastrointestinal conditions. The aim was to validate the identified protease IBD candidates from a previously performed systematic review through a genetic association study and functional follow-up. We performed a genetic association study in a large multicentre cohort of patients with Crohn's disease (CD) and UC from five European IBD referral centres in a total of 2320 CD patients, 2112 UC patients and 1796 healthy controls. Subsequently, we did an extensive functional assessment of the candidate genes to explore their causality in IBD pathogenesis. Ten single nucleotide polymorphisms (SNPs) in four genes were significantly associated with CD: CYLD, USP40, APEH and USP3. CYLD was the most significant gene with the intronically located rs12324931 the strongest associated SNP (p(FDR)=1.74e-17, OR=2.24 (1.83 to 2.74)). Five SNPs in four genes were significantly associated with UC: USP40, APEH, DAG1 and USP3. CYLD, as well as some of the other associated genes, is part of the ubiquitin proteasome system (UPS). We therefore determined if the IBD-associated adherent-invasive Escherichia coli (AIEC) can modulate the UPS functioning. Infection of intestinal epithelial cells with the AIEC LF82 reference strain modulated the UPS turnover by reducing poly-ubiquitin conjugate accumulation, increasing 26S proteasome activities and decreasing protein levels of the NF-κB regulator CYLD. This resulted in IκB-α degradation and NF-κB activation. This activity was very important for the pathogenicity of AIEC since decreased CYLD resulted in increased ability of AIEC LF82 to replicate intracellularly. Our results reveal the UPS, and CYLD specifically, as an important contributor to IBD pathogenesis, which is favoured by both genetic and microbial factors. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Virulence Genes and Antibiotic Susceptibilities of Uropathogenic E. coli Strains.

PubMed

Uzun, Cengiz; Oncül, Oral; Gümüş, Defne; Alan, Servet; Dayioğlu, Nurten; Küçüker, Mine Anğ

2015-01-01

The aim of this study is to detect the presence of and possible relation between virulence genes and antibiotic resistance in E. coli strains isolated from patients with acute, uncomplicated urinary tract infections (UTI). 62 E. coli strains isolated from patients with acute, uncomplicated urinary tract infections (50 strains isolated from acute uncomplicated cystitis cases (AUC); 12 strains from acute uncomplicated pyelonephritis cases (AUP)) were screened for virulence genes [pap (pyelonephritis-associated pili), sfa/foc (S and F1C fimbriae), afa (afimbrial adhesins), hly (hemolysin), cnf1 (cytotoxic necrotizing factor), aer (aerobactin), PAI (pathogenicity island marker), iroN (catecholate siderophore receptor), ompT (outer membrane protein T), usp (uropathogenic specific protein)] by PCR and for antimicrobial resistance by disk diffusion method according to CLSI criteria. It was found that 56 strains (90.3%) carried at least one virulence gene. The most common virulence genes were ompT (79%), aer (51.6%), PAI (51.6%) and usp (56.5%). 60% of the strains were resistant to at least one antibiotic. The highest resistance rates were against ampicillin (79%) and co-trimoxazole (41.9%). Fifty percent of the E. coli strains (31 strains) were found to be multiple resistant. Eight (12.9%) out of 62 strains were found to be ESBL positive. Statistically significant relationships were found between the absence of usp and AMP - SXT resistance, iroN and OFX - CIP resistance, PAI and SXT resistance, cnf1 and AMP resistance, and a significant relationship was also found between the presence of the afa and OFX resistance. No difference between E. coli strains isolated from two different clinical presentations was found in terms of virulence genes and antibiotic susceptibility.

Identification of differentially expressed genes in human breast cancer cells induced by 4-hydroxyltamoxifen and elucidation of their pathophysiological relevance and mechanisms

PubMed Central

Fang, Qi; Yao, Shuang; Luo, Guanghua; Zhang, Xiaoying

2018-01-01

While tamoxifen (TAM) is used for treating estrogen receptor (ER)a-positive breast cancer patients, its anti-breast cancer mechanisms are not completely elucidated. This study aimed to examine effects of 4-hydroxyltamoxifen (4-OH-TAM) on ER-positive (ER+) breast cancer MCF-7 cell growth and gene expression profiles. MCF-7 cell growth was inhibited by 4-OH-TAM dose-dependently with IC50 of 29 μM. 332 genes were up-regulated while 320 genes were down-regulated. The mRNA levels of up-regulated genes including STAT1, STAT2, EIF2AK2, TGM2, DDX58, PARP9, SASH1, RBL2 and USP18 as well as down-regulated genes including CCDN1, S100A9, S100A8, ANXA1 and PGR were confirmed by quantitative real-time PCR (qRT-PCR). In human breast tumor tissues, mRNA levels of EIF2Ak2, USP18, DDX58, RBL2, STAT2, PGR, S1000A9, and CCND1 were significantly higher in ER+- than in ER--breast cancer tissues. The mRNA levels of EIF2AK2, TGM2, USP18, DDX58, PARP9, STAT2, STAT1, PGR and CCND1 were all significantly higher in ER+-tumor tissues than in their corresponding tumor-adjacent tissues. These genes, except PGR and CCND1 which were down-regulated, were also up-regulated in ER+ MCF-7 cells by 4-OH-TAM. Total 14 genes mentioned above are involved in regulation of cell proliferation, apoptosis, cell cycles, and estrogen and interferon signal pathways. Bioinformatics analysis also revealed other novel and important regulatory factors that are associated with these genes and involved in the mentioned functional processes. This study has paved a foundation for elucidating TAM anti-breast cancer mechanisms in E2/ER-dependent and independent pathways. PMID:29416786

Identification of differentially expressed genes in human breast cancer cells induced by 4-hydroxyltamoxifen and elucidation of their pathophysiological relevance and mechanisms.

PubMed

Fang, Qi; Yao, Shuang; Luo, Guanghua; Zhang, Xiaoying

2018-01-05

While tamoxifen (TAM) is used for treating estrogen receptor (ER)a-positive breast cancer patients, its anti-breast cancer mechanisms are not completely elucidated. This study aimed to examine effects of 4-hydroxyltamoxifen (4-OH-TAM) on ER-positive (ER + ) breast cancer MCF-7 cell growth and gene expression profiles. MCF-7 cell growth was inhibited by 4-OH-TAM dose-dependently with IC 50 of 29 μM. 332 genes were up-regulated while 320 genes were down-regulated. The mRNA levels of up-regulated genes including STAT1, STAT2, EIF2AK2, TGM2, DDX58, PARP9, SASH1, RBL2 and USP18 as well as down-regulated genes including CCDN1, S100A9, S100A8, ANXA1 and PGR were confirmed by quantitative real-time PCR (qRT-PCR). In human breast tumor tissues, mRNA levels of EIF2Ak2, USP18, DDX58, RBL2, STAT2, PGR, S1000A9, and CCND1 were significantly higher in ER + - than in ER - -breast cancer tissues. The mRNA levels of EIF2AK2, TGM2, USP18, DDX58, PARP9, STAT2, STAT1, PGR and CCND1 were all significantly higher in ER + -tumor tissues than in their corresponding tumor-adjacent tissues. These genes, except PGR and CCND1 which were down-regulated, were also up-regulated in ER + MCF-7 cells by 4-OH-TAM. Total 14 genes mentioned above are involved in regulation of cell proliferation, apoptosis, cell cycles, and estrogen and interferon signal pathways. Bioinformatics analysis also revealed other novel and important regulatory factors that are associated with these genes and involved in the mentioned functional processes. This study has paved a foundation for elucidating TAM anti-breast cancer mechanisms in E2/ER-dependent and independent pathways.

Disrupting effects of antibiotic sulfathiazole on developmental process during sensitive life-cycle stage of Chironomus riparius.

PubMed

Park, Kiyun; Kwak, Ihn-Sil

2018-01-01

Antibiotics in the environment are a concern due to their potential to harm humans and interrupt ecosystems. Sulfathiazole (STZ), a sulfonamide antibiotic, is commonly used in aquaculture and is typically found in aquatic ecosystems. We evaluated the ecological risk of STZ by examining biological, molecular and biochemical response in Chironomus riparius. Samples were exposed to STZ for 12, 24 and 96 h, and effects of STZ were evaluated at the molecular level by analyzing changes in gene expression related to the endocrine system, cellular stress response and enzyme activity of genes on antioxidant and detoxification pathways. STZ exposure induced significant effects on survival, growth and sex ratio of emergent adults and mouthpart deformity in C. riparius. STZ caused concentration and time-dependent toxicity in most of the selected biomarkers. STZ exposure leads to significant heat-shock response of protein genes (HSP70, HSP40, HSP90 and HSP27) and to disruption by up-regulating selected genes, including the ecdysone receptor gene, estrogen-related receptors, ultraspiracle and E74 early ecdysone-responsive gene. Furthermore, STZ induced alteration of enzyme activities on antioxidant and detoxification responses (catalase, superoxide dismutase, glutathione peroxidase and peroxidase) in C. riparius. By inducing oxidative stress, antibiotic STZ disturbs the endocrine system and produces adverse effects in growth processes of invertebrates. Copyright © 2017 Elsevier Ltd. All rights reserved.

BEND3 represses rDNA transcription by stabilizing a NoRC component via USP21 deubiquitinase

PubMed Central

Khan, Abid; Giri, Sumanprava; Wang, Yating; Chakraborty, Arindam; Ghosh, Archit K.; Anantharaman, Aparna; Aggarwal, Vasudha; Sathyan, Kizhakke M.; Ha, Taekjip; Prasanth, Kannanganattu V.; Prasanth, Supriya G.

2015-01-01

Ribosome biogenesis dictates the translational capacity of cells. Several mechanisms establish and maintain transcriptional output from eukaryotic ribosomal DNA (rDNA) loci. rDNA silencing is one such mechanism that ensures the inactivity and hence the maintenance of a silenced state of a subset of rRNA gene copies. Whereas oncogenic agents stimulate rRNA gene transcription, tumor suppressors decrease rRNA gene transcription. We demonstrate in mammalian cells that BANP, E5R, and Nac1 (BEN) domain 3 (BEND3), a quadruple BEN domain-containing protein, localizes in nucleoli and binds to ribosomal RNA gene promoters to help repress rRNA genes. Loss of BEND3 increases histone H3K4 trimethylation and, correspondingly, decreases rDNA promoter DNA methylation, consistent with a role for BEND3 in rDNA silencing. BEND3 associates with the nucleolar-remodeling complex (NoRC), and SUMOylated BEND3 stabilizes NoRC component TTF-1–interacting protein 5 via association with ubiquitin specific protease 21 (USP21) debiquitinase. Our results provide mechanistic insights into how the novel rDNA transcription repressor BEND3 acts together with NoRC to actively coordinate the establishment of rDNA silencing. PMID:26100909

BEND3 represses rDNA transcription by stabilizing a NoRC component via USP21 deubiquitinase.

PubMed

Khan, Abid; Giri, Sumanprava; Wang, Yating; Chakraborty, Arindam; Ghosh, Archit K; Anantharaman, Aparna; Aggarwal, Vasudha; Sathyan, Kizhakke M; Ha, Taekjip; Prasanth, Kannanganattu V; Prasanth, Supriya G

2015-07-07

Ribosome biogenesis dictates the translational capacity of cells. Several mechanisms establish and maintain transcriptional output from eukaryotic ribosomal DNA (rDNA) loci. rDNA silencing is one such mechanism that ensures the inactivity and hence the maintenance of a silenced state of a subset of rRNA gene copies. Whereas oncogenic agents stimulate rRNA gene transcription, tumor suppressors decrease rRNA gene transcription. We demonstrate in mammalian cells that BANP, E5R, and Nac1 (BEN) domain 3 (BEND3), a quadruple BEN domain-containing protein, localizes in nucleoli and binds to ribosomal RNA gene promoters to help repress rRNA genes. Loss of BEND3 increases histone H3K4 trimethylation and, correspondingly, decreases rDNA promoter DNA methylation, consistent with a role for BEND3 in rDNA silencing. BEND3 associates with the nucleolar-remodeling complex (NoRC), and SUMOylated BEND3 stabilizes NoRC component TTF-1-interacting protein 5 via association with ubiquitin specific protease 21 (USP21) debiquitinase. Our results provide mechanistic insights into how the novel rDNA transcription repressor BEND3 acts together with NoRC to actively coordinate the establishment of rDNA silencing.

76 FR 65756 - Privacy Act of 1974; System of Records

Federal Register 2010, 2011, 2012, 2013, 2014

2011-10-24

... 600.400, Administrative Litigation Records; USPS 810.100, http://www.usps.com Registration; USPS 810... Records; USPS 870.200, Postage Meter and PC Postage Customer Data and Transaction Records; USPS 880.000....400 SYSTEM NAME: Administrative Litigation Records USPS 810.100 SYSTEM NAME: http://www.usps.com...

Characterization of the transcriptome of fast and slow muscle myotomal fibres in the pacu (Piaractus mesopotamicus).

PubMed

Mareco, Edson A; Garcia de la Serrana, Daniel; Johnston, Ian A; Dal-Pai-Silva, Maeli

2015-03-14

The Pacu (Piaractus mesopotamicus) is a member of the Characiform family native to the Prata Basin (South America) and a target for the aquaculture industry. A limitation for the development of a selective breeding program for this species is a lack of available genetic information. The primary objectives of the present study were 1) to increase the genetic resources available for the species, 2) to exploit the anatomical separation of myotomal fibres types to compare the transcriptomes of slow and fast muscle phenotypes and 3) to systematically investigate the expression of Ubiquitin Specific Protease (USP) family members in fast and slow muscle in response to fasting and refeeding. We generated 0.6 Tb of pair-end reads from slow and fast skeletal muscle libraries. Over 665 million reads were assembled into 504,065 contigs with an average length of 1,334 bp and N50 = 2,772 bp. We successfully annotated nearly 47% of the transcriptome and identified around 15,000 unique genes and over 8000 complete coding sequences. 319 KEGG metabolic pathways were also annotated and 380 putative microsatellites were identified. 956 and 604 genes were differentially expressed between slow and fast skeletal muscle, respectively. 442 paralogues pairs arising from the teleost-specific whole genome duplication were identified, with the majority showing different expression patterns between fibres types (301 in slow and 245 in fast skeletal muscle). 45 members of the USP family were identified in the transcriptome. Transcript levels were quantified by qPCR in a separate fasting and refeeding experiment. USP genes in fast muscle showed a similar transient increase in expression with fasting as the better characterized E3 ubiquitin ligases. We have generated a 53-fold coverage transcriptome for fast and slow myotomal muscle in the pacu (Piaractus mesopotamicus) significantly increasing the genetic resources available for this important aquaculture species. We describe significant differences in gene expression between muscle fibre types for fundamental components of general metabolism, the Pi3k/Akt/mTor network and myogenesis, including detailed analysis of paralogue expression. We also provide a comprehensive description of USP family member expression between muscle fibre types and with changing nutritional status.

Biofilm formation by the periodontopathic bacteria Treponema denticola and Porphyromonas gingivalis.

PubMed

Kuramitsu, Howard K; Chen, Wen; Ikegami, Aki

2005-11-01

Periodontitis develops as a result of the interaction of the host with subgingival plaque bacteria. Both Porphyromonas gingivalis and Treponema denticola are frequently associated together in these oral biofilms. The molecular basis for in vitro biofilm formation was investigated for P. gingivalis 381, T. denticola 35405, and mixtures of the two organisms using microtiter plate assays. In addition, the biofilms were examined following confocal laser scanning microscopy. P. gingivalis 381, but not T. denticola strains, formed biofilms in vitro. This property was dependent, in part, on the strain 381 fimA, ppk, and usp genes. Microarray and Northern blot analyses suggested that the expression of the ppk gene was required for maximal expression of the uspA gene. P. gingivalis 381 formed synergistic biofilms when incubated with T. denticola strains. This process was dependent upon the strain 381 rgpB and fimA genes as well as the T. denticola flgE and cfpA genes. P. gingivalis 381 formed synergistic biofilms with T. denticola 35405. These results may be relevant to the previous observations that the two organisms are frequently observed together in subgingival plaque with the spirochetes localized to the exterior of the oral biofilms. It is suggested that other such synergistic effects may also occur between other plaque bacteria.

Biofilm Formation by the Periodontopathic Bacteria Treponema denticola and Porphyromonas gingivalis.

PubMed

Kuramitsu, Howard K; Chen, Wen; Ikegami, Aki

2005-11-01

Periodontitis develops as a result of the interaction of the host with subgingival plaque bacteria. Both Porphyromonas gingivalis and Treponema denticola are frequently associated together in these oral biofilms. The molecular basis for in vitro biofilm formation was investigated for P. gingivalis 381, T. denticola 35405, and mixtures of the two organisms using microtiter plate assays. In addition, the biofilms were examined following confocal laser scanning microscopy. P. gingivalis 381, but not T. denticola strains, formed biofilms in vitro. This property was dependent, in part, on the strain 381 fimA, ppk, and usp genes. Microarray and Northern blot analyses suggested that the expression of the ppk gene was required for maximal expression of the uspA gene. P. gingivalis 381 formed synergistic biofilms when incubated with T. denticola strains. This process was dependent upon the strain 381 rgpB and fimA genes as well as the T. denticola flgE and cfpA genes. P. gingivalis 381 formed synergistic biofilms with T. denticola 35405. These results may be relevant to the previous observations that the two organisms are frequently observed together in subgingival plaque with the spirochetes localized to the exterior of the oral biofilms. It is suggested that other such synergistic effects may also occur between other plaque bacteria. © 2005 American Academy of Periodontology.

Mechanisms of mitophagy: PINK1, Parkin, USP30 and beyond.

PubMed

Bingol, Baris; Sheng, Morgan

2016-11-01

Mitochondrial quality control is central for maintaining a healthy population of mitochondria. Two Parkinson's disease genes, mitochondrial kinase PINK1 and ubiquitin ligase Parkin, degrade damaged mitochondria though mitophagy. In this pathway, PINK1 senses mitochondrial damage and activates Parkin by phosphorylating Parkin and ubiquitin. Activated Parkin then builds ubiquitin chains on damaged mitochondria to tag them for degradation in lysosomes. USP30 deubiquitinase acts as a brake on mitophagy by opposing Parkin-mediated ubiquitination. Human genetic data point to a role for mitophagy defects in neurodegenerative diseases. This review highlights the molecular mechanisms of the mitophagy pathway and the recent advances in the understanding of mitophagy in vivo. Copyright © 2016 Elsevier Inc. All rights reserved.

USP17 is upregulated in osteosarcoma and promotes cell proliferation, metastasis, and epithelial-mesenchymal transition through stabilizing SMAD4.

PubMed

Song, Chenyang; Liu, Wenge; Li, Jiandong

2017-07-01

USP17 is upregulated in several cancers, indicating that USP17 might play essential functions in tumor development. However, the function of USP17 in osteosarcoma is still unknown. Our work aimed to investigate the function of USP17 in osteosarcoma. We found that the expression of USP17 was upregulated in osteosarcoma tissues and cell lines, including MG-63 and U2OS. Several functional experiments, such as colony formation analysis, Cell Counting Kit-8 assay, wound healing analysis, and transwell assay, showed that USP17 promoted cell proliferation, migration, and invasion. Moreover, we found that USP17 facilitated migration and invasion through promoting epithelial-mesenchymal transition. SMAD4 has been found to regulate epithelial-mesenchymal transition, co-immunopurification, and glutathione S-transferase pull-down analysis demonstrated that USP17 interacted with SMAD4. Furthermore, USP17 stabilized SMAD4 through its deubiquitinase activity. In conclusion, this study shows that USP17 enhances osteosarcoma cell proliferation and invasion through stabilizing SMAD4.

Replication of a low-pathogenic avian influenza virus is enhanced by chicken ubiquitin-specific protease 18.

PubMed

Tanikawa, Taichiro; Uchida, Yuko; Saito, Takehiko

2017-09-01

Previous research revealed the induction of chicken USP18 (chUSP18) in the lungs of chickens infected with highly pathogenic avian influenza viruses (HPAIVs). This activity was correlated with the degree of pathogenicity of the viruses to chickens. As mammalian ubiquitin-specific protease (USP18) is known to remove type I interferon (IFN I)-inducible ubiquitin-like molecules from conjugated proteins and block IFN I signalling, we explored the function of the chicken homologue of USP18 during avian influenza virus infection. With this aim, we cloned chUSP18 from cultured chicken cells and revealed that the putative chUSP18 ORF comprises 1137 bp. Comparative analysis of the predicted aa sequence of chUSP18 with those of human and mouse USP18 revealed relatively high sequence similarity among the sequences, including domains specific for the ubiquitin-specific processing protease family. Furthermore, we found that chUSP18 expression was induced by chicken IFN I, as observed for mammalian USP18. Experiments based on chUSP18 over-expression and depletion demonstrated that chUSP18 significantly enhanced the replication of a low-pathogenic avian influenza virus (LPAIV), but not an HPAIV. Our findings suggest that chUSP18, being similar to mammalian USP18, acts as a pro-viral factor during LPAIV replication in vitro.

USP7 Is a Suppressor of PCNA Ubiquitination and Oxidative-Stress-Induced Mutagenesis in Human Cells.

PubMed

Kashiwaba, Shu-ichiro; Kanao, Rie; Masuda, Yuji; Kusumoto-Matsuo, Rika; Hanaoka, Fumio; Masutani, Chikahide

2015-12-15

Mono-ubiquitinated PCNA activates error-prone DNA polymerases; therefore, strict regulation of PCNA mono-ubiquitination is crucial in avoiding undesired mutagenesis. In this study, we used an in vitro assay system to identify USP7 as a deubiquitinating enzyme of mono-ubiquitinated PCNA. Suppression of USP1, a previously identified PCNA deubiquitinase, or USP7 increased UV- and H2O2-induced PCNA mono-ubiquitination in a distinct and additive manner, suggesting that USP1 and USP7 make different contributions to PCNA deubiquitination in human cells. Cell-cycle-synchronization analyses revealed that USP7 suppression increased H2O2-induced PCNA ubiquitination throughout interphase, whereas USP1 suppression specifically increased ubiquitination in S-phase cells. UV-induced mutagenesis was elevated in USP1-suppressed cells, whereas H2O2-induced mutagenesis was elevated in USP7-suppressed cells. These results suggest that USP1 suppresses UV-induced mutations produced in a manner involving DNA replication, whereas USP7 suppresses H2O2-induced mutagenesis involving cell-cycle-independent processes such as DNA repair. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Function of ubiquitin (Ub) specific protease 15 (USP15) in HIV-1 replication and viral protein degradation.

PubMed

Pyeon, Dohun; Timani, Khalid Amine; Gulraiz, Fahad; He, Johnny J; Park, In-Woo

2016-09-02

HIV-1 Nef is necessary and may be sufficient for HIV-1-associated AIDS pathogenicity, in that knockout of Nef alone can protect HIV-infected patients from AIDS. We therefore investigated the feasibility of physical knockout of Nef, using the host ubiquitin proteasome system in HIV-1-infected cells. Our co-immunoprecipitation analysis demonstrated that Nef interacted with ubiquitin specific protease 15 (USP15), and that USP15, which is known to stabilize cellular proteins, degraded Nef. Nef could also cause decay of USP15, although Nef-mediated degradation of USP15 was weaker than USP15-mediated Nef degradation. Direct interaction between Nef and USP15 was essential for the observed reciprocal decay of the proteins. Further, USP15 degraded not only Nef but also HIV-1 structural protein, Gag, thereby substantially inhibiting HIV-1 replication. However, Gag did not degrade USP15, indicating that the Nef and USP15 complex, in distinction to other viral proteins, play an integral role in coordinating viral protein degradation and hence HIV-1 replication. Moreover, Nef and USP15 globally suppressed ubiquitylation of cellular proteins, indicating that these proteins are major determinants for the stability of cellular as well as viral proteins. Taken together, these data indicate that Nef and USP15 are vital in regulating degradation of viral and cellular proteins and thus HIV-1 replication, and specific degradation of viral, not cellular proteins, by USP15 points to USP15 as a candidate therapeutic agent to combat AIDS by eliminating viral proteins from the infected cells via USP15-mediated proteosomal degradation. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Transcriptional landscape of human cancers.

PubMed

Li, Mengyuan; Sun, Qingrong; Wang, Xiaosheng

2017-05-23

The homogeneity and heterogeneity in somatic mutations, copy number alterations and methylation across different cancer types have been extensively explored. However, the related exploration based on transcriptome data is lacking. In this study we explored gene expression profiles across 33 human cancer types using The Cancer Genome Atlas (TCGA) data. We identified consistently upregulated genes (such as E2F1, EZH2, FOXM1, MYBL2, PLK1, TTK, AURKA/B and BUB1) and consistently downregulated genes (such as SCARA5, MYOM1, NKAPL, PEG3, USP2, SLC5A7 and HMGCLL1) across various cancers. The dysregulation of these genes is likely to be associated with poor clinical outcomes in cancer. The dysregulated pathways commonly in cancers include cell cycle, DNA replication, repair, and recombination, Notch signaling, p53 signaling, Wnt signaling, TGFβ signaling, immune response etc. We also identified genes consistently upregulated or downregulated in highly-advanced cancers compared to lowly-advanced cancers. The highly (low) expressed genes in highly-advanced cancers are likely to have higher (lower) expression levels in cancers than in normal tissue, indicating that common gene expression perturbations drive cancer initiation and cancer progression. In addition, we identified a substantial number of genes exclusively dysregulated in a single cancer type or inconsistently dysregulated in different cancer types, demonstrating the intertumor heterogeneity. More importantly, we found a number of genes commonly dysregulated in various cancers such as PLP1, MYOM1, NKAPL and USP2 which were investigated in few cancer related studies, and thus represent our novel findings. Our study provides comprehensive portraits of transcriptional landscape of human cancers.

Transcriptional landscape of human cancers

PubMed Central

Li, Mengyuan; Sun, Qingrong; Wang, Xiaosheng

2017-01-01

The homogeneity and heterogeneity in somatic mutations, copy number alterations and methylation across different cancer types have been extensively explored. However, the related exploration based on transcriptome data is lacking. In this study we explored gene expression profiles across 33 human cancer types using The Cancer Genome Atlas (TCGA) data. We identified consistently upregulated genes (such as E2F1, EZH2, FOXM1, MYBL2, PLK1, TTK, AURKA/B and BUB1) and consistently downregulated genes (such as SCARA5, MYOM1, NKAPL, PEG3, USP2, SLC5A7 and HMGCLL1) across various cancers. The dysregulation of these genes is likely to be associated with poor clinical outcomes in cancer. The dysregulated pathways commonly in cancers include cell cycle, DNA replication, repair, and recombination, Notch signaling, p53 signaling, Wnt signaling, TGFβ signaling, immune response etc. We also identified genes consistently upregulated or downregulated in highly-advanced cancers compared to lowly-advanced cancers. The highly (low) expressed genes in highly-advanced cancers are likely to have higher (lower) expression levels in cancers than in normal tissue, indicating that common gene expression perturbations drive cancer initiation and cancer progression. In addition, we identified a substantial number of genes exclusively dysregulated in a single cancer type or inconsistently dysregulated in different cancer types, demonstrating the intertumor heterogeneity. More importantly, we found a number of genes commonly dysregulated in various cancers such as PLP1, MYOM1, NKAPL and USP2 which were investigated in few cancer related studies, and thus represent our novel findings. Our study provides comprehensive portraits of transcriptional landscape of human cancers. PMID:28427185

Official USP Reference Standards: metrology concepts, overview, and scientific issues and opportunities.

PubMed

Williams, Roger L

2006-01-23

The United States Pharmacopeia (USP) is a private standards-setting body created in 1820 by practitioners who wished to promote the quality of therapeutic products in commerce. The principal product of USP, then and now, is the United StatesPharmacopeia (USP), to which was added the National Formulary (NF) in 1975. The two compendia are published as a combined text annually (USP-NF). Originally a book of process standards, USP-NF evolved over time into compendia containing primarily product standards that are expressed in monographs for therapeutic ingredients, products, and excipients. As a public health service, USP supplies official USP Reference Standards to manufacturers and others who wish to test an article according to selected procedures of a monograph or General Chapter. During the past decade, understanding of USP monographs and official USP Reference Standards as a means of controlling the quality of a therapeutic article has evolved, based on advances in metrology, on activities in the International Conference on Harmonization of Technical Requirements for the Registration of Pharmaceuticals for Human Use (ICH), and on considerations by the USP Council of Experts and its Expert Committees and USP staff. This article discusses the evolution of this understanding, focusing on drug substances and excipients for well-characterized small molecules and their corresponding dosage forms.

Mice lacking the USP2 deubiquitinating enzyme have severe male subfertility associated with defects in fertilization and sperm motility.

PubMed

Bedard, Nathalie; Yang, Yaoming; Gregory, Mary; Cyr, Daniel G; Suzuki, João; Yu, Xiaomin; Chian, Ri-Cheng; Hermo, Louis; O'Flaherty, Cristian; Smith, Charles E; Clarke, Hugh J; Wing, Simon S

2011-09-01

The ubiquitin-proteasome system plays an important role in spermatogenesis. However, the functions of deubiquitinating enzymes in this process remain poorly characterized. We previously showed that the deubiquitinating enzyme USP2 is induced in late elongating spermatids. To identify its function, we generated mice lacking USP2. Usp2 -/- mice appeared normal, and the weights of major organs, including the testis, did not differ from wild type (Usp2 +/+). However, although the numbers of testicular spermatids and epididymal spermatozoa were normal in Usp2 -/- males, these animals had a severe defect in fertility, yielding only 12% as many offspring as Usp2 +/+ littermates. Spermatogenesis in Usp2 -/- mice was morphologically normal except for the presence of abnormal aggregations of elongating spermatids and formation of multinucleated cells in some tubules. The epididymal epithelium was morphologically normal in Usp2 -/- mice, but some abnormal cells other than sperm were present in the lumen. Usp2 -/- epididymal spermatozoa manifested normal motility when incubated in culture media, but rapidly became immotile when incubated in PBS in contrast to Usp2 +/+ spermatozoa, which largely maintained motility under this condition. Usp2 -/- and +/+ spermatozoa underwent acrosome reactions in vitro with similar frequency. In vitro fertilization assays demonstrated a severe defect in the ability of Usp2 -/- spermatozoa to fertilize eggs. This could be bypassed by intracytoplasmic sperm injection or removal of the zona pellucida, which resulted in fertilization rates similar to that of Usp2 +/+ mice. We demonstrate for the first time, using mouse transgenic approaches, a role for the ubiquitin system in fertilization.

Secreted Expression of the Cap Gene of Porcine Circovirus Type 2 in Classical Swine Fever Virus C-Strain: Potential of C-Strain Used as a Vaccine Vector

PubMed Central

Zhang, Lingkai; Li, Yongfeng; Xie, Libao; Wang, Xiao; Gao, Xulei; Sun, Yuan; Qiu, Hua-Ji

2017-01-01

Bivalent vaccines based on live attenuated viruses expressing a heterologous protein are an attractive strategy to address co-infections with various pathogens in the field. Considering the excellent efficacy and safety of the lapinized live attenuated vaccine C-strain (HCLV strain) of classical swine fever virus (CSFV), we proposed that C-strain has the potential as a viral vector for developing bivalent vaccines. To this end, we generated three recombinant viruses based on C-strain, one expressing the capsid (Cap) gene of porcine circovirus type 2 (PCV2) with the nuclear localization signal (NLS) (rHCLV-2ACap), and the other two expressing the PCV2 Cap gene without the NLS yet containing the signal peptide of the prolactin gene (rHCLV-pspCap) or that of the ubiquitin-specific peptidase gene (rHCLV-uspCap). All the recombinant viruses exhibited phenotypes similar to those of the parental virus and produced high-level anti-CSFV neutralizing antibodies (NAbs) in rabbits. Interestingly, rHCLV-uspCap and rHCLV-pspCap, but not rHCLV-2ACap, elicited detectable anti-Cap and -PCV2 NAbs in rabbits. Taken together, our data demonstrate that C-strain can be used as a viral vector to develop bivalent vaccines. PMID:29035292

Secreted Expression of the Cap Gene of Porcine Circovirus Type 2 in Classical Swine Fever Virus C-Strain: Potential of C-Strain Used as a Vaccine Vector.

PubMed

Zhang, Lingkai; Li, Yongfeng; Xie, Libao; Wang, Xiao; Gao, Xulei; Sun, Yuan; Qiu, Hua-Ji

2017-10-16

Bivalent vaccines based on live attenuated viruses expressing a heterologous protein are an attractive strategy to address co-infections with various pathogens in the field. Considering the excellent efficacy and safety of the lapinized live attenuated vaccine C-strain (HCLV strain) of classical swine fever virus (CSFV), we proposed that C-strain has the potential as a viral vector for developing bivalent vaccines. To this end, we generated three recombinant viruses based on C-strain, one expressing the capsid ( Cap ) gene of porcine circovirus type 2 (PCV2) with the nuclear localization signal (NLS) (rHCLV-2ACap), and the other two expressing the PCV2 Cap gene without the NLS yet containing the signal peptide of the prolactin gene (rHCLV-pspCap) or that of the ubiquitin-specific peptidase gene (rHCLV-uspCap). All the recombinant viruses exhibited phenotypes similar to those of the parental virus and produced high-level anti-CSFV neutralizing antibodies (NAbs) in rabbits. Interestingly, rHCLV-uspCap and rHCLV-pspCap, but not rHCLV-2ACap, elicited detectable anti-Cap and -PCV2 NAbs in rabbits. Taken together, our data demonstrate that C-strain can be used as a viral vector to develop bivalent vaccines.

Evaluation and Validation of Housekeeping Genes as Reference for Gene Expression Studies in Pigeonpea (Cajanus cajan) Under Drought Stress Conditions

PubMed Central

Sinha, Pallavi; Singh, Vikas K.; Suryanarayana, V.; Krishnamurthy, L.; Saxena, Rachit K.; Varshney, Rajeev K.

2015-01-01

Gene expression analysis using quantitative real-time PCR (qRT-PCR) is a very sensitive technique and its sensitivity depends on the stable performance of reference gene(s) used in the study. A number of housekeeping genes have been used in various expression studies in many crops however, their expression were found to be inconsistent under different stress conditions. As a result, species specific housekeeping genes have been recommended for different expression studies in several crop species. However, such specific housekeeping genes have not been reported in the case of pigeonpea (Cajanus cajan) despite the fact that genome sequence has become available for the crop. To identify the stable housekeeping genes in pigeonpea for expression analysis under drought stress conditions, the relative expression variations of 10 commonly used housekeeping genes (EF1α, UBQ10, GAPDH, 18SrRNA, 25SrRNA, TUB6, ACT1, IF4α, UBC and HSP90) were studied on root, stem and leaves tissues of Asha (ICPL 87119). Three statistical algorithms geNorm, NormFinder and BestKeeper were used to define the stability of candidate genes. geNorm analysis identified IF4α and TUB6 as the most stable housekeeping genes however, NormFinder analysis determined IF4α and HSP90 as the most stable housekeeping genes under drought stress conditions. Subsequently validation of the identified candidate genes was undertaken in qRT-PCR based gene expression analysis of uspA gene which plays an important role for drought stress conditions in pigeonpea. The relative quantification of the uspA gene varied according to the internal controls (stable and least stable genes), thus highlighting the importance of the choice of as well as validation of internal controls in such experiments. The identified stable and validated housekeeping genes will facilitate gene expression studies in pigeonpea especially under drought stress conditions. PMID:25849964

Evaluation and validation of housekeeping genes as reference for gene expression studies in pigeonpea (Cajanus cajan) under drought stress conditions.

PubMed

Sinha, Pallavi; Singh, Vikas K; Suryanarayana, V; Krishnamurthy, L; Saxena, Rachit K; Varshney, Rajeev K

2015-01-01

Gene expression analysis using quantitative real-time PCR (qRT-PCR) is a very sensitive technique and its sensitivity depends on the stable performance of reference gene(s) used in the study. A number of housekeeping genes have been used in various expression studies in many crops however, their expression were found to be inconsistent under different stress conditions. As a result, species specific housekeeping genes have been recommended for different expression studies in several crop species. However, such specific housekeeping genes have not been reported in the case of pigeonpea (Cajanus cajan) despite the fact that genome sequence has become available for the crop. To identify the stable housekeeping genes in pigeonpea for expression analysis under drought stress conditions, the relative expression variations of 10 commonly used housekeeping genes (EF1α, UBQ10, GAPDH, 18SrRNA, 25SrRNA, TUB6, ACT1, IF4α, UBC and HSP90) were studied on root, stem and leaves tissues of Asha (ICPL 87119). Three statistical algorithms geNorm, NormFinder and BestKeeper were used to define the stability of candidate genes. geNorm analysis identified IF4α and TUB6 as the most stable housekeeping genes however, NormFinder analysis determined IF4α and HSP90 as the most stable housekeeping genes under drought stress conditions. Subsequently validation of the identified candidate genes was undertaken in qRT-PCR based gene expression analysis of uspA gene which plays an important role for drought stress conditions in pigeonpea. The relative quantification of the uspA gene varied according to the internal controls (stable and least stable genes), thus highlighting the importance of the choice of as well as validation of internal controls in such experiments. The identified stable and validated housekeeping genes will facilitate gene expression studies in pigeonpea especially under drought stress conditions.

Identification of interleukin-1 beta as a key mediator in the upregulation of Cav3.2–USP5 interactions in the pain pathway

PubMed Central

Stemkowski, Patrick L; Garcia-Caballero, Agustin; Gadotti, Vinicius M; M’Dahoma, Said; Chen, Lina; Souza, Ivana A

2017-01-01

We recently reported that nerve injury or peripheral inflammation triggers an upregulation of the deubiquitinase, USP5 in mouse dorsal root ganglion and spinal dorsal horn. This leads to dysregulated ubiquitination of Cav3.2 T-type calcium channels, thus increasing Cav3.2 channel plasma membrane expression and nociceptive signaling in the primary afferent pain pathway. This phenomenon could be recapitulated by noninvasive, optogenetic activation of transient receptor potential vanilloid-1–expressing nociceptors, indicating that neuronal activity is a key player in this process. Given the relevance of the pro-inflammatory cytokine interleukin-1 beta in many forms of pathological pain, we hypothesized that interleukin-1 beta may be a critical cofactor required to drive upregulation of interactions between USP5 and Cav3.2 channels. Here, we report that gene expression, as well as protein levels for interleukin-1 beta and the endogenous interleukin-1 receptor-I antagonist, IL-1Ra are unaltered following conditioning stimulation of optogenetically targeted cutaneous nociceptors, indicating that neuronal activity is not a driver of interleukin-1 beta signaling. In contrast, co-immunoprecipitation experiments revealed that intrathecal administration of interleukin-1 beta in wild-type mice led to an increase in the interaction between USP5 and Cav3.2 in the spinal dorsal horn. Moreover, disruption of the interaction between USP5 and Cav3.2 with TAT peptides suppressed acute nocifensive responses produced by interleukin-1 beta, which was similar to that achieved by elimination of T-type channel activity with the channel blockers, mibefradil, or TTA-A2. Finally, this upregulation could be maintained in dorsal root ganglion neuron cultures exposed overnight to interleukin-1 beta, while the copresence of interleukin-1 receptor antagonist or the dampening of neuronal cell activity with tetrodotoxin attenuated this response. Altogether, our findings identify interleukin-1 beta as an upstream trigger for the upregulation of interactions between USP5 and Cav3.2 channels in the pain pathway, presumably by triggering increased firing activity in afferent fibers. PMID:28741432

USP22 acts as an oncogene by regulating the stability of cyclooxygenase-2 in non-small cell lung cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xiao, Haibo; Tian, Yue; Yang, Yang

2015-05-08

The histone ubiquitin hydrolase ubiquitin-specific protease 22 (USP22) is an epigenetic modifier and an oncogene that is upregulated in many types of cancer. In non-small cell lung cancer (NSCLC), aberrant expression of USP22 is a predictor of poor survival, as is high expression of cyclooxygenase-2 (COX-2). Despite its oncogenic role, few substrates of USP22 have been identified and its mechanism of action in cancer remains unclear. Here, we identified COX-2 as a direct substrate of USP22 and showed that its levels are modulated by USP22 mediated deubiquitination. Silencing of USP22 downregulated COX-2, decreased its half-life, and inhibited lung carcinoma cellmore » proliferation by directly interacting with and modulating the stability and activity of COX-2 through the regulation of its ubiquitination status. The findings of the present study suggest a potential mechanism underlying the oncogenic role of USP22 mediated by the modulation of the stability and activity of COX-2. - Highlights: • USP22 interacts with COX-2. • USP22 deubiquitinates and stabilizes COX-2. • USP22 is required for COX-2-mediated upregulation of prostaglandin E2.« less

CDDO-Me reveals USP7 as a novel target in ovarian cancer cells.

PubMed

Qin, Dongjun; Wang, Weiwei; Lei, Hu; Luo, Hao; Cai, Haiyan; Tang, Caixia; Wu, Yunzhao; Wang, Yingying; Jin, Jin; Xiao, Weilie; Wang, Tongdan; Ma, Chunmin; Xu, Hanzhang; Zhang, Jinfu; Gao, Fenghou; Wu, Ying-Li

2016-11-22

Deubiquitinating enzyme USP7 has been involved in the pathogenesis and progression of several cancers. Targeting USP7 is becoming an attractive strategy for cancer therapy. In this study, we identified synthetic triterpenoid C-28 methyl ester of 2-cyano-3, 12-dioxoolen-1, 9-dien-28-oic acid (CDDO-Me) as a novel inhibitor of USP7 but not of other cysteine proteases such as cathepsin B and cathepsin D. CDDO-Me inhibits USP7 activity via a mechanism that is independent of the presence of α, β-unsaturated ketones. Molecular docking studies showed that CDDO-Me fits well in the ubiquitin carboxyl terminus-binding pocket on USP7. Given that CDDO-Me is known to be effective against ovarian cancer cells, we speculated that CDDO-Me may target USP7 in ovarian cancer cells. We demonstrated that ovarian cancer cells have higher USP7 expression than their normal counterparts. Knockdown of USP7 inhibits the proliferation of ovarian cancer cells both in vitro and in vivo. Using the cellular thermal shift assay and the drug affinity responsive target stability assay, we further demonstrated that CDDO-Me directly binds to USP7 in cells, which leads to the decrease of its substrates such as MDM2, MDMX and UHRF1. CDDO-Me suppresses ovarian cancer tumor growth in an xenograft model. In conclusion, we demonstrate that USP7 is a novel target of ovarian cancer cells; targeting USP7 may contribute to the anti-cancer effect of CDDO-Me. The development of novel USP7 selective compounds based on the CDDO-Me-scaffold warrants further investigation.

CDDO-Me reveals USP7 as a novel target in ovarian cancer cells

PubMed Central

Cai, Haiyan; Tang, Caixia; Wu, Yunzhao; Wang, Yingying; Jin, Jin; Xiao, Weilie; Wang, Tongdan; Ma, Chunmin; Xu, Hanzhang; Zhang, Jinfu; Gao, Fenghou; Wu, Ying-Li

2016-01-01

Deubiquitinating enzyme USP7 has been involved in the pathogenesis and progression of several cancers. Targeting USP7 is becoming an attractive strategy for cancer therapy. In this study, we identified synthetic triterpenoid C-28 methyl ester of 2-cyano-3, 12-dioxoolen-1, 9-dien-28-oic acid (CDDO-Me) as a novel inhibitor of USP7 but not of other cysteine proteases such as cathepsin B and cathepsin D. CDDO-Me inhibits USP7 activity via a mechanism that is independent of the presence of α, β-unsaturated ketones. Molecular docking studies showed that CDDO-Me fits well in the ubiquitin carboxyl terminus-binding pocket on USP7. Given that CDDO-Me is known to be effective against ovarian cancer cells, we speculated that CDDO-Me may target USP7 in ovarian cancer cells. We demonstrated that ovarian cancer cells have higher USP7 expression than their normal counterparts. Knockdown of USP7 inhibits the proliferation of ovarian cancer cells both in vitro and in vivo. Using the cellular thermal shift assay and the drug affinity responsive target stability assay, we further demonstrated that CDDO-Me directly binds to USP7 in cells, which leads to the decrease of its substrates such as MDM2, MDMX and UHRF1. CDDO-Me suppresses ovarian cancer tumor growth in an xenograft model. In conclusion, we demonstrate that USP7 is a novel target of ovarian cancer cells; targeting USP7 may contribute to the anti-cancer effect of CDDO-Me. The development of novel USP7 selective compounds based on the CDDO-Me-scaffold warrants further investigation. PMID:27780924

Identification of candidate substrates of ubiquitin-specific protease 13 using 2D-DIGE

PubMed Central

Wang, Jianmin; Liu, Yingli; Tang, Lijuan; Qi, Sufen; Mi, Yingjun; Liu, Dianwu; Tian, Qingbao

2017-01-01

The present study aimed to identify candidate substrates of ubiquitin-specific protease (USP)13 using two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). USP13 is a well-characterized member of the USP family, which regulates diverse cellular functions by cleaving ubiquitin from ubiquitinated protein substrates. However, existing studies indicate that USP13 has no detectable hydrolytic activity in vitro. This finding implies that USP13 likely has different substrate specificity. In this study, a USP cleavage assay was performed using two different types of model substrates (glutathione S-transferase-Ub52 and ubiquitin-β-galactosidase) to detect the deubiquitinating enzyme (DUB) activity of USP13. In addition, a proteomic approach was taken by using 2D-DIGE to detect cellular proteins whose expressoin is significantly altered in 293T cell lines following the overexpression of USP13 or its C345S mutant (the catalytically inactive form). The data indicated that USP13 still has no detectable DUB activity in vitro nor does C345S. The results of 2D-DIGE demonstrated that the expression of several proteins increased or decreased significantly in 293T cells following the overexpression of USP13. Mass spec troscopy analysis of gel spots identified 7 proteins, including 4 proteins with an increased expression, namely vinculin, thimet oligopeptidase, cleavage and polyadenylation specific factor 3, and methylosome protein 50, and 3 proteins with a decreased expression, namely adenylosuccinate synthetase, annexin and phosphoglycerate mutase. In addition, in the samples of 293T cell lines after the overexpression of USP13 and USP13 C345S, vinculin exhibited an increased expression, suggesting that it may be a candidate substrate of USP13. However, sufficient follow-up validation studies are required in order to determine whether vinculin protein directly interacts with USP13. PMID:28498477

Inactivation of USP14 Perturbs Ubiquitin Homeostasis and Delays the Cell Cycle in Mouse Embryonic Fibroblasts and in Fruit Fly Drosophila.

PubMed

Lee, Jung Hoon; Park, Seoyoung; Yun, Yejin; Choi, Won Hoon; Kang, Min-Ji; Lee, Min Jae

2018-01-01

The 26S proteasome is the key proteolytic complex for recognition and degradation of polyubiquitinated target substrates in eukaryotes. Among numerous proteasome-associated proteins, a deubiquitinating enzyme (DUB) USP14 has been identified as an endogenous inhibitor of the proteasome. Here, we explored the complex regulatory functions of USP14 that involve ubiquitin (Ub) homeostasis and substrate degradation in flies and mammals. USP14-null primary and immortalized mouse embryonic fibroblasts (MEFs) and USP14 knocked-down Drosophila were analyzed in this study. We measured proteasome and DUB activities using fluorogenic reporter substrates and adduct-forming probes. To examine the levels of ubiquitin, we performed immunoblotting and immunohistochemistry. Mass spectrometry (MS) was used to examine polyUb chain linkages and USP14-interacing proteins. Cell cycle was analyzed by flow cytometry, BrdU labeling, and phospho-histone H3 staining. The homeostasis of Ub in USP14-/-MEFs was markedly perturbed because of facilitated clearance of Ub. This phenomenon was recapitulated in muscles of USP14-deficient Drosophila with old ages. Absolute quantitation using MS also revealed that USP14-/- MEFs contained significantly increased amounts of Ub, compared with wild-type. The key phenotype of USP14-/- MEFs was their delayed proliferation originated from prolonged interphase possibly through aberrant degradation of cyclins A and B1. We found that knocking down USP14 in Drosophila resulted in delayed eye development associated with reduced mitotic activity. Our study identifies novel cellular functions of USP14 not only in cellular Ub hometostasis but also in cell cycle progression. USP14 was also essential for proper Drosophila eye development. These results strongly suggest that the USP14-mediated proteasome activity regulation may be directly related to various human diseases including cancer. © 2018 The Author(s). Published by S. Karger AG, Basel.

USP1 targeting impedes GBM growth by inhibiting stem cell maintenance and radioresistance

PubMed Central

Lee, Jin-Ku; Chang, Nakho; Yoon, Yeup; Yang, Heekyoung; Cho, Heejin; Kim, Eunhee; Shin, Yongjae; Kang, Wonyoung; Oh, Young Taek; Mun, Gyeong In; Joo, Kyeung Min; Nam, Do-Hyun; Lee, Jeongwu

2016-01-01

Background Clinical benefits from standard therapies against glioblastoma (GBM) are limited in part due to intrinsic radio- and chemoresistance of GBM and inefficient targeting of GBM stem-like cells (GSCs). Novel therapeutic approaches that overcome treatment resistance and diminish stem-like properties of GBM are needed. Methods We determined the expression levels of ubiquitination-specific proteases (USPs) by transcriptome analysis and found that USP1 is highly expressed in GBM. Using the patient GBM-derived primary tumor cells, we inhibited USP1 by shRNA-mediated knockdown or its specific inhibitor pimozide and evaluated the effects on stem cell marker expression, proliferation, and clonogenic growth of tumor cells. Results USP1 was highly expressed in gliomas relative to normal brain tissues and more preferentially in GSC enrichment marker (CD133 or CD15) positive cells. USP1 positively regulated the protein stability of the ID1 and CHEK1, critical regulators of DNA damage response and stem cell maintenance. Targeting USP1 by RNA interference or treatment with a chemical USP1 inhibitor attenuated clonogenic growth and survival of GSCs and enhanced radiosensitivity of GBM cells. Finally, USP1 inhibition alone or in combination with radiation significantly prolonged the survival of tumor-bearing mice. Conclusion USP1-mediated protein stabilization promotes GSC maintenance and treatment resistance, thereby providing a rationale for USP1 inhibition as a potential therapeutic approach against GBM. PMID:26032834

Deubiquitylases USP5 and USP13 are recruited to and regulate heat-induced stress granules through their deubiquitylating activities.

PubMed

Xie, Xuan; Matsumoto, Shunsuke; Endo, Akinori; Fukushima, Toshiaki; Kawahara, Hiroyuki; Saeki, Yasushi; Komada, Masayuki

2018-04-12

Stress granules are transient cytoplasmic foci induced by various stresses that contain translation-stalled mRNAs and RNA-binding proteins. They are proposed to modulate mRNA translation and stress responses. Here, we show that the deubiquitylases USP5 and USP13 are recruited to heat-induced stress granules. Heat-induced stress granules also contained K48- and K63-linked ubiquitin chains. Depletion of USP5 or USP13 resulted in elevated ubiquitin chain levels and accelerated assembly of heat-induced stress granules, suggesting that these enzymes regulate the stability of the stress granules through their ubiquitin isopeptidase activity. Moreover, disassembly of heat-induced stress granules after returning the cells to normal temperatures was markedly repressed by individual depletion of USP5 or USP13. Finally, overexpression of a ubiquitin mutant lacking the C-terminal diglycine motif caused the accumulation of unanchored ubiquitin chains and the repression of the disassembly of heat-induced stress granules. As unanchored ubiquitin chains are preferred substrates for USP5, we suggest that USP5 regulates the assembly and disassembly of heat-induced stress granules by mediating the hydrolysis of unanchored ubiquitin chains while USP13 regulates stress granules through deubiquitylating protein-conjugated ubiquitin chains.This article has an associated First Person interview with the first author of the paper. © 2018. Published by The Company of Biologists Ltd.

Structural and Functional Investigations of the N-Terminal Ubiquitin Binding Region of Usp25.

PubMed

Yang, Yuanyuan; Shi, Li; Ding, Yiluan; Shi, Yanhong; Hu, Hong-Yu; Wen, Yi; Zhang, Naixia

2017-05-23

Ubiquitin-specific protease 25 (Usp25) is a deubiquitinase that is involved in multiple biological processes. The N-terminal ubiquitin-binding region (UBR) of Usp25 contains one ubiquitin-associated domain, one small ubiquitin-like modifier (SUMO)-interacting motif and two ubiquitin-interacting motifs. Previous studies suggest that the covalent sumoylation in the UBR of Usp25 impairs its enzymatic activity. Here, we raise the hypothesis that non-covalent binding of SUMO, a prerequisite for efficient sumoylation, will impair Usp25's catalytic activity as well. To test our hypothesis and elucidate the underlying molecular mechanism, we investigated the structure and function of the Usp25 N-terminal UBR. The solution structure of Usp25 1-146 is obtained, and the key residues responsible for recognition of ubiquitin and SUMO2 are identified. Our data suggest inhibition of Usp25's catalytic activity upon the non-covalent binding of SUMO2 to the Usp25 SUMO-interacting motif. We also find that SUMO2 can competitively block the interaction between the Usp25 UBR and its ubiquitin substrates. Based on our findings, we have proposed a working model to depict the regulatory role of the Usp25 UBR in the functional display of the enzyme. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Juvenile Hormone Prevents 20-Hydroxyecdysone-induced Metamorphosis by Regulating the Phosphorylation of a Newly Identified Broad Protein*

PubMed Central

Cai, Mei-Juan; Liu, Wen; Pei, Xu-Yang; Li, Xiang-Ru; He, Hong-Juan; Wang, Jin-Xing; Zhao, Xiao-Fan

2014-01-01

The steroid hormone 20-hydroxyecdysone (20E) initiates insect molting and metamorphosis. By contrast, juvenile hormone (JH) prevents metamorphosis. However, the mechanism by which JH inhibits metamorphosis remains unclear. In this study, we propose that JH induces the phosphorylation of Broad isoform Z7 (BrZ7), a newly identified protein, to inhibit 20E-mediated metamorphosis in the lepidopteran insect Helicoverpa armigera. The knockdown of BrZ7 in larvae inhibited metamorphosis by repressing the expression of the 20E response gene. BrZ7 was weakly expressed and phosphorylated during larval growth but highly expressed and non-phosphorylated during metamorphosis. JH regulated the rapid phosphorylation of BrZ7 via a G-protein-coupled receptor-, phospholipase C-, and protein kinase C-triggered pathway. The phosphorylated BrZ7 bound to the 5′-regulatory region of calponin to regulate its expression in the JH pathway. Exogenous JH induced BrZ7 phosphorylation to prevent metamorphosis by suppressing 20E-related gene transcription. JH promoted non-phosphorylated calponin interacting with ultraspiracle protein to activate the JH pathway and antagonize the 20E pathway. This study reveals one of the possible mechanisms by which JH counteracts 20E-regulated metamorphosis by inducing the phosphorylation of BrZ7. PMID:25096576

Isolation and Characterization of Two Proteins from Moraxella catarrhalis That Bear a Common Epitope

PubMed Central

McMichael, John C.; Fiske, Michael J.; Fredenburg, Ross A.; Chakravarti, Deb N.; VanDerMeid, Karl R.; Barniak, Vicki; Caplan, Jeffrey; Bortell, Eric; Baker, Steven; Arumugham, Rasappa; Chen, Dexiang

1998-01-01

The UspA1 and UspA2 proteins of Moraxella catarrhalis are potential vaccine candidates for preventing disease caused by this organism. We have characterized both proteins and evaluated their vaccine potential using both in vitro and in vivo assays. Both proteins were purified from the O35E isolate by Triton X-100 extraction, followed by ion-exchange and hydroxyapatite chromatography. Analysis of the sequences of internal peptides, prepared by enzymatic and chemical cleavage of the proteins, revealed that UspA1 and UspA2 exhibited distinct structural differences but shared a common sequence including an epitope recognized by the monoclonal antibody 17C7. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), purified UspA1 exhibited a molecular weight of approximately 350,000 when unheated and a molecular weight of 100,000 after being heated for 10 min at 100°C. In contrast, purified UspA2 exhibited an apparent molecular weight of 240,000 by SDS-PAGE that did not change with the length of time of heating. Their sizes as determined by gel filtration were 1,150,000 and 830,000 for UspA1 and UspA2, respectively. Preliminary results indicate the proteins have separate functions in bacterial pathogenesis. Purified UspA1 was found to bind HEp-2 cells, and sera against UspA1, but not against UspA2, blocked binding of the O35E isolate to the HEp-2 cells. UspA1 also bound fibronectin and appears to have a role in bacterial attachment. Purified UspA2, however, did not bind fibronectin but had an affinity for vitronectin. Both proteins elicited bactericidal antibodies in mice to homologous and heterologous disease isolates. Finally, mice immunized with each of the proteins, followed by pulmonary challenge with either the homologous or a heterologous isolate, cleared the bacteria more rapidly than mock-immunized mice. These results suggest that UspA1 and UspA2 serve different virulence functions and that both are promising vaccine candidates. PMID:9712790

USP <800>: Key Considerations and Changes for Health Systems

PubMed Central

Sahadeo, Priya; Weber, Robert J.

2015-01-01

On March 28, 2014, The United States Pharmacopeia and The National Formulary (USP-NF) published USP General Chapter <800> Hazardous Drugs–Handling in Healthcare Settings, as open for public comment in the USP Pharmacopeial Forum (PF) 40(3). Pharmacy directors must be proactive in understanding the impact that USP <800> will have on their processes for preparing sterile products. USP General Chapter <797> pertains to the compounding of both hazardous and nonhazardous drugs. USP <800> serves as a new standard to guide the handling of hazardous drugs in order to protect patients, health care personnel, and the environment. USP <800> describes hazardous drug handling related to the receipt, storage, compounding, dispensing, administration, and disposal of both sterile and nonsterile products and preparations. Regardless of all of the requirements listed in USP <800>, there is no substitute for disciplined, consistent work practices regarding proper sterile technique. This point should be emphasized with all compounding personnel. Even if one is compounding in the most compliant USP <800> cleanroom, improper technique can negate all the benefits of the physical structures. Pharmacy leaders at every level will play a key role in assisting an organization to achieve timely compliance with USP <800> standards. Until the standard becomes official, it is important for pharmacists to become familiarized with the latest draft to identify potential barriers to compliance and to strategize a plan to overcome barriers. Although complying with USP <800> may seem to be a daunting task, it can be manageable if approached in a systematic organized way. PMID:27729683

27 CFR 21.50 - Formula No. 25.

Code of Federal Regulations, 2011 CFR

2011-04-01

... pounds of iodine, U.S.P., and 15 pounds of either potassium iodide, U.S.P., or sodium iodide, U.S.P. (b) Authorized uses. (1) As a solvent: 230.Tinctures of iodine. 249.Miscellaneous external pharmaceuticals, U.S.P...

27 CFR 21.50 - Formula No. 25.

Code of Federal Regulations, 2010 CFR

2010-04-01

... pounds of iodine, U.S.P., and 15 pounds of either potassium iodide, U.S.P., or sodium iodide, U.S.P. (b) Authorized uses. (1) As a solvent: 230.Tinctures of iodine. 249.Miscellaneous external pharmaceuticals, U.S.P...

Divergence in Ubiquitin Interaction and Catalysis among the Ubiquitin-Specific Protease Family Deubiquitinating Enzymes.

PubMed

Tencer, Adam H; Liang, Qin; Zhuang, Zhihao

2016-08-23

Deubiquitinating enzymes (DUBs) are responsible for reversing mono- and polyubiquitination of proteins and play essential roles in numerous cellular processes. Close to 100 human DUBs have been identified and are classified into five families, with the ubiquitin-specific protease (USP) family being the largest (>50 members). The binding of ubiquitin (Ub) to USP is strikingly different from that observed for the DUBs in the ubiquitin C-terminal hydrolase (UCH) and ovarian tumor domain protease (OTU) families. We generated a panel of mutant ubiquitins and used them to probe the ubiquitin's interaction with a number of USPs. Our results revealed a remarkable divergence of USP-Ub interactions among the USP catalytic domains. Our double-mutant cycle analysis targeting the ubiquitin residues located in the tip, the central body, and the tail of ubiquitin also demonstrated different crosstalk among the USP-Ub interactions. This work uncovered intriguing divergence in the ubiquitin-binding mode in the USP family DUBs and raised the possibility of targeting the ubiquitin-binding hot spots on USPs for selective inhibition of USPs by small molecule antagonists.

Protein expression vector and secretion signal peptide optimization to drive the production, secretion, and functional expression of the bacteriocin enterocin A in lactic acid bacteria.

PubMed

Borrero, Juan; Jiménez, Juan J; Gútiez, Loreto; Herranz, Carmen; Cintas, Luis M; Hernández, Pablo E

2011-10-20

Replacement of the leader sequence (LS) of the bacteriocin enterocin A (LS(entA)) by the signal peptides (SP) of the protein Usp45 (SP(usp45)), and the bacteriocins enterocin P (SP(entP)), and hiracin JM79 (SP(hirJM79)) permits the production, secretion, and functional expression of EntA by different lactic acid bacteria (LAB). Chimeric genes encoding the SP(usp45), the SP(entP), and the SP(hirJM79) fused to mature EntA plus the EntA immunity genes (entA+entiA) were cloned into the expression vectors pNZ8048 and pMSP3545, under control of the inducible P(nisA) promoter, and in pMG36c, under control of the constitutive P(32) promoter. The amount, antimicrobial activity, and specific antimicrobial activity of the EntA produced by the recombinant Lactococcus lactis, Enterococcus faecium, E. faecalis, Lactobacillus sakei and Pediococcus acidilactici hosts varied depending on the signal peptide, the expression vector, and the host strain. However, the antimicrobial activity and the specific antimicrobial activity of the EntA produced by most of the LAB transformants was lower than expected from their production. The supernatants of the recombinant L. lactis NZ9000 (pNZUAI) and L. lactis NZ9000 (pNZHAI), overproducers of EntA, showed a 1.2- to 5.1-fold higher antimicrobial activity than that of the natural producer E. faecium T136 against different Listeria spp. Copyright © 2011 Elsevier B.V. All rights reserved.

Onco-Regulon: an integrated database and software suite for site specific targeting of transcription factors of cancer genes

PubMed Central

Tomar, Navneet; Mishra, Akhilesh; Mrinal, Nirotpal; Jayaram, B.

2016-01-01

Transcription factors (TFs) bind at multiple sites in the genome and regulate expression of many genes. Regulating TF binding in a gene specific manner remains a formidable challenge in drug discovery because the same binding motif may be present at multiple locations in the genome. Here, we present Onco-Regulon (http://www.scfbio-iitd.res.in/software/onco/NavSite/index.htm), an integrated database of regulatory motifs of cancer genes clubbed with Unique Sequence-Predictor (USP) a software suite that identifies unique sequences for each of these regulatory DNA motifs at the specified position in the genome. USP works by extending a given DNA motif, in 5′→3′, 3′ →5′ or both directions by adding one nucleotide at each step, and calculates the frequency of each extended motif in the genome by Frequency Counter programme. This step is iterated till the frequency of the extended motif becomes unity in the genome. Thus, for each given motif, we get three possible unique sequences. Closest Sequence Finder program predicts off-target drug binding in the genome. Inclusion of DNA-Protein structural information further makes Onco-Regulon a highly informative repository for gene specific drug development. We believe that Onco-Regulon will help researchers to design drugs which will bind to an exclusive site in the genome with no off-target effects, theoretically. Database URL: http://www.scfbio-iitd.res.in/software/onco/NavSite/index.htm PMID:27515825

Genetic Variations in the TP53 Pathway in Native Americans Strongly Suggest Adaptation to the High Altitudes of the Andes.

PubMed

Jacovas, Vanessa Cristina; Rovaris, Diego Luiz; Peréz, Orlando; de Azevedo, Soledad; Macedo, Gabriel Souza; Sandoval, José Raul; Salazar-Granara, Alberto; Villena, Mercedes; Dugoujon, Jean-Michel; Bisso-Machado, Rafael; Petzl-Erler, Maria Luiza; Salzano, Francisco Mauro; Ashton-Prolla, Patricia; Ramallo, Virginia; Bortolini, Maria Cátira

2015-01-01

The diversity of the five single nucleotide polymorphisms located in genes of the TP53 pathway (TP53, rs1042522; MDM2, rs2279744; MDM4, rs1563828; USP7, rs1529916; and LIF, rs929271) were studied in a total of 282 individuals belonging to Quechua, Aymara, Chivay, Cabanaconde, Yanke, Taquile, Amantani, Anapia, Uros, Guarani Ñandeva, and Guarani Kaiowá populations, characterized as Native American or as having a high level (> 90%) of Native American ancestry. In addition, published data pertaining to 100 persons from five other Native American populations (Surui, Karitiana, Maya, Pima, and Piapoco) were analyzed. The populations were classified as living in high altitude (≥ 2,500 m) or in lowlands (< 2,500 m). Our analyses revealed that alleles USP7-G, LIF-T, and MDM2-T showed significant evidence that they were selected for in relation to harsh environmental variables related to high altitudes. Our results show for the first time that alleles of classical TP53 network genes have been evolutionary co-opted for the successful human colonization of the Andes.

Structure and catalytic regulatory function of ubiquitin specific protease 11 N-terminal and ubiquitin-like domains.

PubMed

Harper, Stephen; Gratton, Hayley E; Cornaciu, Irina; Oberer, Monika; Scott, David J; Emsley, Jonas; Dreveny, Ingrid

2014-05-13

The ubiquitin specific protease 11 (USP11) is implicated in DNA repair, viral RNA replication, and TGFβ signaling. We report the first characterization of the USP11 domain architecture and its role in regulating the enzymatic activity. USP11 consists of an N-terminal "domain present in USPs" (DUSP) and "ubiquitin-like" (UBL) domain, together referred to as DU domains, and the catalytic domain harboring a second UBL domain. Crystal structures of the DU domains show a tandem arrangement with a shortened β-hairpin at the two-domain interface and altered surface characteristics compared to the homologues USP4 and USP15. A conserved VEVY motif is a signature feature at the two-domain interface that shapes a potential protein interaction site. Small angle X-ray scattering and gel filtration experiments are consistent with the USP11DU domains and full-length USP11 being monomeric. Unexpectedly, we reveal, through kinetic assays of a series of deletion mutants, that the catalytic activity of USP11 is not regulated through intramolecular autoinhibition or activation by the N-terminal DU or UBL domains. Moreover, ubiquitin chain cleavage assays with all eight linkages reveal a preference for Lys(63)-, Lys(6)-, Lys(33)-, and Lys(11)-linked chains over Lys(27)-, Lys(29)-, and Lys(48)-linked and linear chains consistent with USP11's function in DNA repair pathways that is mediated by the protease domain. Our data support a model whereby USP11 domains outside the catalytic core domain serve as protein interaction or trafficking modules rather than a direct regulatory function of the proteolytic activity. This highlights the diversity of USPs in substrate recognition and regulation of ubiquitin deconjugation.

Ubiquitin-specific protease 8 links the PTEN-Akt-AIP4 pathway to the control of FLIPS stability and TRAIL sensitivity in glioblastoma multiforme.

PubMed

Panner, Amith; Crane, Courtney A; Weng, Changjiang; Feletti, Alberto; Fang, Shanna; Parsa, Andrew T; Pieper, Russell O

2010-06-15

The antiapoptotic protein FLIP(S) is a key suppressor of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in human glioblastoma multiforme (GBM) cells. We previously reported that a novel phosphatase and tensin homologue (PTEN)-Akt-atrophin-interacting protein 4 (AIP4) pathway regulates FLIP(S) ubiquitination and stability, although the means by which PTEN and Akt were linked to AIP4 activity were unclear. Here, we report that a second regulator of ubiquitin metabolism, the ubiquitin-specific protease 8 (USP8), is a downstream target of Akt, and that USP8 links Akt to AIP4 and the regulation of FLIP(S) stability and TRAIL resistance. In human GBM xenografts, levels of USP8 correlated inversely with pAkt levels, and genetic or pharmacologic manipulation of Akt regulated USP8 levels in an inverse manner. Overexpression of wild-type USP8, but not catalytically inactive USP8, increased FLIP(S) ubiquitination, decreased FLIP(S) half-life, decreased FLIP(S) steady-state levels, and decreased TRAIL resistance, whereas short interfering RNA (siRNA)-mediated suppression of USP8 levels had the opposite effect. Because high levels of the USP8 deubiquitinase correlated with high levels of FLIP(S) ubiquitination, USP8 seemed to control FLIP(S) ubiquitination through an intermediate target. Consistent with this idea, overexpression of wild-type USP8 decreased the ubiquitination of the FLIP(S) E3 ubiquitin ligase AIP4, an event previously shown to increase AIP4-FLIP(S) interaction, whereas siRNA-mediated suppression of USP8 increased AIP4 ubiquitination. Furthermore, the suppression of FLIP(S) levels by USP8 overexpression was reversed by the introduction of siRNA targeting AIP4. These results show that USP8, a downstream target of Akt, regulates the ability of AIP4 to control FLIP(S) stability and TRAIL sensitivity.

Identification of a novel higher molecular weight isoform of USP7/HAUSP that interacts with the Herpes simplex virus type-1 immediate early protein ICP0.

PubMed

Antrobus, Robin; Boutell, Chris

2008-10-01

The Herpes simplex virus type-1 (HSV-1) regulatory protein ICP0, a RING-finger E3 ubiquitin ligase, stimulates the onset of viral lytic replication and the reactivation of quiescent viral genomes from latency. Like many ubiquitin ligases ICP0 induces its own ubiquitination, a process that can lead to its proteasome-dependent degradation. ICP0 counteracts this activity by recruiting the cellular ubiquitin-specific protease USP7/HAUSP. Here we show that ICP0 can also interact with a previously unidentified isoform of USP7 (termed here USP7(beta)). This isoform is not a predominantly ubiquitinated, SUMO-modified, or phosphorylated species of USP7 but is constitutively expressed in a number of different cell types. Like USP7, USP7(beta) binds specifically to an electrophilic ubiquitin probe, indicating that it contains an accessible catalytic core with potential ubiquitin-protease activity. The interaction formed between ICP0 and USP7(beta) requires ICP0 to have an intact USP7-binding domain and results in its susceptibility to ICP0-mediated degradation during HSV-1 infection.

Usp5 links suppression of p53 and FAS levels in melanoma to the BRAF pathway

PubMed Central

Potu, Harish; Peterson, Luke F.; Pal, Anupama; Verhaegen, Monique; Cao, Juxiang; Talpaz, Moshe; Donato, Nicholas J.

2014-01-01

Usp5 is a deubiquitinase (DUB) previously shown to regulate unanchored polyubiquitin (Ub) chains, p53 transcriptional activity and double-strand DNA repair. In BRAF mutant melanoma cells, Usp5 activity was suppressed by BRAF inhibitor (vemurafenib) in sensitive but not in acquired or intrinsically resistant cells. Usp5 knockdown overcame acquired vemurafenib resistance and sensitized BRAF and NRAS mutant melanoma cells to apoptosis initiated by MEK inhibitor, cytokines or DNA-damaging agents. Knockdown and overexpression studies demonstrated that Usp5 regulates p53 (and p73) levels and alters cell growth and cell cycle distribution associated with p21 induction. Usp5 also regulates the intrinsic apoptotic pathway by modulating p53-dependent FAS expression. A small molecule DUB inhibitor (EOAI3402143) phenocopied the FAS induction and apoptotic sensitization of Usp5 knockdown and fully blocked melanoma tumor growth in mice. Overall, our results demonstrate that BRAF activates Usp5 to suppress cell cycle checkpoint control and apoptosis by blocking p53 and FAS induction; all of which can be restored by small molecule-mediated Usp5 inhibition. These results suggest that Usp5 inhibition can provide an alternate approach in recovery of diminished p53 (or p73) function in melanoma and can add to the targeted therapies already used in the treatment of melanoma. PMID:24980819

A Role for USP7 in DNA Replication

PubMed Central

Jagannathan, Madhav; Nguyen, Tin; Gallo, David; Luthra, Niharika; Brown, Grant W.; Saridakis, Vivian

2014-01-01

The minichromosome maintenance (MCM) complex, which plays multiple important roles in DNA replication, is loaded onto chromatin following mitosis, remains on chromatin until the completion of DNA synthesis, and then is unloaded by a poorly defined mechanism that involves the MCM binding protein (MCM-BP). Here we show that MCM-BP directly interacts with the ubiquitin-specific protease USP7, that this interaction occurs predominantly on chromatin, and that MCM-BP can tether USP7 to MCM proteins. Detailed biochemical and structure analyses of the USP7–MCM-BP interaction showed that the 155PSTS158 MCM-BP sequence mediates critical interactions with the TRAF domain binding pocket of USP7. Analysis of the effects of USP7 knockout on DNA replication revealed that lack of USP7 results in slowed progression through late S phase without globally affecting the fork rate or origin usage. Lack of USP7 also resulted in increased levels of MCM proteins on chromatin, and investigation of the cause of this increase revealed a defect in the dissociation of MCM proteins from chromatin in mid- to late S phase. This role of USP7 mirrors the previously described role for MCM-BP in MCM complex unloading and suggests that USP7 works with MCM-BP to unload MCM complexes from chromatin at the end of S phase. PMID:24190967

rigor mortis encodes a novel nuclear receptor interacting protein required for ecdysone signaling during Drosophila larval development.

PubMed

Gates, Julie; Lam, Geanette; Ortiz, José A; Losson, Régine; Thummel, Carl S

2004-01-01

Pulses of the steroid hormone ecdysone trigger the major developmental transitions in Drosophila, including molting and puparium formation. The ecdysone signal is transduced by the EcR/USP nuclear receptor heterodimer that binds to specific response elements in the genome and directly regulates target gene transcription. We describe a novel nuclear receptor interacting protein encoded by rigor mortis (rig) that is required for ecdysone responses during larval development. rig mutants display defects in molting, delayed larval development, larval lethality, duplicated mouth parts, and defects in puparium formation--phenotypes that resemble those seen in EcR, usp, E75A and betaFTZ-F1 mutants. Although the expression of these nuclear receptor genes is essentially normal in rig mutant larvae, the ecdysone-triggered switch in E74 isoform expression is defective. rig encodes a protein with multiple WD-40 repeats and an LXXLL motif, sequences that act as specific protein-protein interaction domains. Consistent with the presence of these elements and the lethal phenotypes of rig mutants, Rig protein interacts with several Drosophila nuclear receptors in GST pull-down experiments, including EcR, USP, DHR3, SVP and betaFTZ-F1. The ligand binding domain of betaFTZ-F1 is sufficient for this interaction, which can occur in an AF-2-independent manner. Antibody stains reveal that Rig protein is present in the brain and imaginal discs of second and third instar larvae, where it is restricted to the cytoplasm. In larval salivary gland and midgut cells, however, Rig shuttles between the cytoplasm and nucleus in a spatially and temporally regulated manner, at times that correlate with the major lethal phase of rig mutants and major switches in ecdysone-regulated gene expression. Taken together, these data indicate that rig exerts essential functions during larval development through gene-specific effects on ecdysone-regulated transcription, most likely as a cofactor for one or more nuclear receptors. Furthermore, the dynamic intracellular redistribution of Rig protein suggests that it may act to refine spatial and temporal responses to ecdysone during development.

USP1 targeting impedes GBM growth by inhibiting stem cell maintenance and radioresistance.

PubMed

Lee, Jin-Ku; Chang, Nakho; Yoon, Yeup; Yang, Heekyoung; Cho, Heejin; Kim, Eunhee; Shin, Yongjae; Kang, Wonyoung; Oh, Young Taek; Mun, Gyeong In; Joo, Kyeung Min; Nam, Do-Hyun; Lee, Jeongwu

2016-01-01

Clinical benefits from standard therapies against glioblastoma (GBM) are limited in part due to intrinsic radio- and chemoresistance of GBM and inefficient targeting of GBM stem-like cells (GSCs). Novel therapeutic approaches that overcome treatment resistance and diminish stem-like properties of GBM are needed. We determined the expression levels of ubiquitination-specific proteases (USPs) by transcriptome analysis and found that USP1 is highly expressed in GBM. Using the patient GBM-derived primary tumor cells, we inhibited USP1 by shRNA-mediated knockdown or its specific inhibitor pimozide and evaluated the effects on stem cell marker expression, proliferation, and clonogenic growth of tumor cells. USP1 was highly expressed in gliomas relative to normal brain tissues and more preferentially in GSC enrichment marker (CD133 or CD15) positive cells. USP1 positively regulated the protein stability of the ID1 and CHEK1, critical regulators of DNA damage response and stem cell maintenance. Targeting USP1 by RNA interference or treatment with a chemical USP1 inhibitor attenuated clonogenic growth and survival of GSCs and enhanced radiosensitivity of GBM cells. Finally, USP1 inhibition alone or in combination with radiation significantly prolonged the survival of tumor-bearing mice. USP1-mediated protein stabilization promotes GSC maintenance and treatment resistance, thereby providing a rationale for USP1 inhibition as a potential therapeutic approach against GBM. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Stratifin regulates stabilization of receptor tyrosine kinases via interaction with ubiquitin-specific protease 8 in lung adenocarcinoma.

PubMed

Kim, Yunjung; Shiba-Ishii, Aya; Nakagawa, Tomoki; Iemura, Shun-Ichiro; Natsume, Tohru; Nakano, Noriyuki; Matsuoka, Ryota; Sakashita, Shingo; Lee, SangJoon; Kawaguchi, Atsushi; Sato, Yukio; Noguchi, Masayuki

2018-06-07

Previously we have reported that stratifin (SFN, 14-3-3 sigma) acts as a novel oncogene, accelerating the tumor initiation and progression of lung adenocarcinoma. Here, pull-down assay and LC-MS/MS analysis revealed that ubiquitin-specific protease 8 (USP8) specifically bound to SFN in lung adenocarcinoma cells. Both USP8 and SFN showed higher expression in human lung adenocarcinoma than in normal lung tissue, and USP8 expression was significantly correlated with SFN expression. Expression of SFN, but not of USP8, was associated with histological subtype, pathological stage, and poor prognosis. USP8 stabilizes receptor tyrosine kinases (RTKs) such as EGFR and MET by deubiquitination, contributing to the proliferative activity of many human cancers including non-small cell lung cancer. In vitro, USP8 binds to SFN and they co-localize at the early endosomes in lung adenocarcinoma cells. Moreover, USP8 or SFN knockdown leads to downregulation of tumor cellular proliferation and upregulation of apoptosis, p-EGFR or p-MET, which are related to the degradation pathway, and accumulation of ubiquitinated RTKs, leading to lysosomal degradation. Additionally, mutant USP8, which is unable to bind to SFN, reduces the expression of RTKs and p-STAT3. We also found that interaction with SFN is critical for USP8 to exert its autodeubiquitination function and avoid dephosphorylation by PP1. Our findings demonstrate that SFN enhances RTK stabilization through abnormal USP8 regulation in lung adenocarcinoma, suggesting that SFN could be a more suitable therapeutic target for lung adenocarcinoma than USP8.

USP1 deubiquitinase: cellular functions, regulatory mechanisms and emerging potential as target in cancer therapy

PubMed Central

2013-01-01

Reversible protein ubiquitination is emerging as a key process for maintaining cell homeostasis, and the enzymes that participate in this process, in particular E3 ubiquitin ligases and deubiquitinases (DUBs), are increasingly being regarded as candidates for drug discovery. Human DUBs are a group of approximately 100 proteins, whose cellular functions and regulatory mechanisms remain, with some exceptions, poorly characterized. One of the best-characterized human DUBs is ubiquitin-specific protease 1 (USP1), which plays an important role in the cellular response to DNA damage. USP1 levels, localization and activity are modulated through several mechanisms, including protein-protein interactions, autocleavage/degradation and phosphorylation, ensuring that USP1 function is carried out in a properly regulated spatio-temporal manner. Importantly, USP1 expression is deregulated in certain types of human cancer, suggesting that USP1 could represent a valid target in cancer therapy. This view has gained recent support with the finding that USP1 inhibition may contribute to revert cisplatin resistance in an in vitro model of non-small cell lung cancer (NSCLC). Here, we describe the current knowledge on the cellular functions and regulatory mechanisms of USP1. We also summarize USP1 alterations found in cancer, combining data from the literature and public databases with our own data. Finally, we discuss the emerging potential of USP1 as a target, integrating published data with our novel findings on the effects of the USP1 inhibitor pimozide in combination with cisplatin in NSCLC cells. PMID:23937906

Ubiquitin-specific protease 11 (USP11) functions as a tumor suppressor through deubiquitinating and stabilizing VGLL4 protein

PubMed Central

Zhang, Encheng; Shen, Bing; Mu, Xingyu; Qin, Yan; Zhang, Fang; Liu, Yong; Xiao, Jiantao; Zhang, Pingzhao; Wang, Chenji; Tan, Mingyue; Fan, Yu

2016-01-01

VGLL4 is a transcriptional repressor that interacts with transcription factors TEADs and inhibits YAP-induced overgrowth and tumorigenesis. VGLL4 protein was dramatically reduced in various types of human cancers. But how VGLL4 protein is post-transcriptional regulated is poorly understood. In this study, we identify deubiquitinating enzyme USP11 as a novel VGLL4 interactor. We reveal that the USP domain of USP11 and the N-terminal region of VGLL4 are required for mutual binding. USP11 controls VGLL4 protein stability by promoting its deubiquitination. Furthermore, our results show that knockdown of USP11 promotes cell growth, migration, and invasion in a YAP-dependent manner. Together, our results suggest that USP11 may exert its tumor suppressor role by modulating VGLL4/YAP-TEADs regulatory loop. PMID:28042509

Nucleophosmin/B23 regulates ubiquitin dynamics in nucleoli by recruiting deubiquitylating enzyme USP36.

PubMed

Endo, Akinori; Kitamura, Naomi; Komada, Masayuki

2009-10-09

The nucleolus is a subnuclear compartment with multiple cellular functions, including ribosome biogenesis. USP36 is a deubiquitylating enzyme that localizes to nucleoli and plays an essential role in regulating the structure and function of the organelle. However, how the localization of USP36 is regulated remains unknown. Here, we identified a short stretch of basic amino acids (RGKEKKIKKFKREKRR) that resides in the C-terminal region of USP36 and serves as a nucleolar localization signal for the protein. We found that this motif interacts with a central acidic region of nucleophosmin/B23, a major nucleolar protein involved in various nucleolar functions. Knockdown of nucleophosmin/B23 resulted in a significant reduction in the amount of USP36 in nucleoli, without affecting the cellular USP36 level. This was associated with elevated ubiquitylation levels of fibrillarin, a USP36 substrate protein in nucleoli. We conclude that nucleophosmin/B23 recruits USP36 to nucleoli, thereby serving as a platform for the regulation of nucleolar protein functions through ubiquitylation/deubiquitylation.

USP10 Is an Essential Deubiquitinase for Hematopoiesis and Inhibits Apoptosis of Long-Term Hematopoietic Stem Cells.

PubMed

Higuchi, Masaya; Kawamura, Hiroki; Matsuki, Hideaki; Hara, Toshifumi; Takahashi, Masahiko; Saito, Suguru; Saito, Kousuke; Jiang, Shuying; Naito, Makoto; Kiyonari, Hiroshi; Fujii, Masahiro

2016-12-13

Self-renewal, replication, and differentiation of hematopoietic stem cells (HSCs) are regulated by cytokines produced by niche cells in fetal liver and bone marrow. HSCs must overcome stresses induced by cytokine deprivation during normal development. In this study, we found that ubiquitin-specific peptidase 10 (USP10) is a crucial deubiquitinase for mouse hematopoiesis. All USP10 knockout (KO) mice died within 1 year because of bone marrow failure with pancytopenia. Bone marrow failure in these USP10-KO mice was associated with remarkable reductions of long-term HSCs (LT-HSCs) in bone marrow and fetal liver. Such USP10-KO fetal liver exhibited enhanced apoptosis of hematopoietic stem/progenitor cells (HSPCs) including LT-HSCs but not of lineage-committed progenitor cells. Transplantation of USP10-competent bone marrow cells into USP10-KO mice reconstituted multilineage hematopoiesis. These results suggest that USP10 is an essential deubiquitinase in hematopoiesis and functions by inhibiting apoptosis of HSPCs including LT-HSCs. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Selective and cell-active inhibitors of the USP1/UAF1 deubiquitinase complex reverse cisplatin resistance in non-small cell lung cancer cells

PubMed Central

Chen, Junjun; Dexheimer, Thomas S.; Ai, Yongxing; Liang, Qin; Villamil, Mark A.; Inglese, James; Maloney, David J; Jadhav, Ajit; Simeonov, Anton; Zhuang, Zhihao

2012-01-01

Ubiquitin-specific proteases (USPs) have in recent years emerged as a promising therapeutic target class. We identified selective small-molecule inhibitors against a deubiquitinase complex, the human USP1/UAF1, through quantitative high throughput screening (qHTS) of a collection of bioactive molecules. The top inhibitors, pimozide and GW7647, inhibited USP1/UAF1 noncompetitively with a Ki of 0.5 and 0.7 μM respectively, and displayed selectivity against a number of deubiquitinases, deSUMOylase and cysteine proteases. The USP1/UAF1 inhibitors act synergistically with cisplatin in inhibiting cisplatin-resistant non-small cell lung cancer (NSCLC) cell proliferation. USP1/UAF1 represents a promising target for drug intervention because of its involvement in translesion synthesis and Fanconi anemia pathway important for normal DNA damage response. Our results support USP1/UAF1 as a potential therapeutic target and provide the first example of targeting the USP/WD40 repeat protein complex for inhibitor discovery. PMID:22118673

The Human Papillomavirus E6 Oncoprotein Targets USP15 and TRIM25 To Suppress RIG-I-Mediated Innate Immune Signaling.

PubMed

Chiang, Cindy; Pauli, Eva-Katharina; Biryukov, Jennifer; Feister, Katharina F; Meng, Melissa; White, Elizabeth A; Münger, Karl; Howley, Peter M; Meyers, Craig; Gack, Michaela U

2018-03-15

Retinoic acid-inducible gene I (RIG-I) is a key pattern recognition receptor that senses viral RNA and interacts with the mitochondrial adaptor MAVS, triggering a signaling cascade that results in the production of type I interferons (IFNs). This signaling axis is initiated by K63-linked ubiquitination of RIG-I mediated by the E3 ubiquitin ligase TRIM25, which promotes the interaction of RIG-I with MAVS. USP15 was recently identified as an upstream regulator of TRIM25, stabilizing the enzyme through removal of degradative K48-linked polyubiquitin, ultimately promoting RIG-I-dependent cytokine responses. Here, we show that the E6 oncoprotein of human papillomavirus type 16 (HPV16) as well as of other HPV types form a complex with TRIM25 and USP15 in human cells. In the presence of E6, the K48-linked ubiquitination of TRIM25 was markedly increased, and in line with this, TRIM25 degradation was enhanced. Our results further showed that E6 inhibited the TRIM25-mediated K63-linked ubiquitination of RIG-I and its CARD-dependent interaction with MAVS. HPV16 E6, but not E7, suppressed the RIG-I-mediated induction of IFN-β, chemokines, and IFN-stimulated genes (ISGs). Finally, CRISPR-Cas9 gene targeting in human keratinocytes showed that the TRIM25-RIG-I-MAVS triad is important for eliciting an antiviral immune response to HPV16 infection. Our study thus identifies a novel immune escape mechanism that is conserved among different HPV strains and further indicates that the RIG-I signaling pathway plays an important role in the innate immune response to HPV infection. IMPORTANCE Persistent infection and tumorigenesis by HPVs are known to require viral manipulation of a variety of cellular processes, including those involved in innate immune responses. Here, we show that the HPV E6 oncoprotein antagonizes the activation of the cytoplasmic innate immune sensor RIG-I by targeting its upstream regulatory enzymes TRIM25 and USP15. We further show that the RIG-I signaling cascade is important for an antiviral innate immune response to HPV16 infection, providing evidence that RIG-I, whose role in sensing RNA virus infections has been well characterized, also plays a crucial role in the antiviral host response to small DNA viruses of the Papillomaviridae family. Copyright © 2018 American Society for Microbiology.

78 FR 18352 - Determination That QUESTRAN (Cholestyramine for Oral Suspension, USP), Equivalent to 4 Grams, and...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-03-26

...] Determination That QUESTRAN (Cholestyramine for Oral Suspension, USP), Equivalent to 4 Grams, and QUESTRAN LIGHT (Cholestyramine for Oral Suspension, USP), Equivalent to 4 Grams, Were Not Withdrawn From Sale for Reasons of...), equivalent to (EQ) 4 grams (g), and QUESTRAN LIGHT (cholestyramine for oral suspension, USP), EQ 4 g, were...

77 FR 50702 - Ranbaxy Laboratories Limited; Withdrawal of Approval of 27 Abbreviated New Drug Applications

Federal Register 2010, 2011, 2012, 2013, 2014

2012-08-22

... Tablets USP, 1 gram. 065043 Cefuroxime Axetil Tablets USP, 125 mg, 250 mg, and 500 mg. 065080 Dispermox (amoxicillin tablets for oral suspension USP), 200 mg and 400 mg. 065092 Raniclor (cefaclor chewable tablets USP), 125 mg, 187 mg, 250 mg, and 375 mg. 065100 Panixine Disperdose (cephalexin tablets for oral...

Pathogenic variants screening in seventeen candidate genes on 2p15 for association with ankylosing spondylitis in a Han Chinese population.

PubMed

Wang, Mengmeng; Xin, Lihong; Cai, Guoqi; Zhang, Xu; Yang, Xiao; Li, Xiaona; Xia, Qing; Wang, Li; Xu, Shengqian; Xu, Jianhua; Shuai, Zongwen; Ding, Changhai; Pan, Faming

2017-01-01

Previous studies have found the association between rs10865331 in 2p15 area and ankylosing spondylitis (AS). This study aimed to identify additional functional genetic variants in 2p15 region associated with AS susceptibility. We used next generation sequencing (NGS) in 100 AS cases and 100 healthy controls to screen AS susceptible genetic variants, and validated these variants in 620 cases and 620 controls by using imLDRTM technique for single nucleotide polymorphism (SNP) genotyping. Totally, we identified 12 SNPs that might confer susceptibility to AS. Of those SNPs, three (rs14170, rs2123111 and rs1729674) were nominally associated (P<0.05) with AS, but were no longer statistically significant after Bonferroni correction. After stratified by gender, another two SNPs (rs11428092 and rs10208769 in USP34) were associated with AS in males but not females, though this was not statistically significant after Bonferroni correction. In addition, rs1729674, rs14170, rs2123111 and rs10208769 were in strong linkage disequilibrium (LD) and were further enrolled in haplotype analysis. A novel haplotype TAGA was found to be associated with a decreased risk of AS (odds ratio (OR) (95% confidence interval (CI)) = 0.832 (0.705-0.982)). Beyond that, we also demonstrated a strong relationship between rs10865331 and AS susceptibility (OR (95% CI) = 1.303(1.111-1.526)). rs14170 and rs2123111 inUSP34 and rs1729674 in C2orf74 may be associated with AS susceptibility in Han Chinese population. USP34 and C2orf74 in 2p15 region may be AS novel susceptibility genes.

CAPNS1 Regulates USP1 Stability and Maintenance of Genome Integrity

PubMed Central

Cataldo, Francesca; Peche, Leticia Y.; Klaric, Enio; Brancolini, Claudio; Myers, Michael P.

2013-01-01

Calpains regulate a wide spectrum of biological functions, including migration, adhesion, apoptosis, secretion, and autophagy, through the modulating cleavage of specific substrates. Ubiquitous microcalpain (μ-calpain) and millicalpain (m-calpain) are heterodimers composed of catalytic subunits encoded, respectively, by CAPN1 and CAPN2 and a regulatory subunit encoded by CAPNS1. Here we show that calpain is required for the stability of the deubiquitinating enzyme USP1 in several cell lines. USP1 modulates DNA replication polymerase choice and repair by deubiquitinating PCNA. The ubiquitinated form of the USP1 substrate PCNA is stabilized in CAPNS1-depleted U2OS cells and mouse embryonic fibroblasts (MEFs), favoring polymerase-η loading on chromatin and increased mutagenesis. USP1 degradation directed by the cell cycle regulator APC/Ccdh1, which marks USP1 for destruction in the G1 phase, is upregulated in CAPNS1-depleted cells. USP1 stability can be rescued upon forced expression of calpain-activated Cdk5/p25, previously reported as a cdh1 repressor. These data suggest that calpain stabilizes USP1 by activating Cdk5, which in turn inhibits cdh1 and, consequently, USP1 degradation. Altogether these findings point to a connection between the calpain system and the ubiquitin pathway in the regulation of DNA damage response and place calpain at the interface between cell cycle modulation and DNA repair. PMID:23589330

Tandem UIMs confer Lys48 ubiquitin chain substrate preference to deubiquitinase USP25

PubMed Central

Kawaguchi, Kohei; Uo, Kazune; Tanaka, Toshiaki; Komada, Masayuki

2017-01-01

Ubiquitin-specific protease (USP) 25, belonging to the USP family of deubiquitinases, harbors two tandem ubiquitin-interacting motifs (UIMs), a ~20-amino-acid α-helical stretch that binds to ubiquitin. However, the role of the UIMs in USP25 remains unclear. Here we show that the tandem UIM region binds to Lys48-, but not Lys63-, linked ubiquitin chains, where the two UIMs played a critical and cooperative role. Purified USP25 exhibited higher ubiquitin isopeptidase activity to Lys48-, than to Lys63-, linked ubiquitin chains. Mutations that disrupted the ubiquitin-binding ability of the tandem UIMs resulted in a reduced ubiquitin isopeptidase activity of USP25, suggesting a role for the UIMs in exerting the full catalytic activity of USP25. Moreover, when mutations that convert the binding preference from Lys48- to Lys63-linked ubiquitin chains were introduced into the tandem UIM region, the USP25 mutants acquired elevated and reduced isopeptidase activity toward Lys63- and Lys48-linked ubiquitin chains, respectively. These results suggested that the binding preference of the tandem UIMs toward Lys48-linked ubiquitin chains contributes not only to the full catalytic activity but also to the ubiquitin chain substrate preference of USP25, possibly by selectively holding the Lys48-linked ubiquitin chain substrates in the proximity of the catalytic core. PMID:28327663

The Advantages and Challenges of Unannounced Standardized Patients Methodology to Assess Healthcare Communication

PubMed Central

Siminoff, Laura A.; Rogers, Heather L.; Waller, Allison C.; Harris-Haywood, Sonja; Esptein, Ronald M.; Borrell Carrio, Francesc; Gliva-McConvey, Gayle; Longo, Daniel R.

2011-01-01

Objective This paper provides an overview of the implementation of using Unannounced Standardized Patients (USPs) to conduct health communication research in clinical settings. Methods Certain types of health communication situations are difficult to capture because of their rarity or unpredictable nature. In primary care the real reasons for a visit are frequently unknown until the consultation is well under way. Therefore, it is logistically difficult for communication studies to capture many real-time communications between patients and their physicians. Although the USP methodology is ideal for capturing these communication behaviors, challenges to using this method include developing collaborative relationships with clinical practices, logistical issues such as safeguarding the identity of the USP, training USPs and creating their identities, maintaining fidelity to the role, and analyzing the resultant data. Results This paper discusses the challenges and solutions to USP implementation. We provide an example of how to implement a USP study using an on-going study being conducted in primary care practices. Conclusion This paper explores the advantages and challenges as well as strategies to overcome obstacles to implementing a USP study. Practice Implications Despite the challenges, USP methodology can contribute much to our understanding of health communication and practice. PMID:21316182

The deubiquitination enzyme USP46 functions as a tumor suppressor by controlling PHLPP-dependent attenuation of Akt signaling in colon cancer

PubMed Central

Li, Xin; Stevens, Payton D.; Yang, Haihua; Gulhati, Pat; Wang, Wei; Evers, B. Mark; Gao, Tianyan

2012-01-01

PHLPP is a family of Ser/Thr protein phosphatases that serve as tumor suppressors by negatively regulating Akt. Our recent studies have demonstrated that the ubiquitin proteasome pathway plays an important role in the downregulation of PHLPP in colorectal cancer. In this study, we show that the deubiquitinase USP46 stabilizes the expression of both PHLPP isoforms by reducing the rate of PHLPP degradation. USP46 binds to PHLPP and directly removes the polyubiquitin chains from PHLPP in vitro and in cells. Increased USP46 expression correlates with decreased ubiquitination and upregulation of PHLPP proteins in colon cancer cells, whereas knockdown of USP46 has the opposite effect. Functionally, USP46-mediated stabilization of PHLPP and the subsequent inhibition of Akt result in a decrease in cell proliferation and tumorigenesis of colon cancer cells in vivo. Moreover, reduced USP46 protein level is found associated with poor PHLPP expression in colorectal cancer patient specimens. Taken together, these results indentify a tumor suppressor role of USP46 in promoting PHLPP expression and inhibiting Akt signaling in colon cancer. PMID:22391563

Molecular mechanism for USP7-mediated DNMT1 stabilization by acetylation

NASA Astrophysics Data System (ADS)

Cheng, Jingdong; Yang, Huirong; Fang, Jian; Ma, Lixiang; Gong, Rui; Wang, Ping; Li, Ze; Xu, Yanhui

2015-05-01

DNMT1 is an important epigenetic regulator that plays a key role in the maintenance of DNA methylation. Here we determined the crystal structure of DNMT1 in complex with USP7 at 2.9 Å resolution. The interaction between the two proteins is primarily mediated by an acidic pocket in USP7 and Lysine residues within DNMT1's KG linker. This intermolecular interaction is required for USP7-mediated stabilization of DNMT1. Acetylation of the KG linker Lysine residues impair DNMT1-USP7 interaction and promote the degradation of DNMT1. Treatment with HDAC inhibitors results in an increase in acetylated DNMT1 and decreased total DNMT1 protein. This negative correlation is observed in differentiated neuronal cells and pancreatic cancer cells. Our studies reveal that USP7-mediated stabilization of DNMT1 is regulated by acetylation and provide a structural basis for the design of inhibitors, targeting the DNMT1-USP7 interaction surface for therapeutic applications.

USP38, FREM3, SDC1, DDC, and LOC727982 Gene Polymorphisms and Differential Susceptibility to Severe Malaria in Tanzania.

PubMed

Manjurano, Alphaxard; Sepúlveda, Nuno; Nadjm, Behzad; Mtove, George; Wangai, Hannah; Maxwell, Caroline; Olomi, Raimos; Reyburn, Hugh; Drakeley, Christopher J; Riley, Eleanor M; Clark, Taane G

2015-10-01

Populations exposed to Plasmodium falciparum infection develop genetic mechanisms of protection against severe malarial disease. Despite decades of genetic epidemiological research, the sickle cell trait (HbAS) sickle cell polymorphism, ABO blood group, and other hemoglobinopathies remain the few major determinants in severe malaria to be replicated across different African populations and study designs. Within a case-control study in a region of high transmission in Tanzania (n = 983), we investigated the role of 40 new loci identified in recent genome-wide studies. In 32 loci passing quality control procedures, we found polymorphisms in USP38, FREM3, SDC1, DDC, and LOC727982 genes to be putatively associated with differential susceptibility to severe malaria. Established candidates explained 7.4% of variation in severe malaria risk (HbAS polymorphism, 6.3%; α-thalassemia, 0.3%; ABO group, 0.3%; and glucose-6-phosphate dehydrogenase deficiency, 0.5%) and the new polymorphisms, another 4.3%. The regions encompassing the loci identified are promising targets for the design of future treatment and control interventions. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America.

Genetic Variations in the TP53 Pathway in Native Americans Strongly Suggest Adaptation to the High Altitudes of the Andes

PubMed Central

Peréz, Orlando; de Azevedo, Soledad; Macedo, Gabriel Souza; Sandoval, José Raul; Salazar-Granara, Alberto; Villena, Mercedes; Dugoujon, Jean-Michel; Bisso-Machado, Rafael; Petzl-Erler, Maria Luiza; Salzano, Francisco Mauro; Ashton-Prolla, Patricia; Ramallo, Virginia; Bortolini, Maria Cátira

2015-01-01

The diversity of the five single nucleotide polymorphisms located in genes of the TP53 pathway (TP53, rs1042522; MDM2, rs2279744; MDM4, rs1563828; USP7, rs1529916; and LIF, rs929271) were studied in a total of 282 individuals belonging to Quechua, Aymara, Chivay, Cabanaconde, Yanke, Taquile, Amantani, Anapia, Uros, Guarani Ñandeva, and Guarani Kaiowá populations, characterized as Native American or as having a high level (> 90%) of Native American ancestry. In addition, published data pertaining to 100 persons from five other Native American populations (Surui, Karitiana, Maya, Pima, and Piapoco) were analyzed. The populations were classified as living in high altitude (≥ 2,500 m) or in lowlands (< 2,500 m). Our analyses revealed that alleles USP7-G, LIF-T, and MDM2-T showed significant evidence that they were selected for in relation to harsh environmental variables related to high altitudes. Our results show for the first time that alleles of classical TP53 network genes have been evolutionary co-opted for the successful human colonization of the Andes. PMID:26382048

Juvenile hormone prevents 20-hydroxyecdysone-induced metamorphosis by regulating the phosphorylation of a newly identified broad protein.

PubMed

Cai, Mei-Juan; Liu, Wen; Pei, Xu-Yang; Li, Xiang-Ru; He, Hong-Juan; Wang, Jin-Xing; Zhao, Xiao-Fan

2014-09-19

The steroid hormone 20-hydroxyecdysone (20E) initiates insect molting and metamorphosis. By contrast, juvenile hormone (JH) prevents metamorphosis. However, the mechanism by which JH inhibits metamorphosis remains unclear. In this study, we propose that JH induces the phosphorylation of Broad isoform Z7 (BrZ7), a newly identified protein, to inhibit 20E-mediated metamorphosis in the lepidopteran insect Helicoverpa armigera. The knockdown of BrZ7 in larvae inhibited metamorphosis by repressing the expression of the 20E response gene. BrZ7 was weakly expressed and phosphorylated during larval growth but highly expressed and non-phosphorylated during metamorphosis. JH regulated the rapid phosphorylation of BrZ7 via a G-protein-coupled receptor-, phospholipase C-, and protein kinase C-triggered pathway. The phosphorylated BrZ7 bound to the 5'-regulatory region of calponin to regulate its expression in the JH pathway. Exogenous JH induced BrZ7 phosphorylation to prevent metamorphosis by suppressing 20E-related gene transcription. JH promoted non-phosphorylated calponin interacting with ultraspiracle protein to activate the JH pathway and antagonize the 20E pathway. This study reveals one of the possible mechanisms by which JH counteracts 20E-regulated metamorphosis by inducing the phosphorylation of BrZ7. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Ti:sapphire/KrF hybrid laser system generating trains of subterawatt subpicosecond UV pulses

NASA Astrophysics Data System (ADS)

Zvorykin, V. D.; Ionin, A. A.; Levchenko, A. O.; Mesyats, G. A.; Seleznev, L. V.; Sinitsyn, D. V.; Ustinovskii, N. N.; Shutov, A. V.

2014-05-01

The GARPUN-MTW Ti:sapphire/KrF hybrid laser system is used to investigate different multipass schemes for amplifying trains of ultrashort pulses (USPs) of subpicosecond duration. It is shown that, for an USP repetition period of 3 - 5 ns, which exceeds the gain-medium recovery time (~2 ns), trains are amplified in the same way as single USPs. Due to this, a train can efficiently extract pump energy from the amplifier and sum energies of individual USPs. The energy of a four-USP train, extracted during four passes through the preamplifier and two passes through the final KrF amplifier (4 + 2 scheme), is saturated at a level of 1.6 J and corresponds to maximum USP peak powers of about 0.6 TW. The energy of amplified spontaneous emission (ASE), on the contrary, rapidly increases at a large total gain length Leff ≈ 6 m and is approximately equal to the USP energy. In the (4 + 1) and (2 + 2) schemes the USP energy decreases only slightly, to Eout = 1.3 and 1.2 J, and the ASE fraction is reduced to about 10% and 3%, respectively. USP self-focusing leads to multiple laser beam filamentation and a 200-fold local increase in the radiation intensity in filaments, to ~2 × 1011 W cm-2, a level at which the nonlinear loss in the output CaF2 windows of the KrF amplifier, caused by three-photon absorption, nonlinear scattering, and broadening of the radiation spectrum to a value exceeding the gain band of the KrF laser transition, becomes the main factor determining the saturation of the USP output energy.

Ubiquitin-specific Protease 7 Regulates Nucleotide Excision Repair through Deubiquitinating XPC Protein and Preventing XPC Protein from Undergoing Ultraviolet Light-induced and VCP/p97 Protein-regulated Proteolysis*

PubMed Central

He, Jinshan; Zhu, Qianzheng; Wani, Gulzar; Sharma, Nidhi; Han, Chunhua; Qian, Jiang; Pentz, Kyle; Wang, Qi-en; Wani, Altaf A.

2014-01-01

Ubiquitin specific protease 7 (USP7) is a known deubiquitinating enzyme for tumor suppressor p53 and its downstream regulator, E3 ubiquitin ligase Mdm2. Here we report that USP7 regulates nucleotide excision repair (NER) via deubiquitinating xeroderma pigmentosum complementation group C (XPC) protein, a critical damage recognition factor that binds to helix-distorting DNA lesions and initiates NER. XPC is ubiquitinated during the early stage of NER of UV light-induced DNA lesions. We demonstrate that transiently compromising cellular USP7 by siRNA and chemical inhibition leads to accumulation of ubiquitinated forms of XPC, whereas complete USP7 deficiency leads to rapid ubiquitin-mediated XPC degradation upon UV irradiation. We show that USP7 physically interacts with XPC in vitro and in vivo. Overexpression of wild-type USP7, but not its catalytically inactive or interaction-defective mutants, reduces the ubiquitinated forms of XPC. Importantly, USP7 efficiently deubiquitinates XPC-ubiquitin conjugates in deubiquitination assays in vitro. We further show that valosin-containing protein (VCP)/p97 is involved in UV light-induced XPC degradation in USP7-deficient cells. VCP/p97 is readily recruited to DNA damage sites and colocalizes with XPC. Chemical inhibition of the activity of VCP/p97 ATPase causes an increase in ubiquitinated XPC on DNA-damaged chromatin. Moreover, USP7 deficiency severely impairs the repair of cyclobutane pyrimidine dimers and, to a lesser extent, affects the repair of 6-4 photoproducts. Taken together, our findings uncovered an important role of USP7 in regulating NER via deubiquitinating XPC and by preventing its VCP/p97-regulated proteolysis. PMID:25118285

USP7 promotes cell proliferation through the stabilization of Ki-67 protein in non-small cell lung cancer cells.

PubMed

Zhang, Chao; Lu, Jing; Zhang, Quan-Wu; Zhao, Wei; Guo, Jia-Hui; Liu, Shan-Ling; Wu, Ying-Li; Jiang, Bin; Gao, Feng-Hou

2016-10-01

The Ki-67 antigen (Ki-67) is the most reliable immunohistochemical marker for evaluation of cell proliferation in non-small cell lung cancer. However, the mechanisms underlying the regulation of protein levels of Ki-67 in non-small cell lung cancer have remained elusive. In this study, we found that Ki-67 and ubiquitin-specific processing protease 7 (USP7) protein were highly expressed in the nucleus of non-small cell lung cancer cells. Furthermore, statistical analysis uncovered the existence of a strong correlation between Ki-67 and USP7 levels. We could also show that the protein levels of Ki-67 in non-small cell lung cancer cells significantly decreased after treatment with P22077, a selective chemical inhibitor of USP7, while the Ki-67 mRNA levels were unperturbed. Similar results were obtained by knocking down USP7 using short hairpin RNA (shRNA) in lung cancer cells. Interestingly, we noticed that ubiquitination levels of Ki-67 increased dramatically in USP7-silenced cells. The tests in vitro and vivo showed a significant delay in tumor cell growth upon knockdown of USP7. Additionally, drug sensitivity tests indicated that USP7-silenced A549 cells had enhanced sensitivity to paclitaxel and docetaxel, while there was no significant change in sensitivity toward carboplatin and cisplatin. Taken together, these data strongly suggest that the overexpression of USP7 might promote cell proliferation by deubiquitinating Ki-67 protein, thereby maintaining its high levels in the non-small cell lung cancer. Our study also hints potential for the development of deubiquitinase-based therapies, especially those targeting USP7 to improve the condition of patients diagnosed with non-small cell lung cancer. Copyright © 2016 Elsevier Ltd. All rights reserved.

A High-Throughput Screening Method for Identification of Inhibitors of the Deubiquitinating Enzyme USP14

PubMed Central

Lee, Byung-Hoon; Finley, Daniel; King, Randall W.

2013-01-01

Deubiquitinating enzymes (DUBs) reverse the process of ubiquitination, and number nearly 100 in humans. In principle, DUBs represent promising drug targets, as several of the enzymes have been implicated in human diseases. The isopeptidase activity of DUBs can be selectively inhibited by targeting the catalytic site with drug-like compounds. Notably, the mammalian 26S proteasome is associated with three major DUBs: RPN11, UCH37 and USP14. Because the ubiquitin ‘chain-trimming’ activity of USP14 can inhibit proteasome function, inhibitors of USP14 can stimulate proteasomal degradation. We recently established a high-throughput screening (HTS) method to discover small-molecule inhibitors specific for USP14. The protocols in this article cover the necessary procedures for preparing assay reagents, performing HTS for USP14 inhibitors, and carrying out post-HTS analysis. PMID:23788557

DUB3 and USP7 de-ubiquitinating enzymes control replication inhibitor Geminin: molecular characterization and associations with breast cancer.

PubMed

Hernández-Pérez, S; Cabrera, E; Salido, E; Lim, M; Reid, L; Lakhani, S R; Khanna, K K; Saunus, J M; Freire, R

2017-08-17

Correct control of DNA replication is crucial to maintain genomic stability in dividing cells. Inappropriate re-licensing of replicated origins is associated with chromosomal instability (CIN), a hallmark of cancer progression that at the same time provides potential opportunities for therapeutic intervention. Geminin is a critical inhibitor of the DNA replication licensing factor Cdt1. To properly achieve its functions, Geminin levels are tightly regulated through the cell cycle by ubiquitin-dependent proteasomal degradation, but the de-ubiquitinating enzymes (DUBs) involved had not been identified. Here we report that DUB3 and USP7 control human Geminin. Overexpression of either DUB3 or USP7 increases Geminin levels through reduced ubiquitination. Conversely, depletion of DUB3 or USP7 reduces Geminin levels, and DUB3 knockdown increases re-replication events, analogous to the effect of Geminin depletion. In exploring potential clinical implications, we found that USP7 and Geminin are strongly correlated in a cohort of invasive breast cancers (P<1.01E-08). As expected, Geminin expression is highly prognostic. Interestingly, we found a non-monotonic relationship between USP7 and breast cancer-specific survival, with both very low or high levels of USP7 associated with poor outcome, independent of estrogen receptor status. Altogether, our data identify DUB3 and USP7 as factors that regulate DNA replication by controlling Geminin protein stability, and suggest that USP7 may be involved in Geminin dysregulation during breast cancer progression.

RNF20 and USP44 regulate stem cell differentiation by modulating H2B monoubiquitylation

PubMed Central

Fuchs, Gilad; Shema, Efrat; Vesterman, Rita; Kotler, Eran; Wolchinsky, Zohar; Wilder, Sylvia; Golomb, Lior; Pribluda, Ariel; Zhang, Feng; Haj-Yahya, Mahmood; Feldmesser, Ester; Brik, Ashraf; Yu, Xiaochun; Hanna, Jacob; Aberdam, Daniel; Domany, Eytan; Oren, Moshe

2012-01-01

Summary Embryonic stem cells (ESC) maintain high genomic plasticity, essential for their capacity to enter diverse differentiation pathways. Post-transcriptional modifications of chromatin histones play a pivotal role in maintaining this plasticity. We now report that one such modification, monoubiquitylation of histone H2B on lysine 120 (H2Bub1), catalyzed by the E3 ligase RNF20, increases during ESC differentiation and is required for efficient execution of this process. This increase is particularly important for the transcriptional induction of relatively long genes during ESC differentiation. Furthermore, we identify the deubiquitinase USP44 as a negative regulator of H2B ubiquitylation, whose downregulation during ESC differentiation contributes to the increase in H2Bub1. Our findings suggest that optimal ESC differentiation requires dynamic changes in H2B ubiquitylation patterns, which must occur in a timely and well-coordinated manner. PMID:22681888

Absence of a Metallicity Effect for Ultra-short-period Planets

DOE Office of Scientific and Technical Information (OSTI.GOV)

Winn, Joshua N.; Sanchis-Ojeda, Roberto; Isaacson, Howard

Ultra-short-period (USP) planets are a newly recognized class of planets with periods shorter than one day and radii smaller than about 2  R {sub ⊕}. It has been proposed that USP planets are the solid cores of hot Jupiters that have lost their gaseous envelopes due to photo-evaporation or Roche lobe overflow. We test this hypothesis by asking whether USP planets are associated with metal-rich stars, as has long been observed for hot Jupiters. We find the metallicity distributions of USP-planet and hot-Jupiter hosts to be significantly different ( p = 3 × 10{sup −4}) based on Keck spectroscopy ofmore » Kepler stars. Evidently, the sample of USP planets is not dominated by the evaporated cores of hot Jupiters. The metallicity distribution of stars with USP planets is indistinguishable from that of stars with short-period planets with sizes between 2 and 4  R {sub ⊕}. Thus, it remains possible that the USP planets are the solid cores of formerly gaseous planets that are smaller than Neptune.« less

The deubiquitinating enzyme USP36 controls selective autophagy activation by ubiquitinated proteins.

PubMed

Taillebourg, Emmanuel; Gregoire, Isabel; Viargues, Perrine; Jacomin, Anne-Claire; Thevenon, Dominique; Faure, Mathias; Fauvarque, Marie-Odile

2012-05-01

Initially described as a nonspecific degradation process induced upon starvation, autophagy is now known also to be involved in the degradation of specific ubiquitinated substrates such as mitochondria, bacteria and aggregated proteins, ensuring crucial functions in cell physiology and immunity. We report here that the deubiquitinating enzyme USP36 controls selective autophagy activation in Drosophila and in human cells. We show that dUsp36 loss of function autonomously inhibits cell growth while activating autophagy. Despite the phenotypic similarity, dUSP36 is not part of the TOR signaling pathway. Autophagy induced by dUsp36 loss of function depends on p62/SQSTM1, an adaptor for delivering cargo marked by polyubiquitin to autophagosomes. Consistent with p62 requirement, dUsp36 mutant cells display nuclear aggregates of ubiquitinated proteins, including Histone H2B, and cytoplasmic ubiquitinated proteins; the latter are eliminated by autophagy. Importantly, USP36 function in p62-dependent selective autophagy is conserved in human cells. Our work identifies a novel, crucial role for a deubiquitinating enzyme in selective autophagy.

USP7 is a SUMO deubiquitinase essential for DNA replication.

PubMed

Lecona, Emilio; Rodriguez-Acebes, Sara; Specks, Julia; Lopez-Contreras, Andres J; Ruppen, Isabel; Murga, Matilde; Muñoz, Javier; Mendez, Juan; Fernandez-Capetillo, Oscar

2016-04-01

Post-translational modification of proteins by ubiquitin (Ub) and Ub-like modifiers regulates DNA replication. We have previously shown that chromatin around replisomes is rich in SUMO and poor in Ub, whereas mature chromatin exhibits an opposite pattern. How this SUMO-rich, Ub-poor environment is maintained at sites of DNA replication in mammalian cells remains unexplored. Here we identify USP7 as a replisome-enriched SUMO deubiquitinase that is essential for DNA replication. By acting on SUMO and SUMOylated proteins, USP7 counteracts their ubiquitination. Inhibition or genetic deletion of USP7 leads to the accumulation of Ub on SUMOylated proteins, which are displaced away from replisomes. Our findings provide a model explaining the differential accumulation of SUMO and Ub at replication forks and identify an essential role of USP7 in DNA replication that should be considered in the development of USP7 inhibitors as anticancer agents.

Transcriptional, translational, and physiological signatures of undernourished honey bees (Apis mellifera) suggest a role for hormonal factors in hypopharyngeal gland degradation.

PubMed

Corby-Harris, Vanessa; Meador, Charlotte A D; Snyder, Lucy A; Schwan, Melissa R; Maes, Patrick; Jones, Beryl M; Walton, Alexander; Anderson, Kirk E

2016-02-01

Honey bee colonies function as a superorganism, where facultatively sterile female workers perform various tasks that support the hive. Nurse workers undergo numerous anatomical and physiological changes in preparation for brood rearing, including the growth of hypopharyngeal glands (HGs). These glands produce the major protein fraction of a protein- and lipid-rich jelly used to sustain developing larvae. Pollen intake is positively correlated with HG growth, but growth in the first three days is similar regardless of diet, suggesting that initial growth is a pre-determined process while later HG development depends on nutrient availability during a critical window in early adulthood (>3 d). It is unclear whether the resultant size differences in nurse HG are simply due to growth arrest or active degradation of the tissue. To determine what processes cause such differences in HG size, we catalogued the differential expression of both gene transcripts and proteins in the HGs of 8 d old bees that were fed diets containing pollen or no pollen. 3438 genes and 367 proteins were differentially regulated due to nutrition. Of the genes and proteins differentially expressed, undernourished bees exhibited more gene and protein up-regulation compared to well-nourished bees, with the affected processes including salivary gland apoptosis, oogenesis, and hormone signaling. Protein secretion was virtually the only process up-regulated in well-nourished bees. Further assays demonstrated that inhibition of ultraspiracle, one component of the ecdysteroid receptor, in the fat body caused larger HGs. Undernourished bees also had higher acid phosphatase activity, a physiological marker of cell death, compared to well-nourished bees. These results support a connection between poor nutrition, hormonal signaling, and HG degradation. Published by Elsevier Ltd.

Proteasome-associated deubiquitinase ubiquitin-specific protease 14 regulates prostate cancer proliferation by deubiquitinating and stabilizing androgen receptor.

PubMed

Liao, Yuning; Liu, Ningning; Hua, Xianliang; Cai, Jianyu; Xia, Xiaohong; Wang, Xuejun; Huang, Hongbiao; Liu, Jinbao

2017-02-02

Androgen receptor (AR) is frequently over-expressed and plays a critical role in the growth and progression of human prostate cancer. The therapy attempting to target AR signalling was established in decades ago but the treatment of prostate cancer is far from being satisfactory. The assignable cause is that our understanding of the mechanism of AR regulation and re-activation remains incomplete. Increasing evidence suggests that deubiquitinases are involved in the regulation of cancer development and progression but the specific underlying mechanism often is not elucidated. In the current study, we have identified ubiquitin-specific protease 14 (USP14) as a novel regulator of AR, inhibiting the degradation of AR via deubiquitinating this oncoprotein in the androgen-responsive prostate cancer cells. We found that (i) USP14 could bind to AR, and additionally, both genetic and pharmacological inhibition of USP14 accelerated the ubiquitination and degradation of AR; (ii) downregulation or inhibition of USP14 suppressed cell proliferation and colony formation of LNcap cells and, conversely, overexpression of USP14 promoted the proliferation; and (iii) reduction or inhibition of USP14 induced G0/G1 phase arrest in LNcap prostate cancer cells. Hence, we conclude that USP14 promotes prostate cancer progression likely through stabilization of AR, suggesting that USP14 could be a promising therapeutic target for prostate cancer.

27 CFR 21.51 - Formula No. 25-A.

Code of Federal Regulations, 2010 CFR

2010-04-01

... solution composed of 20 pounds of iodine, U.S.P.; 15 pounds of either potassium iodide, U.S.P., or sodium iodide, U.S.P.; and 15 pounds of water. (b) Authorized uses. (1) As a solvent: 230.Tinctures of iodine...

miR-132 suppresses the migration and invasion of lung cancer cells by blocking USP9X-induced epithelial-mesenchymal transition

PubMed Central

Guo, Huihui; Zhang, Xilin; Chen, Qiuqiang; Bao, Ying; Dong, Chaohui; Wang, Xiang

2018-01-01

miR-132, a microRNA, has been reported to be down-regulated in several human cancers and is related with tumor progression; however, its function in non-small cell lung cancer (NSCLC) progression remains unclear. This study aimed to investigate the putative role of miR-132 in the metastasis of NSCLC. We determined the function of miR-132 in the migration and invasion of a NSCLC cell line in vitro using a miR-132 inhibitor and mimic. Our results showed overexpression of miR-132 significantly inhibited the migration and invasion of NSCLC cells in vitro. We then identified USP9X as a potential target of miR-132, and demonstrated miR-132 could regulate the expression of USP9X at both the mRNA and protein level. miR-132 could directly bind to the 3’ untranslated region (3’-UTR) of USP9X. Inhibition of USP9X by its inhibitor WP1130 reduced the migration and invasion of NSCLC cells. Furthermore, USP9X inhibition also reversed the increased migration and invasion mediated by miR-132 inhibition. We found USP9X inhibition up-regulated expression of the epithelial-mesenchymal transition (EMT) marker E-cadherin, but down-regulated vimentin expression. A similar effect was seen with miR-132 overexpression, while the opposite effect occurred with miR-132 knockdown. USP9X inhibition reversed the miR-132 inhibitor-induced vimentin up-regulation and E-cadherin down-regulation. Taken together, these results indicate miR-132 prohibits the migration and invasion of NSCLC cells via targeting USP9X-induced EMT. Our data provides further evidence for the critical role of miR-132 and USP9X in regulating cell invasion and migration of NSCLC. PMID:29423007

miR-132 suppresses the migration and invasion of lung cancer cells by blocking USP9X-induced epithelial-mesenchymal transition.

PubMed

Guo, Huihui; Zhang, Xilin; Chen, Qiuqiang; Bao, Ying; Dong, Chaohui; Wang, Xiang

2018-01-01

miR-132, a microRNA, has been reported to be down-regulated in several human cancers and is related with tumor progression; however, its function in non-small cell lung cancer (NSCLC) progression remains unclear. This study aimed to investigate the putative role of miR-132 in the metastasis of NSCLC. We determined the function of miR-132 in the migration and invasion of a NSCLC cell line in vitro using a miR-132 inhibitor and mimic. Our results showed overexpression of miR-132 significantly inhibited the migration and invasion of NSCLC cells in vitro . We then identified USP9X as a potential target of miR-132, and demonstrated miR-132 could regulate the expression of USP9X at both the mRNA and protein level. miR-132 could directly bind to the 3' untranslated region (3'-UTR) of USP9X. Inhibition of USP9X by its inhibitor WP1130 reduced the migration and invasion of NSCLC cells. Furthermore, USP9X inhibition also reversed the increased migration and invasion mediated by miR-132 inhibition. We found USP9X inhibition up-regulated expression of the epithelial-mesenchymal transition (EMT) marker E-cadherin, but down-regulated vimentin expression. A similar effect was seen with miR-132 overexpression, while the opposite effect occurred with miR-132 knockdown. USP9X inhibition reversed the miR-132 inhibitor-induced vimentin up-regulation and E-cadherin down-regulation. Taken together, these results indicate miR-132 prohibits the migration and invasion of NSCLC cells via targeting USP9X-induced EMT. Our data provides further evidence for the critical role of miR-132 and USP9X in regulating cell invasion and migration of NSCLC.

Biosynthesis of nucleotide sugars by a promiscuous UDP-sugar pyrophosphorylase from Arabidopsis thaliana (AtUSP).

PubMed

Liu, Jun; Zou, Yang; Guan, Wanyi; Zhai, Yafei; Xue, Mengyang; Jin, Lan; Zhao, Xueer; Dong, Junkai; Wang, Wenjun; Shen, Jie; Wang, Peng George; Chen, Min

2013-07-01

Nucleotide sugars are activated forms of monosaccharides and key intermediates of carbohydrate metabolism in all organisms. The availability of structurally diverse nucleotide sugars is particularly important for the characterization of glycosyltransferases. Given that limited methods are available for preparation of nucleotide sugars, especially their useful non-natural derivatives, we introduced herein an efficient one-step three-enzyme catalytic system for the synthesis of nucleotide sugars from monosaccharides. In this study, a promiscuous UDP-sugar pyrophosphorylase (USP) from Arabidopsis thaliana (AtUSP) was used with a galactokinase from Streptococcus pneumoniae TIGR4 (SpGalK) and an inorganic pyrophosphatase (PPase) to effectively synthesize four UDP-sugars. AtUSP has better tolerance for C4-derivatives of Gal-1-P compared to UDP-glucose pyrophosphorylase from S. pneumoniae TIGR4 (SpGalU). Besides, the nucleotide substrate specificity and kinetic parameters of AtUSP were systematically studied. AtUSP exhibited considerable activity toward UTP, dUTP and dTTP, the yield of which was 87%, 85% and 84%, respectively. These results provide abundant information for better understanding of the relationship between substrate specificity and structural features of AtUSP. Copyright © 2013 Elsevier Ltd. All rights reserved.

USP1 regulates AKT phosphorylation by modulating the stability of PHLPP1 in lung cancer cells.

PubMed

Zhiqiang, Zhang; Qinghui, Yang; Yongqiang, Zhang; Jian, Zhang; Xin, Zhao; Haiying, Ma; Yuepeng, Guo

2012-07-01

Hyperactivation of phosphatidylinositol 3-kinase/Akt signaling is commonly associated with human tumors including lung cancers. PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1), which terminates Akt signaling by directly dephosphorylating and inactivating Akt, has been identified as a tumor suppressor. The protein level of PHLPP1 is regulated by E3 ligase beta-TRCP, however, the deubiquitinase for PHLPP1 is still not known. The mRNA levels of USP1 and PHLPP1 in lung cancer cells and tissues were determined by real-time PCR. The half-life of PHLPP1 was detected by CHX assay. The interaction between USP1 and PHLPP1 was examined by immunoprecipitation and GST pull-down assay. Both USP1 and PHLPP1 are low expressed in lung cancer cells and tissues and silencing of USP1 by RNA interference significantly decreased the half-life of PHLPP1, which in turn amplified Akt1 phosphorylation. Our data identified a novel USP1-PHLPP1-Akt signaling axis, and decreased USP1 level in lung cancer cells may play an important role in lung cancer progress.

Deubiquitylation of histone H2A activates transcriptional initiation via trans-histone cross-talk with H3K4 di- and trimethylation

PubMed Central

Nakagawa, Takeya; Kajitani, Takuya; Togo, Shinji; Masuko, Norio; Ohdan, Hideki; Hishikawa, Yoshitaka; Koji, Takehiko; Matsuyama, Toshifumi; Ikura, Tsuyoshi; Muramatsu, Masami; Ito, Takashi

2008-01-01

Transcriptional initiation is a key step in the control of mRNA synthesis and is intimately related to chromatin structure and histone modification. Here, we show that the ubiquitylation of H2A (ubH2A) correlates with silent chromatin and regulates transcriptional initiation. The levels of ubH2A vary during hepatocyte regeneration, and based on microarray expression data from regenerating liver, we identified USP21, a ubiquitin-specific protease that catalyzes the hydrolysis of ubH2A. When chromatin is assembled in vitro, ubH2A, but not H2A, specifically represses the di- and trimethylation of H3K4. USP21 relieves this ubH2A-specific repression. In addition, in vitro transcription analysis revealed that ubH2A represses transcriptional initiation, but not transcriptional elongation, by inhibiting H3K4 methylation. Notably, ubH2A-mediated repression was not observed when H3 Lys 4 was changed to arginine. Furthermore, overexpression of USP21 in the liver up-regulates a gene that is normally down-regulated during hepatocyte regeneration. Our studies revealed a novel mode of trans-histone cross-talk, in which H2A ubiquitylation controls the di- and trimethylation of H3K4, resulting in regulation of transcriptional initiation. PMID:18172164

The USP1-UAF1 complex interacts with RAD51AP1 to promote homologous recombination repair.

PubMed

Cukras, Scott; Lee, Euiho; Palumbo, Emily; Benavidez, Pamela; Moldovan, George-Lucian; Kee, Younghoon

2016-10-01

USP1 deubiquitinating enzyme and its stoichiometric binding partner UAF1 play an essential role in promoting DNA homologous recombination (HR) repair in response to various types of DNA damaging agents. Deubiquitination of FANCD2 may be attributed to the key role of USP1-UAF1 complex in regulating HR repair, however whether USP1-UAF1 promotes HR repair independently of FANCD2 deubiquitination is not known. Here we show evidence that the USP1-UAF1 complex has a FANCD2-independent function in promoting HR repair. Proteomic search of UAF1-interacting proteins revealed that UAF1 associates with RAD51AP1, a RAD51-interacting protein implicated in HR repair. We show that UAF1 mediates the interaction between USP1 and RAD51AP1, and that depletion of USP1 or UAF1 led to a decreased stability of RAD51AP1. Protein interaction mapping analysis identified some key residues within RAD51AP1 required for interacting with the USP1-UAF1 complex. Cells expressing the UAF1 interaction-deficient mutant of RAD51AP1 show increased chromosomal aberrations in response to Mitomycin C treatment. Moreover, similar to the RAD51AP1 depleted cells, the cells expressing UAF1-interaction deficient RAD51AP1 display persistent RAD51 foci following DNA damage exposure, indicating that these factors regulate a later step during the HR repair. These data altogether suggest that the USP1-UAF1 complex promotes HR repair via multiple mechanisms: through FANCD2 deubiquitination, as well as by interacting with RAD51AP1.

The USP1-UAF1 complex interacts with RAD51AP1 to promote homologous recombination repair

PubMed Central

Cukras, Scott; Lee, Euiho; Palumbo, Emily; Benavidez, Pamela; Moldovan, George-Lucian; Kee, Younghoon

2016-01-01

ABSTRACT USP1 deubiquitinating enzyme and its stoichiometric binding partner UAF1 play an essential role in promoting DNA homologous recombination (HR) repair in response to various types of DNA damaging agents. Deubiquitination of FANCD2 may be attributed to the key role of USP1-UAF1 complex in regulating HR repair, however whether USP1-UAF1 promotes HR repair independently of FANCD2 deubiquitination is not known. Here we show evidence that the USP1-UAF1 complex has a FANCD2-independent function in promoting HR repair. Proteomic search of UAF1-interacting proteins revealed that UAF1 associates with RAD51AP1, a RAD51-interacting protein implicated in HR repair. We show that UAF1 mediates the interaction between USP1 and RAD51AP1, and that depletion of USP1 or UAF1 led to a decreased stability of RAD51AP1. Protein interaction mapping analysis identified some key residues within RAD51AP1 required for interacting with the USP1-UAF1 complex. Cells expressing the UAF1 interaction-deficient mutant of RAD51AP1 show increased chromosomal aberrations in response to Mitomycin C treatment. Moreover, similar to the RAD51AP1 depleted cells, the cells expressing UAF1-interaction deficient RAD51AP1 display persistent RAD51 foci following DNA damage exposure, indicating that these factors regulate a later step during the HR repair. These data altogether suggest that the USP1-UAF1 complex promotes HR repair via multiple mechanisms: through FANCD2 deubiquitination, as well as by interacting with RAD51AP1. PMID:27463890

Autocatalytic Activity of the Ubiquitin-Specific Protease Domain of Herpes Simplex Virus 1 VP1-2▿†

PubMed Central

Bolstad, M.; Abaitua, F.; Crump, C. M.; O'Hare, P.

2011-01-01

The herpes simplex virus (HSV) tegument protein VP1-2 is essential for virus entry and assembly. VP1-2 also contains a highly conserved ubiquitin-specific protease (USP) domain within its N-terminal region. Despite conservation of the USP and the demonstration that it can act on artificial substrates such as polyubiquitin chains, identification of the relevance of the USP in vivo to levels or function of any substrate remains limited. Here we show that HSV VP1-2 USP can act on itself and is important for stability. VP1-2 N-terminal variants encompassing the core USP domain itself were not affected by mutation of the catalytic cysteine residue (C65). However, extending the N-terminal region resulted in protein species requiring USP activity for accumulation. In this context, C65A mutation resulted in a drastic reduction in protein levels which could be stabilized by proteosomal inhibition or by the presence of normal C65. The functional USP domain could increase abundance of unstable variants, indicating action at least in part, in trans. Interestingly, full-length variants containing the inactive USP, although unstable when expressed in isolation, were stabilized by virus infection. The catalytically inactive VP1-2 retained complementation activity of a VP1-2-negative virus. Furthermore, a recombinant virus expressing a C65A mutant VP1-2 exhibited little difference in single-step growth curves and the kinetics and abundance of VP1-2 or a number of test proteins. Despite the absence of a phenotype for these replication parameters, the USP activity of VP1-2 may be required for function, including its own stability, under certain circumstances. PMID:21715485

USP37 deubiquitinates Cdt1 and contributes to regulate DNA replication.

PubMed

Hernández-Pérez, Santiago; Cabrera, Elisa; Amoedo, Hugo; Rodríguez-Acebes, Sara; Koundrioukoff, Stephane; Debatisse, Michelle; Méndez, Juan; Freire, Raimundo

2016-10-01

DNA replication control is a key process in maintaining genomic integrity. Monitoring DNA replication initiation is particularly important as it needs to be coordinated with other cellular events and should occur only once per cell cycle. Crucial players in the initiation of DNA replication are the ORC protein complex, marking the origin of replication, and the Cdt1 and Cdc6 proteins, that license these origins to replicate by recruiting the MCM2-7 helicase. To accurately achieve its functions, Cdt1 is tightly regulated. Cdt1 levels are high from metaphase and during G1 and low in S/G2 phases of the cell cycle. This control is achieved, among other processes, by ubiquitination and proteasomal degradation. In an overexpression screen for Cdt1 deubiquitinating enzymes, we isolated USP37, to date the first ubiquitin hydrolase controlling Cdt1. USP37 overexpression stabilizes Cdt1, most likely a phosphorylated form of the protein. In contrast, USP37 knock down destabilizes Cdt1, predominantly during G1 and G1/S phases of the cell cycle. USP37 interacts with Cdt1 and is able to de-ubiquitinate Cdt1 in vivo and, USP37 is able to regulate the loading of MCM complexes onto the chromatin. In addition, downregulation of USP37 reduces DNA replication fork speed. Taken together, here we show that the deubiquitinase USP37 plays an important role in the regulation of DNA replication. Whether this is achieved via Cdt1, a central protein in this process, which we have shown to be stabilized by USP37, or via additional factors, remains to be tested. Copyright © 2016 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Integrative mouse and human mRNA studies using WGCNA nominates novel candidate genes involved in the pathogenesis of major depressive disorder.

PubMed

Malki, Karim; Tosto, Maria Grazia; Jumabhoy, Irfan; Lourdusamy, Anbarasu; Sluyter, Frans; Craig, Ian; Uher, Rudolf; McGuffin, Peter; Schalkwyk, Leonard C

2013-12-01

This study aims to identify novel genes associated with major depressive disorder and pharmacological treatment response using animal and human mRNA studies. Weighted gene coexpression network analysis was used to uncover genes associated with stress factors in mice and to inform mRNA probe set selection in a post-mortem study of depression. A total of 171 genes were found to be differentially regulated in response to both early and late stress protocols in a mouse study. Ten human genes, orthologous to mouse genes differentially expressed by stress, were also found to be dysregulated in depressed cases in a human post-mortem brain study from the Stanley Foundation Brain Collection. Several novel genes associated with depression were uncovered, including NOVA1 and USP9X. Moreover, we found further evidence in support of hippocampal neurogenesis and peripheral inflammation in major depressive disorder.

USP7 is a SUMO deubiquitinase essential for DNA replication

PubMed Central

Lecona, Emilio; Rodriguez-Acebes, Sara; Specks, Julia; Lopez-Contreras, Andres J; Ruppen, Isabel; Murga, Matilde; Muñoz, Javier; Mendez, Juan; Fernandez-Capetillo, Oscar

2016-01-01

Post-translational modification of proteins by ubiquitin (Ub) and Ub-like modifiers regulates various aspects of DNA replication. We previously showed that the chromatin around replisomes is rich in SUMO and depleted in Ub, whereas an opposite pattern is observed in mature chromatin. How this SUMO-rich/Ub-low environment is maintained at sites of DNA replication is not known. Here we identify USP7 as a replisome-enriched SUMO deubiquitinase that is essential for DNA replication. By acting on SUMO and SUMOylated proteins, USP7 counteracts their ubiquitination. Chemical inhibition or genetic deletion of USP7 leads to the accumulation of Ub on SUMOylated proteins, which are displaced to chromatin away from replisomes. Our findings provide a model to explain the differential accumulation of SUMO and Ub at replication forks, and identify an essential role of USP7 in DNA replication that should be taken into account for the use of USP7 inhibitors as anticancer agents. PMID:26950370

USP19-Mediated Deubiquitination Facilitates the Stabilization of HRD1 Ubiquitin Ligase.

PubMed

Harada, Kumi; Kato, Masako; Nakamura, Nobuhiro

2016-11-02

In the endoplasmic reticulum (ER), misfolded and unfolded proteins are eliminated by a process called ER-associated protein degradation (ERAD) in order to maintain cell homeostasis. In the ERAD pathway, several ER-localized E3 ubiquitin ligases target ERAD substrate proteins for ubiquitination and subsequent proteasomal degradation. However, little is known about how the functions of the ERAD ubiquitin ligases are regulated. Recently, USP19, an ER-anchored deubiquitinating enzyme (DUB), has been suggested to be involved in the regulation of ERAD. In this study, HRD1, an ERAD ubiquitin ligase, is shown to be a novel substrate for USP19. We demonstrate that USP19 rescues HRD1 from proteasomal degradation by deubiquitination of K48-linked ubiquitin chains. In addition, the altered expression of USP19 affects the steady-state levels of HRD1. These results suggest that USP19 regulates the stability of HRD1 and provide insight into the regulatory mechanism of the ERAD ubiquitin ligases.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Wei; Sartori, Maria A.; Makhnevych, Taras

Post-translational modification of the p53 signaling pathway plays an important role in cell cycle progression and stress-induced apoptosis. Indeed, a large body of work has shown that dysregulation of p53 and its E3 ligase MDM2 by the ubiquitin-proteasome system (UPS) promotes carcinogenesis and malignant transformation. Thus, drug discovery efforts have focused on the restoration of wild-type p53 activity or inactivation of oncogenic mutant p53 by targeted inhibition of UPS components, particularly key deubiquitinases (DUBs) of the ubiquitin-specific protease (USP) class. However, development of selective small-molecule USP inhibitors has been challenging, partly due to the highly conserved structural features of themore » catalytic sites across the class. To tackle this problem, we devised a protein engineering strategy for rational design of inhibitors for DUBs and other UPS proteins. We employed a phage-displayed ubiquitin variant (UbV) library to develop inhibitors targeting the DUBs USP7 and USP10, which are involved in regulating levels of p53 and MDM2. We were able to identify UbVs that bound USP7 or USP10 with high affinity and inhibited deubiquitination activity. We solved the crystal structure of UbV.7.2 and rationalized the molecular basis for enhanced affinity and specificity for USP7. Finally, cell death was increased significantly by UbV.7.2 expression in a colon cancer cell line that was treated with the chemotherapy drug cisplatin, demonstrating the therapeutic potential of inhibiting USP7 by this approach« less

78 FR 62712 - Rate Adjustment

Federal Register 2010, 2011, 2012, 2013, 2014

2013-10-22

....e., in an across-the-board fashion. Id. at 2, 12, 35. The Postal Service states that the Governors... Services Worksheets USPS-R2010-4R/7 Product Cost & Contribution Estimation Model (Public Version) USPS... Product Cost & Contribution Estimation Model (Non- Public Version) USPS-R2010-4R/NP2 Cost Factor...

39 CFR 241.3 - Discontinuance of USPS-operated retail facilities.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 39 Postal Service 1 2013-07-01 2013-07-01 false Discontinuance of USPS-operated retail facilities... ESTABLISHMENT CLASSIFICATION, AND DISCONTINUANCE § 241.3 Discontinuance of USPS-operated retail facilities. (a... of whether an existing retail Post Office, station, or branch should be discontinued. The rules cover...

39 CFR 241.3 - Discontinuance of USPS-operated retail facilities.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 39 Postal Service 1 2014-07-01 2014-07-01 false Discontinuance of USPS-operated retail facilities... ESTABLISHMENT CLASSIFICATION, AND DISCONTINUANCE § 241.3 Discontinuance of USPS-operated retail facilities. (a... of whether an existing retail Post Office, station, or branch should be discontinued. The rules cover...

39 CFR 241.3 - Discontinuance of USPS-operated retail facilities.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 39 Postal Service 1 2012-07-01 2012-07-01 false Discontinuance of USPS-operated retail facilities... ESTABLISHMENT CLASSIFICATION, AND DISCONTINUANCE § 241.3 Discontinuance of USPS-operated retail facilities. (a... of whether an existing retail Post Office, station, or branch should be discontinued. The rules cover...

The deubiquitinase Usp27x stabilizes the BH3-only protein Bim and enhances apoptosis.

PubMed

Weber, Arnim; Heinlein, Melanie; Dengjel, Jörn; Alber, Claudia; Singh, Prafull Kumar; Häcker, Georg

2016-05-01

Bim is a pro-apoptotic Bcl-2 family member of the BH3-only protein subgroup. Expression levels of Bim determine apoptosis susceptibility in non-malignant and in tumour cells. Bim protein expression is downregulated by proteasomal degradation following ERK-dependent phosphorylation and ubiquitination. Here, we report the identification of a deubiquitinase, Usp27x, that binds Bim upon its ERK-dependent phosphorylation and can upregulate its expression levels. Overexpression of Usp27x reduces ERK-dependent Bim ubiquitination, stabilizes phosphorylated Bim, and induces apoptosis in PMA-stimulated cells, as well as in tumour cells with a constitutively active Raf/ERK pathway. Loss of endogenous Usp27x enhances the Bim-degrading activity of oncogenic Raf. Overexpression of Usp27x induces low levels of apoptosis in melanoma and non-small cell lung cancer (NSCLC) cells and substantially enhances apoptosis induced in these cells by the inhibition of ERK signalling. Finally, deletion of Usp27x reduces apoptosis in NSCLC cells treated with an EGFR inhibitor. Thus, Usp27x can trigger via its proteolytic activity the deubiquitination of Bim and enhance its levels, counteracting the anti-apoptotic effects of ERK activity, and therefore acts as a tumour suppressor. © 2016 The Authors.

Absence of a Metallicity Effect for Ultra-short-period Planets

NASA Astrophysics Data System (ADS)

Winn, Joshua N.; Sanchis-Ojeda, Roberto; Rogers, Leslie; Petigura, Erik A.; Howard, Andrew W.; Isaacson, Howard; Marcy, Geoffrey W.; Schlaufman, Kevin C.; Cargile, Phillip; Hebb, Leslie

2017-08-01

Ultra-short-period (USP) planets are a newly recognized class of planets with periods shorter than one day and radii smaller than about 2 R ⊕. It has been proposed that USP planets are the solid cores of hot Jupiters that have lost their gaseous envelopes due to photo-evaporation or Roche lobe overflow. We test this hypothesis by asking whether USP planets are associated with metal-rich stars, as has long been observed for hot Jupiters. We find the metallicity distributions of USP-planet and hot-Jupiter hosts to be significantly different (p = 3 × 10-4) based on Keck spectroscopy of Kepler stars. Evidently, the sample of USP planets is not dominated by the evaporated cores of hot Jupiters. The metallicity distribution of stars with USP planets is indistinguishable from that of stars with short-period planets with sizes between 2 and 4 R ⊕. Thus, it remains possible that the USP planets are the solid cores of formerly gaseous planets that are smaller than Neptune. Based on observations obtained at the W. M. Keck Observatory, which is operated jointly by the University of California and the California Institute of Technology. Keck time was granted by NASA, the University of California, the California Institute of Technology, and the University of Hawaii.

The ubiquitin ligase TRIM25 targets ERG for degradation in prostate cancer.

PubMed

Wang, Shan; Kollipara, Rahul K; Humphries, Caroline G; Ma, Shi-Hong; Hutchinson, Ryan; Li, Rui; Siddiqui, Javed; Tomlins, Scott A; Raj, Ganesh V; Kittler, Ralf

2016-10-04

Ets related gene (ERG) is a transcription factor that is overexpressed in 40% of prostate tumors due to a gene fusion between ERG and TMPRSS2. Because ERG functions as a driver of prostate carcinogenesis, understanding the mechanisms that influence its turnover may provide new molecular handles to target the protein. Previously, we found that ERG undergoes ubiquitination and then is deubiquitinated by USP9X in prostate cancer cells to prevent its proteasomal degradation. Here, we identify Tripartite motif-containing protein 25 (TRIM25) as the E3 ubiquitin ligase that ubiquitinates the protein prior to its degradation. TRIM25 binds full-length ERG, and it also binds the N-terminally truncated variants of ERG that are expressed in tumors with TMPRSS2-ERG fusions. We demonstrate that TRIM25 polyubiquitinates ERG in vitro and that inactivation of TRIM25 resulted in reduced polyubiquitination and stabilization of ERG. TRIM25 mRNA and protein expression was increased in ERG rearrangement-positive prostate cancer specimens, and we provide evidence that ERG upregulates TRIM25 expression. Thus, overexpression of ERG in prostate cancer may cause an increase in TRIM25 activity, which is mitigated by the expression of the deubiquitinase USP9X, which is required to stabilize ERG.

The ubiquitin ligase TRIM25 targets ERG for degradation in prostate cancer

PubMed Central

Wang, Shan; Kollipara, Rahul K.; Humphries, Caroline G.; Ma, Shi-Hong; Hutchinson, Ryan; Li, Rui; Siddiqui, Javed; Tomlins, Scott A.; Raj, Ganesh V.; Kittler, Ralf

2016-01-01

Ets related gene (ERG) is a transcription factor that is overexpressed in 40% of prostate tumors due to a gene fusion between ERG and TMPRSS2. Because ERG functions as a driver of prostate carcinogenesis, understanding the mechanisms that influence its turnover may provide new molecular handles to target the protein. Previously, we found that ERG undergoes ubiquitination and then is deubiquitinated by USP9X in prostate cancer cells to prevent its proteasomal degradation. Here, we identify Tripartite motif-containing protein 25 (TRIM25) as the E3 ubiquitin ligase that ubiquitinates the protein prior to its degradation. TRIM25 binds full-length ERG, and it also binds the N-terminally truncated variants of ERG that are expressed in tumors with TMPRSS2-ERG fusions. We demonstrate that TRIM25 polyubiquitinates ERG in vitro and that inactivation of TRIM25 resulted in reduced polyubiquitination and stabilization of ERG. TRIM25 mRNA and protein expression was increased in ERG rearrangement-positive prostate cancer specimens, and we provide evidence that ERG upregulates TRIM25 expression. Thus, overexpression of ERG in prostate cancer may cause an increase in TRIM25 activity, which is mitigated by the expression of the deubiquitinase USP9X, which is required to stabilize ERG. PMID:27626314

Structure of the USP15 N-terminal domains: a β-hairpin mediates close association between the DUSP and UBL domains.

PubMed

Harper, Stephen; Besong, Tabot M D; Emsley, Jonas; Scott, David J; Dreveny, Ingrid

2011-09-20

Ubiquitin specific protease 15 (USP15) functions in COP9 signalosome mediated regulation of protein degradation and cellular signaling through catalyzing the ubiquitin deconjugation reaction of a discrete number of substrates. It influences the stability of adenomatous polyposis coli, IκBα, caspase-3, and the human papillomavirus type 16 E6. USP15 forms a subfamily with USP4 and USP11 related through a shared presence of N-terminal "domain present in ubiquitin specific proteases" (DUSP) and "ubiquitin-like" (UBL) domains (DU subfamily). Here we report the 1.5 Å resolution crystal structure of the human USP15 N-terminal domains revealing a 80 Å elongated arrangement with the DU domains aligned in tandem. This architecture is generated through formation of a defined interface that is dominated by an intervening β-hairpin structure (DU finger) that engages in an intricate hydrogen-bonding network between the domains. The UBL domain is closely related to ubiquitin among β-grasp folds but is characterized by the presence of longer loop regions and different surface characteristics, indicating that this domain is unlikely to act as ubiquitin mimic. Comparison with the related murine USP4 DUSP-UBL crystal structure reveals that the main DU interdomain contacts are conserved. Analytical ultracentrifugation, small-angle X-ray scattering, and gel filtration experiments revealed that USP15 DU is monomeric in solution. Our data provide a framework to advance study of the structure and function of the DU subfamily. © 2011 American Chemical Society

Excitation of atoms and ions in plasmas by ultra-short electromagnetic pulses

NASA Astrophysics Data System (ADS)

Astapenko, V. A.; Sakhno, S. V.; Svita, S. Yu; Lisitsa, V. S.

2017-02-01

The problem of atoms and ions diagnostics in rarefied and dense plasmas by ultrashort laser pulses (USP) is under consideration. The application of USP provides: 1) excitation from ground states due to their carrier frequency high enough, 2) penetration into optically dense media due to short pulses duration. The excitation from ground atomic states increases sharply populations of excited atomic states in contrast with standard laser induced fluorescence spectroscopy based on radiative transitions between excited atomic states. New broadening parameter in radiation absorption, namely inverse pulse duration time 1/τ appears in addition to standard line-shape width in the profile G(ω). The Lyman-beta absorption spectra for USP are calculated for Holtsmark static broadening mechanism. Excitation of highly charged H-like ions in hot plasmas is described by both Gaussian shapes for Doppler broadening and pulse spectrum resulting in analytical absorption line-shape. USP penetration into optically thick media and corresponding excitation probability are calculated. It is shown a great effect of USP duration on excitation probabilities in optically thick media. The typical situations for plasma diagnostics by USP are discussed in details.

Ubiquitin-specific Protease 11 (USP11) Deubiquitinates Hybrid Small Ubiquitin-like Modifier (SUMO)-Ubiquitin Chains to Counteract RING Finger Protein 4 (RNF4)*

PubMed Central

Hendriks, Ivo A.; Schimmel, Joost; Eifler, Karolin; Olsen, Jesper V.; Vertegaal, Alfred C. O.

2015-01-01

Ring finger protein 4 (RNF4) is a SUMO-targeted ubiquitin E3 ligase with a pivotal function in the DNA damage response (DDR). SUMO interaction motifs (SIMs) in the N-terminal part of RNF4 tightly bind to SUMO polymers, and RNF4 can ubiquitinate these polymers in vitro. Using a proteomic approach, we identified the deubiquitinating enzyme ubiquitin-specific protease 11 (USP11), a known DDR-component, as a functional interactor of RNF4. USP11 can deubiquitinate hybrid SUMO-ubiquitin chains to counteract RNF4. SUMO-enriched nuclear bodies are stabilized by USP11, which functions downstream of RNF4 as a counterbalancing factor. In response to DNA damage induced by methyl methanesulfonate, USP11 could counteract RNF4 to inhibit the dissolution of nuclear bodies. Thus, we provide novel insight into cross-talk between ubiquitin and SUMO and uncover USP11 and RNF4 as a balanced SUMO-targeted ubiquitin ligase/protease pair with a role in the DDR. PMID:25969536

A Fat-Facets-Dscam1-JNK Pathway Enhances Axonal Growth in Development and after Injury

PubMed Central

Koch, Marta; Nicolas, Maya; Zschaetzsch, Marlen; de Geest, Natalie; Claeys, Annelies; Yan, Jiekun; Morgan, Matthew J.; Erfurth, Maria-Luise; Holt, Matthew; Schmucker, Dietmar; Hassan, Bassem A.

2018-01-01

Injury to the adult central nervous systems (CNS) can result in severe long-term disability because damaged CNS connections fail to regenerate after trauma. Identification of regulators that enhance the intrinsic growth capacity of severed axons is a first step to restore function. Here, we conducted a gain-of-function genetic screen in Drosophila to identify strong inducers of axonal growth after injury. We focus on a novel axis the Down Syndrome Cell Adhesion Molecule (Dscam1), the de-ubiquitinating enzyme Fat Facets (Faf)/Usp9x and the Jun N-Terminal Kinase (JNK) pathway transcription factor Kayak (Kay)/Fos. Genetic and biochemical analyses link these genes in a common signaling pathway whereby Faf stabilizes Dscam1 protein levels, by acting on the 3′-UTR of its mRNA, and Dscam1 acts upstream of the growth-promoting JNK signal. The mammalian homolog of Faf, Usp9x/FAM, shares both the regenerative and Dscam1 stabilizing activities, suggesting a conserved mechanism. PMID:29472843

Comparison of central corneal thickness measurements using ultrasound pachymetry, ultrasound biomicroscopy, and the Artemis-2 VHF scanner in normal eyes

PubMed Central

Al-Farhan, Haya M; Al-Otaibi, Wafa’a Majed

2012-01-01

Purpose To compare the precision of central corneal thickness (CCT) measurements taken with the handheld ultrasound pachymeter (USP), ultrasound biomicroscopy (UBM), and the Artemis-2 very high frequency ultrasound scanner (VHFUS) on normal subjects. Design Prospective study. Methods One eye from each of 61 normal subjects was randomly selected for this study. The measurements of the CCT were taken with the USP, VHFUS, and UBM. Results were compared statistically using repeated-measures analysis of variance (ANOVA), Pearson’s correlation coefficient, and limits of agreement. Results The average CCT (± standard deviation) was 530.1 ± 30.5 μm, 554.9 ± 31.7 μm, and 559.5 ± 30.7 μm for UBM, VHFUS, and USP respectively. The intraobserver repeatability analyses of variance are not significant for USP, UBM, and VHFUS. P-values were 0.17, 0.19, and 0.37 respectively. Repeated-measures ANOVA showed a significant difference between the three different methods of measuring CCT (P = 0.0001). The ANOVA test revealed no statistically significant difference between USP and VHFUS (P > 0.05), yet statistical significant differences with UBM versus USP and UBM versus VHFUS (P < 0.001). There were high correlations between the three instruments (P < 0.0001). The mean differences (and upper/lower limits of agreement) for CCT measurements were 29.4 ± 14.3 (2.7/56), 4.6 ± 8.6 (−14.7/23.8), and −24.8 ± 13.1 (−50.4/0.8) for USP versus UBM, USP versus VHFUS, and UBM versus VHFUS, respectively. Conclusion The UBM produces CCT measurements that vary significantly from those returned by the USP and the VHFUS, suggesting that the UBM may not be used interchangeably with either equipment for monitoring the CCT in the clinical setting. PMID:22848145

27 CFR 21.65 - Formula No. 38-B.

Code of Federal Regulations, 2012 CFR

2012-04-01

.... Cinnamic aldehyde, N.F. IX. Cinnamon oil, N.F. Citronella oil, natural. Clove oil, N.F. Coal tar, U.S.P..., terpeneless. Spike lavender oil, natural. Storax, U.S.P. Thyme oil, N.F. XII. Thymol, N.F. Tolu balsam, U.S.P... the denaturant for analysis. (b) Authorized uses. (1) As a solvent: 111.Hair and scalp preparations...

27 CFR 21.65 - Formula No. 38-B.

Code of Federal Regulations, 2010 CFR

2010-04-01

.... Cinnamic aldehyde, N.F. IX. Cinnamon oil, N.F. Citronella oil, natural. Clove oil, N.F. Coal tar, U.S.P..., terpeneless. Spike lavender oil, natural. Storax, U.S.P. Thyme oil, N.F. XII. Thymol, N.F. Tolu balsam, U.S.P... the denaturant for analysis. (b) Authorized uses. (1) As a solvent: 111.Hair and scalp preparations...

27 CFR 21.65 - Formula No. 38-B.

Code of Federal Regulations, 2013 CFR

2013-04-01

.... Cinnamic aldehyde, N.F. IX. Cinnamon oil, N.F. Citronella oil, natural. Clove oil, N.F. Coal tar, U.S.P..., terpeneless. Spike lavender oil, natural. Storax, U.S.P. Thyme oil, N.F. XII. Thymol, N.F. Tolu balsam, U.S.P... the denaturant for analysis. (b) Authorized uses. (1) As a solvent: 111.Hair and scalp preparations...

27 CFR 21.65 - Formula No. 38-B.

Code of Federal Regulations, 2011 CFR

2011-04-01

.... Cinnamic aldehyde, N.F. IX. Cinnamon oil, N.F. Citronella oil, natural. Clove oil, N.F. Coal tar, U.S.P..., terpeneless. Spike lavender oil, natural. Storax, U.S.P. Thyme oil, N.F. XII. Thymol, N.F. Tolu balsam, U.S.P... the denaturant for analysis. (b) Authorized uses. (1) As a solvent: 111.Hair and scalp preparations...

27 CFR 21.65 - Formula No. 38-B.

Code of Federal Regulations, 2014 CFR

2014-04-01

.... Cinnamic aldehyde, N.F. IX. Cinnamon oil, N.F. Citronella oil, natural. Clove oil, N.F. Coal tar, U.S.P..., terpeneless. Spike lavender oil, natural. Storax, U.S.P. Thyme oil, N.F. XII. Thymol, N.F. Tolu balsam, U.S.P... the denaturant for analysis. (b) Authorized uses. (1) As a solvent: 111.Hair and scalp preparations...

USP7 small-molecule inhibitors interfere with ubiquitin binding.

PubMed

Kategaya, Lorna; Di Lello, Paola; Rougé, Lionel; Pastor, Richard; Clark, Kevin R; Drummond, Jason; Kleinheinz, Tracy; Lin, Eva; Upton, John-Paul; Prakash, Sumit; Heideker, Johanna; McCleland, Mark; Ritorto, Maria Stella; Alessi, Dario R; Trost, Matthias; Bainbridge, Travis W; Kwok, Michael C M; Ma, Taylur P; Stiffler, Zachary; Brasher, Bradley; Tang, Yinyan; Jaishankar, Priyadarshini; Hearn, Brian R; Renslo, Adam R; Arkin, Michelle R; Cohen, Frederick; Yu, Kebing; Peale, Frank; Gnad, Florian; Chang, Matthew T; Klijn, Christiaan; Blackwood, Elizabeth; Martin, Scott E; Forrest, William F; Ernst, James A; Ndubaku, Chudi; Wang, Xiaojing; Beresini, Maureen H; Tsui, Vickie; Schwerdtfeger, Carsten; Blake, Robert A; Murray, Jeremy; Maurer, Till; Wertz, Ingrid E

2017-10-26

The ubiquitin system regulates essential cellular processes in eukaryotes. Ubiquitin is ligated to substrate proteins as monomers or chains and the topology of ubiquitin modifications regulates substrate interactions with specific proteins. Thus ubiquitination directs a variety of substrate fates including proteasomal degradation. Deubiquitinase enzymes cleave ubiquitin from substrates and are implicated in disease; for example, ubiquitin-specific protease-7 (USP7) regulates stability of the p53 tumour suppressor and other proteins critical for tumour cell survival. However, developing selective deubiquitinase inhibitors has been challenging and no co-crystal structures have been solved with small-molecule inhibitors. Here, using nuclear magnetic resonance-based screening and structure-based design, we describe the development of selective USP7 inhibitors GNE-6640 and GNE-6776. These compounds induce tumour cell death and enhance cytotoxicity with chemotherapeutic agents and targeted compounds, including PIM kinase inhibitors. Structural studies reveal that GNE-6640 and GNE-6776 non-covalently target USP7 12 Å distant from the catalytic cysteine. The compounds attenuate ubiquitin binding and thus inhibit USP7 deubiquitinase activity. GNE-6640 and GNE-6776 interact with acidic residues that mediate hydrogen-bond interactions with the ubiquitin Lys48 side chain, suggesting that USP7 preferentially interacts with and cleaves ubiquitin moieties that have free Lys48 side chains. We investigated this idea by engineering di-ubiquitin chains containing differential proximal and distal isotopic labels and measuring USP7 binding by nuclear magnetic resonance. This preferential binding protracted the depolymerization kinetics of Lys48-linked ubiquitin chains relative to Lys63-linked chains. In summary, engineering compounds that inhibit USP7 activity by attenuating ubiquitin binding suggests opportunities for developing other deubiquitinase inhibitors and may be a strategy more broadly applicable to inhibiting proteins that require ubiquitin binding for full functional activity.

The DUSP–Ubl domain of USP4 enhances its catalytic efficiency by promoting ubiquitin exchange

PubMed Central

Clerici, Marcello; Luna-Vargas, Mark P. A.; Faesen, Alex C.; Sixma, Titia K.

2014-01-01

Ubiquitin-specific protease USP4 is emerging as an important regulator of cellular pathways, including the TGF-β response, NF-κB signalling and splicing, with possible roles in cancer. Here we show that USP4 has its catalytic triad arranged in a productive conformation. Nevertheless, it requires its N-terminal DUSP–Ubl domain to achieve full catalytic turnover. Pre-steady-state kinetics measurements reveal that USP4 catalytic domain activity is strongly inhibited by slow dissociation of ubiquitin after substrate hydrolysis. The DUSP–Ubl domain is able to enhance ubiquitin dissociation, hence promoting efficient turnover. In a mechanism that requires all USP4 domains, binding of the DUSP–Ubl domain promotes a change of a switching loop near the active site. This ‘allosteric regulation of product discharge’ provides a novel way of regulating deubiquitinating enzymes that may have relevance for other enzyme classes. PMID:25404403

The deubiquitinases USP33 and USP20 coordinate beta2 adrenergic receptor recycling and resensitization.

PubMed

Berthouze, Magali; Venkataramanan, Vidya; Li, Yi; Shenoy, Sudha K

2009-06-17

Agonist-induced ubiquitination of the beta(2) adrenergic receptor (beta(2)AR) functions as an important post-translational modification to sort internalized receptors to the lysosomes for degradation. We now show that this ubiquitination is reversed by two deubiquitinating enzymes, ubiquitin-specific proteases (USPs) 20 and 33, thus, inhibiting lysosomal trafficking when concomitantly promoting receptor recycling from the late-endosomal compartments as well as resensitization of recycled receptors at the cell surface. Dissociation of constitutively bound endogenously expressed USPs 20 and 33 from the beta(2)AR immediately after agonist stimulation and reassociation on prolonged agonist treatment allows receptors to first become ubiquitinated and then deubiquitinated, thus, providing a 'trip switch' between degradative and recycling pathways at the late-endosomal compartments. Thus, USPs 20 and 33 serve as novel regulators that dictate both post-endocytic sorting as well as the intensity and extent of beta(2)AR signalling from the cell surface.

Leveraging blood serotonin as an endophenotype to identify de novo and rare variants involved in autism.

PubMed

Chen, Rui; Davis, Lea K; Guter, Stephen; Wei, Qiang; Jacob, Suma; Potter, Melissa H; Cox, Nancy J; Cook, Edwin H; Sutcliffe, James S; Li, Bingshan

2017-01-01

Autism spectrum disorder (ASD) is one of the most highly heritable neuropsychiatric disorders, but underlying molecular mechanisms are still unresolved due to extreme locus heterogeneity. Leveraging meaningful endophenotypes or biomarkers may be an effective strategy to reduce heterogeneity to identify novel ASD genes. Numerous lines of evidence suggest a link between hyperserotonemia, i.e., elevated serotonin (5-hydroxytryptamine or 5-HT) in whole blood, and ASD. However, the genetic determinants of blood 5-HT level and their relationship to ASD are largely unknown. In this study, pursuing the hypothesis that de novo variants (DNVs) and rare risk alleles acting in a recessive mode may play an important role in predisposition of hyperserotonemia in people with ASD, we carried out whole exome sequencing (WES) in 116 ASD parent-proband trios with most (107) probands having 5-HT measurements. Combined with published ASD DNVs, we identified USP15 as having recurrent de novo loss of function mutations and discovered evidence supporting two other known genes with recurrent DNVs ( FOXP1 and KDM5B ). Genes harboring functional DNVs significantly overlap with functional/disease gene sets known to be involved in ASD etiology, including FMRP targets and synaptic formation and transcriptional regulation genes. We grouped the probands into High-5HT and Normal-5HT groups based on normalized serotonin levels, and used network-based gene set enrichment analysis (NGSEA) to identify novel hyperserotonemia-related ASD genes based on LoF and missense DNVs. We found enrichment in the High-5HT group for a gene network module (DAWN-1) previously implicated in ASD, and this points to the TGF-β pathway and cell junction processes. Through analysis of rare recessively acting variants (RAVs), we also found that rare compound heterozygotes (CHs) in the High-5HT group were enriched for loci in an ASD-associated gene set. Finally, we carried out rare variant group-wise transmission disequilibrium tests (gTDT) and observed significant association of rare variants in genes encoding a subset of the serotonin pathway with ASD. Our study identified USP15 as a novel gene implicated in ASD based on recurrent DNVs. It also demonstrates the potential value of 5-HT as an effective endophenotype for gene discovery in ASD, and the effectiveness of this strategy needs to be further explored in studies of larger sample sizes.

A Content Analysis of Unique Selling Propositions of Tobacco Print Ads.

PubMed

Johnson Shen, Megan; Banerjee, Smita C; Greene, Kathryn; Carpenter, Amanda; Ostroff, Jamie S

2017-03-01

We describe the unique selling propositions (USPs) (propositions used to convince customers to use a particular brand/product by focusing on the unique benefit) of print tobacco ads. A qualitative content analysis was conducted of print tobacco ads (N = 171) selected from August 2012 to August 2013 for cigarettes, moist snuff, e-cigarettes, cigars, and snus to determine the content and themes of USPs for tobacco ads. Cigarette ad USP themes focused on portraying the product as attractive; moist snuff ads focused on portraying product as masculine; cigar ads focused on selling a "high end product;" and new and emerging tobacco products (e-cigarette, snus) focused on directly comparing these products to cigarettes. Whereas traditional tobacco product ads used USPs focused on themes of enjoyment and pleasure (eg, attractive for cigarettes, "high end product" for cigars), new and emerging tobacco product ads offered the unique benefit (USP) of their product being a better and "safer" alternative to traditional tobacco products. Snuff's USPs focused nearly exclusively on the masculinity of their products. Our results provide targets for potential tobacco regulatory actions that could be implemented to reduce demand for tobacco products by reducing their perceived unique benefits.

Saturation scanning of ubiquitin variants reveals a common hot spot for binding to USP2 and USP21.

PubMed

Leung, Isabel; Dekel, Ayelet; Shifman, Julia M; Sidhu, Sachdev S

2016-08-02

A detailed understanding of the molecular mechanisms whereby ubiquitin (Ub) recognizes enzymes in the Ub proteasome system is crucial for understanding the biological function of Ub. Many structures of Ub complexes have been solved and, in most cases, reveal a large structural epitope on a common face of the Ub molecule. However, owing to the generally weak nature of these interactions, it has been difficult to map in detail the functional contributions of individual Ub side chains to affinity and specificity. Here we took advantage of Ub variants (Ubvs) that bind tightly to particular Ub-specific proteases (USPs) and used phage display and saturation scanning mutagenesis to comprehensively map functional epitopes within the structural epitopes. We found that Ubvs that bind to USP2 or USP21 contain a remarkably similar core functional epitope, or "hot spot," consisting mainly of positions that are conserved as the wild type sequence, but also some positions that prefer mutant sequences. The Ubv core functional epitope contacts residues that are conserved in the human USP family, and thus it is likely important for the interactions of Ub across many family members.

Data integration in physiology using Bayes’ rule and minimum Bayes’ factors: deubiquitylating enzymes in the renal collecting duct

PubMed Central

Xue, Zhe; Chen, Jia-Xu; Zhao, Yue; Medvar, Barbara

2017-01-01

A major challenge in physiology is to exploit the many large-scale data sets available from “-omic” studies to seek answers to key physiological questions. In previous studies, Bayes’ theorem has been used for this purpose. This approach requires a means to map continuously distributed experimental data to probabilities (likelihood values) to derive posterior probabilities from the combination of prior probabilities and new data. Here, we introduce the use of minimum Bayes’ factors for this purpose and illustrate the approach by addressing a physiological question, “Which deubiquitylating enzymes (DUBs) encoded by mammalian genomes are most likely to regulate plasma membrane transport processes in renal cortical collecting duct principal cells?” To do this, we have created a comprehensive online database of 110 DUBs present in the mammalian genome (https://hpcwebapps.cit.nih.gov/ESBL/Database/DUBs/). We used Bayes’ theorem to integrate available information from large-scale data sets derived from proteomic and transcriptomic studies of renal collecting duct cells to rank the 110 known DUBs with regard to likelihood of interacting with and regulating transport processes. The top-ranked DUBs were OTUB1, USP14, PSMD7, PSMD14, USP7, USP9X, OTUD4, USP10, and UCHL5. Among these USP7, USP9X, OTUD4, and USP10 are known to be involved in endosomal trafficking and have potential roles in endosomal recycling of plasma membrane proteins in the mammalian cortical collecting duct. PMID:28039431

Data integration in physiology using Bayes' rule and minimum Bayes' factors: deubiquitylating enzymes in the renal collecting duct.

PubMed

Xue, Zhe; Chen, Jia-Xu; Zhao, Yue; Medvar, Barbara; Knepper, Mark A

2017-03-01

A major challenge in physiology is to exploit the many large-scale data sets available from "-omic" studies to seek answers to key physiological questions. In previous studies, Bayes' theorem has been used for this purpose. This approach requires a means to map continuously distributed experimental data to probabilities (likelihood values) to derive posterior probabilities from the combination of prior probabilities and new data. Here, we introduce the use of minimum Bayes' factors for this purpose and illustrate the approach by addressing a physiological question, "Which deubiquitylating enzymes (DUBs) encoded by mammalian genomes are most likely to regulate plasma membrane transport processes in renal cortical collecting duct principal cells?" To do this, we have created a comprehensive online database of 110 DUBs present in the mammalian genome (https://hpcwebapps.cit.nih.gov/ESBL/Database/DUBs/). We used Bayes' theorem to integrate available information from large-scale data sets derived from proteomic and transcriptomic studies of renal collecting duct cells to rank the 110 known DUBs with regard to likelihood of interacting with and regulating transport processes. The top-ranked DUBs were OTUB1, USP14, PSMD7, PSMD14, USP7, USP9X, OTUD4, USP10, and UCHL5. Among these USP7, USP9X, OTUD4, and USP10 are known to be involved in endosomal trafficking and have potential roles in endosomal recycling of plasma membrane proteins in the mammalian cortical collecting duct. Copyright © 2017 the American Physiological Society.

Artificial Recruitment of UAF1-USP Complexes by a PHLPP1-E1 Chimeric Helicase Enhances Human Papillomavirus DNA Replication

PubMed Central

Gagnon, David; Lehoux, Michaël

2015-01-01

ABSTRACT The E1 helicase from anogenital human papillomavirus (HPV) types interacts with the cellular WD repeat-containing protein UAF1 in complex with the deubiquitinating enzyme USP1, USP12, or USP46. This interaction stimulates viral DNA replication and is required for maintenance of the viral episome in keratinocytes. E1 associates with UAF1 through a short UAF1-binding site (UBS) located within the N-terminal 40 residues of the protein. Here, we investigated if the E1 UBS could be replaced by the analogous domain from an unrelated protein, the pleckstrin homology domain and leucine-rich repeat protein phosphatase 1 (PHLPP1). We found that PHLPP1 and E1 interact with UAF1 in a mutually exclusive manner and mapped the minimal PHLPP1 UBS (PUBS) to a 100-amino-acid region sufficient for assembly into UAF1-USP complexes. Similarly to the E1 UBS, overexpression of PUBS in trans inhibited HPV DNA replication, albeit less efficiently. Characterization of a PHLPP1-E1 chimeric helicase revealed that PUBS could partially substitute for the E1 UBS in enhancing viral DNA replication and that the stimulatory effect of PUBS likely involves recruitment of UAF1-USP complexes, as it was abolished by mutations that weaken UAF1-binding and by overexpression of catalytically inactive USPs. Although functionally similar to the E1 UBS, PUBS is larger in size and requires both the WD repeat region and C-terminal ubiquitin-like domain of UAF1 for interaction, in contrast to E1, which does not contact the latter. Overall, this comparison of two heterologous UBSs indicates that these domains function as transferable protein interaction modules and provide further evidence that the association of E1 with UAF1-containing deubiquitinating complexes stimulates HPV DNA replication. IMPORTANCE The E1 protein from anogenital HPV types interacts with the UAF1-associated deubiquitinating enzymes USP1, USP12, and USP46 to stimulate replication of the viral genome. Little is known about the molecular nature of the E1-UAF1 interaction and, more generally, how UAF1-USP complexes recognize their substrate proteins. To address this question, we characterized the UAF1-binding site (UBS) of PHLPP1, a protein unrelated to E1. Using a PHLPP1-E1 chimeric helicase, we show that the PHLPP1 UBS (PUBS) can partially substitute for the E1 UBS in stimulating HPV DNA replication. This stimulation required conserved sequences in PUBS that meditate its interaction with UAF1, including a motif common to the E1 UBS. These results indicate that UAF1-binding sequences function as transferable protein interaction modules and provide further evidence that UAF1-USP complexes stimulate HPV DNA replication. PMID:25833051

Artificial Recruitment of UAF1-USP Complexes by a PHLPP1-E1 Chimeric Helicase Enhances Human Papillomavirus DNA Replication.

PubMed

Gagnon, David; Lehoux, Michaël; Archambault, Jacques

2015-06-01

The E1 helicase from anogenital human papillomavirus (HPV) types interacts with the cellular WD repeat-containing protein UAF1 in complex with the deubiquitinating enzyme USP1, USP12, or USP46. This interaction stimulates viral DNA replication and is required for maintenance of the viral episome in keratinocytes. E1 associates with UAF1 through a short UAF1-binding site (UBS) located within the N-terminal 40 residues of the protein. Here, we investigated if the E1 UBS could be replaced by the analogous domain from an unrelated protein, the pleckstrin homology domain and leucine-rich repeat protein phosphatase 1 (PHLPP1). We found that PHLPP1 and E1 interact with UAF1 in a mutually exclusive manner and mapped the minimal PHLPP1 UBS (PUBS) to a 100-amino-acid region sufficient for assembly into UAF1-USP complexes. Similarly to the E1 UBS, overexpression of PUBS in trans inhibited HPV DNA replication, albeit less efficiently. Characterization of a PHLPP1-E1 chimeric helicase revealed that PUBS could partially substitute for the E1 UBS in enhancing viral DNA replication and that the stimulatory effect of PUBS likely involves recruitment of UAF1-USP complexes, as it was abolished by mutations that weaken UAF1-binding and by overexpression of catalytically inactive USPs. Although functionally similar to the E1 UBS, PUBS is larger in size and requires both the WD repeat region and C-terminal ubiquitin-like domain of UAF1 for interaction, in contrast to E1, which does not contact the latter. Overall, this comparison of two heterologous UBSs indicates that these domains function as transferable protein interaction modules and provide further evidence that the association of E1 with UAF1-containing deubiquitinating complexes stimulates HPV DNA replication. The E1 protein from anogenital HPV types interacts with the UAF1-associated deubiquitinating enzymes USP1, USP12, and USP46 to stimulate replication of the viral genome. Little is known about the molecular nature of the E1-UAF1 interaction and, more generally, how UAF1-USP complexes recognize their substrate proteins. To address this question, we characterized the UAF1-binding site (UBS) of PHLPP1, a protein unrelated to E1. Using a PHLPP1-E1 chimeric helicase, we show that the PHLPP1 UBS (PUBS) can partially substitute for the E1 UBS in stimulating HPV DNA replication. This stimulation required conserved sequences in PUBS that meditate its interaction with UAF1, including a motif common to the E1 UBS. These results indicate that UAF1-binding sequences function as transferable protein interaction modules and provide further evidence that UAF1-USP complexes stimulate HPV DNA replication. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Human Cytomegalovirus Protein pUL38 Prevents Premature Cell Death by Binding to Ubiquitin-Specific Protease 24 and Regulating Iron Metabolism.

PubMed

Sun, Yamei; Bao, Qunchao; Xuan, Baoqin; Xu, Wenjia; Pan, Deng; Li, Qi; Qian, Zhikang

2018-07-01

Human cytomegalovirus (HCMV) protein pUL38 has been shown to prevent premature cell death by antagonizing cellular stress responses; however, the underlying mechanism remains unknown. In this study, we identified the host protein ubiquitin-specific protease 24 (USP24) as an interaction partner of pUL38. Mutagenesis analysis of pUL38 revealed that amino acids TFV at positions 227 to 230 were critical for its interaction with USP24. Mutant pUL38 TFV/AAA protein did not bind to USP24 and failed to prevent cell death induced by pUL38-deficient HCMV infection. Knockdown of USP24 suppressed the cell death during pUL38-deficient HCMV infection, suggesting that pUL38 achieved its function by antagonizing the function of USP24. We investigated the cellular pathways regulated by USP24 that might be involved in the cell death phenotype by testing several small-molecule compounds known to have a protective effect during stress-induced cell death. The iron chelators ciclopirox olamine and Tiron specifically protected cells from pUL38-deficient HCMV infection-induced cell death, thus identifying deregulated iron homeostasis as a potential mechanism. Protein levels of nuclear receptor coactivator 4 (NCOA4) and lysosomal ferritin degradation, a process called ferritinophagy, were also regulated by pUL38 and USP24 during HCMV infection. Knockdown of USP24 decreased NCOA4 protein stability and ferritin heavy chain degradation in lysosomes. Blockage of ferritinophagy by genetic inhibition of NCOA4 or Atg5/Atg7 prevented pUL38-deficient HCMV infection-induced cell death. Overall, these results support the hypothesis that pUL38 binds to USP24 to reduce ferritinophagy, which may then protect cells from lysosome dysfunction-induced cell death. IMPORTANCE Premature cell death is considered a first line of defense against various pathogens. Human cytomegalovirus (HCMV) is a slow-replicating virus that encodes several cell death inhibitors, such as pUL36 and pUL37x1, which allow it to overcome both extrinsic and intrinsic mitochondrion-mediated apoptosis. We previously identified HCMV protein pUL38 as another virus-encoded cell death inhibitor. In this study, we demonstrated that pUL38 achieved its activity by interacting with and antagonizing the function of the host protein ubiquitin-specific protease 24 (USP24). pUL38 blocked USP24-mediated ferritin degradation in lysosomes, which could otherwise be detrimental to the lysosome and initiate cell death. These novel findings suggest that iron metabolism is finely tuned during HCMV infection to avoid cellular toxicity. The results also provide a solid basis for further investigations of the role of USP24 in regulating iron metabolism during infection and other diseases. Copyright © 2018 American Society for Microbiology.

USP21 regulates Hippo pathway activity by mediating MARK protein turnover.

PubMed

Nguyen, Hung Thanh; Kugler, Jan-Michael; Loya, Anand C; Cohen, Stephen M

2017-09-08

The Hippo pathway, which acts to repress the activity of YAP and TAZ trancriptional co-activators, serve as a barrier for oncogenic transformation. Unlike other oncoproteins, YAP and TAZ are rarely activated by mutations or amplified in cancer. However, elevated YAP/TAZ activity is frequently observed in cancer and often correlates with worse survival. The activity and stability of Hippo pathway components, including YAP/TAZ, AMOT and LATS1/2, are regulated by ubiquitin-mediated protein degradation. Aberrant expression of ubiquitin ligase complexes that regulate the turnover of Hippo components and deubiquitylating enzymes that counteract these ubiquitin ligases have been implicated in human cancer. Here we identify the USP21 deubiquitylating enzyme as a novel regulator of Hippo pathway activity. We provide evidence that USP21 regulates YAP/TAZ activity by controlling the stability of MARK kinases, which promote Hippo signaling. Low expression of USP21 in early stage renal clear cell carcinoma suggests that USP21 may be a useful biomarker.

Can laboratory and pilot recycling trials predict adhesive removal in commercial recycling systems? : results from the USPS environmentally benign stamp project

Treesearch

Carl Houtman; Daniel Seiter; Nancy Ross Sutherland; Donald Donermeyer

2002-01-01

The ultimate goal of the US Postal Service (USPS) Environmentally Benign Stamp Program is to develop stamp laminates, i.e., face paper, adhesive and siliconized liner, that do not cause difficulties in recycling mills. The criterion for success, and the USPS definition of benignity, is the avoidance of process and product quality hardships when such PSA laminates are...

A Content Analysis of Unique Selling Propositions of Tobacco Print Ads

PubMed Central

Shen, Megan Johnson; Banerjee, Smita C.; Greene, Kathryn; Carpenter, Amanda; Ostroff, Jamie S.

2017-01-01

Objectives The present study described the unique selling propositions (USPs) (propositions used to convince customers to use a particular brand/product by focusing on the unique benefit) of print tobacco ads. Methods A qualitative content analysis was conducted of print tobacco ads (N = 171) selected from August 2012-August 2013 for cigarettes, moist snuff, e-cigarettes, cigars, and snus to determine the content and themes of USPs for tobacco ads. Results Cigarette ad USP themes focused on portraying the product as attractive; moist snuff ads focused on portraying product as masculine; cigar ads focused on selling a “high end product;” and new and emerging tobacco products (e-cigarette, snus) focused on directly comparing these products to cigarettes. Conclusions Whereas traditional tobacco product ads used USPs focused on themes of enjoyment and pleasure (eg, attractive for cigarettes, “high end product” for cigars), new and emerging tobacco product ads offered the unique benefit (USP) of their product being a better and “safer” alternative to traditional tobacco products. Snuff’s USPs focused nearly exclusively on the masculinity of their products. Results of this study provide targets for potential tobacco regulatory actions that could be implemented to reduce demand for tobacco products by reducing their perceived unique benefits. PMID:28452697

USP4 inhibits SMAD4 monoubiquitination and promotes activin and BMP signaling.

PubMed

Zhou, Fangfang; Xie, Feng; Jin, Ke; Zhang, Zhengkui; Clerici, Marcello; Gao, Rui; van Dinther, Maarten; Sixma, Titia K; Huang, Huizhe; Zhang, Long; Ten Dijke, Peter

2017-06-01

SMAD4 is a common intracellular effector for TGF-β family cytokines, but the mechanism by which its activity is dynamically regulated is unclear. We demonstrated that ubiquitin-specific protease (USP) 4 strongly induces activin/BMP signaling by removing the inhibitory monoubiquitination from SMAD4. This modification was triggered by the recruitment of the E3 ligase, SMURF2, to SMAD4 following ligand-induced regulatory (R)-SMAD-SMAD4 complex formation. Whereas the interaction of the negative regulator c-SKI inhibits SMAD4 monoubiquitination, the ligand stimulates the recruitment of SMURF2 to the c-SKI-SMAD2 complex and triggers c-SKI ubiquitination and degradation. Thus, SMURF2 has a role in termination and initiation of TGF-β family signaling. An increase in monoubiquitinated SMAD4 in USP4-depleted mouse embryonic stem cells (mESCs) decreased both the BMP- and activin-induced changes in the embryonic stem cell fate. USP4 sustained SMAD4 activity during activin- and BMP-mediated morphogenic events in early zebrafish embryos. Moreover, zebrafish depleted of USP4 exhibited defective cell migration and slower coordinated cell movement known as epiboly, both of which could be rescued by SMAD4. Therefore, USP4 is a critical determinant of SMAD4 activity. © 2017 The Authors.

Trauma team utilization of universal precautions: if you see something, say something.

PubMed

Peponis, T; Cropano, M C; Larentzakis, A; van der Wilden, M G; Mejaddam, Y A; Sideris, C A; Michailidou, M; Fikry, K; Bramos, A; Janjua, S; Chang, Y; King, D R

2017-02-01

The risks deriving from the lack of compliance with universal safety precautions (USPs) are unequivocal. However, the adoption of these prophylactic precautions by healthcare providers remains unacceptably low. We hypothesized that trauma teams are not routinely adhering to USPs and that a brief educational intervention, followed by real-time peer feedback, would substantially improve compliance rates. This before-and-after interventional study took place in the resuscitation bay of a Level I Trauma Center during trauma team activations. Six USPs were examined: hand washing (before and after patient contact), use of gloves, gowns, eye protection, and masks. Surgery and Emergency Medicine attending physicians, residents, and nurses, who had direct patient contact, were included. Following 162 baseline observations, an educational intervention in the form of brief lectures was conducted, emphasizing the danger to self from dereliction of USPs. Subsequently, 167 post-intervention observations were made after a one-month period of knowledge decay. Finally, real-time feedback was provided by trauma team leaders and study staff. Adherence to prophylactic measures was recorded again. Baseline compliance rates were dismal. Only hand washing prior to patient interaction, the use of eye protection, and the use of masks improved significantly (p < 0.05) after the educational initiative. However, compliance rates remained suboptimal. No difference was noted regarding the three other USPs. Impressively, following real-time behavioral corrections, compliance improved to nearly 90 % for all USPs (p < 0.05). Compliance with OSHA-required USPs during trauma team activations is unacceptably low, but can be dramatically improved through simple educational interventions, combined with real-time peer feedback.

Scattering of ultrashort electromagnetic pulses on metal clusters

DOE Office of Scientific and Technical Information (OSTI.GOV)

Astapenko, V. A., E-mail: astval@mail.ru; Sakhno, S. V.

We have calculated and analyzed the probability of ultrashort electromagnetic pulse (USP) scattering on small metal clusters in the frequency range of plasmon resonances during the field action. The main attention is devoted to dependence of the probability of scattering on the pulse duration for various detunings of the USP carrier frequency from the plasmon resonance frequency. Peculiarities of the USP scattering from plasmon resonances with various figures of merit are revealed.

Scattering of ultrashort electromagnetic pulses on metal clusters

NASA Astrophysics Data System (ADS)

Astapenko, V. A.; Sakhno, S. V.

2016-12-01

We have calculated and analyzed the probability of ultrashort electromagnetic pulse (USP) scattering on small metal clusters in the frequency range of plasmon resonances during the field action. The main attention is devoted to dependence of the probability of scattering on the pulse duration for various detunings of the USP carrier frequency from the plasmon resonance frequency. Peculiarities of the USP scattering from plasmon resonances with various figures of merit are revealed.

Genomic characterization of pediatric T-cell acute lymphoblastic leukemia reveals novel recurrent driver mutations

PubMed Central

Spinella, Jean-François; Cassart, Pauline; Richer, Chantal; Saillour, Virginie; Ouimet, Manon; Langlois, Sylvie; St-Onge, Pascal; Sontag, Thomas; Healy, Jasmine; Minden, Mark D.; Sinnett, Daniel

2016-01-01

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy with variable prognosis. It represents 15% of diagnosed pediatric ALL cases and has a threefold higher incidence among males. Many recurrent alterations have been identified and help define molecular subgroups of T-ALL, however the full range of events involved in driving transformation remain to be defined. Using an integrative approach combining genomic and transcriptomic data, we molecularly characterized 30 pediatric T-ALLs and identified common recurrent T-ALL targets such as FBXW7, JAK1, JAK3, PHF6, KDM6A and NOTCH1 as well as novel candidate T-ALL driver mutations including the p.R35L missense mutation in splicesome factor U2AF1 found in 3 patients and loss of function mutations in the X-linked tumor suppressor genes MED12 (frameshit mutation p.V167fs, splice site mutation g.chrX:70339329T>C, missense mutation p.R1989H) and USP9X (nonsense mutation p.Q117*). In vitro functional studies further supported the putative role of these novel T-ALL genes in driving transformation. U2AF1 p.R35L was shown to induce aberrant splicing of downstream target genes, and shRNA knockdown of MED12 and USP9X was shown to confer resistance to apoptosis following T-ALL relevant chemotherapy drug treatment in Jurkat leukemia cells. Interestingly, nearly 60% of novel candidate driver events were identified among immature T-ALL cases, highlighting the underlying genomic complexity of pediatric T-ALL, and the need for larger integrative studies to decipher the mechanisms that contribute to its various subtypes and provide opportunities to refine patient stratification and treatment. PMID:27602765

39 CFR 266.9 - Exemptions.

Code of Federal Regulations, 2010 CFR

2010-07-01

... and evaluative purposes, it is impossible to determine in advance what information might be of... Records, USPS 100.300; Personnel Research Records, 100.600; and Emergency Management Records, USPS 500.300...

39 CFR 266.9 - Exemptions.

Code of Federal Regulations, 2013 CFR

2013-07-01

... and evaluative purposes, it is impossible to determine in advance what information might be of... Records, USPS 100.300; Personnel Research Records, 100.600; and Emergency Management Records, USPS 500.300...

39 CFR 266.9 - Exemptions.

Code of Federal Regulations, 2014 CFR

2014-07-01

... and evaluative purposes, it is impossible to determine in advance what information might be of... Records, USPS 100.300; Personnel Research Records, 100.600; and Emergency Management Records, USPS 500.300...

39 CFR 266.9 - Exemptions.

Code of Federal Regulations, 2011 CFR

2011-07-01

... and evaluative purposes, it is impossible to determine in advance what information might be of... Records, USPS 100.300; Personnel Research Records, 100.600; and Emergency Management Records, USPS 500.300...

39 CFR 266.9 - Exemptions.

Code of Federal Regulations, 2012 CFR

2012-07-01

... and evaluative purposes, it is impossible to determine in advance what information might be of... Records, USPS 100.300; Personnel Research Records, 100.600; and Emergency Management Records, USPS 500.300...

Development of the antimicrobial effectiveness test as USP chapter <51>.

PubMed

Sutton, Scott V W; Porter, David

2002-01-01

The antimicrobial effectiveness test first appeared as a USP General Chapter in the 18th revision, official September 1, 1970. This chapter, at the beginning, was designed to evaluate the performance of antimicrobials added to inhibit the growth of microorganisms that might be introduced during or subsequent to the manufacturing process. As Good Manufacturing Practices (GMPs) became a governing principal in pharmaceutical manufacturing, the purpose of the test was refined to focus on activity of the preservative system as a protection against inadvertent contamination during storage and usage of the product. This article will review the history of the antimicrobial test; its function, technique, and the background discussions that resulted in the changes from the test that appeared in USP XVIII to that of the current USP 25.

Developing a Terrestrial Biogeochemical Cycle Modeling System to Support the Management of Fort Benning and its Surrounding Areas

DTIC Science & Technology

2010-12-01

Soil Survey Geographic database USDA U.S. Department of Agriculture USLE Universal Soil Loss Equation USPED Unit-Stream-Power Erosion and...2003). A suite of models has been developed to simulate soil erosion and deposition, ranging from empirical (e.g., USLE and MUSLE at http... Soil Erosion and Deposition 4.4.1 USPED The algorithm for the simulation of soil erosion in USPED is similar to that of the USLE or RUSLE model

Particulate-matter content of 11 cephalosporin injections: conformance with USP limits.

PubMed

Parkins, D A; Taylor, A J

1987-05-01

The particulate-matter content of 11 dry-powder cephalosporin injections was determined using a modified version of the official United States Pharmacopeial Convention (USP) method for particulate matter in small-volume injections (SVIs). Ten vials of each cephalosporin product were each constituted with 10 mL of Water for Injections BP that had been filtered through a 0.22-micron membrane. The pooled contents of the 10 vials for each product were allowed to stand under reduced pressure to ensure removal of gas bubbles. Particulate-matter content was determined using a HIAC/Royco particle counter on six 10-mL samples obtained from the pooled solutions for each product. All solution preparation and particle counting was performed in a horizontal-laminar-airflow hood. Modifications of the USP method used in this study included the use of six rather than two samples from each pooled solution, the addition of diluent to the injections through the rubber closure with a needle instead of into the open container, and changes in the degassing method. Particle counts for all products examined were lower than USP limits for SVIs. All but two products contained less than 15% of USP limits for particles greater than or equal to 10 microns in effective diameter and particles greater than or equal to 25 microns in effective diameter. The standard USP method for degassing (standing for two minutes) was inadequate. Application of reduced pressure for up to 10 minutes was necessary for thorough degassing of products.(ABSTRACT TRUNCATED AT 250 WORDS)

Born dry in the photoevaporation desert: Kepler's ultra-short-period planets formed water-poor

NASA Astrophysics Data System (ADS)

Lopez, Eric D.

2017-11-01

Recent surveys have uncovered an exciting new population of ultra-short-period (USP) planets with orbital periods less than a day. These planets typically have radii ≲1.5 R⊕, indicating that they likely have rocky compositions. This stands in contrast to the overall distribution of planets out to ∼100 d, which is dominated by low-density sub-Neptunes above 2 R⊕, which must have gaseous envelopes to explain their size. However, on the USP orbits, planets are bombarded by intense levels of photoionizing radiation and consequently gaseous sub-Neptunes are extremely vulnerable to losing their envelopes to atmospheric photoevaporation. Using models of planet evolution, I show that the rocky USP planets can easily be produced as the evaporated remnants of sub-Neptunes with H/He envelopes and that we can therefore understand the observed dearth of USP sub-Neptunes as a natural consequence of photoevaporation. Critically however, planets on USP orbits could often retain their envelopes if they are formed with very high-metallicity water-dominated envelopes. Such water-rich planets would commonly be ≳2 R⊕ today, which is inconsistent with the observed evaporation desert, indicating that most USP planets likely formed from water-poor material within the snow-line. Finally, I examine the special case of 55 Cancri e and its possible composition in the light of recent observations, and discuss the prospects for further characterizing this population with future observations.

COMMITTEES: SQM2009 - 14th International Conference on Strangeness in Quark Matter SQM2009 - 14th International Conference on Strangeness in Quark Matter

NASA Astrophysics Data System (ADS)

2008-04-01

Local Organizing Committee Takeshi Kodama Chair, UFRJ Jun Takahashi Co-chair, UNICAMP Ignácio Bediaga e Hickman CBPF Eduardo Fraga UFRJ Frederique Grassi USP Yogiro Hama USP Gastão Krein IFT Erasmo Madureira Ferreira UFRJ Marcelo G. Munhoz USP Fernando Navarra USP Sandra Padula IFT Alejandro Szanto de Toledo USP César Augusto Zen Vasconcellos UFRGS International Advisory Committee Jörg Aichelin Nantes Federico Antinori Padova Tamás Biró Budapest Peter Braun-Munzinger GSI Jean Cleymans Cape Town Láaszló Csernai Bergen Timothy Hallman BNL Huan Zhong Huang UCLA Takeshi Kodama Rio de Janeiro Yu-Gang Ma Shanghai Jes Madsen Aarhus Ágnes Mócsy Pratt University Berndt Müller Duke University Grazyna Odyniec LBNL Helmut Oeschler Darmstadt Johann Rafelski Arizona Hans Georg Ritter LBNL Gunther Rolland MIT Karel Šafařík CERN Ladislav Sandor Kosice University Jack Sandweiss Yale University George S F Stephans MIT Horst Stöcker Frankfurt Larry McLerranBNL Helmut Satz Universitä Bielefeld Nu Xu LBNL Fuqiang Wang Purdue University William A. Zajc Columbia University Pengfei Zhuang Tsinghua University

Aberrant phosphorylation of SMAD4 Thr277-mediated USP9x-SMAD4 interaction by free fatty acids promotes breast cancer metastasis

PubMed Central

Wu, Yong; Yu, Xiaoting; Yi, Xianghua; Wu, Ke; Dwabe, Sami; Atefi, Mohammad; Elshimali, Yahya; Kemp, Kevin T.; Bhat, Kruttika; Haro, Jesse; Sarkissyan, Marianna; Vadgama, Jaydutt V

2017-01-01

Obesity increases the risk of distant metastatic recurrence and reduces breast cancer (BC) survival. However, the mechanisms behind this pathology and identification of relevant therapeutic targets are poorly defined. Plasma free fatty acids (FFA) levels are elevated in obese individuals. Here we report that TGF-β transiently activates ERK and subsequently phosphorylates SMAD4 at Thr277, which facilitates a SMAD4-USP9x interaction, SMAD4 nuclear retention, and stimulates TGF-β /SMAD3-mediated transcription of Twist and Snail. USP9x inhibited the E3 ubiquitin-protein ligase TIF1γ from binding and monoubiquitinating SMAD4, hence maintaining SMAD4 nuclear retention. FFA further facilitated TGF-β-induced ERK activation, SMAD4 phosphorylation and nuclear retention, promoting TGF-β-dependent cancer progression. Inhibition of ERK and USP9x suppressed obesity-induced metastasis. Additionally, clinical data indicated that phospho-ERK and -SMAD4 levels correlate with activated TGF-β signaling and metastasis in overweight/obese patient BC specimens. Altogether, we demonstrate the vital interaction of USP9x and SMAD4 for governing TGF-β signaling and dyslipidemia-induced, aberrant TGF-β activation during BC metastasis. PMID:28115363

A high-confidence interaction map identifies SIRT1 as a mediator of acetylation of USP22 and the SAGA coactivator complex.

PubMed

Armour, Sean M; Bennett, Eric J; Braun, Craig R; Zhang, Xiao-Yong; McMahon, Steven B; Gygi, Steven P; Harper, J Wade; Sinclair, David A

2013-04-01

Although many functions and targets have been attributed to the histone and protein deacetylase SIRT1, a comprehensive analysis of SIRT1 binding proteins yielding a high-confidence interaction map has not been established. Using a comparative statistical analysis of binding partners, we have assembled a high-confidence SIRT1 interactome. Employing this method, we identified the deubiquitinating enzyme ubiquitin-specific protease 22 (USP22), a component of the deubiquitinating module (DUBm) of the SAGA transcriptional coactivating complex, as a SIRT1-interacting partner. We found that this interaction is highly specific, requires the ZnF-UBP domain of USP22, and is disrupted by the inactivating H363Y mutation within SIRT1. Moreover, we show that USP22 is acetylated on multiple lysine residues and that alteration of a single lysine (K129) within the ZnF-UBP domain is sufficient to alter interaction of the DUBm with the core SAGA complex. Furthermore, USP22-mediated recruitment of SIRT1 activity promotes the deacetylation of individual SAGA complex components. Our results indicate an important role of SIRT1-mediated deacetylation in regulating the formation of DUBm subcomplexes within the larger SAGA complex.

United States Pharmacopeial Convention

MedlinePlus

... Online USP Compounding USP Donor Portal Chromatographic Columns Social Media Linked In Twitter Facebook You Tube QualityMatters Blog ... Events & Training Get Involved Partner Volunteer Provide Input Social Media Linked In Twitter Facebook You Tube QualityMatters Blog ...

27 CFR 21.51 - Formula No. 25-A.

Code of Federal Regulations, 2013 CFR

2013-04-01

... iodide, U.S.P.; and 15 pounds of water. (b) Authorized uses. (1) As a solvent: 230.Tinctures of iodine. 249.Miscellaneous external pharmaceuticals, U.S.P. or N.F. (2) Miscellaneous uses: 812.Product...

27 CFR 21.51 - Formula No. 25-A.

Code of Federal Regulations, 2012 CFR

2012-04-01

... iodide, U.S.P.; and 15 pounds of water. (b) Authorized uses. (1) As a solvent: 230.Tinctures of iodine. 249.Miscellaneous external pharmaceuticals, U.S.P. or N.F. (2) Miscellaneous uses: 812.Product...

27 CFR 21.51 - Formula No. 25-A.

Code of Federal Regulations, 2014 CFR

2014-04-01

... iodide, U.S.P.; and 15 pounds of water. (b) Authorized uses. (1) As a solvent: 230.Tinctures of iodine. 249.Miscellaneous external pharmaceuticals, U.S.P. or N.F. (2) Miscellaneous uses: 812.Product...

27 CFR 21.51 - Formula No. 25-A.

Code of Federal Regulations, 2011 CFR

2011-04-01

... iodide, U.S.P.; and 15 pounds of water. (b) Authorized uses. (1) As a solvent: 230.Tinctures of iodine. 249.Miscellaneous external pharmaceuticals, U.S.P. or N.F. (2) Miscellaneous uses: 812.Product...

Developing a Spatially Distributed Terrestrial Biogeochemical Cycle Modeling System to Support the Management of Fort Benning and its Surrounding Areas

DTIC Science & Technology

2010-12-01

nitrogen SSURGO Soil Survey Geographic database USDA U.S. Department of Agriculture USLE Universal Soil Loss Equation USPED Unit-Stream-Power...Zaluski et al., 2003). A suite of models has been developed to simulate soil erosion and deposition, ranging from empirical (e.g., USLE and MUSLE at http...Estimating Soil Erosion and Deposition 4.4.1 USPED The algorithm for the simulation of soil erosion in USPED is similar to that of the USLE or RUSLE

Department of the Army Supply Bulletin, Army Medical Department Supply Information, SB8-75-11

DTIC Science & Technology

2001-11-20

SULFATE INJECTION USP 10MG 1ML CARTRIDGE-NEEDLE UNIT10S MFR: SANOFI LOT/SER NO: C790PD NSN: 6505-00-812-2596 NOM: MORPHINE SULFATE INJECTION...100 TABLETS PER BOTTLE MFR: SANOFI LOT/SER NO: SP344 NSN: 6505-01-178-7903 NOM: PYRIDOSTIGMINE BROMIDE TABLETS USP 30MG I.S. 210 TABS...ML 2ML SYRINGE WITH NEEDLE 10/PACKAGE MFR: SANOFI LOT/SER NO: C740RL NSN: 6505-01-274-0951 NOM: DIAZEPAM INJECTION USP 5MG/ML 2ML SYRINGE

27 CFR 21.151 - List of denaturants authorized for denatured spirits.

Code of Federal Regulations, 2010 CFR

2010-04-01

... Denatured Rum (S.D.R.) Acetaldehyde S.D.A. 29. Acetone, U.S.P S.D.A. 23-A, 23-H. Acetaldol C.D.A. 18. Almond... alcohol S.D.A. 39, 39-A, 39-B, 40, 40-A, 40-B, 40-C. Camphor, U.S.P S.D.A. 27, 27-A, 38-B. Caustic soda.... Formaldehyde solution, U.S.P S.D.A. 22, 38-C, 38-D. Gasoline C.D.A. 18, 19; S.D.A. 28-A. Gasoline, unleaded C.D...

27 CFR 21.151 - List of denaturants authorized for denatured spirits.

Code of Federal Regulations, 2011 CFR

2011-04-01

... Denatured Rum (S.D.R.) Acetaldehyde S.D.A. 29. Acetone, U.S.P S.D.A. 23-A, 23-H. Acetaldol C.D.A. 18. Almond... alcohol S.D.A. 39, 39-A, 39-B, 40, 40-A, 40-B, 40-C. Camphor, U.S.P S.D.A. 27, 27-A, 38-B. Caustic soda.... Formaldehyde solution, U.S.P S.D.A. 22, 38-C, 38-D. Gasoline C.D.A. 18, 19; S.D.A. 28-A. Gasoline, unleaded C.D...

Molecular adaptation and resilience of the insect’s nuclear receptor USP

PubMed Central

2012-01-01

Background The maintenance of biological systems requires plasticity and robustness. The function of the ecdysone receptor, a heterodimer composed of the nuclear receptors ECR (NR1H1) and USP (NR2B4), was maintained in insects despite a dramatic divergence that occurred during the emergence of Mecopterida. This receptor is therefore a good model to study the evolution of plasticity. We tested the hypothesis that selection has shaped the Ligand-Binding Domain (LBD) of USP during evolution of Mecopterida. Results We isolated usp and cox1 in several species of Drosophilidae, Tenebrionidae and Blattaria and estimated non-synonymous/synonymous rate ratios using maximum-likelihood methods and codon-based substitution models. Although the usp sequences were mainly under negative selection, we detected relaxation at residues located on the surface of the LBD within Mecopterida families. Using branch-site models, we also detected changes in selective constraints along three successive branches of the Mecopterida evolution. Residues located at the bottom of the ligand-binding pocket (LBP) underwent strong positive selection during the emergence of Mecopterida. This change is correlated with the acquisition of a large LBP filled by phospholipids that probably allowed the stabilisation of the new Mecopterida structure. Later, when the two subgroups of Mecopterida (Amphiesmenoptera: Lepidoptera, Trichoptera; Antliophora: Diptera, Mecoptera, Siphonaptera) diverged, the same positions became under purifying selection. Similarly, several positions of the heterodimerisation interface experienced positive selection during the emergence of Mecopterida, rapidly followed by a phase of constrained evolution. An enlargement of the heterodimerisation surface is specific for Mecopterida and was associated with a reinforcement of the obligatory partnership between ECR and USP, at the expense of homodimerisation. Conclusions In order to explain the episodic mode of evolution of USP, we propose a model in which the molecular adaptation of this protein is seen as a process of resilience for the maintenance of the ecdysone receptor functionality. PMID:23039844

Unstable Roche-Lobe Overflow of Gaseous Planets

NASA Astrophysics Data System (ADS)

Jackson, Brian

The discoveries of more than 100 roughly Earth-sized bodies with orbital periods less than 1 day, ultra-short-period planets or candidates (USPs), have challenged planet formation theories, and evidence suggests USPs may be the remnants of gaseous planets that shed their atmospheres. Indeed, many hot Jupiters are near Roche-Lobe overflow (RLO), and tidal decay can push them the rest of the way in. Recent work has shown stable RLO (atmospheres lost via a steady outflow and thin accretion disk) probably cannot produce USPs on its own but suggested unstable RLO (atmospheres quickly shed on dynamical timescales) may. In fact, stable RLO may drive overflowing hot Jupiters into unstable RLO, and by analogy with the common-envelope binaries, the core that remains can drive off the gaseous envelope at the cost of its orbital energy. Wellestablished mass-radius relations for gaseous planets, coupled to simple energy and angular momentum considerations, provide a connection between the observed masses and periods for USPs and their putative progenitor gaseous planets, with few free parameters. We propose to investigate the hypothesis that USPs originate through tidal decay and a combination of stable and unstable Roche-lobe overflow of short-period gaseous planets through the following studies: -We will explore the planetary masses, orbital periods, etc. that produce unstable RLO using the Modules for Experiments in Stellar Astrophysics (MESA) suite. -We will relate the observed periods and masses of USPs to their putative progenitor masses and periods to see whether they are consistent with the unstable RLO hypothesis. This proposal is directly relevant to the Exoplanets Research Program since it seeks to "understand the ... physical processes of exoplanets" and "improve understanding of [their] origins" through "theoretical studies ... and modeling'". We also expect that it will have broad impacts on a variety of astrophysical topics: -Ultra-short period planets could provide a glimpse inside gas giants. If our study corroborates the unstable RLO hypothesis, the properties of USPs may represent those of gaseous planet cores, which are almost entirely unconstrained. Even in our own solar system, estimates of the core masses for Jupiter (between 0 and 14 Earth masses) and Saturn (between 8 and 25 Earth masses) have large uncertainties. The upcoming Juno mission will clarify the situation for Jupiter, but it's unclear whether these constraints will directly bear on other planetary systems. -The TESS mission will probably find hundreds of ultra-short-period planets. The Transiting Exoplanet Survey Satellite (TESS) will launch in 2017 and look for shortperiod rocky planets around 500,000 nearby stars. Roughly 0.1% of Sun-like stars host USPs, so TESS should find hundreds of USPs. These planets would be ideal for followup, and a clear framework for their origins would motivate and guide additional TESS observations. -Even if we show they are not remnants of gaseous planets, ultra-short-period planets may serve as probes of the innermost protoplanetary gas disk and betray the presence of additional planets in their host systems. As we discuss below, one other obvious origin scenario involves dynamical interactions in a multi-planet system. Many USPs do not have observed siblings, so this scenario requires that there are additional, unseen planets in those systems. The exact location of USPs may be set, in part, by the inner edge of their maternal gas disk, a sensitive function of the host star's youthful properties.

Retigabine ameliorates acute stress-induced impairment of spatial memory retrieval through regulating USP2 signaling pathways in hippocampal CA1 area.

PubMed

Li, Cai; Zhang, Ji; Xu, Haiwei; Chang, Mujun; Lv, Chuntao; Xue, Wenhua; Song, Zhizhen; Zhang, Lizhen; Zhang, Xiaojian; Tian, Xin

2018-06-01

Acute stress could trigger maladaptive changes associated with stress-related cognitive and emotional deficits. Dysfunction of ion channel or receptor in the hippocampal area has been linked to the cognitive deficits induced by stress. It is known that Kv7 channel openers, including FDA-approved drug retigabine, show cognitive protective efficacy. However, the underlying molecular mechanisms remain elusive. Here we showed that exposing adult male rats to acute stress significantly impaired the spatial memory, a cognitive process controlled by the hippocampus. Concomitantly, significantly reduced AMPA receptor expression was found in hippocampal CA1 area from acute stressed rats. This effect relied on the down-regulation of deubiquitinating enzyme USP2 and its upstream regulators (PGC-1α and β-catenin), and the subsequent enhancement of mTOR-related autophagy which is regulated by USP2. These findings suggested that acute stress dampened AMPA receptor expression by controlling USP2-related signaling, which caused the detrimental effect on hippocampus-dependent cognitive processes. We also found that retigabine alleviated acute stress-induced spatial memory retrieval impairment through adjusting the aberrance of USP2, its upstream regulators (PGC-1α, E4BP4 and β-catenin) and its downstream targets (mTOR, autophagy and GluA1). Our results have identified USP2 as a key molecule that mediates stress-induced spatial memory retrieval impairment, which provides a framework for new druggable targets to conceptually treat stress-associated cognitive deficits. Copyright © 2018 Elsevier Ltd. All rights reserved.

Engineering Signal Peptides for Enhanced Protein Secretion from Lactococcus lactis

PubMed Central

Ng, Daphne T. W.

2013-01-01

Lactococcus lactis is an attractive vehicle for biotechnological production of proteins and clinical delivery of therapeutics. In many such applications using this host, it is desirable to maximize secretion of recombinant proteins into the extracellular space, which is typically achieved by using the native signal peptide from a major secreted lactococcal protein, Usp45. In order to further increase protein secretion from L. lactis, inherent limitations of the Usp45 signal peptide (Usp45sp) must be elucidated. Here, we performed extensive mutagenesis on Usp45sp to probe the effects of both the mRNA sequence (silent mutations) and the peptide sequence (amino acid substitutions) on secretion. We screened signal peptides based on their resulting secretion levels of Staphylococcus aureus nuclease and further evaluated them for secretion of Bacillus subtilis α-amylase. Silent mutations alone gave an increase of up to 16% in the secretion of α-amylase through a mechanism consistent with relaxed mRNA folding around the ribosome binding site and enhanced translation. Targeted amino acid mutagenesis in Usp45sp, combined with additional silent mutations from the best clone in the initial screen, yielded an increase of up to 51% in maximum secretion of α-amylase while maintaining secretion at lower induction levels. The best sequence from our screen preserves the tripartite structure of the native signal peptide but increases the positive charge of the n-region. Our study presents the first example of an engineered L. lactis signal peptide with a higher secretion yield than Usp45sp and, more generally, provides strategies for further enhancing protein secretion in bacterial hosts. PMID:23124224

Engineering signal peptides for enhanced protein secretion from Lactococcus lactis.

PubMed

Ng, Daphne T W; Sarkar, Casim A

2013-01-01

Lactococcus lactis is an attractive vehicle for biotechnological production of proteins and clinical delivery of therapeutics. In many such applications using this host, it is desirable to maximize secretion of recombinant proteins into the extracellular space, which is typically achieved by using the native signal peptide from a major secreted lactococcal protein, Usp45. In order to further increase protein secretion from L. lactis, inherent limitations of the Usp45 signal peptide (Usp45sp) must be elucidated. Here, we performed extensive mutagenesis on Usp45sp to probe the effects of both the mRNA sequence (silent mutations) and the peptide sequence (amino acid substitutions) on secretion. We screened signal peptides based on their resulting secretion levels of Staphylococcus aureus nuclease and further evaluated them for secretion of Bacillus subtilis α-amylase. Silent mutations alone gave an increase of up to 16% in the secretion of α-amylase through a mechanism consistent with relaxed mRNA folding around the ribosome binding site and enhanced translation. Targeted amino acid mutagenesis in Usp45sp, combined with additional silent mutations from the best clone in the initial screen, yielded an increase of up to 51% in maximum secretion of α-amylase while maintaining secretion at lower induction levels. The best sequence from our screen preserves the tripartite structure of the native signal peptide but increases the positive charge of the n-region. Our study presents the first example of an engineered L. lactis signal peptide with a higher secretion yield than Usp45sp and, more generally, provides strategies for further enhancing protein secretion in bacterial hosts.

Legibility of USP pictograms by clients of community pharmacies in Portugal.

PubMed

Soares, Maria Augusta

2013-02-01

Effective and safe use of medicines depends on patients' good understanding of the pharmacotherapy. Pictograms are a communication tool, as complement of verbal/written information, to facilitate drug use and, avoiding errors, being useful for drug effectiveness and safety. To assess the ability of pharmacy's clients, in understanding USP pictograms, establishing the relationship with their socio demographic profiles. The study was developed in community pharmacies, in Lisbon region. Cross sectional study, carried out in pharmacies in Lisbon. A structured questionnaire was used in pharmacies clients of 18 years old and above. Legibility of fifteen USP pictograms was studied using ISO and ANSI criteria. Correlation between the legibility of pictograms and pharmacies clients' demographic profile (age, gender, scholarship, frequency of medicines use) was evaluated. SPSS data base version 18 was used for descriptive analysis. Legibility of fifteen United States Pharmacopeia (USP) pictograms and, its relationship with clients' demographic data (scholarship degree, age, frequency of medicines use and, gender) was studied. From 751 responders, ten pictograms were legible by ISO and seven by ANSI. More than 30 % of the responders weren't able to understand five of the pictograms. It was found statistically significant relationships between some the understanding of some pictograms and clients' scholarships degree, age and frequency of medicines use. It was found that not all the fifteen tested USP pictograms were correctly comprehended by Portuguese pharmacies' clients, having found correlations with scholarship degree, daily use of medicines and age group, for some pictograms. To ensure the effectiveness of USP pictograms it is advisable to test patients' comprehension, before their use in general practice.

27 CFR 21.78 - Formula No. 42.

Code of Federal Regulations, 2012 CFR

2012-04-01

...) Eighty grams of potassium iodide, U.S.P., and 109 grams of red mercuric iodide, N.F. XI; or (2) Ninety-five grams of thimerosal, U.S.P.; or (3) Seventy-six grams of any of the following: phenyl mercuric...

27 CFR 21.78 - Formula No. 42.

Code of Federal Regulations, 2014 CFR

2014-04-01

...) Eighty grams of potassium iodide, U.S.P., and 109 grams of red mercuric iodide, N.F. XI; or (2) Ninety-five grams of thimerosal, U.S.P.; or (3) Seventy-six grams of any of the following: phenyl mercuric...

27 CFR 21.78 - Formula No. 42.

Code of Federal Regulations, 2013 CFR

2013-04-01

...) Eighty grams of potassium iodide, U.S.P., and 109 grams of red mercuric iodide, N.F. XI; or (2) Ninety-five grams of thimerosal, U.S.P.; or (3) Seventy-six grams of any of the following: phenyl mercuric...

27 CFR 21.78 - Formula No. 42.

Code of Federal Regulations, 2010 CFR

2010-04-01

...) Eighty grams of potassium iodide, U.S.P., and 109 grams of red mercuric iodide, N.F. XI; or (2) Ninety-five grams of thimerosal, U.S.P.; or (3) Seventy-six grams of any of the following: phenyl mercuric...

27 CFR 21.78 - Formula No. 42.

Code of Federal Regulations, 2011 CFR

2011-04-01

...) Eighty grams of potassium iodide, U.S.P., and 109 grams of red mercuric iodide, N.F. XI; or (2) Ninety-five grams of thimerosal, U.S.P.; or (3) Seventy-six grams of any of the following: phenyl mercuric...

39 CFR 20.4 - Amendments to the International Mail Manual.

Code of Federal Regulations, 2010 CFR

2010-07-01

... incorporated by reference into this part and will be available at http://pe.usps.gov. The text of amendments to... Postal Bulletin, copies of which may be accessed at http://www.usps.com/cpim/ftp/bulletin/pb.htm. ...

39 CFR 20.4 - Amendments to the International Mail Manual.

Code of Federal Regulations, 2012 CFR

2012-07-01

... incorporated by reference into this part and will be available at http://pe.usps.gov. The text of amendments to... Postal Bulletin, copies of which may be accessed at http://www.usps.com/cpim/ftp/bulletin/pb.htm. ...

39 CFR 20.4 - Amendments to the International Mail Manual.

Code of Federal Regulations, 2011 CFR

2011-07-01

... incorporated by reference into this part and will be available at http://pe.usps.gov. The text of amendments to... Postal Bulletin, copies of which may be accessed at http://www.usps.com/cpim/ftp/bulletin/pb.htm. ...

39 CFR 20.4 - Amendments to the International Mail Manual.

Code of Federal Regulations, 2013 CFR

2013-07-01

... incorporated by reference into this part and will be available at http://pe.usps.gov. The text of amendments to... Postal Bulletin, copies of which may be accessed at http://www.usps.com/cpim/ftp/bulletin/pb.htm. ...

39 CFR 20.4 - Amendments to the International Mail Manual.

Code of Federal Regulations, 2014 CFR

2014-07-01

... incorporated by reference into this part and will be available at http://pe.usps.gov. The text of amendments to... Postal Bulletin, copies of which may be accessed at http://www.usps.com/cpim/ftp/bulletin/pb.htm. ...

A New Test Unit for Disintegration End-Point Determination of Orodispersible Films.

PubMed

Low, Ariana; Kok, Si Ling; Khong, Yuet Mei; Chan, Sui Yung; Gokhale, Rajeev

2015-11-01

No standard time or pharmacopoeia disintegration test method for orodispersible films (ODFs) exists. The USP disintegration test for tablets and capsules poses significant challenges for end-point determination when used for ODFs. We tested a newly developed disintegration test unit (DTU) against the USP disintegration test. The DTU is an accessory to the USP disintegration apparatus. It holds the ODF in a horizontal position, allowing top-view of the ODF during testing. A Gauge R&R study was conducted to assign relative contributions of the total variability from the operator, sample or the experimental set-up. Precision was compared using commercial ODF products in different media. Agreement between the two measurement methods was analysed. The DTU showed improved repeatability and reproducibility compared to the USP disintegration system with tighter standard deviations regardless of operator or medium. There is good agreement between the two methods, with the USP disintegration test giving generally longer disintegration times possibly due to difficulty in end-point determination. The DTU provided clear end-point determination and is suitable for quality control of ODFs during product developmental stage or manufacturing. This may facilitate the development of a standardized methodology for disintegration time determination of ODFs. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

Stabilization of the methyl-CpG binding protein ZBTB38 by the deubiquitinase USP9X limits the occurrence and toxicity of oxidative stress in human cells.

PubMed

Miotto, Benoit; Marchal, Claire; Adelmant, Guillaume; Guinot, Nadège; Xie, Ping; Marto, Jarrod A; Zhang, Lingqiang; Defossez, Pierre-Antoine

2018-05-18

Reactive oxygen species (ROS) are a byproduct of cell metabolism, and can also arise from environmental sources, such as toxins or radiation. Depending on dose and context, ROS have both beneficial and deleterious roles in mammalian development and disease, therefore it is crucial to understand how these molecules are generated, sensed, and detoxified. The question of how oxidative stress connects to the epigenome, in particular, is important yet incompletely understood. Here we show that an epigenetic regulator, the methyl-CpG-binding protein ZBTB38, limits the basal cellular production of ROS, is induced by ROS, and is required to mount a proper response to oxidative stress. Molecularly, these functions depend on a deubiquitinase, USP9X, which interacts with ZBTB38, deubiquitinates it, and stabilizes it. We find that USP9X is itself stabilized by oxidative stress, and is required together with ZBTB38 to limit the basal generation of ROS, as well as the toxicity of an acute oxidative stress. Our data uncover a new nuclear target of USP9X, show that the USP9X/ZBTB38 axis limits, senses and detoxifies ROS, and provide a molecular link between oxidative stress and the epigenome.

A High-Confidence Interaction Map Identifies SIRT1 as a Mediator of Acetylation of USP22 and the SAGA Coactivator Complex

PubMed Central

Armour, Sean M.; Bennett, Eric J.; Braun, Craig R.; Zhang, Xiao-Yong; McMahon, Steven B.; Gygi, Steven P.; Harper, J. Wade

2013-01-01

Although many functions and targets have been attributed to the histone and protein deacetylase SIRT1, a comprehensive analysis of SIRT1 binding proteins yielding a high-confidence interaction map has not been established. Using a comparative statistical analysis of binding partners, we have assembled a high-confidence SIRT1 interactome. Employing this method, we identified the deubiquitinating enzyme ubiquitin-specific protease 22 (USP22), a component of the deubiquitinating module (DUBm) of the SAGA transcriptional coactivating complex, as a SIRT1-interacting partner. We found that this interaction is highly specific, requires the ZnF-UBP domain of USP22, and is disrupted by the inactivating H363Y mutation within SIRT1. Moreover, we show that USP22 is acetylated on multiple lysine residues and that alteration of a single lysine (K129) within the ZnF-UBP domain is sufficient to alter interaction of the DUBm with the core SAGA complex. Furthermore, USP22-mediated recruitment of SIRT1 activity promotes the deacetylation of individual SAGA complex components. Our results indicate an important role of SIRT1-mediated deacetylation in regulating the formation of DUBm subcomplexes within the larger SAGA complex. PMID:23382074

Nanosuspension delivery of paclitaxel to xenograft mice can alter drug disposition and anti-tumor activity

NASA Astrophysics Data System (ADS)

Chiang, Po-Chang; Gould, Stephen; Nannini, Michelle; Qin, Ann; Deng, Yuzhong; Arrazate, Alfonso; Kam, Kimberly R.; Ran, Yingqing; Wong, Harvey

2014-04-01

Paclitaxel is a common chemotherapeutic agent that is effective against various cancers. The poor aqueous solubility of paclitaxel necessitates a large percentage of Cremophor EL:ethanol (USP) in its commercial formulation which leads to hypersensitivity reactions in patients. We evaluate the use of a crystalline nanosuspension versus the USP formulation to deliver paclitaxel to tumor-bearing xenograft mice. Anti-tumor efficacy was assessed following intravenous administration of three 20 mg/kg doses of paclitaxel. Paclitaxel pharmacokinetics and tissue distribution were evaluated, and differences were observed between the two formulations. Plasma clearance and tissue to plasma ratio of mice that were dosed with the nanosuspension are approximately 33- and 11-fold higher compared to those of mice that were given the USP formulation. Despite a higher tumor to plasma ratio for the nanosuspension treatment group, absolute paclitaxel tumor exposure was higher for the USP group. Accordingly, a higher anti-tumor effect was observed in the xenograft mice that were dosed with the USP formulation (90% versus 42% tumor growth inhibition). This reduction in activity of nanoparticle formulation appeared to result from a slower than anticipated dissolution in vivo. This study illustrates a need for careful consideration of both dose and systemic solubility prior utilizing nanosuspension as a mode of intravenous delivery.

Aberrant Phosphorylation of SMAD4 Thr277-Mediated USP9x-SMAD4 Interaction by Free Fatty Acids Promotes Breast Cancer Metastasis.

PubMed

Wu, Yong; Yu, Xiaoting; Yi, Xianghua; Wu, Ke; Dwabe, Sami; Atefi, Mohammad; Elshimali, Yahya; Kemp, Kevin T; Bhat, Kruttika; Haro, Jesse; Sarkissyan, Marianna; Vadgama, Jaydutt V

2017-03-15

Obesity increases the risk of distant metastatic recurrence and reduces breast cancer survival. However, the mechanisms behind this pathology and identification of relevant therapeutic targets are poorly defined. Plasma free fatty acids (FFA) levels are elevated in obese individuals. Here we report that TGFβ transiently activates ERK and subsequently phosphorylates SMAD4 at Thr277, which facilitates a SMAD4-USP9x interaction, SMAD4 nuclear retention, and stimulates TGFβ/SMAD3-mediated transcription of Twist and Snail. USP9x inhibited the E3 ubiquitin-protein ligase TIF1γ from binding and monoubiquitinating SMAD4, hence maintaining the SMAD4 nuclear retention. FFA further facilitated TGFβ-induced ERK activation, SMAD4 phosphorylation, and nuclear retention, promoting TGFβ-dependent cancer progression. Inhibition of ERK and USP9x suppressed obesity-induced metastasis. In addition, clinical data indicated that phospho-ERK and -SMAD4 levels correlate with activated TGFβ signaling and metastasis in overweight/obese patient breast cancer specimens. Altogether, we demonstrate the vital interaction of USP9x and SMAD4 for governing TGFβ signaling and dyslipidemia-induced aberrant TGFβ activation during breast cancer metastasis. Cancer Res; 77(6); 1383-94. ©2017 AACR . ©2017 American Association for Cancer Research.

TRAF Family Member-associated NF-κB Activator (TANK) Inhibits Genotoxic Nuclear Factor κB Activation by Facilitating Deubiquitinase USP10-dependent Deubiquitination of TRAF6 Ligase*

PubMed Central

Wang, Wei; Huang, Xuan; Xin, Hong-Bo; Fu, Mingui; Xue, Aimin; Wu, Zhao-Hui

2015-01-01

DNA damage-induced NF-κB activation plays a critical role in regulating cellular response to genotoxic stress. However, the molecular mechanisms controlling the magnitude and duration of this genotoxic NF-κB signaling cascade are poorly understood. We recently demonstrated that genotoxic NF-κB activation is regulated by reversible ubiquitination of several essential mediators involved in this signaling pathway. Here we show that TRAF family member-associated NF-κB activator (TANK) negatively regulates NF-κB activation by DNA damage via inhibiting ubiquitination of TRAF6. Despite the lack of a deubiquitination enzyme domain, TANK has been shown to negatively regulate the ubiquitination of TRAF proteins. We found TANK formed a complex with MCPIP1 (also known as ZC3H12A) and a deubiquitinase, USP10, which was essential for the USP10-dependent deubiquitination of TRAF6 and the resolution of genotoxic NF-κB activation upon DNA damage. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated deletion of TANK in human cells significantly enhanced NF-κB activation by genotoxic treatment, resulting in enhanced cell survival and increased inflammatory cytokine production. Furthermore, we found that the TANK-MCPIP1-USP10 complex also decreased TRAF6 ubiquitination in cells treated with IL-1β or LPS. In accordance, depletion of USP10 enhanced NF-κB activation induced by IL-1β or LPS. Collectively, our data demonstrate that TANK serves as an important negative regulator of NF-κB signaling cascades induced by genotoxic stress and IL-1R/Toll-like receptor stimulation in a manner dependent on MCPIP1/USP10-mediated TRAF6 deubiquitination. PMID:25861989

Accuracy of Cirrus HD-OCT and Topcon SP-3000P for measuring central corneal thickness.

PubMed

Calvo-Sanz, Jorge A; Ruiz-Alcocer, Javier; Sánchez-Tena, Miguel A

2017-02-18

To compare and analyze the interchangeability of three measuring systems, each based on a different technique, for central corneal thickness (CCT) analysis. CCT measurements were measured using optical coherence tomography (OCT), non-contact specular microscopy (NCSM), and ultrasonic pachymetry (USP) in 60 eyes of 60 healthy patients with a mean age of 66.5±15.0 years and a mean spherical equivalent of 0.43±1.14 D. Analysis of variations in measurement concordance and correlation among the three different methods were performed. Comparison of CCT measurements were done using Bland-Altman plots (with bias and 95% confidence intervals), intraclass correlation coefficient (ICC), and paired t-student analysis. Mean CCT values were: 549.20±26.91μm for USP (range 503-618μm), 514.20±27.49μm for NCSM (range 456-586μm) and 542.80±25.56μm for OCT (range 486-605μm). CCT values obtained with NCMS were significantly lower than those obtained with OCT and USP methods. NCMS CCT value was 36.08±10.72μm lower than USP value (p<0.05), and NCMS CCT value was 7.88±8.86μm lower than OCT value (p<0.05). ICC between USP-NCSM pair was 0.488 and 0.909 between USP-OCT pair. OCT and UPS offered highly comparable results, whereas NCSM offered lower mean CCT values compared to the other two methods. Therefore, NCSM should not be considered a reliable method for measuring CCT and should rather be considered for assessing longitudinal changes in the same patient. Copyright © 2017 Spanish General Council of Optometry. Published by Elsevier España, S.L.U. All rights reserved.

Internalization property of intestinal bacteria in colon cancer and HIV/AIDS patients.

PubMed

Wachsmannova, Lenka; Ciernikova, Sona; Majek, Juraj; Mego, Michal; Stevurkova, Viola; Zajac, Vladimir

2016-07-01

Bacteria from the intestinal tract of Slovak and American HIV/AIDS patients and Slovak colon cancer patients were tested for the capacity to be internalized by cells of the HL-60 cell line as well as by normal human lymphocytes. They were anticipated to possess a specific characteristic, i.e. a vigorous ability to be internalized by HL-60 cells and human lymphocytes. This assumption was confirmed by gentamicin protection assay. Internalization of bacteria from HIV/AIDS patients frequently resulted in partial (patients SKM1, SKM22) or complete lysis (patients SKK1-1, SKM12) of HL-60 cells. In comparison with intramucosal bacteria isolated from patients with colorectal cancer (TSG, 883, 660, 838, 536, MZRa), their capacity to internalize HL-60 cells was found to be 15-20 times higher (USP15/7, USP1/4, USP3/3, SK725/5). Partial lysis (patients USP15/7, USP3/3 and SKM22) and complete lysis (patients USP1/4, SKK1-1/1, SKM1/6, SKM12/5) were detected also after internalization of bacteria by normal human lymphocytes. Compared to the amount of intracellular bacteria isolated from patients with HIV/AIDS, the ability of bacteria from patients with colorectal cancer to internalize normal human lymphocytes was significantly lower (10-15 times), yet still higher than that of bacteria isolated from healthy people. Our results present the ability of bacteria of colon cancer patients and HIV/AIDS patients to internalize HL-60 cells and normal human lymphocytes. The findings underline the potentially important function of bacteria in the induction of colorectal cancer and immunodeficiency. The particularly high detection ability of bacteria from HIV/AIDS patients to internalize normal human cells emphasizes their potentially important role in the process of AIDS.

A study of the shortest-period planets found with Kepler

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sanchis-Ojeda, Roberto; Rappaport, Saul; Winn, Joshua N.

2014-05-20

We present the results of a survey aimed at discovering and studying transiting planets with orbital periods shorter than one day (ultra-short-period, or USP, planets), using data from the Kepler spacecraft. We computed Fourier transforms of the photometric time series for all 200,000 target stars, and detected transit signals based on the presence of regularly spaced sharp peaks in the Fourier spectrum. We present a list of 106 USP candidates, of which 18 have not previously been described in the literature. This list of candidates increases the number of planet candidates with orbital periods shorter than about six hours frommore » two to seven. In addition, among the objects we studied, there are 26 USP candidates that had been previously reported in the literature which do not pass our various tests. All 106 of our candidates have passed several standard tests to rule out false positives due to eclipsing stellar systems. A low false positive rate is also implied by the relatively high fraction of candidates for which more than one transiting planet signal was detected. By assuming these multi-transit candidates represent coplanar multi-planet systems, we are able to infer that the USP planets are typically accompanied by other planets with periods in the range 1-50 days, in contrast with hot Jupiters which very rarely have companions in that same period range. Another clear pattern is that almost all USP planets are smaller than 2 R {sub ⊕}, possibly because gas giants in very tight orbits would lose their atmospheres by photoevaporation when subject to extremely strong stellar irradiation. Based on our survey statistics, USP planets exist around approximately (0.51 ± 0.07)% of G-dwarf stars, and (0.83 ± 0.18)% of K-dwarf stars.« less

Fuel Cells Provide Reliable Power to U.S. Postal Service Facility in Anchorage, Alaska

DOE Office of Scientific and Technical Information (OSTI.GOV)

Parker, Steven

2003-01-01

Working together, the U.S. Postal Service (USPS) and Chugach Electric Association, partnering with the Department of Defense (DOD), Department of Energy (DOE), US Army Corps of Engineers Construction Engineering Research Laboratories (USA CERL), Electric Power Research Institute (EPRI), and National Rural Electric Cooperative Association (NRECA), developed and installed one of the largest fuel cell installations in the world. The one-megawatt fuel cell combined heat and power plant sits behind the Anchorage U.S. Postal Service Mail Processing and Distribution Facility. Chugach Electric owns, operates, and maintains the fuel cell power plant, which provides clean, reliable power to the USPS facility. Inmore » addition, heat recovered from the fuel cells, in the form of hot water, is used to heat the USPS Mail Processing and Distribution Facility. By taking a leadership role, the USPS will save over $800,000 in electricity and natural gas costs over the 5 1/2-year contract term with Chugach Electric.« less

Hydrodynamics-induced variability in the USP apparatus II dissolution test.

PubMed

Baxter, Jennifer L; Kukura, Joseph; Muzzio, Fernando J

2005-03-23

The USP tablet dissolution test is an analytical tool used for the verification of drug release processes and formulation selection within the pharmaceutical industry. Given the strong impact of this test, it is surprising that operating conditions and testing devices have been selected empirically. In fact, the flow phenomena in the USP test have received little attention in the past. An examination of the hydrodynamics in the USP apparatus II shows that the device is highly vulnerable to mixing problems that can affect testing performance and consistency. Experimental and computational techniques reveal that the flow field within the device is not uniform, and dissolution results can vary dramatically with the position of the tablet within the vessel. Specifically, computations predict sharp variations in the shear along the bottom of the vessel where the tablet is most likely to settle. Experiments in which the tablet location was carefully controlled reveal that the variation of shear within the testing device can affect the measured dissolution rate.

USP7/HAUSP: A SUMO deubiquitinase at the heart of DNA replication.

PubMed

Smits, Veronique A J; Freire, Raimundo

2016-09-01

DNA replication is both highly conserved and controlled. Problematic DNA replication can lead to genomic instability and therefore carcinogenesis. Numerous mechanisms work together to achieve this tight control and increasing evidence suggests that post-translational modifications (phosphorylation, ubiquitination, SUMOylation) of DNA replication proteins play a pivotal role in this process. Here we discuss such modifications in the light of a recent article that describes a novel role for the deubiquitinase (DUB) USP7/HAUSP in the control of DNA replication. USP7 achieves this function by an unusual and novel mechanism, namely deubiquitination of SUMOylated proteins at the replication fork, making USP7 also a SUMO DUB (SDUB). This work extends previous observations of increased levels of SUMO and low levels of ubiquitin at the on-going replication fork. Here, we discuss this novel study, its contribution to the DNA replication and genomic stability field and what questions arise from this work. © 2016 WILEY Periodicals, Inc.

77 FR 18707 - USPS Package Intercept-New Product Offerings

Federal Register 2010, 2011, 2012, 2013, 2014

2012-03-28

... service that replaced the former recall of mail process. Plans were announced to implement new features.... Additionally, customers using the electronic process will have the option of adding selected extra services to... POSTAL SERVICE 39 CFR Part 111 USPS Package Intercept--New Product Offerings AGENCY: Postal...

Pharmaceutics, Drug Delivery and Pharmaceutical Technology: A New Test Unit for Disintegration End-Point Determination of Orodispersible Films.

PubMed

Low, Ariana; Kok, Si Ling; Khong, Yuetmei; Chan, Sui Yung; Gokhale, Rajeev

2015-11-01

No standard time or pharmacopoeia disintegration test method for orodispersible films (ODFs) exists. The USP disintegration test for tablets and capsules poses significant challenges for end-point determination when used for ODFs. We tested a newly developed disintegration test unit (DTU) against the USP disintegration test. The DTU is an accessory to the USP disintegration apparatus. It holds the ODF in a horizontal position, allowing top-view of the ODF during testing. A Gauge R&R study was conducted to assign relative contributions of the total variability from the operator, sample or the experimental set-up. Precision was compared using commercial ODF products in different media. Agreement between the two measurement methods was analysed. The DTU showed improved repeatability and reproducibility compared to the USP disintegration system with tighter standard deviations regardless of operator or medium. There is good agreement between the two methods, with the USP disintegration test giving generally longer disintegration times possibly due to difficulty in end-point determination. The DTU provided clear end-point determination and is suitable for quality control of ODFs during product developmental stage or manufacturing. This may facilitate the development of a standardized methodology for disintegration time determination of ODFs. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:3893-3903, 2015. Copyright © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

Deubiquitinylase USP47 Promotes RelA Phosphorylation and Survival in Gastric Cancer Cells.

PubMed

Naghavi, Lara; Schwalbe, Martin; Ghanem, Ahmed; Naumann, Michael

2018-05-22

Every year, gastric cancer causes around 819,000 deaths worldwide. The incidence of gastric cancer in the western world is slowly declining, but the prognosis is unpromising. In Germany, the 5-year-survival rate is around 32%, and the average life span after diagnosis is 6 to 9 months. Therapy of gastric cancer patients comprises a gastrectomy and perioperative or adjuvant chemotherapy. However, resistance of gastric cancer cells to these agents is widespread; thus, improved chemotherapeutic approaches are required. Nuclear factor kappa B (NF-κB) transcription factors are associated with anti-apoptosis, carcinogenesis, and chemoresistance, and thus, constitute attractive targets for therapeutic intervention. In immunoblots, we show that ubiquitin specific protease 47 (USP47) promotes β-transducin repeat-containing protein (βTrCP) stability and phosphorylation of RelA. Furthermore, after knockdown of USP47 by RNA interference, we analyzed in gastric cancer cell lines metabolic activity/viability in an MTT assay, and apoptotic cell death by Annexin V staining and poly(ADP-Ribose) polymerase (PARP)-1, caspase 3, and caspase 8 cleavage, respectively. We found that USP47 contributes to cell viability and chemoresistance in NCI-N87 gastric carcinoma cells treated with etoposide and camptothecin. Inhibition of USP47 might be a suitable strategy to downregulate NF-κB activity, and to overcome chemoresistance in gastric cancer.

The Deubiquitinase USP47 Stabilizes MAPK by Counteracting the Function of the N-end Rule ligase POE/UBR4 in Drosophila.

PubMed

Ashton-Beaucage, Dariel; Lemieux, Caroline; Udell, Christian M; Sahmi, Malha; Rochette, Samuel; Therrien, Marc

2016-08-01

RAS-induced MAPK signaling is a central driver of the cell proliferation apparatus. Disruption of this pathway is widely observed in cancer and other pathologies. Consequently, considerable effort has been devoted to understanding the mechanistic aspects of RAS-MAPK signal transmission and regulation. While much information has been garnered on the steps leading up to the activation and inactivation of core pathway components, comparatively little is known on the mechanisms controlling their expression and turnover. We recently identified several factors that dictate Drosophila MAPK levels. Here, we describe the function of one of these, the deubiquitinase (DUB) USP47. We found that USP47 acts post-translationally to counteract a proteasome-mediated event that reduces MAPK half-life and thereby dampens signaling output. Using an RNAi-based genetic interaction screening strategy, we identified UBC6, POE/UBR4, and UFD4, respectively, as E2 and E3 enzymes that oppose USP47 activity. Further characterization of POE-associated factors uncovered KCMF1 as another key component modulating MAPK levels. Together, these results identify a novel protein degradation module that governs MAPK levels. Given the role of UBR4 as an N-recognin ubiquitin ligase, our findings suggest that RAS-MAPK signaling in Drosophila is controlled by the N-end rule pathway and that USP47 counteracts its activity.

A dose-response study in animals to evaluate the anticoagulant effect of the stage 2 unfractionated heparin USP monograph change.

PubMed

Honchel, R; Carraway, J; Gopee, N; Callicott, R; Chen, J; Patton, R; Xu, Q; Zalkkar, J; Laniyonu, A; Krefting, I; Cato, M; Robie-Suh, K; Rieves, R

2011-08-01

The United States Pharmacopeia (USP) monograph for unfractionated heparin (UFH) was revised in October 2009. This revision was anticipated, based upon in vitro tests, to reduce UFH potency by approximately 10%. To study the potential in vivo consequences of the monograph change, we evaluated activated partial thromboplastin time (aPTT) and activated clotting time (ACT) responses in animals. Female mini-pigs and monkeys (n=8/species) were administered intravenously 60, 54, 48, or 42 U/kg and 50, 45, 40, or 35 U/kg "old" (pre-USP revision) UFH, respectively, in a Williams 4×4 crossover design. Blood samples for aPTT and ACT were collected at 15 min after dosing. The same study design was then repeated using "new" (post-USP revision) UFH. Mean "new" UFH aPTT and ACT values were generally lower than those for "old" UFH although individual animal responses varied considerably. The aPTT and ACT response was generally dose-proportional for both "old" and "new" UFH. These studies indicate that the USP monograph alteration for UFH may result in a modest reduction in the anticoagulant response across a population, but the variability in animal responses underscores the importance of individualization of clinical UFH dosing and the importance of anticoagulant test monitoring. Published by Elsevier Inc.

77 FR 38342 - Privacy Act of 1974; System of Records

Federal Register 2010, 2011, 2012, 2013, 2014

2012-06-27

... to adding postage to postage meters. III. Description of Changes to Systems of Records The Postal... Management Records USPS 300.000 SYSTEM NAME: Finance Records USPS 400.000 SYSTEM NAME: Supplier and Tenant...: Postage Meter and PC Postage Customer Data and Transaction Records Accordingly, for the reasons stated...

Recycling evaluation of new-generation environmentally benign pressure sensitive adhesives

Treesearch

Said M. Abubakr; Carl Houtman; Dave Bormett; Nancy Ross Sutherland; Joe Peng

1999-01-01

As a result of a United States Postal Service (USPS) initiative, a work team was formed consisting of representatives from USPS, Forest Products Laboratory (FPL), Specialized Technology Resources, Inc. (STR), and industry. The industries represented included papermakers, paper recyclers, paper collectors, equipment manufacturers, paper users, adhesive manufacturers and...

USP <800> Adds Significant Safety Standards: Facility Upgrades Needed to Protect Employees From Hazardous Drugs.

PubMed

Beans, Bruce E

2017-05-01

The new USP standard for handling hazardous drugs (HDs) will require millions of dollars in capital outlays for facility and equipment upgrades and also requires in-depth assessments of each HD that facilities handle, significant workflow and work practice changes, and thorough staff training.

Rehabilitative treatment of cleft lip and palate: experience of the Hospital for Rehabilitation of Craniofacial Anomalies/USP (HRAC/USP) - Part 4: Oral Rehabilitation

PubMed Central

FREITAS, José Alberto de Souza; de ALMEIDA, Ana Lúcia Pompéia Fraga; SOARES, Simone; NEVES, Lucimara Teixeira das; GARIB, Daniela Gamba; TRINDADE-SUEDAM, Ivy Kiemle; YAEDÚ, Renato Yassutaka Faria; LAURIS, Rita de Cássia Moura Carvalho; OLIVEIRA, Thais Marchini; PINTO, João Henrique Nogueira

2013-01-01

Treatment of patients with cleft lip and palate is completed with fixed prostheses, removable, total, implants and aims to restore aesthetics, phonetics and function and should be guided by the basic principles of oral rehabilitation, such as physiology, stability, aesthetics, hygiene and the expectations of the patient. In order to obtain longevity of a prosthetic rehabilitation, the periodontal and dental tissue as well as the biomechanics of the prosthesis are to be respected. The purpose of this article is to describe the types of prosthetics treatment, which are performed at HRAC/USP for the rehabilitation of cleft area in adult patients. PMID:23857655

Rehabilitative treatment of cleft lip and palate: experience of the Hospital for Rehabilitation of Craniofacial Anomalies/USP (HRAC/USP) - Part 4: oral rehabilitation.

PubMed

Freitas, José Alberto de Souza; Almeida, Ana Lúcia Pompéia Fraga de; Soares, Simone; Neves, Lucimara Teixeira das; Garib, Daniela Gamba; Trindade-Suedam, Ivy Kiemle; Yaedú, Renato Yassutaka Faria; Lauris, Rita de Cássia Moura Carvalho; Oliveira, Thais Marchini; Pinto, João Henrique Nogueira

2013-01-01

Treatment of patients with cleft lip and palate is completed with fixed prostheses, removable, total, implants and aims to restore aesthetics, phonetics and function and should be guided by the basic principles of oral rehabilitation, such as physiology, stability, aesthetics, hygiene and the expectations of the patient. In order to obtain longevity of a prosthetic rehabilitation, the periodontal and dental tissue as well as the biomechanics of the prosthesis are to be respected. The purpose of this article is to describe the types of prosthetics treatment, which are performed at HRAC/USP for the rehabilitation of cleft area in adult patients.

Identification and Validation of Selected Universal Stress Protein Domain Containing Drought-Responsive Genes in Pigeonpea (Cajanus cajan L.)

PubMed Central

Sinha, Pallavi; Pazhamala, Lekha T.; Singh, Vikas K.; Saxena, Rachit K.; Krishnamurthy, L.; Azam, Sarwar; Khan, Aamir W.; Varshney, Rajeev K.

2016-01-01

Pigeonpea is a resilient crop, which is relatively more drought tolerant than many other legume crops. To understand the molecular mechanisms of this unique feature of pigeonpea, 51 genes were selected using the Hidden Markov Models (HMM) those codes for proteins having close similarity to universal stress protein domain. Validation of these genes was conducted on three pigeonpea genotypes (ICPL 151, ICPL 8755, and ICPL 227) having different levels of drought tolerance. Gene expression analysis using qRT-PCR revealed 6, 8, and 18 genes to be ≥2-fold differentially expressed in ICPL 151, ICPL 8755, and ICPL 227, respectively. A total of 10 differentially expressed genes showed ≥2-fold up-regulation in the more drought tolerant genotype, which encoded four different classes of proteins. These include plant U-box protein (four genes), universal stress protein A-like protein (four genes), cation/H(+) antiporter protein (one gene) and an uncharacterized protein (one gene). Genes C.cajan_29830 and C.cajan_33874 belonging to uspA, were found significantly expressed in all the three genotypes with ≥2-fold expression variations. Expression profiling of these two genes on the four other legume crops revealed their specific role in pigeonpea. Therefore, these genes seem to be promising candidates for conferring drought tolerance specifically to pigeonpea. PMID:26779199

38 CFR 76.2 - Assistance allowance.

Code of Federal Regulations, 2011 CFR

2011-07-01

... part of any month in which the veteran is training or competing in any event sponsored by the USP or the IPC; or (2) Residing at a USP training center in connection with any paralympic training or competition for the period certified under § 76.3. (b) In providing this allowance, VA will periodically...

27 CFR 21.60 - Formula No. 33.

Code of Federal Regulations, 2014 CFR

2014-04-01

... pounds of gentian violet or gentian violet, U.S.P. (b) Authorized uses. (1) As a solvent: 052.Inks (not including meat branding inks). 210.External pharmaceuticals, not U.S.P. or N.F. 249.Miscellaneous external...). (c) Conditions governing use. Meat branding inks made with Formula No. 33 do not meet U.S. Department...

27 CFR 21.60 - Formula No. 33.

Code of Federal Regulations, 2012 CFR

2012-04-01

... pounds of gentian violet or gentian violet, U.S.P. (b) Authorized uses. (1) As a solvent: 052.Inks (not including meat branding inks). 210.External pharmaceuticals, not U.S.P. or N.F. 249.Miscellaneous external...). (c) Conditions governing use. Meat branding inks made with Formula No. 33 do not meet U.S. Department...

27 CFR 21.60 - Formula No. 33.

Code of Federal Regulations, 2011 CFR

2011-04-01

... pounds of gentian violet or gentian violet, U.S.P. (b) Authorized uses. (1) As a solvent: 052.Inks (not including meat branding inks). 210.External pharmaceuticals, not U.S.P. or N.F. 249.Miscellaneous external...). (c) Conditions governing use. Meat branding inks made with Formula No. 33 do not meet U.S. Department...

27 CFR 21.60 - Formula No. 33.

Code of Federal Regulations, 2013 CFR

2013-04-01

... pounds of gentian violet or gentian violet, U.S.P. (b) Authorized uses. (1) As a solvent: 052.Inks (not including meat branding inks). 210.External pharmaceuticals, not U.S.P. or N.F. 249.Miscellaneous external...). (c) Conditions governing use. Meat branding inks made with Formula No. 33 do not meet U.S. Department...

75 FR 65515 - NRC Corporation, USPS Help Desk of Customer Care Center, Including On-Site Leased Workers of Volt...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-10-25

... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-74,347] NRC Corporation, USPS Help Desk of Customer Care Center, Including On-Site Leased Workers of Volt Consulting, West Columbia, SC; Notice of Affirmative Determination Regarding Application for Reconsideration By application...

USP7S-dependent inactivation of Mule regulates DNA damage signalling and repair.

PubMed

Khoronenkova, Svetlana V; Dianov, Grigory L

2013-02-01

The E3 ubiquitin ligase Mule/ARF-BP1 plays an important role in the cellular DNA damage response by controlling base excision repair and p53 protein levels. However, how the activity of Mule is regulated in response to DNA damage is currently unknown. Here, we report that the Ser18-containing isoform of the USP7 deubiquitylation enzyme (USP7S) controls Mule stability by preventing its self-ubiquitylation and subsequent proteasomal degradation. We find that in response to DNA damage, downregulation of USP7S leads to self-ubiquitylation and proteasomal degradation of Mule, which eventually leads to p53 accumulation. Cells that are unable to downregulate Mule show reduced ability to upregulate p53 levels in response to DNA damage. We also find that, as Mule inactivation is required for stabilization of base excision repair enzymes, the failure of cells to downregulate Mule after DNA damage results in deficient DNA repair. Our data describe a novel mechanism by which Mule is regulated in response to DNA damage and coordinates cellular DNA damage responses and DNA repair.

Comparative study on deposition of fluorine-doped tin dioxide thin films by conventional and ultrasonic spray pyrolysis methods for dye-sensitized solar modules

NASA Astrophysics Data System (ADS)

Icli, Kerem Cagatay; Kocaoglu, Bahadir Can; Ozenbas, Macit

2018-01-01

Fluorine-doped tin dioxide (FTO) thin films were produced via conventional spray pyrolysis and ultrasonic spray pyrolysis (USP) methods using alcohol-based solutions. The prepared films were compared in terms of crystal structure, morphology, surface roughness, visible light transmittance, and electronic properties. Upon investigation of the grain structures and morphologies, the films prepared using ultrasonic spray method provided relatively larger grains and due to this condition, carrier mobilities of these films exhibited slightly higher values. Dye-sensitized solar cells and 10×10 cm modules were prepared using commercially available and USP-deposited FTO/glass substrates, and solar performances were compared. It is observed that there exists no remarkable efficiency difference for both cells and modules, where module efficiency of the USP-deposited FTO glass substrates is 3.06% compared to commercial substrate giving 2.85% under identical conditions. We demonstrated that USP deposition is a low cost and versatile method of depositing commercial quality FTO thin films on large substrates employed in large area dye-sensitized solar modules or other thin film technologies.

Ubiquitin-specific protease 8 deubiquitinates Sec31A and decreases large COPII carriers and collagen IV secretion.

PubMed

Kawaguchi, Kohei; Endo, Akinori; Fukushima, Toshiaki; Madoka, Yuka; Tanaka, Toshiaki; Komada, Masayuki

2018-05-15

Nascent cargo proteins in the endoplasmic reticulum are transported to the Golgi by COPII carriers. Typical COPII vesicles are 60-70 nm in diameter, and much larger macromolecules, such as procollagen, are transported by atypical large COPII carriers in mammalian cells. The formation of large COPII carriers is enhanced by Cul3 ubiquitin ligase, which mono-ubiquitinates Sec31A, a COPII coat protein. However, the deubiquitinating enzyme for Sec31A was unclear. Here, we show that the deubiquitinating enzyme USP8 interacts with and deubiquitinates Sec31A. The interaction was mediated by the adaptor protein STAM1. USP8 overexpression inhibited the formation of large COPII carriers. By contrast, USP8 knockdown caused the accumulation of COPII coat proteins around the cis-Golgi, promoted the intracellular trafficking of procollagen IV from the endoplasmic reticulum to the Golgi, and increased collagen IV secretion. We concluded that USP8 deubiquitinates Sec31A and inhibits the formation of large COPII carriers, thereby suppressing collagen IV secretion. Copyright © 2018 Elsevier Inc. All rights reserved.

53BP1 and USP28 mediate p53-dependent cell cycle arrest in response to centrosome loss and prolonged mitosis.

PubMed

Fong, Chii Shyang; Mazo, Gregory; Das, Tuhin; Goodman, Joshua; Kim, Minhee; O'Rourke, Brian P; Izquierdo, Denisse; Tsou, Meng-Fu Bryan

2016-07-02

Mitosis occurs efficiently, but when it is disturbed or delayed, p53-dependent cell death or senescence is often triggered after mitotic exit. To characterize this process, we conducted CRISPR-mediated loss-of-function screens using a cell-based assay in which mitosis is consistently disturbed by centrosome loss. We identified 53BP1 and USP28 as essential components acting upstream of p53, evoking p21-dependent cell cycle arrest in response not only to centrosome loss, but also to other distinct defects causing prolonged mitosis. Intriguingly, 53BP1 mediates p53 activation independently of its DNA repair activity, but requiring its interacting protein USP28 that can directly deubiquitinate p53 in vitro and ectopically stabilize p53 in vivo. Moreover, 53BP1 can transduce prolonged mitosis to cell cycle arrest independently of the spindle assembly checkpoint (SAC), suggesting that while SAC protects mitotic accuracy by slowing down mitosis, 53BP1 and USP28 function in parallel to select against disturbed or delayed mitosis, promoting mitotic efficiency.

Phosphorylation and activation of ubiquitin-specific protease-14 by Akt regulates the ubiquitin-proteasome system

PubMed Central

Xu, Daichao; Shan, Bing; Lee, Byung-Hoon; Zhu, Kezhou; Zhang, Tao; Sun, Huawang; Liu, Min; Shi, Linyu; Liang, Wei; Qian, Lihui; Xiao, Juan; Wang, Lili; Pan, Lifeng; Finley, Daniel; Yuan, Junying

2015-01-01

Regulation of ubiquitin-proteasome system (UPS), which controls the turnover of short-lived proteins in eukaryotic cells, is critical in maintaining cellular proteostasis. Here we show that USP14, a major deubiquitinating enzyme that regulates the UPS, is a substrate of Akt, a serine/threonine-specific protein kinase critical in mediating intracellular signaling transducer for growth factors. We report that Akt-mediated phosphorylation of USP14 at Ser432, which normally blocks its catalytic site in the inactive conformation, activates its deubiquitinating activity in vitro and in cells. We also demonstrate that phosphorylation of USP14 is critical for Akt to regulate proteasome activity and consequently global protein degradation. Since Akt can be activated by a wide range of growth factors and is under negative control by phosphoinosotide phosphatase PTEN, we suggest that regulation of UPS by Akt-mediated phosphorylation of USP14 may provide a common mechanism for growth factors to control global proteostasis and for promoting tumorigenesis in PTEN-negative cancer cells. DOI: http://dx.doi.org/10.7554/eLife.10510.001 PMID:26523394

Endothelial RSPO3 Controls Vascular Stability and Pruning through Non-canonical WNT/Ca(2+)/NFAT Signaling.

PubMed

Scholz, Beate; Korn, Claudia; Wojtarowicz, Jessica; Mogler, Carolin; Augustin, Iris; Boutros, Michael; Niehrs, Christof; Augustin, Hellmut G

2016-01-11

The WNT signaling enhancer R-spondin3 (RSPO3) is prominently expressed in the vasculature. Correspondingly, embryonic lethality of Rspo3-deficient mice is caused by vessel remodeling defects. Yet the mechanisms underlying vascular RSPO3 function remain elusive. Inducible endothelial Rspo3 deletion (Rspo3-iECKO) resulted in perturbed developmental and tumor vascular remodeling. Endothelial cell apoptosis and vascular pruning led to reduced microvessel density in Rspo3-iECKO mice. Rspo3-iECKO mice strikingly phenocopied the non-canonical WNT signaling-induced vascular defects of mice deleted for the WNT secretion factor Evi/Wls. An endothelial screen for RSPO3 and EVI/WLS co-regulated genes identified Rnf213, Usp18, and Trim30α. RNF213 targets filamin A and NFAT1 for proteasomal degradation attenuating non-canonical WNT/Ca(2+) signaling. Likewise, USP18 and TRIM5α inhibited NFAT1 activation. Consequently, NFAT protein levels were decreased in endothelial cells of Rspo3-iECKO mice and pharmacological NFAT inhibition phenocopied Rspo3-iECKO mice. The data identify endothelial RSPO3-driven non-canonical WNT/Ca(2+)/NFAT signaling as a critical maintenance pathway of the remodeling vasculature. Copyright © 2016 Elsevier Inc. All rights reserved.

The Prader-Willi syndrome proteins MAGEL2 and necdin regulate leptin receptor cell surface abundance through ubiquitination pathways.

PubMed

Wijesuriya, Tishani Methsala; De Ceuninck, Leentje; Masschaele, Delphine; Sanderson, Matthea R; Carias, Karin Vanessa; Tavernier, Jan; Wevrick, Rachel

2017-11-01

In Prader-Willi syndrome (PWS), obesity is caused by the disruption of appetite-controlling pathways in the brain. Two PWS candidate genes encode MAGEL2 and necdin, related melanoma antigen proteins that assemble into ubiquitination complexes. Mice lacking Magel2 are obese and lack leptin sensitivity in hypothalamic pro-opiomelanocortin neurons, suggesting dysregulation of leptin receptor (LepR) activity. Hypothalamus from Magel2-null mice had less LepR and altered levels of ubiquitin pathway proteins that regulate LepR processing (Rnf41, Usp8, and Stam1). MAGEL2 increased the cell surface abundance of LepR and decreased their degradation. LepR interacts with necdin, which interacts with MAGEL2, which complexes with RNF41 and USP8. Mutations in the MAGE homology domain of MAGEL2 suppress RNF41 stabilization and prevent the MAGEL2-mediated increase of cell surface LepR. Thus, MAGEL2 and necdin together control LepR sorting and degradation through a dynamic ubiquitin-dependent pathway. Loss of MAGEL2 and necdin may uncouple LepR from ubiquitination pathways, providing a cellular mechanism for obesity in PWS. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Globule-size distribution in injectable 20% lipid emulsions: Compliance with USP requirements.

PubMed

Driscoll, David F

2007-10-01

The compliance of injectable 20% lipid emulsions with the globule-size limits in chapter 729 of the U.S. Pharmacopeia (USP) was examined. As established in chapter 729, dynamic light scattering was applied to determine mean droplet diameter (MDD), with an upper limit of 500 nm. Light obscuration was used to determine the size of fat globules found in the large-diameter tail, expressed as the volume-weighted percent fat exceeding 5 microm (PFAT(5)), with an upper limit of 0.05%. Compliance of seven different emulsions, six of which were stored in plastic bags, with USP limits was assessed. To avoid reaching coincidence limits during the application of method II from overly concentrated emulsion samples, a variable dilution scheme was used to optimize the globule-size measurements for each emulsion. One-way analysis of variance of globule-size distribution (GSD) data was conducted if any results of method I or II exceeded the respective upper limits. Most injectable lipid emulsions complied with limits established by USP chapter 729, with the exception of those of one manufacturer, which failed limits as proposed for to meet the PFAT(5) three of the emulsions tested. In contrast, all others studied (one packaged in glass and three packaged in plastic) met both criteria. Among seven injectable lipid emulsions tested for GSD, all met USP chapter 729 MDD requirements and three, all from the same manufacturer and packaged in plastic, did not meet PFAT(5) requirements.

Inhibition of deubiquitinases primes glioblastoma cells to apoptosis in vitro and in vivo

PubMed Central

Karpel-Massler, Georg; Banu, Matei A.; Shu, Chang; Halatsch, Marc-Eric; Westhoff, Mike-Andrew; Bruce, Jeffrey N.; Canoll, Peter; Siegelin, Markus D.

2016-01-01

It remains a challenge in oncology to identify novel drug regimens to efficiently tackle glioblastoma, the most common primary brain tumor in adults. Here, we target deubiquitinases for glioblastoma therapy by utilizing the small-molecule inhibitor WP1130 which has been characterized as a deubiquitinase inhibitor that interferes with the function of Usp9X. Expression analysis data confirm that Usp9X expression is increased in glioblastoma compared to normal brain tissue indicating its potential as a therapeutic. Consistently, increasing concentrations of WP1130 decrease the cellular viability of established, patient-derived xenograft (PDX) and stem cell-like glioblastoma cells. Specific down-regulation of Usp9X reduces viability in glioblastoma cells mimicking the effects of WP1130. Mechanistically, WP1130 elicits apoptosis and increases activation of caspases. Moreover, WP1130 and siRNAs targeting Usp9X reduce the expression of anti-apoptotic Bcl-2 family members and Inhibitor of Apoptosis Proteins, XIAP and Survivin. Pharmacological and genetic interference with Usp9X efficiently sensitized glioblastoma cells to intrinsic and extrinsic apoptotic stimuli. In addition, single treatment with WP1130 elicited anti-glioma activity in an orthotopic proneural murine model of glioblastoma. Finally, the combination treatment of WP1130 and ABT263 inhibited tumor growth more efficiently than each reagent by its own in vivo without detectable side effects or organ toxicity. Taken together, these results suggest that targeting deubiquitinases for glioma therapy is feasible and effective. PMID:26872380

Internet cigarette vendor compliance with credit card payment and shipping bans.

PubMed

Williams, Rebecca S; Ribisl, Kurt M

2014-02-01

Most Internet cigarette sales have violated taxation and youth access laws, leading to landmark 2005 agreements with credit card companies, PayPal, and private shippers (United Parcel Service, Federal Express, DHL) to cease participation in these transactions. Despite their promise at the time, loopholes allowed for check payment and U.S. Postal Service (USPS) shipping. This study assessed actual vendor compliance with the payment and shipping bans using a purchase survey. In late 2007 and early 2008, an adult buyer attempted to order cigarettes from the 97 most popular Internet cigarette vendors (ICVs) using banned payment and shipping methods. When banned payment or shipping methods were unavailable, purchases were attempted with alternate methods (e.g., checks, e-checks, USPS). Twenty-seven of 100 orders were placed with (banned) credit cards; 23 were successfully received. Seventy-one orders were placed with checks (60 successfully received). Four orders were delivered using banned shippers; 79 of 83 successfully received orders were delivered by the USPS. About a quarter of ICVs violated the payment ban, others adapted by accepting checks. Most vendors complied with the shipping ban, perhaps because USPS was an easy substitute shipping option. Better enforcement of the bans is needed; the 2009 Prevent All Cigarette Trafficking Act closed the USPS loophole by making cigarettes nonmailable material; evaluation of enforcement efforts and adaptations by vendors are needed. These sorts of bans are a promising approach to controlling the sale of restricted goods online.

Molecular Weights of Bovine and Porcine Heparin Samples: Comparison of Chromatographic Methods and Results of a Collaborative Survey.

PubMed

Bertini, Sabrina; Risi, Giulia; Guerrini, Marco; Carrick, Kevin; Szajek, Anita Y; Mulloy, Barbara

2017-07-19

In a collaborative study involving six laboratories in the USA, Europe, and India the molecular weight distributions of a panel of heparin sodium samples were determined, in order to compare heparin sodium of bovine intestinal origin with that of bovine lung and porcine intestinal origin. Porcine samples met the current criteria as laid out in the USP Heparin Sodium monograph. Bovine lung heparin samples had consistently lower average molecular weights. Bovine intestinal heparin was variable in molecular weight; some samples fell below the USP limits, some fell within these limits and others fell above the upper limits. These data will inform the establishment of pharmacopeial acceptance criteria for heparin sodium derived from bovine intestinal mucosa. The method for MW determination as described in the USP monograph uses a single, broad standard calibrant to characterize the chromatographic profile of heparin sodium on high-resolution silica-based GPC columns. These columns may be short-lived in some laboratories. Using the panel of samples described above, methods based on the use of robust polymer-based columns have been developed. In addition to the use of the USP's broad standard calibrant for heparin sodium with these columns, a set of conditions have been devised that allow light-scattering detected molecular weight characterization of heparin sodium, giving results that agree well with the monograph method. These findings may facilitate the validation of variant chromatographic methods with some practical advantages over the USP monograph method.

Development and validation of an ICP-MS method for the determination of elemental impurities in TP-6076 active pharmaceutical ingredient (API) according to USP 〈232〉/〈233〉.

PubMed

Chahrour, Osama; Malone, John; Collins, Mark; Salmon, Vrushali; Greenan, Catherine; Bombardier, Amy; Ma, Zhongze; Dunwoody, Nick

2017-10-25

The new guidelines of the United States pharmacopeia (USP), European pharmacopeia (EP) and international conference on harmonization (ICH) regulating elemental impurities limits in pharmaceuticals signify the end of unspecific analysis of metals as outlined in USP 〈231〉. The new guidelines specify both daily doses and concentration/limits of elemental impurities in pharmaceutical final products, active pharmaceutical ingredients (API) and excipients. In chapter USP 〈233〉 method implementation, validation and quality control during the analytical process are described. We herein report the use of a stabilising matrix that overcomes low spike recovery problem encountered with Os and allows the determination of all USP required elemental impurities (As, Cd, Hg, Pb, V, Cr, Ni, Mo, Cu, Pt, Pd, Ru, Rh, Os and Ir) in a single analysis. The matrix was used in the validation of a method to determine elemental impurities in TP-6076 active pharmaceutical ingredient (API) by ICP-MS according to the procedures defined in USP〈233〉 and to GMP requirements. This validation will support the regulatory submission of TP-6076 which is a novel tetracycline analogue effective against the most urgent multidrug-resistant gram-negative bacteria. Evaluation of TP-6076 in IND-enabling toxicology studies has led to the initiation of a phase 1 clinical trial. Copyright © 2017 Elsevier B.V. All rights reserved.

Accelerated in vitro release testing method for naltrexone loaded PLGA microspheres.

PubMed

Andhariya, Janki V; Choi, Stephanie; Wang, Yan; Zou, Yuan; Burgess, Diane J; Shen, Jie

2017-03-30

The objective of the present study was to develop a discriminatory and reproducible accelerated release testing method for naltrexone loaded parenteral polymeric microspheres. The commercially available naltrexone microsphere product (Vivitrol ® ) was used as the testing formulation in the in vitro release method development, and both sample-and-separate and USP apparatus 4 methods were investigated. Following an in vitro drug stability study, frequent media replacement and addition of anti-oxidant in the release medium were used to prevent degradation of naltrexone during release testing at "real-time" (37°C) and "accelerated" (45°C), respectively. The USP apparatus 4 method was more reproducible than the sample-and-separate method. In addition, the accelerated release profile obtained using USP apparatus 4 had a shortened release duration (within seven days), and good correlation with the "real-time" release profile. Lastly, the discriminatory ability of the developed accelerated release method was assessed using compositionally equivalent naltrexone microspheres with different release characteristics. The developed accelerated USP apparatus 4 release method was able to detect differences in the release characteristics of the prepared naltrexone microspheres. Moreover, a linear correlation was observed between the "real-time" and accelerated release profiles of all the formulations investigated, suggesting that the release mechanism(s) may be similar under both conditions. These results indicate that the developed accelerated USP apparatus 4 method has the potential to be an appropriate fast quality control tool for long-acting naltrexone PLGA microspheres. Copyright © 2017 Elsevier B.V. All rights reserved.

TRAF Family Member-associated NF-κB Activator (TANK) Inhibits Genotoxic Nuclear Factor κB Activation by Facilitating Deubiquitinase USP10-dependent Deubiquitination of TRAF6 Ligase.

PubMed

Wang, Wei; Huang, Xuan; Xin, Hong-Bo; Fu, Mingui; Xue, Aimin; Wu, Zhao-Hui

2015-05-22

DNA damage-induced NF-κB activation plays a critical role in regulating cellular response to genotoxic stress. However, the molecular mechanisms controlling the magnitude and duration of this genotoxic NF-κB signaling cascade are poorly understood. We recently demonstrated that genotoxic NF-κB activation is regulated by reversible ubiquitination of several essential mediators involved in this signaling pathway. Here we show that TRAF family member-associated NF-κB activator (TANK) negatively regulates NF-κB activation by DNA damage via inhibiting ubiquitination of TRAF6. Despite the lack of a deubiquitination enzyme domain, TANK has been shown to negatively regulate the ubiquitination of TRAF proteins. We found TANK formed a complex with MCPIP1 (also known as ZC3H12A) and a deubiquitinase, USP10, which was essential for the USP10-dependent deubiquitination of TRAF6 and the resolution of genotoxic NF-κB activation upon DNA damage. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated deletion of TANK in human cells significantly enhanced NF-κB activation by genotoxic treatment, resulting in enhanced cell survival and increased inflammatory cytokine production. Furthermore, we found that the TANK-MCPIP1-USP10 complex also decreased TRAF6 ubiquitination in cells treated with IL-1β or LPS. In accordance, depletion of USP10 enhanced NF-κB activation induced by IL-1β or LPS. Collectively, our data demonstrate that TANK serves as an important negative regulator of NF-κB signaling cascades induced by genotoxic stress and IL-1R/Toll-like receptor stimulation in a manner dependent on MCPIP1/USP10-mediated TRAF6 deubiquitination. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Comparison of Central Corneal Thickness Measured by Standard Ultrasound Pachymetry, Corneal Topography, Tono-Pachymetry and Anterior Segment Optical Coherence Tomography.

PubMed

González-Pérez, Javier; Queiruga Piñeiro, Juan; Sánchez García, Ángelx; González Méijome, José Manuel

2018-04-10

To compare central corneal thickness (CCT) measured by standard ultrasound pachymetry (USP), and three non-contact devices in healthy eyes. A cross-sectional study of CCT measurement in 52 eyes of 52 healthy volunteers was done by a single examiner at Ocular Surface and Contact Lens Laboratory. Three consecutive measurements were done by standard USP, non-contact tono-pachymeter, Pentacam corneal topographer, and Anterior Segment Optical Coherence Tomography (AS-OCT). The mean values were used for assessment. The results were compared using multivariate ANOVA, linear regression, and Pearson correlation. Agreement among the devices was analyzed using mean differences and Bland-Altman analysis with 95% limits of agreement (LoA). Finally, reliability was analyzed using intraclass correlation coefficient (ICC). Mean CCT by ultrasound pachymeter, tono-pachymeter, corneal topographer and AS-OCT were 558.9 ± 31.2 µm, 525.8 ± 43.1 µm, 550.4 ± 30.5 µm, and 545.9 ± 30.5 µm respectively. There was a significant positive correlation between AS-OCT and USP (Pearson correlation = 0.957, p < 0.001), corneal topography and USP (Pearson correlation = 0.965, p < 0.001), and corneal topography and AS-OCT (Pearson correlation = 0.965, p < 0.001). There was a lower correlation between CT-1P tono-pachymeter and the other three modalities. Intraclass correlation coefficients show an excellent reliability between pairs except for CT-1P against the other three instruments that were found moderate. CT-1P tono-pachymeter underestimates CCT measurements compared to Scheimpflug system, AS-OCT device, and USP. Mean CCT among USP, Pentacam and AS-OCT were comparable and had significant linear correlations. In clinical practice, these three modalities could be interchangeable in healthy patients.

Mycobacterium tuberculosis Universal Stress Protein Rv2623 Regulates Bacillary Growth by ATP Binding: Requirement for Establishing Chronic Persistent Infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Drumm, J.; Mi, K; Bilder, P

Tuberculous latency and reactivation play a significant role in the pathogenesis of tuberculosis, yet the mechanisms that regulate these processes remain unclear. The Mycobacterium tuberculosisuniversal stress protein (USP) homolog, rv2623, is among the most highly induced genes when the tubercle bacillus is subjected to hypoxia and nitrosative stress, conditions thought to promote latency. Induction of rv2623 also occurs when M. tuberculosis encounters conditions associated with growth arrest, such as the intracellular milieu of macrophages and in the lungs of mice with chronic tuberculosis. Therefore, we tested the hypothesis that Rv2623 regulates tuberculosis latency. We observed that an Rv2623-deficient mutant failsmore » to establish chronic tuberculous infection in guinea pigs and mice, exhibiting a hypervirulence phenotype associated with increased bacterial burden and mortality. Consistent with this in vivo growth-regulatory role, constitutive overexpression of rv2623 attenuates mycobacterial growth in vitro. Biochemical analysis of purified Rv2623 suggested that this mycobacterial USP binds ATP, and the 2.9-A-resolution crystal structure revealed that Rv2623 engages ATP in a novel nucleotide-binding pocket. Structure-guided mutagenesis yielded Rv2623 mutants with reduced ATP-binding capacity. Analysis of mycobacteria overexpressing these mutants revealed that the in vitro growth-inhibitory property of Rv2623 correlates with its ability to bind ATP. Together, the results indicate that i M. tuberculosis Rv2623 regulates mycobacterial growth in vitro and in vivo, and ii Rv2623 is required for the entry of the tubercle bacillus into the chronic phase of infection in the host; in addition, iii Rv2623 binds ATP; and iv the growth-regulatory attribute of this USP is dependent on its ATP-binding activity. We propose that Rv2623 may function as an ATP-dependent signaling intermediate in a pathway that promotes persistent infection.« less

Hepatitis C virus (HCV) induces formation of stress granules whose proteins regulate HCV RNA replication and virus assembly and egress.

PubMed

Garaigorta, Urtzi; Heim, Markus H; Boyd, Bryan; Wieland, Stefan; Chisari, Francis V

2012-10-01

Stress granules (SGs) are cytoplasmic structures that are induced in response to environmental stress, including viral infections. Here we report that hepatitis C virus (HCV) triggers the appearance of SGs in a PKR- and interferon (IFN)-dependent manner. Moreover, we show an inverse correlation between the presence of stress granules and the induction of IFN-stimulated proteins, i.e., MxA and USP18, in HCV-infected cells despite high-level expression of the corresponding MxA and USP18 mRNAs, suggesting that interferon-stimulated gene translation is inhibited in stress granule-containing HCV-infected cells. Finally, in short hairpin RNA (shRNA) knockdown experiments, we found that the stress granule proteins T-cell-restricted intracellular antigen 1 (TIA-1), TIA1-related protein (TIAR), and RasGAP-SH3 domain binding protein 1 (G3BP1) are required for efficient HCV RNA and protein accumulation at early time points in the infection and that G3BP1 and TIA-1 are required for intracellular and extracellular infectious virus production late in the infection, suggesting that they are required for virus assembly. In contrast, TIAR downregulation decreases extracellular infectious virus titers with little effect on intracellular RNA content or infectivity late in the infection, suggesting that it is required for infectious particle release. Collectively, these results illustrate that HCV exploits the stress granule machinery at least two ways: by inducing the formation of SGs by triggering PKR phosphorylation, thereby downregulating the translation of antiviral interferon-stimulated genes, and by co-opting SG proteins for its replication, assembly, and egress.

38 CFR 76.4 - Amount of allowance.

Code of Federal Regulations, 2011 CFR

2011-07-01

... of the monthly rate to veterans who train or compete in USP or IPC sponsored events for each day of training or competition, or to veterans who reside at a USP training center, for each day of residence, or on a monthly basis at the monthly rate to veterans who train or compete continuously for a full month...

27 CFR 21.54 - Formula No. 27-B.

Code of Federal Regulations, 2012 CFR

2012-04-01

... gallon of lavender oil, N.F., and 100 pounds of green soap, U.S.P. Note. The requirements of this formula may be met by adding 1 gallon of lavender oil, N.F., and 66.5 pounds of U.S.P. quality soap... other biocides. (2) Miscellaneous uses: 812.Product development and pilot plant uses (own use only). ...

27 CFR 21.54 - Formula No. 27-B.

Code of Federal Regulations, 2011 CFR

2011-04-01

... gallon of lavender oil, N.F., and 100 pounds of green soap, U.S.P. Note. The requirements of this formula may be met by adding 1 gallon of lavender oil, N.F., and 66.5 pounds of U.S.P. quality soap... other biocides. (2) Miscellaneous uses: 812.Product development and pilot plant uses (own use only). ...

Development of USPS Laboratory and pilot-scale testing protocols

Treesearch

Carl Houtman; Nancy Ross Sutherland; David Bormett; Donald Donermeyer

2000-01-01

The ultimate goal of the US Postal Service (USPS) Environmentally Benign Stamp Program is to develop stamp adhesives that can be removed by unit operations found in recycling mills. The maintenance of final product quality specifications for a recycling mill while loading the feedstock with a significant quantity of adhesive is the criterion for success of this program...

Compatibility of pressure sensitive adhesives with recycling unit operations

Treesearch

David Bormett; Carl Houtman; Said Abubakr; Joseph Peng

1999-01-01

Removal of pressure sensitive adhesives (PSAs) from recovered paper is a major problem facing the paper recycling industry. As a result of a United States Postal Service (USPS) initiative, which currently purchases about 12% of domestic PSA production, a team was formed consisting of representatives from the USPS, the Forest Products Laboratory, Springborn Testing and...

76 FR 52580 - Procedures To Adjudicate Claims for Personal Injury or Property Damage Arising Out of the...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-08-23

..., Room 2400, St. Louis, MO 63155-9948. FOR FURTHER INFORMATION CONTACT: Ruth A. Przybeck, Chief Counsel... Center, USPS National Tort Center, 1720 Market Street, Room 2400, St. Louis, MO 63155-9948'' in its place... Counsel, Torts, General Law Service Center, USPS National Tort Center, 1720 Market Street, Room 2400, St...

The Ubiquitin-Specific Protease 14 (USP14) Is a Critical Regulator of Long-Term Memory Formation

ERIC Educational Resources Information Center

Jarome, Timothy J.; Kwapis, Janine L.; Hallengren, Jada J.; Wilson, Scott M.; Helmstetter, Fred J.

2014-01-01

Numerous studies have suggested a role for ubiquitin-proteasome-mediated protein degradation in learning-dependent synaptic plasticity; however, very little is known about how protein degradation is regulated at the level of the proteasome during memory formation. The ubiquitin-specific protease 14 (USP14) is a proteasomal deubiquitinating enzyme…

Innovative approach to solving "stickies" problem and developing environmentally benign pressure sensitive adhesives through partnerships

Treesearch

Said M. Abubakr; Joe Peng

1999-01-01

As a result of a United States Postal Service (USPS) initiative, a work team consisting of the USPS, the Forest Products Laboratory (FPL),Springborn Testing and Research (STR), and industry representatives wasformed. The industry representatives include papermakers, paper recyclers,paper collectors, equipment manufacturers, paper users, and adhesive and chemical...

27 CFR 21.54 - Formula No. 27-B.

Code of Federal Regulations, 2013 CFR

2013-04-01

... concentrate containing 25 percent water to 100 gallons of alcohol and, after mixing, by adding thereto 33.5 pounds of water and again mixing. (b) Authorized uses. (1) As a solvent: 141.Shampoos. 210.External pharmaceuticals, not U.S.P. or N.F. 243.Liniments, U.S.P. or N.F. 410.Disinfectants insecticides, fungicides, and...

27 CFR 21.54 - Formula No. 27-B.

Code of Federal Regulations, 2014 CFR

2014-04-01

... concentrate containing 25 percent water to 100 gallons of alcohol and, after mixing, by adding thereto 33.5 pounds of water and again mixing. (b) Authorized uses. (1) As a solvent: 141.Shampoos. 210.External pharmaceuticals, not U.S.P. or N.F. 243.Liniments, U.S.P. or N.F. 410.Disinfectants insecticides, fungicides, and...

77 FR 46685 - In the Matter of: Steven Neal Greenoe, Currently Incarcerated at: Inmate #54450-056, USP Atlanta...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-08-06

... DEPARTMENT OF COMMERCE Bureau of Industry and Security In the Matter of: Steven Neal Greenoe, Currently Incarcerated at: Inmate 54450-056, USP Atlanta, U.S. Penitentiary, P.O. Box 1150160, Atlanta, GA... On January 10, 2012, in the U.S. District Court, District of North Carolina, Steven Neal Greenoe...

20-Hydroxyecdysone upregulates Atg genes to induce autophagy in the Bombyx fat body.

PubMed

Tian, Ling; Ma, Li; Guo, Enen; Deng, Xiaojuan; Ma, Sanyuan; Xia, Qingyou; Cao, Yang; Li, Sheng

2013-08-01

Autophagy is finely regulated at multiple levels and plays crucial roles in development and disease. In the fat body of the silkworm, Bombyx mori, autophagy occurs and Atg gene expression peaks during the nonfeeding molting and pupation stages when the steroid hormone (20-hydroxyecdysone; 20E) is high. Injection of 20E into the feeding larvae upregulated Atg genes and reduced TORC1 activity resulting in autophagy induction in the fat body. Conversely, RNAi knockdown of the 20E receptor partner (USP) or targeted overexpression of a dominant negative mutant of the 20E receptor (EcR (DN) ) in the larval fat body reduced autophagy and downregulated the Atg genes, confirming the importance of 20E-induction of Atg gene expression during pupation. Moreover, in vitro treatments of the larval fat body with 20E upregulated the Atg genes. Five Atg genes were potentially 20E primary-responsive, and a 20E response element was identified in the Atg1 (ortholog of human ULK1) promoter region. Furthermore, RNAi knockdown of 4 key genes (namely Br-C, E74, HR3 and βftz-F1) in the 20E-triggered transcriptional cascade reduced autophagy and downregulated Atg genes to different levels. Taken together, we conclude that in addition to blocking TORC1 activity for autophagosome initiation, 20E upregulates Atg genes to induce autophagy in the Bombyx fat body.

20-hydroxyecdysone upregulates Atg genes to induce autophagy in the Bombyx fat body

PubMed Central

Tian, Ling; Ma, Li; Guo, Enen; Deng, Xiaojuan; Ma, Sanyuan; Xia, Qingyou; Cao, Yang; Li, Sheng

2013-01-01

Autophagy is finely regulated at multiple levels and plays crucial roles in development and disease. In the fat body of the silkworm, Bombyx mori, autophagy occurs and Atg gene expression peaks during the nonfeeding molting and pupation stages when the steroid hormone (20-hydroxyecdysone; 20E) is high. Injection of 20E into the feeding larvae upregulated Atg genes and reduced TORC1 activity resulting in autophagy induction in the fat body. Conversely, RNAi knockdown of the 20E receptor partner (USP) or targeted overexpression of a dominant negative mutant of the 20E receptor (EcRDN) in the larval fat body reduced autophagy and downregulated the Atg genes, confirming the importance of 20E-induction of Atg gene expression during pupation. Moreover, in vitro treatments of the larval fat body with 20E upregulated the Atg genes. Five Atg genes were potentially 20E primary-responsive, and a 20E response element was identified in the Atg1 (ortholog of human ULK1) promoter region. Furthermore, RNAi knockdown of 4 key genes (namely Br-C, E74, HR3 and βftz-F1) in the 20E-triggered transcriptional cascade reduced autophagy and downregulated Atg genes to different levels. Taken together, we conclude that in addition to blocking TORC1 activity for autophagosome initiation, 20E upregulates Atg genes to induce autophagy in the Bombyx fat body. PMID:23674061

Lipid globule size in total nutrient admixtures prepared in three-chamber plastic bags.

PubMed

Driscoll, David F; Thoma, Andrea; Franke, Rolf; Klütsch, Karsten; Nehne, Jörg; Bistrian, Bruce R

2009-04-01

The stability of injectable lipid emulsions in three-chamber plastic (3CP) bags, applying the globule-size limits established by United States Pharmacopeia ( USP ) chapter 729, was studied. A total of five premixed total nutrient admixture (TNA) products packaged in 3CP bags from two different lipid manufacturers containing either 20% soybean oil or a mixture of soybean oil and medium-chain-triglyceride oil as injectable lipid emulsions were tested. Two low-osmolarity 3CP bags and three high-osmolarity 3CP bags were studied. All products were tested with the addition of trace elements and multivitamins. All additive conditions (with and without electrolytes) were tested in triplicate at time 0 (immediately after mixing) and at 6, 24, 30, and 48 hours after mixing; the bags were stored at 24-26 degrees C. All additives were equally distributed in each bag for comparative testing, applying both globule sizing methods outlined in USP chapter 729. Of the bags tested, all bags from one manufacturer were coarse emulsions, showing signs of significant growth in the large-diameter tail when mixed as a TNA formulation and failing the limits set by method II of USP chapter 729 from the outset and throughout the study, while the bags from the other manufacturer were fine emulsions and met these limits. Of the bags that failed, significant instability was noted in one series containing additional electrolytes. Injectable lipid emulsions provided in 3CP bags that did not meet the globule-size limits of USP chapter 729 produced coarser TNA formulations than emulsions that met the USP limits.

Increased acid ceramidase expression depends on upregulation of androgen-dependent deubiquitinases, USP2, in a human prostate cancer cell line, LNCaP.

PubMed

Mizutani, Naoki; Inoue, Minami; Omori, Yukari; Ito, Hiromi; Tamiya-Koizumi, Keiko; Takagi, Akira; Kojima, Tetsuhito; Nakamura, Mitsuhiro; Iwaki, Soichiro; Nakatochi, Masahiro; Suzuki, Motoshi; Nozawa, Yoshinori; Murate, Takashi

2015-10-01

Acid ceramidase (ACDase) metabolizes ceramide to sphingosine, leading to sphingosine 1-phosphate production. Reportedly, ACDase has been upregulated in prostate cancer. However, its regulatory mechanism remains unclear. LNCaP (androgen-sensitive prostate cancer cell line) but not PC3 and DU-145, (androgen-unresponsive cell lines) exhibited the highest ACDase protein. Among three cell lines, ASAH1 mRNA level was not correlated with ACDase protein expression, and the 5'-promoter activity did not show androgen dependency, suggesting the post-transcriptional regulation of ACDase in LNCaP cells. Based on these results, LNCaP was analysed further. Casodex, androgen receptor antagonist, and charcoal-stripped FCS (CS-FCS) decreased ACDase protein and activity, whereas dihydrotestosterone in CS-FCS culture increased ACDase protein and enzyme activity. MG132, a proteasome inhibitor, prevented the decrease of ACDase protein when cultured in CS-FCS, suggesting the involvement of ubiquitin/proteasome system. Reportedly, USP2, a deubiquitinase, plays an important role in LNCaP cells. USP2 siRNA decreased ACDase protein, whereas USP2 overexpression increased ACDase protein of LNCaP cells. However, SKP2, an ubiquitin E3 ligase known to be active in prostate cancer, did not affect androgen-dependent ACDase expression in LNCaP cells. Thus, ACDase regulation by androgen in androgen-sensitive LNCaP cells is mainly due to its prolonged protein half-life by androgen-stimulated USP2 expression. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Chemical Potency and Degradation Products of Medications Stored Over 550 Earth Days at the International Space Station.

PubMed

Wotring, Virginia E

2016-01-01

Medications degrade over time, and degradation is hastened by extreme storage conditions. Current procedures ensure that medications aboard the International Space Station (ISS) are restocked before their expiration dates, but resupply may not be possible on future long-duration exploration missions. For this reason, medications stored on the ISS were returned to Earth for analysis. This was an opportunistic, observational pilot-scale investigation to test the hypothesis that ISS-aging does not cause unusual degradation. Nine medications were analyzed for active pharmaceutical ingredient (API) content and degradant amounts; results were compared to 2012 United States Pharmacopeia (USP) requirements. The medications were two sleep aids, two antihistamines/decongestants, three pain relievers, an antidiarrheal, and an alertness medication. Because the samples were obtained opportunistically from unused medical supplies, each medication was available at only 1 time point and no control samples (samples aged for a similar period on Earth) were available. One medication met USP requirements 5 months after its expiration date. Four of the nine (44% of those tested) medications tested met USP requirements 8 months post expiration. Another three medications (33%) met USP guidelines 2-3 months before expiration. One compound, a dietary supplement used as a sleep aid, failed to meet USP requirements at 11 months post expiration. No unusual degradation products were identified. Limited, evidence-based extension of medication shelf-lives may be possible and would be useful in preparation for lengthy exploration missions. Only analysis of flight-aged samples compared to appropriately matched ground controls will permit determination of the spaceflight environment on medication stability.

21 CFR 139.155 - Enriched noodle products.

Code of Federal Regulations, 2010 CFR

2010-04-01

... contain as an optional ingredient added vitamin D in such quantity that each pound of the finished food contains not less than 250 U.S.P. units and not more than 1000 U.S.P. units of vitamin D; (3) Each such... conform to the specifications of shape and size prescribed for egg macaroni in § 139.150(c). (d) Enriched...

27 CFR 17.132 - U.S.P., N.F., and H.P.U.S. preparations.

Code of Federal Regulations, 2011 CFR

2011-04-01

... NONBEVERAGE PRODUCTS Formulas and Samples Approval of Formulas § 17.132 U.S.P., N.F., and H.P.U.S..., formulas for compounds in which alcohol is a prescribed quantitative ingredient, which are stated in the... formulas and may be used as formulas for drawback products without the filing of TTB Form 5154.1. (b...

27 CFR 17.132 - U.S.P., N.F., and H.P.U.S. preparations.

Code of Federal Regulations, 2010 CFR

2010-04-01

... NONBEVERAGE PRODUCTS Formulas and Samples Approval of Formulas § 17.132 U.S.P., N.F., and H.P.U.S..., formulas for compounds in which alcohol is a prescribed quantitative ingredient, which are stated in the... formulas and may be used as formulas for drawback products without the filing of TTB Form 5154.1. (b...

27 CFR 17.132 - U.S.P., N.F., and H.P.U.S. preparations.

Code of Federal Regulations, 2013 CFR

2013-04-01

... NONBEVERAGE PRODUCTS Formulas and Samples Approval of Formulas § 17.132 U.S.P., N.F., and H.P.U.S..., formulas for compounds in which alcohol is a prescribed quantitative ingredient, which are stated in the... formulas and may be used as formulas for drawback products without the filing of TTB Form 5154.1. (b...

27 CFR 17.132 - U.S.P., N.F., and H.P.U.S. preparations.

Code of Federal Regulations, 2014 CFR

2014-04-01

... NONBEVERAGE PRODUCTS Formulas and Samples Approval of Formulas § 17.132 U.S.P., N.F., and H.P.U.S..., formulas for compounds in which alcohol is a prescribed quantitative ingredient, which are stated in the... formulas and may be used as formulas for drawback products without the filing of TTB Form 5154.1. (b...

27 CFR 17.132 - U.S.P., N.F., and H.P.U.S. preparations.

Code of Federal Regulations, 2012 CFR

2012-04-01

... NONBEVERAGE PRODUCTS Formulas and Samples Approval of Formulas § 17.132 U.S.P., N.F., and H.P.U.S..., formulas for compounds in which alcohol is a prescribed quantitative ingredient, which are stated in the... formulas and may be used as formulas for drawback products without the filing of TTB Form 5154.1. (b...

78 FR 76812 - In the Matter of: Andrew Vincent O'Donnell, Inmate Number-62355-019, USP Atlanta, U.S...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-12-19

... DEPARTMENT OF COMMERCE Bureau of Industry and Security In the Matter of: Andrew Vincent O'Donnell, Inmate Number--62355- 019, USP Atlanta, U.S. Penitentiary, P.O. Box 15060, Atlanta, GA 30315; Order..., Andrew Vincent O'Donnell (``O'Donnell''), was convicted of violating Section 38 of the Arms Export...

Cytoplasmic expression of a thermostable invertase from Thermotoga maritima in Lactococcus lactis.

PubMed

Pek, Han Bin; Lim, Pei Yu; Liu, Chengcheng; Lee, Dong-Yup; Bi, Xuezhi; Wong, Fong Tian; Ow, Dave Siak-Wei

2017-05-01

To evaluate the secretory and cytoplasmic expression of a thermostable Thermogata maritima invertase in Lactococcus lactis. The thermostable invertase from T. maritima was cloned with and without the USP45 secretory peptide into the pNZ8148 vector for nisin-inducible expression in L. lactis. The introduction of an USP45 secretion peptide at the N-terminal of the enzyme led to a loss of protein solubility. Computational homology modeling and hydrophobicity studies indicated that the USP45 peptide exposes a stretch of hydrophobic amino acids on the protein surface resulting in lower solubility. Removal of the USP45 secretion peptide allowed a soluble and functional invertase to be expressed intracellularly in L. lactis. Immobilized metal affinity chromatography purification of the cell lysate with nickel-NTA gave a single protein band on SDS-PAGE, while E. coli-expressed invertase consistently co-purified with an additional band. The yields of the purified invertase from E. coli and L. lactis were 14.1 and 6.3 mg/l respectively. Invertase can be expressed in L. lactis and purified in a functional form. L. lactis is a suitable host for the production of food-grade invertase for use in the food and biotechnology industries.

ProbFAST: Probabilistic functional analysis system tool.

PubMed

Silva, Israel T; Vêncio, Ricardo Z N; Oliveira, Thiago Y K; Molfetta, Greice A; Silva, Wilson A

2010-03-30

The post-genomic era has brought new challenges regarding the understanding of the organization and function of the human genome. Many of these challenges are centered on the meaning of differential gene regulation under distinct biological conditions and can be performed by analyzing the Multiple Differential Expression (MDE) of genes associated with normal and abnormal biological processes. Currently MDE analyses are limited to usual methods of differential expression initially designed for paired analysis. We proposed a web platform named ProbFAST for MDE analysis which uses Bayesian inference to identify key genes that are intuitively prioritized by means of probabilities. A simulated study revealed that our method gives a better performance when compared to other approaches and when applied to public expression data, we demonstrated its flexibility to obtain relevant genes biologically associated with normal and abnormal biological processes. ProbFAST is a free accessible web-based application that enables MDE analysis on a global scale. It offers an efficient methodological approach for MDE analysis of a set of genes that are turned on and off related to functional information during the evolution of a tumor or tissue differentiation. ProbFAST server can be accessed at http://gdm.fmrp.usp.br/probfast.

ProbFAST: Probabilistic Functional Analysis System Tool

PubMed Central

2010-01-01

Background The post-genomic era has brought new challenges regarding the understanding of the organization and function of the human genome. Many of these challenges are centered on the meaning of differential gene regulation under distinct biological conditions and can be performed by analyzing the Multiple Differential Expression (MDE) of genes associated with normal and abnormal biological processes. Currently MDE analyses are limited to usual methods of differential expression initially designed for paired analysis. Results We proposed a web platform named ProbFAST for MDE analysis which uses Bayesian inference to identify key genes that are intuitively prioritized by means of probabilities. A simulated study revealed that our method gives a better performance when compared to other approaches and when applied to public expression data, we demonstrated its flexibility to obtain relevant genes biologically associated with normal and abnormal biological processes. Conclusions ProbFAST is a free accessible web-based application that enables MDE analysis on a global scale. It offers an efficient methodological approach for MDE analysis of a set of genes that are turned on and off related to functional information during the evolution of a tumor or tissue differentiation. ProbFAST server can be accessed at http://gdm.fmrp.usp.br/probfast. PMID:20353576

Under sleeper pads in switches & crossings

NASA Astrophysics Data System (ADS)

Plasek, Otto; Hruzikova, Miroslava

2017-09-01

Under sleeper pads (USPs) have been installed in the Czech railway lines since 2007 into two trial sections in switches and crossings and in a trial section with the aim to reduce rail pitch corrugation. The basic motivation for the construction of the trial track sections in turnouts was an evaluation of USPs’ influence on improvement of track quality as a consequence of ballast bed protection against extreme stress. The paper comprises experience of USPs installation in the Czech Republic in the two trial sections with switches and crossings built in 2007 and 2014. The paper particularly provides an information about USPs applications focusing on the track settlement received by an evaluation of data from the geodetic surveying by precise levelling. Some positive influence of under sleeper pads on the track quality is observed.

100th anniversary of the birth of V L Ginzburg (Scientific session of the Physical Sciences Division of the Russian Academy of Sciences, 5 October 2016)

NASA Astrophysics Data System (ADS)

2017-04-01

A scientific session of the Physical Sciences Division of the Russian Academy of Sciences (RAS) devoted to the centenary of the birth of V L Ginzburg was held on 5 October 2016 in the conference hall of the Lebedev Physical Institute, RAS. The agenda posted on the website of the RAS Physical Sciences Division http://www.gpad.ac.ru comprised the following reports: (1) Ritus V I (Lebedev Physical Institute, RAS, Moscow), "V L Ginzburg and the Atomic project"; (2) Dremin I M (Lebedev Physical Institute, RAS, Moscow), "Unexpected interaction properties between high-energy protons"; (3) Kocharovsky Vl V, Zheleznyakov V V (Institute of Applied Physics, RAS, Nizhny Novgorod), Belyanin A A (Institute of Applied Physics, RAS, Nizhny Novgorod and Texas A \\&M University, USA), Kocharovskaya E R, Kocharovsky V V (Institute of Applied Physics, RAS, Nizhny Novgorod), "Superradiance: the principles of generation and implementation in lasers"; (4) Pudalov V M (Lebedev Physical Institute, RAS, Moscow), "Structure of the superconducting order parameter in high-temperature Fe-based superconductors"; (5) Sadovski M V (Institute of Electrophysics, UB RAS, Ekaterinburg; M N Mikheev Institute of Metal Physics, UB RAS, Ekaterinburg), "High-temperature superconductivity in an FeSe monolayer: Why is T_c so high?" The paper versions of reports 1 and 4 are presented in this Phys. Usp. issue (see pp. 414 and 420, respectively). Report 2, with its content expanded but title unchanged, is to be published as a review paper in Usp. Fiz. Nauk 187 (4) 353 (2017); Phys. Usp. 60 (4) 333 (2017). Report 3, with its content expanded, is to be published as a review paper in Usp. Fiz. Nauk 187 (4) 367 (2017); Phys. Usp. 60 (4) 345 (2017). Report 5, with its content expanded, was published as a review paper in Usp. Fiz. Nauk 186 (10) 1035 (2016); Phys. Usp. 59 (10) 947 (2016). • V L Ginzburg and the Atomic Project, V I Ritus Physics-Uspekhi, 2017, Volume 60, Number 4, Pages 413-418 • On the structure of the superconducting order parameter in high-temperature Fe-based superconductors, T E Kuzmicheva, A V Muratov, S A Kuzmichev, A V Sadakov, Yu A Aleshchenko, V A Vlasenko, V P Martovitsky, K S Pervakov, Yu F Eltsev, V M Pudalov Physics-Uspekhi, 2017, Volume 60, Number 4, Pages 419-429

TCOF1 mutation database: novel mutation in the alternatively spliced exon 6A and update in mutation nomenclature.

PubMed

Splendore, Alessandra; Fanganiello, Roberto D; Masotti, Cibele; Morganti, Lucas S C; Passos-Bueno, M Rita

2005-05-01

Recently, a novel exon was described in TCOF1 that, although alternatively spliced, is included in the major protein isoform. In addition, most published mutations in this gene do not conform to current mutation nomenclature guidelines. Given these observations, we developed an online database of TCOF1 mutations in which all the reported mutations are renamed according to standard recommendations and in reference to the genomic and novel cDNA reference sequences (www.genoma.ib.usp.br/TCOF1_database). We also report in this work: 1) results of the first screening for large deletions in TCOF1 by Southern blot in patients without mutation detected by direct sequencing; 2) the identification of the first pathogenic mutation in the newly described exon 6A; and 3) statistical analysis of pathogenic mutations and polymorphism distribution throughout the gene.

Providing University Education in Physical Geography across the South Pacific Islands: Multi-Modal Course Delivery and Student Grade Performance

ERIC Educational Resources Information Center

Terry, James P.; Poole, Brian

2012-01-01

Enormous distances across the vast South Pacific hinder student access to the main Fiji campus of the regional tertiary education provider, the University of the South Pacific (USP). Fortunately, USP has been a pioneer in distance education (DE) and promotes multi-modal delivery of programmes. Geography has embraced DE, but doubts remain about…

27 CFR 21.92 - Denaturants listed as U.S.P. or N.F.

Code of Federal Regulations, 2010 CFR

2010-04-01

....P. or N.F. 21.92 Section 21.92 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND... for Denaturants § 21.92 Denaturants listed as U.S.P. or N.F. Denaturing materials and products listed in this part as “U.S.P.” or “N.F.” shall meet the specifications set forth in the current United...

27 CFR 21.92 - Denaturants listed as U.S.P. or N.F.

Code of Federal Regulations, 2014 CFR

2014-04-01

....P. or N.F. 21.92 Section 21.92 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND... for Denaturants § 21.92 Denaturants listed as U.S.P. or N.F. Denaturing materials and products listed in this part as “U.S.P.” or “N.F.” shall meet the specifications set forth in the current United...

27 CFR 21.92 - Denaturants listed as U.S.P. or N.F.

Code of Federal Regulations, 2011 CFR

2011-04-01

....P. or N.F. 21.92 Section 21.92 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND... for Denaturants § 21.92 Denaturants listed as U.S.P. or N.F. Denaturing materials and products listed in this part as “U.S.P.” or “N.F.” shall meet the specifications set forth in the current United...

27 CFR 21.92 - Denaturants listed as U.S.P. or N.F.

Code of Federal Regulations, 2013 CFR

2013-04-01

....P. or N.F. 21.92 Section 21.92 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND... for Denaturants § 21.92 Denaturants listed as U.S.P. or N.F. Denaturing materials and products listed in this part as “U.S.P.” or “N.F.” shall meet the specifications set forth in the current United...

27 CFR 21.92 - Denaturants listed as U.S.P. or N.F.

Code of Federal Regulations, 2012 CFR

2012-04-01

....P. or N.F. 21.92 Section 21.92 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND... for Denaturants § 21.92 Denaturants listed as U.S.P. or N.F. Denaturing materials and products listed in this part as “U.S.P.” or “N.F.” shall meet the specifications set forth in the current United...

75 FR 11169 - Reedsport OPT Wave Park Project; Reedsport OPT Wave Park; LLC Notice of Scoping Meetings and...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-03-10

... seabed. The 10 PowerBuoy units would be connected to a single Underwater Substation Pod (USP) via power... transmission cable, buried in the seabed to a depth of 3 to 6 feet, would extend from the USP to an existing... continue within the effluent pipe eastward for approximately 3 miles, where it would connect to the Douglas...

75 FR 32451 - Reedsport OPT Wave Park, LLC; Notice of Application Accepted for Filing, Ready for Environmental...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-06-08

... seabed. The 10 PowerBuoy units would be connected to a single Underwater Substation Pod (USP) via power... transmission cable, buried in the seabed to a depth of 3 to 6 feet, would extend from the USP to an existing... continue within the effluent pipe eastward for approximately 3 miles, where it would connect to the Douglas...

Lessons Learned from the San Diego Urban Systemic Project (USP): Implications for Funders and Future Project Designers

ERIC Educational Resources Information Center

St. John, Mark; Heenan, Barbara; Helms, Jenifer

2007-01-01

This brief draws upon the five-year evaluation study of the San Diego Urban Systemic Project (USP) that Inverness Research Associates conducted from 2001 to 2006. The intended audiences for this brief are those interested in investing in, supporting, or designing initiatives that aim to improve math and science education in large urban districts.…

Gene networks associated with conditional fear in mice identified using a systems genetics approach

PubMed Central

2011-01-01

Background Our understanding of the genetic basis of learning and memory remains shrouded in mystery. To explore the genetic networks governing the biology of conditional fear, we used a systems genetics approach to analyze a hybrid mouse diversity panel (HMDP) with high mapping resolution. Results A total of 27 behavioral quantitative trait loci were mapped with a false discovery rate of 5%. By integrating fear phenotypes, transcript profiling data from hippocampus and striatum and also genotype information, two gene co-expression networks correlated with context-dependent immobility were identified. We prioritized the key markers and genes in these pathways using intramodular connectivity measures and structural equation modeling. Highly connected genes in the context fear modules included Psmd6, Ube2a and Usp33, suggesting an important role for ubiquitination in learning and memory. In addition, we surveyed the architecture of brain transcript regulation and demonstrated preservation of gene co-expression modules in hippocampus and striatum, while also highlighting important differences. Rps15a, Kif3a, Stard7, 6330503K22RIK, and Plvap were among the individual genes whose transcript abundance were strongly associated with fear phenotypes. Conclusion Application of our multi-faceted mapping strategy permits an increasingly detailed characterization of the genetic networks underlying behavior. PMID:21410935

Final Report for Intravenous Fluid Generation (IVGEN) Spaceflight Experiment

NASA Technical Reports Server (NTRS)

McQuillen, John B.; McKay, Terri L.; Griffin, DeVon W.; Brown, Dan F.; Zoldak, John T.

2011-01-01

NASA designed and operated the Intravenous Fluid Generation (IVGEN) experiment onboard the International Space Station (ISS), Increment 23/24, during May 2010. This hardware was a demonstration experiment to generate intravenous (IV) fluid from ISS Water Processing Assembly (WPA) potable water using a water purification technique and pharmaceutical mixing system. The IVGEN experiment utilizes a deionizing resin bed to remove contaminants from feedstock water to a purity level that meets the standards of the United States Pharmacopeia (USP), the governing body for pharmaceuticals in the United States. The water was then introduced into an IV bag where the fluid was mixed with USP-grade crystalline salt to produce USP normal saline (NS). Inline conductivity sensors quantified the feedstock water quality, output water purity, and NS mixing uniformity. Six 1.5-L bags of purified water were produced. Two of these bags were mixed with sodium chloride to make 0.9 percent NS solution. These two bags were returned to Earth to test for compliance with USP requirements. On-orbit results indicated that all of the experimental success criteria were met with the exception of the salt concentration. Problems with a large air bubble in the first bag of purified water resulted in a slightly concentrated saline solution of 117 percent of the target value of 0.9 g/L. The second bag had an inadequate amount of salt premeasured into the mixing bag resulting in a slightly deficient salt concentration of 93.8 percent of the target value. The USP permits a range from 95 to 105 percent of the target value. The testing plans for improvements for an operational system are also presented.

Quality by design (QbD) based development and validation of an HPLC method for amiodarone hydrochloride and its impurities in the drug substance.

PubMed

Karmarkar, S; Yang, X; Garber, R; Szajkovics, A; Koberda, M

2014-11-01

The USP monograph describes an HPLC method for seven impurities in the amiodarone drug substance using a L1 column, 4.6mm×150mm, 5μm packing (PF listed ODS2 GL-Science, Inertsil column) at 30°C with detection at 240nm. The standard contains 0.01mg/mL of amiodarone, and USP specified impurities D and E with a resolution requirement of NLT 3.5 between peaks D and E. Impurities in a 5mg/mL sample are quantitated against the standard. Impurity A peak elutes just before peak D. We observed two problems with the method; the column lot-to-lot variability resulted in unresolved A, D, and E peaks, and peak D in the sample preparation eluted much later than that in the standard solution. Therefore, optimization experiments were conducted on the USP method following the QbD approach with Fusion AE™ software (S-Matrix Corporation). The resulting optimized conditions were within the allowable changes per USP 〈621〉. Lot-to-lot variability was negligible with the Atlantis T3 (Waters Corporation) L1 column. Peak D retention time remained constant from standard to sample. The optimized method was validated in terms of accuracy, precision, linearity, range, LOQ/LOD, specificity, robustness, equivalency to the USP method, and solution stability. The QbD based development helped in generating a design space and operating space with knowledge of all method performance characteristics and limitations and successful method robustness within the operating space. Copyright © 2014 Elsevier B.V. All rights reserved.

TDEM survey in urban environmental for hydrogeological study at USP campus in São Paulo city, Brazil

NASA Astrophysics Data System (ADS)

Porsani, Jorge Luís; Bortolozo, Cassiano Antonio; Almeida, Emerson Rodrigo; Sobrinho, Esther Novais Santos; Santos, Thiago Gomes dos

2012-01-01

In this work, some TDEM (Time Domain Electromagnetic) results at USP ( University of São Paulo) campus in São Paulo city, Brazil, are presented. The data were acquired focusing on two mains objectives: (i) to map geoelectrical stratigraphy of São Paulo sedimentary basin, emphasizing on hydrogeological studies about sedimentary and crystalline aquifers, and (ii) to analyze the viability of TDEM data acquisition use in urban environment. The study area is located in São Paulo basin border, characterized by Resende and São Paulo formations, which are constituted by sand-clays sediments over a granite-gneissic basement. Two equipments were used in order to acquire database: Protem47 (low power), and Protem57-MK2 (high power). Capacitive noise affect obtained data with Protem47 due to the presence of metal pipes buried at IAG/USP (Institute of Astronomy, Geophysics, and Atmospheric Science) test site at USP. On the other hand, capacitive noise did not affect acquired data with Protem57-MK2, and the data present high signal to noise ratio. Surveys helped in determining sedimentary and crystalline aquifers, characterized by a fracture zone with water inside basin basement (conductive zone). Results show good agreement with local geology obtained from lithological boreholes located in the study areas. Moreover, it shows that TDEM method can be used in urban environments with a countless potential in hydrogeological studies, offering great reliability. Studies showed that main TDEM-method limitation at USP was the lack of space for opening the transmitter loop. Results are very promising and open new perspectives for TDEM-method use in urban environments as this area remains unexplored.

Measurements of donor endothelial keratoplasty lenticules prepared from fresh donated whole eyes by using ultrasound and optical coherence tomography.

PubMed

Kanavi, Mozhgan Rezaei; Nemati, Farzan; Chamani, Tahereh; Kheiri, Bahar; Javadi, Mohammad Ali

2017-03-01

This study was conducted to analyze the profile and thickness of endothelial keratoplasty lenticules prepared from fresh donated whole eyes with Visante optical coherence tomography (V-OCT) compared to measurements obtained from ultrasound pachymetry (USP) at the Central Eye Bank of Iran. Microkeratome-assisted precut corneas were prepared for Descemet stripping automated endothelial keratoplasty by using standard eye bank protocol. Central posterior lenticule thickness (CPLT) on fresh whole eye, before excising corneoscleral disc and transferring to Optisol-GS, was measured by USP. V-OCT was used to measure central, paracentral, and midperipheral thicknesses of lenticules after transferring the tissue to Optisol-GS. Chi Square and Bonferroni tests were respectively used to uncover the differences between the USP and V-OCT measurements and also the thickness profile of lenticules. Postoperative reports for the entire transplanted lenticules were recorded. Accordingly, on evaluation of 312 enrolled precut corneas, CPLT measurements by V-OCT versus USP were statistically different (mean: 136 µm vs 165 µm, respectively; P = 0.008). Thickness profile of the posterior lenticules revealed increased thickness from the central to the peripheral parts of the cornea (mean increase of 16 µm at the pericentral and 64.2 µm at the peripheral locations, respectively); however, the increase in the thickness was relatively symmetrical. Postoperative reports of transplanted lenticules were unremarkable, since there were no posterior flap detachments. In essence, V-OCT measurements of microkeratome-assisted precut lenticules prepared from fresh donated whole eyes averaged 29 μm thinner than USP measurements and revealed a significant but symmetric increase of thickness towards the peripheral parts of the corneas. However, the variation in the thickness profile did not affect the attachment or the clarity of transplanted precut lenticlues.

The Endosome-associated Deubiquitinating Enzyme USP8 Regulates BACE1 Enzyme Ubiquitination and Degradation.

PubMed

Yeates, Eniola Funmilayo Aduke; Tesco, Giuseppina

2016-07-22

The β-site amyloid precursor protein-cleaving enzyme (BACE1) is the rate-limiting enzyme in the production of amyloid-β, the toxic peptide that accumulates in the brain of subjects affected by Alzheimer disease. Our previous studies have shown that BACE1 is degraded via the lysosomal pathway and that that depletion of the trafficking molecule Golgi-localized γ-ear-containing ARF-binding protein 3 (GGA3) results in increased BACE1 levels and activity because of impaired lysosomal degradation. We also determined that GGA3 regulation of BACE1 levels requires its ability to bind ubiquitin. Accordingly, we reported that BACE1 is ubiquitinated at lysine 501 and that lack of ubiquitination at lysine 501 produces BACE1 stabilization. Ubiquitin conjugation is a reversible process mediated by deubiquitinating enzymes. The ubiquitin-specific peptidase 8 (USP8), an endosome-associated deubiquitinating enzyme, regulates the ubiquitination, trafficking, and lysosomal degradation of several plasma membrane proteins. Here, we report that RNAi-mediated depletion of USP8 reduced levels of both ectopically expressed and endogenous BACE1 in H4 human neuroglioma cells. Moreover, USP8 depletion increased BACE1 ubiquitination, promoted BACE1 accumulation in the early endosomes and late endosomes/lysosomes, and decreased levels of BACE1 in the recycling endosomes. We also found that decreased BACE1 protein levels were accompanied by a decrease in BACE1-mediated amyloid precursor protein cleavage and amyloid-β levels. Our findings demonstrate that USP8 plays a key role in the trafficking and degradation of BACE1 by deubiquitinating lysine 501. These studies suggest that therapies able to accelerate BACE1 degradation (e.g. by increasing BACE1 ubiquitination) may represent a potential treatment for Alzheimer disease. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Using Tidal Fluctuation-Induced Dynamics of Radium Isotopes (224Ra, 223Ra, and 228Ra) to Trace the Hydrodynamics and Geochemical Reactions in a Coastal Groundwater Mixing Zone

NASA Astrophysics Data System (ADS)

Liu, Yi; Jiao, Jiu Jimmy; Liang, Wenzhao; Luo, Xin

2018-04-01

The reactive transport of radium isotopes (224Ra, 223Ra, and 228Ra) in coastal groundwater mixing zones (CGMZs) is sensitive to shifts of redox conditions and geochemical reactions induced by tidal fluctuation. This study presents a spatial distribution and temporal variation of radium isotopes in the CGMZ for the first time. Results show that the activity of radium isotopes in the upper saline plume (USP) is comparatively low due to a short residence time and mixing loss induced by the infiltration of low radium seawater whereas the activity of radium isotopes in the salt wedge (SW) is comparatively high due to a long residence time in the aquifer. The spatial distribution of radium isotopes is determined by the partitioning of radium isotopes, groundwater residence time, and relative ingrowth rates of radium isotopes. In addition, the variation of radium isotopes in the USP lags slightly (˜0 h) whereas the fluctuation of radium isotopes in the SW lags significantly (˜12 h) behind sea level oscillation. Tidal fluctuation affects the partitioning of radium isotopes through controlling seawater infiltration and subsequently influences the dynamics of radium isotopes in the USP. Concurrently, seawater infiltration significantly affects geochemical processes such as the production of nutrients and total alkalinity. Therefore, radium dynamics in the USP have implications for these geochemical processes. The variation of radium isotopes in the USP also has potential implications for transformation of trace metals such as iron and manganese because of the close affinity of radium isotopes to manganese and iron oxides.

The Endosome-associated Deubiquitinating Enzyme USP8 Regulates BACE1 Enzyme Ubiquitination and Degradation*

PubMed Central

Yeates, Eniola Funmilayo Aduke; Tesco, Giuseppina

2016-01-01

The β-site amyloid precursor protein-cleaving enzyme (BACE1) is the rate-limiting enzyme in the production of amyloid-β, the toxic peptide that accumulates in the brain of subjects affected by Alzheimer disease. Our previous studies have shown that BACE1 is degraded via the lysosomal pathway and that that depletion of the trafficking molecule Golgi-localized γ-ear-containing ARF-binding protein 3 (GGA3) results in increased BACE1 levels and activity because of impaired lysosomal degradation. We also determined that GGA3 regulation of BACE1 levels requires its ability to bind ubiquitin. Accordingly, we reported that BACE1 is ubiquitinated at lysine 501 and that lack of ubiquitination at lysine 501 produces BACE1 stabilization. Ubiquitin conjugation is a reversible process mediated by deubiquitinating enzymes. The ubiquitin-specific peptidase 8 (USP8), an endosome-associated deubiquitinating enzyme, regulates the ubiquitination, trafficking, and lysosomal degradation of several plasma membrane proteins. Here, we report that RNAi-mediated depletion of USP8 reduced levels of both ectopically expressed and endogenous BACE1 in H4 human neuroglioma cells. Moreover, USP8 depletion increased BACE1 ubiquitination, promoted BACE1 accumulation in the early endosomes and late endosomes/lysosomes, and decreased levels of BACE1 in the recycling endosomes. We also found that decreased BACE1 protein levels were accompanied by a decrease in BACE1-mediated amyloid precursor protein cleavage and amyloid-β levels. Our findings demonstrate that USP8 plays a key role in the trafficking and degradation of BACE1 by deubiquitinating lysine 501. These studies suggest that therapies able to accelerate BACE1 degradation (e.g. by increasing BACE1 ubiquitination) may represent a potential treatment for Alzheimer disease. PMID:27302062

AGEs-RAGE system down-regulates Sirt1 through the ubiquitin-proteasome pathway to promote FN and TGF-β1 expression in male rat glomerular mesangial cells.

PubMed

Huang, Kai-Peng; Chen, Cheng; Hao, Jie; Huang, Jun-Ying; Liu, Pei-Qing; Huang, He-Qing

2015-01-01

We previously demonstrated that advanced glycation-end products (AGEs) promote the pathological progression of diabetic nephropathy by decreasing silent information regulator 2-related protein 1 (Sirt1) expression in glomerular mesangial cells (GMCs). Here, we investigated whether AGEs-receptor for AGEs (RAGE) system down-regulated Sirt1 expression through ubiquitin-proteasome pathway and whether Sirt1 ubiquitination affected fibronectin (FN) and TGF-β1, 2 fibrotic indicators in GMCs. Sirt1 was polyubiquitinated and subsequently degraded by proteasome. AGEs increased Sirt1 ubiquitination and proteasome-mediated degradation, shortened Sirt1 half-life, and promoted FN and TGF-β1 expression. Ubiquitin-specific protease 22 (USP22) reduced Sirt1 ubiquitination and degradation and decreased FN and TGF-β1 expression in GMCs under both basal and AGEs-treated conditions. USP22 depletion enhanced Sirt1 degradation and displayed combined effects with AGEs to further promote FN and TGF-β1 expression. RAGE functioned crucial mediating roles in these processes via its C-terminal cytosolic domain. Inhibiting Sirt1 by EX-527 substantially suppressed the down-regulation of FN and TGF-β1 resulting from USP22 overexpression under both normal and AGEs-treated conditions, eventually leading to their up-regulation in GMCs. These results indicated that the AGEs-RAGE system increased the ubiquitination and subsequent proteasome-mediated degradation of Sirt1 by reducing USP22 level, and AGEs-RAGE-USP22-Sirt1 formed a cascade pathway that regulated FN and TGF-β1 level, which participated in the pathological progression of diabetic nephropathy.

Effects of hydrogeological properties on sea-derived benzene transport in unconfined coastal aquifers.

PubMed

Li, Wei-Ci; Ni, Chuen-Fa; Tsai, Chia-Hsing; Wei, Yi-Ming

2016-05-01

This paper presents numerical investigations on quantifying the hydrodynamic effects of coastal environment factors, including tidal fluctuations, beach slopes, hydraulic conductivity, and hydraulic gradients on sea-derived benzene transport in unconfined coastal aquifers. A hydrologic transport and mixed geochemical kinetic/equilibrium reactions in saturated-unsaturated media model was used to simulate the spatial and temporal behaviors of the density flow and benzene transport for various hydrogeological conditions. Simulation results indicated that the tidal fluctuations lead to upper saline plumes (USPs) near the groundwater and seawater interfaces. Such local circulation zones trapped the seaward benzene plumes and carried them down in aquifers to the depth depending on the tide amplitudes and beach slopes across the coastal lines. Comparisons based on different tidal fluctuations, beach slopes, hydraulic conductivity, and hydraulic gradient were systematically conducted and quantified. The results indicated that areas with USPs increased with the tidal amplitude and decreased with the increasing beach slope. However, the variation of hydraulic conductivity and hydraulic gradient has relatively small influence on the patterns of flow fields in the study. The increase of the USP depths was linearly correlated with the increase of the tidal amplitudes. The benzene reactive transport simulations revealed that the plume migrations are mainly controlled by the local flow dynamics and constrained in the USP circulation zones. The self-cleaning process of a coastal aquifer is time-consuming, typically requiring double the time of the contamination process that the benzene plume reach the bottom of a USP circulation zone. The presented systematic analysis can provide useful information for rapidly evaluating seaward contaminants along a coastal line with available hydrogeological properties.

Termite and fungal resistance of in situ polymerized tributyltin acrylate and acetylated Indonesian and USA wood

Treesearch

Rebecca E. Ibach; Yusuf Sudo Hadi; Dodi Nandika; Sulaeman Yusuf; Yuliati Indrayani

2000-01-01

Wood [Indonesian pine (IP), Indonesian Jabon (IJ) and USA southern yellow pine (USP)] was either in situ polymerized with tributyltin acrylate (TBTA) or acetylated and then exposed to termite and fungal degradation both in laboratory tests and field exposure. The TBTA woods had an average weight percent gain (WPG) of 11% for IP, 12% for IJ, and 10% for USP. The...

Constraining Source Terms, Regional Attenuation Models, and Site Effects (Postprint)

DTIC Science & Technology

2012-03-22

YAK ZRNK 10245 10831 11116 11346 12278...MDJ KAR NIL PDG QIZ RAYN SSE TATO TKM2TLG TLY UCHULHL ULN USP VOS WMQ WUS XAN YAK ZRNK 10245 10831 11116 11346 12278 12376 12574 12937 1311713130...LSA LZH MAKZUZ MDJ KAR NIL PDG QIZ RAYN SSE TATO TKM2TLG TLY UCHULHL ULN USP VOS WMQ WUS XAN YAK ZRNK 10245 10831 11116 11346 12278 12376 12574

Comparison of anesthetic efficacy between lidocaine with and without magnesium sulfate USP 50% for inferior alveolar nerve blocks in patients with symptomatic irreversible pulpitis.

PubMed

Shetty, Krishna Prasad; Satish, Sarvepalli Venkata; Kilaru, Krishna Rao; Sardar, Poonam; Luke, Alexander M

2015-04-01

The purpose of this prospective, randomized, double-blind, placebo-controlled study was to compare the anesthetic efficacy between lidocaine with and without magnesium sulfate USP 50% for inferior alveolar nerve (IAN) blocks in patients with symptomatic irreversible pulpitis. One hundred patients with symptomatic irreversible pulpitis of mandibular posterior teeth were selected for the study. The patients received 1 mL magnesium sulfate USP 50% or distilled water (placebo) 1 hour before administration of conventional IAN block. Endodontic access cavity preparation was initiated 15 minutes after the IAN block injection. Lip numbness was recorded for all the patients. Success of IAN block was defined as no or mild pain on the visual analogue scale during access cavity preparation and initial instrumentation. The success rate for the IAN block was 58% for magnesium sulfate group and 32% for the placebo group, with statistically significant difference between the 2 groups (P = .016). In mandibular posterior teeth diagnosed with symptomatic irreversible pulpitis, preoperative administration of 1 mL magnesium sulfate USP 50% resulted in statistically significant increase in success of IAN block compared with placebo. Copyright © 2015 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Effect on moisture permeability of typewriting on unit dose package surfaces.

PubMed

Rackson, J T; Zellhofer, M J; Birmingham, P H

1984-10-01

The effects of typewriting on labels of two unit dose packages with respect to moisture permeability were examined. Using an electric typewriter, a standard label format was imprinted on two different types of class A unit dose packages: (1) a heat-sealed paper-backed foil and cellofilm strip pouch, and (2) a copolyester and polyethylene multiple-cup blister with a heat-sealed paper-backed foil and cellofilm cover. The labels were typed at various typing-element impact settings. The official USP test for water permeation was then performed on typed packages and untyped control packages. The original untyped packages were confirmed to be USP class A quality. The packages for which successively harder impact settings were used showed a corresponding increase in moisture permeability. This resulted in a lowering of USP package ratings from class A to class B and D, some of which would be unsuitable for use in any unit dose system under current FDA repackaging standards. Typing directly onto the label of a unit dose package before it is sealed will most likely damage the package and possibly make it unfit for use. Pharmacists who must type labels for the unit dose packages studied should use the lowest possible typewriter impact setting and test for damage using the USP moisture-permeation test.

Aktiv De-Orbiting Onboard System from Leo of Upper Stages of Launchers

NASA Astrophysics Data System (ADS)

Trushlyakov, V.; Shalay, V.; Shatrov, J.; Jakovlev, M.; Kostantino, A.

2009-03-01

The active de-orbiting onboard system (VDOS) of upper separable parts (USP) stage of launchers from LEO into orbits of utilization with term of existence orbital lifetimes till 25 years is offered. ADOS it is based on use of power resources of not produced rests of liquid fuel onboard USP launchers with liquid propulsion module (LPM). Following systems enter in structure VDOS: the gas jet propulsion system consisting of a system of gasification, chambers of gas engines (GE), a control system. For gasification of the rests of liquid fuel the heat-carrier received in the autonomous gas generator is used. The gasification propellant components from each tank with temperature and the pressure determined by strength of the corresponding tank, move in chambers of the GE established on a top of a fuel compartment. After separation of a payload execute twist USP for preservation of its position in the space by activity of the GE. Ways of increase of a system effectiveness of gasification are offered by superposition on the entered heat-carrier of ultrasonic oscillations, and also introduction in gaseous fuel nanopowder of aluminum. The volume of adaptations of construction USP, connected with introduction VDOS does not exceed 5 % from weight of a dry construction.

Dietary supplements quality analysis tools from the United States Pharmacopeia.

PubMed

Sarma, Nandakumara; Giancaspro, Gabriel; Venema, Jaap

2016-01-01

The United States Food and Drug Administration (FDA) issued the dietary supplement (DS) current good manufacturing practice (GMP) regulations in compliance with the mandate from the Dietary Supplements Health and Education Act (DSHEA), with the intention of protecting public health by ensuring the quality of DS. The GMP regulations require manufacturers to establish their own quality specifications for identity, purity, strength, composition, and absence of contaminants. Numerous FDA-conducted GMP inspections found that the private specifications set by these manufacturers are often insufficient to ensure adequate quality of dietary ingredients and DS. Wider use of the public standards developed by the United States Pharmacopeial Convention (USP), in conjunction with GMP compliance, can help ensure quality and consistency of DS as they do for medicines. Public health protection could be enhanced by strengthening the GMP provisions to require conformance with relevant United States Pharmacopeia-National Formulary (USP-NF) standards, or in the absence of USP standards, other public compendial standards. Another serious concern is the presence of synthetic drugs and drug analogues in products marketed as DS. Use of the new USP General Chapter Adulteration of Dietary Supplements with Drugs and Drug Analogs <2251> may reduce the exposure of consumers to dangerous drugs disguised as DS. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Gender-Specific Gene Expression in Post-Mortem Human Brain: Localization to Sex Chromosomes

PubMed Central

Vawter, Marquis P; Evans, Simon; Choudary, Prabhakara; Tomita, Hiroaki; Meador-Woodruff, Jim; Molnar, Margherita; Li, Jun; Lopez, Juan F; Myers, Rick; Cox, David; Watson, Stanley J; Akil, Huda; Jones, Edward G; Bunney, William E

2011-01-01

Gender differences in brain development and in the prevalence of neuropsychiatric disorders such as depression have been reported. Gender differences in human brain might be related to patterns of gene expression. Microarray technology is one useful method for investigation of gene expression in brain. We investigated gene expression, cell types, and regional expression patterns of differentially expressed sex chromosome genes in brain. We profiled gene expression in male and female dorsolateral prefrontal cortex, anterior cingulate cortex, and cerebellum using the Affymetrix oligonucleotide microarray platform. Differentially expressed genes between males and females on the Y chromosome (DBY, SMCY, UTY, RPS4Y, and USP9Y) and X chromosome (XIST) were confirmed using real-time PCR measurements. In situ hybridization confirmed the differential expression of gender-specific genes and neuronal expression of XIST, RPS4Y, SMCY, and UTY in three brain regions examined. The XIST gene, which silences gene expression on regions of the X chromosome, is expressed in a subset of neurons. Since a subset of neurons express gender-specific genes, neural subpopulations may exhibit a subtle sexual dimorphism at the level of differences in gene regulation and function. The distinctive pattern of neuronal expression of XIST, RPS4Y, SMCY, and UTY and other sex chromosome genes in neuronal subpopulations may possibly contribute to gender differences in prevalence noted for some neuropsychiatric disorders. Studies of the protein expression of these sex- chromosome-linked genes in brain tissue are required to address the functional consequences of the observed gene expression differences. PMID:14583743

Halophytes: Potential Resources for Salt Stress Tolerance Genes and Promoters

PubMed Central

Mishra, Avinash; Tanna, Bhakti

2017-01-01

Halophytes have demonstrated their capability to thrive under extremely saline conditions and thus considered as one of the best germplasm for saline agriculture. Salinity is a worldwide problem, and the salt-affected areas are increasing day-by-day because of scanty rainfall, poor irrigation system, salt ingression, water contamination, and other environmental factors. The salinity stress tolerance mechanism is a very complex phenomenon, and some pathways are coordinately linked for imparting salinity tolerance. Though a number of salt responsive genes have been reported from the halophytes, there is always a quest for promising stress-responsive genes that can modulate plant physiology according to the salt stress. Halophytes such as Aeluropus, Mesembryanthemum, Suaeda, Atriplex, Thellungiella, Cakile, and Salicornia serve as a potential candidate for the salt-responsive genes and promoters. Several known genes like antiporters (NHX, SOS, HKT, VTPase), ion channels (Cl−, Ca2+, aquaporins), antioxidant encoding genes (APX, CAT, GST, BADH, SOD) and some novel genes such as USP, SDR1, SRP etc. were isolated from halophytes and explored for developing stress tolerance in the crop plants (glycophytes). It is evidenced that stress triggers salt sensors that lead to the activation of stress tolerance mechanisms which involve multiple signaling proteins, up- or down-regulation of several genes, and finally the distinctive or collective effects of stress-responsive genes. In this review, halophytes are discussed as an excellent platform for salt responsive genes which can be utilized for developing salinity tolerance in crop plants through genetic engineering. PMID:28572812

Halophytes: Potential Resources for Salt Stress Tolerance Genes and Promoters.

PubMed

Mishra, Avinash; Tanna, Bhakti

2017-01-01

Halophytes have demonstrated their capability to thrive under extremely saline conditions and thus considered as one of the best germplasm for saline agriculture. Salinity is a worldwide problem, and the salt-affected areas are increasing day-by-day because of scanty rainfall, poor irrigation system, salt ingression, water contamination, and other environmental factors. The salinity stress tolerance mechanism is a very complex phenomenon, and some pathways are coordinately linked for imparting salinity tolerance. Though a number of salt responsive genes have been reported from the halophytes, there is always a quest for promising stress-responsive genes that can modulate plant physiology according to the salt stress. Halophytes such as Aeluropus, Mesembryanthemum, Suaeda, Atriplex, Thellungiella, Cakile , and Salicornia serve as a potential candidate for the salt-responsive genes and promoters. Several known genes like antiporters ( NHX, SOS, HKT, VTPase ), ion channels (Cl - , Ca 2+ , aquaporins), antioxidant encoding genes ( APX, CAT, GST, BADH, SOD ) and some novel genes such as USP, SDR1, SRP etc. were isolated from halophytes and explored for developing stress tolerance in the crop plants (glycophytes). It is evidenced that stress triggers salt sensors that lead to the activation of stress tolerance mechanisms which involve multiple signaling proteins, up- or down-regulation of several genes, and finally the distinctive or collective effects of stress-responsive genes. In this review, halophytes are discussed as an excellent platform for salt responsive genes which can be utilized for developing salinity tolerance in crop plants through genetic engineering.

Mapping autosomal recessive intellectual disability: combined microarray and exome sequencing identifies 26 novel candidate genes in 192 consanguineous families.

PubMed

Harripaul, R; Vasli, N; Mikhailov, A; Rafiq, M A; Mittal, K; Windpassinger, C; Sheikh, T I; Noor, A; Mahmood, H; Downey, S; Johnson, M; Vleuten, K; Bell, L; Ilyas, M; Khan, F S; Khan, V; Moradi, M; Ayaz, M; Naeem, F; Heidari, A; Ahmed, I; Ghadami, S; Agha, Z; Zeinali, S; Qamar, R; Mozhdehipanah, H; John, P; Mir, A; Ansar, M; French, L; Ayub, M; Vincent, J B

2018-04-01

Approximately 1% of the global population is affected by intellectual disability (ID), and the majority receive no molecular diagnosis. Previous studies have indicated high levels of genetic heterogeneity, with estimates of more than 2500 autosomal ID genes, the majority of which are autosomal recessive (AR). Here, we combined microarray genotyping, homozygosity-by-descent (HBD) mapping, copy number variation (CNV) analysis, and whole exome sequencing (WES) to identify disease genes/mutations in 192 multiplex Pakistani and Iranian consanguineous families with non-syndromic ID. We identified definite or candidate mutations (or CNVs) in 51% of families in 72 different genes, including 26 not previously reported for ARID. The new ARID genes include nine with loss-of-function mutations (ABI2, MAPK8, MPDZ, PIDD1, SLAIN1, TBC1D23, TRAPPC6B, UBA7 and USP44), and missense mutations include the first reports of variants in BDNF or TET1 associated with ID. The genes identified also showed overlap with de novo gene sets for other neuropsychiatric disorders. Transcriptional studies showed prominent expression in the prenatal brain. The high yield of AR mutations for ID indicated that this approach has excellent clinical potential and should inform clinical diagnostics, including clinical whole exome and genome sequencing, for populations in which consanguinity is common. As with other AR disorders, the relevance will also apply to outbred populations.

Ultrashort Pulse (USP) Laser-Matter Interactions

DTIC Science & Technology

2013-03-05

spectroscopy • Frequency/time transfer • High-capacity comms • Coherent LIDAR • Optical clocks • Calibration Material Science ultrashort, high...Laboratory 41 Laser -driven x-rays generation (0.1 – 10 MeV) • Scattering from a 300 MeV electron beam can Doppler shift a 1-eV energy laser ...1 Integrity  Service  Excellence Ultrashort Pulse (USP) Laser – Matter Interactions 5 MAR 2013 Dr. Riq Parra Program Officer AFOSR/RTB

21 CFR 522.1680 - Oxytocin injection.

Code of Federal Regulations, 2012 CFR

2012-04-01

... as conditions require: mL U.S.P. units Cats 0.25 to 0.5 5 to 10. Dogs 0.25 to 1.5 5 to 30. Ewes, Sows... desirable. The following dosage is recommended and may be repeated as conditions require: mL U.S.P. units Cows 0.5 to 1.0 10 to 20. Sows 0.25 to 1.0 5 to 20. (2) Indications for use. Oxytocin may be used as a...

The first Pan-Podocnemididae turtle egg from the Presidente Prudente Formation (Late Cretaceous, Bauru Group), Brazil.

PubMed

Marsola, Júlio C De A; Grellet-Tinner, Gerald; Montefeltro, Felipe C; Langer, Max C

2014-10-08

Pan-Podocnemididae turtles are ubiquitous in Late Cretaceous rocks of the Bauru Group in southeastern Brazil. This group of side-necked turtles is particularly abundant in a turtle-bearing site of the Presidente Prudente Formation known as Tartaruguito. Here, we describe the first turtle egg (LPRP-USP 0052) from the Tartaruguito site. LPRP-USP 0052 is nearly complete but misses a pole and measures 5,1 and 2,9-2,2 centimeters due to its flattened minor axis. The egg morphology and microstructure were analyzed by observations performed with CT, Optic Microscopy, Scanning Electronic Microscopy and Wave Dispersion Energy analyses. The eggshell ranges from 145 to160 micrometers thick. Considering the matching morphology of the new specimen and its provenance from the stratigraphic horizon that yielded only the podocnemidids Bauruemys and Roxochelys, it is most likely that LPRP-USP 0052 was produced by a podocnemidid turtle. 

Nevada State plan; final approval determination. Occupational Safety and Health Administration (OSHA), U.S. Department of Labor. Final State plan approval--Nevada.

PubMed

2000-04-18

This document amends OSHA's regulations to reflect the Assistant Secretary's decision granting final approval to the Nevada State plan. As a result of this affirmative determination under section 18(e) of the Occupational Safety and Health Act of 1970, Federal OSHA's standards and enforcement authority no longer apply to occupational safety and health issues covered by the Nevada plan, and authority for Federal concurrent jurisdiction is relinquished. Federal enforcement jurisdiction is retained over any private sector maritime employment, private sector employers on Indian land, and any contractors or subcontractors on any Federal establishment where the land is exclusive Federal jurisdiction. Federal jurisdiction remains in effect with respect to Federal government employers and employees. Federal OSHA will also retain authority for coverage of the United States Postal Service (USPS), including USPS employees, contract employees, and contractor-operated facilities engaged in USPS mail operations.

Evaluation of the antimicrobial activity of three irrigating solutions in teeth with pulpal necrosis.

PubMed

Ferreira, C M; Bonifácio, K C; Fröner, I C; Ito, I Y

1999-01-01

The antimicrobial activity of 0.4% papaine gel (FCF-USP), an antibacterial product derived from 3.3% castor oil (IQSC-USP), and 0.5% sodium hypochlorite (FORP-USP) was evaluated in teeth with radiographically visible pulpal necrosis and periapical lesion in vivo. After cavity access, under aseptic conditions, a first harvesting was performed. The 3 irrigating solutions were used for biomechanical preparation. After 72 hours, a second harvesting was performed, also under aseptic conditions. The number of colony forming units (cfu) was counted with a stereomicroscope under reflected light. Castor oil and 0.5% sodium hypochlorite presented similar antimicrobial activities for the reduction of the anaerobe number, S. mutans and streptococci; however, the papaine gel showed lower activity. We conclude that both castor oil and sodium hypochlorite are effective as antimicrobial agents and can be used in the treatment of root canals with pulpal necrosis.

Improvements in soft gelatin capsule sample preparation for USP-based simethicone FTIR analysis.

PubMed

Hargis, Amy D; Whittall, Linda B

2013-02-23

Due to the absence of a significant chromophore, Simethicone raw material and finished product analysis is achieved using a FTIR-based method that quantifies the polydimethylsiloxane (PDMS) component of the active ingredient. The method can be found in the USP monographs for several dosage forms of Simethicone-containing pharmaceutical products. For soft gelatin capsules, the PDMS assay values determined using the procedure described in the USP method were variable (%RSDs from 2 to 9%) and often lower than expected based on raw material values. After investigation, it was determined that the extraction procedure used for sample preparation was causing loss of material to the container walls due to the hydrophobic nature of PDMS. Evaluation revealed that a simple dissolution of the gelatin capsule fill in toluene provided improved assay results (%RSDs≤0.5%) as well as a simplified and rapid sample preparation. Copyright © 2012 Elsevier B.V. All rights reserved.

A novel method for the determination of chemical purity and assay of menaquinone-7. Comparison with the methods from the official USP monograph.

PubMed

Jedynak, Łukasz; Jedynak, Maria; Kossykowska, Magdalena; Zagrodzka, Joanna

2017-02-20

An HPLC method with UV detection and separation with the use of a C30 reversed phase analytical column for the determination of chemical purity and assay of menaquinone-7 (MK7) in one chromatographic run was developed. The method is superior to the methods published in the USP Monograph in terms of selectivity, sensitivity and accuracy, as well as time, solvent and sample consumption. The developed methodology was applied to MK7 samples of active pharmaceutical ingredient (API) purity, MK7 samples of lower quality and crude MK7 samples before purification. The comparison of the results revealed that the use of USP methodology could lead to serious overestimation (up to a few percent) of both purity and MK7 assay in menaquinone-7 samples. Copyright © 2016 Elsevier B.V. All rights reserved.

Mapping the interactome of HPV E6 and E7 oncoproteins with the ubiquitin-proteasome system.

PubMed

Poirson, Juline; Biquand, Elise; Straub, Marie-Laure; Cassonnet, Patricia; Nominé, Yves; Jones, Louis; van der Werf, Sylvie; Travé, Gilles; Zanier, Katia; Jacob, Yves; Demeret, Caroline; Masson, Murielle

2017-10-01

Protein ubiquitination and its reverse reaction, deubiquitination, regulate protein stability, protein binding activity, and their subcellular localization. These reactions are catalyzed by the enzymes E1, E2, and E3 ubiquitin (Ub) ligases and deubiquitinases (DUBs). The Ub-proteasome system (UPS) is targeted by viruses for the sake of their replication and to escape host immune response. To identify novel partners of human papillomavirus 16 (HPV16) E6 and E7 proteins, we assembled and screened a library of 590 cDNAs related to the UPS by using the Gaussia princeps luciferase protein complementation assay. HPV16 E6 was found to bind to the homology to E6AP C terminus-type Ub ligase (E6AP), three really interesting new gene (RING)-type Ub ligases (MGRN1, LNX3, LNX4), and the DUB Ub-specific protease 15 (USP15). Except for E6AP, the binding of UPS factors did not require the LxxLL-binding pocket of HPV16 E6. LNX3 bound preferentially to all high-risk mucosal HPV E6 tested, whereas LNX4 bound specifically to HPV16 E6. HPV16 E7 was found to bind to several broad-complex tramtrack and bric-a-brac domain-containing proteins (such as TNFAIP1/KCTD13) that are potential substrate adaptors of Cullin 3-RING Ub ligases, to RING-type Ub ligases implicated in innate immunity (RNF135, TRIM32, TRAF2, TRAF5), to the substrate adaptor DCAF15 of Cullin 4-RING Ub ligase and to some DUBs (USP29, USP33). The binding to UPS factors did not require the LxCxE motif but rather the C-terminal region of HPV16 E7 protein. The identified UPS factors interacted with most of E7 proteins across different HPV types. This study establishes a strategy for the rapid identification of interactions between host or pathogen proteins and the human ubiquitination system. © 2017 Federation of European Biochemical Societies.

Studies of inactivation mechanism of non-enveloped icosahedral virus by a visible ultrashort pulsed laser

PubMed Central

2014-01-01

Background Low-power ultrashort pulsed (USP) lasers operating at wavelengths of 425 nm and near infrared region have been shown to effectively inactivate viruses such as human immunodeficiency virus (HIV), M13 bacteriophage, and murine cytomegalovirus (MCMV). It was shown previously that non-enveloped, helical viruses such as M13 bacteriophage, were inactivated by a USP laser through an impulsive stimulated Raman scattering (ISRS) process. Recently, enveloped virus like MCMV has been shown to be inactivated by a USP laser via protein aggregation induced by an ISRS process. However, the inactivation mechanism for a clinically important class of viruses – non-enveloped, icosahedral viruses remains unknown. Results and discussions We have ruled out the following four possible inactivation mechanisms for non-enveloped, icosahedral viruses, namely, (1) inactivation due to ultraviolet C (UVC) photons produced by non-linear optical process of the intense, fundamental laser beam at 425 nm; (2) inactivation caused by thermal heating generated by the direct laser absorption/heating of the virion; (3) inactivation resulting from a one-photon absorption process via chromophores such as porphyrin molecules, or indicator dyes, potentially producing reactive oxygen or other species; (4) inactivation by the USP lasers in which the extremely intense laser pulse produces shock wave-like vibrations upon impact with the viral particle. We present data which support that the inactivation mechanism for non-enveloped, icosahedral viruses is the impulsive stimulated Raman scattering process. Real-time PCR experiments show that, within the amplicon size of 273 bp tested, there is no damage on the genome of MNV-1 caused by the USP laser irradiation. Conclusion We conclude that our model non-enveloped virus, MNV-1, is inactivated by the ISRS process. These studies provide fundamental knowledge on photon-virus interactions on femtosecond time scales. From the analysis of the transmission electron microscope (TEM) images of viral particles before and after USP laser irradiation, the locations of weak structural links on the capsid of MNV-1 were revealed. This important information will greatly aid our understanding of the structure of non-enveloped, icosahedral viruses. We envision that this non-invasive, efficient viral eradication method will find applications in the disinfection of pharmaceuticals, biologicals and blood products in the near future. PMID:24495489

Comparison of the influence of nonpreserved oxybuprocaine and a preserved artificial tear (thera tears) on human corneal thickness measured by two pachymeters.

PubMed

Almubrad, Turki M; Alshehri, Fayez H; Ogbuehi, Kelechi C; Osuagwu, Uchechukwu L

2013-06-01

To compare the effect of nonpreserved oxybuprocaine and preserved artificial tears on central corneal thickness (CCT) obtained by 2 pachymeters. In this prospective, placebo-controlled study, involving a random sample of 100 eyes of 50 subjects, aged 24±2.3 years, CCT readings were obtained in 2 separate sessions with the Topcon SP-3000P and ultrasound pachymetry (USP), respectively, before, 5 and 10 min after instillation of a drop each of either oxybuprocaine hydrochloride (oxybuprocaine HCl) (group 1) or carboxymethylcellulose sodium, thera tears (group 2), and placebo. The baseline mean CCT for SP-3000P was 509±38 μm and 542±36 μm for USP. No statistical significant differences between baseline CCTs (P>0.05 for both devices) in both groups. In both group experimental eyes, neither SP-3000P nor USP-measured CCTs varied significantly from the control eyes at 5 (P>0.05) and 10 (P>0.05) mins postinstillation of drops in both sessions. In group one, the 95% confidence intervals (CIs) for the SP-3000P CCTs were similar at 5 (-16 to 17 μm) and 10 min (-16 to 17 μm), but in the USP-measured CCTs, it was wider at 10 min (-41 to 46 μm) than at 5 min (-30 to 41 μm) postinstillation. In group two, the 95% CIs at 5 and 10 mins postinstillation, respectively, ranged between -20 and 47 μm, -21 and 43 μm (SP-3000P) and -29 and 23 μm, -26 and 23 μm (USP). Within groups and between groups, variations in CCT were similar at both times intervals in all comparisons. Although oxybuprocaine HCl and thera tears consistently did not affect the mean CCT obtained by both devices at both time intervals, variation in SP-3000P measured CCT was more consistent in both sessions and narrower in relation to USP-measured CCT. It may be reasonable to suggest that measurements of CCT in normal patients be taken before examinations requiring instillation of anesthetics or such measurements when obtained postinstillation of either oxybuprocaine or preserved artificial tears be interpreted with caution.

A Proven Method for Meeting Export Control Objectives in Postal and Shipping Sectors

DTIC Science & Technology

2015-02-01

Mellon University for the operation of the Software Engineering Institute, a federally funded research and development center sponsored by the United...Export Control at USPS 5 3.3 Objectives for Improving Export Screening at USPS 6 4 Development of the New Screening Process 7 4.1 “Walking the Model...Export Screening Development Process 10 Figure 2: Induction and Processing of International Mail 10 Figure 3: The Export Screening Process 11

Taking the Temperature of a Lava Planet

NASA Astrophysics Data System (ADS)

Kreidberg, Laura; Lopez, Eric; Cowan, Nick; Lupu, Roxana; Stevenson, Kevin; Louden, Tom; Malavolta, Luca

2018-05-01

Ultra-short period rocky planets (USPs) are an exotic class of planet found around less than 1% of stars. With orbital periods shorter than 24 hours, these worlds are blasted with stellar radiation that is expected to obliterate any traces of a primordial atmosphere and melt the dayside surface into a magma ocean. Observations of USPs have yielded several surprising results, including the measurement of an offset hotspot in the thermal phase curve of 55 Cancri e (which may indicate a thick atmosphere has survived), and a high Bond albedo for Kepler-10b, which suggests the presence of unusually reflective lava on its surface. To further explore the properties of USPs and put these results in context, we propose to observe a thermal phase curve of the newly discovered USP K2- 141b. This planet is a rocky world in a 6.7 hour orbit around a bright, nearby star. When combined with optical phase curve measured by K2, our observations will uniquely determine the planet's Bond albedo, precisely measure the offset of the thermal curve, and determine the temperature of the dayside surface. These results will cement Spitzer's role as a pioneer in the study of terrestrial planets beyond the Solar System, and provide a critical foundation for pursuing the optimal follow-up strategy for K2-141b with JWST.

Ion mobility-mass spectrometry reveals conformational flexibility in the deubiquitinating enzyme USP5.

PubMed

Scott, Daniel; Layfield, Robert; Oldham, Neil J

2015-08-01

Many proteins exhibit conformation flexibility as part of their biological function, whether through the presence of a series of well-defined states or by the existence of intrinsic disorder. Ion mobility spectrometry, in combination with MS (IM-MS), offers a rapid and sensitive means of probing ensembles of protein structures through measurement of gas-phase collisional cross sections. We have applied IM-MS analysis to the multidomain deubiquitinating enzyme ubiquitin specific protease 5 (USP5), which is believed to exhibit significant conformational flexibility. Native ESI-MS measurement of the 94-kDa USP5 revealed two distinct charge-state distributions: [M + 17H](+) to [M + 21H](+) and [M + 24H](+) to [M + 29H](+). The collisional cross sections of these ions revealed clear groupings of 52 ± 4 nm(2) for the lower charges and 66 ± 6 nm(2) for the higher charges. Molecular dynamics simulation of a compact form of USP5, based on a crystal structure, produced structures of 53-54 nm(2) following 2 ns in the gas phase, while simulation of an extended form (based on small-angle X-ray scattering data) led to structures of 64 nm(2). These data demonstrate that IM-MS is a valuable tool in studying proteins with different discrete conformational states. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Skin corrosion test: a comparison between reconstructed human epidermis and full thickness skin models.

PubMed

Catarino, Carolina Motter; do Nascimento Pedrosa, Tatiana; Pennacchi, Paula Comune; de Assis, Silvia Romano; Gimenes, Fabrícia; Consolaro, Márcia Edilaine Lopes; de Moraes Barros, Silvia Berlanga; Maria-Engler, Silvya Stuchi

2018-04-01

Currently, there is a strong global trend towards the development of in vitro models to replace the use of animals in safety evaluation tests. Reconstructed Human Epidermis (RHE) models have been employed as an alternative method to animal testing of skin corrosion and irritation potential of chemical compounds. However, the consequences of an absence of the dermal compartment in these models should be considered since the cross-talk between fibroblasts and keratinocytes is fundamental for promoting proper epidermal stratification, homeostasis, inflammatory response and wound healing. In this study, we compare in-house developed models of Reconstructed Human Epidermis (i.e. USP-RHE) and full thickness skin (i.e. USP-FTS) regarding their response when submitted to skin corrosion assays, based on Guideline 431 (OECD). The results show that both models correctly classified the four substances tested (2-phenylethyl bromide, benzylacetone, lactic acid, octanoic acid) as corrosive or non-corrosive. Furthermore, we have demonstrated higher cell viability of the USP-FTS model compared to the USP-RHE model, a sign of its improved barrier function, following the exposure to the substances test on the corrosion assay. This emphasizes the importance of employing in vitro models that are more physiologically relevant and that better mimic the in vivo situation for the toxicological screening of substances. Copyright © 2018 Elsevier B.V. All rights reserved.

Synaptic Strength Is Bidirectionally Controlled by Opposing Activity-Dependent Regulation of Nedd4-1 and USP8

PubMed Central

Scudder, Samantha L.; Goo, Marisa S.; Cartier, Anna E.; Molteni, Alice; Schwarz, Lindsay A.; Wright, Rebecca

2014-01-01

The trafficking of AMPA receptors (AMPARs) to and from synapses is crucial for synaptic plasticity. Previous work has demonstrated that AMPARs undergo activity-dependent ubiquitination by the E3 ubiquitin ligase Nedd4-1, which promotes their internalization and degradation in lysosomes. Here, we define the molecular mechanisms involved in ubiquitination and deubiquitination of AMPARs. We report that Nedd4-1 is rapidly redistributed to dendritic spines in response to AMPAR activation and not in response to NMDA receptor (NMDAR) activation in cultured rat neurons. In contrast, NMDAR activation directly antagonizes Nedd4-1 function by promoting the deubiquitination of AMPARs. We show that NMDAR activation causes the rapid dephosphorylation and activation of the deubiquitinating enzyme (DUB) USP8. Surface AMPAR levels and synaptic strength are inversely regulated by Nedd4-1 and USP8. Strikingly, we show that homeostatic downscaling of synaptic strength is accompanied by an increase and decrease in Nedd4-1 and USP8 protein levels, respectively. Furthermore, we show that Nedd4-1 is required for homeostatic loss of surface AMPARs and downscaling of synaptic strength. This study provides the first mechanistic evidence for rapid and opposing activity-dependent control of a ubiquitin ligase and DUB at mammalian CNS synapses. We propose that the dynamic regulation of these opposing forces is critical in maintaining synapses and scaling them during homeostatic plasticity. PMID:25505317

A virtual clean room to teach USP 797 regulations for intravenous medications.

PubMed

Patel, Sheetal; Vincent, Ashley H; Abel, Steven R; Jacobs, Carolyn M; Dunlop, Steven R; Seibert, Megan

2011-02-10

To provide a virtual environment for pharmacy students to learn United States Pharmacopeia Chapter 797 (USP 797) requirements, while recognizing the role of pharmacists in the safe use of intravenous (IV) medications. A virtual laboratory was created that included stations for IV medications, product verification, medication safety, and patient cases pertaining to high-alert medications. Pharmacy students used 3-D glasses and a wireless controller to navigate through the session and identify violations of USP 797 regulations. Pre-assessments and post-assessments were administered to students who completed the session in each of the 2 years it was offered. In the first year, 88% of students strongly agreed or agreed that the sessions met their expectation. Following their APPE clerkship, 92% of these students felt the virtual IV room prepared them for the IV clean room experience. In the second year, 88% of students felt the experience enhanced their understanding of clean room procedures. After session completion, 75% of participants perceived medication errors to be more significant. Written examinations also were administered and students' mean scores improved significantly compared to those of students' prior to implementation of the session (89.6% in year 0; 91.2% in year 1; and 96.1% in year 2). The immersive virtual environment is a contemporary and effective way to teach USP 797 requirements and enhance the awareness of medication errors.

A Virtual Clean Room to Teach USP 797 Regulations for Intravenous Medications

PubMed Central

Patel, Sheetal; Abel, Steven R.; Jacobs, Carolyn M.; Dunlop, Steven R.; Seibert, Megan

2011-01-01

Objective To provide a virtual environment for pharmacy students to learn United States Pharmacopeia Chapter 797 (USP 797) requirements, while recognizing the role of pharmacists in the safe use of intravenous (IV) medications. Design A virtual laboratory was created that included stations for IV medications, product verification, medication safety, and patient cases pertaining to high-alert medications. Pharmacy students used 3-D glasses and a wireless controller to navigate through the session and identify violations of USP 797 regulations. Assessment Preassessments and postassessments were administered to students who completed the session in each of the 2 years it was offered. In the first year, 88% of students strongly agreed or agreed that the sessions met their expectation. Following their APPE clerkship, 92% of these students felt the virtual IV room prepared them for the IV clean room experience. In the second year, 88% of students felt the experience enhanced their understanding of clean room procedures. After session completion, 75% of participants perceived medication errors to be more significant. Written examinations also were administered and students' mean scores improved significantly compared to those of students' prior to implementation of the session (89.6% in year 0; 91.2% in year 1; and 96.1% in year 2). Conclusion The immersive virtual environment is a contemporary and effective way to teach USP 797 requirements and enhance the awareness of medication errors. PMID:21451759

Nitrogen release from urea with different coatings.

PubMed

Campos, Odirley R; Mattiello, Edson Marcio; Cantarutti, Reinaldo Bertola; Vergütz, Leonardus

2018-01-01

Coatings or urease inhibitors are designed to reduce losses of ammonia [NH 3(g) ] from urea fertilizers. However, nitrogen (N) release and its effects on soil solution have not previously been evaluated under standardized conditions in soils. In this study, the urea fertilizers were incubated in chambers filled with sandy loam soil, adapted for the collection of NH 3(g) and soil solution by centrifugation. In the fast-release N fertilizers, around 93% and 100% of urea-N applied was recovered within the first hours of incubation. In contrast, in the slow-release N fertilizers, less than 40% of urea-N applied, was recovered at 19 days of incubation. The maximum N release from the fertilizers followed the order: UP1 (106%) ≈ UNBPT (102%) ≈ urea (93%) > USP2 (57%) ≈ USP3 (57%) > USP4 (31%) ≈ USP5 (18%). NH 3(g) volatilization accounted for only 3% of the applied N in the slow-release fertilizers, which corresponded to about 88% less than the NH 3(g) loss from prilled urea. This study demonstrated distinct N release patterns, which changed the N dynamics in the soil. Some coatings effectively delayed urea release from granules and reduced NH 3(g) gas losses, while other were not efficient. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Computational hydrodynamic comparison of a mini vessel and a USP 2 dissolution testing system to predict the dynamic operating conditions for similarity of dissolution performance.

PubMed

Wang, Bing; Bredael, Gerard; Armenante, Piero M

2018-03-25

The hydrodynamic characteristics of a mini vessel and a USP 2 dissolution testing system were obtained and compared to predict the tablet-liquid mass transfer coefficient from velocity distributions near the tablet and establish the dynamic operating conditions under which dissolution in mini vessels could be conducted to generate concentration profiles similar to those in the USP 2. Velocity profiles were obtained experimentally using Particle Image Velocimetry (PIV). Computational Fluid Dynamics (CFD) was used to predict the velocity distribution and strain rate around a model tablet. A CFD-based mass transfer model was also developed. When plotted against strain rate, the predicted tablet-liquid mass transfer coefficient was found to be independent of the system where it was obtained, implying that a tablet would dissolve at the same rate in both systems provided that the concentration gradient between the tablet surface and the bulk is the same, the tablet surface area per unit liquid volume is identical, and the two systems are operated at the appropriate agitation speeds specified in this work. The results of this work will help dissolution scientists operate mini vessels so as to predict the dissolution profiles in the USP 2, especially during the early stages of drug development. Copyright © 2018 Elsevier B.V. All rights reserved.

Massively Parallel Sequencing of Patients with Intellectual Disability, Congenital Anomalies and/or Autism Spectrum Disorders with a Targeted Gene Panel

PubMed Central

Brett, Maggie; McPherson, John; Zang, Zhi Jiang; Lai, Angeline; Tan, Ee-Shien; Ng, Ivy; Ong, Lai-Choo; Cham, Breana; Tan, Patrick; Rozen, Steve; Tan, Ene-Choo

2014-01-01

Developmental delay and/or intellectual disability (DD/ID) affects 1–3% of all children. At least half of these are thought to have a genetic etiology. Recent studies have shown that massively parallel sequencing (MPS) using a targeted gene panel is particularly suited for diagnostic testing for genetically heterogeneous conditions. We report on our experiences with using massively parallel sequencing of a targeted gene panel of 355 genes for investigating the genetic etiology of eight patients with a wide range of phenotypes including DD/ID, congenital anomalies and/or autism spectrum disorder. Targeted sequence enrichment was performed using the Agilent SureSelect Target Enrichment Kit and sequenced on the Illumina HiSeq2000 using paired-end reads. For all eight patients, 81–84% of the targeted regions achieved read depths of at least 20×, with average read depths overlapping targets ranging from 322× to 798×. Causative variants were successfully identified in two of the eight patients: a nonsense mutation in the ATRX gene and a canonical splice site mutation in the L1CAM gene. In a third patient, a canonical splice site variant in the USP9X gene could likely explain all or some of her clinical phenotypes. These results confirm the value of targeted MPS for investigating DD/ID in children for diagnostic purposes. However, targeted gene MPS was less likely to provide a genetic diagnosis for children whose phenotype includes autism. PMID:24690944

Stability Analysis of ISS Medications

NASA Technical Reports Server (NTRS)

Wotring, V. E.

2014-01-01

It is known that medications degrade over time, and that extreme storage conditions will hasten their degradation. The temperature and humidity conditions of the ISS have been shown to be within the ideal ranges for medication storage, but the effects of other environmental factors, like elevated exposure to radiation, have not yet been evaluated. Current operational procedures ensure that ISS medications are re-stocked before expiration, but this may not be possible on long duration exploration missions. For this reason, medications that have experienced long duration storage on the ISS were returned to JSC for analysis to determine any unusual effects of aging in the low- Earth orbit environment. METHODS Medications were obtained by the JSC Pharmacy from commercial distributors and were re-packaged by JSC pharmacists to conserve up mass and volume. All medication doses were part of the ISS crew medical kit and were transported to the International Space Station (ISS) via NASA's Shuttle Transportation System (Space Shuttle). After 568 days of storage, the medications were removed from the supply chain and returned to Earth on a Dragon (SpaceX) capsule. Upon return to Earth, medications were transferred to temperature and humidity controlled environmental chambers until analysis. Nine medications were chosen on the basis of their availability for study. The medications included several of the most heavily used by US crewmembers: 2 sleep aids, 2 antihistamines/decongestants, 3 pain relievers, an antidiarrheal and an alertness medication. Each medication was available at a single time point; analysis of the same medication at multiple time points was not possible. Because the samples examined in this study were obtained opportunistically from medical supplies, there were no control samples available (i.e. samples aged for a similar period of time on the ground); a significant limitation of this study. Medications were analyzed using the HPLC/MS methods described in the United States Pharmacopeia (USP) to measure the amount of intact active ingredient, identify degradation products and measure their amounts. Some analyses were conducted by an independent analytical laboratory, but certain (Schedule) medications could not be shipped to their facility and were analyzed at JSC. RESULTS Nine medications were analyzed with respect to active pharmaceutical ingredient (API) and degradant amounts. Results were compared to the USP requirements for API and degradants/impurities content for every FDA-approved medication. One medication met USP requirements at 5 months after its expiration date. Four of the nine (44% of those tested) medications tested met USP requirements up to 8 months post-expiration. Another 3 medications (33% of those tested) met USP guidelines 2-3 months before expiration. One medication, a compound classed by the FDA as a dietary supplement and sometimes used as a sleep aid, failed to meet USP requirements at 11 months post-expiration. CONCLUSION Analysis of each medication at a single time point provides limited information on the stability of a medication stored in particular conditions; it is not possible to predict how long a medication may be safe and effective from these data. Notwithstanding, five of the nine medications tested (56%) met USP requirements for API and degradants/impurities at least 5 months past expiration dates. The single compound that failed to meet USP requirements is not regulated as strictly as prescription medications are during manufacture; it is unknown if this medication would have met the requirements prior to flight. Notably, it was the furthest beyond its expiration date. Only more comprehensive analysis of flight-aged samples compared to appropriate ground controls will permit determination of spaceflight effects on medication stability.

Dose uniformity of scored and unscored tablets: Application of the FDA Tablet Scoring Guidance for Industry.

PubMed

Ciavarella, Anthony; Khan, Mansoor; Gupta, Abhay; Faustino, Patrick

2016-06-20

This FDA laboratory study examines the impact of tablet splitting, the effect of tablet splitters, and the presence of a tablet score on the dose uniformity of two model drugs. Whole tablets were purchased from five manufacturers for amlodipine and six for gabapentin. Two splitters were used for each drug product and the gabapentin tablets were also split by hand. Whole and split amlodipine tablets were tested for content uniformity following the general chapter of the United States Pharmacopeia (USP) Uniformity of Dosage Units <905>, which is a requirement of the new FDA Guidance for Industry on tablet scoring. The USP weight variation method was used for gabapentin split tablets based on the recommendation of the guidance. All whole tablets met the USP acceptance criteria for the Uniformity of Dosage Units. Variation in whole tablet content ranged from 0.5-2.1 standard deviation (SD) of the % label claim. Splitting the unscored amlodipine tablets resulted in a significant increase in dose variability of 6.5-25.4 SD when compared to whole tablets. Split tablets from all amlodipine drug products did not meet the USP acceptance criteria for content uniformity. Variation in the weight for gabapentin split tablets was greater than the whole tablets, ranging from 1.3-9.3 SD. All fully scored gabapentin products met the USP acceptance criteria for weight variation. Size, shape, and the presence or absence of a tablet score can affect the content uniformity and weight variation of amlodipine and gabapentin tablets. Tablet splitting produced higher variability. Differences in dose variability and fragmentation were observed between tablet splitters and hand splitting. These results are consistent with the FDA's concerns that tablet splitting "can affect how much drug is present in the split tablet and available for absorption" as stated in the guidance (1). Copyright © 2016, Parenteral Drug Association.

Visible light-induced water oxidation on mesoscopic alpha-Fe2O3 films made by ultrasonic spray pyrolysis.

PubMed

Duret, Alexis; Grätzel, Michael

2005-09-15

Alpha-Fe(2)O(3) films having a mesoscopic leaflet type structure were produced for the first time by ultrasonic spray pyrolysis (USP) to explore their potential as oxygen-evolving photoanodes. The target of these studies is to use translucent hematite films deposited on conducting fluorine doped tin oxide (FTO) glass as top electrodes in a tandem cell that accomplishes the cleavage of water into hydrogen and oxygen by sunlight. The properties of layers made by USP were compared to those deposited by conventional spray pyrolysis (SP). Although both types of films show similar XRD and UV-visible and Raman spectra, they differ greatly in their morphology. The mesoscopic alpha-Fe(2)O(3) layers produced by USP consist mainly of 100 nm-sized platelets with a thickness of 5-10 nm. These nanosheets are oriented mainly perpendicularly to the FTO support, their flat surface exposing (001) facets. The mesoscopic leaflet structure has the advantage that it allows for efficient harvesting of visible light, while offering at the same time the very short distance required for the photogenerated holes to reach the electrolyte interface before recombining with conduction band electrons. This allows for water oxidation by the valence band holes even though their diffusion length is only a few nanometers. Distances are longer in the particles produced by SP favoring recombination of photoinduced charge carriers. Open-circuit photovoltage measurements indicate a lower surface state density for the nanoplatelets as compared to the round particles. These factors explain the much higher photoactivity of the USP compared to the SP deposited alpha-Fe(2)O(3) layers. Addition of hydrogen peroxide to the alkaline electrolyte further improves the photocurrent-voltage characteristics of films generated by USP indicating the hole transfer from the valence band of the semiconductor oxide to the adsorbed water to be the rate-limiting kinetic step in the oxygen generation reaction.

A mathematical analysis of drug dissolution in the USP flow through apparatus

NASA Astrophysics Data System (ADS)

McDonnell, David; D'Arcy, D. M.; Crane, L. J.; Redmond, Brendan

2018-03-01

This paper applies boundary layer theory to the process of drug dissolution in the USP (United States Pharmacopeia) Flow Through Apparatus. The mass transfer rate from the vertical planar surface of a compact within the device is examined. The theoretical results obtained are then compared with those of experiment. The paper also examines the effect on the dissolution process caused by the interaction between natural and forced convection within the apparatus and the introduction of additional boundaries.

A survey of the interactome of Kaposi's sarcoma-associated herpesvirus ORF45 revealed its binding to viral ORF33 and cellular USP7, resulting in stabilization of ORF33 that is required for production of progeny viruses.

PubMed

Gillen, Joseph; Li, Wenwei; Liang, Qiming; Avey, Denis; Wu, Jianjun; Wu, Fayi; Myoung, JinJong; Zhu, Fanxiu

2015-05-01

The ORF45 protein of Kaposi's sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus-specific immediate-early tegument protein. Our previous studies have revealed its crucial roles in both early and late stages of KSHV infection. In this study, we surveyed the interactome of ORF45 using a panel of monoclonal antibodies. In addition to the previously identified extracellular regulated kinase (ERK) and p90 ribosomal S6 kinase (RSK) proteins, we found several other copurified proteins, including prominent ones of ∼38 kDa and ∼130 kDa. Mass spectrometry revealed that the 38-kDa protein is viral ORF33 and the 130-kDa protein is cellular USP7 (ubiquitin-specific protease 7). We mapped the ORF33-binding domain to the highly conserved carboxyl-terminal 19 amino acids (aa) of ORF45 and the USP7-binding domain to the reported consensus motif in the central region of ORF45. Using immunofluorescence staining, we observed colocalization of ORF45 with ORF33 or USP7 both under transfected conditions and in KSHV-infected cells. Moreover, we noticed ORF45-dependent relocalization of a portion of ORF33/USP7 from the nucleus to the cytoplasm. We found that ORF45 caused an increase in ORF33 protein accumulation that was abolished if either the ORF33- or USP7-binding domain in ORF45 was deleted. Furthermore, deletion of the conserved carboxyl terminus of ORF45 in the KSHV genome drastically reduced the level of ORF33 protein in KSHV-infected cells and abolished production of progeny virions. Collectively, our results not only reveal new components of the ORF45 interactome, but also demonstrate that the interactions among these proteins are crucial for KSHV lytic replication. Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of several human cancers. KSHV ORF45 is a multifunctional protein that is required for KSHV lytic replication, but the exact mechanisms by which ORF45 performs its critical functions are unclear. Our previous studies revealed that all ORF45 protein in cells exists in high-molecular-weight complexes. We therefore sought to characterize the interactome of ORF45 to provide insights into its roles during lytic replication. Using a panel of monoclonal antibodies, we surveyed the ORF45 interactome in KSHV-infected cells. We identified two new binding partners of ORF45: the viral protein ORF33 and cellular ubiquitin-specific protease 7 (USP7). We further demonstrate that the interaction between ORF45 and ORF33 is crucial for the efficient production of KSHV viral particles, suggesting that the targeted interference with this interaction may represent a novel strategy to inhibit KSHV lytic replication. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

fabp4 is central to eight obesity associated genes: a functional gene network-based polymorphic study.

PubMed

Bag, Susmita; Ramaiah, Sudha; Anbarasu, Anand

2015-01-07

Network study on genes and proteins offers functional basics of the complexity of gene and protein, and its interacting partners. The gene fatty acid-binding protein 4 (fabp4) is found to be highly expressed in adipose tissue, and is one of the most abundant proteins in mature adipocytes. Our investigations on functional modules of fabp4 provide useful information on the functional genes interacting with fabp4, their biochemical properties and their regulatory functions. The present study shows that there are eight set of candidate genes: acp1, ext2, insr, lipe, ostf1, sncg, usp15, and vim that are strongly and functionally linked up with fabp4. Gene ontological analysis of network modules of fabp4 provides an explicit idea on the functional aspect of fabp4 and its interacting nodes. The hierarchal mapping on gene ontology indicates gene specific processes and functions as well as their compartmentalization in tissues. The fabp4 along with its interacting genes are involved in lipid metabolic activity and are integrated in multi-cellular processes of tissues and organs. They also have important protein/enzyme binding activity. Our study elucidated disease-associated nsSNP prediction for fabp4 and it is interesting to note that there are four rsID׳s (rs1051231, rs3204631, rs140925685 and rs141169989) with disease allelic variation (T104P, T126P, G27D and G90V respectively). On the whole, our gene network analysis presents a clear insight about the interactions and functions associated with fabp4 gene network. Copyright © 2014 Elsevier Ltd. All rights reserved.

Nonlinear scattering of ultrashort laser pulses on two-level system

NASA Astrophysics Data System (ADS)

Astapenko, Valery A.; Sakhno, Sergey V.

2015-05-01

The presentation is devoted to the theoretical investigation of nonlinear scattering of ultrashort electromagnetic pulses (USP) on two-level quantum system. We consider the scattering of several types of USP, namely, so called corrected Gaussian pulse (CGP) and cosine wavelet pulse. Such pulses have no constant component in their spectrum in contrast with traditional Gaussian pulse. It should be noted that the presence of constant component in the limit of ultrashort pulse durations leads to unphysical results. The main purpose of the present work is the investigation of the change of pulse temporal shape after scattering as a function of initial phase at different distances from the target. Numerical calculations are based on the solution of Bloch equations and expression for scattering field strength via dipole moment of two-level system exposed by the action of incident USP. In our calculation we also account for the influence of refracting index of the air on electric field strength in the pulse after scattering.

Analysis of Temperature Variability in Medication Storage Compartments in Emergency Transport Helicopters.

PubMed

O'Donnell, Margaret A; Whitfield, Justin

The purpose of this study was to determine whether the temperature in medication storage compartments in air medical helicopters was within United States Pharmacopeia (USP)-defined limits for controlled room temperature. This was a prospective study using data obtained from a continuous temperature monitoring device. A total of 4 monitors were placed within 2 medication storage locations in 2 identical helicopters. The data collection period lasted 2 weeks during the summer and winter seasons. Data retrieved from monitors were compared against USP parameters for proper medication storage. Results documented temperatures outside the acceptable range a majority of the time with temperatures above the high limit during summer and below the low limit during winter. The study determined that compartments used for medication storage frequently fell outside of the range for USP-defined limits for medication storage. Flight programs should monitor storage areas, carefully taking actions to keep medication within defined ranges. Copyright © 2016 Air Medical Journal Associates. Published by Elsevier Inc. All rights reserved.

Ubiquitinated Proteins Activate the Proteasomal ATPases by Binding to Usp14 or Uch37 Homologs*

PubMed Central

Peth, Andreas; Kukushkin, Nikolay; Bossé, Marc; Goldberg, Alfred L.

2013-01-01

Degradation of ubiquitinated proteins by 26 S proteasomes requires ATP hydrolysis, but it is unclear how the proteasomal ATPases are regulated and how proteolysis, substrate deubiquitination, degradation, and ATP hydrolysis are coordinated. Polyubiquitinated proteins were shown to stimulate ATP hydrolysis by purified proteasomes, but only if the proteins contain a loosely folded domain. If they were not ubiquitinated, such proteins did not increase ATPase activity. However, they did so upon addition of ubiquitin aldehyde, which mimics the ubiquitin chain and binds to 26 S-associated deubiquitinating enzymes (DUBs): in yeast to Ubp6, which is essential for the ATPase activation, and in mammalian 26 S to the Ubp6 homolog, Usp14, and Uch37. Occupancy of either DUB by a ubiquitin conjugate leads to ATPase stimulation, thereby coupling deubiquitination and ATP hydrolysis. Thus, ubiquitinated loosely folded proteins, after becoming bound to the 26 S, interact with Ubp6/Usp14 or Uch37 to activate ATP hydrolysis and enhance their own destruction. PMID:23341450

Combining Zebrafish and Mouse Models to Test the Function of Deubiquitinating Enzyme (Dubs) Genes in Development: Role of USP45 in the Retina.

PubMed

Toulis, Vasileios; Garanto, Alejandro; Marfany, Gemma

2016-01-01

Ubiquitination is a dynamic and reversible posttranslational modification. Much effort has been devoted to characterize the function of ubiquitin pathway genes in the cell context, but much less is known on their functional role in the development and maintenance of organs and tissues in the organism. In fact, several ubiquitin ligases and deubiquitinating enzymes (DUBs) are implicated in human pathological disorders, from cancer to neurodegeneration. The aim of our work is to explore the relevance of DUBs in retinal function in health and disease, particularly since some genes related to the ubiquitin or SUMO pathways cause retinal dystrophies, a group of rare diseases that affect 1:3000 individuals worldwide. We propose zebrafish as an extremely useful and informative genetic model to characterize the function of any particular gene in the retina, and thus complement the expression data from mouse. A preliminary characterization of gene expression in mouse retinas (RT-PCR and in situ hybridization) was performed to select particularly interesting genes, and we later replicated the experiments in zebrafish. As a proof of concept, we selected ups45 to be knocked down by morpholino injection in zebrafish embryos. Morphant phenotypic analysis showed moderate to severe eye morphological defects, with a defective formation of the retinal structures, therefore supporting the relevance of DUBs in the formation and differentiation of the vertebrate retina, and suggesting that genes encoding ubiquitin pathway enzymes are good candidates for causing hereditary retinal dystrophies.

Stability of total nutrient admixtures with lipid injectable emulsions in glass versus plastic packaging.

PubMed

Driscoll, David F; Silvestri, Anthony P; Bistrian, Bruce R; Mikrut, Bernard A

2007-02-15

The physical stability of two emulsions compounded as part of a total nutrient admixture (TNA) was studied in lipids packaged in either glass or plastic containers. Five weight-based adult TNA formulations that were designed to meet the full nutritional needs of adults with body weights between 40 and 80 kg were studied. Triplicate preparations of each TNA were assessed over 30 hours at room temperature by applying currently proposed United States Pharmacopeia (USP) criteria for mean droplet diameter, large-diameter tail, and globule-size distribution (GSD) for lipid injectable emulsions. In accordance with conditions set forth in USP chapter 729, the higher levels of volume-weighted percent of fat exceeding 5 microm (PFAT(5)) should not exceed 0.05% of the total lipid concentration. Significant differences were noted among TNA admixtures based on whether the lipid emulsion product was manufactured in glass or plastic. The plastic-contained TNAs failed the proposed USP methods for large-diameter fat globules in all formulations from the outset, and 60% had significant growth in large-diameter fat globules over time. In contrast, glass-contained TNAs were stable throughout and in all cases would have passed proposed USP limits. Certain lipid injectable emulsions packaged in plastic containers have baseline abnormal GSD profiles compared with those packaged in glass containers. When used to compound TNAs, the abnormal profile worsens and produces less stable TNAs than those compounded with lipid injectable emulsions packaged in glass containers.

BDE-47 causes developmental retardation with down-regulated expression profiles of ecdysteroid signaling pathway-involved nuclear receptor (NR) genes in the copepod Tigriopus japonicus.

PubMed

Hwang, Dae-Sik; Han, Jeonghoon; Won, Eun-Ji; Kim, Duck-Hyun; Jeong, Chang-Bum; Hwang, Un-Ki; Zhou, Bingsheng; Choe, Joonho; Lee, Jae-Seong

2016-08-01

2,2',4,4'-Tetrabromodiphenyl ether (BDE-47) is a persistent organic pollutant (POP) in marine environments. Despite its adverse effects (e.g. developmental retardation) in ecdysozoa, the effects of BDE-47 on transcription of ecdysteroid signaling pathway-involved-nuclear receptor (NR) genes and metamorphosis-related genes have not been examined in copepods. To examine the deleterious effect of BDE-47 on copepod molting and metamorphosis, BDE-47 was exposed to the harpacticoid copepod Tigriopus japonicus, followed by monitoring developmental retardation and transcriptional alteration of NR genes. The developmental rate was significantly inhibited (P<0.05) in response to BDE-47 and the agricultural insecticide gamma-hexachlorocyclohexane. Conversely, the ecdysteroid agonist ponasterone A (PoA) led to decreased molting and metamorphosis time (P<0.05) from the nauplius stage to the adult stage. In particular, expression profiles of all NR genes were the highest at naupliar stages 5-6 except for SVP, FTZ-F1, and HR96 genes. Nuclear receptor USP, HR96, and FTZ-F1 genes also showed significant sex differences (P<0.05) in gene expression levels over different developmental stages, indicating that these genes may be involved in vitellogenesis. NR gene expression patterns showed significant decreases (P<0.05) in response to BDE-47 exposure, implying that molting and metamorphosis retardation is likely associated with NR gene expression. In summary, BDE-47 leads to molting and metamorphosis retardation and suppresses transcription of NR genes. This information will be helpful in understanding the molting and metamorphosis delay mechanism in response to BDE-47 exposure. Copyright © 2016 Elsevier B.V. All rights reserved.

Physical stability of 20% lipid injectable emulsions via simulated syringe infusion: effects of glass vs plastic product packaging.

PubMed

Driscoll, David F; Ling, Pei-Ra; Bistrian, Bruce R

2007-01-01

The United States Pharmacopeia (USP) has proposed large-globule-size limits to ensure the physical stability of lipid injectable emulsions, expressed as the percent fat >5 microm, or PFAT(5), not exceeding 0.05%. Visibly obvious phase separation as free oil has been shown to occur in some samples if PFAT(5) is >0.4%. We recently found that lipids, newly packaged in plastic (P), exceed the proposed USP limits and seem to produce less stable total nutrient admixtures compared with those made from conventional glass (G), which do meet proposed USP standards. We tested the possible stability differences between 20% lipid injectable emulsions in either P or G in a simulated neonatal syringe infusion study. Eighteen individual syringes were prepared from each 20% lipid injectable emulsion product (n = 36) and attached to a syringe pump set at an infusion rate of 0.5 mL/hour. The starting PFAT(5) levels were measured at time 0 and after 24 hours of infusion, using a laser-based light obscuration technique as described by the USP Chapter <729>. The data were assessed by a 2-way analysis of variance (ANOVA) with Container (G vs P) and Time as the independent variables and PFAT as the dependent variable. At time 0, the starting PFAT(5) level for lipids packaged in G was 0.006% +/- 0.001% vs 0.162% +/- 0.026% for P, whereas at the end of the infusion they were 0.013% +/- 0.003% and 0.328% +/- 0.046%, respectively. Significant differences were noted overall between groups for Container, Time, and Container-Time interaction (all p < .001). Bonferroni tests showed significant differences in PFAT(5) levels between Containers at time 0 (T-0; p < .001) and T-0 vs T-24 for P-based lipids (p < .001), whereas no such differences were noted for Time for the G-based lipids. Similar results were noted for PFAT(10) levels. We confirm that presently available lipid injectable emulsions packaged in newly introduced plastic containers exceed the proposed USP <729> PFAT(5) limits and subsequently become significantly less stable during a simulated syringe-based infusion. Although modest growth (p = NS) in large-diameter fat globules was observed for the glass-based lipids, they remained within proposed USP globule size limits throughout the study. Glass-based lipids seem to be a more stable dosage form and potentially a safer way to deliver lipids via syringe infusion to critically ill neonates.

Identification of host genes linked with the survivability of chickens infected with recombinant viruses possessing H5N1 surface antigens from a highly pathogenic avian influenza virus.

PubMed

Uchida, Yuko; Watanabe, Chiaki; Takemae, Nobuhiro; Hayashi, Tsuyoshi; Oka, Takehiko; Ito, Toshihiro; Saito, Takehiko

2012-03-01

Seventeen recombinant viruses were generated by a reverse genetic technique to elucidate the pathogenicity of highly pathogenic avian influenza viruses (HPAIVs) in chickens. The recombinant viruses generated possessed hemagglutinin (HA) and neuraminidase (NA) genes from an HPAIV. Other segments were combinations of the genes from an HPAIV and two low-pathogenic avian influenza viruses (LPAIVs) derived from chicken (LP) and wild bird (WB). Exchange of whole internal genes from an HPAIV with those of an LPAIV resulted in a significant extension of the survival time following intranasal infection of the chickens with the recombinants. Survival analysis demonstrated that the exchange of a gene segment affected survivability of the chickens with statistical significance. The analysis revealed three groups of recombinants with various gene constellations that depended upon the survivability of the infected chickens. Recombinants where the PA gene was exchanged from LP to WB in the LP gene background, LP (W/PA), did not kill any chickens. LP (W/PA) replicated less efficiently both in vitro and in vivo, suggesting that the intrinsic replication ability of LP (W/PA) affects pathogenicity; however, such a correlation was not seen for the other recombinants. Microarray analysis of the infected chicken lungs indicated that the expression of 7 genes, CD274, RNF19B, OASL, ZC3HAV1 [corrected] , PLA2G6, GCH1, and USP18, correlated with the survivability of the chickens infected (P < 0.01). Further analysis of the functions of these genes in chickens would aid in the understanding of host gene responses following fatal infections by HPAIVs.

AtRD22 and AtUSPL1, Members of the Plant-Specific BURP Domain Family Involved in Arabidopsis thaliana Drought Tolerance

PubMed Central

Harshavardhan, Vokkaliga Thammegowda; Van Son, Le; Seiler, Christiane; Junker, Astrid; Weigelt-Fischer, Kathleen; Klukas, Christian; Altmann, Thomas; Sreenivasulu, Nese; Bäumlein, Helmut; Kuhlmann, Markus

2014-01-01

Crop plants are regularly challenged by a range of environmental stresses which typically retard their growth and ultimately compromise economic yield. The stress response involves the reprogramming of approximately 4% of the transcriptome. Here, the behavior of AtRD22 and AtUSPL1, both members of the Arabidopsis thaliana BURP (BNM2, USP, RD22 and polygalacturonase isozyme) domain-containing gene family, has been characterized. Both genes are up-regulated as part of the abscisic acid (ABA) mediated moisture stress response. While AtRD22 transcript was largely restricted to the leaf, that of AtUSPL1 was more prevalent in the root. As the loss of function of either gene increased the plant's moisture stress tolerance, the implication was that their products act to suppress the drought stress response. In addition to the known involvement of AtUSPL1 in seed development, a further role in stress tolerance was demonstrated. Based on transcriptomic data and phenotype we concluded that the enhanced moisture stress tolerance of the two loss-of-function mutants is a consequence of an enhanced basal defense response. PMID:25333723

Identification of a cis-regulatory region of a gene in Arabidopsis thaliana whose induction by dehydration is mediated by abscisic acid and requires protein synthesis.

PubMed

Iwasaki, T; Yamaguchi-Shinozaki, K; Shinozaki, K

1995-05-20

In Arabidopsis thaliana, the induction of a dehydration-responsive gene, rd22, is mediated by abscisic acid (ABA) but the gene does not include any sequence corresponding to the consensus ABA-responsive element (ABRE), RYACGTGGYR, in its promoter region. The cis-regulatory region of the rd22 promoter was identified by monitoring the expression of beta-glucuronidase (GUS) activity in leaves of transgenic tobacco plants transformed with chimeric gene fusions constructed between 5'-deleted promoters of rd22 and the coding region of the GUS reporter gene. A 67-bp nucleotide fragment corresponding to positions -207 to -141 of the rd22 promoter conferred responsiveness to dehydration and ABA on a non-responsive promoter. The 67-bp fragment contains the sequences of the recognition sites for some transcription factors, such as MYC, MYB, and GT-1. The fact that accumulation of rd22 mRNA requires protein synthesis raises the possibility that the expression of rd22 might be regulated by one of these trans-acting protein factors whose de novo synthesis is induced by dehydration or ABA. Although the structure of the RD22 protein is very similar to that of a non-storage seed protein, USP, of Vicia faba, the expression of the GUS gene driven by the rd22 promoter in non-stressed transgenic Arabidopsis plants was found mainly in flowers and bolted stems rather than in seeds.

Possible roles of the transcription factor Nrf1 (NFE2L1) in neural homeostasis by regulating the gene expression of deubiquitinating enzymes.

PubMed

Taniguchi, Hiroaki; Okamuro, Shota; Koji, Misaki; Waku, Tsuyoshi; Kubo, Kaori; Hatanaka, Atsushi; Sun, Yimeng; Chowdhury, A M Masudul Azad; Fukamizu, Akiyoshi; Kobayashi, Akira

2017-02-26

The transcription factor Nrf1 (NFE2L1) maintains protein homeostasis (proteostasis) by regulating the gene expression of proteasome subunits in response to proteasome inhibition. The deletion of the Nrf1 gene in neural stem/progenitor cells causes severe neurodegeneration due to the accumulation of ubiquitinated proteins in Purkinje cells and motor neurons (Nrf1 NKO mice). However, the molecular mechanisms governing this neurodegenerative process remain unclear. We demonstrate herein that the loss of Nrf1 leads to the reduced gene expression of the deubiquitinating enzymes (DUBs) but not proteasome subunits in Nrf1 NKO mice between P7 and P18. First, we show that K48-linked polyubiquitinated proteins accumulate in Nrf1-deficient Purkinje cells and cerebral cortex neurons. Nevertheless, loss of Nrf1 does not alter the expression and proteolytic activity of proteasome. A significantly reduced expression of deubiquitinating enzymes was also demonstrated in Nrf1-deficient cerebellar tissue using microarray analysis. The genome database further reveals species-conserved ARE, a Nrf1 recognition element, in the regulatory region of certain DUB genes. Furthermore, we show that Nrf1 can activate Usp9x gene expression related to neurodegeneration. Altogether these findings suggest that neurodegeneration in Nrf1 NKO mice may stem from the dysfunction of the ubiquitin-mediated regulation of neuronal proteins. Copyright © 2017 Elsevier Inc. All rights reserved.

Microspheres and nanoparticles from ultrasound

NASA Astrophysics Data System (ADS)

Suh, Won Hyuk

Improved preparations of various examples of monodispersed, porous, hollow, and core-shell metal and semiconductor nanoparticles or nanowires have been developed. Now titania microspheres and nanoparticles and silica microspheres can be synthesized using an inexpensive high frequency (1.7 MHz) ultrasonic generator (household humidifier; ultrasonic spray pyrolysis; USP). Morphology and pore size of titania microspheres were controlled by the silica to Ti(IV) ratio and silica particle size. Fine tuning the precursor ratio affords sub-50 nm titania nanoparticles as well. In terms of silica microspheres, morphology was controlled by the silica to organic monomer ratio. In liquids irradiated with high intensity ultrasound (20 kHz; HIUS), acoustic cavitation produces high energy chemistry through intense local heating inside the gas phase of collapsing bubbles in the liquid. HIUS and USP confine the chemical reactions to isolated sub-micron reaction zones, but sonochemistry does so in a heated gas phase within a liquid, while USP uses a hot liquid droplet carried by a gas flow. Thus, USP can be viewed as a method of phase-separated synthesis using submicron-sized droplets as isolated chemical reactors for nanomaterial synthesis. While USP has been used to create both titania and silica spheres separately, there are no prior reports of titania-silica composites. Such nanocomposites of metal oxides have been produced, and by further manipulation, various porous structures with fascinating morphologies were generated. Briefly, a precursor solution was nebulized using a commercially available household ultrasonic humidifier (1.7 MHz ultrasound generator), and the resulting mist was carried in a gas stream of air through a quartz glass tube in a hot furnace. After exiting the hot zone, these microspheres are porous or hollow and in certain cases magnetically responsive. In the case of titania microspheres, they are rapidly taken up into the cytoplasm of mammalian cells and nearly noncytoxic. Small molecules like Rhodamine and DHED (dehydroevodiamine HCl; Alzheimer's disease therapeutic) can be delivered along with them. Furthermore, synthesis of carbon nanoparticles and titanate nanotube species are possible utilizing these microspheres. Characterizations were done by SEM, (S)TEM, optical/confocal microscopy, XRD, XPS, EDS, SAED, zeta potential, and BET.

Designing Preclinical Perceptibility Measures to Evaluate Topical Vaginal Gel Formulations: Relating User Sensory Perceptions and Experiences to Formulation Properties

PubMed Central

Fava, Joseph L.; Rosen, Rochelle K.; Vargas, Sara; Shaw, Julia G.; Kojic, E. Milu; Kiser, Patrick F.; Friend, David R.; Katz, David F.

2014-01-01

Abstract The effectiveness of any biomedical prevention technology relies on both biological efficacy and behavioral adherence. Microbicide trials have been hampered by low adherence, limiting the ability to draw meaningful conclusions about product effectiveness. Central to this problem may be an inadequate conceptualization of how product properties themselves impact user experience and adherence. Our goal is to expand the current microbicide development framework to include product “perceptibility,” the objective measurement of user sensory perceptions (i.e., sensations) and experiences of formulation performance during use. For vaginal gels, a set of biophysical properties, including rheological properties and measures of spreading and retention, may critically impact user experiences. Project LINK sought to characterize the user experience in this regard, and to validate measures of user sensory perceptions and experiences (USPEs) using four prototype topical vaginal gel formulations designed for pericoital use. Perceptibility scales captured a range of USPEs during the product application process (five scales), ambulation after product insertion (six scales), and during sexual activity (eight scales). Comparative statistical analyses provided empirical support for hypothesized relationships between gel properties, spreading performance, and the user experience. Project LINK provides preliminary evidence for the utility of evaluating USPEs, introducing a paradigm shift in the field of microbicide formulation design. We propose that these user sensory perceptions and experiences initiate cognitive processes in users resulting in product choice and willingness-to-use. By understanding the impact of USPEs on that process, formulation development can optimize both drug delivery and adherence. PMID:24180360

Droplet-size distribution and stability of commercial injectable lipid emulsions containing fish oil.

PubMed

Gallegos, Críspulo; Valencia, Concepción; Partal, Pedro; Franco, José M; Maglio, Omay; Abrahamsson, Malin; Brito-de la Fuente, Edmundo

2012-08-01

The droplet size of commercial fish oil-containing injectable lipid emulsions, including conformance to United States Pharmacopeia (USP) standards on fat-globule size, was investigated. A total of 18 batches of three multichamber parenteral products containing the emulsion SMOFlipid as a component were analyzed. Samples from multiple lots of the products were evaluated to determine compliance with standards on the volume-weighted percentage of fat exceeding 0.05% (PFAT(5)) specified in USP chapter 729 to ensure the physical stability of i.v. lipid emulsions. The products were also analyzed to determine the effects of various storage times (3, 6, 9, and 12 months) and storage temperatures (25, 30, and 40 °C) on product stability. Larger-size lipid particles were quantified via single-particle optical sensing (SPOS). The emulsion's droplet-size distribution was determined via laser light scattering. SPOS and light-scattering analysis demonstrated mean PFAT(5) values well below USP-specified globule-size limits for all the tested products under all study conditions. In addition, emulsion aging at any storage temperature in the range studied did not result in a significant increase of PFAT(5) values, and mean droplet-size values did not change significantly during storage of up to 12 months at temperatures of 25-40 °C. PFAT(5) values were below the USP upper limits in SMOFlipid samples from multiple lots of three multichamber products after up to 12 months of storage at 25 or 30 °C or 6 months of storage at 40 °C.

A new low-cost negative-pressure wound therapy versus a commercially available therapy device widely used to treat complex traumatic injuries: a prospective, randomized, non-inferiority trial.

PubMed

Kamamoto, Fabio; Lima, Ana Lucia Munhoz; Rezende, Marcelo Rosa de; Mattar-Junior, Rames; Leonhardt, Marcos de Camargo; Kojima, Kodi Edson; Santos, Carla Chineze Dos

2017-12-01

Negative-pressure wound therapy has been widely adopted to reduce the complexity of treating a broad range of acute and chronic wounds. However, its cost is high. The objective of this study was to evaluate the following two different methods of negative-pressure wound therapy in terms of healing time: a low-cost method of negative-pressure wound therapy (a pressure stabilizer device connected to a hospital wall-vacuum system with a gauze-sealed dressing, USP) and the standard of care (vacuum-assisted closure, VAC). This is a randomized, controlled, non-inferiority, unblinded trial. Patients admitted with complex injuries to a trauma center in a public referral hospital who were indicated for orthopedic surgery were randomized to a USP or VAC group. The primary outcome was the time required to achieve a "ready for surgery condition", which was defined as a wound bed with healthy granulation tissue and without necrosis or purulent secretion. Wound bed area contraction, granulation tissue growth and the direct costs of the dressings were secondary outcomes. Variation in area and granulation tissue growth were essentially the same between the systems, and healing time was equal between the groups (p=0.379). In both systems, serial debridement increased wound area (p=0.934), and granulation tissue was also increased (p=0.408). The mean treatment cost was US$ 15.15 in the USP group and US$ 872.59 in the VAC group. For treating complex traumatic injuries, USP was non-inferior to and less expensive than VAC.

Designing preclinical perceptibility measures to evaluate topical vaginal gel formulations: relating user sensory perceptions and experiences to formulation properties.

PubMed

Morrow, Kathleen M; Fava, Joseph L; Rosen, Rochelle K; Vargas, Sara; Shaw, Julia G; Kojic, E Milu; Kiser, Patrick F; Friend, David R; Katz, David F

2014-01-01

Abstract The effectiveness of any biomedical prevention technology relies on both biological efficacy and behavioral adherence. Microbicide trials have been hampered by low adherence, limiting the ability to draw meaningful conclusions about product effectiveness. Central to this problem may be an inadequate conceptualization of how product properties themselves impact user experience and adherence. Our goal is to expand the current microbicide development framework to include product "perceptibility," the objective measurement of user sensory perceptions (i.e., sensations) and experiences of formulation performance during use. For vaginal gels, a set of biophysical properties, including rheological properties and measures of spreading and retention, may critically impact user experiences. Project LINK sought to characterize the user experience in this regard, and to validate measures of user sensory perceptions and experiences (USPEs) using four prototype topical vaginal gel formulations designed for pericoital use. Perceptibility scales captured a range of USPEs during the product application process (five scales), ambulation after product insertion (six scales), and during sexual activity (eight scales). Comparative statistical analyses provided empirical support for hypothesized relationships between gel properties, spreading performance, and the user experience. Project LINK provides preliminary evidence for the utility of evaluating USPEs, introducing a paradigm shift in the field of microbicide formulation design. We propose that these user sensory perceptions and experiences initiate cognitive processes in users resulting in product choice and willingness-to-use. By understanding the impact of USPEs on that process, formulation development can optimize both drug delivery and adherence.

Production of Phytase Enzyme by a Bioengineered Probiotic for Degrading of Phytate Phosphorus in the Digestive Tract of Poultry.

PubMed

Pakbaten, Bahareh; Majidzadeh Heravi, Reza; Kermanshahi, Hassan; Sekhavati, Mohammad-Hadi; Javadmanesh, Ali; Mohammadi Ziarat, Masoud

2018-04-21

Probiotics are beneficial microorganisms and have long been used in food production as well as health promotion products. Bioengineered probiotics are used to express and transfer native or recombinant molecules to the mucosal surface of the digestive tract to improve feed efficiency and promote health. Lactococcus lactis is a potential probiotic candidate to produce useful biological proteins. The aim of this investigation was to develop a recombinant Lactococcus lactis with the potential of producing phytase. To enhance the efficiency of expression and secretion of recombinant phytase, usp45 signal peptide was added to the expression vector containing phytase gene (appA2) derived from Escherichia coli. Sequencing of recombinant plasmid containing appA2 showed the correct construction of plasmid. Total length of the phytase insert was 1.25 kbp. A Blast search of the cloned fragment showed 99% similarity to the reported E. coli phytase sequence in the GenBank (accession number: AM946981.2). A plasmid containing usp45 and appA2 electrotransferred into Lactococcus lactis. Zymogram with polyacrylamide gel revealed that the protein extract from the supernatant and the cell pellet of recombinant bacteria had phytase activity. Enzyme activity of 4 U/ml was obtained in cell extracts, and supernatant maximal phytase activity was 19 U/ml. The recombinant L. lactis was supplemented in broiler chicken feed and showed the increase of apparent digestibility on phytate phosphorus in the digestive tract and it was same as performance of E. coli commercial phytase.

Racial and Gender Issues with Physics in the Pacific Region

NASA Astrophysics Data System (ADS)

Aung, Than; Singh, Awnesh; Prasad, Uma

2011-09-01

This paper examines the state of physics teaching and learning in the Pacific Island nations. How have things changed in teaching physics? We believe that some of the goals and many of the challenges faced today have changed very little over the years. This paper is purely based upon the authors' experiences in teaching physics at the first-year level at the University of the South Pacific (USP). USP is a typical medium-sized teaching and research university as compared to universities internationally. In the Pacific, it is the biggest university and is effectively serving the 12 Pacific Island nations. Consequently, the findings described here should represent the overall situation for the Pacific region. Perhaps some of our results will resonate with readers in other locations as well. Worldwide, university students often find studying physics to be very challenging, and only a small fraction of them choose physics as a major for their degree or as a career. Students at USP are not an exception, and here we describe the severity of the problem.

Tailored laser beam shaping for efficient and accurate microstructuring

NASA Astrophysics Data System (ADS)

Häfner, T.; Strauß, J.; Roider, C.; Heberle, J.; Schmidt, M.

2018-02-01

Large-area processing with high material removal rates by ultrashort pulsed (USP) lasers is coming into focus by the development of high-power USP laser systems. However, currently the bottleneck for high-rate production is given by slow and inefficient beam manipulation. On the one hand, slow beam deflection with regard to high pulse repetition rates leads to heat accumulation and shielding effects, on the other hand, a conventional focus cannot provide the optimum fluence due to the Gaussian intensity profile. In this paper, we emphasize on two approaches of dynamic laser beam shaping with liquid crystal on silicon spatial light modulation and acousto-optic beam shaping. Advantages and limitations of dynamic laser beam shaping with regard to USP laser material processing and methods for reducing the influence of speckle are discussed. Additionally, the influence of optics induced aberrations on speckle characteristics is evaluated. Laser material processing results are presented correlating the achieved structure quality with the simulated and measured beam quality. Experimental and analytical investigations show a certain fluence dependence of the necessary number of alternative holograms to realize homogeneous microstructures.

A selective USP1-UAF1 inhibitor links deubiquitination to DNA damage responses

PubMed Central

Liang, Qin; Dexheimer, Thomas S; Zhang, Ping; Rosenthal, Andrew S; Villamil, Mark A; You, Changjun; Zhang, Qiuting; Chen, Junjun; Ott, Christine A; Sun, Hongmao; Luci, Diane K; Yuan, Bifeng; Simeonov, Anton; Jadhav, Ajit; Xiao, Hui; Wang, Yinsheng; Maloney, David J; Zhuang, Zhihao

2014-01-01

Protein ubiquitination and deubiquitination are central to the control of a large number of cellular pathways and signaling networks in eukaryotes. Although the essential roles of ubiquitination have been established in the eukaryotic DNA damage response, the deubiquitination process remains poorly defined. Chemical probes that perturb the activity of deubiquitinases (DUBs) are needed to characterize the cellular function of deubiquitination. Here we report ML323 (2), a highly potent inhibitor of the USP1-UAF1 deubiquitinase complex with excellent selectivity against human DUBs, deSUMOylase, deneddylase and unrelated proteases. Using ML323, we interrogated deubiquitination in the cellular response to UV- and cisplatin-induced DNA damage and revealed new insights into the requirement of deubiquitination in the DNA translesion synthesis and Fanconi anemia pathways. Moreover, ML323 potentiates cisplatin cytotoxicity in non-small cell lung cancer and osteosarcoma cells. Our findings point to USP1-UAF1 as a key regulator of the DNA damage response and a target for overcoming resistance to the platinum-based anticancer drugs. PMID:24531842

Primary osteomyelofibrosis and an XX-male genotype.

PubMed

Schanz, Julie; Haase, Detlef; Steuernagel, Peter; Shirneshan, Katayoo; Bäsecke, Jörg

2015-09-01

A 62-yr-old man with two healthy daughters was diagnosed with osteomyelofibrosis. To our surprise, a female XX-karyotype was observed in bone marrow and confirmed in PHA-stimulated T-lymphocytes from peripheral blood. Further molecular genetic investigation revealed a submicroscopic translocation between the short arm of X and Y, which leads to an XX-male genotype based on an unbalanced translocation X;Y. This rare coincidence was further accentuated as the USP9Y gene, suspected to be to be involved in sperm cell production, was absent, but no azoospermia was present. In general, routine cytogenetics may result in findings that need to be further delineated and, as here, lead to a rare observation. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

X-exome sequencing of 405 unresolved families identifies seven novel intellectual disability genes.

PubMed

Hu, H; Haas, S A; Chelly, J; Van Esch, H; Raynaud, M; de Brouwer, A P M; Weinert, S; Froyen, G; Frints, S G M; Laumonnier, F; Zemojtel, T; Love, M I; Richard, H; Emde, A-K; Bienek, M; Jensen, C; Hambrock, M; Fischer, U; Langnick, C; Feldkamp, M; Wissink-Lindhout, W; Lebrun, N; Castelnau, L; Rucci, J; Montjean, R; Dorseuil, O; Billuart, P; Stuhlmann, T; Shaw, M; Corbett, M A; Gardner, A; Willis-Owen, S; Tan, C; Friend, K L; Belet, S; van Roozendaal, K E P; Jimenez-Pocquet, M; Moizard, M-P; Ronce, N; Sun, R; O'Keeffe, S; Chenna, R; van Bömmel, A; Göke, J; Hackett, A; Field, M; Christie, L; Boyle, J; Haan, E; Nelson, J; Turner, G; Baynam, G; Gillessen-Kaesbach, G; Müller, U; Steinberger, D; Budny, B; Badura-Stronka, M; Latos-Bieleńska, A; Ousager, L B; Wieacker, P; Rodríguez Criado, G; Bondeson, M-L; Annerén, G; Dufke, A; Cohen, M; Van Maldergem, L; Vincent-Delorme, C; Echenne, B; Simon-Bouy, B; Kleefstra, T; Willemsen, M; Fryns, J-P; Devriendt, K; Ullmann, R; Vingron, M; Wrogemann, K; Wienker, T F; Tzschach, A; van Bokhoven, H; Gecz, J; Jentsch, T J; Chen, W; Ropers, H-H; Kalscheuer, V M

2016-01-01

X-linked intellectual disability (XLID) is a clinically and genetically heterogeneous disorder. During the past two decades in excess of 100 X-chromosome ID genes have been identified. Yet, a large number of families mapping to the X-chromosome remained unresolved suggesting that more XLID genes or loci are yet to be identified. Here, we have investigated 405 unresolved families with XLID. We employed massively parallel sequencing of all X-chromosome exons in the index males. The majority of these males were previously tested negative for copy number variations and for mutations in a subset of known XLID genes by Sanger sequencing. In total, 745 X-chromosomal genes were screened. After stringent filtering, a total of 1297 non-recurrent exonic variants remained for prioritization. Co-segregation analysis of potential clinically relevant changes revealed that 80 families (20%) carried pathogenic variants in established XLID genes. In 19 families, we detected likely causative protein truncating and missense variants in 7 novel and validated XLID genes (CLCN4, CNKSR2, FRMPD4, KLHL15, LAS1L, RLIM and USP27X) and potentially deleterious variants in 2 novel candidate XLID genes (CDK16 and TAF1). We show that the CLCN4 and CNKSR2 variants impair protein functions as indicated by electrophysiological studies and altered differentiation of cultured primary neurons from Clcn4(-/-) mice or after mRNA knock-down. The newly identified and candidate XLID proteins belong to pathways and networks with established roles in cognitive function and intellectual disability in particular. We suggest that systematic sequencing of all X-chromosomal genes in a cohort of patients with genetic evidence for X-chromosome locus involvement may resolve up to 58% of Fragile X-negative cases.

X-exome sequencing of 405 unresolved families identifies seven novel intellectual disability genes

PubMed Central

Hu, H; Haas, S A; Chelly, J; Van Esch, H; Raynaud, M; de Brouwer, A P M; Weinert, S; Froyen, G; Frints, S G M; Laumonnier, F; Zemojtel, T; Love, M I; Richard, H; Emde, A-K; Bienek, M; Jensen, C; Hambrock, M; Fischer, U; Langnick, C; Feldkamp, M; Wissink-Lindhout, W; Lebrun, N; Castelnau, L; Rucci, J; Montjean, R; Dorseuil, O; Billuart, P; Stuhlmann, T; Shaw, M; Corbett, M A; Gardner, A; Willis-Owen, S; Tan, C; Friend, K L; Belet, S; van Roozendaal, K E P; Jimenez-Pocquet, M; Moizard, M-P; Ronce, N; Sun, R; O'Keeffe, S; Chenna, R; van Bömmel, A; Göke, J; Hackett, A; Field, M; Christie, L; Boyle, J; Haan, E; Nelson, J; Turner, G; Baynam, G; Gillessen-Kaesbach, G; Müller, U; Steinberger, D; Budny, B; Badura-Stronka, M; Latos-Bieleńska, A; Ousager, L B; Wieacker, P; Rodríguez Criado, G; Bondeson, M-L; Annerén, G; Dufke, A; Cohen, M; Van Maldergem, L; Vincent-Delorme, C; Echenne, B; Simon-Bouy, B; Kleefstra, T; Willemsen, M; Fryns, J-P; Devriendt, K; Ullmann, R; Vingron, M; Wrogemann, K; Wienker, T F; Tzschach, A; van Bokhoven, H; Gecz, J; Jentsch, T J; Chen, W; Ropers, H-H; Kalscheuer, V M

2016-01-01

X-linked intellectual disability (XLID) is a clinically and genetically heterogeneous disorder. During the past two decades in excess of 100 X-chromosome ID genes have been identified. Yet, a large number of families mapping to the X-chromosome remained unresolved suggesting that more XLID genes or loci are yet to be identified. Here, we have investigated 405 unresolved families with XLID. We employed massively parallel sequencing of all X-chromosome exons in the index males. The majority of these males were previously tested negative for copy number variations and for mutations in a subset of known XLID genes by Sanger sequencing. In total, 745 X-chromosomal genes were screened. After stringent filtering, a total of 1297 non-recurrent exonic variants remained for prioritization. Co-segregation analysis of potential clinically relevant changes revealed that 80 families (20%) carried pathogenic variants in established XLID genes. In 19 families, we detected likely causative protein truncating and missense variants in 7 novel and validated XLID genes (CLCN4, CNKSR2, FRMPD4, KLHL15, LAS1L, RLIM and USP27X) and potentially deleterious variants in 2 novel candidate XLID genes (CDK16 and TAF1). We show that the CLCN4 and CNKSR2 variants impair protein functions as indicated by electrophysiological studies and altered differentiation of cultured primary neurons from Clcn4−/− mice or after mRNA knock-down. The newly identified and candidate XLID proteins belong to pathways and networks with established roles in cognitive function and intellectual disability in particular. We suggest that systematic sequencing of all X-chromosomal genes in a cohort of patients with genetic evidence for X-chromosome locus involvement may resolve up to 58% of Fragile X-negative cases. PMID:25644381

The Fanconi anemia family of genes and its correlation with breast cancer susceptibility and breast cancer features.

PubMed

Barroso, E; Pita, G; Arias, J I; Menendez, P; Zamora, P; Blanco, M; Benitez, J; Ribas, G

2009-12-01

Fanconi anemia (FA) family of proteins participates in the DNA repair pathway by homologous recombination, and it is currently formed by 13 genes. Some of these proteins also confer susceptibility to hereditary breast and ovarian cancer (HBOC), since FANCD1 is the BRCA2 breast cancer susceptibility gene, and FANCN/PALB2 and FANCJ/BRIP1 explain 2% of non-BRCA1/2 HBOC families. Thus, there is an important connection between FA and BRCA pathways. In a previous case-control association study analysing FANCA, FANCD2 and FANCL, we reported an association between FANCD2 and sporadic breast cancer (BC) risk (OR = 1.35). In order to know whether variants in other FA genes could also be involved in this association, we have extended our study with the rest of FA genes and some others implicated in the BRCA pathway. We have also analyzed the correlation with survival, nodal metastasis and hormonal receptors (ER- and PR-). A total of 61 SNPs in ten FA genes (FANC-B, -C, -D1, -E, -F, -G, -I, -J, -M, -N) and five FA related genes (ATM, ATR, BRCA1, H2AX and USP1) were studied in a total of 547 consecutive and nonrelated sporadic BC cases and 552 unaffected controls from the Spanish population. Association analyses reported marginal statistically significant results with the minor allele of intronic SNPs in three genes: BRCA1, BRCA2/FANCD1, and ATM. Survival association with SNPs on FANCC and BRCA2/FANCD1 genes were also reported. Sub-group analyses revealed associations between SNPs on FANCI and ATM and nodal metastasis status and between FANCJ/BRIP1 and FANCN/PALB2 and PR- status.

Aerosol-assisted molten salt synthesis of NaInS(2) nanoplates for use as a new photoanode material.

PubMed

Mann, Amanda K P; Wicker, Susanne; Skrabalak, Sara E

2012-12-04

NaInS(2) , a H(2) -evolving photocatalyst, is synthesized as single-crystalline hexagonal plates by coupling a molten salt synthesis with ultrasonic spray pyrolysis (USP) for the first time. USP NaInS(2) films are used as a new photoanode material and have an initial photocurrent of ≈37 μA/cm(2) upon illumination and activities 25 times greater than films made from a standard non-aerosol NaInS(2) sample. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

LASER APPLICATIONS AND OTHER TOPICS IN QUANTUM ELECTRONICS: Application of the Wigner function and matrix optics to describe variations in the shape of ultrashort laser pulses propagating through linear optical systems

NASA Astrophysics Data System (ADS)

Gitin, Andrey V.

2006-04-01

The transformation of the shape of ultrashort laser pulses (USPs) in time can be described similarly to the process of image formation in space. It is shown that the wave description of imaging is simplified by using the Wigner function, this description in the quadratic approximation being identical to the use of the ABCD matrices. The transformation of USPs propagating through linear optical systems was described and these systems were classified by the methods of matrix optics.

Modeling the erosion of tropical volcanic ocean islands : The Tahiti island case (French Polynesia)

NASA Astrophysics Data System (ADS)

Ye, F.; Sichoix, L.; Barriot, J.; Dumas, P.

2009-12-01

In this study, we are interested in modeling the erosion of the Tahiti island, with two main objectives: risk assessment (erodibility of terrains with rainfall, catastrophic runoffs) and estimation of subsidence rate. The Tahiti island created around 1.4 Myears ago by an intraplate hotspot (aerial radiometric dating), is divided into two geological units: the main island Tahiti-Nui to northwest (end of volcanism 200,000 years ago) and the subsidiary Tahiti-Iti to the southeast (end of volcanism 380,000 years ago). It is now volcanically inactive and is deeply dissected by erosion. Tahiti Nui is around 30 km in diameter, and Tahiti Iti around 15 km. Both are linked through the isthmus of Taravao. The highest elevation is 2241 m. The two sub-islands are basaltic edifices, with an overwhelming presence of oxisols (down to tens of meters in some places). Slopes can be divided into three classes: 15° for the global slope of the shield volcanoes, 47° for the incision valleys and 2° for the seashore rim. Rainfalls range from 8,000 mm/year on the East side of Tahiti (trade winds) to 2,000 mm/year on the West side, the humid season of a year is summer. This study is conducted to validate the Unit Stream Power Erosion and Deposition (USPED) model, an enrichment to the Universal Soil Loss Equation (USLE) to calculate average annual soil loss per unit land area resulting from rill and sheet erosion. The USPED model differs from other USLE models on how it handles the influence of topography on the erosion process, because USLE consider erosion only along the flow line without the influence of flow convergence/divergence. As the result, the USPED model predicts both erosion and deposition, while most other USLE-based models are limited to predictions of erosion only. The USLE, USPED equation can be written as A=R*K*LS*C*P where A is the soil loss, R the rainfall-runoff erosivity factor, K a soil erodibility factor, L a slope-length factor, S a slope steepness factor, C a cover-management factor and P a supporting practice factor. However, USPED adds a dimensionless index of sediment transport capacity and a topographic index, representing the change in transport capacity in the flow direction, to estimate the spatial distribution of both erosion and deposition. As an application, we show how this approach permits a better modeling of the soil losses in Tahiti with respect to the basic USLE-only approach, with both societal and risk-assessment benefits. Keywords: erosion, volcano, modeling, USLE, USPED

Phylogenetic relationships, biofilm formation, motility, antibiotic resistance and extended virulence genotypes among Escherichia coli strains from women with community-onset primitive acute pyelonephritis.

PubMed

Pompilio, Arianna; Crocetta, Valentina; Savini, Vincenzo; Petrelli, Dezemona; Di Nicola, Marta; Bucco, Silvia; Amoroso, Luigi; Bonomini, Mario; Di Bonaventura, Giovanni

2018-01-01

The present work set out to search for a virulence repertoire distinctive for Escherichia coli causing primitive acute pyelonephritis (APN). To this end, the virulence potential of 18 E. coli APN strains was genotypically and phenotypically assessed, comparatively with 19 strains causing recurrent cystitis (RC), and 16 clinically not significant (control, CO) strains. Most of the strains belong to phylogenetic group B1 (69.8%; p<0.01), and APN strains showed unique features, which are the presence of phylogroup A, and the absence of phylogroup B2 and non-typeable strains. Overall, the most dominant virulence factor genes (VFGs) were ecpA and fyuA (92.4 and 86.7%, respectively; p<0.05), and the mean number of VFGs was significantly higher in uropathogenic strains. Particularly, papAH and malX were exclusive for uropathogenic strains. APN and RC strains showed a significantly higher prevalence of fyuA, usp, and malX than of CO strains. Compared to RC strains, APN ones showed a higher prevalence of iha, but a lower prevalence of iroN, cnf1, and kpsMT-II. Hierarchical cluster analysis showed a higher proportion of two gene clusters (malX and usp, and fyuA and ecpA) were detected in the APN and RC groups than in CO, whereas iutA and iha clusters were detected more frequently in APN strains. The motility level did not differ among the study-groups and phylogroups considered, although a higher proportion of swarming strains was observed in APN strains. Antibiotic-resistance rates were generally low except for ampicillin (37.7%), and were not associated with specific study- or phylogenetic groups. APN and RC strains produced more biofilm than CO strains. In APN strains, iha was associated with higher biofilm biomass formation, whereas iroN and KpSMT-K1 were associated with a lower amount of biofilm biomass. Further work is needed to grasp the virulence and fitness mechanisms adopted by E. coli causing APN, and hence develop new therapeutic and prophylactic approaches.

Development and validation of a hydrophilic interaction chromatography method coupled with a charged aerosol detector for quantitative analysis of nonchromophoric α-hydroxyamines, organic impurities of metoprolol.

PubMed

Xu, Qun; Tan, Shane; Petrova, Katya

2016-01-25

The European Pharmacopeia (EP) metoprolol impurities M and N are polar, nonchromophoric α-hydroxyamines, which are poorly retained in a conventional reversed-phase chromatographic system and are invisible for UV detection. Impurities M and N are currently analyzed by TLC methods in the EP as specified impurities and in the United States Pharmacopeia-National Formulary (USP-NF) as unspecified impurities. In order to modernize the USP monographs of metoprolol drug substances and related drug products, a hydrophilic interaction chromatography (HILIC) method coupled with a charged aerosol detector (CAD) was explored for the analysis of the two impurities. A comprehensive column screening that covers a variety of HILIC stationary phases (underivatized silica, amide, diol, amino, zwitterionic, polysuccinimide, cyclodextrin, and mixed-mode) and optimization of HPLC conditions led to the identification of a Halo Penta HILIC column (4.6 × 150 mm, 5 μm) and a mobile phase comprising 85% acetonitrile and 15% ammonium formate buffer (100 mM, pH 3.2). Efficient separations of metoprolol, succinic acid, and EP metoprolol impurities M and N were achieved within a short time frame (<8 min). The HILIC-CAD method was subsequently validated per USP validation guidelines with respect to specificity, robustness, linearity, accuracy, and precision, and could be incorporated into the current USP-NF monographs to replace the outdated TLC methods. Furthermore, the developed method was successfully applied to determine organic impurities in metoprolol drug substance (metoprolol succinate) and drug products (metoprolol tartrate injection and metoprolol succinate extended release tablets). Copyright © 2015 Elsevier B.V. All rights reserved.

The diagnosis of nonclassic congenital adrenal hyperplasia due to 21-hydroxylase deficiency, based on serum basal or post-ACTH stimulation 17-hydroxyprogesterone, can lead to false-positive diagnosis.

PubMed

Ambroziak, Urszula; Kępczyńska-Nyk, Anna; Kuryłowicz, Alina; Małunowicz, Ewa Maria; Wójcicka, Anna; Miśkiewicz, Piotr; Macech, Magdalena

2016-01-01

As nonclassic congenital adrenal hyperplasia (NCCAH) needs to be taken into account in women with hyperandrogenism, we aimed to assess whether the recommended level of poststimulated 17OHP ≥30 nmol/l confirms NCCAH. Forty, consecutive women with biochemical and/or clinical hyperandrogenism (aged 25·4, 18-38) suspected of having NCCAH were recruited to the study. In patients with 17OHP level between 5·1 and 29·9 nmol/l an ACTH stimulation test was performed. In patients with basal or poststimulated 17OHP ≥30 nmol/l, twenty-four-hour urinary steroid profile (USP) analysis was performed and CYP21A2 mutation was assessed. In selected patients with poststimulated 17OHP <30 nmol/l USP was also performed. The group was divided into two subgroups with basal or poststimulated 17OHP ≥30 nmol/l (group A) and with poststimulated 17OHP <30 nmol/l (group B). Among 40 patients, basal or poststimulated 17OHP ≥30 nmol/l was found in 21, but NCCAH was confirmed by USP followed by genetic testing only in 5 (24%). Four patients were diagnosed as heterozygotes, and in twelve, no CYP21A2 mutation was detected. The diagnosis of NCCAH based only on serum 17OHP measurements (basal or poststimulated) may lead to false-positive diagnosis when performed by immunoassay with a cut-off value of ≥30 nmol/l. The definitive diagnosis can be established based on USP and/or genetic testing. © 2015 John Wiley & Sons Ltd.

Economic evaluation of epinephrine auto-injectors for peanut allergy.

PubMed

Shaker, Marcus; Bean, Katherine; Verdi, Marylee

2017-08-01

Three commercial epinephrine auto-injectors were available in the United States in the summer of 2016: EpiPen, Adrenaclick, and epinephrine injection, USP auto-injector. To describe the variation in pharmacy costs among epinephrine auto-injector devices in New England and evaluate the additional expense associated with incremental auto-injector costs. Decision analysis software was used to evaluate costs of the most and least expensive epinephrine auto-injector devices for children with peanut allergy. To evaluate regional variation in epinephrine auto-injector costs, a random sample of New England national and corporate pharmacies was compared with a convenience sample of pharmacies from 10 Canadian provinces. Assuming prescriptions written for 2 double epinephrine packs each year (home and school), the mean costs of food allergy over the 20-year model horizon totaled $58,667 (95% confidence interval [CI] $57,745-$59,588) when EpiPen was prescribed and $45,588 (95% CI $44,873-$46,304) when epinephrine injection, USP auto-injector was prescribed. No effectiveness differences were evident between groups, with 17.19 (95% CI 17.11-17.27) quality-adjusted life years accruing for each subject. The incremental cost per episode of anaphylaxis treated with epinephrine over the model horizon was $12,576 for EpiPen vs epinephrine injection, USP auto-injector. EpiPen costs were lowest at Canadian pharmacies ($96, 95% CI $85-$107). There was price consistency between corporate and independent pharmacies throughout New England by device brand, with the epinephrine injection, USP auto-injector being the most affordable device. Cost differences among epinephrine auto-injectors were significant. More expensive auto-injector brands did not appear to provide incremental benefit. Copyright © 2017 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

The diagnostic value of tumor markers in bronchoalveolar lavage fluid for the peripheral pulmonary carcinoma.

PubMed

Zhang, Shi; Zhao, Yun-Feng; Zhang, Ming-Zhou; Wu, Xue-Ling

2017-07-01

To investigate the value of Ubiquitin specific peptidase 8 (USP8), Chitinase 3-like 1 (YKL40), Heat shock protein 90a (HSP90α), glutathione S-transferase P1 (GSTP1), carcinoembryonic antigen (CEA), neuron specific enolase (NSE) and cytokeratin fragment antiogen 21-1 (CYFRA21-1) in bronchoalveolar lavage fluid (BALF) and serum for diagnosis in patients with peripheral lung cancer. The concentration of these markers were measured in 50 patients with peripheral lung cancer and 50 patients with benign lung diseases by using enzyme-linked immuno sorbent assay methods. There were significant differences between the peripheral lung cancer group and the benign lung disease group (P < 0.05) in the BALF of USP8, YKL40, HSP90α, CEA, NSE and CYFRA21-1. There were significant differences between the peripheral lung cancer group and the benign lung disease group (P < 0.05) in the serum of HSP90α and CEA. There were no differences in others. There were no correlation between the concentration of all markers and age, histological type, TNM stage (I-IV). There was a weak correlation between the primary foci diameters and the concentration of YKL40 in BALF. (Pearson's correlation: 0.203, P = 0.048) The diagnostic efficiencies of USP8, YKL40, HSP90α were superior to CYFRA21-1 and NSE, being lower CEA. Detection of tumor markers in BALF was superior to serum specimens. The measurement of USP8, HSP90α and YKL40 in BALF had more clinical value for the diagnosis of peripheral pulmonary carcinoma. © 2015 John Wiley & Sons Ltd.

Multicenter validation of the value of BASFI and BASDAI in Chinese ankylosing spondylitis and undifferentiated spondyloarthropathy patients

PubMed Central

Lin, Zhiming; He, Peigen; Gao, Jiesheng; Zuo, Xiaoxia; Ye, Zhizhong; Shao, Fengmin; Zhan, Feng; Lin, Jinying; Li, Li; Wei, Yanlin; Xu, Manlong; Liao, Zetao; Lin, Qu

2009-01-01

The objectives of this study were to evaluate the reliability of Bath ankylosing spondylitis functional index (BASFI) and Bath ankylosing spondylitis disease activity index (BASDAI) in Chinese ankylosing spondylitis (AS) and undifferentiated spondyloarthropathy (USpA) patients. 664 AS patients by the revised New York criteria for AS and 252 USpA patients by the European Spondyloarthropathy Study Group criteria were enrolled. BASDAI and BASFI questionnaires were translated into Chinese. Participants were required to fill in BASFI and BASDAI questionnaires again after 24 h. Moreover, BASDAI and BASFI were compared in AS patients receiving Enbrel or infliximab before and after treatment. For AS group, BASDAI ICC: 0.9502 (95% CI: 0.9330–0.9502, α = 0.9702), BASFI ICC: 0.9587 (95% CI: 0.9521–0.9645, α = 0.9789). For USpA group, BASDAI ICC: 0.9530 (95% CI: 0.9402–0.9632, α = 0.9760), BASFI ICC: 0.9900 (95% CI: 0.9871–0.9922, α = 0.9950). In the AS group, disease duration, occipital wall distance, modified Schober test, chest expansion, ESR, and CRP showed significant correlation with BASDAI and BASFI (all P < 0.01). In the USpA group, onset age, ESR, and CRP were significantly correlated with BASDAI (all P < 0.05), while modified Schober test, ESR, and CRP were significantly associated with BASFI (all P < 0.05). The change in BASDAI and BASFI via Enbrel or infliximab treatment showed a significant positive correlation (P < 0.01). The two instruments have good reliability and reference value regarding the evaluation of patient’s condition and anti-TNF-α treatment response. PMID:20012866

Initial load-to-failure and failure analysis in single- and double-row repair techniques for rotator cuff repair.

PubMed

Baums, M H; Buchhorn, G H; Gilbert, F; Spahn, G; Schultz, W; Klinger, H-M

2010-09-01

This experimental study aimed to compare the load-to-failure rate and stiffness of single- versus double-row suture techniques for repairing rotator cuff lesions using two different suture materials. Additionally, the mode of failure of each repair was evaluated. In 32 sheep shoulders, a standardized tear of the infraspinatus tendon was created. Then, n = 8 specimen were randomized to four repair methods: (1) Double-row Anchor Ethibond coupled with polyester sutures, USP No. 2; (2) Double-Row Anchor HiFi with polyblend polyethylene sutures, USP No. 2; (3) Single-Row Anchor Ethibond coupled with braided polyester sutures, USP No. 2; and (4) Single-Row Anchor HiFi with braided polyblend polyethylene sutures, USP No. 2. Arthroscopic Mason-Allen stitches were placed (single-row) and combined with medial horizontal mattress stitches (double-row). All specimens were loaded to failure at a constant displacement rate on a material testing machine. Group 4 showed lowest load-to-failure result with 155.7 +/- 31.1 N compared to group 1 (293.4 +/- 16.1 N) and group 2 (397.7 +/- 7.4 N) (P < 0.001). Stiffness was highest in group 2 (162 +/- 7.3 N/mm) and lowest in group 4 (84.4 +/- 19.9 mm) (P < 0.001). In group 4, the main cause of failure was due to the suture cutting through the tendon (n = 6), a failure case observed in only n = 1 specimen in group 2 (P < 0.001). A double-row technique combined with arthroscopic Mason-Allen/horizontal mattress stitches provides high initial failure strength and may minimize the risk of the polyethylene sutures cutting through the tendon in rotator cuff repair when a single load force is used.

De-agglomeration Effect of the US Pharmacopeia and Alberta Throats on Carrier-Based Powders in Commercial Inhalation Products.

PubMed

Leung, Sharon Shui Yee; Tang, Patricia; Zhou, Qi Tony; Tong, Zhenbo; Leung, Cassandra; Decharaksa, Janwit; Yang, Runyu; Chan, Hak-Kim

2015-11-01

The US pharmacopeia (USP) and Alberta throats were recently reported to cause further de-agglomeration of carrier-free powders emitted from some dry powder inhalers (DPIs). This study assessed if they have similar influences on commercially available carrier-based DPIs. A straight tube, a USP throat, and an Alberta throat (non-coated and coated) were used for cascade impaction testing. Aerosol fine particle fraction (FPF ≤ 5 μm) was computed to evaluate throat-induced de-agglomeration. Computational fluid dynamics are employed to simulate airflow patterns and particle trajectories inside the USP and Alberta throats. For all tested products, no significant differences in the in vitro aerosol performance were observed between the USP throat and the straight tube. Using fine lactose carriers (<10 μm), Symbicort(®) and Oxis(™) showed minimal impaction inside the Alberta throat and resulted in similar FPF among all induction ports. For products using coarse lactose carriers (>10 μm), impaction frequency and energy inside the Alberta throat were significant. Further de-agglomeration was noted inside the non-coated Alberta throat for Seretide(®) and Spiriva(®), but agglomerates emitted from Relenza(®), Ventolin(®), and Foradil(®) did not further break up into smaller fractions. The coated Alberta throat considerably reduced the FPF values of these products due to the high throat retention, but they generally agreed better with the in vivo data. In conclusion, depending on the powder formulation (including carrier particle size), the inhaler, and the induction port, further de-agglomeration could happen ex-inhaler and create differences in the in vitro measurements.

An Ultra-short Period Rocky Super-Earth with a Secondary Eclipse and a Neptune-like Companion around K2-141

NASA Astrophysics Data System (ADS)

Malavolta, Luca; Mayo, Andrew W.; Louden, Tom; Rajpaul, Vinesh M.; Bonomo, Aldo S.; Buchhave, Lars A.; Kreidberg, Laura; Kristiansen, Martti H.; Lopez-Morales, Mercedes; Mortier, Annelies; Vanderburg, Andrew; Coffinet, Adrien; Ehrenreich, David; Lovis, Christophe; Bouchy, Francois; Charbonneau, David; Ciardi, David R.; Collier Cameron, Andrew; Cosentino, Rosario; Crossfield, Ian J. M.; Damasso, Mario; Dressing, Courtney D.; Dumusque, Xavier; Everett, Mark E.; Figueira, Pedro; Fiorenzano, Aldo F. M.; Gonzales, Erica J.; Haywood, Raphaëlle D.; Harutyunyan, Avet; Hirsch, Lea; Howell, Steve B.; Johnson, John Asher; Latham, David W.; Lopez, Eric; Mayor, Michel; Micela, Giusi; Molinari, Emilio; Nascimbeni, Valerio; Pepe, Francesco; Phillips, David F.; Piotto, Giampaolo; Rice, Ken; Sasselov, Dimitar; Ségransan, Damien; Sozzetti, Alessandro; Udry, Stéphane; Watson, Chris

2018-03-01

Ultra-short period (USP) planets are a class of low-mass planets with periods shorter than one day. Their origin is still unknown, with photo-evaporation of mini-Neptunes and in situ formation being the most credited hypotheses. Formation scenarios differ radically in the predicted composition of USP planets, and it is therefore extremely important to increase the still limited sample of USP planets with precise and accurate mass and density measurements. We report here the characterization of a USP planet with a period of 0.28 days around K2-141 (EPIC 246393474), and the validation of an outer planet with a period of 7.7 days in a grazing transit configuration. We derived the radii of the planets from the K2 light curve and used high-precision radial velocities gathered with the HARPS-N spectrograph for mass measurements. For K2-141b, we thus inferred a radius of 1.51 ± 0.05 R {}\\oplus and a mass of 5.08 ± 0.41 M {}\\oplus , consistent with a rocky composition and lack of a thick atmosphere. K2-141c is likely a Neptune-like planet, although due to the grazing transits and the non-detection in the RV data set, we were not able to put a strong constraint on its density. We also report the detection of secondary eclipses and phase curve variations for K2-141b. The phase variation can be modeled either by a planet with a geometric albedo of 0.30 ± 0.06 in the Kepler bandpass, or by thermal emission from the surface of the planet at ∼3000 K. Only follow-up observations at longer wavelengths will allow us to distinguish between these two scenarios.

In vitro biorelevant models for evaluating modified release mesalamine products to forecast the effect of formulation and meal intake on drug release.

PubMed

Andreas, Cord J; Chen, Ying-Chen; Markopoulos, Constantinos; Reppas, Christos; Dressman, Jennifer

2015-11-01

Postprandial administration of solid oral dosage forms greatly changes the dissolution environment compared to fasted state administration. The aims of this study were to investigate and forecast the effect of co-administration of a meal on drug release for delayed and/or extended release mesalamine formulations as well as design of in vitro tests to distinguish among formulations in a biorelevant way. Five different mesalamine formulations (Asacol® 400 mg, Mezavant® 1200 mg, Pentasa® 500 mg and Salofalk® in the 250 mg and 500 mg strengths) were investigated with biorelevant dissolution methods using the USP apparatus III and USP apparatus IV (open loop mode) under both fasted and fed state conditions, as well as with the dissolution methods described in pharmacopeia for delayed and extended release mesalamine products. Using the biorelevant experimental conditions proposed in this study, changes in release in the proximal gut due to meal intake are forecast to be minimal for Asacol®, Mezavant®, Pentasa® and Salofalk® 500 mg, while for Salofalk® 250 mg release was predicted to occur much earlier under fed state conditions. The USP apparatus III generally tended to result in faster dissolution rates and forecast more pronounced food effects for Salofalk® 250 mg than the USP apparatus IV. The biorelevant dissolution gradients were also able to reflect the in vivo behavior of the formulations. In vitro biorelevant models can be useful in the comparison of the release behavior from different delayed and extended release mesalamine formulations as well as forecasting effects of concomitant meal intake on drug release. Copyright © 2015 Elsevier B.V. All rights reserved.

The substrate binding domains of human SIAH E3 ubiquitin ligases are now crystal clear

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Qi; Wang, Zhongduo; Hou, Feng

2017-01-01

Seven in absentia homologs (SIAHs) comprise a family of highly conserved E3 ubiquitin ligases that play an important role in regulating signalling pathways in tumorigenesis, including the DNA damage repair and hypoxia response pathways. SIAH1 and SIAH2 have been found to function as a tumour repressor and a proto-oncogene, respectively, despite the high sequence identity of their substrate binding domains (SBDs). Ubiquitin-specific protease USP19 is a deubiquitinase that forms a complex with SIAHs and counteracts the ligase function. Much effort has been made to find selective inhibitors of the SIAHs E3 ligases. Menadione was reported to inhibit SIAH2 specifically. Wemore » used X-ray crystallography, peptide array, bioinformatic analysis, and biophysical techniques to characterize the structure and interaction of SIAHs with deubiquitinases and literature reported compounds. We solved the crystal structures of SIAH1 in complex with a USP19 peptide and of the apo form SIAH2. Phylogenetic analysis revealed the SIAH/USP19 complex is conserved in evolution. We demonstrated that menadione destabilizes both SIAH1 and SIAH2 non-specifically through covalent modification. The SBDs of SIAH E3 ligases are structurally similar with a subtle stability difference. USP19 is the only deubiquitinase that directly binds to SIAHs through the substrate binding pocket. Menadione is not a specific inhibitor for SIAH2. The crystallographic models provide structural insights into the substrate binding of the SIAH family E3 ubiquitin ligases that are critically involved in regulating cancer-related pathways. Our results suggest caution should be taken when using menadione as a specific SIAH2 inhibitor.« less

Improvement of pest resistance in transgenic tobacco plants expressing dsRNA of an insect-associated gene EcR.

PubMed

Zhu, Jin-Qi; Liu, Shumin; Ma, Yao; Zhang, Jia-Qi; Qi, Hai-Sheng; Wei, Zhao-Jun; Yao, Qiong; Zhang, Wen-Qing; Li, Sheng

2012-01-01

The adoption of pest-resistant transgenic plants to reduce yield loss and pesticide utilization has been successful in the past three decades. Recently, transgenic plant expressing double-stranded RNA (dsRNA) targeting pest genes emerges as a promising strategy for improving pest resistance in crops. The steroid hormone, 20-hydroxyecdysone (20E), predominately controls insect molting via its nuclear receptor complex, EcR-USP. Here we report that pest resistance is improved in transgenic tobacco plants expressing dsRNA of EcR from the cotton bollworm, Helicoverpa armigera, a serious lepidopteran pest for a variety of crops. When H. armigera larvae were fed with the whole transgenic tobacco plants expressing EcR dsRNA, resistance to H. armigera was significantly improved in transgenic plants. Meanwhile, when H. armigera larvae were fed with leaves of transgenic tobacco plants expressing EcR dsRNA, its EcR mRNA level was dramatically decreased causing molting defects and larval lethality. In addition, the transgenic tobacco plants expressing H. armigera EcR dsRNA were also resistant to another lepidopteran pest, the beet armyworm, Spodoptera exigua, due to the high similarity in the nucleotide sequences of their EcR genes. This study provides additional evidence that transgenic plant expressing dsRNA targeting insect-associated genes is able to improve pest resistance.

Apoptotic activity and gene responses in Drosophila melanogaster S2 cells, induced by azadirachtin A.

PubMed

Xu, Lin; Li, Sheng; Ran, Xueqin; Liu, Chang; Lin, Rutao; Wang, Jiafu

2016-09-01

Azadirachtin has been used as an antifeedant and growth disruption agent for many insect species. Previous investigations have reported the apoptotic effects of azadirachtin on some insect cells, but the molecular mechanisms are still not clear. This study investigated the underlying molecular mechanisms for the apoptotic effects induced by azadirachtin on Drosophila melanogaster S2 cells in vitro. The results of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay demonstrated that azadirachtin exhibited significant cytotoxicity to S2 cells in a time- and dose-dependent manner. The changes in cellular morphology and the DNA fragmentation demonstrated that azadirachtin induced remarkable apoptosis of S2 cells. Expression levels of 276 genes were found to be significantly changed in S2 cells after exposure to azadirachtin, as detected by Drosophila genome array. Among these genes, calmodulin (CaM) was the most highly upregulated gene. Azadirachtin was further demonstrated to trigger intracellular Ca(2+) release in S2 cells. The genes related to the apoptosis pathway, determined from chip data, were validated by the real-time quantitative polymerase chain reaction method. The results showed that azadirachtin-mediated intracellular Ca(2+) release was the primary event that triggered apoptosis in Drosophila S2 cells through both pathways of the Ca(2+) -CaM and EcR/Usp signalling cascade. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

Noninvasive in situ identification and band assignments of some pharmaceutical excipients inside USP vials with FT-near-infrared spectroscopy

NASA Astrophysics Data System (ADS)

Ali, Hassan Refat H.; Edwards, Howell G. M.; Scowen, Ian J.

2009-05-01

For the manufacture of dosage forms all ingredients must be reliably identified. In this paper, the suitability of FT-NIR spectroscopy to identify potassium sorbate, sodium starch glycollate, calcium ascorbate, calcium carbonate, candelilla wax, maltosextrin, monohydrated and anhydrous lactose inside USP vials was investigated. Differentiation between the anhydrous and monohydrated forms of lactose was found to be possible by studying the regions of the near-infrared spectrum corresponding to the combination and first overtone stretching frequencies of water. The results show unequivocally the potential of FT-NIR spectroscopy for rapid, in situ and non-destructive identification of pharmaceutical excipients.

Bioterrorism and its aftermath: dealing individually and organizationally with the emotional reactions to an anthrax attack.

PubMed

Sugden, Brian W; Katchmar, Rosemary

2005-01-01

From September 2001 through April 2004, the United States Postal Service (USPS) dealt, for the first time, with bioterrorism resulting in employee deaths and the closure of a large mail processing plant in Washington, D.C. The Employee Assistance Program (EAP) partnered with the USPS throughout this tumultuous time to meet the multiple and evolving behavioral health needs of the employees and facilitate the employees' emotional preparedness for their return to work at the closed facility. This paper discusses the reactions manifested by the employees during this extended period, as well as the EAP activities in the recovery process.

Electrochemical behavior and pH stability of artificial salivas for corrosion tests.

PubMed

Queiroz, Gláucia Maria Oliveira de; Silva, Leandro Freitas; Ferreira, José Tarcísio Lima; Gomes, José Antônio da Cunha P; Sathler, Lúcio

2007-01-01

It is assumed that the compositions of artificial salivas are similar to that of human saliva. However, the use of solutions with different compositions in in vitro corrosion studies can lead dissimilar electrolytes to exhibit dissimilar corrosivity and electrochemical stability. This study evaluated four artificial salivas as regards pH stability with time, redox potentials and the polarization response of an inert platinum electrode. The tested solutions were: SAGF medium, Mondelli artificial saliva, UFRJ artificial saliva (prepared at the School of Pharmacy, Federal University of Rio de Janeiro, RJ, Brazil) and USP-RP artificial saliva (prepared at the School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, SP, Brazil). It was observed that pH variations were less than 1 unit during a 50-hour test. The SAGF medium, and the UFRJ and USP-RP solutions exhibited more oxidizing characteristics, whereas the Mondelli solution presented reducing properties. Anodic polarization revealed oxidation of the evaluated electrolytes at potentials below +600 mV SCE. It was observed that the UFRJ and USP-RP solutions presented more intense oxidation and reduction processes as compared to the Mondelli and SAGF solutions.

Rehabilitative treatment of cleft lip and palate: experience of the Hospital for Rehabilitation of Craniofacial Anomalies - USP (HRAC-USP) - Part 2: Pediatric Dentistry and Orthodontics

PubMed Central

FREITAS, José Alberto de Souza; GARIB, Daniela Gamba; OLIVEIRA, Thais Marchini; LAURIS, Rita de Cássia Moura Carvalho; de ALMEIDA, Ana Lúcia Pompéia Fraga; NEVES, Lucimara Teixeira; TRINDADE-SUEDAM, Ivy Kiemle; YAEDÚ, Renato Yassutaka Faria; SOARES, Simone; PINTO, João Henrique Nogueira

2012-01-01

The aim of this article is to present the pediatric dentistry and orthodontic treatment protocol of rehabilitation of cleft lip and palate patients performed at the Hospital for Rehabilitation of Craniofacial Anomalies - University of São Paulo (HRAC-USP). Pediatric dentistry provides oral health information and should be able to follow the child with cleft lip and palate since the first months of life until establishment of the mixed dentition, craniofacial growth and dentition development. Orthodontic intervention starts in the mixed dentition, at 8-9 years of age, for preparing the maxillary arch for secondary bone graft procedure (SBGP). At this stage, rapid maxillary expansion is performed and a fixed palatal retainer is delivered before SBGP. When the permanent dentition is completed, comprehensive orthodontic treatment is initiated aiming tooth alignment and space closure. Maxillary permanent canines are commonly moved mesially in order to substitute absent maxillary lateral incisors. Patients with complete cleft lip and palate and poor midface growth will require orthognatic surgery for reaching adequate anteroposterior interarch relationship and good facial esthetics. PMID:22666849

Transcriptional profile of fibroblasts obtained from the primary site, lymph node and bone marrow of breast cancer patients.

PubMed

Del Valle, Paulo Roberto; Milani, Cintia; Brentani, Maria Mitzi; Katayama, Maria Lucia Hirata; de Lyra, Eduardo Carneiro; Carraro, Dirce Maria; Brentani, Helena; Puga, Renato; Lima, Leandro A; Rozenchan, Patricia Bortman; Nunes, Bárbara Dos Santos; Góes, João Carlos Guedes Sampaio; Azevedo Koike Folgueira, Maria Aparecida

2014-09-01

Cancer-associated fibroblasts (CAF) influence tumor development at primary as well as in metastatic sites, but there have been no direct comparisons of the transcriptional profiles of stromal cells from different tumor sites. In this study, we used customized cDNA microarrays to compare the gene expression profile of stromal cells from primary tumor (CAF, n = 4), lymph node metastasis (N+, n = 3) and bone marrow (BM, n = 4) obtained from breast cancer patients. Biological validation was done in another 16 samples by RT-qPCR. Differences between CAF vs N+, CAF vs BM and N+ vs BM were represented by 20, 235 and 245 genes, respectively (SAM test, FDR < 0.01). Functional analysis revealed that genes related to development and morphogenesis were overrepresented. In a biological validation set, NOTCH2 was confirmed to be more expressed in N+ (vs CAF) and ADCY2, HECTD1, HNMT, LOX, MACF1, SLC1A3 and USP16 more expressed in BM (vs CAF). Only small differences were observed in the transcriptional profiles of fibroblasts from the primary tumor and lymph node of breast cancer patients, whereas greater differences were observed between bone marrow stromal cells and the other two sites. These differences may reflect the activities of distinct differentiation programs.

A Novel Ecdysone Receptor Mediates Steroid-Regulated Developmental Events during the Mid-Third Instar of Drosophila

PubMed Central

Costantino, Benjamin F. B.; Bricker, Daniel K.; Alexandre, Kelly; Shen, Kate; Merriam, John R.; Antoniewski, Christophe; Callender, Jenna L.; Henrich, Vincent C.; Presente, Asaf; Andres, Andrew J.

2008-01-01

The larval salivary gland of Drosophila melanogaster synthesizes and secretes glue glycoproteins that cement developing animals to a solid surface during metamorphosis. The steroid hormone 20-hydroxyecdysone (20E) is an essential signaling molecule that modulates most of the physiological functions of the larval gland. At the end of larval development, it is known that 20E—signaling through a nuclear receptor heterodimer consisting of EcR and USP—induces the early and late puffing cascade of the polytene chromosomes and causes the exocytosis of stored glue granules into the lumen of the gland. It has also been reported that an earlier pulse of hormone induces the temporally and spatially specific transcriptional activation of the glue genes; however, the receptor responsible for triggering this response has not been characterized. Here we show that the coordinated expression of the glue genes midway through the third instar is mediated by 20E acting to induce genes of the Broad Complex (BRC) through a receptor that is not an EcR/USP heterodimer. This result is novel because it demonstrates for the first time that at least some 20E-mediated, mid-larval, developmental responses are controlled by an uncharacterized receptor that does not contain an RXR-like component. PMID:18566664

Transcriptional profile of fibroblasts obtained from the primary site, lymph node and bone marrow of breast cancer patients

PubMed Central

Del Valle, Paulo Roberto; Milani, Cintia; Brentani, Maria Mitzi; Katayama, Maria Lucia Hirata; de Lyra, Eduardo Carneiro; Carraro, Dirce Maria; Brentani, Helena; Puga, Renato; Lima, Leandro A.; Rozenchan, Patricia Bortman; Nunes, Bárbara dos Santos; Góes, João Carlos Guedes Sampaio; Azevedo Koike Folgueira, Maria Aparecida

2014-01-01

Cancer-associated fibroblasts (CAF) influence tumor development at primary as well as in metastatic sites, but there have been no direct comparisons of the transcriptional profiles of stromal cells from different tumor sites. In this study, we used customized cDNA microarrays to compare the gene expression profile of stromal cells from primary tumor (CAF, n = 4), lymph node metastasis (N+, n = 3) and bone marrow (BM, n = 4) obtained from breast cancer patients. Biological validation was done in another 16 samples by RT-qPCR. Differences between CAF vs N+, CAF vs BM and N+ vs BM were represented by 20, 235 and 245 genes, respectively (SAM test, FDR < 0.01). Functional analysis revealed that genes related to development and morphogenesis were overrepresented. In a biological validation set, NOTCH2 was confirmed to be more expressed in N+ (vs CAF) and ADCY2, HECTD1, HNMT, LOX, MACF1, SLC1A3 and USP16 more expressed in BM (vs CAF). Only small differences were observed in the transcriptional profiles of fibroblasts from the primary tumor and lymph node of breast cancer patients, whereas greater differences were observed between bone marrow stromal cells and the other two sites. These differences may reflect the activities of distinct differentiation programs. PMID:25249769

Harvey Cushing Treated the First Known Patient With Carney Complex.

PubMed

Tsay, Cynthia J; Stratakis, Constantine A; Faucz, Fabio Rueda; London, Edra; Stathopoulou, Chaido; Allgauer, Michael; Quezado, Martha; Dagradi, Terry; Spencer, Dennis D; Lodish, Maya

2017-10-01

Carney complex (CNC) is a syndrome characterized by hyperplasia of endocrine organs and may present with clinical features of Cushing syndrome and acromegaly due to functional adrenal and pituitary gland tumors. CNC has been linked to mutations in the regulatory subunit of protein kinase A type I-alpha ( PRKAR1A ) gene. Tissue samples were taken from the hypothalamus or thalamus or tumors of patients with pituitary adenomas seen and operated on by neurosurgeon Harvey Cushing between 1913 and 1932. Following DNA extraction, sequencing for genes of interest was attempted, including PRKAR1A , AIP , USP8 , GNAS1 , and GPR101 , to explore the possibility that these mutations associated with acromegaly, CNC, and Cushing syndrome have been conserved over time. We report a patient described by Dr. Cushing in 1914 with a clinical presentation and postmortem findings suggestive of CNC. Genetic sequencing of the hypothalamus and pituitary adenoma revealed a germline heterozygous p.Arg74His mutation in the PRKAR1A gene, a codon previously described as mutated in CNC, but with a novel amino acid change. This patient is, to our knowledge, the first molecularly confirmed individual with CNC. This case demonstrates the power of modern genetics in studying archived tissues and the importance of recording detailed clinical notes in the diagnosis of disease.

Somatic Genetic Variation in Solid Pseudopapillary Tumor of the Pancreas by Whole Exome Sequencing

PubMed Central

Guo, Meng; Luo, Guopei; Jin, Kaizhou; Long, Jiang; Cheng, He; Lu, Yu; Wang, Zhengshi; Yang, Chao; Xu, Jin; Ni, Quanxing; Yu, Xianjun; Liu, Chen

2017-01-01

Solid pseudopapillary tumor of the pancreas (SPT) is a rare pancreatic disease with a unique clinical manifestation. Although CTNNB1 gene mutations had been universally reported, genetic variation profiles of SPT are largely unidentified. We conducted whole exome sequencing in nine SPT patients to probe the SPT-specific insertions and deletions (indels) and single nucleotide polymorphisms (SNPs). In total, 54 SNPs and 41 indels of prominent variations were demonstrated through parallel exome sequencing. We detected that CTNNB1 mutations presented throughout all patients studied (100%), and a higher count of SNPs was particularly detected in patients with older age, larger tumor, and metastatic disease. By aggregating 95 detected variation events and viewing the interconnections among each of the genes with variations, CTNNB1 was identified as the core portion in the network, which might collaborate with other events such as variations of USP9X, EP400, HTT, MED12, and PKD1 to regulate tumorigenesis. Pathway analysis showed that the events involved in other cancers had the potential to influence the progression of the SNPs count. Our study revealed an insight into the variation of the gene encoding region underlying solid-pseudopapillary neoplasm tumorigenesis. The detection of these variations might partly reflect the potential molecular mechanism. PMID:28054945

An Integrated Human/Murine Transcriptome and Pathway Approach To Identify Prenatal Treatments For Down Syndrome.

PubMed

Guedj, Faycal; Pennings, Jeroen LA; Massingham, Lauren J; Wick, Heather C; Siegel, Ashley E; Tantravahi, Umadevi; Bianchi, Diana W

2016-09-02

Anatomical and functional brain abnormalities begin during fetal life in Down syndrome (DS). We hypothesize that novel prenatal treatments can be identified by targeting signaling pathways that are consistently perturbed in cell types/tissues obtained from human fetuses with DS and mouse embryos. We analyzed transcriptome data from fetuses with trisomy 21, age and sex-matched euploid controls, and embryonic day 15.5 forebrains from Ts1Cje, Ts65Dn, and Dp16 mice. The new datasets were compared to other publicly available datasets from humans with DS. We used the human Connectivity Map (CMap) database and created a murine adaptation to identify FDA-approved drugs that can rescue affected pathways. USP16 and TTC3 were dysregulated in all affected human cells and two mouse models. DS-associated pathway abnormalities were either the result of gene dosage specific effects or the consequence of a global cell stress response with activation of compensatory mechanisms. CMap analyses identified 56 molecules with high predictive scores to rescue abnormal gene expression in both species. Our novel integrated human/murine systems biology approach identified commonly dysregulated genes and pathways. This can help to prioritize therapeutic molecules on which to further test safety and efficacy. Additional studies in human cells are ongoing prior to pre-clinical prenatal treatment in mice.

SARS hCoV papain-like protease is a unique Lys48 linkage-specific di-distributive deubiquitinating enzyme

PubMed Central

Békés, Miklós; Rut, Wioletta; Kasperkiewicz, Paulina; Mulder, Monique P. C.; Ovaa, Huib; Drag, Marcin; Lima, Christopher D.; Huang, Tony T.

2015-01-01

Ubiquitin (Ub) and the ubiquitin-like modifier interferon stimulated gene 15 (ISG15) participate in the host defense of viral infections. Viruses, including the Severe Acute Respiratory Syndrome human coronavirus (SARS hCoV), have co-opted Ub/ISG15-conjugation pathways for their own advantage or have evolved effector proteins to counter pro-inflammatory properties of Ub/ISG15-conjugated host proteins. Here, we compare substrate specificities of the papain-like protease (PLpro) from the recently emerged Middle Eastern Respiratory Syndrome (MERS) hCoV to the related protease from SARS, SARS PLpro. Through biochemical assays, we show that similar to SARS PLpro, MERS PLpro is both a deubiquitinating and a deISGylating enzyme. Further analysis of the intrinsic deubiquitinating enzyme (DUB) activity of these viral proteases revealed unique differences between the recognition and cleavage specificities of polyUb chains. First, MERS PLpro shows broad linkage specificity for the cleavage of polyUb chains, while SARS PLpro prefers to cleave Lys48-linked polyUb chains. Second, MERS PLpro cleaves polyUb chains in a “mono-distributive” manner (one Ub at a time), and SARS PLpro prefers to cleave K48-linked poly-Ub chains by sensing a di-Ub moiety as a minimal recognition element using a “di-distributive” cleavage mechanism. The di-distributive cleavage mechanism for SARS PLpro appears to be uncommon among USP-family DUBs, as related USP family members from humans do not display such a mechanism. We propose that these intrinsic enzymatic differences between SARS and MERS PLpro will help identify pro-inflammatory substrates of these viral DUBs and can guide in the design of therapeutics to combat infection by coronaviruses. PMID:25764917

SARS hCoV papain-like protease is a unique Lys48 linkage-specific di-distributive deubiquitinating enzyme.

PubMed

Békés, Miklós; Rut, Wioletta; Kasperkiewicz, Paulina; Mulder, Monique P C; Ovaa, Huib; Drag, Marcin; Lima, Christopher D; Huang, Tony T

2015-06-01

Ubiquitin (Ub) and the Ub-like (Ubl) modifier interferon-stimulated gene 15 (ISG15) participate in the host defence of viral infections. Viruses, including the severe acute respiratory syndrome human coronavirus (SARS hCoV), have co-opted Ub-ISG15 conjugation pathways for their own advantage or have evolved effector proteins to counter pro-inflammatory properties of Ub-ISG15-conjugated host proteins. In the present study, we compare substrate specificities of the papain-like protease (PLpro) from the recently emerged Middle East respiratory syndrome (MERS) hCoV to the related protease from SARS, SARS PLpro. Through biochemical assays, we show that, similar to SARS PLpro, MERS PLpro is both a deubiquitinating (DUB) and a deISGylating enzyme. Further analysis of the intrinsic DUB activity of these viral proteases revealed unique differences between the recognition and cleavage specificities of polyUb chains. First, MERS PLpro shows broad linkage specificity for the cleavage of polyUb chains, whereas SARS PLpro prefers to cleave Lys48-linked polyUb chains. Secondly, MERS PLpro cleaves polyUb chains in a 'mono-distributive' manner (one Ub at a time) and SARS PLpro prefers to cleave Lys48-linked polyUb chains by sensing a di-Ub moiety as a minimal recognition element using a 'di-distributive' cleavage mechanism. The di-distributive cleavage mechanism for SARS PLpro appears to be uncommon among USP (Ub-specific protease)-family DUBs, as related USP family members from humans do not display such a mechanism. We propose that these intrinsic enzymatic differences between SARS and MERS PLpro will help to identify pro-inflammatory substrates of these viral DUBs and can guide in the design of therapeutics to combat infection by coronaviruses.

Identification of candidate genes for familial early-onset essential tremor.

PubMed

Liu, Xinmin; Hernandez, Nora; Kisselev, Sergey; Floratos, Aris; Sawle, Ashley; Ionita-Laza, Iuliana; Ottman, Ruth; Louis, Elan D; Clark, Lorraine N

2016-07-01

Essential tremor (ET) is one of the most common causes of tremor in humans. Despite its high heritability and prevalence, few susceptibility genes for ET have been identified. To identify ET genes, whole-exome sequencing was performed in 37 early-onset ET families with an autosomal-dominant inheritance pattern. We identified candidate genes for follow-up functional studies in five ET families. In two independent families, we identified variants predicted to affect function in the nitric oxide (NO) synthase 3 gene (NOS3) that cosegregated with disease. NOS3 is highly expressed in the central nervous system (including cerebellum), neurons and endothelial cells, and is one of three enzymes that converts l-arginine to the neurotransmitter NO. In one family, a heterozygous variant, c.46G>A (p.(Gly16Ser)), in NOS3, was identified in three affected ET cases and was absent in an unaffected family member; and in a second family, a heterozygous variant, c.164C>T (p.(Pro55Leu)), was identified in three affected ET cases (dizygotic twins and their mother). Both variants result in amino-acid substitutions of highly conserved amino-acid residues that are predicted to be deleterious and damaging by in silico analysis. In three independent families, variants predicted to affect function were also identified in other genes, including KCNS2 (KV9.2), HAPLN4 (BRAL2) and USP46. These genes are highly expressed in the cerebellum and Purkinje cells, and influence function of the gamma-amino butyric acid (GABA)-ergic system. This is in concordance with recent evidence that the pathophysiological process in ET involves cerebellar dysfunction and possibly cerebellar degeneration with a reduction in Purkinje cells, and a decrease in GABA-ergic tone.

A Methodology for the Development of RESTful Semantic Web Services for Gene Expression Analysis

PubMed Central

Guardia, Gabriela D. A.; Pires, Luís Ferreira; Vêncio, Ricardo Z. N.; Malmegrim, Kelen C. R.; de Farias, Cléver R. G.

2015-01-01

Gene expression studies are generally performed through multi-step analysis processes, which require the integrated use of a number of analysis tools. In order to facilitate tool/data integration, an increasing number of analysis tools have been developed as or adapted to semantic web services. In recent years, some approaches have been defined for the development and semantic annotation of web services created from legacy software tools, but these approaches still present many limitations. In addition, to the best of our knowledge, no suitable approach has been defined for the functional genomics domain. Therefore, this paper aims at defining an integrated methodology for the implementation of RESTful semantic web services created from gene expression analysis tools and the semantic annotation of such services. We have applied our methodology to the development of a number of services to support the analysis of different types of gene expression data, including microarray and RNASeq. All developed services are publicly available in the Gene Expression Analysis Services (GEAS) Repository at http://dcm.ffclrp.usp.br/lssb/geas. Additionally, we have used a number of the developed services to create different integrated analysis scenarios to reproduce parts of two gene expression studies documented in the literature. The first study involves the analysis of one-color microarray data obtained from multiple sclerosis patients and healthy donors. The second study comprises the analysis of RNA-Seq data obtained from melanoma cells to investigate the role of the remodeller BRG1 in the proliferation and morphology of these cells. Our methodology provides concrete guidelines and technical details in order to facilitate the systematic development of semantic web services. Moreover, it encourages the development and reuse of these services for the creation of semantically integrated solutions for gene expression analysis. PMID:26207740

Identifying candidate genes for 2p15p16.1 microdeletion syndrome using clinical, genomic, and functional analysis

PubMed Central

Bagheri, Hani; Badduke, Chansonette; Qiao, Ying; Colnaghi, Rita; Abramowicz, Iga; Alcantara, Diana; Dunham, Christopher; Wen, Jiadi; Wildin, Robert S.; Nowaczyk, Malgorzata J.M.; Eichmeyer, Jennifer; Lehman, Anna; Maranda, Bruno; Martell, Sally; Shan, Xianghong; Lewis, Suzanne M.E.; O’Driscoll, Mark; Gregory-Evans, Cheryl Y.

2016-01-01

The 2p15p16.1 microdeletion syndrome has a core phenotype consisting of intellectual disability, microcephaly, hypotonia, delayed growth, common craniofacial features, and digital anomalies. So far, more than 20 cases of 2p15p16.1 microdeletion syndrome have been reported in the literature; however, the size of the deletions and their breakpoints vary, making it difficult to identify the candidate genes. Recent reports pointed to 4 genes (XPO1, USP34, BCL11A, and REL) that were included, alone or in combination, in the smallest deletions causing the syndrome. Here, we describe 8 new patients with the 2p15p16.1 deletion and review all published cases to date. We demonstrate functional deficits for the above 4 candidate genes using patients’ lymphoblast cell lines (LCLs) and knockdown of their orthologs in zebrafish. All genes were dosage sensitive on the basis of reduced protein expression in LCLs. In addition, deletion of XPO1, a nuclear exporter, cosegregated with nuclear accumulation of one of its cargo molecules (rpS5) in patients’ LCLs. Other pathways associated with these genes (e.g., NF-κB and Wnt signaling as well as the DNA damage response) were not impaired in patients’ LCLs. Knockdown of xpo1a, rel, bcl11aa, and bcl11ab resulted in abnormal zebrafish embryonic development including microcephaly, dysmorphic body, hindered growth, and small fins as well as structural brain abnormalities. Our multifaceted analysis strongly implicates XPO1, REL, and BCL11A as candidate genes for 2p15p16.1 microdeletion syndrome. PMID:27699255

A Methodology for the Development of RESTful Semantic Web Services for Gene Expression Analysis.

PubMed

Guardia, Gabriela D A; Pires, Luís Ferreira; Vêncio, Ricardo Z N; Malmegrim, Kelen C R; de Farias, Cléver R G

2015-01-01

Gene expression studies are generally performed through multi-step analysis processes, which require the integrated use of a number of analysis tools. In order to facilitate tool/data integration, an increasing number of analysis tools have been developed as or adapted to semantic web services. In recent years, some approaches have been defined for the development and semantic annotation of web services created from legacy software tools, but these approaches still present many limitations. In addition, to the best of our knowledge, no suitable approach has been defined for the functional genomics domain. Therefore, this paper aims at defining an integrated methodology for the implementation of RESTful semantic web services created from gene expression analysis tools and the semantic annotation of such services. We have applied our methodology to the development of a number of services to support the analysis of different types of gene expression data, including microarray and RNASeq. All developed services are publicly available in the Gene Expression Analysis Services (GEAS) Repository at http://dcm.ffclrp.usp.br/lssb/geas. Additionally, we have used a number of the developed services to create different integrated analysis scenarios to reproduce parts of two gene expression studies documented in the literature. The first study involves the analysis of one-color microarray data obtained from multiple sclerosis patients and healthy donors. The second study comprises the analysis of RNA-Seq data obtained from melanoma cells to investigate the role of the remodeller BRG1 in the proliferation and morphology of these cells. Our methodology provides concrete guidelines and technical details in order to facilitate the systematic development of semantic web services. Moreover, it encourages the development and reuse of these services for the creation of semantically integrated solutions for gene expression analysis.

Inspection of the grapevine BURP superfamily highlights an expansion of RD22 genes with distinctive expression features in berry development and ABA-mediated stress responses.

PubMed

Matus, José Tomás; Aquea, Felipe; Espinoza, Carmen; Vega, Andrea; Cavallini, Erika; Dal Santo, Silvia; Cañón, Paola; Rodríguez-Hoces de la Guardia, Amparo; Serrano, Jennifer; Tornielli, Giovanni Battista; Arce-Johnson, Patricio

2014-01-01

The RESPONSIVE TO DEHYDRATION 22 (RD22) gene is a molecular link between abscisic acid (ABA) signalling and abiotic stress responses. Its expression has been used as a reliable ABA early response marker. In Arabidopsis, the single copy RD22 gene possesses a BURP domain also located at the C-terminus of USP embryonic proteins and the beta subunit of polygalacturonases. In grapevine, a RD22 gene has been identified but putative paralogs are also found in the grape genome, possibly forming a large RD22 family in this species. In this work, we searched for annotations containing BURP domains in the Vitis vinifera genome. Nineteen proteins were defined by a comparative analysis between the two genome predictions and RNA-Seq data. These sequences were compared to other plant BURPs identified in previous genome surveys allowing us to reconceive group classifications based on phylogenetic relationships and protein motif occurrence. We observed a lineage-specific evolution of the RD22 family, with the biggest expansion in grapevine and poplar. In contrast, rice, sorghum and maize presented highly expanded monocot-specific groups. The Vitis RD22 group may have expanded from segmental duplications as most of its members are confined to a region in chromosome 4. The inspection of transcriptomic data revealed variable expression of BURP genes in vegetative and reproductive organs. Many genes were induced in specific tissues or by abiotic and biotic stresses. Three RD22 genes were further studied showing that they responded oppositely to ABA and to stress conditions. Our results show that the inclusion of RNA-Seq data is essential while describing gene families and improving gene annotations. Robust phylogenetic analyses including all BURP members from other sequenced species helped us redefine previous relationships that were erroneously established. This work provides additional evidence for RD22 genes serving as marker genes for different organs or stresses in grapevine.

Norfloxacin and metronidazole topical formulations for effective treatment of bacterial infections and burn wounds

PubMed Central

Dua, Kamal; Malipeddi, Venkata Ramana; Madan, Jyotsna; Gupta, Gaurav; Chakravarthi, Srikumar; Awasthi, Rajendra; Kikuchi, Irene Satiko; De Jesus Andreoli Pinto, Terezinha

2016-01-01

Introduction Our various previous findings have shown the suitability of norfloxacin in the treatment of bacterial infections and burn wounds in alone as well as in combination with Curcuma longa in various topical (ointments, gels, and creams) and transdermal drug delivery systems. Aims and methods Keeping these facts in consideration, we have made an another attempt to prepare semisolid formulations containing 1% w/w of norfloxacin and metronidazole with different bases like Carbopol, polyethylene glycol, and hydroxypropylmethyl cellulose for effective treatment of bacterial infections and burn wounds. The prepared formulations were evaluated for physicochemical parameters, in vitro drug release, antimicrobial activity, and burn wound healing properties. Results The prepared formulations were compared with Silver Sulfadiazine cream 1%, USP. Antimicrobial activity of norfloxacin semisolid formulations was found to be equally effective against both aerobic and anaerobic bacteria in comparison to a marketed formulation of Silver Sulfadiazine 1% cream, USP. Based on the burn wound healing property, the prepared norfloxacin semisolid formulation was found to be in good agreement with marketed Silver Sulfadiazine 1% cream, USP. Conclusions These findings suggest formulations containing norfloxacin and metronidazole may also prove as an effective alternative for existing remedies in the treatment of bacterial infections and burn wounds. PMID:28386462

The golden age of Drosophila research at the Universidade de São Paulo (USP): A testimonial on the decades 1940–1950

PubMed Central

de Magalhães, Luiz Edmundo; Vilela, Carlos Ribeiro

2014-01-01

This article is a testimonial written by the first author regarding the research work performed with Drosophila between 1943 and 1959, at the Departamento de Biologia Geral of the Universidade de São Paulo (USP), which permitted the building of a nucleus of excellence. This research work, focused on the systematics and population genetics of the Neotropical species, began during the first of Dobzhansky’s six visits to USP. Special attention was given to the multinational megaprojects conducted during his longer stays, from August 1948 to July 1949 and from June 1955 to July 1956. The role played by the Rockefeller Foundation is duly remembered, and so is the undeniable contribution brought by Dobzhansky, to the establishment of several laboratories dedicated to research in the field of natural population genetics and to the qualification of human resources. On the other hand, important “backstage” episodes are retrieved which were omitted in the official history and occurred from the planning to the execution of the project, carried out on the Angra dos Reis islands. Special attention was given to the relationship problems which resulted from Dobzhansky’s, the leader’s, difficult personality and contributed to the failure of the second and last multinational project. PMID:24688301

The golden age of Drosophila research at the Universidade de São Paulo (USP): A testimonial on the decades 1940-1950.

PubMed

de Magalhães, Luiz Edmundo; Vilela, Carlos Ribeiro

2014-03-01

This article is a testimonial written by the first author regarding the research work performed with Drosophila between 1943 and 1959, at the Departamento de Biologia Geral of the Universidade de São Paulo (USP), which permitted the building of a nucleus of excellence. This research work, focused on the systematics and population genetics of the Neotropical species, began during the first of Dobzhansky's six visits to USP. Special attention was given to the multinational megaprojects conducted during his longer stays, from August 1948 to July 1949 and from June 1955 to July 1956. The role played by the Rockefeller Foundation is duly remembered, and so is the undeniable contribution brought by Dobzhansky, to the establishment of several laboratories dedicated to research in the field of natural population genetics and to the qualification of human resources. On the other hand, important "backstage" episodes are retrieved which were omitted in the official history and occurred from the planning to the execution of the project, carried out on the Angra dos Reis islands. Special attention was given to the relationship problems which resulted from Dobzhansky's, the leader's, difficult personality and contributed to the failure of the second and last multinational project.

The effect of excipients on the stability of levothyroxine sodium pentahydrate tablets.

PubMed

Patel, Himanshu; Stalcup, Apryll; Dansereau, Richard; Sakr, Adel

2003-10-02

Levothyroxine tablets, 50 microg, have been marketed for many decades but have had numerous recalls due to degradation and failure to meet potency. These experiments were devised to study the effects of various excipients on the stability of levothyroxine sodium pentahydrate in aqueous slurries and in formulated tablets. The active alone was found to be stable in the solid state for 6 months at 40 degrees C/75% RH whether stored in open or closed containers, and was found to be non-hygroscopic under normal processing conditions (>30% RH). In aqueous slurries with an excipient, the stability of the active improved as the pH of the slurry was increased from pH 3 to 11. Tablets manufactured with lactose anhydrous, starch, or microcrystalline cellulose failed to meet USP assay requirements at 3 months at 40 degrees C/75% RH. Tablets manufactured with dibasic calcium phosphate or mannitol met USP assay requirements at 3, but not 6 months when stored at 40 degrees C/75% RH. Tablets manufactured with dibasic calcium phosphate and a basic pH modifier, such as sodium carbonate, sodium bicarbonate, or magnesium oxide, met the USP assay requirements at both 3 and 6 months. Thus, the use of basic pH modifiers is a potential technique for improving the stability of levothyroxine sodium pentahydrate tablets.

[Translation and linguistic validation in classical Arabic of the urinary symptom profile (USP) questionnaire].

PubMed

Arabi, H; Bendeddouche, I; Khalfaoui, S; Louardi, N; Ameur, A; Lebreton, F; Amarenco, G

2013-04-01

The objective was to translate and linguistically validate in classical Arabic; the French version of the Urinary Symptom Profile (USP), the scale adapted to vesico-sphincter disorders. Prospective study of 30 patients suffering the vesico-sphincter disorders. The translation was obtained by the method: translation back-translation. Patients completed the final questionnaire on day 0 and day 15. The feasibility, acceptability, internal consistency using Cronbach's alpha and test-retest repeatability by the interclass correlation coefficient (ICC) with the confidence interval (CI) were studied. The sample consisted of 30 subjects including 20 men (66.6%) and 10 women (33.3%). The mean age was 48±18, 14 years ranging from 25 to 70 years. The questionnaire was feasible and acceptable. The Cronbach's alpha of the three dimensions, urinary stress incontinence, overactive bladder and voiding difficulties was respectively 0.9880, 0.9774 and 0.9683, respectively; the ICC was 0.9762 (95% CI: 0.9307-0.9919), 0.9558 (CI 95%: 0.8738-0.9849) and 0.9385 (95% CI: 0.8274-0.9789). The Arabic version of the classic USP had excellent internal consistency and excellent repeatability enable a full assessment of all urinary disorders and their severity. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Sensory phenomena related to tics, obsessive-compulsive symptoms, and global functioning in Tourette syndrome.

PubMed

Kano, Yukiko; Matsuda, Natsumi; Nonaka, Maiko; Fujio, Miyuki; Kuwabara, Hitoshi; Kono, Toshiaki

2015-10-01

Sensory phenomena, including premonitory urges, are experienced by patients with Tourette syndrome (TS) and obsessive-compulsive disorder (OCD). The goal of the present study was to investigate such phenomena related to tics, obsessive-compulsive symptoms (OCS), and global functioning in Japanese patients with TS. Forty-one patients with TS were assessed using the University of São Paulo Sensory Phenomena Scale (USP-SPS), the Premonitory Urge for Tics Scale (PUTS), the Yale Global Tic Severity Scale (YGTSS), the Dimensional Yale-Brown Obsessive-Compulsive Scale (DY-BOCS), and the Global Assessment of Functioning (GAF) Scale. USP-SPS and PUTS total scores were significantly correlated with YGTSS total and vocal tics scores. Additionally, both sensory phenomena severity scores were significantly correlated with DY-BOCS total OCS scores. Of the six dimensional OCS scores, the USP-SPS scores were significantly correlated with measures of aggression and sexual/religious dimensions. Finally, the PUTS total scores were significantly and negatively correlated with GAF scores. By assessing premonitory urges and broader sensory phenomena, and by viewing OCS from a dimensional approach, this study provides significant insight into sensory phenomena related to tics, OCS, and global functioning in patients with TS. Copyright © 2015 Elsevier Inc. All rights reserved.

Characterization of currently marketed heparin products: key tests for quality assurance.

PubMed

Keire, David A; Ye, Hongping; Trehy, Michael L; Ye, Wei; Kolinski, Richard E; Westenberger, Benjamin J; Buhse, Lucinda F; Nasr, Moheb; Al-Hakim, Ali

2011-01-01

During the 2007-2008 heparin crisis, it was found that the United States Pharmacopeia (USP) testing monograph for unfractionated heparin sodium (UFH) did not detect the presence of the contaminant, oversulfated chondroitin sulfate (OSCS) in heparin. In response to this concern, new tests and specifications were developed by the Food and Drug Administration (FDA) and USP and put in place to not only detect the contaminant OSCS but also to improve assurance of quality and purity of the drug product. Additional tests were also developed to monitor the heparin supply chain for other possible economically motivated additives or impurities. In 2009, a new USP monograph was put in place that includes 500 MHz (1)H NMR, SAX-HPLC, %galactosamine in total hexosamine, and anticoagulation time assays with purified factor IIa or factor Xa. These tests represent orthogonal approaches for UFH identification, measurement of bioactivity, and for detection of process impurities or contaminants in UFH. The FDA has applied these analytical approaches to the study of UFH active pharmaceutical ingredients in the marketplace. Here, we describe results from a comprehensive survey of UFH collected from seven different sources after the 2009 monograph revision and compare these data with results obtained on other heparin samples collected during the 2007-2008 crisis.

Performance Equivalence and Validation of the Soleris Automated System for Quantitative Microbial Content Testing Using Pure Suspension Cultures.

PubMed

Limberg, Brian J; Johnstone, Kevin; Filloon, Thomas; Catrenich, Carl

2016-09-01

Using United States Pharmacopeia-National Formulary (USP-NF) general method <1223> guidance, the Soleris(®) automated system and reagents (Nonfermenting Total Viable Count for bacteria and Direct Yeast and Mold for yeast and mold) were validated, using a performance equivalence approach, as an alternative to plate counting for total microbial content analysis using five representative microbes: Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Candida albicans, and Aspergillus brasiliensis. Detection times (DTs) in the alternative automated system were linearly correlated to CFU/sample (R(2) = 0.94-0.97) with ≥70% accuracy per USP General Chapter <1223> guidance. The LOD and LOQ of the automated system were statistically similar to the traditional plate count method. This system was significantly more precise than plate counting (RSD 1.2-2.9% for DT, 7.8-40.6% for plate counts), was statistically comparable to plate counting with respect to variations in analyst, vial lots, and instruments, and was robust when variations in the operating detection thresholds (dTs; ±2 units) were used. The automated system produced accurate results, was more precise and less labor-intensive, and met or exceeded criteria for a valid alternative quantitative method, consistent with USP-NF general method <1223> guidance.

Comparative stability of repackaged metoprolol tartrate tablets.

PubMed

Yang, Yongsheng; Gupta, Abhay; Carlin, Alan S; Faustino, Patrick J; Lyon, Robbe C; Ellison, Christopher D; Rothman, Barry; Khan, Mansoor A

2010-01-29

The stability of metoprolol tartrate tablets packaged in original high density polyethylene containers and repackaged in USP Class A unit-dose blister packs was investigated. Studies were conducted at 25 degrees C/60% relative humidity (RH) for 52 weeks and at 40 degrees C/75% RH for 13 weeks. The potency, dissolution, water content, loss on drying and hardness of the drug products were analyzed. Results indicated no differences in the stability between the tablets in both packages stored under 25 degrees C/60% RH. No difference in potency was found in both packages under either condition. However, a significant weight increase due to moisture uptake was observed for the repackaged tablets stored under 40 degrees C/75% RH. The weight increase was accompanied by a decrease in tablet hardness (6.5-0 kp) and a increase in dissolution rate (51-92%) in 5 min. Near-infrared (NIR) chemical imaging also monitored moisture uptake of the tablet non-invasively through the package. The observed changes in product stability may adversely affect the products bioavailability profile, even though the potency of the active drug remained within USP specification range of 90-110%. Study results suggest product quality can be negatively impacted even when using USP Class A repackaging materials. Published by Elsevier B.V.

Effects of suppression and acceptance of sadness on the urge for non-suicidal self-injury and self-punishment.

PubMed

Svaldi, Jennifer; Dorn, Christina; Matthies, Swantje; Philipsen, Alexandra

2012-12-30

The present study wanted to test the course of the urge for non-suicidal self-injury (UNSSI) and the urge for self-punishment (USP) when suppressing or accepting upcoming emotions in response to a sadness-inducing film clip in female participants with borderline personality disorder (BPD). Thirty-six women with BPD were allocated either to a condition in which they were asked to engage in expressive suppression or acceptance while watching a sadness-inducing film clip. Ratings of UNSSI, USP, and positive and negative emotions were assessed prior to the clip (baseline), immediately after it (t1) and after a 5min waiting period (t2), during which participants viewed landscape pictures. Additionally, physiological measures were obtained. Main results revealed a significant increase in UNSSI from baseline to t2 in the acceptance, but not in the suppression group. Furthermore, USP scores significantly increased from baseline to t2 in the acceptance, but not in the suppression condition. However, there was no differential impact on the sympathetic and parasympathetic branch depending on strategy. The results are in line with recent literature showing that expressive suppression in BPD may also have an adaptive function. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Development and application of a biorelevant dissolution method using USP apparatus 4 in early phase formulation development.

PubMed

Fang, Jiang B; Robertson, Vivian K; Rawat, Archana; Flick, Tawnya; Tang, Zhe J; Cauchon, Nina S; McElvain, James S

2010-10-04

Dissolution testing is frequently used to determine the rate and extent at which a drug is released from a dosage form, and it plays many important roles throughout drug product development. However, the traditional dissolution approach often emphasizes its application in quality control testing and usually strives to obtain 100% drug release. As a result, dissolution methods are not necessarily biorelevant and meaningful application of traditional dissolution methods in the early phases of drug product development can be very limited. This article will describe the development of a biorelevant in vitro dissolution method using USP apparatus 4, biorelevant media, and real-time online UV analysis. Several case studies in the areas of formulation selection, lot-to-lot variability, and food effect will be presented to demonstrate the application of this method in early phase formulation development. This biorelevant dissolution method using USP apparatus 4 provides a valuable tool to predict certain aspects of the in vivo drug release. It can be used to facilitate the formulation development/selection for pharmacokinetic (PK) and clinical studies. It may also potentially be used to minimize the number of PK studies, and to aid in the design of more efficient PK and clinical studies.

Chemical-free inactivated whole influenza virus vaccine prepared by ultrashort pulsed laser treatment

NASA Astrophysics Data System (ADS)

Tsen, Shaw-Wei David; Donthi, Nisha; La, Victor; Hsieh, Wen-Han; Li, Yen-Der; Knoff, Jayne; Chen, Alexander; Wu, Tzyy-Choou; Hung, Chien-Fu; Achilefu, Samuel; Tsen, Kong-Thon

2015-05-01

There is an urgent need for rapid methods to develop vaccines in response to emerging viral pathogens. Whole inactivated virus (WIV) vaccines represent an ideal strategy for this purpose; however, a universal method for producing safe and immunogenic inactivated vaccines is lacking. Conventional pathogen inactivation methods such as formalin, heat, ultraviolet light, and gamma rays cause structural alterations in vaccines that lead to reduced neutralizing antibody specificity, and in some cases, disastrous T helper type 2-mediated immune pathology. We have evaluated the potential of a visible ultrashort pulsed (USP) laser method to generate safe and immunogenic WIV vaccines without adjuvants. Specifically, we demonstrate that vaccination of mice with laser-inactivated H1N1 influenza virus at about a 10-fold lower dose than that required using conventional formalin-inactivated influenza vaccines results in protection against lethal H1N1 challenge in mice. The virus, inactivated by the USP laser irradiation, has been shown to retain its surface protein structure through hemagglutination assay. Unlike conventional inactivation methods, laser treatment did not generate carbonyl groups in protein, thereby reducing the risk of adverse vaccine-elicited T helper type 2 responses. Therefore, USP laser treatment is an attractive potential strategy to generate WIV vaccines with greater potency and safety than vaccines produced by current inactivation techniques.

A prognostic mutation panel for predicting cancer recurrence in stages II and III colorectal cancer.

PubMed

Sho, Shonan; Court, Colin M; Winograd, Paul; Russell, Marcia M; Tomlinson, James S

2017-12-01

Approximately 20-40% of stage II/III colorectal cancer (CRC) patients develop relapse. Clinicopathological factors alone are limited in detecting these patients, resulting in potential under/over-treatment. We sought to identify a prognostic tumor mutational profile that could predict CRC recurrence. Whole-exome sequencing data were obtained for 207 patients with stage II/III CRC from The Cancer Genome Atlas. Mutational landscape in relapse-free versus relapsed cohort was compared using Fisher's exact test, followed by multivariate Cox regression to identify genes associated with cancer recurrence. Bootstrap-validation was used to examine internal/external validity. We identified five prognostic genes (APAF1, DIAPH2, NTNG1, USP7, and VAV2), which were combined to form a prognostic mutation panel. Patients with ≥1 mutation(s) within this five-gene panel had worse prognosis (3-yr relapse-free survival [RFS]: 53.0%), compared to patients with no mutation (3-yr RFS: 84.3%). In multivariate analysis, the five-gene panel remained prognostic for cancer recurrence independent of stage and high-risk features (hazard ratio 3.63, 95%CI [1.93-6.83], P < 0.0001). Furthermore, its prognostic accuracy was superior to the American Joint Commission on Cancer classification (concordance-index: 0.70 vs 0.54). Our proposed mutation panel identifies CRC patients at high-risk for recurrence, which may help guide adjuvant therapy and post-operative surveillance protocols. © 2017 Wiley Periodicals, Inc.

Genome-wide Association Study Identifies Candidate Genes for Male Fertility Traits in Humans

PubMed Central

Kosova, Gülüm; Scott, Nicole M.; Niederberger, Craig; Prins, Gail S.; Ober, Carole

2012-01-01

Despite the fact that hundreds of genes are known to affect fertility in animal models, relatively little is known about genes that influence natural fertility in humans. To broadly survey genes contributing to variation in male fertility, we conducted a genome-wide association study (GWAS) of two fertility traits (family size and birth rate) in 269 married men who are members of a founder population of European descent that proscribes contraception and has large family sizes. Associations between ∼250,000 autosomal SNPs and the fertility traits were examined. A total of 41 SNPs with p ≤ 1 × 10−4 for either trait were taken forward to a validation study of 123 ethnically diverse men from Chicago who had previously undergone semen analyses. Nine (22%) of the SNPs associated with reduced fertility in the GWAS were also associated with one or more of the ten measures of reduced sperm quantity and/or function, yielding 27 associations with p values < 0.05 and seven with p values < 0.01 in the validation study. On the basis of 5,000 permutations of our data, the probabilities of observing this many or more small p values were 0.0014 and 5.6 × 10−4, respectively. Among the nine associated loci, outstanding candidates for male fertility genes include USP8, an essential deubiquitinating enzyme that has a role in acrosome assembly; UBD and EPSTI1, which have potential roles in innate immunity; and LRRC32, which encodes a latent transforming growth factor β (TGF-β) receptor on regulatory T cells. We suggest that mutations in these genes that are more severe may account for some of the unexplained infertility (or subfertility) in the general population. PMID:22633400

Early bovine embryos regulate oviduct epithelial cell gene expression during in vitro co-culture.

PubMed

Schmaltz-Panneau, Barbara; Cordova, Amanda; Dhorne-Pollet, Sophie; Hennequet-Antier, Christelle; Uzbekova, Sveltlana; Martinot, Emmanuelle; Doret, Sarah; Martin, Patrice; Mermillod, Pascal; Locatelli, Yann

2014-10-01

In mammals, the oviduct may participate to the regulation of early embryo development. In vitro co-culture of early bovine embryos with bovine oviduct epithelial cells (BOEC) has been largely used to mimic the maternal environment. However, the mechanisms of BOEC action have not been clearly elucidated yet. The aim of this study was to determine the response of BOEC cultures to the presence of developing bovine embryos. A 21,581-element bovine oligonucleotide array was used compare the gene expression profiles of confluent BOEC cultured for 8 days with or without embryos. This study revealed 34 differentially expressed genes (DEG). Of these 34 genes, IFI6, ISG15, MX1, IFI27, IFI44, RSAD2, IFITM1, EPSTI1, USP18, IFIT5, and STAT1 expression increased to the greatest extent due to the presence of embryos with a major impact on antiviral and immune response. Among the mRNAs at least 25 are already described as induced by interferons. In addition, transcript levels of new candidate genes involved in the regulation of transcription, modulation of the maternal immune system and endometrial remodeling were found to be increased. We selected 7 genes and confirmed their differential expression by quantitative RT-PCR. The immunofluorescence imaging of cellular localization of STAT1 protein in BOEC showed a nuclear translocation in the presence of embryos, suggesting the activation of interferon signaling pathway. This first systematic study of BOEC transcriptome changes in response to the presence of embryos in cattle provides some evidences that these cells are able to adapt their transcriptomic profile in response to embryo signaling. Copyright © 2014 Elsevier B.V. All rights reserved.

Opioid vs nonopioid prescribers: Variations in care for a standardized acute back pain case.

PubMed

Hanley, Kathleen; Zabar, Sondra; Altshuler, Lisa; Lee, Hillary; Ross, Jasmine; Rivera, Nicomedes; Marvilli, Christian; Gillespie, Colleen

2017-01-01

Opioid analgesics are effective and appropriate therapy for many types of acute pain. Epidemiologic evidence supports a direct relationship between increased opioid prescribing and increases in opioid use disorders and overdoses. To tailor our residency curriculum, we designed and fielded an unannounced standardized patient (USP) case involving a patient with acute back pain who is requesting Vicodin (5/325 mg). We describe residents' case management and examine whether their management decisions, including opioid prescribing, were related to their core clinical skills. Results are based on 50 (USP) visits with residents in 2 urban primary care clinics. Highly trained USPs portrayed a patient with acute lower back pain who was taking leftover Vicodin with effective pain relief but was running out. We describe how residents managed this case, using both USP report and chart review data, and compare summary clinical skills scores between those who prescribed Vicodin and those who did not. Of the 50 residents, 18 prescribed Vicodin (10-60 pills). Among those who did not prescribe (32/50), most (50%) prescribed ibuprofen. Eighty-three percent of the prescribers and 72% of nonprescribers ordered physical therapy (nonsignificant). Of the 18 prescribers, 13 documented checking the prescription monitoring database. Prescribers had significantly better communication scores than nonprescribers (relationship development: 80% vs. 58% well done, P = .029; patient education: 59% vs. 31% well done, P = .018). Assessment summary scores were also higher (60% vs. 46%) but not significantly (P = .060). Patient satisfaction and activation scores were higher in the prescribers than nonprescribers (71% vs. 39%, P = .004 and 48% vs. 26%, P = .034, respectively). Most Vicodin prescribers did not follow prescribing guidelines, and they demonstrated better communication and assessment skills than the nonprescribers. Results suggest the need to guide residents in using a systematic approach to prescribing opioids safely and to develop an acceptable alternative pain management plan when they decide against prescribing.

Development and validation of an ICP-OES method for quantitation of elemental impurities in tablets according to coming US pharmacopeia chapters.

PubMed

Støving, Celina; Jensen, Henrik; Gammelgaard, Bente; Stürup, Stefan

2013-10-01

May 1, 2014 the United States Pharmacopeia (USP) will implement two new chapters stating limit concentrations of elemental impurities in pharmaceuticals applying inductively coupled plasma methods. In the present work an inductively coupled plasma optical emission spectrometry (ICP-OES) method for quantitation of As, Cd, Cu, Cr, Fe, Hg, Ir, Mn, Mo, Ni, Os, Pb, Pd, Pt, Rh, Ru, V and Zn in tablets according to the new USP chapters was developed. Sample preparation was performed by microwave-assisted acid digestion using a mixture of 65% HNO3 and 37% HCl (3:1, v/v). Limits of detection and quantitation were at least a factor of ten below the USP limit concentrations showing that the ICP-OES technique is well suited for quantitation of elemental impurities. Excluding Os, spike recoveries in the range of 85.3-103.8% were obtained with relative standard deviations (%RSD) ranging from 1.3 to 3.2%. Due to memory effects the spike recovery and %RSD of Os were 161.5% and 13.7%, respectively, thus the method will need further development with respect to elimination of the memory effect of Os. The method was proven to be specific but with potential spectral interference for Ir, Os, Pb, Pt and Rh necessitating visual examination of the spectra. Hg memory effect was handled by using lower spike levels combined with rinsing with 0.1M HCl. The tablets had a content of Fe and Pt of 182.8 ± 18.1 and 2.8 ± 0.2 μg/g, respectively and did therefore not exceed the limit concentration defined by USP. It is suggested that the developed method is applicable to pharmaceutical products with a composition and maximal amount of daily intake (g drug product/day) similar to the tablets used in this work. Copyright © 2013 Elsevier B.V. All rights reserved.

An integrated assessment of soil erosion dynamics with special emphasis on gully erosion: Case studies from South Africa and Iran

NASA Astrophysics Data System (ADS)

Maerker, Michael; Sommer, Christian; Zakerinejad, Reza; Cama, Elena

2017-04-01

Soil erosion by water is a significant problem in arid and semi arid areas of large parts of Iran. Water erosion is one of the most effective phenomena that leads to decreasing soil productivity and pollution of water resources. Especially in semiarid areas like in the Mazayjan watershed in the Southwestern Fars province as well as in the Mkomazi catchment in Kwa Zulu Natal, South Africa, gully erosion contributes to the sediment dynamics in a significant way. Consequently, the intention of this research is to identify the different types of soil erosion processes acting in the area with a stochastic approach and to assess the process dynamics in an integrative way. Therefore, we applied GIS, and satellite image analysis techniques to derive input information for the numeric models. For sheet and rill erosion the Unit Stream Power-based Erosion Deposition Model (USPED) was utilized. The spatial distribution of gully erosion was assessed using a statistical approach which used three variables (stream power index, slope, and flow accumulation) to predict the spatial distribution of gullies in the study area. The eroded gully volumes were estimated for a multiple years period by fieldwork and Google Earth high resolution images as well as with structure for motion algorithm. Finally, the gully retreat rates were integrated into the USPED model. The results show that the integration of the SPI approach to quantify gully erosion with the USPED model is a suitable method to qualitatively and quantitatively assess water erosion processes in data scarce areas. The application of GIS and stochastic model approaches to spatialize the USPED model input yield valuable results for the prediction of soil erosion in the test areas. The results of this research help to develop an appropriate management of soil and water resources in the study areas.

Initial load-to-failure and failure analysis in single- and double-row repair techniques for rotator cuff repair

PubMed Central

Buchhorn, G. H.; Gilbert, F.; Spahn, G.; Schultz, W.; Klinger, H.-M.

2010-01-01

Aim This experimental study aimed to compare the load-to-failure rate and stiffness of single- versus double-row suture techniques for repairing rotator cuff lesions using two different suture materials. Additionally, the mode of failure of each repair was evaluated. Method In 32 sheep shoulders, a standardized tear of the infraspinatus tendon was created. Then, n = 8 specimen were randomized to four repair methods: (1) Double-row Anchor Ethibond® coupled with polyester sutures, USP No. 2; (2) Double-Row Anchor HiFi® with polyblend polyethylene sutures, USP No. 2; (3) Single-Row Anchor Ethibond® coupled with braided polyester sutures, USP No. 2; and (4) Single-Row Anchor HiFi® with braided polyblend polyethylene sutures, USP No. 2. Arthroscopic Mason–Allen stitches were placed (single-row) and combined with medial horizontal mattress stitches (double-row). All specimens were loaded to failure at a constant displacement rate on a material testing machine. Results Group 4 showed lowest load-to-failure result with 155.7 ± 31.1 N compared to group 1 (293.4 ± 16.1 N) and group 2 (397.7 ± 7.4 N) (P < 0.001). Stiffness was highest in group 2 (162 ± 7.3 N/mm) and lowest in group 4 (84.4 ± 19.9 mm) (P < 0.001). In group 4, the main cause of failure was due to the suture cutting through the tendon (n = 6), a failure case observed in only n = 1 specimen in group 2 (P < 0.001). Conclusions A double-row technique combined with arthroscopic Mason-Allen/horizontal mattress stitches provides high initial failure strength and may minimize the risk of the polyethylene sutures cutting through the tendon in rotator cuff repair when a single load force is used. PMID:20049605

Collaborative study for the establishment of the WHO 3(rd) International Standard for Endotoxin, the Ph. Eur. endotoxin biological reference preparation batch 5 and the USP Reference Standard for Endotoxin Lot H0K354.

PubMed

Findlay, L; Desai, T; Heath, A; Poole, S; Crivellone, M; Hauck, W; Ambrose, M; Morris, T; Daas, A; Rautmann, G; Buchheit, K H; Spieser, J M; Terao, E

2015-01-01

An international collaborative study was organised jointly by the World Health Organization (WHO)/National Institute for Biological Standards and Control (NIBSC), the United States Pharmacopeia (USP) and the European Directorate for the Quality of Medicines & HealthCare (EDQM/Council of Europe) for the establishment of harmonised replacement endotoxin standards for these 3 organisations. Thirty-five laboratories worldwide, including Official Medicines Control Laboratories (OMCLs) and manufacturers enrolled in the study. Three candidate preparations (10/178, 10/190 and 10/196) were produced with the same material and same formulation as the current reference standards with the objective of generating a new (3(rd)) International Standard (IS) with the same potency (10 000 IU/vial) as the current (2(nd)) IS, as well as new European Pharmacopoeia (Ph. Eur.). and USP standards. The suitability of the candidate preparations to act as the reference standard in assays for endotoxin performed according to compendial methods was evaluated. Their potency was calibrated against the WHO 2(nd) IS for Endotoxin (94/580). Gelation and photometric methods produced similar results for each of the candidate preparations. The overall potency estimates for the 3 batches were comparable. Given the intrinsic assay precision, the observed differences between the batches may be considered unimportant for the intended use of these materials. Overall, these results were in line with those generated for the establishment of the current preparations of reference standards. Accelerated degradation testing of vials stored at elevated temperatures supported the long-term stability of the 3 candidate preparations. It was agreed between the 3 organisations that batch 10/178 be shared between WHO and EDQM and that batches 10/190 and 10/196 be allocated to USP, with a common assigned value of 10 000 IU/vial. This value maintains the continuity of the global harmonisation of reference materials and unitage for the testing of endotoxins in parenteral pharmaceutical products. Based on the results of the collaborative study, batch 10/178 was established by the European Pharmacopoeia Commission as the Ph. Eur. Endotoxin Biological Reference Preparation (BRP) batch 5. The same batch was also established by the Expert Committee on Biological Standardisation (ECBS) of WHO as the WHO 3(rd) IS for Endotoxin. Batch 10/190 was adopted as the USP Endotoxin Reference Standard, lot H0K354 and vials from this same batch (10/190) will serve as the United States Food and Drug Administration (USFDA) Endotoxin Standard, EC-7.

Equivalence studies for complex active ingredients and dosage forms.

PubMed

Bhattycharyya, Lokesh; Dabbah, Roger; Hauck, Walter; Sheinin, Eric; Yeoman, Lynn; Williams, Roger

2005-11-17

This article examines the United States Pharmacopeia (USP) and its role in assessing the equivalence and inequivalence of biological and biotechnological drug substances and products-a role USP has played since its founding in 1820. A public monograph in the United States Pharmacopeia-National Formulary helps practitioners and other interested parties understand how an article's strength, quality, and purity should be controlled. Such a monograph is a standard to which all manufactured ingredients and products should conform, and it is a starting point for subsequent-entry manufacturers, recognizing that substantial additional one-time characterization studies may be needed to document equivalence. Review of these studies is the province of the regulatory agency, but compendial tests can provide clarity and guidance in the process.

Harvey Cushing Treated the First Known Patient With Carney Complex

PubMed Central

Tsay, Cynthia J.; Stratakis, Constantine A.; Faucz, Fabio Rueda; London, Edra; Stathopoulou, Chaido; Allgauer, Michael; Quezado, Martha; Dagradi, Terry; Spencer, Dennis D.

2017-01-01

Context: Carney complex (CNC) is a syndrome characterized by hyperplasia of endocrine organs and may present with clinical features of Cushing syndrome and acromegaly due to functional adrenal and pituitary gland tumors. CNC has been linked to mutations in the regulatory subunit of protein kinase A type I-alpha (PRKAR1A) gene. Design: Tissue samples were taken from the hypothalamus or thalamus or tumors of patients with pituitary adenomas seen and operated on by neurosurgeon Harvey Cushing between 1913 and 1932. Following DNA extraction, sequencing for genes of interest was attempted, including PRKAR1A, AIP, USP8, GNAS1, and GPR101, to explore the possibility that these mutations associated with acromegaly, CNC, and Cushing syndrome have been conserved over time. Results: We report a patient described by Dr. Cushing in 1914 with a clinical presentation and postmortem findings suggestive of CNC. Genetic sequencing of the hypothalamus and pituitary adenoma revealed a germline heterozygous p.Arg74His mutation in the PRKAR1A gene, a codon previously described as mutated in CNC, but with a novel amino acid change. Conclusions: This patient is, to our knowledge, the first molecularly confirmed individual with CNC. This case demonstrates the power of modern genetics in studying archived tissues and the importance of recording detailed clinical notes in the diagnosis of disease. PMID:29264456

FnrL and Three Dnr Regulators Are Used for the Metabolic Adaptation to Low Oxygen Tension in Dinoroseobacter shibae

PubMed Central

Ebert, Matthias; Laaß, Sebastian; Thürmer, Andrea; Roselius, Louisa; Eckweiler, Denitsa; Daniel, Rolf; Härtig, Elisabeth; Jahn, Dieter

2017-01-01

The heterotrophic marine bacterium Dinoroseobacter shibae utilizes aerobic respiration and anaerobic denitrification supplemented with aerobic anoxygenic photosynthesis for energy generation. The aerobic to anaerobic transition is controlled by four Fnr/Crp family regulators in a unique cascade-type regulatory network. FnrL is utilizing an oxygen-sensitive Fe-S cluster for oxygen sensing. Active FnrL is inducing most operons encoding the denitrification machinery and the corresponding heme biosynthesis. Activation of gene expression of the high oxygen affinity cbb3-type and repression of the low affinity aa3-type cytochrome c oxidase is mediated by FnrL. Five regulator genes including dnrE and dnrF are directly controlled by FnrL. Multiple genes of the universal stress protein (USP) and cold shock response are further FnrL targets. DnrD, most likely sensing NO via a heme cofactor, co-induces genes of denitrification, heme biosynthesis, and the regulator genes dnrE and dnrF. DnrE is controlling genes for a putative Na+/H+ antiporter, indicating a potential role of a Na+ gradient under anaerobic conditions. The formation of the electron donating primary dehydrogenases is coordinated by FnrL and DnrE. Many plasmid encoded genes were DnrE regulated. DnrF is controlling directly two regulator genes including the Fe-S cluster biosynthesis regulator iscR, genes of the electron transport chain and the glutathione metabolism. The genes for nitrate reductase and CO dehydrogenase are repressed by DnrD and DnrF. Both regulators in concert with FnrL are inducing the photosynthesis genes. One of the major denitrification operon control regions, the intergenic region between nirS and nosR2, contains one Fnr/Dnr binding site. Using regulator gene mutant strains, lacZ-reporter gene fusions in combination with promoter mutagenesis, the function of the single Fnr/Dnr binding site for FnrL-, DnrD-, and partly DnrF-dependent nirS and nosR2 transcriptional activation was shown. Overall, the unique regulatory network of the marine bacterium D. shibae for the transition from aerobic to anaerobic growth composed of four Crp/Fnr family regulators was elucidated. PMID:28473807

Characterization of currently marketed heparin products: key tests for LMWH quality assurance.

PubMed

Ye, Hongping; Toby, Timothy K; Sommers, Cynthia D; Ghasriani, Houman; Trehy, Michael L; Ye, Wei; Kolinski, Richard E; Buhse, Lucinda F; Al-Hakim, Ali; Keire, David A

2013-11-01

During the 2007-2008 heparin crisis it was found that the United States Pharmacopeia (USP) testing monograph for heparin sodium or low molecular weight heparins did not detect the presence of the contaminant, oversulfated chondroitin sulfate (OSCS). In response to this concern, new tests and specifications were developed by the Food and Drug Administration (FDA) and USP and put in place to detect not only the contaminant OSCS, but also to improve assurance of quality and purity of these drug products. The USP monographs for the low molecular weight heparins (LMWHs) approved for use in the United States (dalteparin, tinzaparin and enoxaparin) are also undergoing revision to include many of the same tests used for heparin sodium, including; one-dimensional (1D) 500 MHz (1)H NMR, SAX-HPLC, percent galactosamine in total hexosamine and anticoagulation time assays with purified Factor IIa or Factor Xa. These tests represent orthogonal approaches for heparin identification, measurement of bioactivity and for detection of process impurities or contaminants in these drug products. Here we describe results from a survey of multiple lots from three types of LMWHs in the US market which were collected after the 2009 heparin sodium monograph revision. In addition, innovator and generic versions of formulated enoxaparin products purchased in 2011 are compared using these tests and found to be highly similar within the discriminating power of the assays applied. Published by Elsevier B.V.

Effect of crospovidone and hydroxypropyl cellulose on carbamazepine in high-dose tablet formulation.

PubMed

Flicker, Felicia; Betz, Gabriele

2012-06-01

The aim of this study was to develop a high-dose tablet formulation of the poorly soluble carbamazepine (CBZ) with sufficient tablet hardness and immediate drug release. A further aim was to investigate the influence of various commercial CBZ raw materials on the optimized tablet formulation. Hydroxypropyl cellulose (HPC-SL) was selected as a dry binder and crospovidone (CrosPVP) as a superdisintegrant. A direct compacted tablet formulation of 70% CBZ was optimized by a 3² full factorial design with two input variables, HPC (0--10%) and CrosPVP (0--5%). Response variables included disintegration time, amount of drug released at 15 and 60 min, and tablet hardness, all analyzed according to USP 31. Increasing HPC-SL together with CrosPVP not only increased tablet hardness but also reduced disintegration time. Optimal condition was achieved in the range of 5--9% HPC and 3--5% CrosPVP, where tablet properties were at least 70 N tablet hardness, less than 1 min disintegration, and within the USP requirements for drug release. Testing the optimized formulation with four different commercial CBZ samples, their variability was still observed. Nonetheless, all formulations conformed to the USP specifications. With the excipients CrosPVP and HPC-SL an immediate release tablet formulation was successfully formulated for high-dose CBZ of various commercial sources.

Heparin sodium compliance to USP monograph: structural elucidation of an atypical 2.18 ppm NMR signal.

PubMed

Mourier, Pierre A J; Guichard, Olivier Y; Herman, Fréderic; Viskov, Christian

2012-01-01

The ¹H nuclear magnetic resonance (NMR) acceptance criteria in the new heparin US Pharmacopeia (USP) monograph do not take into account potential structural modifications responsible for any extra signals observed in ¹H NMR spectra, some purified heparins may be non-compliant under the proposed new USP guidelines and incorrectly classified as unsuitable for pharmaceutical use. Heparins from the "ES" source, containing an extra signal at 2.18 ppm, were depolymerized under controlled conditions using heparinases I, II, and III. The oligosaccharides responsible for the 2.18 ppm signal were enriched using orthogonal chromatographic techniques. After multiple purification steps, we obtained an oligosaccharide mixture containing a highly enriched octasaccharide bearing the structural modification responsible for the extra signal. Following heparinase I depolymerization, a pure tetrasaccharide containing the fingerprint structural modification was isolated for full structural determination. Using 1D and 2D ¹H NMR spectroscopy, the structural moiety responsible for the extra signal at 2.18 ppm was identified as an acetyl group on the heparin backbone, most likely resulting from a very minor manufacturing process side reaction that esterifies the uronic acid at position 3. Such analytical peculiarity has always been present in this heparin source and it was used safety over the years. Copyright © 2012 Elsevier B.V. All rights reserved.

Exome sequencing in Thai patients with familial obesity.

PubMed

Kaewsutthi, S; Santiprabhob, J; Phonrat, B; Tungtrongchitr, A; Lertrit, P; Tungtrongchitr, R

2016-07-14

Obesity is a major worldwide health issue, with increasing prevalence in adults and children from developed and developing countries. Obesity causes several chronic diseases, including cardiovascular and respiratory diseases, osteoarthritis, hypertension, stroke, type II diabetes, obstructive sleep apnea, and several types of cancer. Previous genome-wide association studies have identified several genes associated with obesity, including LEP, LEPR, POMC, PCSK1, FTO, MC3R, MC4R, GNPDA2, TMEM18, QPCTL/GIPR, BDNF, ETV5, MAP2K5/SKOR1, SEC16B, SIM1, and TNKS/MSRA. However, most of these variants are found in the intronic or intergenic regions, making it difficult to elucidate the underlying mechanisms. Therefore, in this study, we performed a whole exome sequencing of the protein-coding regions in the total genome (exome) of two obese and one normal subject belonging to the same Thai family to identify the genes responsible for obesity. We identified 709 functional variants that were differentially expressed between obese and normal subjects; of these, 65 were predicted to be deleterious to protein structure or function. The minor allele frequency of 14 of these genes (ALOX5AP, COL9A2, DEFB126, GDPD4, HCRTR1, MLL3, OPLAH, OR4C45, PRIM2, RXFP2, TIGD6, TRPM8, USP49, and ZNF596) was low, indicating causal variants that could be associated with complex traits or diseases. Genotyping revealed HCRTR1, COL9A2, and TRPM8 to be associated with the regulation of feeding behavior and energy expenditure. These genes constituted a network of pathways, including lipid metabolism, signaling transduction, immune, membrane transport, and gene regulation pathways, and seemed to play important roles in obesity.

[Edith Magalhães Fraenkel: the greatest figure of Brazilian nursing].

PubMed

Oguisso, Taka; Freitas, Genival Fernandes de; Takashi, Magali Hiromi

2013-10-01

The present study aimed to describe and analyze the career of Edith Magalhães Fraenkel and her important legacy to Brazilian nursing. The methodology chosen was a documentary analysis of her areas of participation based on documents of the Historical Cultural Center of Iberian-American School of Nursing of the University of São Paulo (Universidade de São Paulo, USP) and in the Rectory of the same University. The following thematic topics were categorized: preparation for leadership, moving to São Paulo, creation of the School of Nursing of USP, event organization, and unique developments at the end of her career. We conclude that Mrs. Edith Fraenkel is the most important figure of Brazilian nursing, thanks to her immeasurable work as a leader of the profession, nationally and internationally.

Magnetic resonance imaging of tablet dissolution.

PubMed

Nott, Kevin P

2010-01-01

Magnetic resonance imaging (MRI) is the technique of choice for measuring hydration, and its effects, during dissolution of tablets since it non-invasively maps (1)H nuclei associated with 'mobile' water. Although most studies have used MRI systems with high-field superconducting magnets, low-field laboratory-based instruments based on permanent magnet technology are being developed that provide key data for the formulation scientist. Incorporation of dissolution hardware, in particular the United States Pharmacopeia (USP) apparatus 4 flow-through cell, allows measurements under controlled conditions for comparison against other dissolution methods. Furthermore, simultaneous image acquisition and measurement of drug concentration allow direct comparison of the drug release throughout the hydration process. The combination of low-field MRI with USP-4 apparatus provides another tool to aid tablet formulation. Copyright 2009 Elsevier B.V. All rights reserved.

Phases of QCD3 from non-SUSY Seiberg duality and brane dynamics

NASA Astrophysics Data System (ADS)

Armoni, Adi; Niarchos, Vasilis

2018-05-01

We consider a nonsupersymmetric USp Yang-Mills Chern-Simons gauge theory coupled to fundamental flavors. The theory is realised in type-IIB string theory via an embedding in a Hanany-Witten brane configuration which includes an orientifold and antibranes. We argue that the theory admits a magnetic Seiberg dual. Using the magnetic dual we identify dynamics in field theory and brane physics that correspond to various phases, obtaining a better understanding of three-dimensional bosonization and dynamical breaking of flavor symmetry in USp QCD3 theory. In field theory both phases correspond to magnetic "squark" condensation. In string theory, they correspond to open string tachyon condensation and brane reconnection. We also discuss other phases where the magnetic `squark' is massive. Finally, we briefly comment on the case of unitary gauge groups.

A SUMO and ubiquitin code coordinates protein traffic at replication factories.

PubMed

Lecona, Emilio; Fernandez-Capetillo, Oscar

2016-12-01

Post-translational modifications regulate each step of DNA replication to ensure the faithful transmission of genetic information. In this context, we recently showed that deubiquitination of SUMO2/3 and SUMOylated proteins by USP7 helps to create a SUMO-rich and ubiquitin-low environment around replisomes that is necessary to maintain the activity of replication forks and for new origin firing. We propose that a two-flag system mediates the collective concentration of factors at sites of DNA replication, whereby SUMO and Ubiquitinated-SUMO would constitute "stay" or "go" signals respectively for replisome and accessory factors. We here discuss the findings that led to this model, which have implications for the potential use of USP7 inhibitors as anticancer agents. © 2016 WILEY Periodicals, Inc.

Coexpression and Secretion of Endoglucanase and Phytase Genes in Lactobacillus reuteri

PubMed Central

Wang, Lei; Yang, Yuxin; Cai, Bei; Cao, Pinghua; Yang, Mingming; Chen, Yulin

2014-01-01

A multifunctional transgenic Lactobacillus with probiotic characteristics and an ability to degrade β-glucan and phytic acid (phytate) was engineered to improve nutrient utilization, increase production performance and decrease digestive diseases in broiler chickens. The Bacillus subtilis WL001 endoglucanase gene (celW) and Aspergillus fumigatus WL002 phytase gene (phyW) mature peptide (phyWM) were cloned into an expression vector with the lactate dehydrogenase promoter of Lactobacillus casei and the secretion signal peptide of the Lactococcus lactis usp45 gene. This construct was then transformed into Lactobacillus reuteri XC1 that had been isolated from the gastrointestinal tract of broilers. Heterologous enzyme production and feed effectiveness of this genetically modified L. reuteri strain were investigated and evaluated. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed that the molecular mass of phyWM and celW was approximately 48.2 and 55 kDa, respectively, consistent with their predicted molecular weights. Endoglucanase and phytase activities in the extracellular fraction of the transformed L. reuteri culture were 0.68 and 0.42 U/mL, respectively. Transformed L. reuteri improved the feed conversion ratio of broilers from 21 to 42 days of age and over the whole feeding period. However, there was no effect on body weight gain and feed intake of chicks. Transformed L. reuteri supplementation improved levels of ash, calcium and phosphorus in tibiae at day 21 and of phosphorus at day 42. In addition, populations of Escherichia coli, Veillonella spp. and Bacteroides vulgatus were decreased, while populations of Bifidobacterium genus and Lactobacillus spp. were increased in the cecum at day 21. PMID:25050780

Comparative analysis of selected innate immune-related genes following infection of immortal DF-1 cells with highly pathogenic (H5N1) and low pathogenic (H9N2) avian influenza viruses.

PubMed

Liu, Ai-Ling; Li, Yu-Feng; Qi, Wenbao; Ma, Xiu-Li; Yu, Ke-Xiang; Huang, Bing; Liao, Ming; Li, Feng; Pan, Jie; Song, Min-Xun

2015-04-01

H5N1 and H9N2 viruses are important causes of avian influenza in China. H5N1 is typically associated with severe to fatal disease in poultry, while H9N2 is usually associated with mild disease. Differences in viral virulence prompted us to investigate whether innate immune responses would be differentially regulated following infection by H5N1 and H9N2 viruses. To address this hypothesis, expression of a panel of innate immune-related genes including IFN-α, IFN-β, Mx1, OASL, ISG12, IFIT5, IRF7, USP18, SST, and KHSRP in immortal DF-1 cells following H5N1 and H9N2 infection was analyzed and compared by real-time quantitative RT-PCR. Cells infected by either virus overall exhibited a similar expression profile for four ISGs (Mx1, OASL, ISG12, and IFIT5), IFN-α, IFN-β, and SST gene. However, two immune-regulatory genes (IRF7 and KHSRP) were not responsive to highly pathogenic H5N1 infection but were strongly up-regulated in DF-1 cells infected with low pathogenic H9N2 infection. The subtype-dependent host response observed in this study offers new insights into the potential roles of IRF7 and KHSRP in control and modulation of the replication and virulence of different subtypes or strains of avian influenza A virus.

Hydrological performance of extensive green roofs in New York City: observations and multi-year modeling of three full-scale systems

NASA Astrophysics Data System (ADS)

Carson, T. B.; Marasco, D. E.; Culligan, P. J.; McGillis, W. R.

2013-06-01

Green roofs can be an attractive strategy for adding perviousness in dense urban environments where rooftops are a high fraction of the impervious land area. As a result, green roofs are being increasingly implemented as part of urban stormwater management plans in cities around the world. In this study, three full-scale green roofs in New York City (NYC) were monitored, representing the three extensive green roof types most commonly constructed: (1) a vegetated mat system installed on a Columbia University residential building, referred to as W118; (2) a built-in-place system installed on the United States Postal Service (USPS) Morgan general mail facility; and (3) a modular tray system installed on the ConEdison (ConEd) Learning Center. Continuous rainfall and runoff data were collected from each green roof between June 2011 and June 2012, resulting in 243 storm events suitable for analysis ranging from 0.25 to 180 mm in depth. Over the monitoring period the W118, USPS, and ConEd roofs retained 36%, 47%, and 61% of the total rainfall respectively. Rainfall attenuation of individual storm events ranged from 3 to 100% for W118, 9 to 100% for USPS, and 20 to 100% for ConEd, where, generally, as total rainfall increased the per cent of rainfall attenuation decreased. Seasonal retention behavior also displayed event size dependence. For events of 10-40 mm rainfall depth, median retention was highest in the summer and lowest in the winter, whereas median retention for events of 0-10 mm and 40 +mm rainfall depth did not conform to this expectation. Given the significant influence of event size on attenuation, the total per cent retention during a given monitoring period might not be indicative of annual rooftop retention if the distribution of observed event sizes varies from characteristic annual rainfall. To account for this, the 12 months of monitoring data were used to develop a characteristic runoff equation (CRE), relating runoff depth and event size, for each green roof. When applied to Central Park, NYC precipitation records from 1971 to 2010, the CRE models estimated total rainfall retention over the 40 year period to be 45%, 53%, and 58% for the W118, USPS, and ConEd green roofs respectively. Differences between the observed and modeled rainfall retention for W118 and USPS were primarily due to an abnormally high frequency of large events, 50 mm of rainfall or more, during the monitoring period compared to historic precipitation patterns. The multi-year retention rates are a more reliable estimate of annual rainfall capture and highlight the importance of long-term evaluations when reporting green roof performance.

27 CFR 21.59 - Formula No. 32.

Code of Federal Regulations, 2014 CFR

2014-04-01

....Collodion, U.S.P. 311.Ethyl cellulose compounds (dehydration). 332.Processing miscellaneous food products... solutions. 481.Photoengraving and rotogravure solutions and dyes. (2) As a raw material: 522.Ethyl chloride...

27 CFR 21.59 - Formula No. 32.

Code of Federal Regulations, 2013 CFR

2013-04-01

....Collodion, U.S.P. 311.Ethyl cellulose compounds (dehydration). 332.Processing miscellaneous food products... solutions. 481.Photoengraving and rotogravure solutions and dyes. (2) As a raw material: 522.Ethyl chloride...

27 CFR 21.59 - Formula No. 32.

Code of Federal Regulations, 2012 CFR

2012-04-01

....Collodion, U.S.P. 311.Ethyl cellulose compounds (dehydration). 332.Processing miscellaneous food products... solutions. 481.Photoengraving and rotogravure solutions and dyes. (2) As a raw material: 522.Ethyl chloride...

76 FR 40844 - Changes to Move Update Standards

Federal Register 2010, 2011, 2012, 2013, 2014

2011-07-12

... accuracy standard: a. For computerized lists, Coding Accuracy Support System (CASS)- certified address matching software and current USPS City State Product, within a mailer's computer systems or through an...

39 CFR 501.7 - Postage Evidencing System requirements.

Code of Federal Regulations, 2010 CFR

2010-07-01

... on the Postal Service Web site at http://www.usps.com/postagesolutions/programdoc.html or requests..., is: (1) Consistent with the Postal Service's intent to maintain neutrality on religious, social...

39 CFR 501.7 - Postage Evidencing System requirements.

Code of Federal Regulations, 2011 CFR

2011-07-01

... on the Postal Service Web site at http://www.usps.com/postagesolutions/programdoc.html or requests..., is: (1) Consistent with the Postal Service's intent to maintain neutrality on religious, social...

27 CFR 21.41 - Formula No. 13-A.

Code of Federal Regulations, 2014 CFR

2014-04-01

...). 241.Collodion, U.S.P. 331.Processing pectin. 332.Processing other food products. 342.Processing... and preserving solutions. 481.Photoengraving and rotogravure solutions and dyes. (2) As a raw material...

27 CFR 21.41 - Formula No. 13-A.

Code of Federal Regulations, 2012 CFR

2012-04-01

...). 241.Collodion, U.S.P. 331.Processing pectin. 332.Processing other food products. 342.Processing... and preserving solutions. 481.Photoengraving and rotogravure solutions and dyes. (2) As a raw material...

Third-party reimbursement for generic prescription drugs: The prevalence of below-cost reimbursement in an environment of maximum allowable cost-based reimbursement.

PubMed

Murry, Logan; Gerleman, Brandon; Urick, Benjamin; Urmie, Julie

2018-05-31

To examine average prescription gross margin (GM) for prescriptions and to evaluate the prevalence of below-cost reimbursement for generic prescriptions across different third-party payers and therapeutic categories. A retrospective descriptive study using 2015 dispensing data from a single independently owned pharmacy in Iowa. To calculate GM, the pharmacy's actual acquisition cost was subtracted from the third-party reimbursement rate for each generic prescription. The frequency of negative GMs was calculated for the top 6 plans and the top 10 therapeutic categories by prescription volume. A single, independently owned community pharmacy in Iowa. Prescription dispensing records for the pharmacy's largest private and public payers by prescription volume. Gross margins were calculated on a payer and United States Pharmacopeia (USP) medication category level. GM for generic prescriptions reimbursed under cost for specific payers and USP medication categories. The 2015 prescription volume for the study pharmacy was 70,866 prescriptions, of which 88% were generic. For all prescriptions, the mean GM was $6.63 per prescription, and the median GM was $3.49 per prescription. Generic medications had a mean GM of $4.66 (median, $2.86), and brand name medications had a mean GM of $21.83 (median, $16.15). The percentage of generic prescriptions paid below acquisition cost was 15.1% overall and ranged from 4.1% for Iowa Medicaid to 25.9% for one of the private payers. The most common USP medication category by prescription volume was cardiovascular agents, representing 25.2% of generic prescriptions. For the 10.9% of these prescriptions reimbursed below cost, the mean GM was -$6.80. The 2 USP medication categories with the largest negative mean GM for generic prescriptions were analgesics and anticonvulsants, with mean GMs of -$10.10 and -$11.30, respectively. The current maximum allowable cost-based reimbursement system often results in inadequate payment for generic prescription drugs. The amount of underpayment varies substantially by payer and therapeutic class. Copyright © 2018 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Bauru School of Dentistry Tele-Health League: an educational strategy applied to research, teaching and extension among applications in tele-health

PubMed Central

da SILVA, Andressa Sharllene Carneiro; RIZZANTE, Fabio Antonio Piola; PICOLINI, Mirela Machado; de CAMPOS, Karis; CORRÊA, Camila de Castro; FRANCO, Elen Caroline; PARDO-FANTON, Cássia de Souza; BLASCA, Wanderléia Quinhoneiro; BERRETIN-FELIX, Giédre

2011-01-01

Tele-health is more than an innovative alternative; it is an excellent tool that enables access to health and education in health, making it possible to minimize distances, optimize time and reduce costs. Based on these advantages, some Brazilian Universities have used these actions in strategies of education, research and extension, aiming at the application of Tele-health in Brazil. In that way, the Bauru School of Dentistry - University of São Paulo (FOB-USP) has applied the use of information and communication technologies in health by means of a "Tele-Health League" (TL), in order to diagnose, prevent and treat diseases, in addition to educate the population and health services. Objective The present study aims to introduce the characteristics of the Tele-Health League of FOB-USP, as well as the development of its projects. Material and Methods The Tele-Health League consisted as a Diffusion Course approved by the Provost of Culture and Academic Extension of the University of São Paulo. It is composed as a large group enclosing professoriate coordinator, academician principal, contributing professors and league members, those, diversified between undergraduates students, graduated, health employees, technology and information areas. The participant members are evaluated by the presence frequency (minimum of 85%), and by the performance of tests and paperwork about the theoretical content provided. Results In four years of activities, the TLFOB-USP obtained a high satisfaction index (90%), an increased number of vacancies due to the interest to become a member, more commitment of the professors of the University and the accomplishment of association with other Brazilian leagues. It is emphasized that the approval percentage of the course results in approval from approximately half of its members. Also, it is important to identify and repair the causes related to the quitting of some members. Conclusions The results showed that the TLFOB-USP members, adjoining to the professor's participants, develop projects in Tele-helth, in Tele-aid and Tele-education areas, thus resulting in the involvement of the University and the community. PMID:22230993

Bauru School of Dentistry Tele-Health League: an educational strategy applied to research, teaching and extension among applications in tele-health.

PubMed

Silva, Andressa Sharllene Carneiro da; Rizzante, Fabio Antonio Piola; Picolini, Mirela Machado; Campos, Karis de; Corrêa, Camila de Castro; Franco, Elen Caroline; Pardo-Fanton, Cássia de Souza; Blasca, Wanderléia Quinhoneiro; Berretin-Felix, Giédre

2011-01-01

Tele-health is more than an innovative alternative; it is an excellent tool that enables access to health and education in health, making it possible to minimize distances, optimize time and reduce costs. Based on these advantages, some Brazilian Universities have used these actions in strategies of education, research and extension, aiming at the application of Tele-health in Brazil. In that way, the Bauru School of Dentistry - University of São Paulo (FOB-USP) has applied the use of information and communication technologies in health by means of a "Tele-Health League" (TL), in order to diagnose, prevent and treat diseases, in addition to educate the population and health services. The present study aims to introduce the characteristics of the Tele-Health League of FOB-USP, as well as the development of its projects. The Tele-Health League consisted as a Diffusion Course approved by the Provost of Culture and Academic Extension of the University of São Paulo. It is composed as a large group enclosing professoriate coordinator, academician principal, contributing professors and league members, those, diversified between undergraduates students, graduated, health employees, technology and information areas. The participant members are evaluated by the presence frequency (minimum of 85%), and by the performance of tests and paperwork about the theoretical content provided. In four years of activities, the TLFOB-USP obtained a high satisfaction index (90%), an increased number of vacancies due to the interest to become a member, more commitment of the professors of the University and the accomplishment of association with other Brazilian leagues. It is emphasized that the approval percentage of the course results in approval from approximately half of its members. Also, it is important to identify and repair the causes related to the quitting of some members. The results showed that the TLFOB-USP members, adjoining to the professor's participants, develop projects in Tele-helth, in Tele-aid and Tele-education areas, thus resulting in the involvement of the University and the community.

Meta-analysis of Pentacam vs. ultrasound pachymetry in central corneal thickness measurement in normal, post-LASIK or PRK, and keratoconic or keratoconus-suspect eyes.

PubMed

Wu, Wenjing; Wang, Yan; Xu, Lulu

2014-01-01

The aim of this meta-analysis is to evaluate the central corneal thickness (CCT) measurement differences between Pentacam (Oculus Inc., Germany) and Ultrasound Pachymetry (USP) in normal (unoperated eyes , myopic and astigmatic eyes without corneal disease or topographic irregularity), after laser in situ keratomileusis (LASIK) or photorefractive keratectomy (PRK), and keratoconic or keratoconus suspected eyes. We assess whether Pentacam and USP have similar CCT differences in normal, thinner corneas after LASIK or PRK procedures, and kerotoconic or keratoconus suspected eyes. Data sources, including PubMed, Medline, EMBASE, and Cochrane Central Registry of Controlled Trials on the Cochrane Library, were searched to find the relevant studies. Primary outcome measures were CCT measurement between Pentacam and USP. Three groups of eyes were analyzed: normal; LASIK or PRK eyes; and keratoconus suspected or keratoconic eyes. Nineteen studies describing 1,908 eyes were enrolled in the normal group. Pentacam results were 1.47 μm ,95 % confidence interval (CI) -2.32 to 5.27, higher than USP without statistically significant difference (P = 0.45). Nine studies with total 539 eyes were included in the corneas after LASIK or PRK. The mean difference in the CCT measurement with Pentacam and ultrasound pachymetry was 1.03 μm, with the 95 % CI -3.36 to 5.42, there was no statistically difference (P = 0.64). Four studies with a total of 185 eyes were included in the keratoconic eyes or keratoconus-suspect group, however,the mean difference was -6.33 μm (95 % CI -9.17 to-3.49), which was statistically different between Pentacam and ultrasound pachymetry in the CCT measurement (P < 0.0001). Pentacam offers similar CCT results to ultrasound pachymetry in normal eyes, thinner corneas after LASIK or PRK procedures. However, in keratoconic or keratoconus-suspect eyes, Pentacam slightly underestimates the central corneal thickness than does ultrasound pachymetry, which may result from the difficulty in fixation of keratoconic eyes, misalignment of Pentacam and the variation of ultrasonic velocity due to the histological deformation.

ABJ quadrality

NASA Astrophysics Data System (ADS)

Honda, Masazumi; Pang, Yi; Zhu, Yaodong

2017-11-01

We study physical consequences of adding orientifolds to the ABJ triality, which is among 3d N=6 superconformal Chern-Simons theory known as ABJ theory, type IIA string in AdS 4 × ℂℙ3 and N=6 supersymmetric (SUSY) Vasiliev higher spin theory in AdS 4. After adding the orientifolds, it is known that the gauge group of the ABJ theory becomes O( N 1) × USp(2 N 2) while the background of the string theory is replaced by AdS 4 × ℂℙ3/ Z 2, and the supersymmetries in the both theories reduce to N=5 . We propose that adding the orientifolds to the N=6 Vasiliev theory leads to N=5 SUSY Vasiliev theory. It turns out that the N=5 case is more involved because there are two formulations of the N=5 Vasiliev theory with either O or USp internal symmetry. We show that the two N=5 Vasiliev theories can be understood as certain projections of the N=6 Vasiliev theory, which we identify with the orientifold projections in the Vasiliev theory. We conjecture that the O( N 1) × USp(2 N 2) ABJ theory has the two vector model like limits: N 2 ≫ N 1 and N 1 ≫ N 2 which correspond to the semi-classical N=5 Vasiliev theories with O( N 1) and USp(2 N 2) internal symmetries respectively. These correspondences together with the standard AdS/CFT correspondence comprise the ABJ quadrality among the N=5 ABJ theory, string/M-theory and two N=5 Vasliev theories. We provide a precise holographic dictionary for the correspondences by comparing correlation functions of stress tensor and flavor currents. Our conjecture is supported by various evidence such as agreements of the spectra, one-loop free energies and SUSY enhancement on the both sides. We also predict the leading free energy of the N=5 Vasiliev theory from the CFT side. As a byproduct, we give a derivation of the relation between the parity violating phase in the N=6 Vasiliev theory and the parameters in the N=6 ABJ theory, which was conjectured in [1].

75 FR 56922 - Implementation of the Intelligent Mail Package Barcode

Federal Register 2010, 2011, 2012, 2013, 2014

2010-09-17

... the USPS Intelligent Mail strategy. Packages that currently bear barcodes designed to provide delivery... symbology of the barcode; however the elements within the barcode and layout will change. There are several...

75 FR 12123 - Restrictions on Private Carriage of Letters

Federal Register 2010, 2011, 2012, 2013, 2014

2010-03-15

... 320 Advertising; Computer technology. 0 For the reasons set forth above, the Postal Service amends 39... from the Inspection Service or the Manager, Mailing Standards, USPS Headquarters, unless an appeal is...

Ecdysteroid 7,9(11)-dien-6-ones as potential photoaffinity labels for ecdysteroid binding proteins

PubMed Central

Bourne, Pauline C.; Whiting, Pensri; Dhadialla, Tarlochan S.; Hormann, Robert E.; Girault, Jean-Pierre; Harmatha, Juraj; Lafont, René; Dinan, Laurence

2002-01-01

Three ecdysteroid 7,9(11)-dien-7-ones (dacryhainansterone, 25-hydroxydacryhainansterone and kaladasterone) were prepared by dehydration of the corresponding 11a-hydroxy ecdysteroids (ajugasterone C, turkesterone and muristerone A, respectively). The biological activities of the dienones in the Drosophila melanogaster BII cell bioassay, which reflect the affinity for the ecdysteroid receptor complex, showed that the dienones retain high biological activity. Irradiation at 350 nm of the ecdysteroid dienones (100 nM) with bacterially-expressed dipteran and lepidopteran ecdysteroid receptor proteins (DmEcR/DmUSP or CfEcR/CfUSP), followed by loading with [3H]ponasterone A revealed that irradiation of dacryhainansterone or kaladasterone resulted in blocking of >70% of the specific binding sites. Thus, ecdysteroid dienones show considerable potential as photoaffinity analogues for ecdysteroid binding proteins. PMID:15455045

Ultra-short pulse generation in the hybridly mode-locked erbium-doped all-fiber ring laser with a distributed polarizer

NASA Astrophysics Data System (ADS)

Krylov, Alexander A.; Sazonkin, Stanislav G.; Lazarev, Vladimir A.; Dvoretskiy, Dmitriy A.; Leonov, Stanislav O.; Pnev, Alexey B.; Karasik, Valeriy E.; Grebenyukov, Vyacheslav V.; Pozharov, Anatoly S.; Obraztsova, Elena D.; Dianov, Evgeny M.

2015-06-01

We report for the first time to the best of our knowledge on the ultra-short pulse (USP) generation in the dispersion-managed erbium-doped all-fiber ring laser hybridly mode-locked with boron nitride-doped single-walled carbon nanotubes in the co-action with a nonlinear polarization evolution in the ring cavity with a distributed polarizer. Stable 92.6 fs dechirped pulses were obtained via precise polarization state adjustment at a central wavelength of 1560 nm with 11.2 mW average output power, corresponding to the 2.9 kW maximum peak power. We have also observed the laser switching from a USP generation regime to a chirped pulse one with a corresponding pulse-width of 7.1 ps at the same intracavity dispersion.

Levothyroxine sodium revisited: A wholistic structural elucidation approach of new impurities via HPLC-HRMS/MS, on-line H/D exchange, NMR spectroscopy and chemical synthesis.

PubMed

Ruggenthaler, M; Grass, J; Schuh, W; Huber, C G; Reischl, R J

2017-02-20

The structural elucidation of unknown pharmaceutical impurities plays an important role in the quality control of newly developed and well-established active pharmaceutical ingredients (APIs). The United States Pharmacopeia (USP) monograph for the API Levothyroxine Sodium, a synthetic thyroid hormone, features two high pressure liquid chromatography (HPLC) methods using UV-VIS absorption detection to determine organic impurities in the drug substance. The impurity profile of the first USP method ("Procedure 1") has already been extensively studied, however for the second method ("Procedure 2"), which exhibits a significantly different impurity profile, no wholistic structural elucidation of impurities has been performed yet. Applying minor modifications to the chromatographic parameters of USP "Procedure 2" and using various comprehensive structural elucidation methods such as high resolution tandem mass spectrometry with on-line hydrogen-deuterium (H/D) exchange or two-dimensional nuclear magnetic resonance spectroscopy (NMR) we gained new insights about the complex impurity profile of the synthetic thyroid hormone. This resulted in the characterization of 24 compounds previously unknown to literature and the introduction of two new classes of Levothyroxine Sodium impurities. Five novel compounds were unambiguously identified via isolation or synthesis of reference substances and subsequent NMR spectroscopic investigation. Additionally, Collision-Induced Dissociation (CID)-type fragmentation of identified major impurities as well as neutral loss fragmentation patterns of many characterized impurities were discussed. Copyright © 2016 Elsevier B.V. All rights reserved.

Can dosage form-dependent food effects be predicted using biorelevant dissolution tests? Case example extended release nifedipine.

PubMed

Andreas, Cord J; Tomaszewska, Irena; Muenster, Uwe; van der Mey, Dorina; Mueck, Wolfgang; Dressman, Jennifer B

2016-08-01

Food intake is known to have various effects on gastrointestinal luminal conditions in terms of transit times, hydrodynamic forces and/or luminal fluid composition and can therefore affect the dissolution behavior of solid oral dosage forms. The aim of this study was to investigate and detect the dosage form-dependent food effect that has been observed for two extended-release formulations of nifedipine using in vitro dissolution tests. Two monolithic extended release formulations, the osmotic pump Adalat® XL 60mg and matrix-type Adalat® Eins 30mg formulation, were investigated with biorelevant dissolution methods using the USP apparatus III and IV under both simulated prandial states, and their corresponding quality control dissolution method. In vitro data were compared to published and unpublished in vivo data using deconvolution-based in vitro - in vivo correlation (IVIVC) approaches. Quality control dissolution methods tended to overestimate the dissolution rate due to the excessive solubilizing capabilities of the sodium dodecyl sulfate (SDS)-containing dissolution media. Using Level II biorelevant media the dosage form dependent food effect for nifedipine was described well when studied with the USP apparatus III, whereas the USP apparatus IV failed to detect the positive food effect for the matrix-type dosage form. It was demonstrated that biorelevant methods can serve as a useful tool during formulation development as they were able to qualitatively reflect the in vivo data. Copyright © 2016 Elsevier B.V. All rights reserved.

Hydrodynamic investigation of USP dissolution test apparatus II.

PubMed

Bai, Ge; Armenante, Piero M; Plank, Russell V; Gentzler, Michael; Ford, Kenneth; Harmon, Paul

2007-09-01

The USP Apparatus II is the device commonly used to conduct dissolution testing in the pharmaceutical industry. Despite its widespread use, dissolution testing remains susceptible to significant error and test failures, and limited information is available on the hydrodynamics of this apparatus. In this work, laser-Doppler velocimetry (LDV) and computational fluid dynamics (CFD) were used, respectively, to experimentally map and computationally predict the velocity distribution inside a standard USP Apparatus II under the typical operating conditions mandated by the dissolution test procedure. The flow in the apparatus is strongly dominated by the tangential component of the velocity. Secondary flows consist of an upper and lower recirculation loop in the vertical plane, above and below the impeller, respectively. A low recirculation zone was observed in the lower part of the hemispherical vessel bottom where the tablet dissolution process takes place. The radial and axial velocities in the region just below the impeller were found to be very small. This is the most critical region of the apparatus since the dissolving tablet will likely be at this location during the dissolution test. The velocities in this region change significantly over short distances along the vessel bottom. This implies that small variations in the location of the tablet on the vessel bottom caused by the randomness of the tablet descent through the liquid are likely to result in significantly different velocities and velocity gradients near the tablet. This is likely to introduce variability in the test. (c) 2007 Wiley-Liss, Inc. and the American Pharmacists Association.

Development and validation of an HPLC method for tetracycline-related USP monographs.

PubMed

Hussien, Emad M

2014-09-01

A novel reversed-phase HPLC method was developed and validated for the assay of tetracycline hydrochloride and the limit of 4-epianhydrotetracycline hydrochloride impurity in tetracycline hydrochloride commercial bulk and pharmaceutical products. The method employed L1 (3 µm, 150 × 4.6 mm) columns, a mobile phase of 0.1% phosphoric acid and acetonitrile at a flow rate of 1.0 mL/min, and detection at 280 nm. The separation was performed in HPLC gradient mode. Forced degradation studies showed that tetracycline eluted as a spectrally pure peak and was well resolved from its degradation products. The fast degradation of tetracycline hydrochloride and 4-epianhydrotetracycline hydrochloride in solution was retarded by controlling the autosampler temperature at 4 °C and using 0.1% H3 PO4 as diluent. The robustness of the method was tested starting with the maximum variations allowed in the US Pharmacopeia (USP) general chapter Chromatography <621>. The method was linear over the range 80-120% of the assay concentration (0.1 mg/mL) for tetracycline hydrochloride and 50-150% of the acceptance criteria specified in the individual USP monographs for 4-epianhydrotetracycline hydrochloride. The limit of quantification for 4-epianhydrotetracycline hydrochloride was 0.1 µg/mL, 20 times lower than the acceptance criteria. The method was specific, precise, accurate and robust. Copyright © 2014 John Wiley & Sons, Ltd.

Validation of a stability-indicating hydrophilic interaction liquid chromatographic method for the quantitative determination of vitamin k3 (menadione sodium bisulfite) in injectable solution formulation.

PubMed

Ghanem, Mashhour M; Abu-Lafi, Saleh A; Hallak, Hussein O

2013-01-01

A simple, specific, accurate, and stability-indicating method was developed and validated for the quantitative determination of menadione sodium bisulfite in the injectable solution formulation. The method is based on zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) coupled with a photodiode array detector. The desired separation was achieved on the ZIC-HILIC column (250 mm × 4.6 mm, 5 μm) at 25°C temperature. The optimized mobile phase consisted of an isocratic solvent mixture of 200mM ammonium acetate (NH4AC) solution and acetonitrile (ACN) (20:80; v/v) pH-adjusted to 5.7 by glacial acetic acid. The mobile phase was fixed at 0.5 ml/min and the analytes were monitored at 261 nm using a photodiode array detector. The effects of the chromatographic conditions on the peak retention, peak USP tailing factor, and column efficiency were systematically optimized. Forced degradation experiments were carried out by exposing menadione sodium bisulfite standard and the injectable solution formulation to thermal, photolytic, oxidative, and acid-base hydrolytic stress conditions. The degradation products were well-resolved from the main peak and the excipients, thus proving that the method is a reliable, stability-indicating tool. The method was validated as per ICH and USP guidelines (USP34/NF29) and found to be adequate for the routine quantitative estimation of menadione sodium bisulfite in commercially available menadione sodium bisulfite injectable solution dosage forms.

Influence of dissolution media pH and USP1 basket speed on erosion and disintegration characteristics of immediate release metformin hydrochloride tablets.

PubMed

Desai, Divyakant; Wong, Benjamin; Huang, Yande; Tang, Dan; Hemenway, Jeffrey; Paruchuri, Srinivasa; Guo, Hang; Hsieh, Daniel; Timmins, Peter

2015-01-01

To investigate the influence of the pH of the dissolution medium on immediate release 850 mg metformin hydrochloride tablets. A traditional wet granulation method was used to manufacture metformin hydrochloride tablets with or without a disintegrant. Tablet dissolution was conducted using the USP apparatus I at 100 rpm. In spite of its pH-independent high solubility, metformin hydrochloride tablets dissolved significantly slower in 0.1 N HCl (pH 1.2) and 50 mM pH 4.5 acetate buffer compared with 50 mM pH 6.8 phosphate buffer, the dissolution medium in the USP. Metformin hydrochloride API compressed into a round 1200 mg disk showed a similar trend. When basket rotation speed was increased from 100 to 250 rpm, the dissolution of metformin hydrochloride tablets was similar in all three media. Incorporation of 2% w/w crospovidone in the tablet formulation improved the dissolution although the pH-dependent trend was still evident, but incorporation of 2% w/w croscarmellose sodium resulted in rapid pH-independent tablet dissolution. In absence of a disintegrant in the tablet formulation, the dissolution was governed by the erosion-diffusion process. Even for a highly soluble drug, a super-disintegrant was needed in the formulation to overcome the diffusion layer limitation and change the dissolution mechanism from erosion-diffusion to disintegration.

Analysis of drug content and weight uniformity for half-tablets of 6 commonly split medications.

PubMed

Hill, Shaynan W; Varker, Andrew S; Karlage, Kelly; Myrdal, Paul B

2009-04-01

Cost savings can be achieved with the practice of tablet splitting. Previous research has shown weight nonuniformity within tablet halves. However, limited research to date has found that the potential dose inaccuracy resulting from splitting tablets does not significantly affect clinical outcomes. To determine the drug content and weight in split half-tablets of 6 commonly split medications using drug assay analysis. This study was performed by 2 fourth-year pharmacy students using 30 randomly selected tablets of each of the following 6 medications: warfarin sodium 5 milligrams (mg), simvastatin 80 mg, metoprolol succinate 200 mg, metoprolol tartrate 25 mg, citalopram 40 mg, and lisinopril 40 mg. A randomly selected half of the tablets were split by a single pharmacy student using a tablet cutter, and the remaining tablets were kept whole. Drug content was analyzed for 15 whole tablets and 30 half-tablets for each of the 6 drugs using high performance liquid chromatography, an analytical tool used to identify and quantify substances in solution. Drug content uniformity was assessed by comparing drug content within half-tablets with one-half of the drug content mean found for all whole tablets in the sample. Weight uniformity was assessed by comparing half-tablet weights, as determined by a Mettler analytical balance, with one-half of the mean weight for whole tablets in the sample. The percentages by which each whole tablet's or half-tablet's drug content and weight differed from sample mean values were compared with proxy United States Pharmacopeia (USP) specification ranges for drug content (95%-105% for warfarin sodium and 90%-110% for the other 5 drugs). Additionally, these outcomes were compared for nonscored versus scored tablets. The percent relative standard deviation (%RSD, ratio of the standard deviation to the mean), a commonly used measure of the repeatability and precision of assays used to analyze drug content, was also calculated in order to determine whether the drugs met proxy USP specification for %RSD (less than 6% for all drugs studied). A total of 43 of 180 half-tablets (23.9%) differed from sample mean values by a percentage that fell outside of proxy USP specification for drug content; warfarin sodium (11 of 30 half-tablets, 36.7%), simvastatin (3 of 30 half-tablets, 10.0%) metoprolol succinate (10 of 30 half-tablets, 33.3%), metoprolol tartrate (4 of 30 half-tablets, 13.3%), citalopram (5 of 30 half-tablets, 16.7%), and lisinopril (10 of 30 half-tablets, 33.3%). Half-tablets outside of proxy USP specification for weight included warfarin sodium (10 of 30 half-tablets, 33.3%), metoprolol succinate (6 of 30 half-tablets, 20%), and lisinopril (7 of 30 half-tablets, 23.3%). The %RSDs for drug content and weight fell outside of the proxy USP specification for %RSD for metoprolol succinate (drug content = 8.98%, weight = 7.70%) and lisinopril (drug content = 10.41%, weight = 8.13%). Mean percent weight loss after splitting was less than 1% for all drugs except lisinopril, which had an average weight loss of 1.25%. The total numbers of scored (nonscored) tablet halves that fell outside of proxy USP specification were 20 (23) for drug content and 10 (13) for weight. When measuring drug content, the numbers of out-of-range half-tablets for scored (nonscored) drugs were 36 (44) at 95%-105%, 9 (23) at 90%-110%, 0 (10) at 85%-115%, and 0 (1) at 75%-125%. When measuring weight, the numbers of out-of-range half-tablets for scored (nonscored) drugs were 28 (38) at 95%-105%, 0 (14) at 90%-110%, 0 (3) at 85%-115%, and 0 (0) at 75%-125%. Dose variation exceeded a proxy USP specification for more than one-third of sampled half-tablets of warfarin sodium, metoprolol succinate, and lisinopril and appeared to be greater for nonscored tablets as compared with scored tablets. Drug content variation in half-tablets appeared to be attributable primarily to weight variation occurring when tablets powder or fragment during the splitting process. Therefore, equal daily doses will be determined by the ability of patients to split tablets perfectly in half.

E93 predominantly transduces 20-hydroxyecdysone signaling to induce autophagy and caspase activity in Drosophila fat body.

PubMed

Liu, Hanhan; Wang, Jin; Li, Sheng

2014-02-01

During the larval-prepupal transition in Drosophila, a balancing crosstalk occurs between autophagy and caspase activity in the remodeling fat body: the inhibition of autophagy induces caspase activity and the inhibition of caspases induces autophagy. Both autophagy and caspase activity are induced by a pulse of molting hormone (20-hydroxyecdysone, 20E) via the 20E nuclear receptor complex, EcR-USP. We here demonstrate that E93, a 20E primary-response gene encoding an HTH transcription factor, predominantly transduces 20E signaling to induce autophagy and caspase activity in the remodeling fat body. RNAi knockdown or mutation of E93 blocks autophagy and caspase activity, E93 overexpression induces them both, while E93 overexpression has a better rescuing effect on the inhibition of autophagy than caspase activity caused by EcR(DN) overexpression. At the transcriptional level, E93 not only greatly impacts the 20E-triggered transcriptional cascade, but also upregulates essential autophagy and apoptosis genes. Meanwhile, at the phosphorylational level, E93 blocks the PI3K-TORC1 signaling to initiate autophagy. Taken together, we conclude that autophagy and caspase activity are induced by 20E and predominantly transduced by E93 in the remodeling fat body of Drosophila. Copyright © 2013 Elsevier Ltd. All rights reserved.

Comparative study of diagnostic accuracy of established PCR assays and in-house developed sdaA PCR method for detection of Mycobacterium tuberculosis in symptomatic patients with pulmonary tuberculosis.

PubMed

Nimesh, Manoj; Joon, Deepali; Pathak, Anil Kumar; Saluja, Daman

2013-11-01

Indian contribution to global burden of tuberculosis is about 26%. In the present study we have developed an in-house PCR assay using primers for sdaA gene of Mycobacterium tuberculosis and evaluated against already established primers devR, IS6110, MPB64, rpoB primers for diagnosis of pulmonary tuberculosis. Using universal sample preparation (USP) method, DNA was extracted from sputum specimens of 412 symptomatic patients from Delhi, India. The DNA so extracted was used as template for PCR amplification using primers targeting sdaA, devR, IS6110, MPB64 and rpoB genes. Out of 412, 149 specimens were considered positive based on composite reference standard (CRS) criteria. The in-house designed sdaA PCR showed high specificity (96.5%), the high positive likelihood ratio (28), the high sensitivity (95.9%), and the very low negative likelihood ratio (0.04) in comparison to CRS. Based on our results, the sdaA PCR assay can be considered as one of the most reliable diagnostic tests in comparison to other PCR based detection methods. Copyright © 2013 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

Discovery of Metastatic Breast Cancer Suppressor Genes Using Functional Genome Analysis

DTIC Science & Technology

2012-07-01

1.33 0.0378 341 MATR3 ‐1.33 ‐1.33 0.0378 340 MXD4 ‐1.33 ‐1.33 0.0378 339 PCSK1 ‐1.33 ‐1.33 0.0378 338 ZSCAN4 ‐1.33 ‐1.33 0.0378 347 NHP2L1 ‐1.32 ‐1.32...0.0857 1.1299 ‐0.05108 ‐0.4502 ‐0.1334 0.7847 PCSK1 ‐0.6531 ‐0.5028 ‐0.1573 ‐0.4033 ‐0.2718 ‐0.1104 0.2373 0.03041 ‐0.07899 0.4092 0.6217 0.3825...CES1P1 STX3 LYN PAPD5 OSTM1 MXD4 INHBA PFDN1 NAALAD2 TDGF1P3 USP21 GUCY1B2 OR6V1 NUP107 PRSS42 KCNH2 THOC3 THEM5 ZNF484 SFTPA1 46 ZNF780B ZNF653 PCSK1

21 CFR 172.869 - Sucrose oligoesters.

Code of Federal Regulations, 2014 CFR

2014-04-01

... than 40% Do. Do. (5) Free Sucrose Not more than 0.5% “Free Sucrose Method,” issued by Mitsubishi... Convention, 12601 Twinbrook Pkwy., Rockville, MD 20852 (Internet address http://www.usp.org) (7) Residue on...

27 CFR 21.60 - Formula No. 33.

Code of Federal Regulations, 2010 CFR

2010-04-01

... including meat branding inks). 210.External pharmaceuticals, not U.S.P. or N.F. 249.Miscellaneous external...). (c) Conditions governing use. Meat branding inks made with Formula No. 33 do not meet U.S. Department...

Hovercraft Underwater Noise Measurements in Alaska.

DOT National Transportation Integrated Search

2001-04-02

The United States Postal Service (USPS) is finishing a two-year demonstration project : examining the feasibility of mail delivery via hovercraft to remote Alaskan villages. Prior to this : demonstration project, and in support of the Environmental A...

Genome-wide association study identifies candidate genes for male fertility traits in humans.

PubMed

Kosova, Gülüm; Scott, Nicole M; Niederberger, Craig; Prins, Gail S; Ober, Carole

2012-06-08

Despite the fact that hundreds of genes are known to affect fertility in animal models, relatively little is known about genes that influence natural fertility in humans. To broadly survey genes contributing to variation in male fertility, we conducted a genome-wide association study (GWAS) of two fertility traits (family size and birth rate) in 269 married men who are members of a founder population of European descent that proscribes contraception and has large family sizes. Associations between ∼250,000 autosomal SNPs and the fertility traits were examined. A total of 41 SNPs with p ≤ 1 × 10(-4) for either trait were taken forward to a validation study of 123 ethnically diverse men from Chicago who had previously undergone semen analyses. Nine (22%) of the SNPs associated with reduced fertility in the GWAS were also associated with one or more of the ten measures of reduced sperm quantity and/or function, yielding 27 associations with p values < 0.05 and seven with p values < 0.01 in the validation study. On the basis of 5,000 permutations of our data, the probabilities of observing this many or more small p values were 0.0014 and 5.6 × 10(-4), respectively. Among the nine associated loci, outstanding candidates for male fertility genes include USP8, an essential deubiquitinating enzyme that has a role in acrosome assembly; UBD and EPSTI1, which have potential roles in innate immunity; and LRRC32, which encodes a latent transforming growth factor β (TGF-β) receptor on regulatory T cells. We suggest that mutations in these genes that are more severe may account for some of the unexplained infertility (or subfertility) in the general population. Copyright © 2012 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Metabolomic Quantitative Trait Loci (mQTL) Mapping Implicates the Ubiquitin Proteasome System in Cardiovascular Disease Pathogenesis.

PubMed

Kraus, William E; Muoio, Deborah M; Stevens, Robert; Craig, Damian; Bain, James R; Grass, Elizabeth; Haynes, Carol; Kwee, Lydia; Qin, Xuejun; Slentz, Dorothy H; Krupp, Deidre; Muehlbauer, Michael; Hauser, Elizabeth R; Gregory, Simon G; Newgard, Christopher B; Shah, Svati H

2015-11-01

Levels of certain circulating short-chain dicarboxylacylcarnitine (SCDA), long-chain dicarboxylacylcarnitine (LCDA) and medium chain acylcarnitine (MCA) metabolites are heritable and predict cardiovascular disease (CVD) events. Little is known about the biological pathways that influence levels of most of these metabolites. Here, we analyzed genetics, epigenetics, and transcriptomics with metabolomics in samples from a large CVD cohort to identify novel genetic markers for CVD and to better understand the role of metabolites in CVD pathogenesis. Using genomewide association in the CATHGEN cohort (N = 1490), we observed associations of several metabolites with genetic loci. Our strongest findings were for SCDA metabolite levels with variants in genes that regulate components of endoplasmic reticulum (ER) stress (USP3, HERC1, STIM1, SEL1L, FBXO25, SUGT1) These findings were validated in a second cohort of CATHGEN subjects (N = 2022, combined p = 8.4x10-6-2.3x10-10). Importantly, variants in these genes independently predicted CVD events. Association of genomewide methylation profiles with SCDA metabolites identified two ER stress genes as differentially methylated (BRSK2 and HOOK2). Expression quantitative trait loci (eQTL) pathway analyses driven by gene variants and SCDA metabolites corroborated perturbations in ER stress and highlighted the ubiquitin proteasome system (UPS) arm. Moreover, culture of human kidney cells in the presence of levels of fatty acids found in individuals with cardiometabolic disease, induced accumulation of SCDA metabolites in parallel with increases in the ER stress marker BiP. Thus, our integrative strategy implicates the UPS arm of the ER stress pathway in CVD pathogenesis, and identifies novel genetic loci associated with CVD event risk.

Metabolomic Quantitative Trait Loci (mQTL) Mapping Implicates the Ubiquitin Proteasome System in Cardiovascular Disease Pathogenesis

PubMed Central

Kraus, William E.; Muoio, Deborah M.; Stevens, Robert; Craig, Damian; Bain, James R.; Grass, Elizabeth; Haynes, Carol; Kwee, Lydia; Qin, Xuejun; Slentz, Dorothy H.; Krupp, Deidre; Muehlbauer, Michael; Hauser, Elizabeth R.; Gregory, Simon G.; Newgard, Christopher B.; Shah, Svati H.

2015-01-01

Levels of certain circulating short-chain dicarboxylacylcarnitine (SCDA), long-chain dicarboxylacylcarnitine (LCDA) and medium chain acylcarnitine (MCA) metabolites are heritable and predict cardiovascular disease (CVD) events. Little is known about the biological pathways that influence levels of most of these metabolites. Here, we analyzed genetics, epigenetics, and transcriptomics with metabolomics in samples from a large CVD cohort to identify novel genetic markers for CVD and to better understand the role of metabolites in CVD pathogenesis. Using genomewide association in the CATHGEN cohort (N = 1490), we observed associations of several metabolites with genetic loci. Our strongest findings were for SCDA metabolite levels with variants in genes that regulate components of endoplasmic reticulum (ER) stress (USP3, HERC1, STIM1, SEL1L, FBXO25, SUGT1) These findings were validated in a second cohort of CATHGEN subjects (N = 2022, combined p = 8.4x10-6–2.3x10-10). Importantly, variants in these genes independently predicted CVD events. Association of genomewide methylation profiles with SCDA metabolites identified two ER stress genes as differentially methylated (BRSK2 and HOOK2). Expression quantitative trait loci (eQTL) pathway analyses driven by gene variants and SCDA metabolites corroborated perturbations in ER stress and highlighted the ubiquitin proteasome system (UPS) arm. Moreover, culture of human kidney cells in the presence of levels of fatty acids found in individuals with cardiometabolic disease, induced accumulation of SCDA metabolites in parallel with increases in the ER stress marker BiP. Thus, our integrative strategy implicates the UPS arm of the ER stress pathway in CVD pathogenesis, and identifies novel genetic loci associated with CVD event risk. PMID:26540294

78 FR 76296 - National Institute of Neurological Disorders and Stroke, Interagency Pain Research Coordinating...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-12-17

..., ethnicity, gender, sexual orientation, disability, and cultural, religious, or socioeconomic status. The... Drive, Room 8A03, Bethesda, MD 20892, [email protected] by either USPS mail or email. Nominations...

27 CFR 21.53 - Formula No. 27-A.

Code of Federal Regulations, 2012 CFR

2012-04-01

...-five pounds of camphor, U.S.P., and 1 gallon of clove oil, N.F. (b) Authorized uses. (1) As a solvent... other biocides. (2) Miscellaneous uses: 812.Product development and pilot plant uses (own use only). ...

27 CFR 21.53 - Formula No. 27-A.

Code of Federal Regulations, 2011 CFR

2011-04-01

...-five pounds of camphor, U.S.P., and 1 gallon of clove oil, N.F. (b) Authorized uses. (1) As a solvent... other biocides. (2) Miscellaneous uses: 812.Product development and pilot plant uses (own use only). ...

27 CFR 21.53 - Formula No. 27-A.

Code of Federal Regulations, 2010 CFR

2010-04-01

...-five pounds of camphor, U.S.P., and 1 gallon of clove oil, N.F. (b) Authorized uses. (1) As a solvent... other biocides. (2) Miscellaneous uses: 812.Product development and pilot plant uses (own use only). ...

78 FR 32612 - Collect on Delivery (COD)-Service Features

Federal Register 2010, 2011, 2012, 2013, 2014

2013-05-31

..., will retire the current manual PS Form 3849-D, Notice to Sender of Undelivered COD Mail. The primary function served by PS Form 3849-D can be provided by USPS Package Intercept service, which allows mailers...

USPS Retiree Health Benefits Prepayment Extension Act of 2011

THOMAS, 112th Congress

Rep. Cummings, Elijah E. [D-MD-7

2011-09-12

House - 10/03/2011 Referred to the Subcommittee on Federal Workforce, U.S. Postal Service, and Labor Policy . (All Actions) Tracker: This bill has the status IntroducedHere are the steps for Status of Legislation:

[Nursing care time in a teaching hospital].

PubMed

Rogenski, Karin Emília; Fugulin, Fernanda Maria Togeiro; Gaidzinski, Raquel Rapone; Rogenski, Noemi Marisa Brunet

2011-03-01

This is a quantitative exploratory, descriptive study performed with the objective to identify and analyze the performance of the average time of nursing care delivered to patients of the Inpatient Units of the University Hospital at University of São Paulo (UH-USP), from 2001 to 2005. The average nursing care time delivered to patients of the referred units was identified by applying of a mathematical equation proposed in the literature, after surveying data from the Medical and Statistical Service and based on the monthly working shifts of the nursing professionals. Data analysis was performed using descriptive statistics. The average nursing care time observed in most units, despite some variations, remained stable during the analyzed period. Based on this observed stability, it is concluded that the nursing staff in the referred HU-USP units has been continuously evaluated with the purposes of maintaining the average time of assistance and, thus, the quality of the care being delivered.

Protein-linked Ubiquitin Chain Structure Restricts Activity of Deubiquitinating Enzymes*

PubMed Central

Schaefer, Jonathan B.; Morgan, David O.

2011-01-01

The attachment of lysine 48 (Lys48)-linked polyubiquitin chains to proteins is a universal signal for degradation by the proteasome. Here, we report that long Lys48-linked chains are resistant to many deubiquitinating enzymes (DUBs). Representative enzymes from this group, Ubp15 from yeast and its human ortholog USP7, rapidly remove mono- and diubiquitin from substrates but are slow to remove longer Lys48-linked chains. This resistance is lost if the structure of Lys48-linked chains is disrupted by mutation of ubiquitin or if chains are linked through Lys63. In contrast to Ubp15 and USP7, Ubp12 readily cleaves the ends of long chains, regardless of chain structure. We propose that the resistance to many DUBs of long, substrate-attached Lys48-linked chains helps ensure that proteins are maintained free from ubiquitin until a threshold of ubiquitin ligase activity enables degradation. PMID:22072716

IIB supergravity and the E 6(6) covariant vector-tensor hierarchy

DOE PAGES

Ciceri, Franz; de Wit, Bernard; Varela, Oscar

2015-04-20

IIB supergravity is reformulated with a manifest local USp(8) invariance that makes the embedding of five-dimensional maximal supergravities transparent. In this formulation the ten-dimensional theory exhibits all the 27 one-form fields and 22 of the 27 two-form fields that are required by the vector-tensor hierarchy of the five-dimensional theory. The missing 5 two-form fields must transform in the same representation as a descendant of the ten-dimensional ‘dual graviton’. The invariant E 6(6) symmetric tensor that appears in the vector-tensor hierarchy is reproduced. Generalized vielbeine are derived from the supersymmetry transformations of the vector fields, as well as consistent expressions formore » the USp(8) covariant fermion fields. Implications are further discussed for the consistency of the truncation of IIB supergravity compactified on the five-sphere to maximal gauged supergravity in five space-time dimensions with an SO(6) gauge group.« less

USP5/Leon deubiquitinase confines postsynaptic growth by maintaining ubiquitin homeostasis through Ubiquilin.

PubMed

Wang, Chien-Hsiang; Huang, Yi-Chun; Chen, Pei-Yi; Cheng, Ying-Ju; Kao, Hsiu-Hua; Pi, Haiwei; Chien, Cheng-Ting

2017-05-10

Synapse formation and growth are tightly controlled processes. How synaptic growth is terminated after reaching proper size remains unclear. Here, we show that Leon, the Drosophila USP5 deubiquitinase, controls postsynaptic growth. In leon mutants, postsynaptic specializations of neuromuscular junctions are dramatically expanded, including the subsynaptic reticulum, the postsynaptic density, and the glutamate receptor cluster. Expansion of these postsynaptic features is caused by a disruption of ubiquitin homeostasis with accumulation of free ubiquitin chains and ubiquitinated substrates in the leon mutant. Accumulation of Ubiquilin (Ubqn), the ubiquitin receptor whose human homolog ubiquilin 2 is associated with familial amyotrophic lateral sclerosis, also contributes to defects in postsynaptic growth and ubiquitin homeostasis. Importantly, accumulations of postsynaptic proteins cause different aspects of postsynaptic overgrowth in leon mutants. Thus, the deubiquitinase Leon maintains ubiquitin homeostasis and proper Ubqn levels, preventing postsynaptic proteins from accumulation to confine postsynaptic growth.

Rehabilitative treatment of cleft lip and palate: experience of the Hospital for Rehabilitation of Craniofacial Anomalies/USP (HRAC/USP) - Part 5: institutional outcomes assessment and the role of the Laboratory of Physiology.

PubMed

Freitas, José Alberto de Souza; Trindade-Suedam, Ivy Kiemle; Garib, Daniela Gamba; Neves, Lucimara Teixeira das; Almeida, Ana Lúcia Pompéia Fraga de; Yaedú, Renato Yassukata Faria; Oliveira, Thaís Marchini; Soares, Simone; Lauris, Rita de Cássia Moura Carvalho; Yamashita, Renata Paciello; Trindade, Alceu Sergio; Trindade, Inge Elly Kiemle; Pinto, João Henrique Nogueira

2013-01-01

The Laboratory of Physiology provides support for the diagnosis of functional disorders associated with cleft lip and palate and also conducts studies to assess, objectively, the institutional outcomes, as recommended by the World Health Organization. The Laboratory is conceptually divided into three units, namely the Unit for Upper Airway Studies, Unit for Stomatognathic System Studies and the Unit for Sleep Studies, which aims at analyzing the impact of different surgical and dental procedures on the upper airways, stomatognathic system and the quality of sleep of individuals with cleft lip and palate. This paper describes the main goals of the Laboratory in the assessment of procedures which constitute the basis of the rehabilitation of cleft lip and palate, i.e., Plastic Surgery, Orthodontics and Maxillofacial Surgery and Speech Pathology.

Microwave-assisted digestion using nitric acid for heavy metals and sulfated ash testing in active pharmaceutical ingredients.

PubMed

Pluhácek, T; Hanzal, J; Hendrych, J; Milde, D

2016-04-01

The monitoring of inorganic impurities in active pharmaceutical ingredients plays a crucial role in the quality control of the pharmaceutical production. The heavy metals and residue on ignition/sulfated ash methods employing microwave-assisted digestion with concentrated nitric acid have been demonstrated as alternatives to inappropriate compendial methods recommended in United States Pharmacopoeia (USP) and European Pharmacopoeia (Ph. Eur.). The recoveries using the heavy metals method ranged between 89% and 122% for nearly all USP and Ph. Eur. restricted elements as well as the recoveries of sodium sulfate spikes were around 100% in all tested matrices. The proposed microwave-assisted digestion method allowed simultaneous decomposition of 15 different active pharmaceutical ingredients with sample weigh up to 1 g. The heavy metals and sulfated ash procedures were successfully applied to the determination of heavy metals and residue on ignition/sulfated ash content in mycophenolate mofetil, nicergoline and silymarin.